1.

CLINICAL EVALUATION AND ABNORMALITIES OF REPRODUCTIVE TRACT

IN FARM ANIMALS

A thorough examination of the female and her reproductive tract should be done before purchasing for
breeding purpose or prior to each breeding season. It is essentially being carried out for the following
reasons

 Diagnosis of pregnancy
 Estimation of the gestational age
 Characterization of reproductive physiological and pathologic status
 Allows the clinician to predict important events to come such as
o estrus
o ovulation
o parturition
o abortion
 Allows for a rational approach to therapy
 Allows for establishing a prognosis of conditions of the uterus, uterine tubes, ovaries and
supporting structures

COWS AND HEIFERS

HISTORY

History taking can be done simultaneously while the animal is being examined. The important issues
that need to be addressed include the following:

 Parity (virgin heifer, pregnant heifer, uniparous or multiparous cow).

 Age (including age at first calving).
 Cyclic history (normal or abnormal cycle lengths, anestrus, nymphomania).
 Calving dates and comments (dystocia, twins, retained placenta, surgical or mechanical
intervention, viability of calf).
 Breeding dates and methods (artificial insemination or natural service, estrus detection
methods and personnel, semen supplier and quality, previous record of bull fertility, including
examination for venereal disease).
 Previous treatments (drugs, dosages and routes, treatment intervals, clinical outcome, drug
withdrawal disease).
 Nutritional program (periparturient supplementation of beef cows, dry-period feeding of dairy
cows, body conditions of cows at calving, milk production levels).

PHYSICAL EXAMINATION

Visual inspection gives valuable information about the individual animal and will be an aid to genital
examination. Visual inspection involves the following:

 General conformation
 Conformation of the external genitalia
 Vulvar discharges
 Condition of the mammary gland
 General behavior of the animal

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General Conformation

 Cows with a masculine appearance of the head and shoulder region may be suffering
from Cystic ovarian degeneration Freemartin heifers may exhibit steer-like appearance.

Conformation of the External Genitalia

Physiological alterations

 The vulval labia are normally covered with soft, thin skin and are symmetrical and closely
opposed to ensure closure of the vestibule and vagina
 There is high tonicity of the pelvic diaphragm as well as the vulva in a non-pregnant or early
pregnant cow manifested by firmness of the diaphragm and relatively small and wrinkled
appearance of the vulval lips
 During the last trimester of pregnancy these structures undergo gradual but continuous
relaxation.
 The relaxation becomes marked and the vulva nearly doubles in size during the last 10 to 14
days prior to parturition and this extreme relaxation of the pelvic ligaments causes the tail
head to become elevated
 Within 2 weeks following parturition the vulva returns to normal size

Pathological alterations

Pathological alterations involve the following

 Extreme relaxation of the pelvic diaphragm, enlargement of the vulva and elevation of the tail
head in Cystic ovarian degeneration
 Relaxation of the vulval lips alone in cows with long standing cases of metritis
 Extreme swelling of the vulva associated with edema, but with increased tension, is found as
the first sign of Infectious Pustular Vulvovaginitis (IPV)

Discharges from the Vulva

Discharges observed in normal animals

 During estrus, the vulva may appear edematous with presence of characteristic clear, elastic
mucus that hangs from the ventral commissure. In many cases the mucus may be present
adhering to the tail
 Blood stained mucus may be present in some heifers and cows during the first few days after
the end of estrus referred to as metestrual bleeding
 Reddish grey discharge, consisting of blood elements and debris of endometrium referred to
as lochia, is observed in post parturient cows and increases in amount reaching peak
quantities around the third week of the post partum period

Discharges associated with pathological conditions

 The presence of a mucopurulent (clear mucus discharge with pus flakes) to purulent discharge
indicates
o inflammation of any segment of the reproductive tract
o infection of the urinary system
 It should be noted that apparent purulent discharge may be absent in cases of pyometra while
a heavy purulent discharge may be present in animals with normally progressing pregnancy
 Greyish discharge not containing apparent pus has been observed in certain cases of cystic
ovaries
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Appearance of the Udder

 Edema and enlargement of the mammary gland are normally found in the pre parturient and
post parturient period
 Cows that have failed to conceive over long periods may have a small shrunken vulva

General Behaviour

 The general behavior of the animal can be observed only when the animal is not confined
 Signs of estrus, hyperestrus, bellowing and pawing can be observed

VAGINAL EXAMINATION

 Supplemental information obtained by vaginal examination helps to refine the tentative

diagnosis made after rectal examination of postpartum cows. However, it is seldom employed
in the cow.

Manual Examination

 Manual examination of the vagina and cervix of the early postpartum cow will aid in the
diagnosis of the following conditions
o Vaginal/cervical trauma
o Retention of fetal membranes
o Patency of the cervical canal
 The cow’s vulva and perineum should be carefully washed with a mild disinfectant soap, and
a lubricated disposable plastic sleeve should be worn by the examiner to perform vaginal
examinations

Vaginoscopic Examination

 After washing the vulva and perineum, the speculum is inserted first in a dorsal-cranial
direction until the ischial symphysis has been passed, then in a cranial direction
 Slight resistance will be noticed at the vestibulovaginal junction which can be easily
overcome by gentle pressure
 With a torch light, the vaginal vault should be examined for
o the location of the cervix,
o cervical/vaginal color and secretions,
o cervical anomalies,
o trauma and discharges

VAGINOSCOPIC FINDINGS

Findings Condition
Large quantities of stringy, water-clear Proestrus or estrus phase
mucus
Bloody discharge through cervix into a Metestrus phase
mucus pool in the anterior vagina
A pale mucosa with scant amounts of Diestrus phase
sticky mucus
Findings Condition
Pus in the external os of the cervix or on Endometritis

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 the floor of the anterior vagina
Pool of urine mixed with mucus in the Urovagina. May temporarily result in an irritated,
anterior vagina hyperemic vaginal and cervical mucosa
Presence of papules, pustules or ulcers Infectious Pustular Vulvovaginitis
in the vagina and vestibule

RECTAL EXAMINATION

 At present, the most cost-effective and accurate method of examination of the reproductive
tract is per rectal palpation of the cervix, uterus, ovaries and supporting structures. The
technique of examination of reproductive tract by rectal palpation has been dealt with in detail
under the practicals.
 Rectal examination involves
o Examination for pregnancy
o Examination for non pregnancy

EXAMINATION FOR PREGNANCY

 Pregnancy diagnosis is based on detection of the physiological changes of the genital organs
associated with pregnancy.
 The uterus is the organ mainly involved and the positive signs of pregnancy include
o Palpation of amniotic vesicle
o Palpation of fetal membrane slip
o Palpation of placentomes
o Palpation of fetus

EXAMINATION OF NORMAL NON PREGNANT REPRODUCTIVE TRACT

 The size, muscular tone and contents of the uterus should be assessed. This can be done
simultaneously with the “membrane slip” for pregnancy determination. Commonly used
terms for characterizing uterine tone include the following:
o Estrus tone: a turgid, contracted uterus that is often curled into a rather tight
configuration
o Diestrus (“normal”): a relaxed muscular uterus
o Edematous: a somewhat turgid uterus but without muscular contraction; may be
palpable for a few days after estrus
o Flaccid: a limp, soft, usually thin-walled uterus that does not contract in response to
palpation
o Thickened (“doughy”): a pathologic description, indicating thickening of the
endometrium and possibly the myometrium as well
o Fluctuant: uterus in which there is intra luminal fluid

Post Parturient Involution

 Pregnancy and involution represent the only two clinically appreciable physiological
alterations of size. In pregnancy, the size progressively increases while during the involution
period, the size of the uterus regresses and returns to the non pregnant state

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ABNORMALITIES INVOLVING UTERUS

Palpation for Uterine Disorders

 During routine post partum examinations in cases in which pregnancy diagnosis is negative or
in examination of “problem cows,” the reproductive tract should be examined for palpable
abnormalities. The essential questions for the examiner to answer are the following:
o Is the uterus symmetrical and approximately the size and tone of the non gravid tract?
o Is there a corpus luteum or an ovarian follicle associated with corpus luteum or an
ovarian follicle associated with increased uterine tone that is indicative of cyclicity?
o Are there any palpable lesions of the reproductive tract?

Uterine Inflammation

 It is generally possible to diagnose moderate to severe endometritis, acute metritis or

pyometra by rectal examination.

Adhesions

 On rectal examination it would be possible to detect the presence of uterine or utero-ovarian

adhesions that would interfere with normal retraction of some part of the tract.
 Commonly, the uterus will adhere to the rumen, the omentum or the ovarian bursae.
 Prognosis depends on the severity of adhesions and the degree of involvement of the oviducts
and fimbriae.

Abscesses

 Uterine abscesses can occur

o following dystocia
o as a sequel to the improper use of an intrauterine pipette.
 Location and size of the abscess varies depending on the degree of mechanical insult in the
former and the degree of endometrial/ myometrial insult in the latter.
 Abscess is most often located in the area of the uterine body and is approximately the size of
a golf ball and in either case the abscess is firm and raised and may cause discomfort when
palpated.
 Adhesions of the abscessed portion of the uterus to other abdominal or pelvic organs are
common.

Tumors

 Tumors of the bovine uterus are not common but when seen occur predominantly in older
cows. Uterine lymphosarcoma, leiomyoma and rarely carcinoma have been diagnosed.
 Lymphosarcoma may be detectable as multiple smooth nodular enlargements of the uterine
wall, often with concurrent enlargement of the deep inguinal and iliac lymph nodes.

Fetal Remnants

 Occasionally, a fragment of an autolyzed term fetus may remain in the uterine lumen
following parturition
 Can be detected as a moveable firm mass in the lumen of an involuting uterus. A foul vaginal
discharge will often be noted.
 Cows or heifers that do not calve at the expected time following a positive pregnancy
diagnosis may have either a mummified or macerated fetus.

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  In cases of fetal maceration, a distended uterus with palpably crepitant fetal bones can be felt.
An ipsilateral CL may be present, as well as a fetid vaginal discharge.
 The prognosis for future fertility of such cows is grave due to severe damage to the
endometrium .

Freemartinism

 In Freemartinism the cervix is rudimentary while the uterus is underdeveloped and

characterized by the presence of two thin walled, very narrow tubes occupying the sites of the
normal horns, suspended in ligamentous sheets resembling the broad ligaments
 Lateral exploration along the edge of the broad ligaments leads to location of barely
perceptible thickening indicating the rudimentary ovary
 Failure to locate the normal cervix during the course of rectal examination should always be
followed by a thorough exploration for signs of freemartinism.

White Heifer Disease

 Also known as segmental aplasia of the Mullerian duct

 The extent of aplasia and the number of the missing segments is variable
 Secretion of the normal segments becomes entrapped between the missing segments or
anterior to the missing part, resulting in marked distension of the normal segment associated
with thinning of the wall. Persistence of the “hymen,” one of the forms of white heifer
disease, results in accumulation of secretion in the anterior part of the vagina, with
consequent dilation which elicits tenesmus.

Uterus Unicornis

 This relatively rare abnormality has been found in practically all breeds.
 The horn that is present is functionally normal and conception is possible only during an
estrus when the follicle develops and ovulates occurs in the ovary on the side of the normal
horn
 Reduced fertility can be anticipated.

Cervix Duplex or Double Cervix

 This also is a rare abnormality. The presence of two cervices, resulting in two single tube
genital tracts anterior to the vagina, might cause temporary confusion in the examiner’s mind.
Diagnosis, however, is easy .
 Fertility of the affected animal does not have to be impaired if natural breeding is employed.
Artificial insemination might result in failure to conceive unless the ovary carrying the follicle
ready to ovulate is detected and the semen is deposited in the cervix of the same side.
 Bilateral insemination in the double cervices might also be recommended.

Pyometra

 Characerised by accumulation of pus in the uterus and may occur due to lack of sufficient
relaxation of the cervix or to the presence of cervicitis combined with atony of the uterus and
consequent lack of expulsive force.
 The amount of exudates varies from 25cc., which is barely felt upon examination, to several
liters.
 The uterine walls are thinner than in the non-pregnant uterus, but thicker than the pregnant
uterus
 The condition has to be differentially diagnosed from pregnancy

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Mucometra or Hydrometra

 Both mucometra and hydrometra are similar except for the degree of hydration of mucin
present in the uterus which may vary from a watery fluid to a semisolid mass.
 Condition is observed in heifers or cows following
o arrest in the development of mullerian duct system.
o persistence of hymen
o prolonged hormonal stimulation with estrogens or progestogens
 Cows with mucometra or hydrometra due to defects of genital tract are sterile.
 Cows with pyometra do not cycle, while cows with a hydrometra do.

Chronic Nonproductive Metritis

 This condition is often referred to as chronic endometritis

 On rectal palpation
o the uterus lacks tone,
o has a thin wall, especially in the intercaruncular spaces,
o caruncles, therefore, appear more prominent, and the endometrial surface feels wavy
and uneven.
 Diagnosis is by vaginal examination and histological examination of biopsies.

EXAMINATION OF ABNORMALITIES INVOLVING OVARY

Smooth Ovaries

 Ovaries are smooth

 Repeated palpation confirms the presence of smooth ovaries. Systemic or local causes should
be investigated.

The detection of smooth ovaries at a single examination in cycling cows, especially during the first
few days following ovulation when the developing CL is not palpable is perfectly normal.

Ovarian Cysts

 Ovarian cysts are fluid-filled structures greater than 2.5 cm in diameter.

Should be differentiated from parovarian cysts, which do not involve the ovary but rather involve
remnants of the mesonephric or paramesonephric duct systems.

Ovarian Hypoplasia

 Ovarian hypoplasia is found in all breeds and may be bilateral or unilateral.

 Bilateral hypoplasia is found in heifers only and is always associated with sterility .
 Cows and heifers which are affected unilaterally might reproduce relatively normally.
 The degree of hypoplasia varies and the affected ovary might be recognized as a barely
distinguishable thickening of the mesovarium. In other cases, the ovary might be slightly
larger.
 Whenever the dimensions of the ovary are found to be less than 2 cm x 0.5 cm x 0.5 cm this
disease should be suspected. The affected gonads are are hard and static.
 Functional structures such as follicles or corpus luteum are not present.
 In cases of doubt, re-examination should be recommended, especially for differentiation from
ovarian atrophy.

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  Hypoplasia of ovaries has been found to be hereditary in nature, and it is very important to
detect the unilateral cases which might reproduce and transmit the disease to offspring.

Underdevelopment of Ovaries in Heifers

 Usually bilateral and is found primarily in poorly fed and managed heifers
 Most commonly heifers of the same age are affected
 The ovaries are small and static, consistency varies from flaccid to fibrotic while there is
atrophy of the uterine wall
 It is difficult to differentiate this condition from ovarian hypoplasia based on single
examination. Re-examination, preferably after correction of feeding practices, might be
necessary

Ovaritis or Oophoritis

 Inflamation or infection of the ovary also known as ovaritis or oophoritis occurs

o secondary to trauma
o to infection from the uterus that passes through the oviducts
o by extension of infection through the uterine walls
 Associated with marked enlargement of the ovary.
 In acute ovaritis, enlargement is due to edema.
 Diagnosis of chronic ovaritis is based on enlarged fibrotic ovaries and presence of organized
adhesions to the surrounding structures, primarily to the mesosalpinx.

Miscellaneous Ovarian Conditions

 Include abscesses and tumors. Both of these conditions result in a greatly enlarged, usually
firm ovary and may be associated with bursal and uterine adhesions
 Abscessed ovaries may have a softened area within the firm mass and may cause pain when
palpated
 Unaffected ovary may function normally so that cyclic structures may be palpated

EXAMINATION OF ABNORMALITIES INVOLVING OVIDUCT

 Only those associated with enlargement of the oviduct are detectable clinically.

Hydrosalpinx

 Hydrosalphinx is the local or general enlargement of the oviduct

 Manifests itself in the form of enlarged segments of varying length
 Local enlargements may resemble ovaries in size. When the entire oviduct is involved, it
appears as a conglomerate of a fluctuating tube
 The width of the enlarged oviduct varies from case to case and may reach 2 cm. in diameter
 Impossible to pathologicaly differentiate serosalpinx, pyosalpinx and hemosalpinx by clinical
examination
 Adhesions may be present between the loops of the enlarged oviducts

Pyosalphinx

 Follows severe uterine infection and is less commonly reported than hydrosalphinx
 Associated with severe adhesions of the mesosalphinx and mesovarium.
 May also follow
o removal of retained corpus luteum
o injection of large doses of estrogen

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ABNORMALITIES INVOLVING MESOSALPHINX AND OVARION BURSA

 Clinical differentiation between parasalpingitis, perisalpingitis and ovarian bursitis is

practically impossible.
 For clinical purposes, perisalpingitis appears to be the most correct term to describe the
inflammation and the consequent thickening and adhesion formation involving mesosalpinx,
mesovarium and salpinx. Other structures in the area, such as the ovaries, the horns of the
uterus and others, might also be embedded in the adhesions.
 Very fine adhesions between the ovary and fimbria-the fringes of the edge of the
infundibulum-are present in numerous animals, especially immediately after ovulation. These
do not appear to interfere with the normal function of the oviduct.

ALTERNATIVE METHODS

Laparoscopy

 The reproductive tract can be directly visualized by laparoscopy/endoscopy

Ultrasonographic Examination

 The uterus and ovaries can be indirectly examined by ultrasonographic techniques

 Real-time ultrasound, in which a two-dimensional “sonic picture” is generated from echoes
 The use of ultrasonography to diagnose pregnancy, normal ovarian structures, uterine and
ovarian pathology are described in detail in practical exercise.

MARE

HISTORY

As a rule, there is less recorded history available about mares than cows, and the information which
might be obtained is less complete. If possible, information should be acquired with regard to the
following:

 Age of the animal

 Duration of the present ownership
 Previous pregnancies and foalings, if any
 Date of the last foaling and the rate of growth and development of the foal
 History of any infections
 The month of commencement of the breeding season as well as its length
 Intensity of estrus, the length of the estrus period and the length and regularity of the estrous
cycle
 The client should be asked about the method employed for observing heat

PHYSICAL EXAMINATION

visual Examination

 Conditions that alter the general conformation and the external appearance of the cow are
simply not encountered in the mare. Changes in the appearance of the “tailhead” are not as
apparent in the mare.
 Relaxation of the pelvic diaphragm and vulva associated with gestation is observed during the
last 2 to 5 days of pregnancy, and then not in all animals.
 Copious discharge of estrus mucus is seldom observed in the mare. Thus, information
suggesting physiological events cannot be obtained by visual inspection.

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  Significant information, however, is gained from observing conformation changes involving
o External genital organs
o Abnormal vaginal discharge
o Hoof and leg infirmities such as a rotated third phalanx following acute laminitis that
may make a mare reluctant to stand for breeding or may make her unfit to carry a
pregnancy to term
o Pelvic injuries or abnormalities that may predispose a mare to dystocia
o Small stature
o Hirsutism associated with a pituitary tumor may be the cause of a mare’s unseasonal
anestrus
o Hypertrophic pulmonary osteopathy has been associated with certain types of ovarian
tumors in mares

Examination of Genitalia

 After completing the general physical examination, a detailed evaluation of the reproductive
organs should be undertaken with the tail wrapped in gauze and tied out of the way
 Mammary glands should be examined and palpated for signs of mastitis, abscessation,
neoplasia or injury.
 Vulva should be examined for conformation, apposition, tone and evidence of discharges.
o Mal apposition of the vulvar lips or poor vulvar conformation may lead to
pneumovagina and fecal contamination of the vaginal vault
o Examination of the vulvar area should continue with the examination of the clitoral
fossa and clitoris that harbors the contagious equine metritis organism Hemophilus
equigenitalis.
 The examination then continues with either rectal or vaginal palpation.

FINDINGS

Pneumovagina

 Pneumovagina or” windsucker” condition is the most frequent conformation change observed
in mares.
 In normal mares the long axis of the vulva occupies an almost vertical position. In
pneumovagina it forms an acute angle with the horizontal plane. In extreme cases the vulva
might be found in an almost horizontal position
 The vulvar lips are relaxed, resulting in partial exposure of the vestibulum and clitoris. Scar
tissue and consequent deformation of the vulva lips are frequently observed in mares which
have experienced dystocia and lacerations
 The anus is sunken and enhances the appearance of a more or less horizontally directed vulva.
The relaxed vulva and its position facilitate contamination of the vagina with fecal material
 The relaxation permits aspiration of air into the vagina and also into the uterus, especially
during estrus thus allowing saprophytic and pathogenic bacteria to gain entrance into the
genital tract. Treatment should be directed at correcting the cause of pneumovagina, and
concurrently treating the resulting acute endometritis. The former can be done surgically
by Caslick’s operation.
 Almost all animals with pneumovagina are infertile and show signs of genital infection

Rectovaginal Fistula

 The trauma which results in rectovaginal fistula affects the perineum, vulva, vestibule and
rectum. Localized internal fistulas are rare.
 The lesions are easy to see, and the diagnosis of either type of fistula does not present any
difficulties. This abnormality leads to contamination and consequent infection of the genital
tract.

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Abnormal Development of the Vulva

 The vulva might be too small when compared with general body size
 Asymmetry of the vulva and overlapping of vulvar lips are abnormalities that result in
improper closure of the vestibule and permit aspiration of air

Discharge from the Vulva

 During estrus the vulva is moist, but the copious discharge which is a sign of estrus in the cow
is not present
 The very viscid discharge present in practically all cows in advanced pregnancies is absent in
the mare
 Lochia, the grayish red discharge in the postparturient animal, appears in much lesser
amounts and only for a few days in the postparturient mare
 Postestrual hemorrhage is not seen in the mare. The appearance of an abnormal discharge is,
however, significant
 Abnormal discharges include haemorrhagic and purulent discharge

Haemorrhagic discharge

 Bloody discharge from the vulva is always serious. In the recently serviced mare, it indicates
service injuries
 Bloody discharge from a pregnant animal practically always indicates threatened or
completed abortion

Purulent discharge

 Purulent material in the vaginal discharge may be observed directly, or noticed as crusts on
the thighs and a loss of hair between the thighs if the exudates has been discharging for a
longer period of time.
 Indicates the presence of inflammation in the genital or in the urinary tract, especially in the
bladder.
 Determination of the site of inflammation requires performing a vaginal examination and
occasionally doing a rectal examination and urine analysis.

VAGINAL EXAMINATION

 The vaginal speculum or vaginoscope is more frequently used for vaginal examination in the
mare than in the cow and is always indicated whenever an abnormal discharge has been
observed.

Preparation

 Vulva and perineum are thoroughly cleaned

 The tail is wrapped and tied out of the way
 The area is disinfected using a mild surgical scrub
 Caution is taken to prevent forcing fluid through the vulvar cleft into the vagina

Examination

 After drying the area the vulvar lips are separated and a sterile vaginal speculum, either
tubular or the three-pronged Caslick speculum, is introduced into the vestibular area
 With the aid of a light, the speculum is used to examine

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 o the cervical os for color and tone as soon as possible, since changes occur as cool air
enters through the speculum
o the vaginal wall for color, evidence of congestion or inflammation, tumors,
lacerations and scars
o the vaginal floor for evidence of exudates or fluid accumulation or injury
o the dorsum of the vagina for evidence of injury or fistulation into the rectum.
 Endometrial cultures can be obtained during speculum examination

physiological Findings During Vaginal Examination

Estrous cycle

 Diestrum
o In the luteal phase vaginal mucosa is pale pink and rather dry.
o Speculum examination results in influx of air and ballooning of the vagina thus
favouring visibility. However, exposure to air causes the mucosa to
become congested and hence, the color of the mucosa should be noted immediately
after insertion of the speculum.
o Secretion is absent.
 Estrum
o Vaginal mucosa appears deep pink and glistening with a small amount of clear
secretion on the floor of the anterior vagina
o The external os is relaxed and lies limp on the floor of the vagina but is, however,
extremely sensitive to touch and responds quickly by becoming erect.
o The appearance of the cervix is greatly helpful in determining whether a mare is in
estrus or not. The erect cervical os during the luteal phase has been described as a
“rosebud,” whereas the relaxed external os during estrus has been described as
“wilted rose.”

Pregnancy

 The vaginal mucosa appears dull, anemic, rough and extremely dry, and insertion of the
speculum might be difficult.
 The external os is more or less relaxed and covered with a sticky, grayish secretion.
 The stickly mucus sticks to the vaginoscope and to the vaginal mucosa and resembles rubbery
glue during manual examination of the vagina.
 These findings are encountered in diestrus mares also

Abnormalities Detected During Vaginal Examination

Persistent hymen

 Easily diagnosed during the process of insertion of the speculum

 Septum may be partial, and might be brushed aside during introduction of the speculum
 If complete, it might lead to a condition resembling white heifer disease in cattle. This is more
pronounced in maiden mares which have experienced estrus
 The cervical and vaginal secretion produced during estrus accumulate anterior to the
obstruction formed by the persistent hymen and cause distention of the vagina
 Prolapse of the vagina and marked straining are common observed

Inflamation of the vagina and cervix

 Recognized by the presence of purulent exudates on the floor of the anterior vagina
 The cervix is open and often discharges purulent material during vaginal examination
 The mucosa of the external os of the cervix and the vagina has an unhealthy red appearance.
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  The observations described above, however, always accompany pneumovagina.

Scars, abrasions, ulcers and other defects of mucosa of the vagina

 Observed as complications after service and parturition injuries.

 Diagnosis, as a rule, is easy.

Retention of urine in the anterior vagina

 The history is that of an intermittent discharge of grayish material.

 Vaginal examination reveals vaginitis and cervicitis which is secondary, since the fluid
retained in the anterior vagina is primarily urine with admixture of epithelial debris from the
mucosa.
 The downward and forward slope of the vaginal floor which might be the primary cause of
the condition.

RECTAL EXAMINATION

 Rectal examination is done to rule out pregnancy. If the mare is pregnant, the procedures that
follow will be altered
 Due to the friable nature of mare’s rectum cre is taken when conducting a rectal examination
 A well lubricated, gloved arm and hand is introduced one finger at a time through the anal
sphincter into the rectum. With the fingers held together, the cupped hand should clean out
the feces as far cranial as possible prior to searching for the reproductive tract.
 One may locate the ovary first by reaching up into the sublumbar area ventral to the fourth or
fifth lumbar vertebrae or the uterus first.

EXAMINATION FOR PREGNANCY

 Pregnancy examination should precede all other examinations and should form the first and
basic step of the approach to any form of infertility problem.
 The earliest time during the gestation period when the laboratory tests for pregnancy are
reliable is 60 to 80 days after conception. i.e., between the third and fourth expected estrous
periods after service
 Pregnancy diagnosis might be made by rectal examination as early as 30 to 35 days of
pregnancy, thus providing ample time for treatment, if indicated, and rebreeding a few days
later on the second expected estrus after service

NORMAL CHANGES IN REPRODUCTIVE TRACT

 Unlike seen in the cow, no dramatic and consistent changes in uterus are observed in the
mare
 During diestrus and proestrus, certain mares have a well defined, slightly contracted uterus.
 During the estrus period, the uterus appears edematous and firm. This consistency persists
until ovulation, after which the uterus becomes limp and flaccid.

Seasonal Changes

Stage Findings Terminology

Anestrus The uterus is flaccid, thin walled and quiescentThe CX 1
ovaries are small and firm the vagina is pale and dry .
The cervix is usually in the upper one third of the
vaginal vault, pale and dry and tight. Passage of a
finger through the cervix is difficult, and some time

~ 13 ~
 should be spent allowing it to soften and to dilate.
transition from Estrogens from the follicles cause the uterus to CX 2
anestrus to the become more edematous, congested and heavier. The
normal cervix changes from a CX 1 to a CX 2 in which case
breeding the cervix is pinkish, softer, moister and lower in the
season vaginal vault. Also the cervix develops folds of tissue
extending from the external os towards the vaginal
floor and will readily admit one to two fingers.
Estrus Increasing estrogen levels makes the cervix very soft CX 3
and pink. It is usually located in the lower third of the
anterior vaginal wall and is very edematous, and
glistens with moisture.

The edematous folds of the external os actually touch

the floor of the vagina, yet the cervix is still
recognizable. Two to three fingers may easily be
introduced through the cervix
Ovulation Estrogen levels have peaked, the cervix is at its CX 4
softest, salmon pink in color, very moist and
edematous and often appears as a mass of edematous
folds on the floor of the vagina.

cystic change

 Occasionally, a small pool of clear, serous mucus may be seen just caudal to the external os of
the cervix. At this time, with stimulation, the cervix may dilate completely and readily allow
the passage of the entire hand into the uterus
 Another normal cervical condition that may be encountered in pregnancy is that of a “capped
cervix" where the cervical appearance is similar to a CX 1 but in this case the external os is
not visible because of a cervical plug and the appearance that one of the cervical folds has
covered and sealed off the end of the cervix
 After ovulation and during diestrus the uterus becomes less edematous, less congested and
more tonic. It is easily identifiable as a firm tubular structure.
 The early pregnant uterus is also tonic and can be differentiated from the diestrus uterus by an
amniotic vesicle bulge after about 20 to 25 days of pregnancy.

Postparturient Involution of the Uterus

 Involution of the uterus after normal foaling is extremely rapid. Regression in size is almost
completed at the 1st day of “foal heat”.
 The relatively low conception rate observed from services on the “foal heat” appear to
indicate that the involution of the endometrium is not completed at this time in all mares.

ABNORMALITIES INVOLVING UTERUS

 Uterine abnormalities can be subdivided into

o Abnormalities associated with a uniformaly enlarged uterus
o Abnormalities associated with discrete abnormalities within the uterus
o Parauterine abnormalities

~ 14 ~
Uniformaly Enlarged Uterus

Must be differentiated from pregnancy and a postpartum uterus. Involution of the mares uterus occurs
very rapidly after foaling in comparison to the cows. By the beginning of foal heat, it should be no
more than two to three times its normal size. Other causes of a uniformly enlarged uterus are
pyometra and pneumo uterus.

Discrete Uterine Enlargements

 Must be differentiated from early pregnancy by identifying the embryo as a discrete bulge in
the uterine horn.
 Other enlargements include
o endometrial cysts (result from blocked and dilated endometrial glands),
o lymphatic lacunae (which result from blocked lymph channels),
o abscesses in the uterine wall and corneal dilatation (following atrophy of the uterine
mucosa in older mares)

Parauterine Abnormalities

 Parauterine abnormalities include hematomas in the broad ligament of the uterus that are
usually associated with parturition.
 A fresh hematoma should not be disturbed, and the mare should be treated with systemic
antibiotics to prevent abscessation.
 A chronic hematoma rarely causes a fertility problem and usually regresses over time.

ABNORMALITIE SINVOLVING OVARIES

 Ovarian abnormalities identified on physical examination can be divided into small ovaries
and large ovaries

Small Ovaries

 These may be either normal or abnormal. Prepubertal or juvenile ovaries are small; therefore,
the age and previous cyclic history of the mare is important
 In anestrus the ovaries are inactive and one half the size they will attain during the breeding
season. In some small, docile, chronically anestrus mares a chromosomal anomaly called XO
Gonadal dysgenesis may be the cause of small ovaries. An endometrial biopsy from these
mares often demonstrates glandular insufficiency
 “True” nymphomaniac mares also contain smal ovaries. These mares act as if they are in
persistent estrus, yet often they will not allow mounting; some of these mares will
demonstrate male like behavior. Other than small, firm ovaries, no other abnormalities of the
genital tract are noted in the nymphomaniac mare

Enlarged Ovaries

 May be a seasonal phenomenone. During the transitional periods, follicles may grow to
abnormally large sizes and persist for various lengths of time before ovulating or regressing

 They usually do not suppress activity in the other ovary and resolve themselves and cause no
permanent problem
 In the early transition period they can be treated with 1000 to 5000 IU of Human Chorionic
Gonadotrophin but results are variable. These persistent follicles are often diagnosed as cystic
ovaries by practitioners unaccustomed to palpating the mare’s ovaries

~ 15 ~
  Cystic ovaries, such as those that occur in cows, do not occur in mares. The biggest problem
in dealing with persistent follicles in mares is differentiating them from certain types of
ovarian tumors

Ovarian tumors

 In the mare ovarian tumors are usually classified according to the main type of cell making up
the tumor. Most of these tumors are unilateral and rarely malignant
 The cystadenoma must be differentiated from persistent follicles. These usually enlarge over a
period of time unlike the persistent follicle that remains the same or regresses. They probably
arise from the surface epithelium of the ovary or the rete ovarii and have one or several large
fluid-filled cavities within them. Diagnosis is by ultrasound treatment is only ovariectomy

ranulose-theca cell tumors

 Most common ovarian tumor in the mare

 Grow to very large sizes and are usually unilateral and benign
 Produce a variety of hormones and usually suppress activity of the opposite ovary
 In addition to secreting inhibin, these tumors frequently secrete testosterone causing the mare
to exhibit stallion-like behavior.
 Hormonal analysis and endometrial biopsy may also be helpful in diagnosing this tumor.
 Clinical signs vary from anestrus to nymphomania and even to virility depending on the
predominant hormone produced by these tumors.
 Treatment is removal of the affected ovary, and the prognosis for fertility is fair to good,
depending on the length of time that the tumor has existed and the degree of suppression of
the opposite ovary. Resumption of cycle occurs 1 to 4 month after the tumor is removed.
 Much less common ovarian tumors are teratoma and the dysgerminoma.

Teratoma

o The teratoma is a multiple tissue type tumor that usually has epithelial structures
including cartilage, bone, hair and glandular epithelium.
o Are usually benign and produce no hormones, so the contralateral ovary usually
remains functional, and the mare may continue to cycle. As in the other ovarian
tumors, ovariectomy is indicated.

Dysgerminoma

o Unlike the previously discussed ovarian tumors, the dysgerminoma can be malignant.
o It arises from the germinal epithelium of the ovary and can become very large. The
tumors may be solid or contain fluid-filled multiple cysts. Ovariectomy is the
treatment of choice.

Non-Neoplastic Ovarian Enlargements

 Other causes of ovarian enlargement such as non-neoplastic ovarian abscesses and hematoma
are common and are difficult to differentiate.
 The mare’s temperature and white blood cell count may help identify the ovarian abscess, yet
these abscesses are often encapsulated within the ovary and do not produce a systemic
reaction after they become chronic.
 Ovarian hematomas often feel similar to ovarian abscesses. In both cases the opposite ovary
usually remains functional and the mare continues to cycle. Ovarian hematomas usually
regress over a period of time and cause no fertility problems.
 Hormone stimulation tests may differentiate these from ovarian tumors. An ultrasound
examination may be of some help in differentiating them.
~ 16 ~
  The last cause of ovarian enlargement that should not be overlooked is the unusually large,
normal cyclic follicle. Most cyclic follicles range in size from 2.5 to 6 cm in diameter prior to
ovulation.
 Occasionally, one or several large follicles grow to 10 cm or more before ovulation. In this
and all cases of ovarian enlargement, several examinations over a 15 to 30 day period are a
valuable means of differentiating these ovarian abnormalities.

ABNORMALITIES INVOLVING OVIDUCT

 The incidence and abnormalities of salphingitis and hydrosalpinx seems to be very low when
compared with cattle.
 Fimbrial cysts are not a rare finding in the mare. These are usually small and inconsequential.
Occasionally, they may grow large enough to interfere with the collection of the ovum by the
fimbria.

ABNORMALITIES INVOLVING CERVIX , VAGINA AND VULVA

Cervical Abnormalities

 When evaluating the cervix, the normal pinkness of estrus must be differentiated from the
redness of inflammation.
 Cervicitis may be caused by contagious equine metritis, endometritis or vaginitis or may be
secondary to pneumo vagina or recto vaginal fistula
 The most common non-infectious abnormalities noted are cervical adhesions and scars
secondary to foaling or breeding problems. These abnormalities may prevent the cervix from
opening and/or closing properly dilate thereby
o preventing the stallion from ejaculating into the uterus
o making delivery of a foal difficult
o predisposing the mare to endometritis which may prevent her from carrying a foal to
term.
 Other abnormalities of the cervix include leiomyoma of the cervix and squamous cell
carcinomas.

Vaginal Abnormalities

 Scars, adhesions and lacerations are some of the more common vaginal abnormalities that
may lead to difficulies in foaling and breeding.
 Lacerations subsequent to breeding often occur in the fornix of the vagina; are usually
retroperitoneal and heal well
 Recto vaginal fistula, which is a foaling accident, occurs as a result of the foal sticking a foot
through the dorsal wall of the vagina and through the ventral floor of the rectum. If the foot is
not withdrawn into the vagina, the entire perineal body between the rectum and vagina may
be torn, producing a third degree perineal laceration.
 Other vaginal problems encountered are
o pneumovagina secondary to cervicitis and endometritis. Caslick’s operation is
performed routinely on many farms and may be one of the best management aids to
overcome the problem of the barren mare.
o Vaginal abscesses occasionally occur subsequent to a vaginal laceration. They should
be drained into the vagina, with care taken to avoid the large perivaginal blood
vessels. Systemic and local antibiotics should be used to speed healing.
o Persistent hymen can be identified by vaginoscopy. Correction involves manually or
surgically dilating the hymen. Prepartum vaginal prolapse is rare in the mare.
o Occasionally, a persistent hymen, perivaginal abscess or hematoma may be mistaken
for a vaginal prolapse.

~ 17 ~
Vulvar Abnormalities

 Dorsocranial slope associated with pneumovagina is the most common vulvar abnormality
encountered. At least 70 per cent of the vulvar cleft should be below the brim of the pelvis.
 Abnormal labial apposition can result in the same problems as abnormal slope and should be
corrected by Caslick’s operation. Occasionally, a mare will be encountered that has suffered a
severe vulvar laceration because an episiotomy was not performed on a mare that has had
Caslick’s operation.
 Third degree perineal lacerations involve the vulva. Reconstructive surgery should be
attempted. Clitoral hypertrophy is occasionally seen in fillies. This is usually a manifestation
of pseudohermaphroditism.
 Neoplasia of the vulva include fibromas or fibropapillomas, malignant melanomas and
squamous cell carcinomas

ALTERNATIVE METHODS

Ultrasonography

 Can be used to identify the amniotic vesicle as early as day 15 of gestation and is valuable in
predicting the presence of twins early enough in gestation to correct the problem without
danger to the mare.
 Also helpful in producing an image of various vaginal, uterine and ovarian masses to
determine if they are solid or fluid filled.

Hormonal and Chromosomal Analysis

 Analysis for progesterone, estrogen and/or testosterone may be of value in differentiating the
several causes of enlarged ovaries.
 Detection of pregnancy with pregnant mare serum gonadotrophin requires a blood test.
 Chromosomal analysis may be of value in ruling out specific cases of persistent anestrus in
mares.

SOWS AND GILTS

PHYSICAL EXAMINATION

 Careful physical examination is required for

o selecting potentially fertile breeding animals
o culling gilts with structural or genital abnormalities prior to breeding
o along with a herd history helpful in the diagnosis of reproductive failure in individual
animals or in breeding groups within a herd
 When indicated, internal reproductive organs should be recovered from slaughtered animals
for a thorough examination as useful information can be obtained when other procedures fail

Structural Soundness

 Soundness in replacement gilts is especially significant, since most structural faults and
weaknesses are aggravated with age and confinement rearing.
 Special attention should be given to selecting gilts free from foot, leg and joint problems,
which may impair their future reproductively.
o A moderate slope to the pasterns provides the animal with a cushion to the foot and
leg joints, enabling her to cope with solid surfaces in confinement. Gilts and sows
with hoof cracks, sole bruises or other foot problems should be culled because
attempted treatments are often unsuccessful. Such problems may arise from abrasive
or damp, slick flooring
~ 18 ~
  Too much slope in the rump area tends to make the animal more prone to unsoundness as she
matures. A steep rump also displaces the vulva to a low position and angle so that boars often
experience difficulties in entering the sow during mating.
 Extreme muscling leads to delayed puberty, low conception rate, farrowing difficulty and
poor mothering ability.

Examination of External Genitalia

 Observing the vulva of replacement gilts at 5 ½ to 6 months of age can help detect potentially
sterile or slow-breeding females.
 The most commonly observed abnormality is the
o infantile vulva which is usually accompanied by small, prepubertal ovaries and
uterine horns.
o dorsally “tipped vulva”. Boars may experience difficulty in servicing gilts having this
trait.
o Injures of the vulva may occur from fighting or at parturition. Unless they are severe,
they generally do not contribute to future reproductive problems.
o Atresia ani, or imperforate anus, is a congenital defect observed in all breeds.
 In gilts the rectum and vagina may be joined, forming a recto vaginal fistula
just anterior to the vulva.
 Males die because they are unable to defecate. Gilts defecate via the vulva
opening.
o Occasionally, an unusually large percentage of females within a group is observed to
have red, swollen vulvas, typical of females in estrus. This observation, when coupled
with mammary development in non pregnant females and barrows, indicates the
presence of exogenous estrogenic substances in the feed

Examination of Mammary System

 A sound underline with atleast six functional, well developed and evenly spaced teats on each
side, with three in front of the navel is prefered
 Gilts with a blind teat that does not fully develop, a pin nipple or an inverted nipple should
not be considered as replacement animals

INTERNAL EXAMINATION

 In herds with a high incidence of reproductive failure that are not diagnosed by other
methods, examination at slaughter is recommended. Females should be tattooed prior to
slaughter for identification so that the individual animal’s reproductive history can be related
to the observed reproductive tract disorder. Observe for
o the presence and size of follicles, corpora lutea and cysts in each ovary
o any adhesions surrounding the ovaries or within the ovarian bursa
o size of the uterine horns
 Externally, each oviduct and uterine horn to the vagina should be traced, for occlusions,
missing parts or adhesions.
 After gross examination the tract is opened and is examined and noted for the presence and
characteristics of fluid or embryonic tissue.

ABNORMALITIES - Anatomical Abnormalities

Hydrosalpinx and Pyosalpinx

 Hydrosalpinx and pyosalpinx refer to distention of oviduct with clear fluid and pus like
material, respectively. Occur more frequently in gilts than in sows
 Result from abnormal embryonic development and may be hereditary.

~ 19 ~
  Affected females have regular estrous cycles but are prone to repeat breeding and reduced
litter size. If lesions are bilateral, affected females are sterile.

Segmental Aplasia

 Segmental aplasia may occur at any position along the uterine horn but most commonly
occurs near the uterine body. Occasionally, an entire uterine horn may be absent.
 Afflicted females will cycle normally. It is possible to have pregnancy in the patent side, but
litter size is usually reduced.

Blind, Double and Missing Cervix

 These abnormalities occur infrequently. Females with either condition cycle normally.
 If part or the entire cervix is missing, the female is sterile.
 Pregnancy can be achieved in females with a double cervix.

Infantilism

 This is a common abnormality and is generally but not always associated with confinement-
reared gilts wherein the presence of a very small vulva and the absence of estrus are
suggestive of this condition.
 The infantile tract is approximately 30 per cent of the size of a tract from normally cycling
gilt.
 The ovaries are hypoplastic and nonfunctional with numerous small follicles and no corpora
lutea. This condition is common in gilts with delayed pubertyor gilts less than 6 months of
age.

Adhesions

 Adhesions have been observed in all areas of the reproductive tract but more frequently in the
oviduct and ovarian bursa.
 Cyclic activity is normal. Reduced litter size and infertility are frequently noted.

Intersexuality

 More predominant in the Yorkshire breed in which a portion of the female reproductive tract
has differentiated into its male homolog.
 Mostly inherited, inheritance is thought to be autosomal recessive accompanied by modifier
genes.
 Characterised by presence of an ovotestis, which may be internal or external, whereas others
may have a prominent clitoris and “sky hood” vulva.
 Some intersexes show male characteristics such as tusk development and mounting
behavior.

Cystic Ovaries

 Cystic ovaries originate in a complete or partial failure of ovulation. Therefore, cystic follicles
may appear on the same ovary as normal appearing corpora lutea.
 Cysts may vary in size from 12 to 50 mm.
 Affected females may be anestrus or exhibit near normal estrus cycle patterns.
 Most attempts at treatment are ineffective.

~ 20 ~
EWE AND DOE

 istory is an essential component of the clinical examination, particularly in sheep and goats
due to inaccessibility of the majority of the reproductive tract to palpation or observation.

Season

 Degree of seasonality varies with breeds of sheep and goats and whether a male is present or
not
 A prolonged breeding season indicates increased or earlier months of transition
 Meat goats as a breed are known for their ability to breed more than once per year
 Introduction of a buck or ram during periods of transition hlps in advancing the onset of the
breeding season by 4 to 6 weeks

Nutrition and Size

 Young females should be two thirds of their expected adult body weight before they are bred
 Thin animals with dull hair coats may take months to recover from poor nutritional
conditions.

Social Order

 To the extent possible, the “comfort” of the animal should be determined in terms of whether
it is a dominant or submissive animal in the group.
 Amount of feeding space, access to shelter for all animals and the opportunity for dominant
does to keep timid ones from seeking the buck should be investigated.

PHYSICAL EXAMINATION

 Physical examination should be done to

o evaluate the current body condition and femininity of the animal
o determine the age by examining the dentition in order to avoid mistaking under
grown infertile adults for young ones.
o Determine whether the animal is polled or horned when the potential for the intersex
condition is considered. Intersex animals can present with a wide array of clinical
features.

Examination of External Genitalia

 Includes evaluation of the anogenital distance and whether the clitoris is visible without
parting the lips of the vulva
 Vulva should be examined for possible abnormalities such as pox or herpesvirus lesions,
tumors, pustular dermatitis, and ectopic mammary tissues
 Includes the palpation of abnormal lumps or swellings in the inguinal region
 Vaginal speculum examination or, alternatively, an endoscopic examination to rule out any
membranes or adhesions present
 Presence of any discharges from the cervix or vagina should be noted
 It should be remembered that the normal caprine vaginal discharge turns from clear mucus
early in standing estrus to thick ”cheesy” exudate late in standing estrus. This type of
discharge at this time of the estrous cycle is normal and does not require treatment

ABNORMALITIES

 Pathological lesions of reproductive system in sheep and goats are similar in most respects to
those in calttle.
~ 21 ~
  Vulvitis, vaginitis, cervicitis, metritis, pyometra, perimetritis, and salphingitis may occcur in
sheep and goats as a sequelae to dystocia, embryotomy, difficult parturition, retained
placenta, and delayed involution of the uterus.
 Tumours of the ovaries and genital tract of the ewe are rare.

Abnormalities Involving Uterus - hydrometra

Abnormalities Involving Oviduct

Fimbrial Cyst in Ewe Mesonephric Duct Cyst in Paraovarian Cyst in Ewe

A large fimbrial cyst on the
right. These cysts may block
the oviduct and prevent the
sperm from reaching the
ovum / ova. If the blockage is
unilateral, the ewe is still
capable of conceiving on the
contralateral side.
Ewe
Remnant of the male duct
system (Wolffian duct). These
cysts are capable of occluding
the oviduct. Not so in this case
as the oviduct is not distended
with fluid. This cyst may create
confusion in an ultrasonogram.
A bi-lobed parovarian cyst
is present in the left
mesovarian. These cysts are
mesonephric in origin. The
left oviduct is distended with
fluid due to an obstruction.

Abnormalities involving Cervix, Vagina and Vulva.

Prolapsed Cervix in Vaginal Prolapse in Vaginal Prolapse with Prominant Clitoris in

Ewe Ewe Evisceration in Ewe Ewe
Prepartum prolapse of Prepartum vaginal Fatal evisceration The clitoris is grossly
the cervix is not and rectal prolapse. through the vaginal wall. and abnormally
common. Exposure of Possible causes Previous vaginal prolapse enlarged which could
the cervix and the include short tail with weakening / be due to the presence
vaginal mucosa will dock and overfull rupture of the dorsal of male gonad(s) in an
lead to drying out, abdomen (multiple vaginal wall is a possible intersex individual, or
contamination, fetuses, abdominal fat, cause. due to the rare
infection and injury. low quality roughage). occurrence of
freemartinism
Cervical Leiomyoma in Doe Prolapsed Vagina in Doe
The cervix and the base of the left horn have Prolapsed vagina during late gestation when
been opened. A large, firm tumor (leiomyoma) estrogen concentrations are rising and
is present in the area of the cervix. The tissues are beginning to relax. Straining has
caruncles are prominent indicating that the doe also produced a small rectal prolapse.
has been pregnant, although she was infertile
for the last 3 years.

ULTRASONOGRAPHY

 ransabdominal ultrasonography can be used to examine the animal for pregnancy,

pseudopregnancy (hypoechoic uterine fluid but no cardinal signs of pregnancy), pyometra,
fetal death, fetal maceration or resorption of the pregnancy.
 Clipping the hair in the inguinal region prior to the examination permits the best quality
images to be obtained. Transrectal imaging with 5 to 7.5 MHz linear probes often allows
visualization of the nonpregnant caprine uterus and ovaries, or early cases of the previously
mentioned conditions.
 The quality of transrectal scanning depends to some extent on the size of the animal and
whether the reproductive tract has descended ventrally along the body wall.
 Holding animals off feed for 24 hours may improve the image quality in some case.

~ 22 ~
 LAPROSCOPY , LAPROTOMY , NECROPSY

 A definitive diagnosis of infertility sometimes cannot be made without examination and

palpation of the reproductive tract. Laparoscopy is less invasive and preferred over
laparotomy when the equipment is available.
 The gonads may be aberrantly positioned in intersex animals, or various degrees of
maldevelopment of the three tubular portions of the reproductive tract may be present.
 In animals that have had peritonitis, abdominal surgery, or embryo collections performed, the
presence of abdominal abscesses or adhesions involving the reproductive tract may be
identified.
 Ovarian cysts or reproductive tract tumors may also be diagnosed by these methods.

BITCH

HISTORY

 The differential diagnosis for most infertility disorders is established by obtaining a thorough
history from the owner
 The initial history should include information regarding
o how well the owners know the bitch and does she live indoors with them or away.
o is she hosed alone, with another bitch that recently completed ovarian cycles, with
ovariohysterectomized bitch or with males?
o Is she normal in height and weight for her breed and for her line?
o Is she receiving any medication and is she well or ill?
 Onset of the pubertal estrus occurs at ages ranging from 6.3 to 23 months
 Toy poodles may benefit from evaluation earlier in life than Bull Mastiffs

PHYSICAL EXAMINATION

 he problem area should always be examined last to mk sure that each bitch receives a
complete physical examination prior to an evaluation of the reproductive tract.

Vulva

 Vulva should be examined to check for size and conformation and for presence of any
discharge.
 Small immature vulva or one that is recessed under a fold of tissue owing to body type or
obesity interfere with normal breeding while an obese bitch is prone to perivulvar dermatitis.
 A swollen, turgid vulva is suggestive of proestrus while a swollen and flaccid one can be
consistent with estrus or approaching parturition.

Vaginal Discharges

 The bitch in anestrus or diestrus usually has no vaginal discharge.

Nature of Discharge Probable Causes

Bloody discharge Proestrus, estrus, separation of the placental sites, or
severe vaginitis
Greenish black or dark bloody vaginal discharge Placental separation as well as postpartum “lochia”
Reddish brown yellowish, or grayish, thick, creamy, Open-cervix pyometra, metritis, or severe vaginitis
malodorous vaginal discharge
Straw-colored vaginal discharges Estrus
Clear mucus Normal and precede parturition

~ 23 ~
  Vaginal cytology specimens should be an integral part of any reproductive evaluation and
should be performed in any bitch with a vaginal discharge.

Digital Examination of the Vestibule and Vagina

 A digital examination of the vaginal vault should be performed routinely and should follow
culture and cytology. Masses, foreign bodies, strictures, painful vaginitis, or abnormal tissue
bands all prevent easy and painless examination
 If the digital examination is abnormal but inconclusive, vaginoscopy provides a more
thorough evaluation

Mammary Glands

 The mammary gland should be palpated for the presence of mammary tumors, for evidence of
lactation, mastitis, inverted teats, or benign nodules
 The ventral midline can also be checked for evidence of a previous surgical incision, which
might be a clue suggesting that the bitch has undergone ovariohysterectomy.

Rectal Examination

 A rectal examination ensures that the pelvic canal has been assessed for previous fractures or
other unsuspected abnormalities as compression of the pelvic canal is a potential cause of
dystocia.

Abdominal Palpation

 The abdomen should be palpated in an effort to identify and characterize the uterus. However,
except in pregnancy and pyometra, the uterus almost never can be evaluated with confidence
on abdominal palpation.

ABNORMALITIES OF UTERUS

 Abnormal development of the uterus or uterine tubes may occur in intersex animals
o Male pseudohermaphrodites are animals with testes and female external genitalia
o Female pseudohermaphrodites are animals with normal uterus and ovaries and male
external genitalia
o XX sex reversed cocker spaniels are genetic females with male gonads and abnormal
male external genitalia

 Hydrometra and mucometra are accumulations of sterile serous or mucoid fluid in the uterus
o Incidental findings either at the time of elective OHE or in aged intact female dogs
undergoing diagnostic work-ups for unrelated disorders, such as congestive heart
failure or mammary neoplasia
o Cystic endometrial hyperplasia frequently is associated with hydrometra and
mucometra.
o Pathogenesis is likely to include that of CEH
o Presumptive diagnosis of hydrometra and mucometra is based on presence of uterine
enlargement, documented by abdominal palpation, radiography or ultrasound and
lack of a systemic inflammatory response
o The primary differential diagnoses are pyometra and pregnancy
o Definitive diagnosis requires cytology and culture of the intrauterine fluid
o The fluid varies in character from serous to mucoid and in color from straw colored to
serosanguineous
o The treatment of choice is OHE, especially in bitches not intended for breeding.
Hematometra is sterile accumulation of blood within the uterus
~ 24 ~
  Cystic endometrial hyperplasia – pyometra complex is an acute or chronic post-estrual
disease of adult intact bitches leading to inflammatory exudates in the uterus that is associated
with variable clinical and pathologic signs. It also is called pyometritis, pyometra complex,
catarrhal endometritis, purulent endometritis, chronic cystic endometritis, and chronic
purulent endometritis

ABNORMALITIES OF OVARY

Congenital Abnormalities

 Complete absence of one or both ovaries known as ovarian agenesis may be associated with
other abnormalities of the reproductive tract, such as uterus unicornis.
 Ovarian hypoplasia has been reported in female dogs with abnormal chromosome number.

Female dogs with anomalous numbers of sex chromosomes and abnormal ovaries are usually infertile
and include

 True hermaphrodites
 Pseudohermaphrodites
o Male Pseudohermaphrodities
o Female Pseudohermaphrodites

Ovarian Cysts

 Ovarian cysts are fluid-filled structures with a distinct wall that develop within the ovary.
 Parovarian cysts are similar in morphology to ovarian cysts but lie next to the ovary.
 Follicular cysts are thin – walled structures containing clear, serous fluid, may be single or
multiple and if multiple cysts are present on one ovary, the cysts do not communicate. More
about follicular cyst in dogs

Other Ovarian Cysts

 Other ovarian cysts include luteal cysts, germinal cysts, cystic corpora lutea, cystic rete ovarii
or rete cysts (are small masses of irregular, anastamosing tubules with cystic changes in the
hilus region of the ovary) and parovarian cysts (cystic structures in remnants of the
mesonephric and paramesonephric tubules surrounding the ovary

Ovarian Remnant Syndrome

 Ovarian remnant syndrome occurs when a retained piece of ovarian tissue revascularizes and
becomes functional. The condition may occur following surgeon’s error in incomplete
removal of the ovary. The most common presentation of ovarian remnant syndrome is
recurrent estrus after OHE. More about ovarian remnant syndrome

Oophoritis

 Oophoritis is diffuse infiltration of the ovary with mononuclear inflammatory cells, with
subsequent degeneration of germ cells and fibrosis of surrounding tissues wherein an
autoimmune pathogenesis is hypothesized.

Ovarian Neoplasia

 The three general categories of primary ovarian neoplasms are those arising from epithelial
cells, those tumors of sex cord / stromal origin, and those arising from germ
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  May be palpable per abdomen, and often are visible on radiographs or sonograms.
 Secondary tumors of the canine ovary include lymphosarcoma.

QUEEN

NORMAL

Ovaries

 The ovaries of the adult queen are

o oval structures
o approximately 1.0 x 0.3 x 0.5 cm in size
o 220 mg in weight
o located in the dorsal abdomen caudal to the kidneys attached to the diaphragm by the
suspensory ligament, to the dorsal body wall by the mesovarium, and to the end of the
uterine horn by the short, thick proper ligament of the ovary
o enclosed by the ovarian bursa that has a small slit-like opening on the medial side
 The queen is an induced ovulator.
o Copulation, vaginal stimulation, or gonadotropin administration induces ovulation
within approximately 24 to 32 hours.
o Corpora lutea, which form after ovulation, appear orange-yellow grossly and may
reach 4.5 mm in diameter, peaking in size about 16 days after ovulation.

Oviduct

 The uterine tube (oviduct) of the adult queen is 5 to 6 cm in length

 The wall of the uterine tube is thin, and the lining is thrown up into longitudinal folds or
ridges.

Uterus

 The uterus of the adult queen is a Y-shaped organ consisting of a 2-cm-long body lying
between the descending colon dorsally and the urinary bladder ventrally and two 10-cm
uterine tubes (oviducts). The cervix is the thick-walled neck of the uterus, connecting it to the
vagina.

Vagina

 The vagina and the vestibule are each about 2 cm long , so that the cervix is located about 40
to 45 mm cranial to the vulva; in pregnancy the vagina is stretched cranially by the weight of
the uterus.

Vestibule

 The vestibule extends from just cranial to the external urethral cranially, to the vulva
caudally, a distance of about 2 cm.
 The external urethral orifice in the cat opens into a mucosal groove located on the floor of the
vestibule just caudal to a transverse fold of mucosa that represents the hymen.

Vulva

 The vulva of the adult queen consists of two small, round labia located just below the anus,
which unite at dorsal and ventral commissures

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  The labia are similar in spayed than in intact cats. During estrus the labia are slightly
edematous and reddened, vulvar discharge is negligible

Mammary Glands

 The queen has four pairs of mammary glands, arranged in two bilaterally symmetrical rows
from the ventral thoracic to ventral abdominal region.
 They have been designated as
o the right and left axillary
o thoracic
o abdominal
o and inguinal mammary glands
 Also designated as right and left mammary glands 1,2,3 and 4 when counting from cranial to
caudal

ABNORMALITIES OF OVARY

Ovarian Dysgenesis

 Ovarian dysgenesis refers to underdevelopment of the ovary; ovarian hypoplasia or to

hermaphroditic and streak gonads usually associated with an abnormal sex chromosome
complement, such as XO monosomy or mosacicim.

True Hermaphroditism

 Rare in cats, and has not been reported in phenotypic females where both gonad histology and
chromosome complement are known.
 Reported in cats that are phenotypically male, where both gonad histology and chromosome
compliment are known, suggesting that presence of testicular tissue in the embryo induces
development of (male phenotype) secondary sexual characteristics, regardless of presence of
ovarian tissue or karyotype.
 Diagnosis of ovarian anomalies is based on history of primary anestrus (ovarian agenesis or
dysgenesis) on careful gross evaluation of internal and external genital organs, on histologic
examination of the ovary, and on karyotype of affected queens

Ectopic Adrenocortical Paraovarian Nodules

 Ectopic adrenal gland nodules occur in the broad ligament of the ovary, within 1 to 4 cm of
the ovary, as single, unilateral nodules, as bilateral nodules, or as two nodules on a single side
and range in size from 2 to 5 mm in diameter.

Ovarian Cysts

 Follicular cysts that arise from mature or atretic follicles are common and affected queens
may be asymptomatic or may exhibit prolonged estrus if cells lining the cyst secrete estrogen.
 Prolonged estrus may be hard to distinguish from normal estrus, because the normal queen
may cycle in and out of the follicular phase as frequently as every 4 to 7 days.

The Ovarian Remnant Syndrome

 The ovarian remnent syndrome describes presence of ovarian tissue and signs of estrus in a
female cat after OHE. The causes are
o may be failure to remove all or a normal ovary at OHE

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 o presence of a partial or complete separation of a portion of normal ovary during
development (the fragment may be located near the ovary or in the broad ligament)
that is not detected at OHE
o supernumerary ovary although rare may also be considered as the cause of estrus
signs after bilateral OHE
 Affected queens demonstrate normal signs of estrus, and may allow copulation, but do not
become pregnant if bred.
 Diagnosis is based on confirmation of estrus, on detection of serum progesterone
concentrations exceeding 2 ng/ml 2 to 3 weeks after induction of ovulation at estrus in a
neutered cat.
 Treatment is exploratory laparotomy within 3 to 6 weeks of induction of ovulation at which
time presence of corpora lutea in a “grape cluster” appearance on the surface of the ovarian
remnant may make small remnants easier to identify.

Ovarian Neoplasia

 The granulose cell tumor of sex cord-stromal origin is the most common primary ovarian
tumor in the cat.
 Presence of palpable mass in the cranial or mid abdomen. Abdominal and thoracic
radiographs and abdominal ultrasonography are indicated in all cases to assess tumor size and
location, and evidence, if any, of the presence of metastases. A vaginal cytology specimen
should be examined for cornification as evidence of estrogen secretion in suspect queens.
Measurements of serum estrogen, testosterone, and progesterone are of interest if functional
tumors are suspected based on clinical signs of prolonged estrus, virilization, or pyometra.
Evaluation of the hemogram and serum chemistry profile is indicated prior to exploratory
surgery.
 Adenoma/Cystadenoma, Adenocarcinoma, Dysgerminoma have also been reported in cats.

ABNORMALITIES OF UTERUS AND OVIDUCT

Hyperplasia of the Uterus and Uterine Tubes

 Multiple, broad-based or pedunculated hyperplastic endometrial polyps have been reported in

cats ranging in age from 4 to 15 years and protrude into the uterine lumen.

Hydrometra/ Mucometra

 Hydrometra and mucometra, the accumulation of non-inflammatory, clear to slightly cloudy,

watery to viscid, sterile fluid in the uterine lumen, occurs occasionally in the cat and are
caused by
o lack of patency of vulva, vagina, cervix, or uterus resulting from congenital anomaly,
o neoplasia
o inflammation,
o scarring,
o accidental ligation.
 Fluid volume in the uterine lumen may reach 500 ml, and distention of the uterine body
and/or horns may be diffuse or segmental.
 Treatment is OHE.

Cystic Endometrial Hyperplasia/Pyometra Complex

 Pyometra in cats is a uterine inflammatory disorder characterized by cystic endometrial

hyperplasia (CEH).

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  Clinical signs include purulent vulvar discharge, anorexia, dehydration,
lethargy,pyrexia,vomiting, polyuria/polydipsia, and weight loss. The uterus becomes palpably
enlarged.
 Diagnosis in the intact queen is based on
o signalment,
o history of previous estrus and clinical signs,
o physical examination,
o hemogram,
o presence of a purulent vulvar discharge and /or enlarged uterus in the nonpregnant
animal.
o Abdominal radiography or ultrasonography is indicated to define uterine size and
shape for initial diagnosis, to rule out pregnancy (ultrasonography, after 21 days
following estrus).
 Recommended treatment for CEH/pyometra in the queen is OHE with concurrent fluid and
antibiotic therapy.
 In females with reproductive value and an open-cervix pyometra (diagnosed by the presence
of a purulent vulvar discharge), uterine evacuation can be attempted with
o PGF2 alpha at a dose rate ranging from 0.05 to 0.5 mg/kg subcutaneously (SC) once
or twice daily for 2 to 5 days until uterine size decreases to normal.
o Prostaglandin analogues should not be used in the cat, because safe and effective does
have not been established.
o Within 1 to 60 minutes of drug injection, panting, restlessness, grooming, tenesmus,
salivation, vomition, defecation, or diarrhoea.

Salpingitis

 Inflammation of the feline uterine tube, salpingitis, usually is purulent, and occurs secondary
to uterine inflammation

Neoplasia of the Uterus/Uterine Tubes

 Uterine tumors constitute 1 to 2 per cent of tumors of the female reproductive organs of the
cat including mammary glands), or 0.2 to 0.4 per cent of all feline tumors and include uterine
leiomyomas and leiomyosarcomas.
 Clincal signs of uterine adenocarcinomas depend on tumor size and pattern of metastatsis and
include ascitis, anorexia, weight loss, purulent or hemorrhagic vulvar discharge, vomiting,
constipation, dysuria, and presence of a palpable abdominal mass.
 Diagnosis is based on uterine palpation, abdominal and thoracic radiographs, surgical
exploration, and histopathologic examination of tumor tissue.
 Ultrasonography has been used to detect uterine neoplasia in the diffusely enlarged uterus
with pyometra.
 The recommended treatment for primary uterine neoplasia without metastasis is OHE.
 Tumors of the uterine tubes have not been reported in the queen.

DISORDERS OF VAGINA VESTIBLE AND VULVA

 nomalies of the vagina and vulva that have been described in the cat include
o segmental aplasia of the cranial vaginal (mullerian duct system),
o presence of a common vulvovestibular-anal opening,
o rectovaginal fistula.

Neoplasia

 The most common primary vaginal tumor type in the cat is the leiomyoma, which may
measure up to 7 x7 x 8m.

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 Clinical signs of vaginal tumors include
o bulging of the perineal region,
o prolapse of tumour tissue from the vulva,
o dysuria,
o pollakiuria,
o constipation.
 Initial diagnosis is based on palpation and on retrograde vaginography and/or
cystourethrography to characterize size and extent of the mass. Abdominal and thoracic
radiography to look for tumor metastasis should be performed prior to surgical excision.
Exfoliative cytology may be diagnostic and should be performed on accessible masses of the
vagina and vestibule. Final diagnosis is based on histopathologic examination after core or
excision biopsy.

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 DELAYED PUBERTY AND SEXUAL MATURITY

 Puberty represents the initiation of estrous cycle and is defined as the age or time at which
the female gonads are able to produce gametes and reproduction may occur. It is not a single
event, but a process. In the female, puberty is characterized by the exhibition of estrum and
ovulation.
 Onset of puberty normally occurs at a certain age relative to the animal’s body weight.
Heifers must attain approximately two thirds their adult sizes before they reach puberty.
 The word puberty originated from latin word "pubscere" that means "to be covered with
hair". The original definition that relates to the presence of hair in certain anatomical regions,
obviously does not hold good to other animals.

PUBERTY

HYPOTHALAMUS DEFEMINIZATION METHODS

 t is important to know the fundamental differences in the hypothalamus of the male and
female in order to have a clear understanding of the puberty in both the sexes. To address this
issue, one should first neccessarily understand

Why GnRH surge centre develops only in female and not in males?

 In the male..
o During prenatal development, defeminization of the brain occurs due to testosterone
from the fetal testis.
 In the female...
o Since there is no testis to produce testosterone, GnRH surge centre develops in the
hypothalamus.
o In order to defeminize the hypothalamus, it is most important that, first testosterone
has to be converted into estradiol.

Why then the estradiol produced by the female fetal ovaries does not cause defeminization?

 In order to cause defeminization, the estradiol produced by the fetal ovaries has to cross the
blood-brain barrier and gain access to the hypothalamus. A protein called, alpha-
fetoprotein binds to the estradiol thus preventing it from crossing the blood-brain barrier.
 Alpha-fetoprotein, is a glycoprotein synthesized by the embryonic yolk sac and later the fetal
liver and serves as a fetal blood osmotic regulator and a carrier of fatty acids.

In Female In Male
Alpha fetoprotein prevents estradiol from Testosterone freely enters the brain because alpha
entering the brain. The hypothalamus is thus fetoprotein does not bind to it. Testosterone is aromatized
"feminized" and the surge centre develop in to estradiol and the male brain is "defeminized".
Therefore, a GnRH surge centre does not develop.

CRITERIA USED TO DEFINE PUBERTY

Age at First Estrus (Heat)

 Female becomes sexually receptive and displays her first heat

 Exhibit behavioural signs of sexual receptivity
 In heifers and ewes, silent ovulation is common and generally not accompanied by
behavioural estrus
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  Age at first estrus may not reflect true acquisition of puberty

Age at First Ovulation

 Age at which first ovulation occurs.

 In bovine, it can be assessed by rectal palpation, ultrasound and laparoscopy. Requires
frequent observations for precise determination.
 Although good criterion, often difficult to determine.

Age at which Pregnancy can Occur without Deleterious Effects

 Applicable to all domestic animals.

 Generate highest number of offspring in the shortest time interval without compromising the
well being of the dam or the neonate.
 In biological sense, females cross a “metabolic threshold” before puberty occurs.

MECHANISM OF PUBERTY

ifferences in the LH secretory patterns in the male and female

 There are fundamental differences in the pattern of LH secretion in postbertal male and
female. LH does not surge in the male, but maintains a relatively consistent day -in and day-
out episodic pattern of secretion with episodes occuring every 2-6 hrs. which in turns results
in a steady pulse of LH and in turn testosterone. In contrast LH and Testosterone surge every
21 days in the female. Between these surges, low amplitude repeated LH pulses are present.

Changes in hypothalamic secretion of GnRH before and after puberty

 Before puberty in the female, the GnRH neurons in both the tonic and surge center of the
hypothalamus release low amplitude and low frequency pulses of GnRH
 After puberty, the tonic center controls basal levels of GnRH but they are higher than in the
prepubertal female because the pulse frequency increases. The surge center controls the
preovulatory surge of GnRH.

Mechanism of Puberty

 The onset of puberty is not affected by the performance of gonads or the anterior lobe of the
pituitary
 The failure of the hypothalamus to produce sufficient quantities of GnRH to cause
gonadotropin release is the major factor limiting pubertal onset.
 Prior to onset of puberty the following events take place
o The tonic centre of the hypothalamus produces GnRH in a pulsatile fashion.
However, the frequency of GnRH pulses are much lower than in the post pubertal
female.
o The low frequency GnRH pulses are not sufficient enough to cause production of
FSH and LH from the anterior pituitary in high levels
o Thus,follicle development (even though it occurs before puberty) connot result in
high levels of estrogen secretion.
o Although estrogen production is low, the hypothalamus is highly sensitive to the
negative feed back effects of low estrogen.
o GnRH and gonadotropin levels are low
 Onset of puberty is characterized by two important events
o Decrease in sensitivity of the hypothalamus to the negative feed back effects of
estrogen
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 o Higher and higher amounts of GnRH and gonadotropins are produced that stimulate
the ovary to produce more and more of estrogen
o When estrogen levels reach a threshold, they exert a positive feedback on the surge
center
o Ovulation can take place and puberty ensues.
 Thus, the triggering mechanism for onset of puberty in the female is the decrease in
sensitivity of the hypothalamus to negative feed back

What stimulates GnRH neurons to change its secretory pattern?

 There is a possible influence of metabolic signals upon GnRH neurons

 Adipocytes produce leptin that enters the blood and may stimulate neuropeptide Y neurons or
directly stimulate GnRH neurons. Blood leptin reflects the nutritional status of the animal
because greater the amount of fat, greater the amount of leptin.
 Blood glucose levels, another indicator of metaboloic stimulus probably stimulate glucose
sensing neurons that in turn stimulate GnRH neurons.
 Blood fatty acids may stimulate neurons that in turn stimulate the GnRH neurons. Blood fatty
acids would be an indicator of nutritional status of the animal.

Animal Onset of puberty Optimum breeding age

Mare 10-24 (Av.18) 24-36
Cow 6-18 14-22
Ewe & Doe 6-12 12-18
Sow 5-8 8-9
Bitch 6-12 12-18
Queen 5-18 12-18

FACTORS AFFECTING PUBERTY

 Interaction with the opposite sex.

 Influence of sex: Female attains puberty at an earlier age.
 Breed: Small breeds attain puberty at an earlier stage. eg. Nine months in Jersey and 11
months in Holstein Friesian.
 Climatic influence: Animals in the tropics attain puberty at an early age.
 Seasonal influence: Breeding season affects onset of puberty. Eg. Ewe lambs born in early
spring attain puberty in the fall i.e., within 180 days. But ewe lambs born in late spring and
early summer attain puberty only in the fall of the next breeding season i.e., only after 400
days.
 Plane of nutrition: High plane of nutrition leads to early puberty and undernourishment delays
onset of puberty.
 Body weight: Sheep attain puberty after reaching a body weight of 40 kg.

DELAYED PUBERTY AND SEXUAL MATURITY

CATTLE

 With good nutritional management, most cattle reach puberty between 8 and 13 months of
age. Failure to exhibit estrus beyond this time is called prepubertal anestrus.
 To begin with, the history of the individual or the group of heifers will provide the key to the
etiology.
 If the problem exists in a single heifer of the same age as rest of the cycling heifers in the
group it is probably related to abnormal reproductive tract such as
o freemartinism,

~ 33 ~
 o hermaphrodite
o aplasia of the mullerian duct.
 Cyclicity in rest of the herd mates indicates that the problem has not affected the entire group.
 Similarly, any debilitating disease such as chronic pneumonia can delay puberty by
decreasing rate of gain, which appears to be a functional dietary problem.
 If delayed puberty occurs in a group of heifers of the same ages or a group of mixed ages then
the problem is one of management.
 Since, the onset of puberty is influenced by the available nutrition heifers of the same age that
are fed a suboptimal energy diet will show a prolonged prepubertal anestrus period.
 Similarly, when a group of heifers of different ages are housed together and given a balanced
ration, the larger or more aggressive herd mates will consume a greater portion of the
available nutrition and tend to attain puberty at an earlier age while others may remain
acyclic. However, in due course the entire population begins to cycle. The inherent danger of
breeding these late heifers before they have developed adequate body size must be borne in
mind as they tend to have more dystocia problems and long postpartum anestrus periods
 Infectious diseases like blue tongue, and bovine diarrhoea virus may result in acute ovaritis
and subsequently ovarian atrophy thus causing anestrus. Animals may respond to hormonal
therapy but promptly return to anestrus once therapy is withdrawn.
 The use of growth stimulation implants in prepubertal heifers can delay pubertal estrus and
affect future fertility.
 In hybrid gilts, puberty usually occurs at around 160 days or age but it is breed dependent.
 Puberty may be delayed by poor environmental conditions, cold, sunburn and poor light.
 Overcrowding and the associated bullying and stress may result in delayed puberty.
 Poor nutrition acts by reducing growth rates or by causing deficiencies in particular nutrients.
 Disease may affect the gilt by reducing body condition or by causing pain. Lameness reduces
bodily condition and precludes behaviour associated with estrus.
 Finally, poor management of peer group contacts may delay puberty.
 Puberty may be delayed by housing with young boars or by contact with old boars too early.
As the expected time of first estrus approaches, inadequate boar contact or contact with board
with low levels of boar odour may delay puberty.
 Clinical signs of delayed puberty consist simply of the failure of gilts to show estrus
(enlargement of the vulva, reddening of the vulva, remaining still for back pressure, clustering
round a boar) by the time they would be expected to have reached puberty based on previous
experience with the breed or hybrid on the farm concerned, or industry norms. For this to be
ascertained, records of the chronological age of the animals must be available, or an estimate
of their age made based on weight, size for age, or time since selection for the breeding pool.
Anatomical evidence of hermaphroditism may be obvious Delayed puberty may occur in
individual animals in a group or in whole groups of animals. If estrus has not occurred or
been noted by 240 days of age, it is unlikely that it will occur.
 For the accurate diagnosis, the age of the animals under examination must be known and
individual animals must be identified or removed from the group when estrus is first detected.
 Daily inspection of the group in adequate light for physical signs of estrus, signs of mounting
and should include behaviour towards boars.
 Prevention of delayed puberty depends upon correcting the management factors.
o Gilts should be reared to 5-6 months of age in groups of 6-30, isolated from boars,
with 12-16 hours light of adequate intensity at a temperature of about 20°C and given
food. Disease should be controlled.
o Gilts which have reached 160-210 days of age should be exposed to vasectomised
boars or housed in sight, smell and touch of a smelly older boar. Estrus should then
be observed. If not, then gilts can be tested with another boar daily, taking the gilt to
the boars.
o Treatment with gonadotrophic hormones induces estrus in pre-pubertal gilts. Gilts
should be reared to 5-6 months of age in isolation from boars, injected with
gonadotrophic hormone preparations and then allowed contact with boars for 15-20
minutes per day. Ninety percent of gilts will develop estrus within 5-7 days. They
should not be mated at this estrus.

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EARLY MATURITY IN NON DESCRIPT PIG

IS THIS POSSIBLE????

 Do you think that a 3 month old male piglet impregnating a sow is impossible? Here is
something to prove that sometimes the impossible is also possible!
 The tribal population of North Eastern India follow a unique practice of mating the sow with
a male pig from its own litter to avoid maintenance of boar for breeding. In this system, it is
claimed that the nondescript local male pigs, at very young age (around 3 months),
impregnate the sow.

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 ESTROUS DETECTION IN FARM AND COMPANION ANIMALS

The single most important problem limiting high reproductive efficiency in a herd is poor detection of
estrus. Thus, effective estrus detection is the key to maximize reproductive efficiency through AI.

Herds, with poor estrus detection efficiency, are characterized by one or more of the following:

 Prolonged calving to first service interval. The interval should be no more than 15 to 18 days
longer than the farmer’s goal and/or average no more than 70 days.
 Prolonged intervals between breedings, especially when many are multiples of a normal 18 to
24 –day when many are multiples of a normal 18 – to 24- day cycle.
 Veterinary examinations confirming that cows are cycling normally, although estrus is not
observed.
 More than 10 to 15 per cent of the cows confirmed to be open at a 35 – to 50- day pregnancy
check.
 Heat detection index less than 0.05 or less than 50 per cent of the eligible cows observed in
estrus within a period of 3 to 4 weeks.

There are several aids to detect the heat in animals however they cannot substitute visual observation.

EVALUATING ESTRUS DETECTION EFFICIENCY

Complete and accurate herd records are essential for evaluating the efficiency and accuracy of estrus
detection. Calving dates, heat and breeding dates and the results of prebreeding and pregnancy
examinations are needed. From these data the following measures of reproductive efficiency relating
to estrus detection can be determined.

 Percentage of cows observed in estrus within 60 days after calving.

 Interval from calving to first breeding.
 Intervals between breeding.
 Estrus detection index, which is defined as the average number of breeding per cow.

ESTRUS DETECTION PROBLEMS AND THEIR CAUSES

 Basically, there are two estrus detection problems:

o Missed or unobserved estrous periods
o Estrus detection errors.
 The latter results in the insemination of cows that are not in the proper stage of the estrous
cycle for conception to occur. Some are near estrus but are bred 1 to 2 days too early or too
late, some are open but in the luteal phase of the cycle and some are pregnant.

UNOBSERVES ESTRUM

 If in most herds essentially all cows are cycling normally, why does the dairy farmer have
difficulty “catching the cycling cow”?. There are several reasons: some represent “people
problems,” but there are several “cow factors” that make estrus detection difficult. The major
factors contributing to poor heat detection efficiency are:
o Failure to spend sufficient time on a daily basis for estrus detection.
o Most mounting activity occurs at night in loosely-oused herds.
o Heat periods are short.
o Low levels of estrus activity when few cows are in heat. This can be a significant
problem in small herds and in groups of cows in large herds in which many cows are
either pregnant, not cycling or in the luteal phase of their estrous cycles.

~ 36 ~
 o Mounts last 10 seconds or less. Farmers must concentrate on estrus detection and
should avoid combining it with other activities.
o Feet and leg problems, slippery floors, summer heat, winter cold and other
environmental factors reduce estrus activity.
 Heat detection programs that limit the effects of these “people” and “cow” factors must be
developed in order to maximize heat detection efficiency.

ESTRUS DETECTION ERRORS

 Estrus detection errors must be avoided. Breeding pregnant cows can cause abortion.
Breeding nonestrus, open cows wastes time, semen and money.
 In order to reduce the number of errors, potential causes of the problem must be identified. In
most herds errors result from
o misidentification of cows,
o misinterpretation of the signs of heat,
o misuse or misinterpretation of the signs of heat
o misuse or misinterpretation of heat detection aids
o cows transmitting the wrong signals (up to 10 per cent of pregnant cows may stand to
be mounted).

ESTRUS DETECTION AIDS

 Estrus detection is difficult, so estrus detection aids are needed in many herds to identify
effectively all the cows that must be inseminated.
 The most important consideration for farmers is to remember that they are only aids.
 For best results aids must be used in conjunction with good visual detection programs, not as
a substitute for visual detection.
 The various estrus detection aids are as follows
o Wall charts, Breeding wheels, Herd monitors and Individual cow records.
o Secondary signs of estrus
o Palpation of reproductive organs
o Mount detection
o Heat detector animals
 Lateral deviation of penis
 Caudal epididymectomy
 Dorsal ligament resection
o Vaginal electrical resistance
o Heat check report system
o Prostaglandins
o Pedometers
o Tricks of the trade

Wall charts, Breeding wheels, Herd monitors and Individual cow records.

 These systems are the least expensive and aim at detection of the next heat period. If the farm
workers know when the next possible heat period is they could closely observe individual
cows for signs of estrus. Thus, more short-or weak-heat periods can be identified. The key to
successful use of these management aids are
o The accurate recording of every heat beginning with the first after calving.
o Their daily use to identify those cows that are due to return to estrus.

SECONDARY SIGNS OF ESTRUS

 Secondary signs indicate that a cow is in or near heat.

 They should be used primarily to identify cows that need careful observation for standing
estrus.
~ 37 ~
  A twice-daily walk behind the cows when most of them are lying down provides a good
opportunity to check for the secondary signs of estrus.

PALPATION

 Routine rectal examination of all cows between 30 and 40 days after calving and of individual
problem cows that have not been inseminated within 70 days after calving should be
encouraged to confirm that the reproductive tract is normal and to predict when the next
estrus will occur or to identify cows for prostaglandin treatment when estrous cycles are
occurring, but estrus has not been detected.

MOUNT DETECTION

 Two methods are widely used for mount detection

o pressure – sensitive devices
 paint stick, chalk or paint on the tail head.

Kamar Heat Close-up of Dye Capsule Paint on Tail Head Chalk on Tail Head
Mount
Detector Red dye is contained in a soft Tail head painting as an Tail head painting with
compressible capsule that is connected by aid to estrus detection. an oil based paint as an
A pressure a hairline channel via a hard cylindrical Oil based paint is used, aid to estrus detection.
sensitive heat tube to an outer compartment (not subsequently to be Subsequently a second
mount detector shown). Sustained pressure, as from a covered with a layer, of layer of a different color
patch (KaMar) mounting animal, is required to express a different color, of is applied with a crayon.
is glued to the the contents which are then readily chalk.
tailhead of the visible in a compartment glued to the
cow tailhead of the cow.

 When animals are in estrus, mounting activity changes the color of the detector or erases the
chalk or paint stick markings. With good management and proper interpretation, pressure-
sensitive mount detectors provide excellent results. However, care must be taken to position
the detectors properly and to minimize the opportunities for false activation of the devices.
 False activation of mount detectors can be reduced by removing cows that are in estrus from
the herd. The disadvantage of this practice is that it removes sexually active cows that
stimulate increased mounting behavior in others that may be in estrus but are less active.
 Recent studies show that the accuracy of mount detectors, when used as the sole method of
heat detection, may be as low as 30 to 50 per cent. These results strongly suggest that mount
detectors should be used only to identify cows that require additional observation. Breeding
on the basis of activated mount detectors without additional signs to confirm that cows are in
estrus should be discouraged.
 Chalking the tail head is a less expensive alternative for mount detection. False-positive are
sometimes a problem, and animals must be restrained and marked every few days, since mud
and manure may obscure the chalk or paint stick marking. Paint can be used instead of chalk
or paint stick. When the paint dries, it becomes brittle and flakes off when the cow is
mounted.

Tail Head Painting Tail Head Chalking Tail Head Marking Tail Head Marking with
with Crayon Crayon
Tail head painting as an aid to estrus Tail head marking with
detection. Oil based paint is used, a crayon or "chalk" as Tailhead marked With cows in lock-up
subsequently to be covered with a layer, an aid to estrus with crayon to stanchion tail heads are easily
of a different color, of chalk. detection. facilitate estrus marked with a crayon and
detection. inspected for scuff marks.

~ 38 ~
HEAT DETECTOR ANIMALS

 Sexually active animals can be used to identify estrus cows. They may be fitted with halters
containing ink-filled reservoirs and ball point pen type devices that wil mark animals that are
mounted, or they can be used without these devices to increase sexual activity and make
visual detection programs more effective.

Chinball Marking Harness Chinball Marked

Filling of the chinball marking Marking Heifer
harness with marking fluid or paint. Harness in Heifer
The spring loaded ball is depressed Place marked by a
and paint is poured or squirted in Chinball teaser bull.
marking harness
fitted on a teaser
bull

 Bulls, “cystic” cows, hormone-treated steers and hormone-treated cows and heifers have been
used. Cows with chronic follicular cysts are inconsistent, and there appears to be variation in
effectiveness among hormone- treated steers.
 The marker bull is the most effective detector animal. Copulation must be prevented even in
sterilized animals to ensure against the spread of veneral diseases.
 Use of surgical techniques that prevent sexual contact is preferred. Mechanical devices that
prevent copulation are less desirable because they sometimes fail, cause infection and tend to
reduce the sex drive of the bull.
 Bulls are dangerous. Injuries to cows and farm workers can and do occur. For this reason,
other bulls must be available so that bulls can be replaced when they become too aggressive.
 Hormone-treated heifers and cows are more docile although they may be slightly less
effective, they are the animal of choice on most farms.
 When marker animals are used, cows should be removed from the herd as they come into
estrus. This will stimulate the marker animal to seek out and identify additional cows that
may be in heat. The ratio of cows to markers should be no greater than 40:1.

Disadvantages

 Some cows may be marked when they are not in estrus.

 Others that are coming into estrus may be marked before they stand to be mounted. Therefore,
care must be exercised when interpreting the marks. For these reasons, the best results are
obtained when marker animals are used in addition to a good visual detection program.
 Also, marker animals tend to become too fat if feed intake is not restricted. A possible
solution to the latter problem in loose-housed herds is to put the marker with the herd only at
night or other periods during the day when visual observation is limited.

LATERAL DEVIATION OF THE PENIS

1. Lateral Deviation of the Penis: The bull is heavily sedated to the point he lies down. He is
then placed in dorsal recumbency. The abdomen is clipped and scrubbed from the xiphoid
process to the base of the scrotum. The initial incision is skin deep and encircles the prepuce.
The skin is also incised from the caudal most part of the circle to the base of the scrotum.
2. Lateral Deviation of the Penis: Skin flaps are dissected free on both sides and deflected to
the sides. Occasional bleeders are occluded with hemostats.
3. Lateral Deviation of the Penis: The penis and the sheath are dissected free from the
abdominal fascia.
4. Lateral Deviation of the Penis: The entire prepuce and the sheath, containing the penis, have
now been dissected free.
5. Lateral Deviation of the Penis: The free (bloody) prepuce and sheath are laid at a 45 degree
angle on the surface of the surgically prepared skin to mark its new direction.
~ 39 ~
 6. Lateral Deviation of the Penis: A circle of skin, slightly smaller than the circle of the
prepuce has been removed in the lower flank. It will be the end of the tunnel.
7. Lateral Deviation of the Penis: A large cervical forceps is used to make a subcutaneous
tunnel from the the circle in the lower flank to the base of the scrotum. The free prepuce and
sheath are then pulled through the tunnel to their new location.
8. Lateral Deviation of the Penis: Post-operative swelling at 24 hours. This swelling will
subside over the next two days as circulation of the cutaneous tissues re-establishes itself. It is
important that the bull can urinate freely.

VAGINAL ELECTRICAL RESISTANCE

 This method is based on the concept that the electrical resistance (ER) of vaginal fluids
decreases during proestrus and through the estrus due to increase in the volume and ionic
composition of the cervical and vaginal fluids.
 The estrus probe is designed to monitor these changes wherein “low” probe readings are
associated with estrus. However, this tool is labour intensive since cattle must be probed
frequently to detect significant changes in ER. Care must be taken to wash the probe in
disinfectant and thoroughly rinse and dry it before using in another cow.

Vaginal Probe and Electrical Measuring the Electrical Cleaning of Vaginal Probe
Resistance Meter Resistance of Vaginal Secretions Cleaning the vaginal probe after
Vaginal probe and the Vaginal probe inserted to measure measuring the electrical resistance
electrical resistance meter. the electrical resistance of the of the vaginal secretions. There is a
There is a direct correlation vaginal secretions. There is a direct direct correlation between the
between the electrical correlation between the electrical electrical resistance of the
resistance of the vaginal resistance of the secretions and the secretions and the progesterone
secretions and the progesterone concentration in the concentration in the milk or plasma.
progesterone concentration in milk or plasma.
plasma or milk.

HEAT CHECK REPORT SYSTEM

 A heat check report system for herds experiencing estrus detection problems has been
developed by Eastern AI Cooperative and Cornell University.
 It has been particularly useful in herds in which more than one person routinely reports estrus
cows. In these herds the best “cow person” is given responsibility for the estrus detection and
breeding programs.
 Workers return heat reports to the person in charge, who then makes the decision on whether
or not to breed the cow.

PROSTAGLANDINS

 One of the greatest potential uses of prostaglandins is as estrus detection aid in dairy cows in
which estrus has not been observed.
 Research has shown that prostaglandin treatment of cows with functional corpora lutea will
induce a fertile estrus within 2 to 7 days.
 Approximately 50 per cent will be observed in estrus within 80 hours after treatment and will
demonstrate normal fertility.
 For best results, insemination should be based on estrus observation, but insemination at 80
hours after treatment for cows that have not been observed in estrus by that time has been
recommended.
 In these cases estrus detection efforts should continue because some will come into estrus
after the “80 hour breeding” and will have to be inseminated again.

~ 40 ~
PODOMETERS

 Because cows become more active when they are in estrus, activity monitoring through the
use of pedometers is a potentially valuable method of identifying estrous cows.
 Studies have shown that the cow activity measured by pedometers strapped to the cows’ rear
legs increased approximately 400 per cent in cows housed in free stalls.
 Electronic heat detection by means of a pedometer applied just above the fetlock. The amount
of activity / walking is recorded by the device which is read by a scanner each time the cow
enters the milking parlor. An added advantage, particularly on large farms, is that the cows
are inventoried each time they come into the milking parlor. A disadvantage is the initial cost
of the computerized system.

TRICKS OF THE TRADE

 In certain management situations various tricks can be used to improve estrus detection.
o First, cows in heat can be left with the herd to stimulate activity. Studies have shown
that mounting activity increased 3-to 5-fold when more than one cow was in heat.
However, an argument for removing estrus cows is that animals who are actively
mounting sometimes choose favorites. This can reduce the chances of detecting
additional cows that are in heat but less aggressive.
o Second, questionable cows can be placed with strage animals to stimulate activity.
o Third, simply moving cows as a group form one area to another, such as from
concreate to a dirt lot, sometimes stimulates activity. Heat checking should always
include getting all cows up and moving them if they are in free stalls or outside.
These tricks will not be feasible in all operations, but for those in which they can be
used more heats may be accurately detected.

~ 41 ~
 ABBERATIONS OF ESTROUS AND ESTROUS DETECTION IN FARM ANIMALS

After puberty the female enters a period of reproductive cyclicity, which continues, throughout most
of her productive life.

Estrous cycle is the rhythmic sexual behavioural pattern that is exhibited by the female beginning at
one estrus (heat) and ending at subsequent estrus. The word "Estrous" is derived form a Greek word
"Oistros" meaning "Gad fly" - used to describe the behaviour of cows when attacked by such flies.
They continue throughout the adult female’s life and are interrupted by pregnancy, nursing and by
season in some species.

Estrous cycles provide females with repeated opportunities to copulate and become pregnant. If
conception fails, another estrous cycle begins, providing the female with another opportunity to mate
and conceive. When conception occurs, the female enters a period of anestrus during pregnancy,
which ends after parturition (giving birth) and uterine involution (repair and returning to normal size).

CLASSIFICATION OF ESTROUS CYCLE

 Animals could be categorized based on the occurrence of estrous cycle as:

o Monoestrus: Only one estrous cycle per year. eg. Wild animals.
o Polyestrus: Periodic estrous cycles throughout the year. eg. cow and sow.
o Seasonally polyestrus: Periodic estrous cycles only during a particular season. eg.
Sheep and mare.
 Depending on the ovarian activity again estrous cycle is classified as:
o Regular estrous cycle: Characterised by ovulation and formation of corpus luteum.
eg. Cow, sheep, mare, bitch and sow
o Spontaneous ovulators: Ovulations are spontaneous but the corpus luteum formed
will not be functional until mating has occurred. eg., rat or mouse
o Induced ovulators: Ovulation and corpus luteum formation depends on whether
mating has occurred or not. eg. Cat, rabbit, mink.

PHASES OF ESTROUS CYCLE

 In bovine, the estrous cycle can be divided into two phases depending upon the dominant
structure present on the ovary.

Follicular Phase

 It is the period from the regression of corpora lutea to ovulation. The primary ovarian
structure is the Graafian follicle and the primary reproductive hormone is estrogen.

Luteal Phase

 The luteal phase is much longer than the follicular phase and extends from ovulation to luteal
regression. Predominant ovarian structure is the corpora lutea and the primary reproductive
hormone is progesterone.
 Even though the luteal phase is dominated by corpus luteum, follicles continue to grow and
regress during this phase. However, these follicles do not produce sufficient amount of
estrogen to bring about estrus and ovulation

~ 42 ~
 The follicular phase begins after luteolysis and The luteal phase begins after ovulation and
causes the decline in progesterone. includes the development of corpora lutea
Gonadotrophs (FSH and LH) are therefore that produces progesterone (P4). The luteal
produced that causes follicles to grow and phase also includes luteolysis that is brought
develop and secrete estrogen (E2). The follicular about by prostaglandin F2 alpha.
phase is dominated by estrogen produced by
ovarian follicles. The follicular phase ends at
ovulation. Estrus is designated as day 0.
STAGES OF ESTROUS CYCLE

 The estrous cycle can divided into four stages viz. Proestrus, Estrus, Metestrus and Diestrus.
Each of these stages is a subdivision of the Follicular and Luteal phases of the cycle.

Proestrus Estrus Metestrus Diestrus

Proestrus is When estradiol reaches Following ovulation the cells of the Diestrus is
characterised by a a certain level, the follicle are transformed into luteal characterised by a
significant rise in female exhibits estrus cells that form the corpus luteum fully functional CL
Estradiol (E2) and then ovulates. (CL) during metestrus. and high
produced by progesterone (P4)
developing follicles.

Proestrus

 Proestrus is the "building up period" and begins with luteal regression and ends with onset of
estrus. Duration is 2 to 5 days and is characterised by a major endocrine transition, from a
period of progesterone dominance to a period of estrogen dominance brought about by FSH
and LH.

Estrus

 Estrus is the period of sexual receptivity and mating. Estradiol is the main reproductive
hormone responsible for estrus behaviour and physiological changes in the reproductive tract.
Duration is 18 to 24 hrs.

Metestrus
~ 43 ~
  Metestrus is the period from ovulation and formation of corpora lutea. It is an ill defined
phase. The cellular transformation of the follicle to the corpus luteum is called luteinization.
Duration is 3 to 5 days.

Diestrus

 Diestrus is the longest phase of the estrous cycle characterised by a fully functional corpus
luteum and high levels of progesterone. Duration is 10-12 days. Females do not exhibit sexual
receptivity.

~ 44 ~
~ 45 ~
SYMPTOMS OF ESTRUM

Cow

 Standing to be mounted by other cows

 Attempt to mount other cows
 Stringy mucous hanging from vulva
 Mucus smeared on buttocks
 Increased restlessness
 Drop in milk yield
 Reduced feed intake
 Bellowing
 Chin resting on cow's rump by other cows, tail raising
 Frequent urination
 Vulval edema

Buffalo

 Overt signs of estrus are not pronounced as in cattle

 Heterosexual behaviour, particularly standing to be mounted by a bull is the most reliable sign
 Homosexual behaviour, such as standing to be mounted by other females, is observed only
occasionally
 Signs such as vulval swelling, clear mucoid vulval discharge, spontaneous milk letdown,
bellowing, restlessness, frequent urination and raised tail vary in occurrence and intensity
from animal to animal, and in relation to standing estrus.

In most species ovulation takes place during estrus but in cows, ovulation occurs 12-14 h after the
end of estrus.

ENDOCRINE CONTROL OF ESTROUS SIGNS

~ 46 ~
CHANGES FOLLOWING OVULATION

 Following ovulation the theca interna and the granulosa cells of the follicle undergo
luteinisation, a process governed by LH.
o Immediately after ovulation the walls of the follicle collapse into many folds.
o These folds begin to interdigitate, allowing thecal cells and granulosa cells to mix.
o The luteal tissue consists of large and small luteal cells. The large cells originate from
granulosa cells and secrete oxytocin and progesterone. Thesmall cells originate from
cells of theca interna and secrete progesterone only.
 The functions of progesterone are
o stimulates hypertrophy of endometrial glands,
o increases uterine milk secretions,
o inhibits contractions of uterus
o prevents neutrophilic infiltration,
o plays an important role in endocrine control of reproduction because
 it exerts a strong negative feed back on the hypothalamus thereby preventing
development of preovulatory follicles (follicles develop during luteal phase
but do not reach preovulatory stage unless progesterone decreases),
 production of estrogen,

~ 47 ~
  behavioural estrus,
 preovulatory surge of GnRH and LH.

 If the animal is not pregnant, the CL undergoes luteolysis. Luteal oxytocin and PGF 2 alpha
from endometrium cause luteolysis. Estrogen from the follicular waves causes formation of
oxytocin receptors in the uterus. The oxytocin from the CL binds to oxytocin receptors in the
endometrium and activates the enzyme system necessary for formation of PGF 2 alpha . The
PGF2 alpha reaches the ovaries by a vascular counter current exchange system.

~ 48 ~
Mechanism of Luteolysis

 Luteolysis is brought about by

o Reduction in blood flow to the CL by vasoconstriction.
o PGF2 alpha binds to specific receptors on large luteal cells causing influx of Ca ions.
High intracellular Ca ions are thought to cause apoptotic effects (programmed cell
death).
o PGF2 alpha receptor complex also activates protein kinase C (PK-C) that inhibits
progesterone synthesis.

o The lysis of CL results in decreased progesterone levels. The negative block on

hypothalamus is removed and a new cycle is initiated.

Reproductive Characteristics of Cattle and Buffaloes

Parameter Cattle(mean) Buffalo (mean)
Sexual Season Polyestrus Polyestrus
Age at Puberty (months) 15 (10-24) 21 (15-36)
Estrous Cycle

Length (days) 21 (14-29) 21 (18-22)

Estrus (hour) 18 (12-30) 21 (17-24)

Ovulation

Type Spontaneous Spontaneous

Time of Onset (hour) 30 (18-48) 32 (18-45)

Number of Eggs Shed 1 1

Life Span of CL (days) 16 16

~ 49 ~
HORMONE CONCENTRATION DURING FOLLICULAR PHASE

Estrus
Proestrus
When recruited follicles develop dominance, they
As progesterone(P4) drops, FSH
produce estradiol and inhibin that suppressess FSH
and LH increase in response to
secretion from the anterior lobe of the pituitary. Thus FSH
GnRH. FSH and LH cause
does not surge with the same magnitude as LH. When
production of estradiol(E2) by
estrogen reaches a threshold level, the preovulatory surge
ovarian follicles.
of LH occurs, inducing ovulation.
HORMONE CONCENTRATION DURINF LUTEAL PHASE

~ 50 ~
The luteal phase begins immediately after ovulation. During the early luteal phase the corpus luteum
begins to develop and progesterone begins to increase. During the mid luteal phase, progesterone is
at its peak. During the last 2-3 days of the luteal phase when regression of the CL takes place,
progesterone begins to decline initiating the onset of proestrus.

MARE

 The normal estrous cycle in mare is 21-22 days.

 Estrus, the period when the mare displays behavioural signs of sexual receptivity to the
stallion, lasts for 5 to 7 days
 During diestrus, the luteal phase that lasts for 14-15 days, the mare displays sexual rejection
to the stallion.
 Duration of estrus varies among individuals and also among estrous cycles of the same mare.
 Long duration of estrus in the mare may be due to the fact that :
o The ovary is surrounded mostly by a serous coat and some follicles have to migrate to
reach the ovulation fossa to rupture.
o The ovary is less sensitive to exogenous FSH than other species (eg. cattle and
sheep), so that the preovulatory follicle requires a longer time to reach maximal size.
o The level of LH is low compared with FSH and this delays ovulation.
 The intensity of behavioural estrus varies both throughout the estrus period and among
individual mares at comparable stages of the period.
 Old mares, mares underfed during the early part of the breeding season and during twin
ovulations have a longer duration of estrus.

~ 51 ~
SYMPTOMS

 Restless and irritable.

 Frequently adopts the micturition posture and voids urine with repeated exposure of the
clitoris by prolonged rhythmic contractions. This is known as winking of clitoris.

 Introduction of a stallion or teaser, these postures are accentuated.

 Raises the tail to one side and leans her hind quarters.
 Vulva becomes large and swollen.
 Labial folds are loose and readily open for examination.
 Vulva becomes scarlet or orange, wet, glossy and covered with a film of transparent mucus.
 Variable amount of mucoid discharge.
 If the mare is in estrus, the stallion will usually exhibit “Flehmen”.
 Vaginal mucosa is highly vascular, and thin watery mucus may accumulate in the vagina.
 During estrus cervix dilates enough to admit 2-4 fingers, during diestrus only one finger can
be inserted.
 If the mare is in estrus, the stallion will usually exhibit “Flehmen”.

SOW

 Sow is polyestrus.
 The average length of the cycle is 21 days and may vary from 18-23 days.
 The estrogenic phase of the cycle is 6-7 days.
 The luteal phase is 14 days with corpus luteum regression beginning on day 16.
 The duration of estrum is 1-4 days, with an average of 2-3 days.
 Silent heats occur in about 2% of porcine cycles.
 Senility (after 6-10 years) may affect estrous cycle.
 Estrus symptoms are definite and marked and include
o Reduced appetite
o Restless and nervous
o Often pace back and forth by the fence
o Salivation
o Champing of the jaws
o Sow, if suckling, may ignore her piglets, may try to escape or if free will seek out the
boar and stand for service
o Vulval lips swollen and congested
o Mucous membrane pinkish-red
o Mucous discharge
o Mounting other animals
o Grunting and standing motionless for long periods in a position for service is the most
noticeable feature. This peculiar immobilizing reflex or stance (lordosis response) can
be produced by applying pressure of the hands on the sow’s back or by straddling the
sow.
o erect ears
o immobilisation stance
o seeking the male\

EWE AND DOE

 The normal estrous cycle length in sheep is 17 days and in goats is 21 days.
 Photoperiodism is a characteristic of the reproductive cycle or sexual receptivity in sheep and
goats. A reduction in daylight to about 10 or 11 h, whether it occurs naturally or whether it is
induced artificially by penning the ewes in a darkened area during part of the day, will induce
the onset of estrous cycle in anestrus ewes.
 In both sheep and goats a considerable variation in the cycle length occurs due to
o Breed differences

~ 52 ~
 o Stage of breeding season, and
o Environmental stress
 During early breeding season, premature regression of corpus luteum or anovulation results in
abnormally short cycles.
 Estrus lasts for 24-36 h in ewe and 24-48 h in the doe.
 Duration of estrus is inflenced by
o both species,
o breed,
o age,
o season and
o the presence of male influences

SIGNS

Doe

 Estrus symptoms are more conspicuous in does and include

o Restless
o Frequent bleating
o Tail wagging from side to side and up and down (most reliable sign)
o Reduced appetite
o Decreased milk production
o Vulva edematous and congested
o Clear mucous discharge from the vagina
o Occasionally does exhibit homosexual behaviour

Ewe

 Estrus symptoms are relatively inconspicuous, and is not evident in the absence of ram.
o Ewes seek the ram, and together form a following “harem”
o Vulva edematous and congested
o Clear mucous discharge from the vagina
o Waggles her tail and moves it laterally

BITCHES

 Dogs experience ovarian cycles twice yearly. Breeding seasons depend on both genetic and
management factors. Breeds enter their first heat between 6 and 10 months of age. Some dogs
may not begin to cycle until 18 to 24 months of age. The inter-estrus interval on an average is
7 months and ranges from 3.5 months to 13 months. However, the Basenji breed cycles once
a year only.

Phases of Estrous Cycle

 There are four phases namely

o Proestrus
o Estrus
o Diestrus
o Anestrus

~ 53 ~
Proestrus

 Begins with the appearance of vaginal bleeding (spotting) and ends when the bitch allows a
male dog to mount and breed. Duration is 9 days (average and ranges from 2-3 to 25 days).
 Hormonal changes
 Proestrus is under the influence of estrogen, which is synthesized by the developing follicles.
Early proestrus is associated with estradiol concentration of 25 pg/ ml which may increase to
60 –70 pg/ml in late proestrus. Thereafter estrogen levels start declining to reach basal levels
(5 – 15 pg/ml) at the onset of diestrus.
 Progesterone concentration start increasing from basal levels (< 1ng/ml) at late proestrus to
reach levels of 3 ng/ml at the onset of estrus and 5 ng/ml at the start of ovulation. This
progesterone is secreted by follicle which becomes partially luteinised prior to ovulation and
developing CL.

Estrus

 Begins with the first acceptance of the male and ends with the refusal. The duration is 9 days
but ranges from 1-2 days to 18-20 days.
 Hormonal changes
o The bitch is unique in that standing estrus is exhibited when estrogen levels have
started to decline and progesterone levels are increasing. This triggers two events
namely
1. Exhibition of maximal estrus behaviour
2. Initiation of LH surge in the bitch
 Ovulation
o Ovulation occurs 24 to 48 h after the LH surge and occurs over a span of 72 to 96 h.
The eggs are released as primary oocytes and require 24 to 72 h to mature and
become fertile. Once mature, their lifespan is 2 to 4 days. At the time of ovulation the
serum progesterone concentration is typically in the range of 4 to 10 ng/ml.

~ 54 ~
Diestrus

 Begins with the cessation of standing heat and ends when blood progesterone levels fall to
basal levels. Progesterone levels continue to rise to reach levels of 50 to 60 ng/ml 20 to 30
days after ovulation. The bitch is unique in that whether the bitch is pregnant or not, the CL
continues to remain functional for a particular period of time. There is no pregnancy
recognition system in dogs. Therefore CL functions throughout normal gestational period
regardless of the presence or absence of fetuses.
 In a pregnant bitch the luteal phase ends with the onset of parturition (approximately 65 days)
while in a non pregnant bitch the CL functions for a longer period. ie., 75 to 100 days and
regression is brought about by aging of CL. Thus the uterus is not involved in the regulation
of the lifespan of the CL. Apart form LH; prolactin is the major luteotrophic factor especially
in the second half of the luteal phase.

Anestrus

 Anestrus is the phase in which uterus involutes. Average duration is 4.5 months but may be
variable.
 FSH levels are quiet high as equal at levels at proestrus but since follicles never fully mature
but regress, estrus behaviour is not exhibited.

SYMPTOMS

Proestrus

 The bitch is playful

 Appears sexually attractive to the male but refuses mounting attempts by the male by moving
away, baring of the teeth and snapping.
 The bitch may keep her tail tight against the perineum and cover her vulva. This behaviour
changes as proestrus progresses.
 The female becomes more receptive, seeks males, playing and teasing but still refuses the
male by crouching or lying down.
 In late proestrus, the bitch may sit or stand passively when mounted.
 The bloody discharge fades and becomes transparent to straw coloured and the vulva which is
swollen and hard during proestrus, now becomes small and soft as estrus approaches.

Estrus

 The bitch may crouch and elevate the perineum toward the male.
 The bitch attracts males over long distances due to the presence of potent pheromones.
 The vulva is soft and flaccid and the vaginal discharge is often straw coloured or pink.

QUEEN

Reproductive Cycle

 Cats are polyoestrus

 Several estrous cycles are noticed during each of its 2 or 3 seasons per year
 Short haired breeds come to cycle through out the year. Use of artificial light from September
to March to lengthen the `day light hours’ will make the females to cycle all year around

~ 55 ~
Estrous Cycle

Anestrum

 Females may rebuff approaching Tom cat by hissing and striking out. If she accepts the
tomcat she will flex her spine when he mounts, covers the perineum tightly with her tail,
almost achieving a sitting position instead of lardosis in estrus
 Olfactory signals from vulvar area are repulsive to some tom cats that turn away after
smelling her perineum

Proestrum

 This is a short phase of one to three days

 Increased rubbing against objects with head and neck (may be misjudged by owners to be
increased friendliness)
 Rubbing is very marked in 36 h of onset of proestrum. Progresses to rolling (gently or violent)
with purring, rhythmic opening and closing of the claws, squirming and scratching
 The female begins to call a male using the `heat cry’ which is unique to proestrus and estrus
(monotone howling which lasts for three minutes at one time) and is more prevalent in
Siamese females. The female sprays the urine so that both urine and sebaceous secretions left
by rubbing will attract the males

Estrus

 Change in behaviour towards females is noticed

 The females still roll and rub and do not refuse the males. Attempts to mount and assumes a
crouching lardosis (thorax and abdomen touch the floor with perineum elevated)
 Copulatory stance can also be induced by stroking queens back, thighs or neck
 Tail is laterally displaced and slight amount of serosanguinous discharge on the vulva is
observed
 Lardosis is necessary if intromission is to occur. It is stimulated by the treading of the
mounted male
 While the male performs copulatory thrust the female adjusts position slightly by alternate
treading with hind limbs
 Facial expression is intense and is similar to that seen in aggressive cats. Lasts for 10 seconds
to five minutes
 Post mating behaviour is characteristically dramatic
o As the male starts to withdraw his penis following ejaculation, the female’s pupils
suddenly dilate
o As she is freed she utters a copulatory cry- a small piercing vocalization
o She turns aggressively on the male, striking out and hissing.
o The female proceeds into the `after reaction’- violently rolls on the ground and licks
her vulva
o Mating resumes in 11 to 95 minutes.
o Mate as frequently 8 times in 20 minutes or 10 times in one hour
 Another feature useful in reproductive management of cat is that cats are induced ovulators.
Estrus female does not ovulate unless mating occurs. Ovulation can also be induced by
stimulation with males penile spines or by artificial means such as a glass rod (several
insertions – 10 seconds duration 5 –10 minutes apart over 48 h periods). Successful
stimulation will result in aggressive after reaction
 Female will be in estrus for 4-6 days. Most females are receptive on 3rd and 4th day. Estrus
ends abruptly within 24 h after coitus. If pregnant she will not return to estrus until next
seasonal peak or the next year. Ten per cent pregnant queens display estrus behaviour (3 –
6th week of gestation). Mating at such times will result in superfetation

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  If a sterile mating occurs during estrus, ovulation and CL formation are induced. This luteal
phase can be termed as diestrus. Therefore there are three possibilities for a feline estrous
cycle:
o Proestrus, estrus (nonbred), interestrus
o Proestrus, estrus (sterile mating ),diestrus , interestrus
o Proestrus ,estrus (fertile mating), pregnancy
 If no tomcat is present, female is in estrus for 10-14 days then, returns to estrus in 2-3 weeks.
Estrous cycle averages 29 days long

Metestrus

 Metestrus is actually an interestrus period between two estrous periods if breeding does not
occur.
o A queen in estrus does not always mate even when this is desired by the owner. There
are a number of factors that may prevent mating such as:
 Size incompatibility: A small male may have difficulty mating a large female
 Unfamiliar surroundings: A tom brought to a new area may be more
interested in `marking’ the territory than in breeding
 Personality: Shy or timid females may reject can aggressive male. The owner
can pet the queen and stimulate her to posture for the male. Occasionally a
queen will reject one male but accept another.

DURATION OF DIFFERENT PHASES OF ESTROUS CYCLE

Length of Estrous Cycle in Different Species

Species / Stage Estrus Metestrus Dioestrus Proestrus
Cow 12-24 h 3-5 13 3
Mare 4-7 3-5 6-10 3
Sow 2-4 3-4 9-13 3
Ewe 1-2 3-5 7-10 2
Bitch 9 - 75-90 9

FACTORS AFFECTING ESTROUS CYCLE

Nutrition

 Inanition or starvation caused by lack of energy or nutritional deficiency impairs or prevents

secretions of gonadotropic hormones from pituitary gland thereby preventing the occurance of
cycles.

Seasonal Influences and Light

 Seasonal breeding is regulated by light through the medium of eyes or the hypothalamus and
pituitary gland. The total daily amount of light is important in control of the onset of estrous
cycle. This is called as sexual photoperiodicity.
 In sheep and goat decreased day light influences onset of estrus.
 In mares increased total daily hours of light will hasten the onset of estrous cycle.

Temperature

 Excessive heat during summer months causes decreased thyroid activity which directly
reduces reproductive efficiency.

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  In cattle under hot conditions length of the estrous cycle is increased to 25 days as compared
to 20-22 days in cool weather.

Age

 In cattle and swine the young female usually has a slightly shorter length of estrous cycle than
the adult animals.

Systemic Diseases

 Severe chronic wasting diseases cause debility and emaciation resulting in cessation of
estrous cycle.

Pathology of Uterus and Cervix

 In cattle, conditions like pyometra, foetal maceration, mummification causes persistence of

corpus luteum and cessation of estrous cycle.

Endocrine Disturbances

 Endocrine disturbances leading to cystic ovaries or pituitary, ovarian and adrenal tumors
affect the length of the estrous cycle.

Miscellaneous Causes

 Pregnancy causes physiological cessation of estrous cycle.

 The presence of males hastens onset of estrus.
 Infusion of iodine compounds into the uterus shortens the estrous cycle.

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 ABBERATIONS OF ESTRUS AND ESTROUS CYCLE

COW

SUB ESTROUS / SILENT HEAT / QUIET OVULATORS

 Ovulations occur without behavioural manifestation of estrus. The first and second ovulation
postpartum are often not preeced by behavioural signs of estrus and are thus are truly 'Silent
heats'.
 In heifers, the estrus associated with first ovulation is usually silent.

Causes

 A sub threshold of hormone production or an imbalance between hormones

 Nutritional defficiencies such as defficiency of ß Carotene, Phosphorous, Copper, Cobalt etc

Diagnosis

 Diagnosis is based on
o clinical history
o rectal palpation of the genital system.

Treatment

 If a mature Cl is present, PGF2α or an analogue followed by Timed Artificial Insemination

(TAI) can be carried out. If a CL is at refractory stage, double injection PG regimen at 11
days interval could be used.
 Alternatively PRID or other progesterone implants could be used folllowed by fixed time
insemination.

ANOESTRUM

 Failure of estrum or anestrum is the principle symptom of many conditions that may affect the
estrous cycle. Anestrum is observed most commonly either
o After parturition as post partum or Pre service anestrum
o Following service- Post service anestrum (when conception does not occur).
 It is due to multiple causes and often not directly related to endocrine system.
 Diagnosis is based on
o history
o careful clinical examination of the genital tract and ovaries per rectum and vagina by
a speculum
o physical examination of the cow for accurate differential diagnosis
 The treatment of anestrum has been dealt with in detail under the Module on Anestrum.

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DELAYED OVULATION

 If ovulation occurs more than 18 hrs after the end of heat, then ovulation is said to be delayed.
 Caused by
o endocrine defficiency or imbalance,
o failure of development of hormone receptors at the target tissue
o mechanical factors.
 Delayed ovulation is generally assumed to be one of the causes for failure of conception in
Repeat Breeders and is diagnosed by palpation of mature follicle on the ovary more than 24-
48 hours after the end of estrum.
 Treatment of delayed ovulation has been discussed in detail under the Module on ovulatory
defects.

MARES

IRREGULARITIES OF ESTROUS CYCLE

 Irregularities of the estrous cycle in mares may be associated with

o ovarian pathology,
o uterine pathology
o apparent functional abnormalities of the ovarian hypophyseal axis.
 Abnormalities of endocrinology, cyclicity, or ovulation are uncommon in mares with normal
reproductive tracts, and hence, other causes of infertility should be ruled out before abnormal
cyclicity is implicated as a cause of infertility.
 Cystic ovaries, as seen in cattle, do not occur in mares.
 Apparent estrous cycle irregularities that are not associated with pathology include
o prolonged anovulatory estrus during the transition period,
o “silent heat”,
o prolonged luteal activity,
o estrus during pregnancy,
~ 60 ~
 o “persistent estrus” or behavioral changes attributable to estrus.
 Diagnosis of the cause of estrous cycle irregularities in mares is based on
o history,
o teasing records,
o findings on palpation
o ultrasonography
o determination of hormone concentrations

ASSESMENT OF ESTRUOS CYCLE AND OVULATION

 An important factor to remember when evaluating apparent cycle abnormalities is the normal
variation in length of estrus.
o The normal estrous cycle length ranges from 2 to 12 days.
o The length of cycle is generally repeatable within mares, but is longer at the
beginning and end of the breeding season.
 Teasing is a major determinant of apparent cyclicity.
o Individual teasing with an active stallion is the best method of heat detection.
o Should be performed at least three times weekly. The mare’s behavior should be
scored by a knowledgeable individual.
o “Silent heat” may occur in normally cyclic mares.
 Knowledge of the estrus and diestrus reactions of each individual mare is crucial; a mare in
heat may be less demonstrative than another mare in diestrus.
o Some mares may show signs of estrus immediately on contact with the stallion.
o Some mares may require teasing for 3 to 4 minutes before they respond.
 A mare that shows no change in behavior at all during her cycle should be examined regularly
by transrectal palpation and ultrasonography to detect estrus.
 Estrus behavior can also be seen in mares
o during seasonal anestrus
o in mares that have been ovariectomized
o in mares with gonadal dysgenesis.
 The length of diestrus is more repeatable among mares than is the length of estrus, at 15-20
days.
 A pattern of high progesterone for about 15 days followed by low progesterone four more
days is strongly indicative of normal cyclicity

Ovarian and Uterine Characteristics of Normal Cyclicity

 In assessing the normality of follicle growth and ovulation, the large variation in follicle size
at ovulation should be recognized.
o The size of follicles at ovulation is commonly 35 to 45 mm diameter, but mares can
ovulate much smaller follicles or larger follicles, with normal fertility.
o Follicle size at oulation is often repeatable for a given mare.
o Follicle size at ovulation also decreases toward the middle of the breeding season.
o Numerous large follicles and corpora lutea are normally present on the ovaries of
pregnant mares, especially between 30 and 120 days. These are sometimes mistaken
for ovarian pathology.
 Transrectal palpation and ultrasonography are indispensable tools in the evaluation of the
estrous cycle.
 Uterine changes detectable on ultrasonographic examination are also helpful in estimating the
stage of the estrous cycle in mares with questionable estrus behavior.

PHYSOILOGICAL ESTRUS IRREGULARITY : THE TRANSITIONAL PERIOD

 The mare is a seasonal, long-day breeder

 Mares enter anestrus during the winter months.

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  The period between late anestrus and the first ovulation of the year is termed the transitional
period.
 Follicular growth occurs due to sufficient Pituitary gonadotropin output but normal follicle
maturation and ovulation do not take place.
 Palpation and Ultrasonography may not be helpful in distinguishing from normal follicles;
however, they may be structurally and hormonally abnormal.
 During the transitional period, mares may exhibit constant or irregular estrus, as a response to
rising and falling estrogen from waves of non ovulatory follicles.
 When estrus behavior does subside, it may recur within days; there is no normal diestrus
interval.
 Diagnosis is based on the season of the year, lack of evidence of ovulation and multiple small
to large follicles present on both ovaries.

CYCLE IRREGULARITIES ASSOCIATED WITH AGING

 Mares cycle less efficiently after about 20 years of age.

 Cycles may cease over 25 years .
 These mares may be presented because of erratic or constant heat during the breeding season,
or because multiple breeding have not resulted in pregnancy. They may also be apparently
anestrus during the breeding season.
 Diagnosis is based on the mare’s age; these changes are not usually seen until the mare is near
20 years of age.
 Breedings during this time will not result in pregnancy because the mare does not ovulate
 Treatment with pulsatile gonadotrophin- releasing hormone may be effective in inducing
follicular growth and ovulation.
 Fertility is reduced due to
o decreased oocyte viability and uterine changes,
o longer follicular phase and
o fewer ovulations per year.
 Mares over 25 years of age may cease cycling altogether. These mares may be presented
because of erratic or constant heat during the breeding season, or because multiple breeding
have not resulted in pregnancy. They may also be apparently anestrus during the breeding
season.

PROLONGED LUTEAL ACTIVITY

 A prolonged luteal phase is common in mares.

o normally occurs in 4 to 18 per cent of cycles
o severe damage to the endometrium, as seen in cases of pyometra. If the damage is
severe enough that prostaglandin production is impaired, retention of the primary CL
results.
 Differential diagnosis includes
o pregnancy,
o silent heat,
o poor estrus detection,
o short heats that are missed, especially near the middle of the breeding season.
 Diagnosis is based on finding a normal non-pregnant diestrus reproductive tract associated
with failure to show estrus or failure during examination and to find changes consistent with
estrus for more than 2 weeks after ovulation. Progesterone concentrations will be high for
more than 2 weeks.
 Treatment involves administration of prostaglandin F2α. To assure a response, the
prostaglandin should be given at least 5 days after the most recent ovulation. Return to heat
occurs in about 3 days.

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SHORTENED LUTEAL PHASE

 A decrease in the length of diestrus may be indicative of premature luteolysis.

 May be associated with
o endometritis;
o prostaglandin production associated with uterine inflammation or bacterial endotoxin
production, or both
 If a shortened luteal phase is detected, an endometrial culture and biopsy should be obtained
to determine if endometritis is present, and, if so, which organism may be responsible.
 Resolution of endometritis should result in return of normal diestrus intervals.

PERSISTANT ESTRUS

 “Persistent estrus” is a fairly common complaint in working and racing mares.

 When evaluating cases of persistent estrus, the normal variation of estrus length, from 2 to
over 10 days, should be considered.
 A major confusing issue in these mares is what is considered to be estrus behavior. Such
mares may be characterized by their owner as being irritable, kicking when their sides are
touched, leaning on the handler, striking, urinating, or wringing their tails interpreted as heat;
however, they may be simply signs of agitation.
 To diagnose the cause of the apparent estrus behavior, findings on palpation and
ultrasonography of the reproductive tract are evaluated along with the history, and the mare
should be teased with an active stallion.
 Mares with large follicles may respond to administration of human chorionic gonadotrophin
by ovulating approximately 2 days late and going out of heat in another 1 to 2 days.
 Mares that have constant agitated behavior, rather than true estrous behavior, may be in any
stage of the estrous cycle at the time of examination. When evaluated critically, although
some signs associated with estrus may be present the behavior of the agitated mare is anxious
or guarding. She is resentful of the approach of a stallion when teased and may lean away
from the stallion or appear fearful. This is in contrast to the mare in true estrus who; during
teasing, is typically calm, submissive, and interested in maintaining contact with the stallion.
 Abnormal estrus-type or aggressive behavior may be associated with granulose cell or other
ovarian tumors
 Signs of persistent estrus may be attributable to vaginal inflammation due to aspiration of air
into the vagina when the mare is working. These mares lack perineal fat, which affects tone of
the vulvar lips, the angle of the vulva, and the weight of the perineal body.
 The mare has signs of vaginitis such as frequent urination, hunching the back, dragging the
hind feet, and wringing the tail. Racing mares commonly have a history of “stopping” or
“pulling up” part way through training periods; this may be due to pneumovagina incurred
during work.
 Examination of the tract shows the mare at any stage of the cycle; bright echogenic particles
representing air may be seen in the vagina or uterus or both during ultrasonographic
examination.
 Vaginoscopy may reveal inflammation and the mare may show extreme signs of irritation
after the speculum is inserted or removed. This problem may occur even after Caslick’s
surgery has been performed.
 Abnormalities of the bladder or urethra, such as cystitis or urethral masses, may result in
frequent urination that is interpreted as persistent estrus.

CYCLIC BEHAVIOURAL PROBLEMS ASOCIATED WITH OESTRUS

 Some mares become hard to manage, perform irregularly, or even appear lame when in heat;
this behavior is intermittent and corresponds to specific stage of the estrous cycle.
 Altrenogest is commonly used to suppress problem estrus behavior in mares and may be
effective.

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  Ovariectomies for mares should be reserved only for cases in which the mare has cyclic
behavioral problems corresponding to a specific part of the estrous cycle; this behavior should
seem to improve over the winter.
 Occasionally a mare is presented that has vicious outbursts of aggressive behavior. Such
mares should be approached with caution. Little information is available on the cause of this
behavior but it is unlikely to be related to the reproductive tract.

CYCLIC IRREGULARITIES ASSOCIATED WITH OVARION PATHOLOGY

onadal Dysgenesis

 Gonadal dysgenesis refers to congenital lack of development of the ovaries.

 Only a “streak” gonad is present, with no follicular activity. The remainder of the tract is
intact but juvenile because no ovarian steroids are present to induce secondary development.
 Condition is most commonly associated with defects of the X chromosome, including XO and
XXX; however. It may be seen in mares with apparently normal karyotypes.
 Presenting signs include anestrus, erratic estrus, or constant estrus. Exhibition of estrus
behavior is due to lack of progesterone.
 Diagnosis of gonadal dysgenesis is based on history, karyotype and repeated palpation and
ultrasonography or progesterone determination. Mares with gonadal dysgenesis have never
foaled and have never been pregnant. Palpation and ultrasonography reveal very small or
apparently absent ovaries; if ovaries are present, no follicular activity is seen.
 A chromosomal abnormality on karyotype supports the diagnosis of gonadal dysgenesis. No
treatment is possible for these mares.

ANOVULATION OR FAILURE OF OVULATION\

 Failure of ovulation is often preceded by an anovulatory estrus, with follicle regressing and
become atretic. Sometimes however a follicle does not regress, but having reached its
maximum size of 2.0-2.5 cm in diameter the walls become luteinized. This structure behaves
the same way as CL either regressing after 17-18 days or frequently much earlier so that the
cow returns to estrus at a shorter than normal interval.
 Diagnosis of anovulation can only be made retrospectively by noting on rectal palpation that a
follicle persists longer than one would have suspected. In cases of lutenized follicle, it will
remain for 17-19 days before regressing, the ovary containing it will be rounded, smooth,
fluctuating rather than irregular and solid as it is with a CL.
 Treatment is directed towards ensuring that ovulation occurs at the next estrus. Hence, hCG
or GnRH is given. If ovarobursal adhesions are present, there is no treatment.

SEASONAL BREEDING

 In most wild animals the breeding season is initiated at a time when the environment is
suitable for the maximum survival of the young at their birth.
 Some species have only one period of estrus each year (monoestrous) while some have series
of estrous cycles limited to a portion of the year (Seasonally polyestrous).
 True seasonal breeding are inherent in ewe, does and mares.

SHEEP AND GOAT

 Most breeds of sheep and goat exhibit seasonal breeding pattern. However, those in the
tropics as an exception will cycle throughout the year.
 Sheep are short day breeders
 Breeding season is initiated with decreasing length of daylight and ends when increasing day
length reach a ratio of nearly equal daylight and darkness.
 Ewe-lambs and yearling ewes have shorter breeding seasons than older ewes.

~ 64 ~
  The onset of breeding season can be advanced by
o Artificial manipulation of the photoperiod and by use of hormonal agents.
o Introduction of rams into a flock during the transition from anestrus to estrus will
result in high degree of synchrony in first mating with estrus peaking 15-20 days after
introduction of the male.
 As with sheep, goats are short day breeders with cyclic activity occurring between late June
and early April. Placement of bucks with does just before start of the breeding season will
stimulate estrus and result in good synchrony.
 Both rams and bucks are affected by photoperiod showing highest breeding activity and
fertility in the fall. The day length pattern has a dominant controlling influence on initiation
and termination of breeding season.

HORSES

 Mares are long day breeders whose seasons are initiated as the ratio of daylight to darkness
increases and ends during decreasing day length.
 Behavioural estrus that occurs during the short day months from spring to autumn is not
frequently accompanied by ovulation.
 The seasonal breeding pattern is not as well defined for stallion. Fertile semen can be
collected throughout the year.
 However, during months of short photoperiod there is a decline in sexual activity and semen
production.

PHOTOPERIOD ACTION

 The role of photo period in regulating seasonal breeding activity is well established. As
breeding season approaches there is an increase in frequency and amplitude of episodic surge
of LH.
 The sensor of photo periodic response change in mammals is the retina of the eye. The nerve
impulse from these photic signals is transmitted from the retina along the retino hypothalamic
tract to suprachiasmatic nuclei, located anterior to the hypothalamus, and then to the superior
cervical ganglia near base of the brain from which arise the sympathetic nerves that innervate
the pineal gland.
 The diurnal rhythm of secretory activity of the pineal gland is generated by these
suprachiasmatic nuclei. Darkness causes increased sympathetic activity of pineal activity
which increases the secretion rate of melatonin whose secretion has been demonstrated only
in seasonal breeders.

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 PREGNANCY DIAGNOSIS IN FARM AND COMPANION ANIMALS

ENDOCRINOLOGY OF PREGNANCY

 Accurate and early pregnancy detection is an important part of monitoring or controlling herd
fertility
 Early pregnancy diagnosis will help to detect those that are not pregnant so that they can be
inseminated again or culled from the herd

Hormone pattern in blood of domestic animals

Cow(Cycle 21-22 days; Pregnancy 277-300 days)

 Progesterone
o Values vary from 0.44 ± 0.17 ng/ml at estrus to a peak of 6-7 ng/ml from days 9-16
of the estrous cycle and then falls to base line.
o During first 14 days of pregnancy, values are the same as during 14 days of proestrus.
o Cycling animals show a decline, while pregnant animals attain values at or somewhat
above cycling maxima which is then maintained for most of pregnancy.
 Estrogens
o The major estrogens are estradiol-17 alpha, estrone and estradiol-17 beta.
o Mated and pregnant cows show values less than 5 pg/ml from day 3-39.
o Between days 140 and 245, values were reported to below or about 100 pg/ml which
then rises after day 250.

Sheep (Cycle 16.4-17.5 days; Pregnancy 144-152 days)

 Progesterone
o Serum values vary from 0.12 ng/ml (equivalent to anestrus) to 2.0 ng/ml from 10-14
days of cycle.
o After mating, pregnant and non-pregnant values do not differ till day 16.
o Values rise in pregnant ewes to 2.5 ng/ml at day 50, 12 - > 20 ng/ml at days 125-130
reach a plateau, and finally fall steeply to the day of lambing.
 Estrogens
o Pregnancy levels were low (in comparison with the goat), remaining less than 50
pg/ml for most of the period and rising to 100 pg/ml just 1 day before birth.
 Luteinizing hormone
o Levels are less than 1 ng/ml until 20 th day of pregnancy. At days 123-128, no
maternal blood LH can be detected
 Prolactin
o Levels in blood of pregnant ewes range between 20-80 ng/ml during the first 20 days.
o At 2 days prepartum a sharp rise begins, reaching 400 ng/ml on the day of parturition.

Pig (Sow)(Cycle 20-22 days; Pregnancy 112-115 days)

 Progesterone
o Levels increase from 1 ng/ml to a peak of 35.4 ng/ml on day 12 of pregnancy, then
decline to 17.2 ng/ml on day 24.
o In the final 20 days before birth, the levels are 8-14 ng/ml, with possibly a tendency
to decrease in the last few days, and a fall to less than 1 ng/ml at birth.
 Estrogens
o Estrogens remained around 20 pg/ml for the first 24 days of pregnancy
o Between the 20 th and 10 th day prepartum, the level was about 100 pg/ml, peaked to
300 pg/ml at days -2 to -1, and fell to zero after delivery of piglets.

~ 66 ~
Goat (Doe)(Cycle 21 days; Pregnancy 149 days)

 Progesterone
o Levels rise gradually to 33 ng/ml by the 90 th day and then fall to 7 ng/ml on the 140
th day, 3 or 4 days before parturition.
 Estrogens
o Both estrone and estradiol 17- beta are present throughout pregnancy.
o Levels are 5 pg/ml on days 0-30; 47±15 pg/ml on days 39-48; 272±35 pg/ml on days
79-88; 451±70 pg/ml on days 119-128 and 622±78 pg/ml on days 139 - 148 (term).

Horse (Mare)

 Progesterone
o Beginning with the day of estrus and insemination, progesterone values are as
follows: day 0, 1.1 ng/ml; day 8, 7.5 ng/ml; day 28, 4.9 ng/ml; days 52-120,
plateauing at 11-14 ng/ml with a maximunm of 15.2 ng/ml on day 64; day 150, 7.0
ng/ml; day 180, 2.0 ng/ml. Following 180 days, with minor fluctuations, values
remained below 1 ng/ml until the final 30 days of gestation.
 Estrogens
o For group E-1, constituted by estrone, equilin and equilenin, amounts observed till
day 80 are 10-20 pg/ml; day 90, 40 pg/ml; day 120, 275 pg/ml; day 210, a peak of
828 pg/ml.
o A fall in level to 370 pg/ml on day 300 and about 150 pg/ml close to parturition is
observed.
o Values for group E-2, constituted by estradiol-17 alpha and estradiol-17- beta, a
similar time course at about 10% of the amounts of E-1 are observed .

Methods of pregnancy diagnosis

Different methods of pregnancy diagnosis in Cattle, Sheep and Goats have been dealt with in detail
under the following headings

COWS SHEEP AND GOATS

 Management Method  External Palpation

 Physical Method
 Laboratory Method
 Ultrasonography and Fetal
Electrocardiography
 Prostaglandin Induced Milk
Flow Test (PGIMFT)
 Abdominal Ballotment
 Recto Abdominal Palpation
 Radiography
 Ultrasonography
 Hormonal Assay

PREGNANCY DIAGNOSIS

Cattle

 Dairy cows should calve for the first time approximately 24 months of age and should deliver
subsequent calves at intervals of approximately 13-13.5 months. Thus, dairy cows should
conceive within approximately 4 months or less of calving in order to get the maximum
economic returns.
 An ideal test would accurately detect pregnancy before the first expected estrus after
insemination (about 21 days) so the cow could be re-inseminated without further loss of time.

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  Unfortunately no tests are currently available that are practical to use and allow detection of
pregnancy in cows prior to the first expected estrus.

1. Managementmethods

 A presumptive pregnancy diagnosis by cattle owners is based on history and clinical signs.

a) Exposure to a Bull or Artificial Insemination

 History of a cow with a bull or the observation of mating or AI is not a reliable indicator of
pregnancy status and may sometimes be deceptive. Reasons are
o even with high fertilization rates only about 50% of the inseminations result in
detectable pregnancy
o due to unobserved, unplanned, or unrecorded matings, many cows presented with a
complaint of infertility are found to be pregnant on rectal palpation

b) Cessation of the Estrous Cycle

 Around day 15 to 17 after ovulation, bovine embryos signal their presence as a result the
corpus luteum is maintained, and the maternal estrous cycle is suspended. Thus, failure to
return to estrus approximately 18 - 24 days after mating, suggests that conception has
occurred.

2. Physical method

RECTAL PALPATION

When to Palpate for Pregnancy?

 First palpation should be made between 35 - 42 days after artificial insemination.

 All cows that have not returned to estrus by 60 days post breeding should be palpated a final
time.
 After about 60 days, fetal death rates are low and most, but not all; cows that are found to be
pregnant after this proceed to term and deliver calves.

Hand Measurements
Average measurements of the male and female hand to size up the
reproductive tract or fetus per rectum. CRL (crown rump length)
approximately 9 cm at 2 months, 22 cm at 4 months, 44 cm at 6
months, 80 cm at 8 months, and 100 cm at 9 months.

Palpable Changes Per Rectum

 Although number of changes occur in the size, texture, location and content of the uterus
during pregnancy, there are four positive signs of pregnancy that are detectable by rectal
palpation, and examiner must detect at least one of these four signs before declaring the cow
pregnant.
 The four positive signs of pregnancy in cows are:
o Palpation of the fetal membrane slip
o Palpation of amniotic vesicle
o Palpation of placentomes
o Palpation of fetus

~ 68 ~
Palpation of foetal membrane slip

 t is best performed from 35-90 days of gestation.

 It is valuable in the differential diagnosis of pregnancy from uterine diseases characterized by
fluids causing uterine distension, eg. pyometra or mucometra.

Double Slipping can be performed by two techniques:

 Fincher Technique consists of gently picking up and pinching or compressing either horn of
the uterus and feeling the fetal membranes, the allantois chorion, slip between the thumb and
the fingers before the uterine wall escapes from between the fingers.

 Zemjanis Technique is grasping the entire horn and letting it slip through the fingers so that
the connective tissue band allantoic vessels on the lesser curvature of the uterus which are
three or four times thicker than the chorioallantoic membrane could be palpated.

Palpation of foetal membrane slip

 The amnion contains the developing conceptus and the amniotic fluid and is palpable as early
as 28 days after conception in heifers and by 32-35 days in pluriparous cows.
 The vesicle is recognized as a nearly spherical, turgid, fluid filled structure that is
approximately 1 cm in diameter at 28 days and increases in size as pregnancy advances.
 It is detected by encircling the uterine horn with the thumb on one side and the fingers on the
other.
 The vesicle is free floating within the uterus but is most commonly found at the cranial edge
of the inter-cornual ligament.
 The amniotic vesicle becomes progressively less turgid and is difficult to recognize by about
day 65 of gestation. At that time the vesicle softens and the fetus becomes palpable.
 In a bovine conceptus, the heart is external until approximately day 42; therefore, caution
must be exercised when attempting to detect early pregnancies and undue pressure must not
be applied to the amniotic vesicle, as rupture of the embryonic heart or other fragile organs
may result.

Palpation of placentomes

 Placentomes begin to form early in gestation and are of sufficient size to be palpable by 75-80
days of gestation
 The size of the placentomes varies with the stage of gestation and their location in the uterus
 Placentomes are progressively larger near the middle of the gravid horn and are smaller at the
cervical and ovarian poles
 They are most consistent in size immediately cranial to the cervix and are palpated at that
location to estimate the stage of pregnancy
 Placentomes are identified by grasping a longitudinal fold of the uterine wall and rolling it
between the thumb and fingers
 In more advanced pregnancies, placentomes can be palpated by passing the flattened hand
over the uterine wall

Palpation of foetus

 Fetus becomes palpable at approximately 65 days when the amniotic membrane loses its
turgidity and remains theoretically palpable for the balance of gestation.

 In early stages of gestation fetus can be grasped directly. Later, the fetus is detected by
ballottement; the examiner sets the fetal fluids in motion by rocking the hand against the
uterine wall and recognizes the fetus as it rebounds against the hand

~ 69 ~
  The fetus is easily palpable as a free floating firm object within the fluid-filled uterus during
the first 4 months of gestation
 As pregnancy advances, increased weight of the fetus and fluids pulls the uterus ventrally and
cranially until the fetus comes to rest on the abdominal floor during the fifth and sixth
months. Continued growth of the fetus positions it closer to the maternal pelvis during the last
trimester (period of ascent) and palpation of the fetus is facilitated.

Supporting signs of pregnancy

 Asymmetry of the uterine horns

 Resilience and fluctance of the uterine wall
 Fixation of the cervix
 Ovarian changes

3. Laboratory methods

Hormone Estimation

 Progesterone in milk and plasma

 Estrone sulfate in milk

Proteins

 Bovine Pregnancy Specific Protein -B

 Immunosuppressive Early Pregnancy Factor

Progesterone in milk and plasma

 Robertson and Sarda (1971) described a method of diagnosing pregnancy by determination of

progesterone concentration in the plasma of cows.
 Measured using Radioimmunoassay (RIA) or by various assay kits that are available for on-
farm use.

Basis

 In cows, progesterone (P4) from corpus luteum is required to maintain pregnancy. If pregnant,
the cow does not return to estrus and P4 concentrations in blood and milk are elevated at 20-
24 days post insemination. Conversely, if pregnancy fails, P4 concentration is elevated until
approximately day 17 when luteolysis is followed by a sharp decline in P4 concentration by
day 20 and return to estrus.
 Optimum time for collecting the milk sample: 24 days after breeding
 If P4 concentrations are low in blood or milk samples assumed to be non-pregnant, if elevated
assumed to be pregnant.

Cow Side Tests

 Developed to obtain results within 1 h of collecting milk sample.

 All necessary reagents and equipment are provided in kit form.
 Semi quantitative or fully quantitative tests are available which requires minimum equipments
and some expertise.
 Both tests are based on the enzyme linked immunosorbent assay (ELISA).

~ 70 ~
Assay Procedure

 Milk sample to be tested (containing unlabelled P4 if the cow is pregnant or in dioestrus) is

added to the plastic well of the microtitre plates precoated with a specific P 4 antibody,
together with a fixed quantity of P4 labelled with an enzyme (usually alkaline phosphatase).
 After a period of incubation, the contents of the wells are washed; however, P4 will remain
bound to the antibody in the well.
 A substrate reagent is then added to the well which, after the second incubation period, reacts
with the enzyme-labelled P4 to produce a colour reaction.

Interpretation

 Colour is assessed visually or using a spectrophotometer by comparison with those produced

by known standard solutions of P4.
 The amount of labelled P4 that remains bound to the antibody on the wells is inversely
proportional to the amount of unlabelled P4 in the milk sample.
 Light colour reaction indicates high the concentration of P4 in the unknown milk sample.
 Most intense colour reaction indicates Zero P4 in the unknown milk sample

Accuracy

 Pregnant: 80 and 88%

 Non- pregnant: Nearly 100%.

Limitations in Using the Assay On-Farm

 Instructions are not readily understood by non-technical personnel’s.

 Requires skill and ease in using simple equipments.
 Timing of incubation and quantities of reagents should be strictly followed.
 Kits should be stored at 4 °C and before use allowed to warm to room temperature, and not be
heated.
 Interpretation of the colour differences can be difficult for some persons.
 Milk samples should be kept at 2-8 °C until assayed and the recommended preservative
tablets must be used.

REASONS FOR FALSE -NEGATIVE AND FALSE POSITIVE DIAGNOSIS

Sl.No FALSE -NEGATIVE FALSE-POSITIVE

1 Mistaken identity of the animal Cows with shorter than average inter-estrus
either on the farm or in the lab intervals. When milk samples obtained 24 days
post breeding; if the cow is non-pregnant, or in
the luteal phase of the next cycle.

2 Milk storage problems due to Early embryonic death

excessive heat or ultra-violet light.

3 Low P 4 production by the CL. Incorrect timing of insemination

4 Inadequate mixing of milk so that a Luteal cysts and pathological prolongation of

low fat sample is obtained. the life span of the CL
Estrone sulphate in milk

 Estrone sulfate is a product of the placenta and is present in the milk of pregnant cows in
concentrations sufficient to differentiate between the pregnant and non-pregnant cows after
approximately day 100 of gestation.

~ 71 ~
  Practically, however, assays for estrone sulfate are not useful for early detection of pregnancy
and offer no substantial advantage over other methods except in the case of a few cows in
which rectal palpation cannot be performed.

Bovine pregnancy specific protein –B

 in bovines a pregnancy-specific protein (bPSPB) secreted by the trophoblastic cells has been
isolated and purified.
 RIA for measurement of bPSPB have been developed and used to differentiate pregnant from
non-pregnant cows.
 Concentrations of bPSPB are detectable in a few cases as early as 15 days after insemination
and in nearly all pregnant cows by 24 days after insemination.
 The protein increases in concentration as gestation advances and is detectable until
parturition.
 Concentrations of bPSPB are higher in twin pregnancies than in single pregnancies, but
individual variations did not permit accurate prediction of fetal numbers.

Immune suppressive early pregnancy factor

 An assay has been developed for detection of a glycoprotein immunosuppressive early

pregnancy factor in the serum of pregnant cows.
 Blood samples collected from dairy cows within 24 h of ovulation can be assayed for
presence of immunosuppressive early pregnancy factor.
 The assay is able to diagnose pregnancy in 87.5% cows at less than 24 hours of gestation
and 12.5% inaccurate in the identification of non-pregnant cows.

ULTRASONOGRAPHY

 In animals, transducers of 5 MHz and 7.5 MHz frequencies are most widely used for
transrectal ultrasonography.
 Lower frequency transducers are capable of penetrating greater depths of tissue but are not
capable of resolving small structures.
 Higher frequency transducers are capable of resolving smaller structures but do not penetrate
deeply through tissues.
 Under practical conditions, ultrasonography with 5 MHz transducer is an accurate method for
pregnancy diagnosis after approximately day 24. A 3.5 MHz transducer is found to be reliable
after day 30.

FETAL ECHOCARDIOGRAPHY

 It is not applicable before 5 months of gestation, but might have application for the diagnosis
of multiple pregnancies. Refer to practical module on Ultrasonography for further details on
instuments, technique etc.

Ultrasonographic observations

 The embryo proper is first detected within the amniotic vesicle on day 20, when it is 3.5 mm
length. By day 60, the embryo grows to 66.1 mm.
 Between days 28-31, fore limb buds become visible and hind limb buds approximately 2 days
later.
 Two claws become visible on the hooves between days 42-49.
 Movements of the fetal head and feet are first detected between days 42-50.
 Ribs could be visualized beginning on days 51-55.
 Placentomes are first visualized between days 33-38 in the area of the embryo and then can be
seen throughout the uterine horn by day 60.
~ 72 ~
  Fetal gender can be determined.
o Scrotal swellings and teats are detected between days 73-120 and the gender of the
fetus can be determined with an accuracy of 94%.
o Visualization of fetal genital tubercle.
o By days 48-49, the tubercle is located between the hind limbs and moves towards the
umbilical cord in males and towards the tail in females.
o In male fetuses, by approximately day 56 the tubercle is immediately caudal to the
umbilical cord.
o In females the tubercle is under the tail by approximately day 54.
o Experienced ultrasonographers can accurately determine fetal gender between days
55-60.

PROSTAGLANDIN INDUCED MILK FLOW TEST

 Labussiere et.al. (1992) reported diagnosis of pregnancy in cows based on the observation of
milk ejection which in the case of CL maintenance results from the release of luteal oxytocin
induced by intravenous administration of a non-luteolytic dose of PGF2 alpha .
o Examine all cows per rectum on day 18 post insemination to assess ovarian status.
o Perform PG-IMF test on the same day 3 hours after evening milking.

Preparation of Non-Luteolytic Dose of PGF2 alpha

 One ml of PGF2 alpha (lutalyse) which contains 5000 micro gram is reconstituted in 39 ml of
distilled water to arrive at a final concentration of 125 micro gram/ml.

Protocol

 After washing the udder and teat, a sterile cannula is placed in the left fore teat to empty the
cistern milk.
 Subsequently a non-luteolytic dose of 125 micro gram of PGF2 alpha is injected through the
ear vein.
 After the injection the time duration of milk flow is recorded.

Inference

 Elicitation of milk let down reflex with free flow of alveolar milk within a few seconds after
injection and lasting for 3-5 minutes is considered to have a functional CL/presence of
conceptus.
 Absence of milk flow is indicative of non-functional CL.

Reason

 The prostaglandin induced milk let down response observed in pregnant animals could be
attributed to the release of endogenous luteal oxytocin that was actively synthesised by the
luteal tissue and was available for immediate release in response to PGF 2 alpha resulting in
alveolar milk ejection immediately by increasing the intra-mammary pressure within the
cistern and enlargement of cisternal volume.

~ 73 ~
SUMMARY OF METHODS OF PREGNANCY DIAGNOSIS IN COW

Methods Earliest time (in

days)

1. Early pregnancy factor (EPF) / early conception factor (ECF) 3

2. Real-time ultrasound (direct imaging) 13

3. Failure of return to estrus and persistence of corpus luteum 21

4. Progesterone concentration in plasma and milk 21-24

5. Assay of pregnancy specific protein -B 24

6. Palpation of allantochorion (membrane slip) 33

7. Unilateral cornual enlargement and disparity in size, thinning of the uterine wall, 35
fluid filled fluctuation of enlarged horns

8. Palpation of the early fetus when the amnion loses its turgidity 45-60

9. Palpation of the caruncles/cotyledons 80

10. Hypertrophy of the middle uterine artery until presence of fremitus 85

11. Estrone sulphate in milk 105

12. Palpation of the fetus 120

~ 74 ~
DIFFERENTIAL DIAGNOSIS

 Uterine enlargement is usually associated with pregnancy. It should not be always construed
that it is the only cause.
 The ability to make an accurate, early diagnosis is required of most successful large animal
practitioners. Hence, it is imperative to differentiate physiological uterine enlargement
(gravid) at each stage of pregnancy from that of one or more of the other causes.
 While performing a rectal palpation to diagnose pregnancy, a careful consideration of
anatomical structure and relationships of the organs and their consistency, will help to prevent
erroneous diagnoses.

Anatomical structures to be differentiated:

1. Distended urinary bladder 2. Pendulous left kidney 3. Rumen

Pathological conditions Characteristic features
Pyometra: accumulation
of pus in a sealed uterus.
Stage of
pregnancy (in
days)
 Uterine wall is thicker, spongy and less 45-120
resilient.
 Uterine horns unequal in size
 Absence of dorsal bulging
 Pus is more viscous than the fluid of
pregnancy and frequently can be moved
from one horn to the other.
 Absence of placentomes and fetus.
 Absence of fremitus

Endometritis: a non-  Absence of pus. 30-45

specific infection of the  Uterine wall thickened and spongy.
endometrium
Metritis: a non-specific  Presence of visible pus. 35-40
infection of the uterus  Uterine wall thickened and spongy.

Mummified fetus  Absence of placentomes and fetal fluids. 90-240

 Absence of fremitus.
 Thick uterine wall tightly contracted around
a hard, firm fetus.
 Presence of Persistent corpus luteum

Tumors  Lymphocytoma, granulose cell tumor of the 45-120

ovary, and fat necrosis in the mesentry

Maceration  Similar to pyometra, with exception that 45-120

fetal death occurring after fourth month
results in presence of fetal bones causing
crepitation when palpated.

Mucometra or  Mucus varies in consistency 45-120

Hydrometra  Uterine wall fairly thin
 Absence of placentomes and fetal fluids.
 Absence of fremitus.
 Failure of double slipping

~ 75 ~
EWE AND DOE

Methods Commonly Used for Pregnancy Diagnosis

Clinical Techniques

 External palpation
 Abdominal ballotment
 Recto-abdominal palpation technique
(Hulet's technique)

Other Techniques

 Radiography
 Ultrasonography
 Hormonal assays

 None of these clinical methods give reliable diagnosis before 3 months of gestation, while
technological methods have little application in developing countries, especially under field
conditions.

BIMANUAL PALPATION TECHNIQUE

 This method involves digital palpation per rectum combined with abdominal manipulation
 Does are examined in the morning before feeding and watering
 Obese animals are fasted over night
 Restrain the animal in standing position by an attendant holding the head
 The urinary bladder is emptied before examination
 Sitting at the level of pelvic region on the right side of the animal, the examiner's pre
lubricated, gloved index finger of the left hand is introduced in to the rectum
 Fecal pellets are removed and a distended urinary bladder is evacuated by gentle recto-
abdominal pressure
 The right palm is held vertically, with the finger tips touching the ventral floor of the posterior
abdomen; it is then lifted upwards to move abdominal organs forward
 Then, using regulated forward, upward, and backward movements, the reproductive tract
within the pelvic cavity could be held in palm of the right hand
 Examination is performed per rectum using the left index finger assisted by the fingers of the
right hand

 The size, shape, consistency and surface characteristics of the vagina, cervix, uterine horns
and adjoining structures are then assessed
 Palpation of the ovaries is performed in the same manner, pressing the index finger per
rectum against the right fingers
 Once the uterine horns are palpated, the ovaries are easily located and palpated lateral to the
center of the coils as small oval bodies on each side
 Palpation is continued to assess their size, shape, mobility and any other gross structural
abnormalities

RECTO – ABDOMINAL PALPATION

 This method was described by Hulet and hence the name, Hulet's Technique.

~ 76 ~
  This technique involves use of a glass or steel rod (50 cms long and 1.5 cms diameter)
inserted in rectum.
 A soap enema is given 5 minutes before examination to evacuate the rectum.
 The ewe or doe is turned on her back.
 The probe is lubricated with paraffin oil and carefully inserted approximately 30 cms inside
the rectum.
 Left palm is placed on the abdominal wall and the rod is moved to and fro in a horizontal
plane with the right hand.

Inference

 If the rod is palpable as it moves slowly with no obstruction to its passage across the abdomen
from side to side, the ewe is considered non-pregnant. If a palpable mass is detected with the
free hand through the abdominal wall on one or both sides, the ewe is pregnant and the
number of fetuses assessed according to the size and position of masses, wherever possible.
 The rectum and uterus should be carefully examined for injury or damage following
examination.

ULTRASONOGRAPHY

 Ultrasonographic determination of pregnancy has been well documented in sheep and has
been reported to some extent in goats and has been discussed under the following headings
o Preparation and restraint
o Speed and accuracy of the procedure
o Transrectal ultrasonography
o Transabdominal ultrasonography
 The choice of the transducer is typically between the linear array and sector
 Transducers of 3 to 5 MHz frequencies are most commonly employed.
 Quite reliable from 30 days of gestation to term with transabdominal use of sector scanners.
 Identification of single vs multiple fetuses are most accurate from 45 to 90 days of gestation.

PREPARATION AND RESTRAINT

 Sheep are most commonly scanned in the right inguinal wool-less area in the standing
position.
 Goats have some hair in this area and may need to be clipped for best results. When large
numbers of goats are being checked, acceptable scans can be obtained without clipping if a
thick coupling lubricant is used.
 Dairy goats are usually restrained in stanchions or against wall.
 Large numbers of sheep and goats are usually moved through a chute designed for pregnancy
testing.
 Restrict feed for 12-24 h before they are scanned to reduce gas and ingesta in the intestinal
tract, which may interfere with visualization of pregnant uteri.
 In advanced pregnancy or in marginal nutrition animals, care must be taken not to induce
pregnancy toxemia.

Speed

 Experienced sonologist with good facilities can pregnancy test 300 or more females per hour
 The separation of females into open, single or multiple groups is more time consuming, with
60-120 animals per hour
 Sonologist should not sacrifice accuracy for speed

Accuracy
~ 77 ~
  The accuracy of identifying single vs. multiple fetuses typically approaches or even exceeds
90% if females are within the 45–90 day range of gestation
 The identification of females carrying three or more fetuses is much less accurate, usually
only about 50%

TRANSRECTAL ULTRASONOGRAPHY

 A small amount of lubricant is applied to the end of the linear - array transducer, which is
then inserted in to the rectum.
 The operator’s index finger maintains contact with the transducer and is used to orient the
direction of the scan.
 The transducer is directed ventrally and then rotated approximately 45° to each side.
 Some operators like to stiffen the cord behind the transducer when it is used in this manner by
taping a rigid tube around it to provide leverage.
 Urinary bladder is identified as a non-echogenic landmark.
 Gravid uteri are identified by non-echogenic areas, a fluid filled structure anterior and ventral
to the urinary bladder.
 Transrectal imaging allows visualization of pregnancies as early as 15 days post-breeding; but
routinely observed by 19-22 days post-breeding.
 After approximately 50 days of gestation, fetuses are too large to be completely visualized on
the screen. After this stage of gestation, placentomes are the most easily identified cardinal
sign of pregnancy.

TRANSABDOMINAL ULTRASONOGRAPHY

 Accurate estimations of gestation length can be made by measuring the biparietal diameter of
fetuses between 40-100 days of gestation.
 Gestational age can be estimated within approximately one week by visualization of the
diameter of the uterine lumen, crown-to-rump length of the embryo or young fetus or thoracic
or skull size of the fetus up to approximately 90 days of gestation.
 Placentome size provides some guidance, but will vary depending on the position of the
placentomes scanned in relation to the umbilicus of the fetus.

PREGNANCY DIAGNOSIS IN MARE AND SOW

METHODS OF PREGNANCY DIAGNOSIS

MARE SOW

o Management o Management methods

o Clinical o Transrectal palpation
o Ultrasonography o Ultrasonography
o Laboratory o Laboratory methods

MARE

Management

 Failure of return to estrus is a good sign that a mare is pregnant

 This requires the presence of a teaser stallion
 It is preferable that mares should be accustomed to the teasing routine
 Teasing should commence 16 days after service and continue for a further 6 days

~ 78 ~
False Positives will Occur:

 If the mare has a silent heat, a common problem when the foal is with mother
 If the mare becomes anestrus as a result of lactation or environmental factors
 If the mare has a prolonged diestrus and yet has not conceived
 If the mare has a prolonged luteal phase associated with embryonic death; this is referred to
as'pseudopregnancy'.

False Negatives will Occur:

 A few mares will show estrus although they are pregnant.

CLINICAL METHODS

Rectal Palpation

 Uterine tone is marked at 17-21 days of pregnancy when the cornua can be palpated as
resilient tubular organs
 Palpation of the conceptus is first possible at 17-21 days when it is a small soft swelling of
2.4-2.8 cm
 At about 100 days it is often possible to ballot the fetus as it floats in the fetal fluids of the
uterine body
 False positive:
 confused with pyometra
 incomplete involution
 pseudopregnancy
 False negative:
 confusion over service date
 uterus not palpated completely
 A portion of the corpus luteum is only palpable for a few days after ovulation in the region of
the ovulation fossa before it is covered by the dense fibrous ovarian tunic
 Ovulation occurs commonly, 52-63 % in the left ovary, about 60 % or more of the fetuses
develop in the right horn
 Based on the ovarian changes the gestation period may be divided in to 4 periods as follows:
o Period-I: (ovulation to 40 days) characterized by the presence of a single CL of
pregnancy and a number of various sized follicles on both ovaries.
o Period-II: (40-150 days) characterized by marked ovarian activity with as many as
10-15 follicles (over 1 cm in diameter) and formation of 3-5 or more accessory
corpora lutea in each ovary. This ovarian activity with follicle and corpora lutea
formation is probably produced by the high level of gonadotrophic hormones secreted
by the uterine endometrium from 40-120 days of gestation.
o Period-III: (150-210 days) characterized by regression of the corpora lutea or absence
of follicles.
o Period-VI: (210 days to foaling) no corpora lutea or follicles are present.
 During these latter two periods steroid hormones produced in the placenta maintain gestation.

CHANGE IN GRAVID UTERUS

Gravid Uterus 60 Days Gravid Uterus 3months Gravid Uterus 120 Days
60-day pregnant uterus. The Gravid uterus at 3 months Gravid uterus at 120 days. The
enlargement is about the size of a opened up to show the right horn is the pregnant horn
softball fetus
Gravid Uterus 5 Months Gravid Uterus 150 Days Gravid Uterus 10 Months
The diameter of the body of the Right horn pregnancy at The mare was 10 months
uterus is ~35 cm. The fetus 150 days. pregnant when she died of colic.
~ 79 ~
 develops largely in the body of The fetus is in dorso-pubic
the uterus [size of the tile is 15 position [size of the tile is 15 cm
cm square]. square].

EMBRYO OR FOETUS AT DIEFFERENT STAGES OF GESTATION

Embryo 30 Days Embryo 35Days Fetus 95 Days

30-day old embryo. Crown-Rump 35-day old embryo, Crown- 95 day old fetus
length ~ 2.5 cm Rump length ~3.5 cm
Fetal Filly 120 Days Fetus 5 Months Fetus 215 Days
120-day old female fetus showing the 5-month old fetus [size of 215 - Day old fetus. Crown-
anus and external genitalia. The the tile is 15 cm square] rump length 65 cm
ultrasonographic appearance of the
clitoris and its position relative to the
anus are used in fetal gender
determination between days 120 and
240

ULTRASOUND

 In mares, three types of ultrasound are used for pregnancy diagnosis.

o Ultrasonic fetal pulse detector
o Ultrasonic amplitude depth analyser (A-Mode)
o Brightness Mode (B-Mode) ultrasound

Day 120 Male Fetus Day 120 Female Fetus

Male fetus at 120 days of gestation. The
large, pendulous prepuce can be seen
directly behind the area where the
umbilical cord (represented by the
distinct, round, anechoic area) meets the
fetal abdomen
Female fetus at 120 days of gestation. The
teats of the mammary gland become visible
after Day 118. In this image, two halves of the
mammary gland can be seen along the
midline with two very small, hyperechoic teats
at their lateral edges.

LAB METHOD

 Milk or Blood Progesterone.

 Blood Estrogens.
 Immunologic-Gel Diffusion or Haemagglutination-Inhibition Techniques.
 Biological Methods
o There are two tests performed for detecting pregnancy in mares.
 Ascheim-Zondek test
 Mucin Test or Kursowa Method
 Chemical Tests
o Chemical methods are used for detecting the presence of estrogenic hormone in the
urine of pregnant mares.
o There are two tests:
 Cuboni's Test
 Lunaa's Test

Ascheim-Zondek test

~ 80 ~
  Used for detecting the presence of gonadotrophic hormones in the mare serum.
 The gonadotrophic hormone from the endometrial cups is first found in the serum from 40-
120 days of gestation, reaches its maximum between days 50-80, gradually declines and is
absent after 150 days.

Normal Values

 At 40 days : 21,000 IU and 0.1-0.6 IU/ml

 At 80 days : 73,000 IU and 6-296 IU/ml
 At 120 days : 42,000 IU and less than 0.5-106 IU/ml

Materials Required

 About 10-20 ml of blood drawn from the jugular vein, after clotting and separation of serum
at room temperature, the serum should be removed and refrigerated. Avoid overheating of the
sample.
 One or preferably two or three immature female rats of 22 days of age.

Test Procedure

 Two ml of fresh blood or serum from the mare to be tested are injected intraperitoneally or
0.5 ml of serum can be injected s/c daily for 2-4 days.
 The animals are sacrificed 72 h later (when injected intraperitoneally) or 96-120 h later (when
injected s/c).

Result

 In positive cases: Haemorrhagic spots or corpora haemorrhagica on the ovaries, edema of the
uterine horns (2-4 times the normal size).
 In negative cases: No definite changes in the ovaries or uterus.
 False positive cases: May be due to over heating of the serum or storage for too long a period
at room temperature.

Accuracy of the Test

 42-50 days of pregnancy : Good

 50-80 days of pregnancy : High
 80-120 days of pregnancy : Good

Testing before 40 days and after 120 days of gestation may be inaccurate due to low level of
circulating gonadotrophins in the blood.

MUCIN TEST OR KUROSAWA METHOD

 A pregnancy test that utilizes changes in the cervical-vaginal mucus was developed by
Japanese workers in the 1920s and 1930s.
 Applicable over a long period of pregnancy.
 The test is as follows
o Spread a sample of mucin from the cervical os on to a glass slide
o The mucous smear is fixed in alcohol, dried, and stained with methylene blue or
hematoxylin

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Inference

 Smears from pregnant mares are thick and dark and contain globules of mucous and epithelial
cells
 Smears from non-pregnant mares are thin and pale and do not contain globules of mucous
 Kurosawa method must be used with caution during the anestrous season. At that time the
smears contain mucous globules, but differentiation can be made between anestrus and
pregnancy by the absence of epithelial cells in the anestrus condition
 The reliability of the test in pseudopregnancy has not been determined
 False positives are likely

CUBONI’S TEST

 To 15 ml of urine add 3 ml of concentrated Hydrochloric acid in a 100 ml conical flask.

 Place in a boiling water bath for 10 minutes and cool under a tap.
 Add 18 ml of benzene with vigorous shaking for at least half a minute and the supernatant
solution mainly the benzene is poured off.
 Then add 3-10 ml of concentrated sulfuric acid.
 Place in a boiling water bath at 80°C for 5 minutes and shake at intervals during this time.
 The mixture is then cooled.

Result

 Positive result: Presence of dark, oily green fluorescent colour in the lower layer of the
sulfuric acid.
 Negative result:Absence of fluorescence and presence of brownish colour.

Accuracy of the test

 Highly accurate: 120-150 days or preferably 150-290 days of pregnancy.

 Not accurate: 75-120 days of pregnancy.
 After 250-290 days: Amount of urinary estrogens fall as end of gestation period approaches.

LUNAA’S TEST

 This test is a refinement of Cuboni's test.

o Add 1 ml of urine to 10 ml of distilled water in 100 ml flask.
o Add 15 ml of concentrated sulfuric acid.
o After 3-5 minutes the flask is cooled.
o A strong, narrow, 3-5 mm flash light beam is placed close to the flask in a dark room.
o Positive: Presence of light green fluorescence.

SOW

 Different methods have been used to detect pregnancy in the sow. They are as follows:
o Management Methods
o Clinical Method - Transrectal Palpation
o Ultrasonographic Methods
o Laboratory Methods
 Vaginal biopsy
 Plasma progesterone assay
 Plasma estrogen assay

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MANAGEMENT

 Failure to return to estrus at 18-22 days after service or artificial insemination is considered as
pregnant.
 This may also be due to a reluctance to show signs, anestrus or ovarian cysts.
 Early identification of pregnant sow or gilt is essential so that breeders can certify before sale,
she can be served again, treated or culled.

TRANSRECTAL

 Need less restraint

 Preferably to be done when the animal is feeding
 Not possible to perform in gilts because they are too small, and even in large sows a slender
arm is advantageous
 Reliable on per rectum palpable changes of the cervix, uterus and middle uterine arteries

0-21 Days of Gestation

 Cervix and uterus feel very similar to their state at diestrus

 Bifurcation of the cornua becomes less distinct and the uterus becomes slightly enlarged, with
soft walls
 Middle uterine artery (MUA) increases to approximately 5 mm in diameter towards the third
week. It is located as it passes across the external iliac artery (the latter can be identified as it
runs along the anteromedial border of the ilium towards the hind leg, ventrally and slightly
posteriorly; it is about 1 cm in diameter in the adult sow) running forwards towards the
abdominal cavity.

21-30 Days of Gestation

 Cornual bifurcation is less distinct, the cervix and uterine walls are flaccid and thin.
 MUA is 5-8 mm in diameter and more easily identified.

31-60 Days of Gestation

 Cervix feels like a soft-walled tubular structure.

 Uterus is ill defined and thin-walled.
 MUA enlarged to about the same size as the external iliac. Fremitus can be first identified at
35-37 days; the pulse pattern comparable with that of the external iliac artery.

60 Days to Term

 MUA is greater in diameter than the external iliac and it has strong fremitus; it crosses the
external iliac artery more dorsally than before.
 Only towards the end of gestation it is possible to palpate piglets at the level of the cornual
bifurcation.

Accuracy

 Improves with experience and advancing pregnancy. Between 30-60 days of gestation:
o Positive: 94% and Negative: 97%
o Positive: 99% and Negative: 86%

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ULTRASOUND

Fetal Pulse Detector (Doppler)

 Earliest diagnosis using a rectal probe - about 25 days of gestation.

 Accuracy
o Pregnant sows: 92-100%
o Non-pregnant sows: 25-100%

Ultrasonic Amplitude-Depth Analysis (A-mode ultrasound)

 More reliable

B-Mode Direct Imaging

 Very successful.
 The transducer probe is applied to the abdominal wall of the standing sow about 5 cm caudal
to the umbilicus, to the right of the midline and just lateral to the teats, and is directed towards
the caudal abdomen; a coupling medium is always required.

LAB METHOD

 Laboratory methods include

o Vaginal Biopsy
o Plasma Progesterone Assay
o Plasma Estrogen Assay

Vaginal Biopsy

 Histological assessment of the number of layers of the stratified squamous epithelium of the
vaginal mucosa obtained by biopsy can be used as a method of diagnosing pregnancy.
 Between 30-90 days of pregnancy, the accuracy is over 90%.
 Sections taken erroneously from the cervix or posterior vagina are unsatisfactory for
diagnosis.
 Although the technique is a satisfactory one, the big disadvantage is the cost of the procedure
and the time taken to perform the test.

Histological Assessment
Reproductive status No. of layers Thickness (in μ)
Pregnant 2-4 12-15
Diestrum 4-5 20-24
Estrum Greatly thickened

Plasma Progesterone Assay

 In non- pregnant cyclical animals, from about day 16, there is a decline in progesterone
concentrations in the peripheral blood.
 Estimation of progesterone concentrations from this time after service would be useful.
 Accuracy at 16-24 days after service: 96%
 Plasma values ≥ 7.5 ng/ml are indicative of pregnancy.
 More reliable to identify non pregnant sows because of the irregularity of the interestrus
interval.
 The major problem is difficulty in obtaining blood samples.

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Plasma Estrogen Assay

 In pregnant animals, estrogens are detectable from day 20 of gestation.

 A small volume of blood sufficient for the assay can be collected from the ear vein.
 Optimum time to perform the assay is about 24—28 days.

PREGNANCY DIAGNOSIS IN BITCH AND QUEEN

 In most domestic species, pregnancy interrupts normal cyclicity by increasing the length of
the luteal phase and delaying the return to estrus.
 However, in the bitch the length of luteal phase is similar in both pregnancy and non-
pregnancy. This is the reason why pseudopregnancy is a common and normal event in
bitches. Due to the peculiarities of the estrous cycle of the bitch, endocrinological methods of
pregnancy diagnosis from other species cannot be simply extrapolated.
 In canines, the following methods are employed for pregnancy diagnosis:
o Absence of Estrus
o Behavioural Changes
o Physical Changes
o Abdominal Palpation
o Identification of Fetal Heart Beats
o Radiography
o Endocrine Tests
o Acute Phase Proteins
o Ultrasonography

ABSENCE OF ESTRUS

 The failure to return to estrus is not a reliable indicator of pregnancy as the bitch is not
polycyclic.
 Further, the interestrus interval is identical in pregnant and non-pregnant cycles.

BEHAVIOURAL CHANGES

 Behavioural changes typical of pregnancy are observed in both pregnant and non-pregnant
bitches.
 Changes not specific for pregnancy are associated with an increase in plasma prolactin
concentration.
 During the second half of pregnancy, there is approximately 50% increase in food intake.
Some bitches may show a brief period of reduced appetite approximately 3-4 weeks after
mating.

PHYSICAL CHANGES AT DIFFERENT STAGES OF PREGNANCY

DAY CHANGES
Approximately one  A slight mucoid, vulval discharge is noticed in pregnant, and also
month after mating noticed in non-pregnant bitches
 Teats become pink and erect.

From day 35  Body weight begins to increase and may go to up 50% of normal.
onwards
From day 40  Abdominal swelling may be noticed.
onwards  Appreciable mammary gland enlargement and serous fluid can be
expressed from the glands. Between primigravida and multigravida,

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 changes may vary considerably.

From day 50  The abdominal swelling may progress to abdominal distension. In

onwards primigravida or bitches with small litters, changes not appreciable

During the last 7 days  Colostrum may be present in the teats. Exercise care while assessing
of pregnancy changes in mammary size and secretion, since pseudopregnant bitches
also exhibit similar features.

ABDOMINAL PALPATION

 Technique of abdominal palpation can be highly accurate if performed between day 26 and 30
of pregnancy which is at a time when the uterine swellings are approximately 2 inches in
lengthBeyond day 35, pregnancy diagnosis by abdominal palpation becomes difficult to
perform as swellings become larger, more elongated, nearly confluent, pliable rather than
firm.
 Counting the number of fetuses by abdominal palpation is difficult except when performing
an examination at approximately day 28 in a relaxed and thin bitch.
 Difficult to perform in obese or nervous animals, in bitches with tense abdomen, in bitches
carrying single pup or a few pups in cranial abdomen.

Changes Observed by Abdominal Palpation at Different Days of Pregnancy

DAY CHANGES
26- o Conceptuses are spherical in outline.
30 o Diameter varies between 15 and 30 mm.
o They are tense fluid filled structures.
o Readily palpated in a relaxed bitch.

35 o Conceptuses become elongated, and enlarged.

o Tend to lose their tenseness.
o Less easy to palpate at this time.

45 o Uterine horns tend to fold upon themselves, so that the caudal portion of each
horn gets positioned against the ventral abdominal wall, and the cranial portion
of the same horn being positioned dorsally.

55 o Fetuses can often be palpated with the forequarters of the bitch elevated and the
uterus manipulated caudally towards the pelvis.
o Difficult to count accurately the number of conceptuses, except at
approximately day 28 in a relaxed and thin bitch.

IDENTIFICATION OF FOETAL HEART BEAT

 In late pregnancy, possible to auscultate fetal heart beats using a stethoscope.

 Fetal hearts may also be detected by recording a fetal ECG.

RADOIGRAPHY

STAGE CHANGES
Day 30  Uterine enlargement can be detected.

~ 86 ~
  Readily identified in the caudal abdomen, originating dorsal to the bladder
and ventral to the rectum; it frequently produces cranial displacement of the
small intestine.
 Early pregnant uterus has soft tissue opacity and it cannot be differentiated
from pyometra, which occurs at the same stage of the estrous cycle.

After day 45  Mineralization of the fetal skeleton is detectable, progressive mineralization

results in an increasing number of bones that can be identified.
 It is unlikely that the fetuses will be damaged by ionizing radiation; however
sedation or anesthesia of the dam may be required and is a potential risk.

In late  Number of puppies can reliably be estimated by counting the number of fetal
pregnancy skulls.

ENDOCRINE TEST

 Plasma concentrations of progesterone are not useful for the diagnosis of pregnancy in the
bitch.
 A significant elevation of plasma prolactin occurs in pregnant bitches compared with non-
pregnant bitches, and it is possible that prolactin assays may become useful as methods of
pregnancy diagnosis.
 Measurement of the hormone relaxin is diagnostic of pregnancy.

ACUTE PHASE PROTEINS

 Approximately at the time of implantation, an acute phase response occurs, appears to be

unique to the pregnant bitch.
 Measurement of fibrinogen, C-reactive protein, or other acute phase proteins is sensitive
markers for pregnancy.
 The initial rise occurs from day 20 onwards with a peak at approximately day 40. Methods
appear to be reliable, although false positive diagnoses may result from inflammatory
conditions such as pyometra.
 The rise in fibrinogen concentration is the basis of commercial pregnancy test. Estimation of
plasma fibrinogen level by 17 days after mating can be taken as an index for detecting
pregnancy and pseudopregnancy in bitches.

ULTRASONOGRAPHY

 Diagnostic B-mode ultrasonography can be used for early pregnancy diagnosis.

 It is a non-invasive imaging modality, which is safe both for the operator and the animal.
 Most accurate time to perform is generally one month after the last mating.
 Sound frequencies in the range of 2-10 MHz are commonly employed in diagnostic
examinations.
o Small dogs (< 10 Kg) : 7.5 or 10 MHz.
o Medium sized dogs : 5.0 MHz
o Large breed dogs : 3.0 MHz or lower frequencies.
 To visualize the entire reproductive tract, multiple positions and scanning planes may be
required

~ 87 ~
STAGE CHANGES
From 15 days after  Homogeneous uterus can be identified dorsal to the bladder.
ovulation  Conceptuses may be visualized, and appear as spherical anechoic
structures approximately 2 mm in diameter.

From day 20 after  Conceptus is approximately 7 mm in diameter and 15 mm in length

ovulation and the embryo can be visualized.

Approximately 22 days  Embryonic heart beat can be detected

after ovulation
Between days 32-55  The limb buds become apparent and there is clear differentiation of
the head, trunk and abdomen.

From 40 days onwards  Fetal skeleton becomes evident, fetal bone appears hyperechoic, and
casts acoustic shadows.

In late pregnancy  The head, spinal column and ribs produce intense reflections and
become more easily identifiable.

TECHNIQUE

 The dog should be placed in dorsal recumbency, including right or left lateral recumbency
and scanned from the dependent or non-dependent side or with the animal standing.
 Standing on the floor is advantageous for large or giant-breed dogs.
 Clipping the ventral abdominal hair is the standard protocol to obtain the best image.
 Application of alcohol or other wetting agents prior to applying acoustic gel to an unclipped
hair coat may improve image quality by reducing air between the transducer and skin.

FELINE

Abdominal palpation

 Satisfactory time to perfom: 16-26 days post breeding

 Conceptuses are readily identifiable as individual turgid spherical swellings
 Conceptuses can be palpated as early as 13 days post breeding, but can be confused with fecal
mass
 After 6 weeks - conceptus swelling increase markedly in size, elongate and merge, making
palpation more difficult
 Abdominal enlargement at this stage will be appreciable

B- MODE ULTRASOUND

 Enlarged uterus can be confirmed as early as first week of pregnancy

 Gestational sacs can be identified from second week which is more reliable
 Fetal viability can be assessed by cardiac activity detected from third week onwards

DIFFERENT CLINICAL METHODS OF PREGNANCY DIAGNISIS IN FARM ANIMALS

~ 88 ~
 SPECIES DIAGNOSTIC TECHNIQUE DAYS FROM MATING
Cattle and buffalo EPF First week
Buffalo Failure to return to estrus 21-14
Progesterone 22-24
Real-time ultrasonography 24
Bpspb 24-30
Rectal palpation 35-70
Estrone sulfate 72
EPF (sheep)
Sheep and Goat Within 24h
Return to esturs 16-21
Real-time ultrasonography 35-40
P4 (goat) 20
Estrone sulfate 40-50
Horse Real-time ultrasonography 9-16
Return to estrus 16-21
P4 16-22
Rectal palpation 17-25
eCG 40-120
Estrone sulfate 40-100
Pig Return to esturs 18-25
P4 21
Real time ultrasonography 24
Esreone sulfate 26
Fremitus in middle uterine 28
A-mode ultrasonography 30-90
ULTRASONOOGRAPHIC METHOD OF PD IN FARM ANIMALS

Species Technique Placement Earliest day after Diagnostic Accurancy

of transducer mating criteria

Horse B-mode Transrectal 9 Embryonic vesicle 100

RT
Cattle B-mode Transrectal 12 Embryonic vesicle 33
RT

20 Embryo, heart, 100

fluids
Buffalo B-mode Transrectal 30 Embryo, heart, ?
RT fluids
Sheep and Doppler Transabdominal 60 Fetal heart sounds 90
Goats
A-mode Transabdominal 45-50 Fetal fluids 70-90

B-mode Transabdominal 20-22 Fetus (es), 100

RT placentomes
Transrectal Transrectal <20
Pig Doppler Transabdominal 60 Fetal heart sounds 80
A-mode Transbdominal 60 Fetal fluids 70-90
B-mode Transbdominal 22 Allantoic fluid
RT

~ 89 ~
 SUPERFETATION AND SUPERFECUNDATION

Superfetation is a condition that occurs when a pregnant female carrying one or more live fetuses,
comes into estrus, ovulates, is bred again and conception occurs.

Superfecundation occurs when a female ovulates two or more ova during one estrus and copulates
with two or more males during the same estrus with the resultant that the ova gets fertilized with
spermatozoa from each male.

SUPERFETATION

 Condition occurs when a pregnant female carrying one or more live fetuses, comes into
estrus, ovulates, is bred again and conception occurs.
 Superfoetation is theoretically not possible in cows for the following reason
o Even though there are chances that cows may be bred during gestational heat, there
are no evidences that ovulations occur during pregnancy.
 However, in mares ovulations do occur in pregnancy, and hence, in this species
superfoetation is theoretically possible.
 In both the species even if ovulation did occur, the spermatozoa would not be able to pass
through a cervix that is closed by a cervical seal and enter the oviduct through the pregnant
uterus; even if the ovum did get fertilized and reached the uterus, the, endometrial area of
both the uterine horns would already be occupied by the first embryo or fetus. If by chance,
the second zygote does develop in the horn opposite to the one containing the embryo, when
the earlier fetus was expelled, the latter would also be expelled at the same time. Hence, the
possibility of superfetation in uniparous animals is highly questionable.
 In multiparous animals with a poorly defined cervix and cervical seal, superfetation is more
likely to occur. There have been reports of pregnant cats developing mature follicles and
ovulating as late as six weeks after conception. All the fetuses of one service might develop in
one horn and subsequently an estrum may occur with ova fertilized from the second service.
Parturitions could occur at different times from each horn.
 Although superfetation is almost possible in multipara and rare in unipara, it appears highly
improbable in most reported instances where the reports on superfetation are obviously
incorrectly diagnosed.

SUPERFECUNDATION

 Superfecundation occurs when a female ovulates two or more ova during one estrus and
copulates with two or more males during the same estrus with the resultant that the ova gets
fertilized with spermatozoa from each male.
 One may suspect the condition based on the breeding history. Following parturition the
condition becomes obvious with offspring resembling each sire.
 Superfecundation occurs more commonly in dogs and cats, because these species have long
heat periods, multiple ovulations and opportunities of being served by different males.
 Condition has also been reported in unipara where there has been birth of twin horse and mule
foals and twin Holstein and Hereford calves.

~ 90 ~
 INFERTILITY IN FARM AND COMPANION ANIMALS

Regular breeding depends upon the normal function of the reproductive system. In order to breed
regularly, the female has to have functional ovaries, display estrus behavior, mate, conceive, sustain
the embryo through gestation, calve, and resume estrus activity and restore uterine function after
calving. Each of these functions can be affected by management, disease and genetic makeup of the
animal. Impairment in reproductive tract function affects the calf per year programme in bovines.

Fertility

The term fertility as applied to the female denotes the desire and ability to mate, the capacity to
conceive and nourish the embryo and finally the power to expel a normal young one and fetal
membranes.

Infertility

Sometimes considered as synonymous with sterility or it implies a failure or delay in producing the
annual live young one. The term sub fertility is a more appropriate term.

Sterility

Sterility refers to absolute inability to reproduce.

HEREDITARY OR CONGENITAL ANATOMICAL DEFECTS OF THE REPRODUCTIVE

TRACT THAT AFFECT FERTILITY

 Both congenital and acquired abnormalities of the genital system can influence fertility.
 Anatomical abnormalities usually affect individual cows or heifers and therefore may not
influence the fertility of a herd.
 In some cows, because of the severity of the abnormalities, sterility is manifested at the time
of first service period while in some, where the defect is less severe, it may not be detected
until late in life.

ABNORMALITIES OF REPRODUCTIVE TRACT AND OVARIES

Ovarian hypoplasia

Ovarian hypoplasia caused by a single autosomal gene with incomplete penetration is a condition in
which one or both ovaries are small, narrow and functionless. The affected ovary may be partially or
totally hypoplastic and undergoes incomplete development as a part or whole lacking the normal
number of primordial follicles. Depending on the severity of the hypoplasia and whether condition is
unilateral or bilateral, infertility or sterility will result.
Segmental aplasia of the Mullerian ducts and Imperforate hymen

Segmental aplasia of the Mullerian ducts and imperforate hymen are developmental defects of the
Mullerian ducts that lead to various anomalies of the vagina, cervix and uterus. A single, recessive,
sex-limited gene with linkage to the gene for white coat color is considered to be the cause for this
condition.

Congenital lack of endometrial glands

This condition has been observed in few heifers where they exhibited failure of estrus and a retained or
persistent corpus luteum apparently due to a failure of the endometrium to produce the PGF2α

~ 91 ~
required for regression of the corpus luteum.
Double external os of the cervix

Double external os of the cervix in cattle is due to failure of the Mullerian ducts to fuse. The lesion
often occurs as a band of tissue 0.5 to 2 inches in width and 0.5 to 1 inch in thickness, caudal to the
external os of the cervix. On speculum examination it may appear as if there were a double os. In other
cases there may be a true double external os of the cervix and the band of tissue separating the 2
openings may extend a short way into the caudal part of the cervical canal. These seldom interfere
with conception. Affected cows usually conceive and calve normally. Occasionally a portion of the
fetus will pass on either side of the band of tissue and result in dystocia that is easily relieved by
incising the band.
Uterus Didelphys

In true uterus didelphys with a double cervix, conception may be delayed when the semen is deposited
in the cervix opposite of the ovary from which ovulation has to take place. Since, only one horn takes
part in the placentation of the fetus, abortions, premature births, retained placenta, and infertility are
more common.
Abnormal Wolffian or Gartner’s ducts

Multiple cysts may develop along the course of the ducts or the duct may form a long, sometimes
rather coiled, cord 0.5-1.5 cm in diameter distended with fluid.
Intersexuality and Freemartinism

Freemartinism is a distinct form of intersexuality which arises as a result of a vascular anastomosis of

the adjacent chorioallantoic sacs of heterozygous fetuses in twin pregnancies. As a result, although the
external genitalia of freemartin heifers appear normal the internal genitalia frequently show
masculinization.

ACQUIRED DEFECTS OF REPRODUCTIVE TRACT

LESIONS OF THE OVARY

Tumours of the Ovary

Granulosa cell tumours are the commonest neoplasm of the bovine ovary but carcinomas, fibromas,
thecomas and sarcomas have also been described. Most of the large and cystic neoplasms of the
bovine ovary reported in pregnant as well as non-pregnant cattle are granulosa cell tumours. In the
early stages of the tumour it presumably secretes estrogen, for the affected animal is often
nymphomanical. Later, most of the tumour tissue undergoes luteinization and then anestrus usually
occurs. Virilism has been reported in long-standing cases.
Ovaritis, Inflammation or Infection of the Ovary

Inflammation or infection of the ovary may occur secondary to an ascending infection from the uterus
by extension of infection through the uterine walls causing a peritonitis and perimetritis, trauma
produced by rough handling or massage of the ovary. Enucleating the corpus luteum or manual
rupturing of cysts may lead to formation of star shaped or transverse scars on the ovary.
Para Ovarian Cysts

Paraovarian cysts, vestiges of the Wolffian or Mullerian duct system have been occasionally found in
the broad ligament of the cow around the ovary and oviduct but reports are less common in the cow
when compared to dog, sheep and horse.

They may be 0.5 to 2 or more inches in diameter and round or oval in shape located most commonly

~ 92 ~
near or in the fimbria of the oviduct.

LESIONS OF THE OVIDUCT AND ADNEXA

Ovarobursal Adhesions

Lesions between the ovary and the ovarian bursa are known as ovarobursal adhesions. The extent of the
adhesions may vary and may consist of fine web-like strands in the depth of the bursa which does not
involve the uterine tube while in others the ovary may be completely enveloped by the bursa. Conception
is unlikely to occur due to ovulations from the affected side. Where there are extensive adhesions of the
bursa with the ovary, ovulation may not occur and the follicle undergoes luteinization. In some cases
ovarian cysts can develop. The condition is rarely seen in heifers but its incidence increases with the age
of the cow.
Hydrosalphinx and Pyosalphinx

Hydrosalphinx has been reported secondary to segmental aplasia of the paramesonephric duct or to
adhesions of proximal and distal portions of the oviduct. The oviduct gets distended to a diameter of 0.5
to 1 cm or more with clear watery mucus and may appear as elongated, coiled, thin walled and
fluctuating on palpation. Pyosalphinx, commonly associated with extensive adhesions of the
mesosalphinx and mesovarium may follow severe uterine infection. The condition is less common than
hydrosalphinx. In pyosalphinx, extensive perimetritis is not unusual.

LESIONS OF THE UTERUS

Adhesions of the Uterus and Parturient Trauma of the Tubular Genital Tract

A troublesome sequel to the caesarean operation is adhesion of the uterus to the omentum, rectum,
intestines or abdominal wall which is frequently associated with sterility.

Dystocia due to fetal oversize is common in cattle, particularly in the Friesian breed. Delivery of large
calves by heavy traction frequently damages the birth canal to such an extent that the animal is rendered
sterile.
Endometritis

Endometritis due to uterine infection and secondary inflammation has been shown to be a cause for
infertility in cattle.The condition has been dealt in detail in the module on specific and non specific
infections of the reproductive tract.
Pyometra

Pyometra in cattle is characterized by accumulation of pus or mucopurulent material, persistent corpus

luteum and failure of estrum. The corpus luteum persists due to failure of PGF2α secretion as a result of
severe endometritis. The condition usually follows an abnormal parturition, uterine infection, and
delayed involution of the uterus following abortion, premature birth twin birth, dystocia, retained
placenta, septic metritis, or post partum metritis.
Mucometra or Hydrometra

Mucometra or hydrometra almost similar in condition are occasionally seen in cattle with the mucin
present in the uterus varying from a watery fluid to a semisolid mass. The condition usually follows long
standing cases of cystic ovaries, cystic endometrial hyperplasia, arrests in the development of Mullerian
duct system or segmental aplasia of the paramesonephric ducts and persistent hymen.The genetic or
congenital defects may result in distension of both horns with watery, viscous or even rather solid
coagulated masses of mucus and cellular debris that may be confused with pregnancy. In these cows the
ovaries and endometrium are normal and estrum therefore occurs normally. Cows with mucometra or
~ 93 ~
hydrometra are usually sterile unless the case is a uterine unicornis where pregnancy can occur in the
normal horn, or a simple imperforate hymen that can be opened. In cases that are associated with cystic
ovaries treatment should be aimed to resolve the cystic ovarian condition. Cases associated with
persistent corpus luteum need to be treated with prostaglandins.
Perimetritis and Parametritis

Perimetritis and parametritis may be occasionally observed in the cow and may be characterized by
adhesions between uterus and broad ligaments and other pelvic and abdominal organs. The adhesions
may occur due to severe septic metritis, douching and perforation of the uterus with a catheter releasing
irritant material into that area, nonfatal rupture of the rectum due to carelessness in rectal examination
or therapy, perforation of the vaginal wall by the penis of the bull, occasional trauma, severe
haemorrhage or rupture of the uterus at the time of calving due to torsion of the uterus, embryotomy,
forced extraction, severe cervical or vaginal lacerations, cesarean section, extrauterine fetus, excessive
bleeding following manual removal of the corpus luteum or excessive massage or rough handling of the
uterus during removal of the placenta. Lesions may vary from a few thin fibrous strands of connective
tissue to firm adhesions between the uterus, broad ligaments, rumen, omentum, intestines, rectum,
bladder and other organs.
Abscess of the Uterine Wall

Abscess of the uterine wall is occasionally observed in the cow and may appear as a round or oval
structure that is tense and firm on palpation. The size may vary from 1-15 cm in diameter changing the
normal contour or outline of the uterine horn. The condition may usually follow severe metritis,
improper removal of the placenta, trauma caused during insemination or douching. The condition can
be easily diagnosed on rectal palpation and should be differentiated from tumour, cyst or hematoma.
Symptoms are usually absent and in most cases the cow fails to conceive. Treatment is usually
impossible. In rare instances the abscess may rupture into the rectum, bladder or vagina. Rupturing the
abscess into the uterine lumen may be attempted as the last resort.

LESIONS OF THE CERVIX, VAGINA AND VULVA

Cervicitis and Vaginitis

Cervicitis or inflammation of the cervix of the cow is a condition that is commonly associated with
metritis and follows abnormal parturitions such as abortions, premature births, dystocia resulting in
lacerations or trauma to the cervix during forced extractions and retained placenta.

Vaginitis or inflammation of the vagina is observed as a primary condition or secondary to metritis or

cervicitis and may follow trauma, lacerations, bacterial, viral or protozoal infections, abortions,
dystocia, fetotomy, retained placenta, prolapsed of the vagina and postpartum metritis.
Tumours of the Cervix, Vagina and Vulva

Tumours of the cervix are rare in cattle and the few recorded cases have been benign.
Fibropapillomata of the vagina and vulva of cattle have been reported. They do not cause infertility
but may interfere with birth. They are usually pedunculated and may be removed surgically. There is a
possibility that one form of vaginal fibropapilloma is of viral origin and that it is transmitted
venereally. It occurs in young cattle and undergoes spontaneous resolution. Tumours of the vulva
include fibromas, angiomas, carcinomas, and fibropapillomas. The prognosis on squamous cell
carcinomas are guarded to poor as it affects all the regional lymph nodes. Slaughter is usually
recommended.
Parturient Laceration or Bruising of the Vulva

Condition may be followed by cicatrization and distortion with imperfect closure of the vulval
sphincter and aspiration of air.

The sequel is similar to but less severe than those of rupture of the perineum. Some of these cows are
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infertile to natural service but conceive to intrauterine insemination. Dystocia owing to fibrosis of the
vulva may arise at subsequent parturition.
Rupture of the Perineum

A third-degree perineal rupture may occur at calving following dystocia wherein the whole thickness
of the vagina and rectal wall ruptures so that the rectum and vagina are confluent. Healing may not
occur as a result of which air and faeces are aspirated into the vagina causing vaginitis and metritis.

Affected cows have a chronic mucopurulent vulval discharge without impairment of general health.
Normal cyclic behaviour resumes but conception may not occur because of the metritis. Surgical
reconstruction of the perineum may be the choice of treatment. Rupture of the perineum may be
prevented by sound obstetric technique, including episiotomy.

Other Conditions of Cervix, Vagina and Vulva

Cirrhosis of the Cervix

 Rarely, parturient laceration of the cervix is followed by fibrosis and obstruction of the
cervical canal, with infertility. Occasionally, cirrhosis of the cervix may prevent proper
dilation of the organ at parturition, but most cases of failure of cervical dilatation are of
functional origin.
 Prolapsed of one or more of the cervical folds is commonly seen in the pluriparous cow. It is a
physiological hazard of parturition and is not a cause of infertility.

Gross Fibrosis of the Vagina

 This may follow laceration and pyogenic infection causing a narrowing of the birth canal and
dystocia. Caesarean section may then be required.

HORMONAL CAUSES OF INFERTILITY

 Hormonal or functional forms of infertility mostly affect individual animals within a herd.
However, when a larger group of animals in a herd are affected, they frequently reflect some
other problem such as inherited factors; nutritional deficiencies or excesses; social influences
which may arise from modern husbandry methods.
 Hormonal diseases may include cystic ovarian degeneration, failure of estrum or anestrum
and repeat breeders.

Cystic ovarian degeneration

 Cystic ovaries, one of the most common condition causing infertility in dairy cattle is
characterized by follicular cysts, luteal cysts and cystic corpora lutea.
 Follicular and luteal cysts are basically anovulatory cysts while cystic corpora lutea are
ovulatory cysts.

The classification, etiology, clinical signs, diagnosis and treatment of cystic ovarian dysfunction has
been dealt with in detail in Module 13 under the title Ovultory Dysfunction

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Anestrum

 Following puberty, the cow should cycle regularly at approximately 21-day intervals unless
interrupted by pregnancy and for a short period in the puerperium. The occurrence of estrus
signs is the only way to tell that the animal is cycling. It is possible that estrus signs are
occurring but are not being observed which is purely a management problem. The other
possible causes why the animal may not exhibit estrus signs are.
o The ovaries may be quiescent and inactive; this is referred to as true anestrus.
o There may be cyclic ovarian activity but the cow is not showing the normal
behavioural signs; this is described as sub estrus or silent heat.
o There may be a progesterone-producing structure in the ovary which is exerting an
inhibitory effect upon the hypothalamus and anterior pituitary; this may be
a persistent corpus luteum or a cyst.

True Anestrus

 In true anestrus the ovaries are quiescent and nonfunctional. The reasons for the failure of
cyclicity may be insufficient release or production of gonadotrophins to cause follicle
development, or it may reflect the failure of the ovaries to respond, but the latter is unlikely.

 The clinical history will be

o That a cow or heifer has not been seen in estrus;
o Rectal palpation will reveal small, flat and smooth ovaries and absence of either a
developing, mature or regressing corpus luteum. In buffaloes the ovaries may appear
spindle like.
o Re-examination of the cow per rectum after 10 days will help confirming the case
where
 in true anestrus ovaries will be virtually unchanged;
 in late diestrus or early diestrus (metestrus) the ovaries will have a distinctly
palpable corpus luteum.
o Milk or blood progesterone determinations are helpful in confirming diagnosis; two
samples can be taken at 10-day intervals where
 low-low values are indicative of anestrus
 high-low or low-high values are indicative of diestrus

Anestrum due to Subestrus or Silent Estrus

 Subestrus or silent estrus most commonly occurs when the first and second ovulations
postpartum are not preceded by behavioural signs of estrus.
o The causes for silent estrus or subestrus could be due to
 improper heat detection,
 genetic predisposition,
 climates where incidence is more common in temperate climates and more
common in the winter than in the summer months,
 nutritional deficiencies such as deficiency of ß-carotene, phosphorus, copper
cobalt and
 overweight.
o Diagnosis is based on the clinical history and rectal palpation of the genital system.
o One cannot differentiate from unobserved estrus, since the clinician will be checking
for evidence of cyclic ovarian activity.
o The corpus luteum must be differentiated from a cyst; it may be persistent or the cow
may be pregnant. In the event of doubt then a re-examination done in 10 days would
help in confirmation.

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 o Treatment involves administration of prostaglandin F2 α or an analogue followed by
fixed-time insemination if a mature corpus luteum is present and the cow is not
pregnant. If the corpus luteum is at a refractory stage (refer module on estrus
synchronization) a double injection prostaglandin regimen at an 11 day interval could
be used. Alternatively a PRID or other progestogen implant could be used followed
by fixed-time insemination.

Anestrum due to Persistent Corpus Luteum

 Persistent corpus luteum occurs when there is failure in the production or release of
endogenous luteolysin. Conditions that result in persistence of corpus luteum are
o Pregnancy
o Pyometra
o Mucometra or hydrometra
o Mummification
o Maceration
 It is important to remember that persistent corpus luteum does not occur in the presence of a
normal non-pregnant uterus. Many veterinarians tend to call wrongly a cyclic CL as
persistent CL.
 The condition, once diagnosed, can be readily treated with PGF2 α or a synthetic analogue,
provided that the clinician is confident that the cow is not pregnant; estrus will occur in 3-5
days.

MANAGERIAL CAUSES OF INFERTILITY

 Managerial deficiencies are a common cause for lowered infertility and repeat breeding in
herds and must be differentiated from infectious form of infertility caused by Vibriosis and
Trichomoniasis which are also herd problems. The common managerial deficiencies leading
to infertility are
o Poor heat detection
o Improper time of insemination
 The average length of the estrus period is 18 to 24 hrs and ovulations occur 12 hrs after the
end of estrus period. Since, the sperm survivability is 48 hrs and the ovum survivability is
only for 12-24 hrs, the sperms should be present in the female reproductive tract about six
hours before ovulation for optimum fertilization. Therefore, the best time to do AI is 12 to 18
hrs after onset of estrus.

Nutritional Deficiencies

 The effect of nutrition, especially TDN intake and need for a positive energy balance for high
fertility has been discussed in detail in this module under Nutritional Causes of Infertility.

Heat Stress

 The effect of stress on lowered bovine fertility is associated with high ambient temperatures
of over 80°F for long periods in summer months. Under hot climatic conditions the duration
of estrus was shorter and signs of estrus were reduced. High temperatures also resulted in
depressed feed consumption and milk production. Similarly, lowered conception rates due to
low fertilization rates and high embryonic mortality rates were also observed.

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Improper Artificial Insemination Technique

In natural service the bull deposits the semen in the proper site, while in artificial insemination there
are many possibilities for man to render infertile the bovine sperm cells. These include

 Improper extension, freezing and storage of fertile semen.

 Improper thawing of frozen semen.
 Insemination should take place promptly, within several minutes after thawing. Thawing
semen and keeping it at 5 ° C or 40 ° F for an hour or more is very detrimental to sperm cells.
 Proper insemination technique should be followed
 Palpation of the ovaries during estrus should be avoided as it may result in rupture of the
graffian follicle. Extreme caution should be taken to perform palpation in a gentle, skilled
manner so as to avoid manual rupturing of the thin walled follicle.

EFFECTS OF NUTRITION UPON REPRODUCTION

 The major contributors to infertility and poor reproductive performance are

o deficiencies of various trace minerals
o inadequate vitamin intakes
o energy protein imbalances
o excessive protein intakes
 Infertility or sterility due to nutritional causes is usually characterised by
o a failure of estrum or a cessation of estrous cycle
o a failure of conception or early embryonic death
 The effects of nutrition on fertility has been dealt with in detail under the following headings
o Effects of Energy Intake on Fertility
o Effects of Protein Intake on Fertility
o Effects of Vitamins on Fertility
o Effects of Micronutrients on Fertility

EFFECTS OF ENERGY INTAKE UPON FERTILITY

 Inadequate energy intake in heifers and early lactation cows reduces reproductive
performance.
o inadequate amounts of energy in heifers delays onset of puberty
o If energy deficient rations are fed to heifers that have begun to have normal estrous
cycles, they may stop cycling.
o Negative energy balance during early lactation affects reproduction because they
cannot consume adequate feed to meet the nutrient requirements for high levels of
milk production. Energy stores in body tissues are mobilized and weight losses occur.
 Excessive energy intake during late lactation and the dry period can cause “fat cow” problems
which lower reproductive efficiency in the next lactation.
 The most severe consequence of inadequate nutrition is
o the cessation of cyclical activity (anestrus)
o silent estrus
o ovulatory defects
o fertilization failure
o embryonic or fetal death.
 The two main ways in which energy deficiency is believed to affect pregnancy rates is via
o GnRH system
o metabolic regulators of ovarian function.
 Negative energy balance does not affect FSH secretion but LH secretion is impaired.

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  Circulating concentrations of glucose, insulin and insulin like growth factor 1 are lower in
cows in negative energy balance than in fully fed animals, whilst concentrations of non-
esterified fatty acids are higher. All of these might be expected to affect follicle development.

Fatty Liver Syndrome

 Excessive energy intakes during the late lactation and dry periods can lead to “fat cow”
problems.
 Over conditioned cows have a higher incidence of retained placenta, more uterine infections,
and more cystic ovaries.
 Cows have poor appetite and body fat reserves are mobilized to meet the energy deficit for
lactation.
 Inadequate supplies of endogenous and exogenous protein exacerbate the syndrome.
 The liver becomes infiltrated with fat and the cow often develops ketosis.

Diagnosis

 Biopsy
 Estimation of blood parameters to diagnose impaired liver function.
 Eight weeks before calving
o There is an increase in
 non-esterified fatty acids
 bilirubin
 aspartate aminotransferase
 ß-hydroxybutyrate concentrations
o There is a decrease in
 glucose
 cholesterol
 albumin
 magnesium
 insulin
 Impaired liver functions will affect albumin production, whilst if fat has replaced glycogen in
the liver parenchyma total glycogen reserves will be reduced.
 In cows with fatty liver
o basal concentrations of LH are lower
o fewer pulses of LH in affected
o pre ovulatory concentrations of LH are lower in cows with fatty liver
o the LH response to administered GnRH is lower
o luteal progesterone concentrations are lower than in normal cows

Treatment

 Treatment is not possible, and usually there will be eventual recovery. Attempts to prevent the
disease can be made by ensuring that cows are not excessively fat at calving and receive
adequate energy thereafter to exclude the need for excess fat mobilization.

EFFECTS OF PROTEIN INTAKE UPON FERTILITY

 Deficiency of protein is seldom encountered except in severe inanition or underfeeding where

Vitamin A and phosphorous are often complicating factors.
 Prolonged deficiency of protein reduces reproductive performance.
 High levels of protein in the diet also affect reproduction due to the following facts
o Degradation of excess Rumen Degradable Protein leads to increased circulating
concentrations of ammonia and urea which when present in high levels in the uterus

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 may be toxic to spermatozoa, oocytes or embryos or may adversely affect uterine
function
o In addition, abnormally high circulating concentrations of urea may also have an
effect upon the hypothalamic-pituitary axis by
 Increasing basal blood LH concentrations, and an exaggerated LH response
to GnRH stimulation
 reducing blood progesterone concentrations and luteal progesterone synthesis

FFECT OF VITAMINS ON FERTILITY

 Adequate intake should be ensured by feeding vitamins and minerals in small amounts of low
energy concentrates or mixing them in a complete dry cow ration. Vitamin deficiencies in
cattle are mainly limited to vitamin A while deficiencies of other vitamins are not likely to
cause any reproductive failure.

Vitamin A

 Vitamin A is required for maintaining healthy tissue in the reproductive tract. Deficiency in
cattle causes delayed sexual maturity, abortion, birth of dead or weak calves, retained
placenta and metritis. The recommended daily supplementation for dairy cows is 30,000-
50,000 units. Dry cows fed only poor quality hay for extended periods without additional
supplementation may benefit from vitamin A injections.
 β-carotene is a substance found in many plants. The cow converts this into vitamin A. It is
known to be in high concentrations in fresh green roughages while grains contain relatively
low amounts. Silages, especially alfalfa, contain moderate levels while corn silage is a poor
source. Dry hays, especially alfalfa, are excellent sources of carotene. Despite high levels at
harvest, β-carotene levels decrease during storage, with the extent of destruction being
dependent on storage conditions.
 Deficiency of β-carotene in diet causes
o Delayed uterine involution
o Delayed first estrus after calving
o Delayed ovulation
o Increased incidence of cystic ovaries
o Early embryonic death and abortion

Vitamin B – Complex

 Deficiency of vitamin B has been demonstrated in animals to produce the same inhibitory
effects on reproduction as reduced food intake or starvation. However, as B complex vitamins
are synthesised in the rumen, cows are in no danger of suffering from vitamin B deficiencies.
Vitamin B12 requires cobalt synthesis and therefore cows in cobalt deficient areas may suffer
from severe inanition due to lack of appetite and insufficient intake of feed brought about by
cobalt or B12 deficiency.

Vitamin D

 Vitamin D is required for normal calcium and phosphorus metabolism. However, deficiencies
are seldom encountered as it is present in roughage of any quality. Cows receiving a normal
amount of natural light manufacture their own Vitamin D. Most commercial concentrates
contain supplemental vitamin D in amounts sufficient to meet the cow’s requirement of
10,000 IU per day.

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Selenium and Vitamin E

 Selenium deficiency in dry cows has been reported to cause retained placenta, abortions, a
high incidence of embryonic-fetal loss, poor fertility, and increased incidence of metritis, a
higher level of general infection and the birth of dead or weak calves in some problem herds.
It is difficult to separate the effects of selenium and/or vitamin E deficiency since both have a
ubiquitous antioxidant function which protects a wide range of biological systems from
oxidative degradation.
 Deficiency occurs when soils contain < 0.5 mg/kg, or diets < 0.05mg/kg selenium. Vitamin E
deficiency occurs when animals graze post-mature pasture, receive other diet components that
contain < 0.7 mg/kg of the vitamin, or are fed diets that are high in polyunsaturated or rancid
fats. Vitamin E deficiency does not affect the estrous cycle or ovarian function.
 Diagnosis of selenium deficiency can be made by measuring circulating concentrations of
selenium or, better, by measuring selenium stores in the liver. Measurement of levels in feed,
pasture or soil is often also indicated. Supplementation is widely practiced, especially in areas
where soils are known to be marginal or deficient. However, it should be remembered that
excessive selenium is toxic, especially where it has been given by injection.

EFFECTS OF MICRONUTRIENTS UPON FERTILITY

 Micronutrient deficiencies affect reproduction by

o depressing the activity of rumen microflora;
o reducing enzyme activity
o affecting energy and protein metabolism and the synthesis of hormones;
o altering the integrity of rapidly dividing cells within the reproductive system.

Cobalt

 Cobalt deficiency causes

o anaemia
o inappetance
o poor bodily condition
o thriftiness
o loss of condition
 Fertility is affected by
o increased number of ‘silent’ estruses
o poor pregnancy rates
o irregular inter estrus intervals.
 Deficiency occurs when diets contain < 0.07 mg/kg D.M. cobalt and is due to failure of
vitamin B12 synthesis, which is an essential co-factor for carbohydrate metabolism.

Copper

 Copper deficiency results in

o anestrus
o sub estrus
o poor pregnancy rates.
 When this occurs in association with other signs of hypocuprosis, such as anaemia, poor
growth, bleached coat colour and diarrhoea, a diagnosis is likely. Hypocuprosis can be either
direct or indirect.
 Indirect deficiency occurs due to excessive molybdenum, iron or sulphur intake and, possibly,
calcium or zinc. Copper deficiency occurs when cattle are fed diets containing <3mg/kg
copper, if the molybdenum content is <3mg/kg; 3 to 10 mg/kg; or >10mg/kg if the
molybdenum content is >10 mg/kg.

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Iodine

 Reproductive failure resulting from iodine deficiency is invariably related to impaired thyroid
function in the dam, embryo or fetus, which in the last two can cause embryonic death,
abortion, stillbirth or weak goitrous calves.
 There is good evidence that treatment with iodised oil injection can improve the deficient
status. Simple iodine deficiency can occur because of an intake below 0.8 mg/kg D.M.
considering a level of 2.0 mg/kg D.M. to be the threshold for deficiency.
 Disturbance of thyroid function can also be due to goitrogenic substance present in lentils,
soya bean and linseed. High levels of goitrogenic substance can produce anestrus in heifers.
Since iodine is needed for thyroxine synthesis, iodine deficiency is largely manifested through
the effects of a lack of thyroxine.
 Thyroxine is a general metabolic regulator and, in particular, a regulator of mitochondrial
activity. Thyroxine deficiency is associated with non-specific signs of poor growth and poor
‘doing’ together with loss of libido and inhibition of estrous behaviour.

Manganese

 Manganese has a ubiquitous role in reproductive function, being involved in steroid synthesis.
Both the pituitary gland and ovaries are relatively rich in this trace element.
 A variety of reproductive disorders which depress fertility in cows have been blamed on
manganese deficiency; these include anoestrus, poor follicular development, delayed
ovulation, silent estrus and reduced conception rates it also causes joint and limb deformities
in calves.
 Under normal circumstances it is likely that normal pasture will provide the necessary
requirement of 80 mg/kg D.M. in the food although some foods are low in manganese. In
addition, there is an interaction with the calcium: phosphorus ratio in the diet, with some
evidence that high liming of pasture can cause manganese deficiency.
 Manganese is a cofactor in a number of enzymes that are responsible for gluconeogenesis and
has a significant role as an antioxidant. Manganese is also involved in cholesterol synthesis
and, hence, affects steroidogenesis.

Phosphorus

 It has been estimated that the normal requirements for phosphorus in the cow for the
maintenance of pregnancy are about 13 g/day, with about 7 g extra for each 4.5 liters of milk.
Providing that forage contains adequate levels of phosphorus, normal diets should contain
adequate phosphorus to ensure normal fertility.
 However, deficiencies can occur where forages have inadequate levels and, perhaps, because
of the interaction between calcium and phosphorus. However, phosphorus – deficient pastures
are often deficient in many other micronutrients making assessment of the role of phosphorus
difficult.
 The evidence for the importance of hypo phosphataemia as a cause of infertility is conflicting.
The provision of supplementary phosphorus has been shown to improve the breeding
performance of grazing cattle.
 A number of authors have described infertility, which was characterized by anoestrus, sub
estrus, irregular cycles and low conception rates in the absence of other clinical signs of
phosphorus deficiency. If hypo phosphataemia is suspected, a rapid response can follow the
feeding of dicalcium phosphate or bone meal. It is important to ensure that the ratio of
calcium to phosphorus is 1:1.

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Zinc

 Zinc deficiency has been shown to have an adverse effect upon reproductive function in the
male of many species. Its influence on reproductive function in the cow and heifer is not
clear.
 Uptake of zinc is impaired by copper, calcium, iron, molybdenum and cadmium. Excessive
levels of zinc supplementation can lead to perturbation of essential fatty acid metabolism,
which affects prostaglandin synthesis. Its potential role as an antioxidant is considered below.

Phyto-Estrogens

 When cows ingest large quantities of these substances they become anestrous, with large
ovarian cysts, vulval and cervical enlargement and poor conception rates Such substances are
found in subterranean clover, certain strains of red and white clover and Lucerne.

SPECIFIC INFECTIOUS DISEASES CAUSING INFERTILITY IN CATTLE

 Infectious diseases of cattle adversely affect reproductive performance, either directly or

indirectly.
 Direct effects upon the embryo include infections that result in early embryonic death, and
those that infect the more advanced fetus or its placenta, resulting in abortion, stillbirths or the
birth of weak calves.
 Indirect effects upon embryo survival include infections that adversely affect uterine function
and infect the maternal component of the placenta leading to embryonic death and fetal
stillbirth.
 Reproduction is also affected by systemic illness causing fetal losses or via a direct
impairment of reproductive cyclicity.
 The various infectious diseases that affect cattle have been dealt with in detail in the module
on Specific Infectious Diseases Causing Infertility.

EWE

 Fertility can be defined as the number of lambs born per 100 ewes put to the ram (i.e. true
lambing percentage).
 The introduction of rams marks the begening of breeding season, and all physical and
financial performance should be calculated from this point, taking into consideration ewes
that die, those that are culled and those that abort or are barren.
 The three factors that influence the number of lambs sold are
o Fertility, i. e. whether the ewes are pregnant and lamb
o Fecundity, i.e. the number of lambs born per pregnancy
o Survival rate to weaning
 Ewes are generally allowed to run with the ram during the breeding season and not
segregated; thus estrus detection problems are not encountered.
 Most breeds of sheep remain acyclici for longer periods after parturition than the cow, thus
allowing the reproductive system time to recover from the effects of pregnancy.
 The main factors responsible for infertility in sheep are specific infectious agents that usually
result in abortion.
 Structural, functional and management factors are of limited importance.

Structural Defects

 Structural defects of ovine genital organs are uncommon. Most defects involve the ovaries
and their associated bursae, with fibrin tags and paraovarian cysts being most frequently
identified.

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  Other lesions identified that would have caused infertility or sterility are ovarian aplasia,
ovarian hypoplasia, bilateral hydrosalpinx, aplasia of the para mesonephric ducts,
freemartinism and hermaphroditism. Owing to the rarity of anastomoses of the adjacent
allantoic vessels of twins, the freemartin condition is likely to be rare.
 Cases of intersexuality are seen, mainly at lamb castration. They are male
pseudohermaphrodites referred to by shepherds as ‘wilgils’. The fact that several may be seen
at once in a flock tends to point to a possible hereditary cause.

FUNCTIONAL CAUSES OF INFERTILITY

 Except in the case of unthrifty ewes (which are usually culled), anestrus is uncommon is
sheep. In fact, when there rams are turned out with the flock it is usual for most of the ewes to
be mated within a month.
 The first estrus of the breeding season in some ewes is anovulatory and, more frequently ewes
fail to become fertilized at these early matings compared to later one.
 Ovarian follicular cysts are occasionally seen while luteal cysts are rare.
 Embryonic death, or resorption, is a conspicuous feature of sheep infertility. Early embryonic
death has been associated with infectious diseases such as toxoplasmosis and Border disease.
 Abortion and of fetal mummification are occasionally seen.
 A specific environmental cause of sheep infertility, due to grazing on pastures of subterranean
clover which contains large amounts of the estrogenic substance genistein, is the cystic
degeneration of the endometrium and permanent sterility.
 Asynchrony or imbalance of the hormonal changes that occur around the time of estrus and
during the early luteal phase probably results in embryonic death.

MANAGEMENT FACTORS

Estrus Detection and Artificial Insemination

 The best method of estrus detection is with a raddled, vasectomised ram. Artificial
insemination in sheep has not assumed the popularity achieved in cattle. A number of factors
have been responsible, notably the disappointing results using frozen/thawed semen deposited
intracervically. The spermatozoa are unable to colonize or traverse the length of the cervix
and are rapidly lost from the ewe’s reproductive tract. However, the use of intrauterine
insemination by laparoscopy has been much more successful. Artificial insemination is best
used in mid estrus, or 12-14 hours after its onset.

Teasing

 The introduction of vasectomised teasers into the flock, before fertile rams, had no effect on
pregnancy (conception) rates but had a profound effect upon the onset of cyclical activity and
hence a compact lambing season. Teasing caused ewes to exhibit estrus in the first 16 days
after exposure to the fertile ram, whilst two cycles were required for the unteased ewes to
show comparable activity.

Ram: Ewe Ratio

 The number of rams per ewe will vary depending upon a number of factors: age of the ram;
age of the ewes; whether more than one ram is to be used with the group of ewes; and terrain
and size of the enclosure. Ram: ewe ratios of 1:25 to 1:40 are suitable in non-synchronized
flocks. However, where synchronization is attempted, a ratio of at least 1:10 should be
available.

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Nutrition

 It is important that ewes are in good bodily condition at tupping. Increasing the energy intake
several weeks before tupping, so that the ewes are gaining weight (flushing), will increase the
fecundity in those ewes with the genetic potential. Provided the level of feeding is maintained
for a month after mating this should ensure good pregnancy rates. Some reduction in food
intake is reasonable during the second and third months of gestation, but feeding should be
increased in the last 6-8 weeks before lambing.

Increasing Fecundity

 Increased ovulation rates can be achieved by the administration of equine chorionic

gonadotrophin (eCG) on the 12th or 13th day of the estrous cycle. Good results have been
obtained by immunization against androstenedione.

INFECTIOUS AGENTS

 Non-specific infections of the genital tract, especially the uterus, are of minimal importance in
ewes, probably because in most breeds of sheep there is a long period of anestrus following
lambing. In the small number of ewes in which bacterial contamination occurs at lambing or
postpartum, which is less than 20% they are rapidly eliminated within a week and thus before
the genital tract can be exposed to a period of progesterone influence: this will occur at the
next diestrus which will normally be many months away.
 In the cow, retention of the fetal membranes (RFM) postpartum is quite common, and this is a
major risk factor in the development of endometritis and subfertility. RFM is relatively
uncommon in ewes; where it does occur, attempted removal by applying traction to the
exposed portions of the membranes can be attempted. If left, they will usually separate and be
shed within 5-6 days. If an affected ewe shows signs of systemic illness due to the
development of metritis, then she should be treated with an appropriate broad-spectrum
antibiotic.

Specific infectious diseases that cause infertility in ewes are

 Enzootic Abortion in Ewes (EAE)

 Toxoplasmosis
 Campylobacteriosis
 Salmonellosis
 Listeriosis
 Border Disease
 Leptospirosis
 Brucellosis
 Q Fever
 Ureaplasmosis
 Tick-borne Fever

GOATS

 In the absence of any major infectious cause of abortion, infertility in the goat is generally not
a major problem, normally with only a small number of barren does remaining at the end of
the breeding season.

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Structural Defects

 Abnormal sexual differentiation during embryological and fetal development, resulting in

intersexes is relatively common in the goat, especially in breeds such as the Alpine, Saanen
and Toggenburg. It is much more prevalent in polled individuals, where ‘polledness’ is a
simple dominant character with full penetrance, but it is also associated with a recessive
hermaphrodite effect with incomplete penetrance. Intersexes can also occur as a result of
Freemartinism, where placental fusion occurs in twins of dissimilar sex. However, the
incidence of hermaphroditism appears to be higher than that of freemartinism in this species.
 Hermaphrodites that have been described in goats are mainly male pseudohermaphrodites,
having testes and the accessory reproductive organs of the female; they are genetic females.
 Intersexes vary in the degree of external structural abnormality. Most are generally female-
like in appearance at birth but, as they grow and mature, there will be evidence of an enlarged
clitoris, perhaps testes in the inguinal region and the development of male secondary sex
characteristics, including the typical male odour.

FUNCTIONAL FACTORS

 The goat is a seasonal breeder responding to the effects of declining day length. It is not
unusual to have irregular estrous cycles at the beginning and end of the breeding season,
especially in goatlings, with short cycles of between 5 and 7 days.
 Anestrus may be due to starvation, parasitism or mineral deficiencies. In the case of the latter,
phosphorus and the trace elements copper, iodine and manganese as well as vitamin E have
been implicated. It can also be influenced by chronic debilitating diseases.

HYDROMETRA OR PSEUDOPREGNANCY ("CLOUD BURST")

 Hydrometra is the accumulation of sterile secretions within the uterine lumen. The etiology of
the condition is not known precisely, but it is always associated with high progesterone levels
secreted by a persistent, functional CL, cessation of cyclical activity, and variable degrees of
abdominal distension. The incidence of the disease varies between herds and within the same
herd from year to year. One possible cause of hydrometra is early, embryonic loss. However
not all animals will have been mated by the buck prior to pseudopregnancy.
 Two types of pseudopregnancy occur:
o After mating there is fertilization, followed by early embryonic death, the CL persists
and the doe acts as if pregnant. The abdomen becomes enlarged, and in some there is
a degree of udder development if not the onset of lactogenesis. Those that are
lactating may have a fall in yield. This type of false pregnancy generally lasts for the
duration of the gestation period, or even longer, until the CL has regressed
spontaneously. The term ‘cloud burst’ is used to describe the voiding of large
volumes of fluid from the uterus as the pseudopregnancy is terminated. Following
this, the abdominal distension disappears; some does may search for the ‘missing’
kids.
o Following estrus, when the doe was not mated, there is cessation of cyclical ovarian
activity but there is no marked hydrometra. At the end of the period of acyclicity,
affected does expel a bloody discharge.Therefore, any unbred does that do not return
to estrus after their first estrous cycle in the autumn, should be treated with PGF 2α
for possible pseudopregnancy.
 Differentiation of hydrometra from normal pregnancy can be made using transabdominal B-
mode ultrasound imaging, demonstrating the presence of a fluid-filled uterus in the absence of
a fetus or placentomes. After 50 days of anestrus, pregnancy and pseudopregnancy can be
differentiated on the basis of serum estrone sulphate levels. Treatment with 2.5 mg PGF2α
will be followed by expulsion of the fluid and estrus will occur in approximately 4 days. By
using a second injection 12 days after the first, good levels of fertility can be achieved with
85% conceiving, compared with 95% of unaffected animals.

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 CYSTIC OVARIAN DISEASE

 Cystic ovarian disease has been described in dairy breeds and has been particularly evident
where they have grazed estrogenic clovers and legumes.
 A history of nymphomania may suggest follicular degeneration, and the typical clinical sings
are those of continuous estrus and short inter estrus intervals with a failure to conceive. They
should be treated with 1500 – 2500 IU of Human chorionic gonadotrophin (hCG);
Gonadotrophin-releasing hormone or Progesterone for 18 days

MANAGEMENT FACTORS

Timing of Service or Artificial Insemination

 Optimum pregnancy rates are obtained when does are mated towards the end of estrus (which
lasts 12-36 hours) and just before ovulation.
 Some goat owners serve their does only in the first 12 hours of estrus supposedly to increase
the number of female kids but with a consequential reduction in pregnancy rates.

Nutrition

 Vitamin A, certain minerals (manganese and iodine) and energy deficiencies reduce fertility,
and may be associated with abortion when the deficiency is chronic.

Stress

 Stress-induced abortion has been described above in Angora goats. However, there is
evidence that other breeds of goat will abort if subjected to stress. This can result from being
chased by dogs, inadequate feeding, transportation and adverse weather, particularly during
the fourth month of gestation.

INFECTIOUS AGENTS

Non-specific infections appear to play a minor role in causing infertility in does, probably for similar
reasons discussed above for the ewe. However, specific infectious agents as listed below are
important in causing abortion and have been discussed in detail under the Module on Specific
Infectious Diseases.

 Brucellosis
 Campylobacteriosis
 Chlamydial (enzootic) abortion
 Leptospirosis
 Listeriosis
 Salmonellosis
 Toxoplasmosis
 Q- Fever
 Mycoplasmosis

SOW AND GILT

 People working in pig production expect very high levels of fertility, and any shortfalls
represent a serious economic loss. The efficiency of a pig operation is always described in
terms of the number of pigs sold per sow per place per year, or the number of kilograms of
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 pig meat sold per square metre of pig unit. However, there are certain fertility parameters that
determine the efficiency of a pig industry. They are
o Farrowing Rate: The number of sows that farrow to a given number of services,
normally expressed as a percentage.
o Farrowing Index: The number of farrowings per sow per year.
o Conception Rate (or non-return rate): The number of sows that conceive to service
expressed as a percentage of those served. The conception rate is usually estimated as
the non-return rate to estrus (28days after service) or is identified by pregnancy
diagnosis at 30 days or more, after service. This term does not necessarily equate to
the farrowing rate, as pregnancy can end at any time, but it can provide an earlier
warning of a problem.
o Non-Productive or Empty Days: The number of days in which a sow is not pregnant.
There are, of course, days during which it is not possible for a sow to be pregnant
(e.g. in lactation, and during the weaning to estrus interval), which should be taken
into account.
o Piglets Born Per Sow Per Year: This figure can be divided into two components:
total numbers born, and numbers born live.
 All fertility parameters interrelate and each producer must establish targets for reproductive
performance. Any discrepancy between the targets and the reality represents an economic loss
resulting from suboptimal fertility. Targets set for a particular unit must take into account all
management factors that influence fertility.

FACTORS THAT AFFECT FERTILITY

 An investigation of a fertility problem in the pig is rarely, if ever, considered on an individual

sow basis. While attempting to solve a fertility problem in sows, the importance of
management and stockmanship should be borne in mind. Any investigation of herd infertility
should take into account management factors before making a detailed study of other issues.
The quality of stockmanship will be reflected in such basic procedures as
o Estrus detection
o Supervision of service
o General hygiene
o Record-keeping
 Good stockmanship should involve a combination of
o Sound basic knowledge
o Patience
o Empathy
o Sensitivity
o Organizational skills
o An appreciation of priorities.
 As units grow larger and as management systems change in response to consumer demands,
stockmanship characteristics and requirements may have to be adjusted, but they remain,
none the less, of paramount importance.
 Sows that deviate from the normal or required parameters of reproductive performance are
invariably removed from the herd rather than being treated or being allowed to continue in a
sub fertile way. Any investigation of herd infertility must begin with an evaluation of the
problem.
 Parameters of relevance to the investigation include:
o Herd size
o Age profile replacement rate
o Return rate/conception rate
o Distribution of return intervals
o Weaning-to-estrus interval
o Farrowing rate
o Total numbers born
o Total numbers born live
o Lactation length
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 o Number of non-productive (empty) days.
 Examination of records should provide a definition of the nature and extent of the problem.
This exercise should be followed by a clinical appraisal of the stock, post-mortem
examination and laboratory diagnosis. At the outset it should be recognized that the route of a
fertility problem is often multi factorial
 The problem will probably fall into one or more of the following categories:
o Anestrus
o Conception Failure
o Pregnancy Failure

ANESTRUS

 Anestrus is one of the most common reproductive disorders in sows and is defined as ‘the
absence of estrus behavior (standing to a boar or to a riding test) but excludes the normal
interval (diestrus) between two successive estrus periods’. By definition, delayed estrus is
also included in this category.
 Anestrus is inevitable at certain stages in a sow’s life (e.g. before puberty, and during
pregnancy and lactation), and this should be taken into account in any investigation.
 The term ‘sub estrus’ refers to a condition in which cyclic animals show no obvious external
signs of estrus and is characterized by the presence of corpora lutea. On rectal palpation the
cervix is relatively small and firm in anestrus and softer in sub estrus under the influence of
this luteal tissue.
 Firstly, it is important to establish that the problem is truly one of anestrus and not simply
improper heat detection. Accurate estrus detection involves time and effort, and strategic use
of boar presence, in conjunction with good record keeping.

Investigation

 Once it is established that the problem on hand is due to anestrus, it is necessary to investigate
the situation by either
o ovarian function tests,
o post mortem examination or
o real time ultrasound.
 Ovarian function tests
o The ovaries of sows that fail to exhibit signs of estrus may be truly inactive
(anestrus), active with in apparent (sub estrus) estrus or may have CL due to
pregnancy.
o Tests for ovarian function such as changes in plasma progesterone concentration will
differentiate between these conditions. Progesterone may be measured in plasma or
whole sow’s blood by enzyme-linked immunoassay (ELISA) and weekly blood
progesterone assays will help differentiate between truly inactive, pregnant and
normally cyclical ovaries.
 Post-mortem examination
o Ovarian function may be assessed by post-mortem examination of the reproductive
tracts of sows culled from the herd under investigation and can reveal the following
abnormalities.
 Acyclic ovaries: inactive, with some small follicles (<5mm diameter),
absence of corpora lutea.
 Cystic ovaries: Multiple large cysts (usually <14 mm in diameter), generally
containing some luteal tissue that produces progesterone. Cysts may regress,
but some persist and can inhibit estrus.Multiple small cysts that often produce
estrogens and result in sows having markedly irregular cycle lengths and
exhibiting intense signs of estrus (nymphomania). Single cysts which rarely
affect sow fertility and tend to be incidental findings at post-mortem.
 Real time ultrasound

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 o This technique can be used to observe changes in the size and shape of the ovaries by
a non-invasive method in the live animal. Such investigations are time-consuming;
require expensive equipment and a considerable level of expertise.

Treatment

 Anestrus is of economic significance and must be treated promptly by identifying and

remedying all contributing factors.
o Injection of a preparation containing 200 IU hCG and 400 IU of eCG induces estrus
within 3-8 days.
o In sows that have normal cyclical ovaries, but are apparently sub estrus, the condition
may result from inadequate estrus detection. Hence, ideally, the producer should be
encouraged to observe for estrus in the presence of the boar at least once (and
preferably twice) each day from the day of weaning.

CONCEPTION FAILURE

 Failure of conception is recognized by an increased number of regular returns to service (i.e.

returns at 18-24 days after service). The conception rate (or non-return rate) in breeding herds
should be at least 90% and an incidence of return of higher than 10% should be regarded as
abnormal and unacceptable. Conception failure suggests that viable ova did not come into
contact with viable spermatozoa at the appropriate time. The result of this will be either total
conception failure (i.e. regular return to service) or partial conception failure (i.e. reduction in
litter size). Assuming that ovulation has taken place, conception failure must be due to one or
more of the following factors:
o Timing of Service
o Quality of Service
o Semen Quality

Timing of Service

 High embryonic survival rate and large litter size at birth can be ensured by a single mating at
the appropriate stage of estrus resulting in a high proportion of ova fertilized at the optimum
time. However, identifying this ideal time is most difficult.
o The sow ovulates, on average, 36-44hours after the onset of standing estrus
o Spermatozoa can survive for approximately 24 hours inside the sow’s reproductive
tract
o A service regimen must take all these factors into account and aim to ensure that the
uterus contains viable spermatozoa prior to the arrival of the ova
o Inappropriate timing of service results in conception failure
 Keeping this in mind, the service management regimen should ensure that each sow is served
on the day of onset of standing estrus and at least once more, 18-24 hours later.

Quality of Service

 Service pen design, particularly with reference to the floor surface, is of direct relevance to
the quality of service. Young boars need to be trained to natural service, and should be well
supervised each time they mount a sow or gilt.

Semen Quality

 Semen quality can be affected in terms of ejaculate volume, sperm count, sperm motility or
morphology by a wide range of factors such as age, environmental temperature, frequency of
use and disease.

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  Where boars are used for natural service, it may be that sub fertile or infertile individuals
remain unidentified. The physical breeding soundness, paying particular attention to feet and
leg conformation of the boar should also be taken into account.

PREGNANCY FAILURE

 Pregnancy failure may be divided into two main sections:

o Failure to Establish Pregnancy
o Failure of an Established Pregnancy

Failure to Establish Pregnancy

 Unattached conceptuses within the uterus are susceptible to damage by many factors. Where
pregnancy fails around the time of maternal recognition of pregnancy (i.e. around days 12-13)
sows tend to return to estrus outside the normal range for ‘regular returns’
 The problem may be associated with failure of maternal recognition of pregnancy related to
o Stress
o Infectious challenge
 Failure of pregnancy at this stage may be total (resulting in an irregular return to service) or
partial (resulting in resorption of some embryos, and consequent reduction in litter size)

Failure of an Established Pregnancy

 Death of conceptuses during the embryonic stage tends to result in resorption if abortion does
not occur
 Dissolution of embryos in the absence of anaerobic bacteria is an aseptic, autolytic process
resulting in complete disappearance of the products or a vaginal discharge.Reduction in
numbers born due to partial resorption of litter will be the only presenting sign
 Once pregnancy has been confirmed (e.g. by the Doppler ultrasound technique on days 28-
35), fetal death is more likely to result from an infectious disease
 Beyond 35 days, fetal death will result in mummified fetuses at farrowing. Mummification is
the most common clinical manifestation of a viral infection (e.g. Aujeszky’s disease, porcine
parvovirus, porcine reproductive respiratory syndrome or swine fever) at this time, although
only a proportion of the fetuses may be affected.

INFECTIOUS FORMS OF INFERTILITY

 An infectious form of infertility can be of great economic significance to a unit. It is vital,

however, to ensure that management and stockmanship are adequate before searching for an
infectious agent in any investigation into infertility. A whole range of management factors
(e.g. environment, stress and nutrition) may lower the natural defense mechanisms, rendering
an animal population more susceptible to disease.
 Various infectious causes of infertility have been dealt with in detail under the Module on
Specific Infectious Diseases Causing Infertility.

VULVAL DISCHARGES

 Vulval discharges are the most obvious clinical sign of bacterial genital infections
 Return intervals are not usually affected. In late pregnancy uterine infection can lead to
abortion
 Investigation of an outbreak of vulval discharge involves identification of the source of the
discharge by
o speculum examination per vaginam. Discharges may originate from the vestibule, the
vagina, the uterus or the bladder

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 o cytological examination
 The consistency of the discharge may be
o thin pale yellow fluid without blood or mucus or
o necrotic debris and mucus with or without blood

Differential Diagnosis

 Abnormal vaginal discharges should be differentiated from

o watery or slightly cloudy discharge of proestrus and estrus
o seminal fluids, including gel expelled after mating
o discharge during pregnancy
o lochial discharge following parturition that will normally persist for up to 5 days

Treatment

 Treatment involves
o improved hygiene, particularly in the service house
o antibiotic injection of sows at weaning
o a programme of in-feed medication

STRUCTURAL ABNORMALITIES OF THE FEMALE REPRODUCTIVE TRACT

 Anatomical defects of the female genitalia include intersexuality, gonadal hypoplasia and
other miscellaneous abnormalities.

Inherited Hypoplasia of the Gonads

 Both sexes may be affected but it is more readily apparent in the male.

Intersexuality

 Such abnormalities are common and appear to be a hereditary condition determined by

recessive genes. Male pseudohermaphrodites are more common and have testes that may be
subanal or intra-abdominal. Externally, intersexes resemble the female and micturate through
the vulva, although a phallus may be present. The animal may be considered a gilt until
puberty, when it starts to demonstrate male behavior.

Bilateral Uterine Tubal Lesions

 Structural sterility resulting from bilateral tubal lesions (e.g. hydrosalpinx, pyosalpinx and
ovarobursal adhesions) has been shown to occur in up to 33.3% of sows and gilts that failed
to breed.
 Apart from the uterine tubes, other parts of the tubular genital tract may show aplasia or
duplication but only when the whole tubular system is aplastic, or when the vagina, cervix or
uterine body is imperforate, will sterility result. The condition of uterus unicornis will lead
only to lowered fecundity.

Absence of One or Both Ovaries

 The absence of one or both ovaries and a generalized underdevelopment of the whole
reproductive tract (infantilism) occur occasionally. Other lesions include double vagina,
septae or ‘strings’ in the vagina and hymenal residues

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 SEASONAL INFERTILITY

 Reduction in fertility in pigs in the summer and early autumn has been reported in many
countries and appears to manifest as a range of problems including delayed puberty in gilts,
delayed post-weaning estrus in sows, regular and irregular returns to estrus, delayed return to
estrus, reduction in the farrowing rate, embryonic death, ovarian cysts and silent estrus.
 Autumn abortion syndrome may also be connected to seasonal infertility.
 It has been suggested that heat stress is particularly damaging during the first 8-14 days post
mating.
 Improved management of sows to avoid stressful and overheated conditions during the hot
summer months can reduce the problem.

MARE

 The objective of the veterinarian working in any horse-breeding enterprise, regardless of size;
should be to produce the maximum number of live, healthy foals from the mares bred during
the previous season. However, the biggest difficulty in reaching this aim is the infertile or
problem breeding mare.
 Very few mares are permanently and completely infertile, but subfertility of varying degrees
is a major problem.
 It might take several cycles to establish a pregnancy, and even then, there is an increased
possibility of pregnancy failure.
 Commitment from both mare owner and veterinarian is needed; the owner should be made
aware of this at the outset and be given a realistic expectation as to the chance of success.
 Infertility in Mares has been dealt under the following headings
1. Protocol for clinical evaluation of a mare
2. Causes of infertility and sub-fertility
3. Structural abnormalities of the female reproductive tract
4. Functional forms of infertility
5. Puerperal metritis
6. Pyometra
7. Retained fetal membranes
8. Endometritis
9. Diagnostic aids used in equine endometritis

PROTOCOL FOR CLINICAL EVALUATION OF A MARE

 The clinician should be aware of how to investigate the problem breeding mare. A protocol
for such an investigation of an infertile or subfertile mare is outlined in table given below.

Outline of a Step - by Step Protocol of the Clinical Examination of

an Infertile Mare

Steps Instruction
1 Obtain the mare's previous breeding history
2 Assess her physical condition, general health and perineal conformation
3 Culture swab samples collected from the vestibule, clitoral fossa and sinuses
4 Examination per vaginum using a speculum and collection of endometrial swabs for
bacterial culture and stained cytological smear.
5 Manual vaginal examination
6 Examine the reproductive tract by rectal palpation
7 Transrectal real time ultrasound examination of the reproductive tract
8 Endometrial biopsy

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 9 Endoscopic examination of the endometrium
10 Peripheral venous blood sample for hormone analysis
11 Peripheral venous blood sample or hair follicle for chromosome analysis.

CAUSES OF INEFERTILITY AND SUBFERTILITY

 Many factors, either alone or in combination with others, cause infertility or subfertility and
can broadly be categorized into
o Infectious (Refer to Module on Specific Infectious Causes of Infertility)
o Non-infectious factors
 Structural Abnormalities
 Functional Aberrations

STRUCTURAL ABNORMALITIES

Vulva

 In the normal mare, the vulva provides the first effective barrier to protect the uterus from
ascending infection. The `normal' mare has three functional genital seals forming a barrier
between the external environment and the uterine lumen:
o the vulva,
o the vulvo-vaginal constriction
o the cervix.
 During estrus, the vulva and cervix relax, leaving the vulvo-vaginal constriction as the only
seal.

 The vulval lips should be full and firm and meet evenly in the mid line with 80% or more of
the vulval opening below the brim of the pelvis.
o If the vulval seal is high (more than 4 cm of length dorsal to the pelvic floor) in
relation to the pelvic brim, the vestibular seal is incompetent and there will be
aspiration of air with bacteria and contaminated material into the vagina
(pneumovagina ; also called `windsucking'). The initial vaginitis may lead to
cervicitis and acute endometritis resulting in sub fertility.
o Contamination of the caudal reproductive tract with bacteria during pregnancy can
result in embryonic death, and in late pregnancy can result in the development of
placentitis and lead to abortion.
o Furthermore, the penumovagina may lead to urovagina (urine pooling within the
vagina) when the vestibule and urethral opening are displaced cranially.
o The more severe conformational abnormalities are more likely to result in failure of
the vulval seal, and to increased faecal contamination since the vulva forms a shelf on
to which feces may collect. The vulval lips may be angled at 25 or even 50° to the
vertical in these cases.
 Defective vulval confirmation can be congenital, which is very rare, or acquired, which is
seen in (1) vulval stretching following repeated foalings, (2) injury to perennial tissue, or (3)
poor bodily condition (old, thin mares).
 Older multiparous mares are more commonly affected with pneumovagina. However, young
mares that are in work and have little body fat and / or poor vulval conformation can develop
pneumovagina. In some mares, pneumovagina may only occur during estrus when the
perineal tissues are more relaxed. Some mares make an obvious noise whilst walking, but in
other mares the diagnosis may be more difficult.
 Diagnosis is by
o The presence of frothy exudate in the anterior vagina on examination with a speculum
o Rectal palpation of a ballooned vagina or uterus from which air can be expelled
confirms the diagnosis.
o Real time ultrasound examination of uterus may reveal the presence of air as
hyperechoic (white) foci sometimes seen as a line at the opposed luminal surfaces.
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 o Cytological and histological examination of the endometrium may demonstrate
significant numbers of neutrophils indicative of an endometritis. Rarely, eosinophils
are also found in association with pneumovagina.
o Treatment involves Caslick's Vulvoplasty Operation.

Vulvo-Vaginal Constriction

 Immediately in front of the external urethral opening is the vulvo-vaginal constriction or

vestibular seal. In genitally healthy mares this forms the second line of defense against
aspirated air and faecal material.

Hymen

 Manual vaginal examination of maiden mares often reveals the presence of hymen tissue
which generally breaks down with pressure. A complete persistent hymen can also occur,
which can result in the accumulation of fluid within the vagina and uterus due to impaired
natural drainage. Sometimes the hymen may be so tough that it can only be ruptured using a
guarded scalpel blade or scissors. The small incision can then be enlarged using the fingers
and hand. Rarely, failure of proper fusion of the Mullerian ducts may result in the presence of
dorso ventral bands of fibrous tissue in the anterior vagina and fornix. They do not interfere
with fertility and are easily broken down manually.

VAGINA, PERINEAL LACERATIONS AND RECTO- VAGINAL FISTULAS

Vagina

 Urovagina urine pooling, is the retention of incompletely voided urine in the cranial vagina
due to an exaggerated downward cranial slope of the reproductive tract. Pneumovagina from
a defective vulval conformation also predisposes to the condition. Transient urine pooling,
which is sometimes found in postpartum mares, usually resolves after uterine involution has
occurred. Clinical signs can include urine dripping from the vulva, urine scalding and a
history of failure to conceive.
 Diagnosis is easiest using a speculum examination during estrus to detect urine in the cranial
vagina. Uterine infection with an accumulation of exudates in the vagina can be confused
with the condition.
 In severe cases, urine pooling should be surgically corrected by vaginoplasty (perhaps more
correctly termed caudal relocation of the transverse fold; as surgical intervention is in the
vestibule), urethral or perineal resection.
 Vaginal bleeding from varicose veins in the remnants of the hymen at the dorsal
vestibulovaginal junction is occasionally seen in older mares, particularly during estrus and
the second half of pregnancy. Although diathermy can be used, treatment is not usually
necessary as the varicose veins normally shrink spontaneously.

Third - Degree Perineal Lacerations and Recto-Vaginal Fistulas

 Both conditions are most often seen in young, primiparous mares where the rigidity of the
birth canal, especially the vulvo-vaginal junction, is important in its pathogenesis.
 In most cases, the veterinarian becomes involved only after the foal is born and the damage
already exists. For treatment in the acute situation, it is difficult to estimate the amount of
devitalized tissue. Even though the edges of the wound may look fresh and clean, much more
tissue is damaged and bruised. This is why immediate repair is not performed, unless one is
present within 2 hours of the injury, and even then most clinicians advise delaying surgery.
 First aid treatment should include:
o debridement of non-viable tissue
o provision of haemostasis and general cleaning of the area

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 o parenteral broad-spectrum antibiotics for 5 days
o NSAIDs and tetanus prophylaxis
o daily cleaning
o monitoring of uterine involution.
 Elective surgery is performed after atleast 10 weeks, and if the foal survives, the operation is
best performed after weaning.

Cervix

 The cervix is the important third (and last) protective physical barrier to protect the uterus
from the external environment. The cervix must also relax during estrus to allow intrauterine
ejaculation and drainage of uterine fluid. An inflammation of the cervix is usually associated
with endometritis and / or vaginitis.
 Anatomically, the cervix is a thick-walled sphincter. Expansion and contractions are possible
due to the action of the longitudinal and circular smooth muscle, which is rich in elastic
fibres. A distinctive feature of the equine cervix is its dilatability, and the absence of rigid,
annular constricting rings seen in farm animals. This means that the uterine body can be
entered by a relatively large-diameter instrument.

 Often an older maiden mare has an abnormally tight cervix due to fibrosis. The cervix fails to
relax properly during estrus, so that fluid is unable to drain and accumulates in the uterine
lumen. In many cases this fluid is sterile and contains no neutrophils. Once the mare is bred,
the fluid accumulation will be exacerbated due to poor lymphatic drainage and impaired
myometrial contraction compounded by the tight cervix.
 Failure of the cervix to open during estrus can lead to unwillingness of the stallion to
complete mating or ejaculate intravaginally. Artificial insemination has been used
successfully in mares with an abnormally narrow cervix. Mares with a fibrosed cervix that
become pregnant do not normally have any difficulties at foaling.
 Failure of the cervix to close during diestrus can lead to persistent endometritis and failure to
conceive, or early embryonic death. Failure to maintain closure during pregnancy can lead to
gestational failure.
 Assessment of the cervix must form a part of the routine pre breeding examination of a mare,
either directly using a speculum per vagina and / or by digital exploration, preferably during
diestrus when it is more tightly closed under the influence of progesterone.
 Injury, resulting in cervical incompetence or fibrosis, most often occurs during parturition
when fetotomy is performed by an inexperienced clinician, or without adequate
instrumentation., during vigorous mating by an oversized stallion, especially if the mare was
not in full physiological and behavioural estrus, although usually it is not too severe, or by
irritant chemicals such as povidone-iodine.
 If severe, cervical lacerations may need surgical repair to restore normal cervical shape and
function.
 Developmental abnormalities of the cervix include aplasia and a double cervix.

Uterine Cysts

 Uterine cysts are the most common type of uterine lesion identified in the mare. The two
distinct morphological types are
o endometrial cysts, which are usually 2 cm or less in diameter
o lymphatic cysts, which are generally larger
 Cysts can be confused with an early conceptus and give rise to false positive early pregnancy
diagnosis or the incorrect diagnosis of twin pregnancies during ultrasound scanning.
Differentiation is based on
o previous cyst mapping
o early mobility of the conceptus
o the conceptus's spherical appearance
o growth rate
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  Some cysts can be very difficult to distinguish from pregnancies. Reassessment of the
irregular structure based on ultrasound will confirm that the fluid is contained, and does not
extend up or down the horn as would be found with free fluid. The appearance of an embryo
around 22-24 days of pregnancy provides a definitive diagnosis. Thorough identification of
cysts at the beginning of the breeding season minimizes the chance of false positive
pregnancy diagnosis
 Larger lymphatic cysts may interfere with the mobility phase of the early conceptus and thus
prevent luteolysis (failure of maternal recognition of pregnancy). Later in pregnancy, the
absorption of nutrients and the development of chorionic villi may be diminished in places of
contact between cysts and fetal membranes leading to an increased risk of embryonic death
 The need for treating endometrial cysts is uncertain
o If a mare is found at the beginning of the breeding season with a large number of
cysts, it is generally best to continue to attempt to get the mare in foal that reason
o If she fails to become pregnant, some form of treatment should be attempted and an
endometrial biopsy should be taken to help determine the likelihood of her carrying a
foal to term because of risks such as uterine haemorrhage
o Larger cysts can be punctured using an endometrial biopsy instrument or manually if
the cervix allows passage of one hand
o Chemical curettage has equivocal results; the cysts may disappear but scar tissue may
form
o Thermocautery, in conjunction with endoscopy involving looping and subsequent
burning of cysts, is possible. Wounds after cautery appear to heal very quickly,
usually within -6 weeks. Because endoscopy should be done while the mare is in
diestrus when the cervix is relatively closed, prostaglandin F2α should be given after
cauterization and the uterus should be lavaged with saline
 Most uterine cysts involve the endometrium. Occasionally an extra luminal cyst lying external
to the endometrium can be identified on ultrasound examination. Its location should be
verified by identification of the uterine lumen. Extra luminal cysts usually have no adverse
effects on fertility

Partial Dilatation of the Uterus

 Ventral uterine enlargement originated by one of four mechanisms:

o mucosal atrophy,
o myometrial atony,
o lymphatic lacunae
o endometrial cysts.
 Their precise relationship to subfertility is not clear, but mares that fail to eliminate the fluid
and debris that accumulate in these sacculations after mating are susceptible to the
establishment of chronic endometritis
 Treatment is the same as for mares with defective uterine clearance

Uterine Adhesions

 Uterine adhesions are most frequently diagnosed on endoscopic examinations of the uterus.
Multiple adhesions adversely affect fertility by causing fluid accumulation or by affecting the
mobility of the conceptus
 It is possible to remove the obstruction endoscopically by either cautery or laser techniques,
starting at the thin membranous parts of the obstruction. It is important not to `burn' too
deeply in the uterine wall, as in these cases more severe damage to the uterine wall might
occur. After removing the obstruction, the uterus should be flushed to remove any debris and
the mare treated with PGF2α
 In addition to an assessment of an endometrial biopsy, the prognosis for future breeding also
depends on the severity of the obstruction and to what extent the obstruction could be
removed

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Uterine Foreign Bodies

 Uterine foreign bodies (e.g. fetal remnants) may act as a nidus for the establishment of
chronic endometritis but are uncommon. Other foreign bodies that have been reported include
straws following AI, and the tips of uterine swabs
 Uterine neoplasia, abscesses and haematomata are rarely reported in the mare

Uterine Tubes and Periovarian Structures

 Uterine tube abnormalities are usually due to remains of embryological structures, and are
rarely reported in the mare. The presence of collagenous masses within the uterine tube that
might occlude the lumen has been documented
 Dye tests are used in cattle to test tubule patency, but this is difficult in the mare, and
occlusion is very rare
 Cysts lying within the ovarian stroma near the ovulation fossa of the ovary arise from the
surface epithelium and are often seen in older mares during examination of the ovary. They
are known as `retention', `inclusion' or `fossa' cysts and generally have no adverse effect upon
fertility
 Periovarian cysts, are not endocrinologically active, do not usually interfere with the process
of ovulation and do not generally affect fertility
 Occasionally, large cysts may be palpated or imaged with ultrasonography and may be
confused with follicles. However, the lack of change in size or appearance of these structures
is usually diagnostic

OVARY

Ovarian Neoplasia

 Reasons for the presence of a large ovary in a mare include

o a normal ovary during the transition or breeding season with large follicles, as
frequently detected during early spring, persistent luteal phase and early pregnancy;
o a solid neoplastic lesion, such as a granulose theca cell tumour, teratoma,
dysgerminoma, cystadenoma and carcinoma
o haematomata of the ovary;
o abscesses and
o haemorrhagic and luteinised follicles.
 Ovarian neoplasia is uncommon in the mare although many types of tumour have been
described, with the granulose theca cell tumors (GTCTs) being by far the common.

Gonadal Dysgenesis

 This condition is not common. However, in a maiden mare, once winter anoestrus has been
eliminated as a cause of acyclicity, XY ovarian dysgenesis must be considered as a possible
cause with small, inactive ovaries and an immature tubular genital tract. Examination of the
reproductive system detects very small ovaries (<1 cm in diameter) and a poorly developed
tubular genital tract, which is difficult to palpate. This is similar to mares with XO
chromosomes (Turner's syndrome). There is no treatment, and the mare is sterile.

FUNCTIONAL INFERTILITY AS A CAUSE OF SUBFERTILITY

 Mares are seasonally polyestrus, and environmental and other factors can exert a profound
effect on reproductive function, particularly during the transitional period between winter
anestrus and the onset of cyclical activity in the spring. Although irregularities of follicular
development, ovulation and behavioural patterns are also observed during the normal
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 breeding season, they are not as common. However, endometritis can also cause cyclical
irregularities.
 Functional infertility can be dealt under the following headings:
o Anestrus due to Ovarian Acyclicity
o Anestrus caused by a Prolonged Luteal Phase
o Behavioural Anestrus - Silent Estrus
o Anestrus caused by a Shortened Luteal Phase
o Irregular or Prolonged Estrus
o Ovulatory Dysfunction

ANESTRUS DUE TO OVARIAN ACYCLICITY

Winter Anestrus

 The onset of cyclical activity is stimulated by increased day length. During winter months
mares are normally acyclical
 Diagnosis
o On rectal palpation or transrectal ultrasound imaging both ovaries will be small (<3 x
2 x 2 cm), and in some mares there will be a number of small follicles. Plasma
progesterone concentrations are > 1 ng/ml
 Treatment
o Although increasing day length is the primary controlling factor, ensuring freedom
from disease and good body condition by stabling, adequate nutrition, anti helminthic
therapy and attention to dental conditions can hasten the onset of cyclical ovarian
activity. Thus, prolonged anestrus can be prevented by good management.
Progesterone / progestogen withdrawal therapy has been used successfully
o Progesterone can be administered as an oil-based intramuscular injection, orally as
the synthetic progestogen altrenogest (Equine Regumate) or by using a silastic
progesterone - releasing intravaginal device (PRID). However, such therapy is
effective only in anestrus mares that are already well into the transitional phase to the
resumption of normal cyclical ovarian activity
o Repeated daily injections of equine pituitary gland extract to mares in winter anestrus
lead to follicular development. In aged mares, the delayed initiation of normal
cyclical ovarian activity may reduce the number of estrus cycles during the breeding
season and, therefore, it is particularly important to prevent poor body condition from
occurring in such animals
 Pituitary abnormalities
o Rarely Cushing's syndrome caused by adenomatous hyperplasia of the intermediate
pituitary has been associated with anestrus in aged mares. This is presumably due to
destruction of the cells secreting luteinising hormone and follicle stimulating
hormone

Lactation - related Anestrus

 Lactation - related anestrus is commonest in mares foaling early in the season. Affected mares
may have a normal postpartum estrus after 6-12 days, but fail to return to estrus at the end of
the first diestrus. Alternatively they may not even have a normal `foal heat'
 Diagnosis
o The ovaries resemble those of a mare in deep winter anestrus. ie. small and inactive,
the condition can last for several months. Originally it was thought to be due to
prolactin suppressing pituitary gonadotrophin release, but this is now in doubt.
Affected mares should be teased and examined weekly per rectum to assess their
ovarian status
 Treatment

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 o Treatments similar to those described above for winter anestrus have been used, but
with little success. Twice-daily injections of 0.04mg (10 ml) of a synthetic GnRH
analogue (buserelin ; Receptal) have been found to induce the development of a
follicle within 7-14 days of commencing therapy

ANESTRUS CAUSED BY A PROLONGED LUTEAL PHASE

Persistence of Luteal Activity

 Persistennce of luteal activity in the non-pregnant mare is a major cause of subfertility.

Traditionally, the term `prolonged diestrus' has been used to describe a condition where the
corpus luteum persists beyond its normal cyclical life span of 15/16 days, resulting in the
maintenance of elevated circulating progesterone concentrations for longer than expected
 These occur in upto 20% of estrous cycles in thoroughbred mares (less frequently in ponies)
and are not accompanied by estrus; the cervix will remain pale in colour, dry and tightly
closed. If diestrus ovulations occur late in the luteal phase, they will be refractory to the effect
of endogenous luteolysins, resulting in a persistent luteal phase
 True persistence of the corpus luteum occurs in approximately 20% of ovulations. These
mares present great difficulty to the stud manager as they can be assumed incorrectly to be
pregnant
 Diagnosis
o Plasma progesterone profiles are indistinguishable from those of pregnant animals.
The uterus becomes firm and tubular (tonic) and the cervix is typical of that of
pregnancy. Transrectal ultrasound imaging fails to detect a conceptus
 Treatment
o Failure of synthesis and or release of PGF2 α at the end of diestrus is the most likely
cause of persistence of the corpus luteum. Failure of the corpus luteum to respond to
PGF2 α or failure of PGF2 α to reach the corpus luteum could also be a reason for CL
persistence
o Treatment is by the injection of a luteolytic dose of PGF2 α or a synthetic analogue.
The interval between treatment and ovulation varies considerably depending upon the
size of follicles at the time of treatment. Therefore it is invisible always to examine
mares using ultrasonography before treatment in order to assess the status of
folliculogenesis

Pyometra

 Pyometra is the accumulation of substantial quantities of inflammatory exudates in the uterus

causing its distention. When the endometrium is severely damaged, there is extensive loss of
surface epithelium, severe endometrial fibrosis and glandular atrophy causing a prolonged
luteal phase, presumably due to interference with the synthesis or release of PGF2α.
 This is in contrast to mild endometritis with the collection of small amounts of intra luminal
uterine fluid, which is more likely to cause premature release of PGF2 α and luteolysis

Pregnancy and Pseudopregnancy

 Pseudopregnancy is a term used to describe a syndrome in which non-pregnant mares that

have been mated do not return to estrus. It occurs if there is early embryonic death after 15
days of gestation with persistence of the corpus luteum veerum resulting in a prolonged luteal
phase
 The cervix remains tightly closed, and the uterus is tense and tubular. It is differentiated from
pregnancy by the absence of a conceptus on ultrasound examination. If early fetal death
occurs after endometrial cup formation at 36 days, mares will either become anestrus or come
into estrus. However, in the latter, follicular luteinization without ovulation is thought to
occur and therefore the estrus is not fertile; this will last until the endometrial cups regress
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 spontaneously at 90-150 days. There is currently no practical way of destroying endometrial
cups prematurely

BEHAVIOURAL ANESTRUS-SILENT ESTRUS

 In silent estrus, mares either do not show estrus, or are slow to show detectable signs using
standard teasing methods despite the fact that ovulation occurs
 The degree of reduced expression of estrus varies from partial (sub estrus) to complete
(anestrus)
 The incidence of silent estrus is higher in maiden mares early in the breeding season and in
mares with a young foal `at foot'
 Other factors that affect estrus behaviour include being at grass with very dominant mares,
and stallion preference.Fillies that are in training and have been treated with anabolic steroids
may be more likely to suffer from the condition due to `androgenisation'.
 In many cases, it is a failure of the estrus detection system rather than a true reproductive
disorder of individual mares. However, it has been associated with reduced estradiol
concentrations in the peripheral circulation and a shorter interval from luteolysis to ovulation

Diagnosis

 Rectal and vaginal examinations confirm that the mare is in estrus and has follicles of an
ovulatory size. It is essential to distinguish the condition from a prolonged luteal phase in
which there is also follicular development

Treatment

 The treatment is based on thorough and careful teasing. Frequent and persistent teasing may
persuade the mare to show estrus. Alternatively, placing the mare in a stable next to a stallion
may be helpful
 If permissible, artificial insemination can be used. To breed mares naturally during a silent
estrus, some form of restraint may be necessary; many mares approaching ovulation accept
the stallion when twitched and hobbled
 An intramuscular injection of estradiol benzoate (10-20 mg) 6 hours before breeding can be
tried as a last resort
 The veterinary surgeon must ensure that the mare is physiologically ready to be bred. In some
cases when the mare is not psychologically prepared for breeding, estrogens are of little
value, and tranquilizers may be more appropriate

SHORTENED LUTEAL PHASE

Endometritis

 At coitus, the mare's uterine lumen becomes contaminated with microorganisms and debris
leading to a transient endometritis that usually resolves spontaneously within 24-72 hours so
that the environment of the uterine lumen is compatible with embryonic and fetal life
 This endometritis should not be regarded as a pathological condition. However, if the
endometritis persists after day 4 or 5 of diestrus, in addition to being incompatible with
embryonic survival, the premature release of PGF2α may result in luteolysis and a rapid
decline of progesterone and an early return to estrus
 These mares are referred to as susceptible and they develop a persistent endometritis

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IRREGULAR OR PROLONGED ESTRUS

 True persistent estrus appears to be rare in mares other than during the transitional period
from winter anestrus, or in association with steroid hormone-producing ovarian tumours.
Some cases that are presented as having a persistent estrus may actually represent normal
behavior.
 Frequent urination due to hind limb or back pain, or a urogenital problem may be mistaken
for persistent estrus.

TRANSITIONAL 'SPRING' ESTRUS

 Pressure to breed mares early in the year before onset of their natural breeding season can
pose problems for the veterinarian. Because of considerable variation in the duration of estrus
during the transitional period, efficient handling of the mare can be difficult. Shortly after
winter solstice, changes in the pineal/hypothalamic pituitary axes result in some follicular
growth; however, follicles remain small, do not ovulate, and regress. Eventually, after a
variable transitional period of upto 2 months, larger follicles (> 35 mm) will develop and
ovulate, usually adding the onset of normal cyclical ovarian activity.

Diagnosis

 The diagnosis is by thorough ultrasonographic examination and rectal palpation, which can
diagnose transitional follicles reaching a preovulatory size of > 30 mm. Visual identification
of a corpus luteum or progesterone levels above 4 ng/ml indicate that the first ovulation has
occurred and the onset of normal ovarian cyclical activity.

Treatment

 The treatment of mares in the transitional stage is based on progesterone or progestogens,

with or without the addition of estradiol esters. Progesterone can be administered as an oil-
based intramuscular injection, orally the synthetic progestogen altrenogest or by using a
silastic progesterone releasing intravaginal device.
 Progesterone exerts a negative feedback on gonadotrophin secretion which is followed by an
decreased release of FSH and luteinising hormone (LH). When the source of progesterone is
withdrawn or its effect wanes, because of the withdrawal of the negative feedback effect there
is follicular growth, maturation and ovulation.
 Progesterone treatment is more effective in mares that are in late transitional stage and is
ineffective in mares with minimal follicular activity, particularly during deep anestrus.
 Currently, the most effective treatment is the use of in-feed medication with the potent
progestogen altrenogest (Equine Regumate).
o This liquid contains 2.2 mg/ml of the active substance and should be added to the
food once per day at a dose rate of 0.044 mg/kg body weight for 10 consecutive days.
o Estrus occurs within 6 days and ovulation between 7 and 13 days after the last
treatment.
o Because of the possibility of ovulation occurring during treatment, an injection of
PGF2α on the last day of in-feed medication may be necessary to cause luteolysis of
any corpus luteum that may be present.
 The use of intramuscular injections of progesterone and estradiol-17β in oil for 10 days
produces a similar response to altrenogest, but the interval to estrus is longer due to the
suppression of follicular development by the estradiol.
 The use of 0.04 mg of buserelin (Receptal) given twice daily by intramuscular injection is
also quite successful. It is expensive, as treatment is necessary for atleast 1-2 weeks.
 Regardless of the hormones used, mares undergoing treatment early in the season need 16
hours of adequate light and good housing and nutrition to ensure success.

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  During the transitional period before the first ovulation of the year, mares demonstrate erratic
estrus behaviour of varying intensity.
o The presence of multiple large follicles, possibly as large as 30 mm, makes detection
of ovulation difficult by palpation alone.
o Even outside this transitional period, misinterpretation of ovulation, even by
experienced clinicians, has been shown to be as high as 50%.
o Visualization of the corpus haemorrhagicum/early corpus luteum
ultrasonographically when the anechoic follicle is replaced by an intensely echoic
area representing the early corpus luteum is much easier.
o It is recommended that the interval between matings should not exceed 2 or 3 days,
although there have been no critical studies on the survival time of sperm in the mares
genital tract. It is important not to begin breeding too early or this will result in the
mare being mated many times.
o The appearance of uterine edema is an indication that the follicle should ovulate
within a few days.

Cystic Ovarian Disease

 Cystic ovarian disease as comparable to the condition described in the cow does not occur in
the mare. The persistent follicles that occur during the transitional and other periods are
structurally normal; however, their presence may explain why this condition has been
diagnosed in the past.

Ovarian Neoplasia

 This has been considered earlier under structural infertility.

CHROMOSOMAL ABNORMALITIES

 The normal chromosome complement for the domestic horse is 2n = 64. Various sex
chromosome anomalies have been described in the horse, but are not common. The incidence
of chromosomal abnormalities is difficult to assess, but must be suspected in the maiden
mares with small, inactive ovaries and an immature tubular genital tract once winter anestrus
has been eliminated as a cause of acyclicity. However, some genetically normal young fillies
in training can be acyclic and thus they must be given more time to mature reproductively;
karyotyping must be performed before making a final diagnosis.
 The main karyotypic abnormality of such mares is the 63, XO (Turner’s syndrome)
genotypes. Examination detects very small ovaries (<1 cm in diameter) and a poorly
developed tubular genital tract that is difficult to palpate. These mares are usually small for
their age and do not cycle, although occasionally they may show passive estrus signs. There is
no treatment and the mare is sterile.
 Other chromosome abnormalities include ovarian hypoplasia and testicular feminisation.
Thee are also rare, but must be considered in female horses with irregular cycles and small
ovaries during the breeding season.

OVULATORY DYSFUNCTION

Anovulatory Haemorrhagic Follicles

 A form of apparent ovulatory failure has been described in the mare wherein
o preovulatory follicle grows to an unusually large size (7-10 cm)
o Fails to rupture and ovulate, but fills with blood and then gradually regresses.
o These haematomata persist for a variable period of time, often beyond the next
ovulation and corpus luteum formation and normal cyclic ovarian activity continues.
o Spontaneously resolve and no treatment is required.
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 o The condition is known as 'haemorrhagic anovulatory follicle syndrome’.
o The condition can be diagnosed ultrasonographically where the preovulatory follicle
filled with blood is initially recognized during transrectal ultrasound, by the presence
of scattered free-floating echogenic spots within the follicular antrum. As the blood
coagulates, the ultrasonic appearance varies from honeycomb or `net-like’ to a
uniformly echogenic mass.
o These structures can be as large as 8-10 cm, occasionally much larger, and develop an
outer wall of luteal tissue.
o Functionally, they gradually regress in the same way as a normal corpus luteum, but
they remain visible ultrasonically over subsequent estrous cycles.
o No treatment is usually necessary. Sometimes they may also fail to regress around
day 14-15 of the cycle and persist.
o Haemorrhagic follicles may be difficult to diagnose.
 The rise in plasma progesterone is not useful for detecting ovulation since
most haemorrhagic follicles tend to luteinise, thus producing progesterone
and hence their alternative name luteinised unruptured follicle.
 These structures cannot be detected by the behavioural responses of the mare,
since estrogen concentrations are initially elevated, and subsequently,
progesterone concentrations may increase and terminate estrus behaviour
similar to that following ovulation.
 On palpation, they are smooth with varying degrees of firmness. This can be
confusing, since they may feel like preovulatory follicles or corpora
haemorrhagica, or they may increase in size and become very large.
 The most obvious difference in their appearance is when they are examined
ultrasonographically. Commonly, there are multiple echoes from within the
follicular cavity, giving a net-like appearance within the follicular fluid.
 The structures may have a similar appearance to that of a Granulose Theca
Cell Tumour (GTCT); the anechoic areas are separated by trabeculae and are
similar to those of a multicystic GTCT.
 The diagnosis of a haemorrhagic follicle may be made on the basis of clinical signs: namely,
maintenance of cyclicity, a normal contralateral ovary, the presence of an ovulation fossa and
speed of enlargement and regression of the ovary with time.
 Their significance is that the oocyte is not released but remains within the large unruptured
haemorrhagic follicle. The abrupt decrease in follicle diameter normally associated with
ovulation is not noted, but rather a steady increaese in size and shape; stigma formation due to
follicle softening is not seen. However, one cannot unequivocally state that they did not form
by rapid filling between examinations.
 The cause of these haemorrhagic follicles is not known. Similar structures are seen under
continued equine chorionic gonadotrophin (eCG) stimulation during days 40-15 of pregnancy.

Anovulatory Follicles in Aged Mares

 While there is no documented menopause in mares, an age-related ovulation failure has been
documented.
 Some aged mares, particularly over 20 years age, fail to ovulate despite showing estrus
behaviour.
 On ultrasound examination their ovaries resemble those of seasonally anovulatory mares with
a few small (<10 mm) follicles.
 Endometrial biopsy shows evidence of gland atrophy.
 Currently there is no treatment, but identification of such mares is important to avoid
unnecessary coverings.

Multiple Ovulation

 Double ovulations occur during 8-25% of estrous cycles, the frequency depending upon the
breed and type of the mare (thoroughbreds, highest rate; ponies, lowest rate).
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  Accurate detection of such ovulations is important as twinning is highly undesirable;
o first, because it often results in abortion and,
o secondly, even if both fetuses survive and are carried to term, many are dysmature,
resulting in a high neonatal mortality rate.
o A further complication is that if embryonic / fetal death occurs after the formation of
the endometrial cups, these latter structures persist until they spontaneously regress as
if pregnancy had been maintained, resulting in psedopregnancy.
 Rectal palpation alone can be misleading in detecting a double ovulation, particularly when
the two follicles are on the same ovary.
 The use of ultrasound examination of the ovaries, which should routinely be performed in
conjunction with a thorough transrectal palpation helps in detection of a double ovulation.
Sometimes the ovulatory area can appear indistinct for the first 24 hours; in these cases the
mare should be re-examined 2 days later when it can be seen more easily whether there is
more than one corpus luteum.

MANAGEMENT OF TWIN OVULATION

 Multiple ovulations in the mare should not be regarded as a reason for withholding breeding.
Instead, pregnancy rates are improved after twin ovulation. Although accurate interpretation
of the ultrasound image of early pregnancies in the mare and the technique of crushing a
conceptus are skills that require experience, the advent of B-mode ultrasound imaging has
provided a method of more readily managing a twin pregnancy in the mare
 There are two approaches to dealing with twins :
o If the initial examination of the mare occurs before fixation (day 16/17) the twin
embryos are reduced to a singleton by the manual destruction of one, either by
pressure with the transducer or by the use of the hand. When the conceptual vesicles
are of dissimilar sizes, the smaller one should be ruptured. This is easier at days 14-16
when they are 14-20 mm in diameter than days 11-13 when they are 5-11 mm in
diameter.
o The disadvantage of this method is that it is more expensive, in that all mares are
scanned before the time of return to estrus.
o In addition, if ovulations that occur more than 3 days apart have not been detected, a
mistaken diagnosis of a single pregnancy may be made if the second vesicle is too
small to detect
o If initial examination is done after fixation but before day 30, and if both conceptuses
are in one horn, one option is to terminate a pregnancy using PGF2α.
o It is advisable to re-examine the mare 5 days later in case reduction has occurred, or
transvaginal ultrasound-guided allantocentesis can be attempted. Management of twin
pregnancies after this period is complicated by the formation of endometerial cups at
approximately day 37, 38 of gestation.
o Endometrial cups remain functional until around days 90-130 of gestation in the
presence or absence of viable fetus. Therefore, if twin pregnancies are not
successfully managed before the cups are formed or both embryos die after ay 37, the
mare usually will not return to a fermale iestrus for a prolonged period of time
 After day 37 of gestation, reduction methods are unreliable. They include dietary energy
restriction, surgical removal of one vesicle, intra-cardiac injection and transvaginal ultrasound
guided needle puncture

PREGNANCY FAILURE

 Pregnancy failure is a source of major economic loss to the equine industry. Embryonic death
occurs before 40 days of gestation when organogenesis is complete, with early embryonic
death (EED) occurring before the maternal recognition of pregnancy. Early fetal death occurs
before 150 days of gestation, and late fetal death occurs after that. Abortion is defined as
expulsion of the fetus and its membranes from day 300 onward.
o Embryonic Death

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 o Fetal Death and Resorption

EMBRYONIC DEATH

 In normal fertile mares the fertilization rate is more than 90%, which is comparable with other
domestic species, with estimates of the Early Embryonic Death (EED) rate at between 5 and
24%. In subfertile mares, the rate is higher.
 The period of greatest embryonic death in subfertile mares occurs in the interval before
pregnancy can be detected with ultrasound (day 11), particularly at the time the embryo enters
the uterus. Between days 14 and 40, the rate of embryonic death varies between 8 and 17%.
EED is multifactorial, in which external factors such as environment and management as well
as pathophysiological factors are involved. The factors involved in embryonic death are
o External Factors
o Maternal Factors
o Embryonic Factors

External Factors

 External factors involved in embryonic death include stress, nutrition, season of the year,
climate, sire effects and transrectal palpation
 Maternal stress due to severe pain, malnutrition and transport has been implicated as a cause
of EED. Transporting pregnant mares for a distance of 300 miles (500 km) in less than 9
hours of traveling time can be stressful, but should not result in embryonic death. If a longer
journey is necessary, the journey should be broken after 8 hours. Waiting until the fifth week
of pregnancy or later to transport brood mares may be advisable when critical events such as
descent of the embryo into the uterus and transition from the yolk sac to the chorioallantoic
placentation have occurred. The common practice of transporting mares to stud for mating
and returning home the same day should not be detrimental to their fertility, as long as the
transport is safe and comfortable
 Far from being avoided, regular exercise is important during pregnancy, although during the
latter half, forced exercise should be decreased. Rectal palpation and ultrasound examinations
should be considered safe procedures when performed correctly and there is no indication that
ultrasound examination is detrimental to the embryo

Maternal Factors

A number of abnormal maternal factors including hormone deficiencies and imbalances, uterine
environment, age and lactation have been implicated

 Hormonal Deficiencies and Imbalances

o Progesterone is critical for the maintenance of pregnancy in mares. The only source
of progesterone during the embryonic period is the primary corpus luteum (corpus
luteum verum). On the assumption that luteal insufficiency is important in EED,
many mares are given exogenous progesterone or progestogens in an attempt to
prevent it from occurring.Many dosage regimens do not effectively elevate or
maintain plasma progesterone levels. A single injection of 40 µg of the GnRH agonist
buserelin has also been shown to reduce the incidence of EED when given 10 days
after ovulation
 Uterine Environment
o An abnormal uterine environment is detrimental to embryonic survival. Acute
endometritis may result in EED by inducing premature luteolysis, or because of its
direct embryopathic effect
o Severe periglandular fibrosis of the uterine glands may reduce the chances of embryo
survival. Not only is this a response to persistent endometritis, but it also increases

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 with age. This is one of the reasons for the reduced fertility of mares over 12 years of
age
 Foal Heat Breeding
o Mares normally resume cyclical ovarian activity very shortly after parturition so that
they are sometimes bred as early as 7 to 10 days postpartum (at the foal heat). There
is conflicting evidence about the level of embryonic death if fertilisation occurs at this
time, with some studies showing a higher rate and others no effect. An advantage of
breeding at the first estrus post-partum is that the foaling-conception interval is
significantly shorter. The reason for the apparent decreased fertility in mares mated at
the foal heat is the hostile uterine environment due to delayed uterine involution or
persistent endometritis. However, pregnancy rates are clearly influenced by how strict
the selection criteria are for mating at the foal heat. Traditionally, such factors as a
normal foaling, placental expulsion, minimal vaginal bruising and absence of
infection have been used. Endometrial cytology and ultrasonic scanning of the genital
tract of each mare may be more reliable methods on which to base a decision
 Lactation
o More pregnancy failures are detected in lactating than non-lactating (maiden or
barren) mares; this phenomenon also increases with the age of the mare

Embryonic Factors

 Embryonic abnormalities are also important to consider in- relation to embryonic death.
Embryos recovered from sub-fertile mares are smaller and have more morphological defects
than embryos from fertile mares; however, this may be due to an abnormal uterine
environment
 Ultrasonic scanning has provided a valuable tool in studying embryonic death. Because
pregnancy is often diagnosed at an early stage, it is important to inform owners that not all
pregnancies detected with ultrasound will survive, even in apparently normal mares
 There are certain morphological features detected with ultrasound that are typical of mares in
which embryonic death is occurring. Some of the consistent features include :
o Presence of fluid within the uterine lumen
o Prominent endometrial edema
o Decreased or prolonged conceptus mobility
o Undersized or irregularly shaped conceptus
o Cessation of embryonic heart beat
o Reduced volume of placental fluids
o Disorganization of placental membrances
o Hyperechogenic areas in the embryo and membranes

FETAL DEATH AND ABORTION

 The causes of equine abortion can be broadly divided into non-infectious (70%), infectious
(15%) and unknown (15%). In practice, it is important to distinguish infectious from non-
infectious causes. Vaginal discharge, premature lactation and colic in pregnant mares may
indicate an impending or recent abortion.
 When abortion occurs, the mare should be isolated, a history obtained and the fetus sent to an
approved laboratory for necropsy. If one wishes to perform a post-mortem examination, small
but representative samples of liver, lung, thymus, spleen and chorioallantois (two samples,
one of which is from the cervical star) should be sent in formal saline for histological
examination. In addition, frozen samples of fresh fetal liver and lung should be stored in a
deep freeze at –20°C should viral isolation investigation be required at a later stage. Paired
serum samples from the mare and close companions should also be taken for serological
investigation. Swabs from fetal heart or liver and the cervical pole of the chorion are used to
screen for bacterial infection.
 The fetus and fetal membranes (amnion, chorioallantois and umbilical cord) must be carefully
examined for the presence of abnormalities and areas of discoloration.
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Infectious Causes of Abortion

 The causal agents of infectious abortion are viruses, bacteria, fungi and more rarely
mycoplasma and protozoa and have been dealt with in detail under the module Specific
infections that affect the genital tract in mares.

Non-infectious Causes of Abortion and Stillbirth

Twinning

 Historically, twins have been the single most important cause of abortion in thoroughbreds.
However, they are now much less common due to the widespread use of ultrasonography.
The diagnosis of twin pregnancy can be made even if only one fetus is found as examination
of the placenta reveals an area devoid of villi where the two placentas were in contact. Twins
should still be submitted to a diagnostic laboratory as twin pregnancies are not protected from
equine herpesvires (EHV) infection.

Umblical Cord Abnormalities

 In mares, the umbilical cord is twisted, usually on a clockwise spiral. The normal length
ranges from 36 to 83 cm. Increased cord length has been associated with excessive cord
torsion, which can cause twisting of the umbilical blood vessels. This twisting causes
increased resistance to blood flow on both directions and the resulting poor placental
perfusion can lead to fetal death. This can result in abortion of an autolysed fetus. Decreased
cord length can cause premature tearing of fetal membranes, leading to fetal asphyxia.
Twisting and vascular compromise currently constitute the commonest single cause of
observed non-infectious abortion.

Premature Placental Separation

 In mares, the interdigitating microvilli are connected by an unidentified electron –dense

material. Placental separation is largely unknown, although maternal stress and endophyte-
contaminated tall fescue have been implicated. When placental separation occurs shortly
before parturition, the thickened placenta often does not rupture through the cervical star, and
the allantochorion bulges out of the vulva (`redbag’ delivery). The foal can become hypoxic,
resulting in the neonatal maladjustment syndrome.

Body Pregnancy

 In this condition almost the entire chorionic surface of the placenta contained within the
uterine body is without villi, while that contained within the horns is covered with an
excessive number of villi. The portion of the placenta corresponding to the two uterine horns
is small, and the fetus is situated entirely within the uterine body. The fetus is frequently
aborted completely contained within its placenta; its growth has been retarded. The abortion
occurs when the nutritional demands of the fetus exceed the ability of the placenta to meet
them.

Fetal Abnormalities

 Severe developmental anomalies involving the central nervous system and development of
body cavities have been reported in aborted fetuses.

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Maternal Disease

 Pyrexia and malnutrition during pregnancy have been implicated as causes of abortion.

PUERPERAL METRITIS

 Metritis is inflammation of the entire thickness of the uterine wall. It occurs when there is
massive contamination of the uterus, frequently in association with trauma during foaling or
RFM
 The prognosis is grave, particularly in heavy horses, since the absorption of toxins from the
uterine lumen into the general circulation results in systemic signs such as pyrexia, depresson,
loss of appetite and laminitis
 The rapid bacterial growth most commonly involving Gram-negative orgaisms leads to toxin
production
 Treatment involves
o repeated lavage of the uterus with warm sterile saline (2-3 litres) several times per
day until it is free of inflammatory exudates and placental debris
o Bacterial growth should be controlled, so as to limit toxin production, with a broad-
spectrum antibiotic effective against E.coli
o Supportive therapy with parenteral antibiotics, antihistamines (in cases of retained
fetal membranes), oxytocin and intravenous fluid therapy is indicated in many cases
 Systemic signs such as pulse rate and mucous membrane colour are used to monitor the
response to therapy in conjunction with examination of the uterine fluid
 Despite all efforts, some mares die due to toxaemia or irreversible changes in the foot
following laminitis such as pedal-bone rotation

PYOMETRA

 Pyometra is the accumulation of large quantities of inflammatory exudates in the uterus

causing its distention.
 It must be distinguished from the smaller, and intermittent, accumulations of fuid that can be
detected by ultrasonography in acute endometritis.
 Pyometra occurs because of interference with natural drainage of fluid from the uterus, which
may be due to cervical adhesions or an abnormally constricted, tortuous or irregular cervix. In
some cases, the fluid accumulates in the absence of cervical lesions presumably due to an
impaired ability to eliminate the exudates. Other predisposing factors are chronic infection
with P.aeruginosa or fungi.
 Damage to endometrium leads to
o extensive loss of surface epithelium,
o severe endometrial fibrosis
o glandular atrophy.
 As a result the luteal phase becomes prolonged presumably due to interference with the
synthesis or release of PGF2α. This is in contrast to mild endometritis with the collection of
small amounts of intraluminal uterine fluid, which is more likely to cause premature release
of PGF2α and luteolysis.
 Some clinicians restrict the term `pyometra’ to cases where, in addition to the accumulation of
exudates within the uterine lumen, the corpus luteum persists beyond its normal life span.
 Some mares with pyometra have normal regular cyclical ovarian activity. Persistence of the
corpus luteum is probably due to failure of the synthesis and or release of prostaglandins from
the uterus.
 Mares that have prolonged luteal phase have the greatest endometral damage.
 The mare with pyometra seldom shows over signs of systemic disease even when there is
upto 60 litres of exudate in the uterine lumen. Very occasionally there is weight loss,
depression and anorexia.
 Pyometra has been classified into two categories in mares. Open and closed.
o In a case of closed pyometra, the fluid accumulates due to a closed cervix.
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 o In open pyometra, the cervix remains open, but purulent material accumulates
because of impaired uterine clearance. A vulval discharge is often observed in open
pyometra, especially at estrus, which may vary inconsistency from watery to cream
like.
 Although the culture of endometrial swabs can sometimes result in the growth of mixed
organisms or sometimes no bacterial growth at all. In most cases the organism isolated is
S.zooepidemicus.

Diagnosis

 The diagnosis of pyometra is based upon rectal palpation, ultrasonic examination of an

enlarged fluid – filled uterus and analysis of the uterine fluid. Pregnancy must be eliminated
together with rare conditions such as mucometra and pneumo uterus.
 Due to the lack of systemic illness, cases of pyometra have often become chronic before
treatment is sought. In such cases the prognosis is poor because of severe endometrial
damage, which is unlikely to be able to sustain a normal pregnancy.

Treatment

 The aim of treating pyometra is to expel the purulent material from the uterus.
 In the absence of systemic illness or an unsightly vulval discharge treatment of chronic
pyometra may not be indicated, although some mares can show signs of discomfort during
exercise.
 Many cases can be significantly improved by repeated large volume lavage with several litres
of warm saline via a wide- bore tube such as a nasogastric tube.
 Initially PGF2α can be used to induce luteolysis of the corpus luteum if present, which should
allow the cervix to relax sufficiently for digital exploration for the presence of any adhesions.
Estradiol or PGF2α may also help relax the cervix.
 The broad-spectrum combination of antibiotics and crystalline benzylpenicillin should be
infused after repeated large volume lavage and oxytocin to achieve drainage of exudates, and
an endometrial biopsy is useful in assessing the degree of endometrial damage.
 Monitoring the uterus by a combination of rectal palpation and ultrasound provides
information on the response to treatment. Even if successfully treated, the mare must be
considered a susceptible mare if she is to be bred and managed accordingly.
 In non responsive cases, hysterectomy can be performed following aspiration of the exudates
from the uterus although great care has to be taken to prevent contamination of the peritoneal
cavity.

RETAINED FETAL MEMBRANES (RFM)

 Retention of the fetal membranes (RFM) is properly regarded by veterinary surgeons as a

potentially more serious affection than the same condition in cattle. This has originated from
the times when draught horses predominated in the horse population and was invariably
followed by serious sequelae; as a result early manual removal was the rule.
 Complications include acute metritis, septicaemia, laminitis and even death. With prompt and
effective treatment these sequelae can be avoided.
 In many cases, uterine involution is delayed even if these more serious complications do not
develop. The riding horses and ponies of today are less likely to suffer form these
complications, but RFM should be treated as an emergency.
 The average time taken for the fresh membranes to be expelled is about 1 hour, and should
not exceed 2 hours, although there is debate amongst equine clinicians about the latter. RFM
is one of the most common peripartum problems in the mare, with an incidence in the range
of 2% to 10%.

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Aetiology

 The precise cause of retained placenta remains unclear. The most likely is uterine inertia due
to hormonal imbalance. Oxytocin has an important role in postpartum uterine contractions,
and low levels of this hormone in the circulation may result in abnormal myometrial activity.
This in turn leads to placental retention.

Clinical Signs

 The most obvious sign of RFM is the presence of a variable portion of tissue protruding from
the vulva; less commonly nothing is visible. Either this means that no parts of the fetal
membranes have been expelled or, more likely, portions remain attached.

Treatment

 Initially, the protruding membranes should be tied in a knot to prevent them touching the
hocks.
 As uterine contractility plays an important role in the dehiscence of the fetal membranes,
administration of oxytocin is recommended as a first and most successful method of treatment
in up to 90% of cases.
 It is good rule not to wait longer than 6 hours after delivery of the foal; the time interval
should be shorter in heavy breeds. This method of treatment avoids manipulation within the
uterus, with the risk of introducing micro-organisms. Oxytocin can be given via the
intramuscular route (20-40 IU), which can be repeated after 1 hour if the membranes have not
been expelled.Altenatively, slow intravenous infusion of 50 IU oxytocin in 1 litre of
physiologic saline over 1 hour could help. Symptoms of colic often follow injections of
oxytocin and commonly precede natural expulsion so that pain-relieving drugs and sedation
may be required.
 Only if this treatment fails and the membranes are almost detached but retained within the
uterus should one attempt gentle manual removal. This interference should be carried out with
scrupulous regard to asepsis, and no undue force should be applied, for even moderate
traction on the after birth may cause the uterus to become inverted and prolapsed.

 In most cases of retention some separation of the allantochorion has occurred and
consequently a variable amount of the after birth hangs down from the vulva.
o The mare is effectively restrained and measures should be taken to protect the
operator from being kicked.
o The tail is bandaged and held to one side by the attendant while the obstetrician
thoroughly washes the perineum and rear of the mare.
o With the hand and arm protected by a clean plastic sleeve, the extruded mass, or
failing that the freed part lying within the vagina, is grasped and twisted into a rope.
o The gloved hand anointed with lubricant is gently introduced along the `rope’ to the
area of circumferential attachment in the uterus. As the `rope’ is gently pulled and
twisted, the tips of the fingers are pressed between the endometrium and the chorion.
o The villi are easily detached, and as the allantochorion is gradually freed it is taken up
by further twisting of the detached mass.
o The allantochorionic membrane is gently separated from the endometrium by moving
one of the hands between them. The tightest attachment is usually at the tip of the
horn. The process of separation usually goes quite smoothly, and the complete sac of
allantochorion can be gradually detached from the pregnant horn. There is a tendency
for attachment to be firmer in the non-pregnant horn, and occasionally retention is
confined to this horn. If it is found impossible to detach the apical portions of the
allantochorionic sac without tearing the membranes it is better to desist and to try
again in 4-6 hours, by which time a successful outcome will be likely.

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 o Unwanted side – effects of this manual removal may be serious haemorrhage,
invagination of one of the horns and a higher chance of retention of microvilli in the
endometrium.
o During a difficult manual removal only the central branches of the chorionic villi are
removed while practically all the microvilli are broken off and retained ; rupture of
endometrial and subendometrial capillaries may also occur.
o The consequences of different removal are increased puerperal exudates, containing
much tissue debris; endometritis and laminitis; uterine spasm and delayed involution
of the uterus.
o A third method described in the literature, and which may be successful under some
circumstances, is the placement of some 10 titre of warm, sterile saline inside the
chorioallantoic membrane. Stretching of the uterine wall stimulates uterine
contractions, via endogenous oxytocin release, and may assist in the separation of the
microvili from their endometrial crypts. This treatment should be used in combination
with exogenous oxytocin administration.
o After removal, it is always important to examine the membranes for completeness
confirming that al the allantochorion has been removed. If necessary, the uterus
should be flushed and siphoned to remove any fluid exudates remaining in the uterus
by using a stomach tube and funnel. After care includes (depending on the severity of
the case) regular general clinical examination, particularly the uterus (for involution
and contents) and, if indicated, flushing and siphoning the uterus once or twice daily
for a few days in combination with further injections of oxytocin. The rationale for
uterine lavage is to remove both debris and bacteria from the uterus. Warm, sterile
physiologic saline should be used in 2-4 litre flushes (until the recovered fluid is
clear).
o Special attention is paid for signs of laminitis, and non-steroidal anti-inflammatory
drugs are given when laminitis is a suspected complication.
o Tetanus antitoxin is recommended and, if indicated, treatment with antibiotics.
o If there is a risk of the mare developing a toxic metritis, she should be treated with
systemic and intrauterine antibiotics. The dominant infective organism is
oftenStreptococcus zooepidemicus initially, but infection with Gram-negative bacteria
such as Escherischia coli frequently develops. The antibiotics chosen should have
broad-spectrum activity and should be effective against endotoxin-producing
organisms. Cyclo-oxygenase inhibitors such as flunixin meglumine should be given
to either treat or minimize the risk of development of endotoxaemia.
 Provided treatment is begun at the correct time and no secondary complications develop, the
prognosis for a case of retained placenta is good.

ENDOMETRITIS

 The term `endometritis’ refers to the acute or chronic inflammatory process involving the
endometrium brought about by microbial infection or may occur due to non-infectious causes.
One of the main obstacles to producing the maximum number of live, healthy foals from
mares bred during the previous season is the mare, which is susceptible to persistent acute
endometritis following breeding.

Cause and Pathogenesis

 The underlying etiology of the specific cause of endometritis determines the type of treatment
to be used, and the following classification system for equine endometritis is useful:
o Venereal Infection
o Chronic Infectious Endometritis
o Endometriosis (chronic degenerative endometritis)
o Persistent Mating-induced Endometritis (delay in uterine clearance).

VENEREAL INFECTION AND CHRONIC INFECTIOUS ENDOMETRITIS

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  It is generally assumed that the uterine lumen of the normal fertile mare is bacteriologically
sterile or may have a temporary, non-resident microflora. This is despite the fact that the
mare’s reproductive tract is often contaminated with bacteria from the act of coitus, foaling
and veterinary procedures. Mares with a defective vulval conformation can also aspirate air
and bacteria into the vagina that can develop into endometritis
 The bacterial species that cause bacterial endometritis are numerous, and can be classified as
follows:
o Contaminants and commensal
o Opportunist
o Venereally transmitted.
 Normally, the vestibular and clitoral area has a harmless and constantly fluctuating bacterial
population. In association with benign saprophytic organisms, opportunistic organisms such
as Streptococcus zooepidemicus, E.coli and Staphylococcus spp. can be found. The stallion’s
penis is colonized by similar organisms. S. zooepidemicus is the most commonly isolated
bacterial species from acute endometritis, particularly in the initial stages. E. coli is the next
most common isolate.
 The uterus responds to these bacteria with a rapid influx of neutrophils. Normally these
neutrophils phagocitize and kill the bacteria rapidly (<24 hours). The inflammatory
byproducts are then mechanically removed and the endometritis resolves itself expect when
the mare suffers from pneumovagina or is a ‘susceptible’ mare. Susceptible mares have a
delay in uterine clearance, and the inflammatory byproducts accumulate as uterine fluid. Such
mares have a reduced pregnancy rate due to a hostile environment for the early developing
conceptus
 In addition to opportunist pathogens, there are three bacteria that are venereally
transmitted: Taylorella equigenitalis (contagious equine metritis organism,
CEMO), Klebsiella pneumoniae and Pseudomonas aeruginosa (some strains)
 Symptomless carriers of both sexes allow persistence within the horse population. Carrier
mares, which may or may not have shown signs of previous endometritis, harbour the
organisms in the vestibular area, particularly the clitoral fossa and sinuses. Mating or
gynaecological examination may result in their transfer into the uterus. Stallions may harbour
the organisms over the entire surface of the penis and in the distal urethra. Control is by
laboratories experienced in the isolation and identification of these specific organisms

DIAGNOSIS

Venereal Disease Screening

 Before the breeding season, swabs should be taken from the clitoral fossa, clitoral sinuses
(only the central sinus may be obvious ) and the vestibule. The perineal area of the mare
should not be cleaned except for the removal of gross contamination of the vulva with faeces
using a dry paper towel.
 A protective disposable glove should be worn by the veterinary surgeon on the hand used to
evert the ventral commissure of the vulva and expose the clitoris. The swabs should be placed
in transport medium, clearly labeled with the mare’s name and sent to an approved laboratory.

 It is important to penetrate the clitoral sinus, and therefore a large swab tip should not be
used. Swabs are cultured aerobically on blood and Mc Conkey agar to screen for the presence
of K. pneumonia and P. aeruginosa. Microaerophilic culture on chocolate blood agar (with
and without streptomycin) must also be done for the detection of CEMO.
 In addition, in stallions, two sets of swabs must be taken from the pre-ejaculatory fluid (if
possible), penile sheath, urethra and urethral fossa.

ENDOMETRIAL CULTURE AND CYTOLOGY

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  A diagnosis of endometritis can be made by collection of concurrent endometrial swab and
smear samples during early estrus for bacteriological culture and cytological examination,
respectively. This allows time for resolution prior to mating, and maximizes the chances of
pregnancy.

 The ideal technique should ensure that the swab enters the uterus and collects bacteria from
the uterine lumen only. It is important to ensure that the method of swabbing does not
introduce bacteria into a previously normal uterus. Two methods can be used:
o A non-guarded endometrial swab on a sterile extension rod is carefully passed via a
sterile speculum through the cervix into the uterine body and, after withdrawal, is
placed in transport medium. A second swab is taken immediately afterwards for the
endometrial smear.
o A guarded swab is passed into the uterine lumen using a sterile speculum or enclosed
in a disposable plastic arm-length glove. The swab tip is exposed only when it is in
the uterine lumen. A swab for cytological examination should again be taken. To
reduce the risk of contamination, the use of guarded swabs is advised.
 Swabs for culture should be plated on blood and Mc Conkey agar, and incubated at 37°C for
48 hours. Cultures should be examined at 24 and 48 hours.

 An air-dried smear is made by gently rolling the second swab on a clean dry microscope
slide.The smear can be differentially stained with a rapid stain such as Diff-Quick (American
Hospital Supplies).The stained smear should then be examined for the presence of
inflammatory and endometrial cells, the latter confirming contact of the swab with the
endometrium. The veterinarian must ensure that the mare is not pregnant before passing a
swab through the cervix.
o A positive culture result, with no evidence of inflammatory cells in the smear (usually
neutrophils), is likely to be due to contamination during collection.
o Diagnosis of acute endometritis is based on the presence or absence of significant
numbers of neutrophils in the smear. Mares that have > 5 neutrophils / high power
field (x40) on a cytology smear should be considered to have active endometritis.

ENDOMETRIAL HISTOLOGY

 In some cases, endometrial biopsy may be a useful diagnostic aid. The technique involves the
insertion of a biopsy instrument through the cervix and into the uterus. The instrument most
commonly used today is the Yeoman (basket-jawed) biopsy forceps, ideally 60-70 cm in
length, with which tissue specimens 2 x 3 x 1 cm (about 0.2% of the whole endometrial
surface) are obtained. If the uterus appears normal on palpation, the sample should be taken
from one of the areas of embryo fixation, i.e., the uterine horn-body junction on either side.
Single samples are usually representative of the entire endometrium.
 If the uterus is abnormal on palpation per rectum, biopsy samples should be taken from both
the affected area and a normal area. Biopsy specimens should be fixed in Bouin’s followed by
sectioning and staining with haematoxylin and eosin. The endometrial biopsy sample should
be sent to a laboratory that is experienced in evaluating samples.

UTERINE LUMINAL FLUID

 The detection of uterine fluid during both estrus and diestrus has been reported. Endometrial
secretions and the formation of the small volume of free fluid may be associated with the
same mechanism that causes normal estrual edema
 In many cases, the uterine luminal fluid that accumulates before mating is sterile and contains
no neutrophils. The importance of these sterile fluid accumulations is that though initially
sterile, the fluid may act as a medium for bacteria that gain entry into the uterus at mating to
multiply and may be spermicidal

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  The amount of fluid that should be considered significant is not clear and it may be that
quantity is more important than nature. This is particularly true of fluid appearing during
estrus
 The significance depends to some extent on when during estrus the fluid is observed. Fluid
detected early in estrus may have disappeared when the mare is further advanced in estrus and
the cervix relaxes more
 Small volumes of intrauterine fluid during estrus do not affect pregnancy rates, in contrast to
mare with larger (>2 cm depth) collections of fluid. In mares that are susceptible to
endometritis there is an accumulation of more fluid than in non-susceptible mares
 Generally if there is more than 1 cm of fluid during estrus, some attempt should be made to
remove this before breeding using oxytocin treatment. If the volume is above 2 cms, the fluid
may need to be drained and investigated for the presence of inflammatory cells and bacteria.
The mare may then need to have a large-volume uterine lavage
 Intrauterine fluid during diestrus is indicative of inflammation, and associated with
subfertility, due to early embryonic death and a shortened luteal phase
 Intraluminal uterine fluid can be graded I to IV according to the degree of echogenicity. The
more echoic the fluid, the more likely the fluid is contaminated with debris including white
blood cells. However, fluid containing cells can appear relatively anechoic so care is needed
in interpretation. Inspissated pus can be so echoic that it is overlooked. It may be that the
actual appearance of the fluid and the ultrasonographic appearance are not as closely linked as
once thought. Ultrasonographic appearance may be proportional to the size and concentration
of particulate matter within the fluid, rather than the viscosity of the fluid; for example,
purulent exudates can appear non-echogenic. Air has hyperechoic foci, and fluid with air
bubbles appears cellular. Urine in the bladder can appear echoic, despite being a watery liquid

Detection of Intraluminal Uterine Fluid Using Transrectal Ultrasound Imaging

 Transrectal ultrasonography provides a rapid, non-invasive method of assessment of the

uterus. In mares that are particularly susceptible to endometritis and in which vaginal contact
should be minimized, endometritis can often be diagnosed on the basis of intrauterine fluid
accumulation. This is more meaningful when the mare has already been swabbed and cleared
of potential venereal diseases. If fluid is present in the uterus, there is vulvar discharge, or the
mare has abnormally short luteal phases, uterine swabs should be taken to determine the cause
of these symptoms

TREATMENT OF VENEREAL INFECTIONS AND CHRONIC INFECTIOUS

ENDOMETRITIS

 Any mare that is suspected of having a venereal infection must not be bred. In the case of
clitoral or vestibular infections, topical treatment is used. This involves
o Thorough cleaning with chlorhexidine surgical scrub followed by the application of
 0.2% nitrofurazone ointment for T.equigenitalis,
 0.3% gentamicin cream for K. pneumoniae
 silver nitrate and gentamicin cream for P. aeruginosa
o Clitoral sinusectomy or clitorectomy may have to be used in refractory cases. A broth
culture containing a mixture of growing organisms prepared from the normal clitoral
flora can suppress venereal pathogens in some cases
o Chronic infectious endometritis is found most frequently in older mares that have had
several foals. Such mares have compromised uterine defense mechanisms that allow
the normal vestibular and vaginal flora to colonise the uterus, thus inducing a
persistent endometritis
o The most favoured approach to treatment has been the infusion of various antibiotics,
dissolved or suspended in water or saline, into the uterine lumen during estrus
o The intrauterine route is preferable to systemic therapy as most acute endometritis
cases are localized. Systemic treatment alone, or in combination with local
application, is suitable in a few circumstances
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 o Ideally, the choice of antibiotic of local treatment should be based on in vitro
antibiotic sensitivity tests. However, in many cases this is not possible and a broad-
spectrum combination should be used that is effective against the mixed aerobic and
anaerobic infections that commonly occur. A particularly successful preparation has
been a buffered, water-soluble mixture of neomycin sulfate (1g), polymyxin B (40
000 IU), furaltadone (600 mg); and crystalline benzyl penicillin dissolved in 40 ml of
sterile water and then instilled through the cervix into the uterus via a sterile irrigation
catheter. A larger volume (upto 100 ml) may be better in older, pluriparous mares to
ensure distribution throughout the uterus. The use of this extremely broad-spectrum,
non-irritant, soluble preparation has not resulted in super infection with Pseudomonas
sp., Klebsiella sp., yeasts or fungi. The number of treatments required depends on
individual circumstances, but daily infusions for 3-5 days during estrus work well in
most cases. The success of this treatment can be monitored using ultrasonography to
identify the presence of intrauterine fluid
o When antibiotics are combined with oxytocin a single daily treatment for 3 days has,
in many cases, proved successful. Repeated endometrial swab/smear examinations
may be used to monitor the response to therapy; however, every time the cervix is
breached there is the risk of introducing more bacteria
o An indwelling intrauterine device has been used that can retain a narrow-diameter
infusion catheter within the cervix ; however, there is a risk of ascending infection
 In addition to the antibiotic therapy, repeated treatment with PGF2α increases the frequency
of the follicular phases, thus allowing intrauterine therapy to be used more readily. In
addition, it also reduces the duration of the luteal phase where progesterone increases the
susceptibility to infection
 Predisposing causes to the persistent endometritis, such as defective vulval conformation,
should also be attended to

ENDOMETRIOSIS

 Endometriosis is a collective term used to describe a wide range of degenerative changes

(fibrosis and glandular degenerative changes) that can be diagnosed by endometrial biopsy.
 Successful treatment of endometriosis is difficult. Improved fertility after endometrial
curettage has been reported. This has involved the use of mechanical and chemical agents
(namely povidone-iodine and kerosene) that cause endometrial necrosis. This treatment apart
from being of questionable efficacy can cause irreversible damage such as adhesions.
 Repeated daily lavage with 2-3 litres of hot (50°C), sterile, isotonic saline has been suggested
as a method of reducing the size of the lymphatics and thereby the whole uterus.
 The prognosis for fertility remains poor whatever treatment is used.

PERSISTENT MATING-INDUCED ENDOMETRITIS

Uterine Defense Mechanisms

 At coitus, the mare's uterine lumen becomes contaminated with micro-organisms and debris
leading to endometritis. Even if mares are bred by artificial insemination, semen is deposited
directly into the uterus. In most mares, this transient endometritis resolves spontaneously
within 24-72 hours so that the environment of the uterine lumen is compatible with
embryonic and fetal life. It is important not to regard this endometritis as pathological
condition. Rather it is a physiological reaction to large numbers of sperm, seminal plasma and
inflammatory debris from the uterus before the embryo descends from the uterine tube to the
uterine lumen 5.5 days after fertilization. However, if the endometritis persists after day 4 to 5
of diestrus, in addition to being incompatible with embryonic survival, the premature lease of
PGF2α results in luteolysis, a rapid decline of progesterone and an early return to estrus
making these mares susceptible and they develop a persistent endometritis.
 In general, reduced resistance to endometritis is associated with advancing age and
multiparity.
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  The physical ability of the uterus to eliminate bacteria, inflammatory debris and fluid is now
known to be the critical factor in uterine defense and any mpairment of this function leads to
uterine infection.
 Lymphatic drainage could also play an important role in the persistence of post-breeding
inflammation, and lymphatic lacunae (lymph stasis) is a common finding in endometrial
biopsies taken from susceptible mares.

Treatment

 The aim of treatment should be to assist the uterus to expel the normal inflammatory products
arising from the response to breeding. Since within 4 hours of mating the spermatozoa
necessary for fertilization are present within the uterine tube, and since the embryo does not
descend into the uterus for about 5.5 days, mares may be treated safely from 4 hours after
mating until 3 days from ovulation, providing non-irritant therapy is used. However,
progesterone concentrations rise rapidly following ovulation in the mare, and it is preferable
to avoid treatment involving uterine interference beyond 2 days after ovulation. Both natural
mating and artificial insemination can be a source of uterine contamination.
 The successful management of susceptible mares should logically require some form of
postmating therapy such as intrauterine antibiotic infusion, uterine lavage and intravenous
oxytocin; these may be used alone or in combination. The emphasis should be on treatment in
relation to breeding and not ovulation.

UTERINE LAVAGE

 The technique of uterine lavage involves the mechanical suction or siphonage of 2-3 litres of
previously warmed (to 42°C), sterile physiological (buffered) saline or lactated Ringer’s
solution infused into the uterus via a catheter that has been retained within the cervix via a
cuff. The most convenient is a large-bore (30 French) (80 cm) autoclavable equine embryo
flushing catheter. The cuff is useful as it effectively seals the internal cervical os. The catheter
should only be inserted after thorough cleansing of the perineum. The rationale for such an
approach is :
o To remove accumulated uterine fluid and inflammatory debris that may interfere with
neutrophil function and the efficacy of antibiotics
o Stimulation of uterine contractility
o Recruitment of fresh neutrophils through mechanical irritation of the endometrium
 The saline is infused by gravity flow 1 litre at a time, and the washings are inspected to
provide immediate information concerning the nature of the uterine contents. The lavage
should be repeated until the fluid that is recovered is clear. In most cases, the fluid is evenly
distributed in both horns, making transrectal massage of the uterus unnecessary. If a rectal
examination is performed whilst the catheter is in the uterus care must be taken to avoid
contaminating the catheter. The fluid should be recovered in the same container from which it
was infused, thereby preventing air being aspirated into the uterus via the catheter.
Measurement of the recovered fluid and ultrasonographic examination of the uterus should be
performed after flushing to ensure that all the fluid has been recovered. This is necessary
because you are dealing with a mare with an impaired ability to drain the uterus
spontaneously
 For this reason the process is usually combined with oxytocin injection. Ideally these mares
will be bred only once, but if repeated matings are necessary, uterine lavage should be
performed after each mating
 Large-volume lavage is beneficial in many cases, particularly the mare with a relatively large
(above 2 cm depth) accumulation of fluid after breeding. The process is time-consuming and
there is the possibility of further contamination of the uterus by passage of a drainage tube

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Oxytocin

 The ideal method of treatment will be the use of a non-invasive technique with early and
complete elimination of any intrauterine fluid
 Oxytocin stimulates uterine contractions in the cyclical, pregnant and postpartum mare and
hence can be used as a method to promote uterine drainage in mares with defective uterine
clearance. However, its use was discouraged because of the worry that it would cause severe
colic

Prostaglandin Analogues

 Prostaglandin analogue cloprostenol given at a dose rate of 500 µg IM increased myometrial

activity and assisted in uterine clearance. Of the prostaglandins administered (PGF2 α,
cloprostenol and fenprostalene) cloprostenol produced the most consistent response.
Cloprostenol would seem to be indicated in mares that have lymphatic stasis as shown by
excessive fluid within the endometrium or large lymphatic cysts. Cloprostenol should not be
given more than 24 hours after ovulation in case of inducing premature luteal regression

Intrauterine Plasma Infusions

 Intrauterine plasma has been used in the susceptible mares and had an enhancing effect on
phagocytosis by uterine neutorphils. However, its use may only apply to mares without a
mechanical clearance problem and thereby repeatedly fail to become pregnant, but have no
history of fluid accumulation

MANAGEMENT PROTOCOL USEFUL IN THE HIGHLY SUSCEPTIBLE MARE

Overall management of mares known to produce a large amount (several centimeters depth) of
luminal fluid after mating should be excellent prior to breeding.

 Good hygiene at foaling is essential and all mares should be thoroughly examined postpartum
for the presence of trauma that might compromise the physical barriers to uterine
contamination
 Gynaecological examinations, particularly of the vagina, should be performed as aseptically
as possible
 Thorough digital examination of the cervix can identify fibrosis, lacerations or adhesions that
may need treatment before breeding
 Since, air in the vagina can cause irritation of the mucosa it should be expelled by applying
downward pressure with the hand through the rectal wall
 Attention should be paid to hygiene at mating by using a tail bandage and washing the mare’s
vulva and perineal area with clean water (ideally from a spray nozzle which avoids the need
for buckets)
 Breeding should occur at the optimal time, and the number of breedings should be minimized.
This means that these mares need very close monitoring of the estrus period by rectal
palpation and ultrasonography
 The use of hCG is strongly recommended in such mares in an attempt to ensure they are bred
only once. Prediction of ovulation can also be made easier by not breeding these mares too
early in the year, i.e., before they have begun to cycle regularly
 If feasible, the use of artificial insemination can be helpful to reduce (but not eliminate) the
inevitable post-breeding endometritis.
 A single breeding must be arranged 1-2 (or even 3) days before the anticipated time of
ovulation
 Ultrasound examination of the uterus 3-12 hours after mating is performed to assess the
amount and echogenicity of any intrauterine fluid
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 After 20 minutes the mare should be re-examined and any fluid pooling in the vagina
removed. This is followed by infusion of low volume (30 ml) of water-soluble, broad-
spectrum antibiotics such as already described into the uterus via a sterile irrigation catheter
 2 x 25 IU of oxytocin should be given by the stud farm personnel that evening and again in
the morning, by the intramuscular route
 In mares with lymphatic stasis, the slower release of prostaglandin (cloprostenol 500 µg im.)
may be useful. In addition cloprostenol should be given 6-8 hours after the first oxytocin
injection
 The mare is re-examined the following day and oxytocin treatment repeated if fluid is still
present. Only rarely will a second infusion of antibiotics or lavage procedure be performed
due to the risk of uterine contamination

 Both the Kalayjian and the Knudsen are designed to protect the swab as it is passed through
the vagina and cervix hence providing the opportunity to secure a true culture of the uterine
lumen.
 The Kalayjian swab is protected by a plastic cap, attached to the casing. This cap can be used
to obtain a scraping of the endometrium suitable for staining.
 When the entire instrument is withdrawn the small, formerly protective, little cap is filled
with uterine fluid.

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BITCH

DEVELOPING A PROBLEM LIST

 Infertility or apparent infrertility problems in the bitch are common. Veterinary advice is
often sought after a bitch fails to conceive. If she fails to exhibit “normal” breeding behavior,
when her cycles appear to be unusual, or for myriad other distrubances.
 “Infertility”therefore, is a huge category comprising a long list of anatomic, physiologic, and
behavioral problems as well as a number of apparent husbandry misunderstandings.
 Further, a championship or other important title may be earned by the bitch, ensuring demand
for and value of any puppies before any attempt has been made at breeding

ASSESMENT OF MALE

 Before embarking on an investigation into the potential causes of infertility in a bitch, the
male should be assessed. Males are so much easier to study than females as the male is
continuously fertile while a female is usually fertile only 1 to 3 weeks per year.
 Male fertility can be established by reviewing the males previous breeding history. Any male
siring a litter or litters within the preceding 1 to 4 months can usually be assumed to be fertile.
It is also helpful to know if the male sired any litters at the time the bitch in question was in
heat and bred. However, even if the responses to these enquiries are affirmative, the fertility
of the male should be demonstrated with a complete semen analysis.

Click here to know more about evaluation of a subfertile male.

 All active stud dogs should be tested for brucellosis every 6 months. Less active studs should
be checked yearly and immediately prior to use. A male that has not sired a litter or has sired
litters in the past but not in the preceding 6 to 12 months must be viewed with suspicion.
 Whenever the male’s fertility is questionable, the owner of the bitch has three main
alternatives:
o have a semen analysis and brucella titer performed on the male,
o utilized an alternative, proven sire on the next heat, and
o evaluate the bitch, realizing she may not be at fault.
 A normal semen analysis is a major step toward ensuring that the male is not at fault.
 Abnormal semen, or an inability to obtain an ejaculate, leaves some suspicion directed at the
male.

EXAMINATION FOR PREGNANCY

History

OBTAINING A “COMPLETE” HISTORY. Before the bitch is examined by the veterinarian, the
various potential causes for infertility must be reduced to a workable number. In other words, the
differential diagnosis for most infertility disorders is established by obtaining a thorough history from
the owner. The initial history should include information such as

 How well the owners know the bitch?

 Does she live indoors with them or 200 miles away at a hunt club?
 Is she housed alone, with another bitch that recently completed ovarian cycles, with
ovariohysterectomized bitch or with males?
 Is she normal in height and weight for her breed and for her line?
 Is she receiving any medication and is she well or ill?

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Items can always be forgotten in reviewing a cases history during a busy workday, and the question
sheet helps to avoid this problem.

AGE AND BREED

 Small dogs reach sexual maturity at a younger age than large dogs.
 Onset of the pubertal estrus in the bitch has been reported to occur at ages ranging from 6.3 to
23 months, with mean ages of 9.6 to 13.9 months.
 Almost all healthy bitches begin cycling by 24 to 30 months of age.
 The first and second cycles may be irregular, unusual, short, or long.
 Infertility evaluations are delayed in most dogs until they are 24 to 30 months of age.
 Toy poodles may benefit from evaluation earlier in life than Bull Mastiffs.
 Each breed does have distinct average interestrus intervals, but the interestrus interval varies
within a breed.
 As a general rule, almost all breeds cycle once every 4.5 to 10 months. The African breeds
cycle once yearly.

PHYSICAL EXAMINATION

EXAMINE THE PROBLEM AREA LAST. As with any serious problem, the area of concern
should be the last to be evaluated on physical examaination. This approach ensures that each bitch
receives a complete physical examination prior to an evaluation of the reproductive tract.

Vulva

 Examination of the reproductive tract usually begins with an external inspection of the vulva,
checking the size and conformation and for presence of any discharge.
o The small immature vulva or one that is recessed under a fold of tissue owing to body
type or obesity may present impediments to normal breeding.
o The obese bitch is prone to perivulvar dermatitis.
o A swollen, turgid vulva is suggestive of proestrus
o A swollen and flaccid vulva can be consistent with estrus or approaching parturition.

Vaginal Discharges

 The bitch in anestrus or diestrus usually has no vaginal discharge.

 A bloody discharge is most suggestive of proestrus, estrus, separation of the placental sites, or
severe vaginitis.
 Greenish black or dark bloody vaginal discharges are associated with placental separation as
well as postpartum “lochia”.

 Reddish brown yellowish, or grayish, thick, creamy, malodorous vaginal discharges are often
seen in open-cervix pyometra, metritis, or severe vaginitis.

 Straw-colored vaginal discharges are sometimes seen when bitches are in estrus.
 Clear mucus can precede parturition and is rarely worrisome.

A vaginal cytology specimen should be an integral part of any reproductive evaluation because it is
easy to obtain the sample, inexpensive, and can be extremely informative. Vaginal cytology should be
performed in any bitch with a vaginal discharge.

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Digital Examination of the Vestibule and Vagina

 A digital examination of the vaginal vault should be performed routinely on any bitch
examined for breeding soundness.
 If a culture or cytology is needed, it should be obtained prior to the digital examination.Most
bitches are easy to examine.
 The gloved and lubricated index finger should pass easily into the vaginal vault, allowing
assessment of the lumen, the urethral opening, and clitoral size and shape. Masses, foreign
bodies, strictures, painful vaginitis, or abnormal tissue bands all prevent easy and painless
examination.
 If the digital examination is abnormal but inconclusive, vaginoscopy provides a more
thorough evaluation.
 The use of an otoscope or a vaginal speculum provides an extremely limited view of the
vaginal vault and is of little value in most clinical situations.
 Pediatric proctoscopes are easy to use for vaginoscopy and are relatively inexpensive, and can
be used in all but the smallest of miniature breeds. A more expensive but smaller diameter
alternative is use of an endoscope which provides far better visualization of the area than an
otoscope.

Mammary Glands

 The mammary gland should be palpated in the bitch examined for breeding soundness.The
primary concern is the presence of mammary tumors.
 The glands can also be checked for evidence of lactation, mastitis, inverted teats, or benign
nodules.
 The ventral midline can also be checked for evidence of a previous surgical incision, which
might be a clue suggesting that the bitch has undergone ovariohysterectomy.

Rectal Examination

 A rectal examination ensures that the pelvic canal has been assessed for previous fractures or
other unsuspected abnormalities.
 Compression of the pelvic canal is a potential cause of dystocia.
 One can also attempt to palpate the vagina ventrally, although the vagina would have to be
extremely abnormal to reveal anything suspicious on palpation.

Abdominal Palpation

 The abdomen should be palpated in an effort to identify and characterize the uterus. However,
except in pregnancy and pyometra, the uterus almost never can be evaluated with confidence
on abdominal palpation.

General Health of the Bitch

 In the clinical evaluation of the infertile bitch, one underlying question is her overall health
status.
 Complete blood counts, chemistry panels, urinalysis, thyroid function, and adrenocortical
function studies can be carried out as an initial step in evaluating the potentially infertile
bitch. However, such extensive diagnostic evaluations are not required unless the history
and/or physical examination dictates that aggressive diagnostic testing is warranted.
 The bitch that appears healthy to an owner, appears healthy on physical examination, and has
normal ovarian cycles does not have thyroid failure or adrenocortical disease and rarely has
other significant organ disease. Therefore, obtaining a complete blood count, urinalysis, and
blood urea nitrogen provides a sufficient data base. However, this approach depends on
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 completing a thorough history and a competent physical examination. If abnormalities are
identified on history or physical examination, appropriate testing can then be completed
which may clarify the nature of the problem or specifically demonstrate the cause of
infertility.

OWNER MANAGEMENT PRACTICES

 Improper management practices are the cause for a large majority of apparent infertility
problems. A bitch that is bred or attempted to be bred at incorrect times may be totally
normal. She may fail to conceive as a result of being brought to the male when she is not
fertile.
 The common errors in breeding management have already been discussed in detail under the
module on "Breeding and Artificial Insemination in Dogs". These and similar parctices do not
consistently result in conception.
 They may work in a majority of bitches, but some normal bitches fail to conceive if bred
according to such criteria.

BITCH IN NORMAL ESTRUS, CYCLES

 Management problems are the most common cause of apparent infertility in the bitch with a
normal cycle. The entire question regarding proper management for an individual bitch can
be answered through obtaining a thorough history with corrections made as needed in past
practices, behavior observation, vaginal cytology review, and monitoring plasma
progesterone concentrations.
 This approach answers the following question:
o How is the owner managing this bitch?
o When does standing heat begin?
o How long does standing heat persist?
o What is the first day of true diestrus?
o When is the bitch truly fertile?
o What are her ideal breeding dates?
o Does she ovulate?
o When does she ovulate?
o Does she have the luteal function necessary to support pregnancy?

MANAGEMENT PROBLEMS

 Adoption of a reliable breeding schedule while simultaneously studying follicular function

and the time of ovulation which may help to correct management related problems. If the
problem is physiologic, it may be identified and treated appropriately. A thorough review of
breeding practices have already been dealt in the module on “Breeding Management and
Artificial Insemination in Dogs”.
 The ovarian function during proestrus and estrus can be monitored using Vaginal
cytology and Progesterone estimations. Vaginal smears can be used to identify day of onset of
estrus and one can count back six days from Day 1 of diestrus to predict the day of ovulation.
Obtaining vaginal smears, staining and examining them.
o The evaluation of the bitch can be enhanced by serial monitoring of serum
progesterone levels to accurately predict the progression of the bitch through
proestrus and estrus and to predict the time of ovulation.
o The ovarian function during diestrus to determine whether ovulations have occurred
can be monitored by identifying day 1 of diestrus based on vaginal exfoliative
cytology coupled with plasma progesterone concentrations obtained between 10 and
20th day of diestrus.
 Shipping or transport practices should be reviewed. It is not known wheter transporting or
stress affects ovulation or conception in the bitch, hower, it is better to avoid during one cycle

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 to see whether the infertility problem can be resolved. the bitch can be bred locally to avoid
transportation.
 All previously or currently used medications must be reviewed. Previous use of
gonadotropins may have long-term deleterious effects on pituitary function while previous
progesterone or estrogen administration may result in subclinical cystic endometrial
hyperplasia with infertility being the only outward effect seen by the owner or veterinarian.
 Hypothyroidism is often described as “common” with signs such as
o persistent anestrus,
o prolonged interestrus interval, and
o prolonged proestrus,
o some bitches demonstrate normal reproductive activity, pregnancy, and parturition.
The diagnosis of hypothyroidism should always be viewed with suspicion, not
because the disease does not exist, but simply because most dogs treated for the
disease are not so afflicted.

INFECTIONS

Brucella Infection

 Brucella canis classically causes abortion late in gestation, resorption in early gestation, birth
of still born puppies, and infertility. All bitches included in breeding programs, especially
those with an infertility problem, should be evaluated for canine brucellosis. The rapid slide
agglutination test is an excellent screening test. False negative test results are unlikely, and a
negative result can be trusted. Bitches that are seropositive should be retested using the tube
agglutination method, because false-positive results do occur.

Other Infections

 Bacterial infections have been implicated as a cause of infertility in the bitch. These infections
are thought to be subclinical in the infertile bitch, only occasionally resulting in obvious
vaginitis, metritis, pyometra, or systemic infection.
 Most normal bitches have bacterial flora present in the anterior vagina, and similar types of
aerobic bacteria are present in the vaginal vaults of infertile bitches. Hence, it is difficult to
establish the role of bacterial infections in canine infertility.
 Treatment with vaginal douches for 2 to 3 weeks, with or without systemic antibiotics, may
be beneficial but such therapies should be reserved for bitches with obvious clinical signs of
infection, such as purulent vaginal discharge.

Mycoplasma and Ureaplasma

 A syndrome of poor conception, early embryonic death, embryonal or fetal resorption,

abortion, stillborn pups, weak newborns, and neonatal death has been suggested to be caused
by mycoplasma and ureaplasma. However, these organisms are present in the vaginal tract of
the normal bitch and hence cannot always be considered as a cause for infertility. If large
numbers of these organisms are identified in pure or nearly pure growth from the vaginal
vault of a breeding bitch with an infertility problem, these microorganisms may be at fault.
Management includes isolation of the animal and tetracycline or chloramphenicol therapy for
10 to 14 days.

Viral Infections

 Viral infections, specifically herpes virus have been isolated in dogs – that had abortions and
stillbirths. However, viral infections as a cause of infertility are not well documented.

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CHRONIC ENDOMETRITIS - CYSTIC ENDOMETRIAL HYPERLASIA

 The bitch with chronic endometrial disease is likely to be infertile. These dogs could
experience normal ovarian cycles, ovulate, and have fertilized eggs, but fail to support
pregnancy because of the abnormal uterine environment that prevents implantation or that
would result in fetal resorption.
 Chronic endometritis or CEH can be extremely difficult to confirm. The diagnosis is
suspected if the non-pregnant uterus is thickened or abnormally large in anestrus or diestrus.
Although a thickened uterine wall is a potentially palpable abnormality, it is difficult to be
certain that one is palpating the uterus.
 Visualizing the non-pregnant uterus using abdominal ultrasonography is a potential method
for documenting the presence of a thickened endometrium or of intraluminal fluid. Uterine
biopsy is the only method of confirming a diagnosis, a procedure usually requiring
laparotomy.

EARLY FETAL RESORPTION

 Early fetal resorption usually appears to both owner and veterinarian as primary infertility
because early pregnancy is so difficult to confirm. Pregnancy cannot be recognized by
palpation until after 21 days of gestation, and then the diagnosis is subjective.
 Radiographically, pregnancy cannot be confirmed until 42 to 45 days of gestation.
 The earliest that pregnancy can be identified is approximately 16 days after first breeding,
using ultrasonography. This tool has been helpful in recognizing early fetal resorption.
 Early fetal resorption suggests an endometrial disorder failure of corpora lutea to support
pregnancy infectious disease such as brucellosis, fetal defects or some less common disorder.

HYPOELUTEOIDSM

 Plasma progesterone concentrations begin to rise prior to the onset of standing heat and
decline to basal levels immediately prior to parturition. The first 6 to 7 weeks of diestrus are
usually associated with progesterone concentrations of 50 ng/ml. Any bitch diagnosed as
having an infertility problem should be evaluated with a plasma progesterone concentration
10 to 20 days after termination of standing heat and then once or twice weekly thereafter.
These studies should be completed in conjunction with evaluation by abdominal
ultrasonography.
 If the progesterone concentration is below 1.0 ng/ml, either the bitch never ovulated or the
corpora lutea have failed to synthesize and /or secrete progesterone. Serum progesterone
concentrations above 2-4 ng/ml should be sufficient to maintain pregnancy. If the
progesterone concentration is less than 2 ng/ml, the amount of progesterone secreted may be
insufficient to maintain pregnancy and abortion or fetal resorption may result. If fetuses are
observed on abdominal ultrasonography early in gestation, abortion or fetal resorption should
become demonstrable with repeated ultrasound examinations. Progesterone therapy can be
given but should be recommended only with great caution.

OCCLUSIN OF UTERUS

 Bilateral segmental aplasia or other causes of obstruction of the uterine horns, or occlusion of
both oviducts, could result in a bitch that cycles, ovulates, and breeds normally, but fails to
conceive. Bilateral occlusion prevents the sperm from ever reaching the egg.
 Diagnosis can be made by
o Hysterosalpingography which involves passing radio opaque dye from the vagina
through the cervix and uterus into both oviducts. It is an excellent theoretic tool but is
difficult to employ on a practical basis.
o An alternative to the radiographic study is direct visualization. Laparoscopy is not a
good tool because the oviducts are not visible with a laparoscope. Laparotomy is the

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 only realistic remaining tool. Surgery also allows uterine biopsies and cultures to be
obtained.
 Little can be done if a bitch is diagnosed as having bilateral uterine or oviductal occlusion.
Unilateral occlusion does not result in infertility. Opening an occluded uterine horn or oviduct
has not been described, and such bitches are permanently infertile.

MISCELLANEOUS

 Among the recognized causes of infertility in species other than the dog, when the female has
normal cycles and the male is fertile,are
o antisperm antibodies produced by the female or spermicidal substances within
secretions of the cervix.
o antiegg zona pellucida antibodies have been developed in bitches through
immunization procedures. Such antibodies do result in infertility.

BITCH WITH SHORTENED INTERESTRUS INTERVAL

Idiopathic shortened Ovarian Cycles

 Bitches normally have an interestrus interval of 5 to 11 months. The German Shepherd dog
and the Rottweiler are breeds that often have fertile cycles every 4.5 months.
 Apparent infertility occurs when a bitch enters proestrus prior to completion of the uterine
repair. Infertility could be the result of implantation failure caused by an abnormal
endometrium that has not recovered from the previous effects of progesterone.
 Prior to making a diagnosis or instituting therapy, a complete history of the bitch must be
obtained and studied. The bitch that cycles at less than 4 month intervals is typically normal
in all respects and is infertile only as a result of incomplete uterine involution.
 Young bitches often have irregular, frequent, or silent ovarian cycles. By the age of 2 to 3
years, ovarian cycles should be regular. Thus, it is recommended not to treat any bitch for
frequent cycles until she is at least 2.5 to 3 years of age
 Treatment for the bitch older than 3 years of age that cycles too frequently is to medically
induce a normal anestrus period. This can usually be accomplished by treating the bitch with
mibolerone drops for a period of 6 months. Medication is started 6 to 8 weeks after the end of
the previous standing heat. One must ascertain that the bitch is not pregnant prior to
beginning mibolerone therapy because this potent synthetic androgen causes urogenital
defects in female fetuses and the bitch also may undergo some virilization, but these signs are
reversible and the drug is not thought to alter future reproductive performance. The bitch
should be bred during the first estrus that follows therapy. This estrus can begin immediately
or as long as 6 to 9 months after discontinuation of therapy.

Follicular Cysts

 Ovarian follicular cysts have been implicated as a cause for shortened interestrus intervals in
the bitch. Follicular cysts are well recognized in association with prolongation of proestrus
and/or estrus. Abdominal ultrasonography is the only practical means of diagnosing an
ovarian cyst. Treatment includes surgical removal of the cyst or the cyst and ovary.

Uterine Disease

 Uterine disease has been suggested as a cause for shortened inter estrous intervals and a
diagnosis of this condition requires histologic evaluation of uterine tissue obtained by uterine
biopsy.

Split Heats
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  Split heats are observed in young pubertal bitches but can occur at any time in life. In split
heat, follicles develop, produce estrogen and the bitch exhibits all signs of proestrus.
However, ovulations do not occur. Four to 10 weeks later, the bitch once again enters into
estrus. The second half of split heat is always an ovulatory heat. Diagnosis is by vaginal
cytology and serum progesterone estimations.

Ovulation Failure

 Failure to ovulate may result in failure to form corpora lutea and failure to synthesize
progesterone. The entire diestrus phase of the ovarian cycle is skipped, and, therefore, the
phase of uterine involution is also brief. Diagnosis is based on serial serum progesterone
determinations. It is not known how this diagnosis differs from that of split heats. In the bitch
less than 3 years of age, no treatment is recommended. In the bitch older than 3 years of age,
an attempt to stimulate ovulation can be undertaken with luteinizing hormone or human
chorionic gonadotrophin administered the day before or the day after first breeding.

BITCH WITH SHORTENED INTERESTRUS INTERVAL

Idiopathic Prolongation of the Interestrous Interval

 A thorough history should be obtained to make sure that, outside of the reproductive tract, the
bitch is healthy. Interestrous intervals increase as the bitch becomes older. An interval of 10
to 13 months for a bitch older than 6 to 8 years of age is not worrisome. However, such
prolonged intervals are not typical of the 2 to 6 year-old bitch.
 It will help to know how closely an owner watches his dog and detects proestrus and estrus.
Sometimes, a heat may simply be missed by the owner if no males are present and the owner
does not specifically examine the vulva once or twice each week. Further evaluations involve
o Physical Examination
o Breed
o In-House Examination
o General Approach
o Hypothyroidism

Physical Examination

 Thorough physical examination is should follow only after obtaining a thorough history. A
physical examination need not be too elaborate, but neither should it be omitted. The finding
of a serious heart murmur, unsuspected organomegaly, a mass that was not expected, or a
variety of other problems must be investigated in order to better treat the patient and to
understand any potential cause for delay in an ovarian cycle.

Breed

 Certain breeds like Basenji and the wolf-hybrid cycle on a yearly basis. The bitch that cycles
less often than every 10 months and appears infertile is of greatest concern.

In-Hospital Evaluation

 Increase in length between ovarian cycles in the bitch can occur secondary to
o An underlying illness
o Any major medical disorder has the potential for delaying the onset of an ovarian
cycle.

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  Hypothyroidism is a disorder most often associated with long interestrous
periods is hypothyroidism.
 Ovarian Cysts or Neoplasm
 Silent Heat

BITCH WITH PRIMARY OR SECONDARY ANESTRUS

 Primary anestrus is referred to a condition of a bitch that has never had an ovarian cycle.
Secondary anestrus is that of a bitch that has had one or more ovarian cycles but subsequently
fails to cycle.
 Current history such as the dog’s age, breed, past history, and physical examination should be
assessed before any major tests are undertaken. Failure to cycle, is a problem that is usually
not pursued until the bitch is older than 2 to 3 years of age. Evaluation of dogs with secondary
anestrus should include all suggested approaches in the previous section on prolonged
interestrous intervals.
 Secondary anestrus can occur after the onset of thyroid, other endocrine or nonendocrine
disease. These bitches should be thoroughly evaluated with history, physical examination, and
laboratory testing and if all testing is normal, it is wise to wait at least 16 to 20 months from
the previous cycle, in case one or two heat cycles were silent and, therefore, missed. The dog
should be closely monitored during this time by the owner, and the veterinarian can
recommend serial testing.

Previous Ovariohysterectomy

 If the past history of a bitch is not known, one cause for failure to cycle is previous
ovariohysterectomy. Examination of the ventral midline for an incision scar provides initial
evidence for an earlier spay. One may need to clip hair away from this area to be certain. The
condition can be confirmed by plasma estimations for LH and FSH determinations. The
ovario-hysterectomized female has persistent elevation in LH and FSH concentrations.

Silent Heat

 Silent heat can be difficult to detect. Bitches in this condition may not have vulvar
enlargement or a sanguineous vaginal discharge, or may not attract or allow breeding by
males. Silent heats should be considered a possible cause for primary anestrus, especially if
the owners of a bitch have little or no experience with an intact female, if the bitch is housed
separately from any contact with a male dog, or if the bitch is not closely observed.
 Diagnosis of silent heat can be done by
o Trying to bring the bitch into contact with a male once weekly to help recognize
estrus.
o Close visual examination of the vulva once or twice weekly as it is an excellent
method for detecting silent heat.
o Close observation which allows the owner to develop some experience with the
anestrus appearance of the vulva. Mild enlargement of the vulva or a slight bloody
discharge is easier to see, and the owner is more comfortable identifying signs of
early proestrus.
o Adopting more aggressive methods of evaluating bitches suspected of having silent
heats that include weekly reading of vaginal cytology smears or monthly serum
progesterone assessments.

Drug-Induced Anestrus

 Anestrus may be induced by drugs specifically marketed for that purpose and by drugs that
result in anestrus as a side effect. Marketed drugs include androgens, which might be used by

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 an owner interested in increasing the strength and/or endurance of his or her pet, without
realizing effects on the hypothalamic pituitary-ovarian axis. Progestagens are used in the
treatment of a variety of maladies, with prolongation of anestrus as a side effect.
Glucocorticoids can have negative feedback effects on the pituitary, suppressing
gonadotrophin activity and preventing ovarian cycles.

Underlying Disease

 Any illness, mild as well as severe, can interfere with ovarian cycle activity in the bitch.
Obtaining a thorough history as well as performing a complete and competent physical
examination is important. Abnormalities identified in these areas must be pursued as potential
explanations for the infertility problem and to avoid the mistake of separating the
reproductive tract from the rest of the animal.
 When silent heats, previous ovariohysterectomy, and owner error are considered unlikely
explanations for apparent failure to cycles, blood and urine testing is advisable. It is
recommended that a CBC, serum chemistry profile, urinalysis, and serum thyroid
concentration be obtained and reviewed. Another integral component of screening a bitch for
unsuspected problems is abdominal ultrasonography. This is a noninvasive means of
evaluating abdominal structures, including the uterus for thickening and/or fluid and the
ovaries for masses or cysts.

Hypothyroidism

 Hypothyroidism has become a “popular” diagnosis in explaining the cause for a bitch failing
to cycle. Although the potential exists for a hypothyroid bitch to exhibit primary or secondary
anestrus, these dogs should have signs of hypothyroidism. The alert, active, vibrant bitch is
rarely hypothyroid. As previously stated, hypothyroidism is over diagnosed by the profession
and by breeders / trainers.
 Hypothyroid dogs rapidly respond to thyroid replacement by becoming more alert, active, and
responsive within days of initiating replacement therapy. Their appetite quickly improves and
weight loss follows. Improvement in hair coat may take weeks. These dogs typically begin
ovarian cycles within 3 to 6 months of initiating therapy. If these responses are not observed,
the thyroid hormone replacement dose is inadequate, a second medical problem exists, or the
diagnosis is not correct.

Glucocorticoid Excess

 Glucocorticoids are used in the treatment of numerous small animal problems.

Glucocorticoids have negative feedback effects on pituitary adrenocorticotropin secreting and
similar effects on suppressing secretion of both FSH and LH. A bitch receiving glucocorticoid
therapy may not exhibit ovarian cycles unless the steroid dosages are kept to a minimum or
administration is discontinued. Naturally occurring hyperadrenocorticism is not usually a
major consideration in the noncycling female because most bitches with Cushing’s syndrome
are older than 8 years of age. Therefore, their failure to cycle is not recognized as a problem
by the owners, whereas the other major signs of Cushing’s syndrome are more obvious, and
worrisome.

Premature Ovarian Failure/Ovarian Aplasia

 A bitch that has never exhibited ovarian cycles may not have ovaries. Other causes of the
aplasia may exist. Secondary hypoplasia simply suggests that one or more ovarian cycles
preceded premature ovarian failure.
 The diagnosis of ovarian failure may be suspected when all other differential diagnoses are
excluded and attempts to induce estrus fail. One method of confirming such a diagnosis is

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 random evaluation of plasma FSH and LH concentrations. Premature ovarian failure is
associated with extremely increased plasma FSH and LH concentrations due to absence of
negative feedback to the pituitary and hypothalamus Alternatively, exploratory surgery can be
undertaken to inspect the reproductive tract and to biopsy the uterus and ovaries for
confirmation of abnormalities.

Progesterone-Secreting Ovarian Cyst

 This is a well –recognized but uncommon syndrome resulting in prolongation of diestrus. To

the owner, the condition usually appears to be a prolongation of anestrus. By definition such a
bitch must have had an ovarian cycle and would be classified as having secondary anestrus.
However, if this follows a silent heat, the prolonged anestrus may appear to be primary. In
either situation, abdominal ultrasonography, together with assessment of the plasma
progesterone concentration, is diagnostic in most bitches. Treatment with prostaglandins to
lyse these cysts is not usually successful, and their surgical removal is recommended.
 Induction of estrus in anestrus dogs have been dealt with in detail in the module on Induction
of estrus.

ANESTRUS ALOGRITHM

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PERSISTANT PROESTRUS AND/ ESTRUS

 Persistent estrus is defined as a bitch willing to breed for longer than 21 to 28 consecutive
days in any one ovarian cycle. Alternatively, and less directly, persistent estrus is defined as
more than 21 to 28 consecutive days of greater than 80 to 90% superficial cells observed on
vaginal cytology. Persistent presence of a large percentage of superficial ells on vaginal
cytology is strong evidence for continued increases in serum estrogen concentration.
 Bitches with persistent proestrus have an enlarged vulva and persistent vaginal bleeding,
attract males, and demonstrate estrogen effects on the reproductive tract. Although the causes
for persistent proestrus/estrus may vary among bitches, the final common denominator is
continued exposure to increases in serum estrogen concentration.

Exogenous estrogen excess

 The use of parenteral estrogen to prevent pregnancy and in some bitches to prevent urinary
incontinence may lead to this condition. If estrogens were administered, the type and dosage
should be ascertained. In addition to associations between exogenous estrogen and
development of pyometra or bone marrow aplasia, ovarian cysts are recognized sequel to such
medication.

Endogenous estrogen excess

 Rarely does the young bitch in her first or second ovarian cycle fails to ovulate and may
exhibit prolonged proestrus or estrus activity due to continued follicular estrogen secretion.
This would probably due to inadequate amounts of estrogen to induce the LH surge or a
failure in LH to induce ovulation leading to development of follicular cysts.

DISORDER OF SEXUAL DEVELOPMENT

Normal Sexual Development

 The sex chromosome constitution of the sperm determines the sex of mammals at
fertilization. The embryo develops as a male if the fertilizing sperm has a Y chromosome. If
the sperm contains an X chromosome, the embryo develops as a female. The genital system
of early developing embryos is neither male nor female. Eventually, the embryo without a Y
chromosome develops an ovary from the undifferentiated gonad, and the Mullerian system
persists as the fallopian tubes, uterus, and cranial vagina. The urogenital sinus and external
genitalia develop in a female pattern.
 In the presence of a Y chromosome the indifferent gonad develops into a testis, which
produces both testosterone and Mullerian inhibiting substance (MIS), secreted by sertoli cells,
causes regression of the Mullerian duct system. Testosterone secreted by Leydig cells
stimulates formation of the epididymis and vas deferens from the Wolffian duct system as
well as the male urethra, penis., prostate, and scrotum.
 Normal sexual development occurs in three steps
o Step 1: Establishment of chromosomal sex
o Step 2: Development of gonadal sex
o Step 3: Development of phenotypic sex.
 An error in any one of these steps can result in a disorder of sexual development which may
be occult or obvious to the owner and/or veterinarian. Normal dogs have a total chromosome
number of 78. These 78 chromosomes include 38 pairs of non-sex chromosomes and 2 sex
chromosomes. The sex chromosome constitution of females is XX, whereas that of males is
XY.

Abnormal Sexual Development

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  Dogs with obvious abnormalities in their external genitalia may be quickly recognized, but
abnormalities may occur at any step in development. Intersex conditions include congenital
malformations of the genital system such that the gender of the individual is ambiguous.
“Interesx” is a generic term including numerous disorders. True hermaphrodites are
individuals with both testicular and ovarian tissue, either combined in one gonad or existing
as separate gonads. Pseudo-hermaphrodites have the gonads of one gender but have
reproductive organs having characteristics of the opposite gender. Male
pseudohermaphrodites have testes but have some female features, such as the presence of a
uterus or external genitalia that are primarily female. Female pseudohermaphrodites have
ovaries but are masculinized to some degree.
 Gonadal gender is best determined by histology of the gonads. Phenotypic sex can be
established following review of a thorough description of both internal and external genitalia.
It is necessary to determine (1) whether the vulva or prepuce is appropriate in form and
position; (2) whether a clitoris or penis is present; (3) what is the location of the urethral
opening; and (4) whether the dog has a prostate or caudal vagina.

Abnormalities of Chromosomal Sex

 Dogs and cats with abnormalities of chromosomal sex are normal-appearing males or females
that have underdeveloped rather than ambiguous genitalia.
 Animals that are chimeras or mosaics may be exceptions to this rule. Most animals with
chromosomal sex abnormalities are sterile, with no treatment advised. Abnormalities of
chromosal sex include
o The XXY Syndrome
o The XO Syndrome
o The XXX Syndrome
o Chimeras and Mosaics
o True Hermaphrodites

Abnormalities of Gonadal Sex

 Abnormalities of Gonadal Sex include

o XX Sex Reversal
o XX True Hermaphrodites
o XX Male Syndrome

Abnormalities of Phenotypic Sex

 Abnormalities of Phenotypic Sex include

o Pseudohermaphrodites-Female and male pseudohermaphrodites have some degree of
sexual ambiguity in the genitalia. Their chromosomal sex and gonadal sex agree, but
their external appearance is reversed; that is, they are phenotypically abnormal.
 Female Pseudohermaphrodites
 Male Pseudohermaphrodites

Hypospadias

 This condition was defined in the previous section the XX male. Hypospadias may occur
from a variety of causes and the result is an incomplete fusion of the urethral folds leading to
the formation of the male urethra.
 The Boston Terrier may have a familial predisposition to hypospadias. Cryptorchidism is the
most common defect associated with hypospadias.

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FAILURE TO PERMIT BREEDING

Mismanagement

 The most common cause for bitches to refuse attempts at mounting by a male is an owner
choosing incorrect breeding dates. Another potential cause of failing to permit breeding,
however, is vaginal defects.

Behavior

 Bitches may be managed properly but still consistently refuse to breed with a particular male.
Mate preference appears to be one potential cause for this problem. Therefore, if no other
cause is evident the owner should attempt to breed the bitch to another, more dominant male
before investigating unusual problems.

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QUEEEN

 Infertility in the queen is a nonspecific, historical complaint that relies on historical

reproductive information for consideration of appropriate causes and appropriate diagnostic
plans. Infertile queens can be assigned to one of the four groups
o Failure to cycle.
o Those with prolonged interestrus intervals.
o Those with prolonged sexual receptivity.
o Cycling queens that refuse copulation with the male.

FAILURE TO CYCLE

Previous Ovariohysterectomy

 If a queen displays no estrus activity whatsoever, one should check to see if she has
previously undergone an OVH. Usually, checking for a “spay” incision is all that is needed

General Health

The cat’s general health must be thoroughly evaluated. This usually involves a good history, complete
physical examination, and a routine blood and urine data base. Estrous cycles can be interrupted or
can cease in an animal under the stress of

 a poor diet
 compromising illness
 overcrowding
 exposure to extremes in temperature
 inadequate exposure to light
 the stress of a show circuit
 traveling
 drug therapy, especially progestagens and glucocorticoids
 variety of ovarian and uterine neoplasias

Silent Heat

 Perhaps the best example of silent heat is the cat housed with a number of other cats. If a cat
is low on the “pecking order” or if overcrowding exists, its cycles may be completely
undetectable, that is silent, to humans and apparently to other cats.
 Diagnostic methods used to diagnose this condition is to teach an owner how to obtain
vaginal smears from the cat.
o Follicular phases are reflected as an increase in the percentage of superficial cells
present.
o Alternatively, once-or twice-weekly plasma samples can be assayed for estrogen
concentration.
 If either study suggests normal follicular function, the cat should be completely isolated and
maintained on 14 hours of light and 10 hours of darkness. Usually, her estrus activity is more
apparent on this regimen and removal from the other cats. One could also attempt to induce
estrus medically.

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Premature Ovarian Failure

 The functional longevity of the ovaries in queens is not known, although many queens do not
continue estrous cycle activity beyond 11 to 13 years of age.
 Queens beyond 8 years of age are not usually used in breeding programs. The ovaries,
abnormally, may cease functioning earlier. This results in a permanent condition interpreted
as prolonged anestrus by the owner.
 Premature ovarian failure is suspected when all other differentials are excluded from the list
of potential diagnoses and induction procedures fail. One could assay LH and FSH
concentrations in the plasma to confirm diagnosis. Persistent elevation of these hormones is
consistent with nonfunctioning ovaries.

Disorders of Sexual Development

 Phenotypically normal females may not have functional ovaries secondary to chromosomal
abnormalities. Several such cats have been reported.
 Karyotyping can be performed to recognize these disorders. Exploratory surgery can be
performed to examine the reproductive tract and to do biopsy or remove any abnormal tissue.
One can also attempt to induce estrus medically to rule out any likelihood of a disorder in
sexual development.

Induction of Estrus

 Once the veterinarian is certain that no anatomic defect, organic illness, or medication would
explain failure to cycle, an attempt can be made to induce a cycle medically. This procedure
can be used in queens that have never cycled, queens with prolonged acquired anestrus, or
queens with highly irregular cycles. Because the queen is an induced ovulator, one must rely
on the male to induce ovulation once estrus is induced, or an attempt can be made to induce
ovulation medically.

PROLONGED INTERESTUS INTERVAL

Pseudopregnancy

 A queen that enters estrus every 30 to 60 days may be ovulating and experiencing repeated
pseudopregnancies. This has been observed in queens that have never been bred. Ovulation in
some queens can be induced by petting, obtaining vaginal cytology smears, or less obvious
factors. These queens are typically healthy and are fertile if breed. The diagnosis of
pseudopregnancy can be confirmed by demonstrating an elevation in the plasma progesterone
concentration 1 to 3 weeks after estrus.
 Queens must receive adequate food, housing, light, and general care if they are to cycle
normally. Owner observation is also valuable to be certain that some cycles are not missed. A
general health examination and laboratory evaluation are also worthwhile because an
underlying illness may interrupt cyclic ovarian activity.

Cystic Follicles

 Cystic follicles could result in either prolonged estrus behavior or a prolonged interestrous
interval. Although difficult to diagnose, functioning follicles create persistent estrus via
production of estrogen, which can be assayed. The most reliable and easiest method of
diagnosing cystic follicles is abdominal ultrasonography. Cystic structures are usually easily
identified and remain relatively static in size and shape. If the plasma estrogen concentration
is persistently increased the diagnosis is further supported.

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PROLONGED SEXUAL RECEPTIVITY – NYMPHOMANIA

 Normal queens may exhibit prolonged sexual receptivity despite having normal waves of
follicular function. In other words, their estrus behavior overlaps interestrous intervals and
persistent estrus results. In most cats this is considered a normal phenomenon not requiring
treatment.The ideal therapy, if any, is to induce ovulation via breeding to a normal or
vasectomized tomcat. Artificial vaginal stimulation could also be used to induce ovulation
and cause an end to persistent behavioral estrus.
 Follicular cysts often produce signs of persistent estrus. A persistent follicle becomes a
persistent source of estrogen and any queen under a constant influence of estrogen displays
continuous estrus behavior. The diagnosis is made by demonstrating increased plasma
estrogen concentrations for more than 3 weeks without evidence of normal cyclicity in a
queen with a cyst associated with one ovary on abdominal ultrasonography.
 Persistent estrus in cats older than 5 years is consistent with the presence of granulose cell
tumors. This is the most common ovarian neoplasm in cats. Such tumors are more likely to be
malignant in cats than in other species.
 Treatment could consist of attempts at breeding to ovulate the cyst. One could attempt to
induce rupture of the follicle(s) by administering 250IU of hCG IM once daily for 2 days. The
recommended treatment is surgical removal of the cyst, with or without the associated ovary.
Usually it is difficult to remove the cyst without the ovary. These cats remain fertile with one
ovary.

FAILURE TO PERMIT BREEDING

 A similar group of differential diagnoses exist in the queen, including management problems,
behavior disorders, vaginal or vulvar defects, and miscellancous obstructions.
 Vulvar and vaginal and vaginal atresias have been diagnosed in our practices. Vaginal
strictures are rare but must be considered in a queen that fails to permit breeding.

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 ANESTRUS

 Anestrus meaning “without cyclicity” is a condition when the female does not exhibit regular
estrous cycles due to insufficient GnRH release from the hypothalamus to stimulate and
maintain gonadotrophin secretion. The ovaries are relatively inactive and neither ovulatory
follicles nor corpus luteum are present.
 Anestrus is observed more commonly either after parturition as postpartum or pre service
anestrus and following service as post service anestrus when conception does not occur.
 There are two categories:
o Class I or False anestrus - with functional CL.
o Class II or True anestrus - with no functional CL.

FALSE ANESTRUM

 False Anestrus may be classified as

o Anestrus due to pregnancy.
o Anestrus due to persistent corpus luteum (CL) – Conditions associated with uterine
pathology such as pyometra, mummified fetus, fetal maceration, other disease states,
mucometra and hydrometra.
o Anestrus associated with CL of pregnancy that terminated early and not recognized.
o Subestrus, weak or silent estrus and unobserved estrus.
 Normal cyclical changes in the genital organs but the signs of heat are not
exhibited or not observed.Most common especially in buffalo cows. Common
during post partum period.
o Anestrus following fetal maceration wherein most of the fetal parts appear to have
been expelled.

Note: Persistent CL does not occur in the presence of a normal non-pregnant uterus. Many
veterinarians tend to call wrongly a cyclic CL as persistent CL.

CAUSES

 Physiological basis is not known, but it may be due to a lack of estrogen and a potentiating
action of progesterone and is seen associated with
o Advanced age
o Arthritis
o Poor nutrition
o Seasonal stress
o Suckling
 Unobserved estrum may be due to managerial deficiencies and short period of estrus.

TREATMENT

In unobserved estrus

 Improving the managerial practice.

 Increased regular observation thrice a day
Provision of adequate lighting to improve estrus detection.
 The use of estrus detection aids.
 Use of teaser bulls
 Careful and frequent examination of cows, prediction and confirmation of estrus and breeding
 Specific treatment using prostaglandin or progesterone therapy and fixed time insemination -
Highly effective.

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TRUE ANESTRUS

 Small inactive ovaries - no functional CL.

 May be due to an insufficient release of
gonadotropins or failure of ovaries to
respond.

PREPUBERTAL ANESTRUS

 Pubertal estrus represents the initiation of the reproductive cycle, and this first estrus
generally occurs by a certain age relative to the animal’s weight. Heifers must attain
approximately two thirds of their adult size before they will reach puberty. With good
nutritional management, most Bos Taurus heifers attain their pubertal weight between 8 and
13 months of age. Failure of estrus expression past this time is prepubertal anestrus.
 Clinically, heifers generally fall into one of two categories:
o The acyclic heifer of the same age as the rest of a cycling group
 Related to an abnormal reproductive tract. Freemartins, hermaphrodites and
aplasia are readily diagnosed by palpation
 Cyclicity of herd mates indicates that the problem does not affect the entire
group. Debilitating disease such as chronic pneumonia can delay puberty by
decreasing rate of gain, and this appears to be a functional dietary problem
o Several acyclic heifers in a group of the same age or a group of mixed ages.
 Management practices play a vital role in the second category, which
comprises acyclic heifers of similar or diverse ages. Since the onset of
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 puberty is influenced by the level of available nutrition, heifers of similar
ages that are fed a suboptimal energy diet will show a prolonged prepubertal
anestrus period.
 Similarly, groups of heifers of diverse ages that are housed together and
given a balanced ration may contain several acyclic animals. Larger or more
aggressive herd mates consume a greater portion of the available nutrition
and tend to cycle first. Puberty, however, is not postponed indefinitely, and
eventually the entire population cycle. There is an inherent danger in
breeding these late heifers before they have developed adequate body size as
they tend to have more dystocia problems and are prone to very long
postpartum anestrous periods.
o Certain infectious diseases can also produce anestrus in heifers. Blue tongue and
bovine diarrhea virus are capable of causing an acute ovaritis, which leads to varying
degrees of ovarian atrophy. Animals with complete atrophy are anestrus unless
stimulated with exogenous hormones. They promptly return to the anestrus state
when hormone therapy is withdrawn.
o Growth –stimulating implants must be used with caution in prepubertal heifers that
will be used as breeding animals. Synovex-H and Zeranol (Ralgro) can delay pubertal
estrus and may affect future fertility.

POSTPARTUM ANESTRUS

 A period of anestrus following parturition is a normal physiological event, and ovarian

cyclicity resumes as the uterus involutes. The anestrous period becomes abnormal when its
duration extends past the accepted average. The duration of the average anestrus interval is
influenced by age, breed, environmental factors and genetic background.
 The normal postpartum cow undergoes surges of Follicle Stimulating Hormone (FSH) which
promote follicular growth that may be detected as early as 9 to 15 days postpartum in the
dairy cow.
 The estradiol surges interact with the neuroendocrine centers, which results in increased
sensitivity to gonadotropin-releasing hormone (GnRH).
 Concurrently, the plasma luteinizing hormone (LH) level rise, and the number and magnitude
of episodic LH peaks tend to increase during the first 2 weeks postpartum. This correlates
with the fact that the quantity of LH at the pituitary level increases after parturition and that
GnRH sensitivity returns at about 8 to 10 days postpartum.
 At approximately 2 to 3 weeks postpartum the LH levels are able to induce ovulation of one
of the ovarian follicles.
 The corpus luteum of this first ovulation has a lower progesterone content and may not be as
responsive to LH, which results in a shortest lifespan. Also, due to the absence of
progesterone prior to this ovulation the estrus in generally silent. Normal behavioral estrus
generally develops at successive heats.
 Factors that modify this course of events can be divided into three categories: lactational
effects, nutritional effects and organic disease. Anestrus may be due to a combination of the
above factors.
 A clinical examination of a true anestrus animal typically reveals ovaries that range from
small and firm with no palpable structures to those that have multiple medium size follicles (5
to 10 mm). Although most cases of anestrus will eventually resolve themselves, this leads to a
prolonged calving interval, which is economically unsound.
 Lactation has been shown to cause an extension of the anestrus period and that suckling
decreased the average plasma LH concentrations and lengthened the interval to the
development of episodic ovulation and blocked the return to cyclicity.
 Extension of the normal anestrus period can be caused by a deficient diet, particularly one
low in energy. The low producers would still cycle first; however, the length of anestrus
would become protracted.
 Organic dysfunction in the postpartum cow that causes anestrus may be related to a primary
uterine disease of a secondary systematic problem.

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  Placental retention, metritis, chronic debilitating diseases, such as leg injuries, displaced
abomasums are associated with the anestrus state. The etiologic agent may be chronic stress,
or it may be related to the reduction in nutrient intake due to a decrease in the animal’s desire
and ability to eat properly. This is further supported by a concurrent decrease in milk
production, indicating the inability to support lactation.

POST SERVICE

 Post service anestrus is a normal event following insemination if the animal has conceived.
Approximately 5 per cent of the pregnant cows or heifers may exhibit behavioural signs of
estrus early in the gestation period.
 Following breeding, the animals are closely observed for estrus activity, which should occur
18 to 23 days after breeding if they failed to conceive or to maintain the embryo past day 12
aft5er ovulation. If the animal remains anestrus she is presented for a pregnancy examination
at 35 to 40 days after breeding. If the cow or heifer is nongravid at this time, the next estrus is
expected in a few days, and reinsemination is advised.
 Cases of anestrus other than pregnancy most frequently are due to estrus detection failure,
cystic follicular degeneration, pyometra, early embryonic death and uterus unicornis, rarely
granulos cell tumors, or leimyomas.
 True cases of postservice anestrus, in which the ovaries are nonfunctional or only have
multiple small follicles, are uncommon and reflect a severe nutiritional deficienty and/or
systemic disease. These animals tend to remain in the anestrus state until the underlying
illness is resolved.

DIAGNOSIS

Based on Rectal Examination

 The ovaries appear small and smooth. In buffaloes the ovaries appear spindle like.
 Should be confirmed by repeated examinations at 10 days interval.
 Ultrasound examinations at regular intervals can be done to diagnose and confirm anestrus.
 Progesterone estimations at intervals of 10 days would aid in confirmation. Low progesterone
levels at both times indicate true anestrus.

CLINICAL TREATMENT

 Therapy for the induction of cyclicity in the anestrus animal has been attempted with a variety
of exogenous hormones and management practices. For economic consideration it is
important to have heifers calve at 2 years of age and cows calve every 12 months.
 Many hormone treatments have been utilized to hasten the onset of puberty or to decrease the
interval from calving to conception. Unfortunately, due to the number of variables, including
age, weight, diet and management hormones do not always give consistent results.
 The overriding considerations for correcting an anestrus problem are that the animal be
healthy, have palpable follicular development and have access to good feed.
 Induction of a pubertal estrus in heifers depends largely on the weight of the animal after she
reaches 13 months of age. Optimum results will be obtained if the heifer is near the average
weight at puberty for her breed. If a heifer is very light she may not continue to cycle after an
attempt at puberty induction.
o The treatment Syncro-Mate B, which combines a 6 mg Norgestomet ear implant with
an injection of 3 mg Norgestomet plus 5 mg estradiol valerate, has given the best
results. The implant is removed 9 days later, and estrus ensues in 50 to 94 per cent of
the animals within 120 hours. Pregnancy rates following insemination during this
induced estrus have been reported to be as high as 50 per cent. The mechanism
behind this therapy attempts to mimic the short luteal phase associated with the

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 “silent” pubertal estrus. The estradiol valerate causes luteal regression if a functional
corpus luteum is present.
o The injectable Norgestomet prevents luteinization of additional follicles by the
progesterone negative feedback mechanism. The 9-day period of implantation is
important in promoting estrus expression by causing progestogen priming necessary
for a psychic estrus. Also the implant helps decrease the incidence of induced corpora
lutea having a reduced lifespan by promoting a normal LH release pattern following
implant removal.
o The use of hormonal therapy in the anestrus suckling beef or lactating dairy cow are
usually done 45 to 90 days postpartum and as with heifers, success depends on their
nutritional status and body condition. Hormone therapy is the same as that described
for acyclic heifers; however, best results are obtained by removing the nursing calves
for 48 hours at the time of implant removal (Shang treatment). Estrus occurs 24 to 48
hours later, and first service conception rates have been reported to range from 40 to
70 per cent under ideal conditions. As with heifers, the progestogen implants decrease
the incidence of short luteal lifespan following the induced estrus, although the
implants do not completely eliminate this phenomenon.
o Weaning without prior hormone therapy results in a higher expression of estrus when
compared with nonweaned cows.
 In general, the protocol involved involves the following steps:
o Improve Nutrition
 Extra feeding of a concentrate mixture or grains like maize, cholam, kambu,
etc., and at least small amount of green fodder along with other roughages.
o Supplement Minerals
 Specific patent preparations which contain important minerals.
 Standard mineral mixture.
o Improve Managerial Practice
 Eradication of internal and external parasitism.
 Proper housing.
 Elimination of stressful factors.
o Specific
 GnRH 0.5 mg IM. to induce estrus. May be repeated after 10 days
 GnRH analogue Buserelin 0.02 mg IM.
 PMSG or FSH is not advisable as they can cause superovulation
 Short term progestogens- CIDR, PRID or Ear implant induces heat even
in anestrus animals.
 Progesterone injection followed by hCG or combination of progesterone +
PMSG + estrogen.
 Clomiphene citrate. 300 mg. daily for 5 days after drenching with
CuSO4 solution.

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 REPEAT BREEDING

A repeat breeder cow is defined as one that,

 Has experienced three or more unsuccessful services.

 Have normal estrous cycles with approximately 21 days intervals.
 Is free from palpable abnormalities.
 Shows no abnormal vaginal discharges.
 Has calved at least once before.
 Is less than 10 years old.

However, this definition is quite restrictive and may not fit in all cases. All the major causes can be
grouped into two categories of those causing

 Fertilization Failure
 Early Embryonic Death

FAILURE OF FERTILIZATION

 Fertilization failure may result from death of the egg before sperm entry, structural and
functional abnormality in the egg or sperm, physical barriers in the female genital tract
preventing gamete transport to the site of fertilization, or ovulatory failure.

Causes

Abnormal eggs

 Several types of morphologic and functional abnormalities have been observed in unfertilized
eggs, e.g., giant egg, oval shaped egg, lentil-shaped egg, and ruptured zone pellucida.
 Failure to undergo fertilization and normal embryonic development may be due to inherent
abnormalities of the egg or to environmental factors.
 In sheep, some of the conception failures at the beginning of the breeding season are
associated with a high incidence of abnormal ova.

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Abnormal Sperms

 The physiologic significance of abnormal sperm in relation to fertilization failure has not been
studied in animals other than cattle. Certain forms of male infertility are related to structural
defects of the DNA protein complex. Sperm aging and injury may cause
o Alterations in the acrosomal cap that may prevent defective spermatozoa from
fertilizing the egg. In bull, ram, and boar, a good correlation exists between fertility
and acrosomal integrity.
o Leakage of vital intracellular constituents such as cyclic AMP or the formation of
lipid peroxides from sperm plasmalogen when sperm are stored under anaerobic
conditions.
o A gradual decrease in the fertilizing capacity of aging of spermatozoa in the female
genital tract.

Structural Barriers to Fertilization

 Congenital or acquired defects of the female genital tract interfere with transport of the sperm
and/or the ovum to the site of fertilization
o Congenital defects are the result of arrested development of the different segments of
the Mullerian ducts or of an incomplete fusion of these ducts caudally. A classic
congenital anomaly associated with the gene for white coat color is “white heifer
disease” in cattle, in which the prenatal, development of the Mullerian ducts is
arrested, and the vaginal canal is obstructed by the presence of an abnormally
developed hymen. It can be differentiated from the freemartin syndrome by the
presence of normal ovaries, vulva, and labia.
o Common anatomic abnormalities are adhesions of the infundibulum to the ovary or
uterine horns; this interferes with the pick-up of the egg or causes a mechanical
obstruction of one part of the reproductive duct system. Bilateral or unilateral missing
segments of the reproductive tract also cause anatomic sterility.

Phytoestrogens

 Reproductive failure occurs more in sheep than in cattle grazing on plants that contain
compounds with estrogenic activity, e.g., subterranean clover and red clover.
 The estrogenic activity is due to plant isoflavones and related substance with hydroxyl
groups. Cows and ewes fed estrogenic forage may suffer impaired ovarian function, often
accompanied by reduced conception rates and increased embryonic loss. In cows, clinical
signs resemble those associated with cystic ovaries.
 The infertility is temporary, normally resolving within one month after removal from the
estrogenic feed. Ewes grazed on estrogenic pastures around the time of joining, shed fewer
ova and have a reduced chance of conception.
 Fertility is improved within 3 weeks, after the ewes are moved into nonestrogenic pastures.
The pathologic changes in temporary infertility are due to actions of estrogen on the
hypophyseal – ovarian axis and on sperm transport.
 Ewes grazed for several seasons on estrogenic pastures mate and ovulate, but fertilization rate
is depressed as a result of failure of sperm transport caused by severe changes occurring in the
cervix.

EARLY EMBRYONIC MORTALITY

 Embryonic mortality denotes the death of fertilized ova and embryos up to the end of
implantation. About 25 to 40% of embryos are normally lost in farm species. It is also noted
in large litters of swine and during multiple pregnancies in cattle and sheep.

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  Mortality is more common during the early than the late embryonic period. Early embryonic
mortality should be regarded as a normal process of eliminating unfit genotypes in each
generation, particularly in large litters of swine and multiple pregnancies in cattle and sheep.
 In the past it was believed that the bovine conceptus was resorbed but transrectal ultrasound
examination has demonstrated that that the conceptus and its breakdown products apparently
are eliminated by expulsion through the cervix, which either goes unnoticed or appears as a
vulval discharge of clear mucus.
 Embryonic mortality after natural breeding or artificial insemination accounts for the majority
of reproductive failures in the cattle, with a mortality rate of up to 40% of all fertilized eggs.
In cattle, most embryonic deaths occur between days 8 and 16 during hatching of the
blastocyst and implantation without affecting cycle lengths. Since, most embryos die between
days 9 and 15 infertile ewes may experience normal as well as prolonged cycles.

Cattle

 Most of the embryonic loss in cattle occurs between days 8 and 16 after insemination.
 The timing of insemination is important as insemination too late in the estrous period leads to
ovum ageing and embryonic death. Artificial insemination during pregnancy will induce loss,
either through mechanical trauma to fetal membranes or the introduction of infection.
 Nutritional causes such as B-carotene, selenium, phosphorus and copper deficiencies have all
been implicated in embryonic loss, but unequivocal data are not available. High intakes of
crude protein, in particular rumen – degradable protein have been associated with reduced
fertility. This is said to be due to the toxic effects of blood urea or ammonia on the embryo.
 Stress, e.g. heat stress, has also been shown to result in embryonic loss. A high rate of
increase in milk yield and high milk yield per se in early lactation are negatively correlated
with fertility and this could be considered a metabolic stress.

Horses

 The commonest cause of embryonic loss in mares is twin conceptions as competition for
placental space usually results in one fetus growing more slowly than the other and the
smaller fetus, with a smaller placenta, dies. Death of one fetus often results in the loss of the
second.
 Other intrinsic factors which are thought to be related to embryonic loss in the mare include
oviductal secretions, embryonic vesicle mobility and uterine environment. Since, the mare’s
embryo is at a more advanced stage whilst still in the uterine tube, the environment may be
relatively more important in this species than others.
 In addition the embryonic vesicle remains free in the lumen of the uterine horn much longer
in the mare than in other species and the degree of mobility of this vesicle is thought to be
important in maternal recognition of pregnancy. Greater mobility enhances the suppression of
luteolysis and results in higher levels of progesterone. As regards the uterine environment,
recurrent endometritis and post-service infection lead to perivascular fibrosis, and this is a
common cause of embryonic and fetal death between 40 and 90 days of gestation. Increased
maternal age has also been associated with increased embryonic loss, but this may merely
reflect increased chronic uterine pathology.
 Other factors such as lactation and service at the foal heat also result in higher embryonic
death rates, although the latter may be due to lactational stress. Stress, due to transportation, is
thought to cause embryonic losses in the mare. However, recent studies failed to confirm this
even though transport did result in raised plasma ascorbic acid levels, which have been
associated with prolonged stress. Nutritional stress, in the form of restricted energy intake,
does increase embryonic loss.

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Sheep

 Nutrition, specifically energy level, is known to affect embryonic survival in sheep in a

complex manner. Low body condition at mating is detrimental to embryo survival,
irrespective of post mating nutrition. However, in ewes that lose weight post – service,
embryo mortality is increased. Prolonged, moderate under nutrition has more effect on ewe
lambs than adult ewes.
 Nutritional energy may exert its effect via peripheral blood progesterone between food intake
and progesterone levels. Other nutrients important in embryo survival are vitamin E and
selenium. Certain plants, such as kale and Veratrum californicum, will cause embryonic
death.
 The latter is also a teratogenic agent. The effects of nutrition may be exaggerated or
confounded by differences in ovulation rate since losses have often been reported to be
disproportionately high in twin ovulations. In breeds with very high ovulation rates the
embryonic death rate rises proportionately, but this is probably due to limitations of uterine
space.
 High environmental temperature, particularly in the first week after mating, has been shown
experimentally to increase the embryonic death rate dramatically. This could be important in
climatic heat waves. However, if there is diurnal variation, as would occur naturally, the loss
is much lower.
 Physiological stress, such as that produced by overcrowding or handling of sheep, also
increases embryonic loss. This may be due to excess secretion of progesterone by the adrenals
and/or raised corticosteroid levels. The age of the ewe is also important, since there is some
evidence that ewe lambs have a higher incidence of embryonic loss than mature ewes.

Goats

 Goats are particularly susceptible to non-infectious fetal loss, and this is particularly true of
the Angora breed. Losses are also common in poorly fed animals of any breed.
 Another reported cause of fetal loss is dosing with anthelmintics such as carbon tetrachloride
and phenothiazine.

Pigs

 Ovulation rate is not usually a limiting factor in productivity in the pig but, in general, as
ovulation rates increase, the embryo survival rate decreases. This can be demonstrated in
gilts, where the ovulation rate can be artificially increased but embryo survival rate decreases.
Even if early embryonic death does not occur with high ovulation rates, a problem may arise
later in pregnancy with competition for uterine space. It has been suggested that a higher fetal
death rate exists when there are more than five fetuses per horn, with those embryos in the
middle of the horn being smaller.
 Apart from the above intrinsic factors, extrinsic factors such as nutrition and stress play an
important part in embryonic loss in the pig. For example, it is well documented that high
energy levels after service result in reduced embryo survival. Stress, associated with extremes
of temperature, or certain management systems such as sow stalls or tethers, is also known to
result in increased embryo mortality. Other husbandry policies such as lactation length, also
affect embryonic death rates, and lactation lengths of less than 3 weeks produce a marked rise
in embryonic mortality presumably due to a poor uterine environment.

Sequel to Embryonic or Fetal Death

 Following early embryonic death the embryonic tissue are usually resorbed, and the animal
returns to estrus if there is no other conceptus in the uterus. If death occurs before there has

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 been maternal recognition of pregnancy the estrous cycle is not prolonged. If it occurs after
recognition has taken place, the estrous cycle will be prolonged.
 If death of the embryo is due to an infection then, even though the embryonic material may be
absorbed, a pyometra may follow. In cattle this condition is characterized by persistence of
the corpus luteum, closed cervix and pus accumulation in the uterine body and horns. It is a
particular characteristic of infection with Tritrichomonas fetus. If fetal death occurs after
ossification of the bones has begun, complete resorption of fetal material cannot take place,
instead, fetal mummification occurs.

CAUSES

Causes for early embryonic death

 Embryonic mortality can be due to maternal factors, embryonic factors, or to embryonic-

maternal interactions. Maternal failure tends to affect an entire litter, resulting in complete
loss of pregnancy.
 In contrast, embryonic failure affects embryos individually, often leaving others in litter
unharmed. In other cases the maternal environment may be insufficient, allowing the support
of only a few strong embryos.

Endocrine Factors

 Accelerated or delayed transport of the egg, as a result of estrogen – progesterone imbalance,

leads to preimplantation death. An abnormally undersized conceptus might not be able to
counteract the uterine luteolytic effect, with consequent regression of the CL and termination
of pregnancy. In swine, as stated previously, at least four living blastocysts are needed by day
10 of pregnancy to counteract the uterine luteolytic effects.
 A critical period of embryonic survival is the late blastocyst stage. Normally, the developing
CL secretes progesterone, which acts on the female tract in close synchrony with the

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 development of the embryos. The cause and effect relationship between luteolysis and
embryonic deaths is controversial. Apparently, embryonic mortality in cattle is not caused by
a progesterone deficiency during the luteal phase of the cycle; luteal regression follows rather
than precedes embryonic mortality. However, a diminished response to circulating
luteotrophic hormones may contribute to embryo mortality in subfertile cows.

Lactation

 Embryonic mortality occurs during lactation in cattle, sheep, and horses and is characterized
by prolonged estrous cycles after breeding. Mating of mares at foal heat leads to early
embryonic mortality, which has been attributed to reduce effectiveness of uterine defense
mechanisms, stress of lactation, and incomplete regeneration of the endometrium.
 Sows bred after weaning at 7 days of lactation suffer high embryonic losses between days 9
and 20 of pregnancy.

Nutrition of the Dam

 Caloric intake and specific nutritional deficiencies affect ovulation rate and fertilization rate,
as well as cause embryonic death. Also extremes in the level of feeding are detrimental to
embryo survival, so too are extremes in the supply of specific dietary nutrients.
 In dairy cows, high intakes of rumen degradable protein may lead to embryonic mortality.
This effect may be mediated through a reduction in the pH of the uterine environment during
the luteal phases of the cycle in which the embryo must grow.
 In swine, high caloric intake or continuous unlimited feeding increases ovulation rate, thereby
increasing the incidence of embryonic mortality before implantation. However, following
implantation, unlimited feeding decreases fetal death.
 In sheep, full feeding before breeding also increases ovulation rate as well as embryonic
mortality. Poor body condition of ewes at mating increases the incidence of embryonic
mortality, whereas moderate feed restriction from day 20 to 100 of pregnancy is less likely to
reduce lambing percentages. Under nutrition affects twin ovulators more than single ovulators
because both embryos are lost in the former, while a single embryo survives in the latter.
Thus, more twin than single ovulating ewes are barren.
 In the mare, the critical period for embryonic resorption is between 25 and days after
ovulation. No resorption occurs if mares are maintained on an adequate plane of nutrition
until 35 days after service.

Age of the Dam

 A higher incidence of embryonic mortality is observed in gilts and in sows after the fifth
gestation. In the ewe, the incidence of late embryonic loss is higher in ewe lambs and ewes
over 6 years than it is in mature ewes, which is due to factors associated with the embryo
rather than the uterine environment.

Overcrowding in Utero

 Because the degree of placental development is primarily influenced by the availability of

space and vascular supply within the uterus, increasing the number of implantations decreases
the vascular supply to each site and restricts placental development. This results in a high
embryonic and fetal mortality rate and probably explains the higher incidence of embryonic
mortality in cattle and sheep following twin rather than single ovulation. It should be noted,
however, that uterine capacity does not limit the ability of the cow and ewe to carry twins,
provided they are located in separate uterine horns. In cattle, embryo transfer experiments
have shown a higher embryonic mortality rate in recipients which received two embryos in a

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 single uterine horn. This loss may be due to overcrowding and intrauterine competition for
nutrients.
 In cattle and sheep with multiple ovulations, the number of embryos surviving is reduced to a
fairly constant number within the first 3 or 4 weeks of pregnancy, which implies that
embryonic loss increases as the number of eggs shed increases. Mortality does not seem to be
due to a deficiency of progesterone. In prolific breeds of sheep, late embryonic deaths occur
in ewes with more than five ovulations.
 Transuterine migration of embryos is of importance for equal distribution of embryos in the
two horns of the uterus in polytocous species such as swine. In its absence, there is a high
incidence of embryonic mortality in swine.

Thermal Stress

 Embryonic mortality increases in a number of species following exposure of the mother to

elevated ambient temperatures, especially in tropical areas the effects of thermal stress on the
early embryo are not apparent until the later stage of its development. Fertilized eggs of sheep
and cattle, when subjected to high temperatures either in vitro or in vivo are damaged but
continue to develop, only to die during the critical stages of implantation.
 Reduced fertility of summer heat-stressed dairy cows may result from decreased viability and
developmental capacity of 6 day –old to 8 day-old embryos and may account for the well-
documented seasonal reduction in the efficiency of artificial insemination during summer.
Heat stress between days 8 and 17 of pregnancy may also alter the uterine environment as
well as growth and secretory activity of the conceptus. Apparently heat stress antagonizes the
inhibitory effects of the embryo on the uterine secretion of PGF2a.
 Several studies have demonstrated that the pig embryo is most susceptible to heat stress
before day 18 of pregnancy particularly during implantation. A greater incidence of
embryonic deaths was noted among gilts exposed to high temperatures 8 to 16 days post
breeding than among those exposed during 0 to 8 days post breeding.

Semen

 A portion of all embryonic mortality is attributable to the male and the mating system.
Genetic factors that are transmitted by the male to the embryo may be inherited, may arise
from testicular tissue, or may occur in spermatozoa after they are released from the testis.
 Infertile matings by highly fertile bulls are primarily due to embryonic mortality, while those
of bulls with low fertility are due to fertilization failure and embryonic deaths. In swine,
semen stored for 3 days before insemination produced zygotes much more susceptible to early
embryonic death, presumably owing to the reduced DNA content in aged spermatozoa.

Incompatibility

 The inherited genotype of the male may include a variety of genetic factors that lead to
incompatibility and early embryonic loss. There may be incompatibility between spermatozoa
and mother, between spermatozoa and egg, or between zygote and mother.
 Immunologic incompatibilities may block fertilization or cause embryonic, fetal, or neonatal
mortality. In cattle, homozygosity for certain blood groups and certain substances related to
transferring and J-antigen in sera are associated with increased embryonic loss as well as
decreased fertilization rate.

Diagnosis

Based on reproductive history and clinical examination

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Treatment

Specific treatments for conditions like delayed ovulation, endometritis may be carried out. Since most
of the cases do not reveal any specific condition the following guidelines may be adopted.

 Bring the animal into positive energy balance.

 Do AI twice at each estrus preferably at 12 or 24 h interval.
 Check the semen quality - use only high quality semen - (This is often taken for granted and
ignored).
 Clitorial stimulation for at least three seconds at AI.
 Administration of 100 µg of GnRH or 1000-1500 IU of LH at the time of AI.
 Administration of 500 mg of depot progesterone on the 5th day of AI.
 Skipping of AI, administration of PGF 2 alpha after 9-10 days and fixed time AI twice at 72
and 96 h.
 Intrauterine infusion of 1 million units of procaine penicillin diluted in saline three times at
the onset of estrus, 8 h after AI and 24 h later.
 Skipping of AI and intrauterine infusion of 1 to 1.5 million units of procaine penicillin in 20
ml of sterile saline daily for 3-4 days.
 Skipping of AI and intrauterine infusion of 2 ml of Lugol’s solution diluted in 8 ml of sterile
saline.
 Flushing of the uterus with normal saline - under moderate pressure as being done in embryo
transfer (to remove cellular debris and also mild block in the uterine tubes).
 Administration of different hormones and antibiotics may preferably be tried at separate
estrus.

INTRAPERITONIAL INSEMINATION

 Mammalian sperm interactions with the female reproductive tract determine the drastic
reduction in the number of spermatozoa that occurs between the site at which the ejaculate is
deposited and the site at which fertilization takes place. Physiological and pathological events
may occur in the tubular genital organs, which could act unfavourably on the mechanisms of
sperm transport and result in infertility. Under normal conditions, sperm transport is of an
ascending nature from the site of semen deposition to the fallopian tubes.
 It is generally accepted that some spermatozoa (perhaps in low numbers) pass through the
oviduct in to the peritoneal cavity after natural mating or insemination. The fate of these
spermatozoa is not known, but on their earlier travel through the genital tract, they become
separated from the rest of the components of the seminal plasma, thus reducing the chances of
an immune response. However, spermatozoa can also approach the site of fertilization from
the peritoneal cavity.

Procedure

Intraperitoneal insemination procedure as outlined by Lopez-Gatius (1995).

 A sterile 19 gauge needle 4 cm long and a glass tube 44 cm long and 10 mm in diameter and
with a conic tip are used as additional accessories to standard AI equipment.
 Immediately before insemination, the vulva and the perineal region of the heifer and the glass
tube are washed with disinfectant solution.
 For insemination, the glass tube is carefully inserted into the vagina, guided by the hand, and
the conic tip positioned to the dorsal fornix.
 The AI catheter with the needle affixed to the end of the plastic sheath is then inserted in to
the glass tube, and the vaginal wall is pierced in cranial direction through the fornix by the
needle.
 Next, semen is slowly injected in to the peritoneal cavity.
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  Frozen semen in 0.25 ml French straws is diluted in Triladyl (Minitub) so that each unit of
semen contains at least 45 million spermatozoa.

Though intraperitoneal insemination appears to a useful technique for the treatment of a wide scope of
infertility problems in human medicine, only 2 previous experiments have been performed in cattle
using this technique. Lopez-Gatius (1995) recorded 9 pregnancies following intraperitoneal
insemination of 62 repeat breeder cows. The pregnancy rate was not different for intraperitoneal or
uterine inseminations. Further, the results suggest that intraperitoneal insemination could be an
alternative procedure to the usual deposition of semen in to the uterus in repeat breeder cows.

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 OVARIAN DYSFUNCTION

CYSTIC OVARIAN DEGENARATION

Among the Ovulatory defects viz. Cystic Ovary, Delayed ovulation and Anovulation, the economic
losses due to cystic ovarian degeneration is the highest and is due to a prolonged calving interval,
increased culling rates and costs of veterinary intervention.

The traditional definition of a follicular cyst has been a structure of at least 2.5 cm diameter that
persists for ten days or more in the absence of a corpus luteum. However, recent findings suggest that
follicular cysts may be smaller than 2.5 cm, especially when several cystic structures are present.
Luteal cysts are typically 3 cm or more in diameter and persist for at least 14 days.

CLASSIFICATION

Pathological

 Follicular cysts: These are anovulatory follicle (s) which may be single or multiple, on one or
both ovaries and are usually thin walled.
 Luteal cysts: These are also anovulatory follicles which are thicker walled due to partial or
incomplete luteinisation and usually single in structure.

Non-pathological

 Cystic corpora lutea – a corpus luteum which has a fluid filled cavity. It is not pathological
and does not alter the length of the estrous cycle
o Also known as cystic corpora lutea as they contain a cavity, an encystment.
o These cavitated CL produce the same concentration of progesterone as a solid CL,
hence they function normally

CLINICAL SIGNS

 Affected cows may exhibit nymphomania or anestrus

 Due to aggressive sexual behaviour, they are called Buller’s
 Relaxation of sacrosciatic ligaments and upward displacement of the coccygeal bones or
elevation of the tail head called as sterility hump develop in the long standing cases
 Cows with sterility hump are more prone for hip dislocation and pelvic fracture as well as
endometritis.
 Other signs include tendency for vaginal prolapse, pneumovagina, hydrometra and
mucometra. They gain a steer- like appearance.
 Microscopically the cystic dilation of the endometrial glands and hyperplasia of the uterine
mucosa take place and in the follicle the thecal layer and/ or granulosa layer may be affected

Features Follicular cyst Luteal cyst

Nature of cyst Anovulatory Anovulatory
Persistence in > 10 days Prolonged period
ovary
Diameter >2.5 cm >2.5 cm
Presence in ovary Multiple in both ovaries Often single

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 Symptoms Nymphomania – frequent,
irregular, prolonged or continuous
oestrus, prolonged period accept
riding of another cow, frequent
attempts to mount on other cows–
sexually aggressive– “ Bullers ”,
relaxation of the sacrosciatic
ligaments -upward displacement
of coccyx - “ Sterility hump ”.
Anestrus – no sign of estrus, if
untreated for a prolonged period some
become virilized develop masculine
conformation, attempt to mount other
cows, and may not allow mounting by
others.

 Based on clinical signs

 Based on rectal examination

Follicular Cyst Luteal Cyst

Both the ovaries greatly enlarged. One of the ovaries greatly enlarged.
Multiple cysts in both ovaries. Single cyst in one of the ovaries
Follicle wall thin, fluid filled, fluctuate and Cystic wall thickened, fluid filled, fluctuate
smooth surface. and smooth surface.
Readily rupture. Difficult to rupture.
Vagina, clitoris and vulva: swollen. No change.
Cervix: Large and dilated. Closed.
Uterus: Thickened, large, tonic and Flaccid.
oedematous.
Accurate diagnosis of cystic conditions is possible with single examination. If doubtful,
repeat examination after 10 days.

 Milk or plasma progesterone estimation

o Milk or Plasma Progesterone estimation may aid in diagnosis.
o Plasma or milk progesterone levels vary according to type of cyst.
o In milk, follicular cysts typically show levels less than 5 ng/ml, with luteal cysts
showing levels above 5 ng/ml.
 Ultrasonography
o Most accurate and practical diagnostic technique to identify and differentiate between
follicular and luteal cysts
o Wall thickness is much less in follicular cysts (average of 2.5 mm, range 1-6 mm)
than in luteal cysts (5.3 mm, range 3-9 mm).
o With follicular cysts, there appears to be a correlation between ultrasonographic wall
thickness and hormone concentrations within that cyst, with thicker-walled cysts
(average 2.5 mm) showing high progesterone and low estradiol concentrations, and
vice versa in thinner-walled cysts.

Prognosis

 Good in early cases.

 Poor in long standing cases, where severe cystic degeneration of the endometrium and
atrophy of the uterine wall has taken place.

Treatment

 If diagnosed as cystic irrespective of the type any one of the following treatment line may be
attempted.
 LH: 2500-5000 IU I/V – optimum and economic
 GnRH:
o 100-250 µg I/M to luteinize
o 0.5 to 1.5 mg for ovulation

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  Synthetic analogue, Buserelin: 0.02 mg I/M
 Following LH or GnRH treatment, the cysts undergo luteinization and most of the cows re-
establish ovarian cycle and exhibit estrus in 18-23 days.
 Following LH or GnRH, PGF2 α- 25 mg may be administered after 9-12 days to cut short the
cycle length
 GnRH or PGF 2 α may be preferred for luteal cyst which is however difficult to differentiate
from follicular cyst, in which PGF2α alone is ineffective
 Progestogens: CIDR, PRID or Ear implant are also effective.
 Progesterone: 100mg intramuscular for 14 days.
 Corticosteroids: 10-40 mg Betamethasone or 10-20 mg Dexamethasone.Found to be as
effective as LH or GnRH. Repeated if necessary (average 1.9 injections). Suppresses the
release of ACTH and also LH and upon the release of exogenous block, LH is released in
bulk.
 Potassium iodide: 30 Gm–divided into 6 doses. Daily oral administration reported to be
successful.
 Other lines of treatment tried include: clomiphene citrate, oxytocin, testosterone, estrogen,
etc.

Reasons for Reduced Recovery Rate

 Inability of the cystic structures to respond to GnRH induced LH release because of fibrosis,
degeneration of the theca and granulosa cells in the cyst.
 Insufficiency of the LH receptors
 Decreased sensitivity to LH
 Low pituitary responsiveness to GnRH or low activity of the secreted LH

DELAYED OVULATION

 Ovulation in the cow is atypical since it occurs 10-12 h after the end of estrus and 18-26 h
after the ovulatory LH peak.
 Ovulatory defects may be due to endocrine deficiency or imbalance and mechanical factors.
 Incidence: 2 to 18%
 Delayed ovulation is generally assumed to be one of the causes of failure of conception.
 Certain cows have prolonged estrus. However, this is opined to be re lated to a delay in
corpus luteum (CL) assuming normal steroidogenesis rather than to the delayed ovulation.
 Conception rate is reduced in cows that ovulated by the second day after oestrus.

Diagnosis

 Diagnosis is difficult and requires sequential rectal palpation of the ovaries.

Treatment

 Repeated AI at 24 h interval two or three times.

 GnRH: Encouraging results.Dose: Natural GnRH, 100 µg i/m. Buserelin, 5-10 µg i/m.
 Luteinizing hormone ( LH ): Dose- 1000-1500 IU. i/m or i/v

ANOVULATION

 Associated with those conditions which can predispose to true anestrus.

 May occur when the cow goes into anestrus or during the first cycle after parturition.
 Diagnosis of anovulation can only be made retrospectively by noting on rectal palpation that a
follicle persists longer than one would have suspected.
 Anovulatory follicle undergoes luteinization and regresses like a normal CL after 17-18 days.
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 ENDOMETRITIS

 Endometritis is a localized inflammation of the uterine lining, associated with chronic

postpartum infection of the uterus with pathogenic bacteria Arcanobacterium
pyogenes (Bondurant, 1999).

Etiology

 The causal organisms usually reach the uterus from the vagina at coitus, insemination,
parturition or postpartum, although it is possible in some circumstances for infection to arrive
by the circulation. The great majority of cows suffer from bacterial contamination of the
uterus after calving, but under normal circumstance this flora is rapidly eliminated. In cows
that develop endometritis, the bacterial flora is not eliminated from the uterus, causing the
endometrium to become inflamed.

Factors Associated with the Development of Endometritis

o Retained fetal membranes.

o Abortion.
o Induced calving.
o Multiple births.
o Dystocia.
o Management factors – state of nutrition, hypocalcaemia, season.
o Return of ovarian cyclicity.
o Bacterial loading.
 The endometritis is almost invariably a sequel to invasion with A. pyogenes. There is good
evidence that there is synergism between A. pyogenes and Fusobacterium necrophorum, the
latter organism producing a leucocidal endotoxin which interferes with the host’s ability to
eliminate A.pyogenes. Similarly Bacteroides sp. also produces substances that interfere with
the phagocytosis and killing of bacteria.

CLINICAL SIGNS

 The presence of a white or whitish-yellow mucopurulent vaginal discharge in the post partum
cow.
 The volume of discharge is variable, but frequently increases at the time of estrus when the
cervix dilates and there is copious vaginal mucus.
 The cows rarely show any signs of systemic illness, although in a few cases milk yield and
appetite may be slightly reduced.
 Rectal palpation frequently shows a poorly involved uterus which has a doughy feel.
 Pus flakes present in the discharge indicating endometritis

DIAGNOSIS

 Clinical signs
 Rectal examination
 Vaginal examination
 Whiteside test
 Uterine biopsy
 Bacterial culture

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RECTAL EXAMINATION

 Transrectal palpation of the uterus. However, this method is subjective and often fails to
account for normal events and variability in uterine involution or to have any association with
reproductive performance.
 By rectal examination cervical diameter, location of the uterus, symmetry of the uterine
horns, diameter of the uterine horns, texture of the uterine wall, palpable uterine lumen are
noted.

VAGINAL EXAMINATION

 At examination, cows are first inspected for the presence of fresh discharge on the vulva,
perineum, or tail. If discharge is not visible externally cows are examined using vaginal
speculum.
 The speculum is inserted into the vagina up to the level of the external os of the cervix.
 Inspection of the cervix and vagina is performed with illumination from a penlight.
 The nature of the discharge may be clear mucus with flakes of pus, mucopurulent, purulent
but not foul smelling.

WHITE SIDE TEST

This test is used to detect sub-clinical endometritis in repeat breeding cows.

Procedure

 The uterine discharges (cervical mucus) is collected aseptically with sterile sheath and
syringe.
 Mixed with equal volume of 5% NaoH in a test tube.
 The mixture is heated up to the boiling point, and
 The intensity of colour changes is graded.

Color Degree
Turbid Normal
Light yellow Mild
Yellow Moderate
Dark yellow Severe

ENDOMETRIAL BIOPSY

 A relatively easy and safe procedure for the practicing veterinarian to perform.
 Its use in conjunction with a detailed history, rectal and vaginal examinations and microbial
cultures can lead to a more accurate prognosis of difficult breeders and greater therapeutic
efficiency.
 Repeated biopsies do not cause adverse effects on cow’s reproductive capacity.
 Biopsy lesions heal rapidly.
 Hemorrhages are of little or no clinical significance and are quickly resorbed.
 Biopsy specimen should be of sufficient size (4 x 6 mm).
 Specimens should be taken from both the horns and the body of the uterus due to variability
of pathology in each section.

BIOPSY CATHETER

 To obtain in vivo uterine endometrial samples.

 It consists of an outer casing and piston of length 57.5 cm and, diameter of 0.7 cm.

~ 175 ~
  Distal end of the catheter has a rounded tip to prevent injury to the reproductive tract and to
facilitate the easy entry of the tip through the cervical canal.\

TECHNIQUE

Step-1  Proper care, disinfection and sterilization of the

biopsy instrument are necessary to prevent
microbial contamination. Before taking biopsy,
thoroughly scrub and clean the vulva and
surrounding perineal area.
 Evert the vulval lips and introduce the biopsy
instrument in closed position through the vagina
and cervix in to the uterus (Step-1).
 Gently push the piston to open the cutting edge.
Press a portion of the uterine wall in to the
cavity of the cutting edge (Step-2). Pull the
Step-2 piston caudally to close the cutting edge so as to
remove a piece of the endometrium.
 Withdraw the instrument out of the reproductive
tract in closed position (Step-3).
 Remove the endometrial tissue from the
instrument and immediately transfer it into 10%
neutral buffered formalin solution at room
temperature.
 Tissues are trimmed, dehydrated, cleared and
embedded in paraffin sections and cut at a
Step-3 thickness of 5-6µ and stained with H&E stain
for histological examination.

INTERPRETATION

 Bovine endometrium is evaluated histologically for:

o periglandular fibrosis.
o cystic glandular changes, and
o cellular infiltration of endometrial stroma.
 Cellular infiltration is the most striking feature of acute endometritis.
 Moderate and severe cases of endometritis are much easier to diagnose on the basis of the
increased number of inflammatory cells spread throughout the stratum compactum and
spongiosum layers compared with few cells seen in mild endometritis.
 Neutrophils may be present in high numbers during normal estrum – erroneously suggesting
acute endometritis.
 Neutrophils present during the luteal phase – definitely indicative of an acute endometritis.
 Initial phases of endometritis: diffuse and possibly the periglandular and perivascular cellular
infiltrations are dominated by neutrophils and lymphocytes

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TREATMENT

 Treatment of endometritis is the subject of considerable controversy among veterinary

practitioners, particularly with respect to which therapy to use, and to a lesser extent, which
cows to treat or whether to treat at all (Gilbert, 1992).
 The general therapy of endometritis is to halt and reverse inflammatory changes that impair
fertility practically, treatments aim to reduce the load of pathogenic bacteria and enhance the
processes of uterine defence and repair.
 A wide range of antiseptics antimicrobial agents and hormones have been used as treatments
for endometritis. Objective studies of the effectiveness of these agents have been difficult
because of the multifactorial nature of the disease and many cases of endometritis are self-
limiting and resolve after the resumption of oestrous cyclicity.
o Antibiotic therapy
o Hormones
o Intrauterine infusion of disinfectants
o Immunomodulators

ANTIBIOTIC

 Local vs systemic administration - For antimicrobial treatment to be effective, an effective,

concentration of drug must be achieved and maintained of the site of infection for an adequate
period. Several antimicrobial agents are absorbed from the uterus (sulfonamides, tetracylines,
streptomycin, penicillin, ampicillin, gentamicin and chloromphenicol). The absorption in the
immediate postpartum period is considerably less than that after complete uterine involution.
Uterine pathologic changes (endometritis) result in further decrease of absorption. Poor
absorption results in a high concentration of the drug in the uterine cavity and on the
endometrium on the other hand, adequate concentrations frequently are not achieved in the
subendometrial tissues, vagina, cervix, or ovaries and oviducts.
 Systemic administration usually results in uterine tissue and lumen antibiotic concentrations
equal to blood plasma concentrations. The concentrations are the same in the normal and the
pathologic uterus. The systemic administration gives a better distribution in the tubular genital
tract and to the ovaries. Furthermore, fetal membranes and abnormal exudate cannot
mechanically influence the distribution. Also, systemic administration eliminates the risk for
damage to the endometrium. Repeated treatment can be carried out relatively simple and
without introduction of new infections. Because, there are reasons to assume that a moderate
to severe uterine infection seldom is localized only to the superficial layer of the
endometrium, therapeutic strategies would have to consider systemic treatment.
 In the treatment of chronic endometritis with antimicrobial substances, it is preferable to
administer the substance by the intrauterine route provided an adequate dose rate is used, this
will result in effective minimum inhibitory concentrations (MICs) reaching the endometrium
and being established in the intraluminal secretions. The latter point is important for the
effective treatment of the disease, since sub therapeutic dose rates are frequently used.
 Several antibiotics are inappropriate for the treatment of uterine infections Nitrofurazone is an
irritant and has an adverse effect on fertility. Aminoglycosides are not effective in the
predominantly anaerobic environment of the infected uterus. Sulphonamides are ineffective
because of the presence of para-aminobenzoic acid metabolites in the lumen of the infected
uterus. Penicillins are susceptible to degradation by the large numbers of penicillinase
producing bacteria that are present.
 A broad-spectrum antibiotic, such as oxytetracycline, used at a dose rate of up to 22 mg/kg,
will provide effective MICs in the lumen and uterine tissues for intra uterine treatment with
oxytetracycline total doses of 0.5 to 5 g may be used. The lower dose (0.5 to 2 g) is unlikely
to yield adequate concentrations in the large postpartum cavity. Systemic administration of
penicillin results in genital tract tissue and lumen concentrations similar to blood plasma
concentrations in the cows. Other antibiotics such as Penicillins, Metronidazole,
Ciprofloxacin and Cephalosporins are administered systemically as well as intrauterine for the
treatment of uterine infections.

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HORMONES PGF2 ALPHA

 When there is a palpable mature CL on the ovary it is arguable that the best-method of
treating clinical endometritis is with PGF2α or its synthetic analogues. When a CL is present,
PGF2α causes luteolysis, thereby stimulating the return to estrus and reducing the high
progesterone concentrations.

I/U INFUSION

 Intrauterine infusions with various disinfectants such as lugols’ iodine and povidone iodine
are relatively common for treatment of postpartum infections. Although positive results
occasionally have been reported, few controlled evaluations have been made. Because
intrauterine use of disinfectants may suppress the uterine defense mechanisms eg.
phagocytosis, the use of intrauterine infusions in the postpartum cow is not recommended.

ROLE OF IMMUNOMODULATORS

Lipopolysaccharides of E.coli

 Serotype 026:B6.
 Dissolve 100 μg in 20 ml of PBS (pH 7.4).
 Administer on day 0 (estrum) through intra uterine route.

Oyster glycogen

 PMN migration into the uterine lumen of healthy cows is stimulated after intrauterine
administration of oyster glycogen, up to 90% of all cells identified in uterine secretions being
neutrophils.
 Variable concentrations of oyster glycogen between 0.1-10% all in 60 ml of vehicle produced
identical responses with a peak PMN concentration 12 h after administration.

Leukotriene B4

 Leukotriene B4 (LTB4) is an effective chemo-attractant, stimulating preferential migration of

PMNs into the lumen of the bovine uterus.
 A single intrauterine treatment of a 30 nmol/L solution increased the intrauterine leucocyte
count 5-10 times within 24 h.

Autologous plasma

 Collect ~300 ml of blood from oestrus animal in JML blood bag. Keep in ice and transport to
the lab. Transfer in to 50 ml of sterile centrifuge plastic vials; centrifuge at 3000 RPM for 15
min, separate the plasma and stored at -20 °C.
 Administer 50 ml of plasma through intra uterine route on days 1, 2, and 3 (day 0 – estrum).

ENDOMETRITIS IN MARE

 Reduced fertility associated with endometritis, both acute and chronic, has been recognized
for many years in brood mares. This subfertility is due to a hostile environment for the
developing conceptus, and in some cases, the endometritis causes early regression of the CL.
 The term `endometritis’ refers to the acute or chronic inflammatory process involving the
endometrium. These changes frequently occur as a result of microbial infection, but they can
also be due to non-infectious causes.

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  One of the main obstacles to producing the maximum number of live, healthy foals from
mares bred during the previous season is the mare, which is susceptible to persistent acute
endometritis following breeding.

ENDOMETRITIS IN SSOW, EWE AND GOAT

 A form of endometritis characterized by profuse vaginal discharge at the onset of estrus has
been described in Europe and other regions. The causative agent is usually Staphylococcus
hyicus or E coli , and the disease seems to be transmitted at mating or artificial insemination;
signs are seen 15-25 days later during the subsequent proestrus or estrus. Infection may be of
long duration with signs recurring at each estrus.
 There is some correlation with serum concentration of progesterone and endometritis. When
inseminations are done at the time when progesterone levels begin to rise after ovulation, the
possibility of inducing endometritis is more. Some sows recover spontaneously, but there
does not seem to be any effective treatment for those that do not. At necropsy, copious
quantities of purulent exudate may be found in the uterus, making this condition more akin to
pyometra. Some virgin gilts also show endometritis. The cause is unclear but may occur
secondary to vulval biting in finishing houses or due to the fact that many gilts attain puberty
prior to transport with infection if the uterus occurring at gilt finishing houses. Endometritis
also occurs following parturition due to dystocia, traumatic injury, abortion or unhygienic
manipulations.
 Endometritis has been seen in sheep and goats.In commercial sheep and goat flocks, diagnosis
is seldom made antemortem, and treatment is generally impractical. In animals with a
persistent uterine discharge, remnants of a macerated fetus should be considered as a nidus of
chronic infection.

VAGINITIES

COW

Contagious vesicular vaginitis/vesicular exanthema/bull burn

 Contagious Vesicular Vaginitis also known as Vesicular exanthema or Bull Burn vaginitis is a
specific inflammation of the vulo-vaginal mucous membrane of cattle and the glans penis and
prepuce of the bull. It is of a highly contagious nature and is ordinarily transmitted by coitus,
but possibly spread also by contact with soiled bedding, by attendants’ hands, etc. The
condition is confined to bovines and its exact cause is unknown; a streptococcus has been
incriminated, but the evidence is inconclusive.

Clinical Signs

 Its onset is sudden and acute; 24-48 hours after mating. Heifers are often more severely
affected than cows. The vulval labia become swollen and tender and the mucous membrane
is deeply congested. This is quickly followed by the development of numerous small red
vesicles on the mucosa. These may rupture early or they may develop to pustules before
doing so, leaving a small, hemorrhagic erosion, 1/8 inch or so in diameter. There is an
abundant muco-purulent discharge which tends to mat the vulval tuft and tail.
 The condition is most painful. The animal swishes its tail; urination may be associated with
marked straining. There may be some fever, reduction in appetite and milk yield, although
systemic symptoms are not severe and there is seldom much loss of flesh.

Prognosis

 Prognosis is good. The infection does not prevent conception, nor is the development of the
fetus adversely affected. Recovery occurs in the course of 2-3 weeks and is generally

~ 179 ~
 complete. In very severe cases, however, stricture of the vulva and posterior vagina may
result from the deposition of scar tissue.

Treatment

 Spread should be controlled by sexual and general isolation. On no account should affected
animals be bred until they have completely recovered. The communal bull is the chief cause
of spread. Artificial insemination of unaffected animals should be considered.
 Daily irrigation of the affected parts using some mild, non-irritating antiseptic, taking care to
sterilize all apparatus immediately after use, is generally recommended. 1/200 acriflavine or
1/250 zinc sulphate can be used. Slightly hypertonic saline or 1 per cent formalin twice or
thrice weekly could help. Similar treatment of the bull should be applied. All bedding should
be burnt and adequate disinfection of byres and attendants’ hands ensured.

Nodular or Granular Vaginitis

 Nodular or Granular Vaginitis is characterized by the presence on the vulval and vaginal
mucosa of small nodular or granular elevations, 1-2 mm. in diameter, surrounded by a
congested base and generally having a linear arrangement. The elevations comprise
accumulations of lymphoid cells. The condition is transmitted by coitus, and that the bull
may suffer from a similar nodular balano-posthitis. The ubiquitous streptococcus has been
incriminated as the cause, but the finding of streptococci of the viridians group is by no means
conclusive evidence that they are pathogenic.
 Of recent years the condition has been ascribed to T. fetus infection, but here again the
evidence is quite inconclusive, and attempts to transmit it experimentally have not thrown any
light on the subject. Nevertheless, many clinicians have reported a peculiar “rasp-like”
feeling of the vaginal mucosa in infected animals.
 There is no doubt that a condition indistinguishable from the one described is frequently
found in animals whose breeding histories are normal. Moreover, there is no satisfactory
evidence that lymphoid elevations on the glans penis of the bull are the outcome of infection
either by protozoa, bacteria or viruses.
 Numerous forms of treatment have been recommended: irrigation with such agents as zinc
sulphate, 1-200; iodine, 1-1000 ; acriflavine, 1-1000.

MARES

 Vaginitis due to a variety of infections is commonly observed in mares. The vagina is more
resistant to irritation and permanent damage caused by infections than does the cervix and
uterus. In severe metritis and cervicitis with a catarrhal exudate the vagina is usually
involved. Mares that develop the habit of pneumovagina usually exhibit vaginitis often with
mucopurulent exudate.
 Fecal material and urine may usually be seen in the cranial part of the vagina. Injuries,
trauma, lacerations or severe infections of the vulva and vagina at parturition may cause
chronic vaginitis, pnemovagina and in some cases stenosis of the vagina. Occasionally the
cloudy, turbid light coloured urine of the mare may be confused with vaginitis. Cystitis may
develop secondary to vaginitis or metritis.

SOWS

 Vulvovaginitis, characterised by swelling of the vulva with mucus discharge, enlarged

mammary glands and occassional prolapse of the rectum commonly occurs due to feeding of
mouldy corn or barley in which estrogenic compounds were present.

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  A mycotoxin from F.graminearum caused vulvovaginitis, vaginal prolapse, perineal
relaxation, and ovarian atrophy in young guilts. The action of mycotoxin was identical to
estrogen.

DOGS

Juvenile or Puppy vaginitis

 Juvenile, or puppy, vaginitis is defined as vaginitis in bitches less than 1 year of age and may
be seen in females as young as 8 weeks of age. Juvenile vaginitis may be an incidental finding
during physical examination in puppies with vulvar discharge. Vulvar discharge is almost
always present, and ranges in volume and character from scant and mucoid to copious and
mucopurulent. Presence of vaginal irritation is variable. Affected bitches usually are not
systemically ill. Cytology of the vaginal discharge usually consists of polymorphonuclear
leukocytes, with or without bacteria. Treatment with antibiotics, either topically or
systematically, may not be successful. Antibiotic treatment is warranted if the vaginal
discharge is cytologically purulent or the bitch is showing signs of discomfort, such as
excessive licking of the vulva. Antibiotic choice should be based on culture and sensitivity
testing of a sample retrieved from the cranial vagina, and should be continued for 4 weeks.
 Conservative treatment is indicated for bitches with juvenile vaginitis that is not causing the
bitch discomfort. Allowing bitches with juvenile vaginitis to go through an estrous cycle may
hasten resolution.

Adult-Onset vaginitis

 Adult-onset vagintis occurs, by definition, in bitches greater than 1 year of age. It is termed
chronic if it has been present for greater than 1 month.
 Vulvar discharge is the most common presenting complaint with other complaints of
pollakiuria, pain when urinating, and vulvar licking. Other clinical signs that have been
reported were dependent on the inciting cause of the vaginitis, and included
polyuria/polydipsia, urinary incontinence, pruritus, and infertility, none of which are signs of
vaginitis but instead reflect concurrent disease.
 On physical examination, vulvar discharge usually is present, either dripping from the vulva
or caught in the perivulvar hair. Vulvar hyperemia may be present. Vaginoscopic examination
often reveals diffuse hyperemia of the vestibular and vaginal mucosa and luminal exudate.
Follicular lesions may be present in the vaginal mucosa. Localized erythema at the urethral
papilla or within the clitoral fossa my be seem occasionally. Vaginoscopic examination may
reveal presence of congenital vaginal abnormalities, vaginal neoplasia, or foreign body.
 Cytology of vaginal specimens collected from mature bitches with vaginitis is more often
indicative of septic inflammation than are samples from immature dogs. Vaginal culture
rarely yields heavy growth of a single organism.Organisms cultured most commonly are
E.coli, Streptococcus species, and Staphylococcus intermedius, all of which are normal
vaginal flora in the bitch.
 Complete blood count (CBC) and serum chemistry profile usually are normal is bitches with
vagnitis. No specific changes were noted on CBC in 18 of 23 mature bitches with
vaginitis.Changes in the CBC, serum chemistry profile, or urinalysis may help pinpoint a
primary disease process, such as diabetes mellitus is strongly recommended for all bitfches
with persistent vulvar discharge.
 Vaginitis must be differentiated from disease of the uterus in intact dogs or uterine stump to
spayed female dogs, and urinary tract diseases; bitches with any of these condition may
present with vular discharge. Vaginitis may occur in animals infected with B.canis or canine
herpesvirus.
 Treatment of ault-onest vaginitis requires careful evaluation of the history and physical
examination findings and any laboratory work performed to try to determine if a predisposing
cause of vaginitis is present. Treatment of the underlying problem usually is curative.

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  Antibiotics should be used if culture of a specimen collected from the anterior vagina reveals
heavy growth of a single organism. Systematic therapy is preferable to topical treatment.
Antibiotic choice should be based on culture and sensitivity testing and should be continued
for 4 weeks. Vaginal douches with antibiotics or antiseptic agents are ineffective in flushing
out significant amounts of vaginal discharge, and may be irritating to the vaginal mucosa,
worsening the vaginitis.
 Treatment with low doses of oral estrogen (diethylstilbestrol (DES; 1mg daily per as for dogs
greater than 20 pounds ,0.5 mg daily per os for dogs less than 20 pounds for 7 days, tapering
the dose over 2 weeks and maintaining lifelong therapy with the minimal affective dose) may
be beneficial in spayed bitches with vagnitis because DES will increase thickness of the
vaginal mucosa and promote resistance by invasion of the atrophic vaginal epithelium by
normal vaginal flora. As many as a third of dogs presenting with adult-onset vaginitis have no
identifiable underlying problem.
 Vagintis in adult dogs may be primary or secondary. Primary vaginitis may be caused by
infection with Brucella canis or Canine Herpesivirus. Secondary vaginitis may occur
subsequent to vaginal atrophy following OHE, to urine or mucus pooling with a congenital
vaginal anomaly, to therapy with drugs such as mibolerone, to the presence of a vaginal
neoplasm or foreign body, or secondary to urinary tract disease or systematic disease, such as
diabetes mellitus. The most common factors underlying vaginitis in adult dogs are estrogen
deprivation following OHE and congential vaginal abnormalities, urinary tract disease and
vaginal neoplasia.

QUEEN

 Primary vaginitis is rare in the cat. Clinical signs include pollakirueia, dysuria, frequent
cleaning of the vulva, and vulvar discharge. Diagnosis is made by inspection, culture, and
biopsy of the vaginal mucosa using a natoscope spectulum in the anesthetized cat. Primary
vaginitis is reported to be rare and self-limiting, with no treatment indicated; secondary
vaginitis may occur following obstetric or coital trauma, pyometra, or viral rhinotracheitis
infection.
 Differential diagnoses include urinary tract disorder, pyometra, or uterine stump granuloma,
which are ruled out on the basis of urinalysis (cystocentesis sample) and palpation,
radiographic and ultrasonic inflammation.

CERVICITIS

COW

 Inflammation of the cervix is called cervicitis and is caused by a variety of infections and
more commonly an injury to the cervical mucous membrane.

Symptoms

 Vaginal speculum examination will help to diagnose cervicitis.

o The external os of the affected cervix is appears edematous and swollen.
o External folds are often prolapsed
o The cervical mucosa appears cherry-red to dark-purple color
o Mucopurulent exudates is seen in and on the cervix.

 Mild cervicitis without metritis does not normally interfere with conception or causes sterility.
Even with severe forms of cervicitis, conception may still occur. Since, cervicitis and
endometritis are closely associated and the former can usually be observed clinically,
cervicitis may often be erroneously blamed for infertility.
 If a severe cervicitis is present endometritis or metritis is probably also present and should be
treated. Occasionally if pregnancy does occur even though a cervicitis is present the gestation
may be insecure, with the possibility that abortion, retained placenta, and other uterine
~ 182 ~
 pathology may develop. Vigorous manipulation of the cervix will produce a severe hyperemia
and congestion of the mucus membrane that should not be confused with infectious cervicitis.
 Occasionally in severe chronic cervicitis the cervix may become very thick and sclerotic and
cervical stenosis may follow. Cervical atresia or obstruction is very rare in the cow and if it
occurs the uterus becomes distended with the retained mucus and debris. Depending on the
nature of the uterine contents a uterine abscess or mucometra develops. Cervical stenosis may
occur due to severe cervical inflammation, or an enlarged cervical ring may extend into the
cervical canal, making a sharp bend in the lumen so that passage of a catheter is difficult or
impossible. These cows frequently conceive promptly when bred naturally or when semen is
deposited artificially in the cervix. In rare instances severe sclerotic, fibrosed, indurated
cervix may result in cervical stenosis at the time of calving causing dystocia.

Prognosis

 The prognosis in most cases of cervicitis is good and spontaneous recovery usually occurs as
the metritis and vaginitis improve. As long as a metritis or severe vaginitis is present,
recovery from a cervicitis cannot occur. The cervical infection usually is overcome by the
natural body-defense mechanisms particularly if the estrus periods are regular. The more
severe forms of cervicitis may take longer to respond. Cervical stenosis or sclerosis frequently
fails to respond to treatment. If the cow conceives these conditions are apt to cause dystocia at
the time of parturition.

Treatment

The entire reproductive tract must be treated if the cervicitis is accompanied by vaginitis or metritis.
The same treatments usually are useful for all 3 conditions.

Ordinarily recovery from cervicitis is usually spontaneous but the following treatments may aid and
hasten recovery.

 Warm or hot mild antiseptic douches of the vagina every 3 to 4 days as described for
endometritis are useful in flushing out the mucopurulent exudates and stimulating circulation
in the mucosa of the vagina, cervix and uterus.
 Swabbing the cervix with one of the following not only stimulates mucous flow, and causes
hyperemia, it also apparently hastens recovery. However, complete elimination of infection
does not occur.
o Lugol’s solution;
o A cervicitis mixture such as phenol 1 part, tincture of iodine 1 part and glycerine 2
parts, or menthol crystals 1 dram, tincture of iodine 4 ounces and glycerine 4 ounces
 If cervicitis is secondary to vaginitis and pneumovagina, a vulva-suturing operation should be
performed.
 In chronic cervicitis of the external os that is associated with severely prolapsed cervical
rings, the external cervical rings may be amputated. This may be done readily with large
serrated scissors. Following amputation of the cervical rings the vaginal mucosa may be
sutured to the cervical mucosa to help control hemorrhage and promote rapid healing.
Epidural anesthesia is not required as there are no sensory nerves in the cervix.
 Trachelorrhaphy is the suturing of cervical lacerations after calving and partial trachelectomy
is the amputation of a portion of the cervix. However, this operation is probably not necessary
since cervical infections of the external os seldom prevent conception and the internal portion
of the cervix provides a firm seal even though the external os may be ectropic and inflamed.
 Regular estrus periods have as definite a healing effect on cervicitis as they have on
endometritis.
 If cervicitis is severe, sexual rest is indicated for two or more estrus periods.
 Cervicitis may be prevented by the same precautions that can be taken to prevent endometritis
or vaginitis.
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  Trauma to the cervix should be avoided whenever possible.

MARE

 Cervicitis due to a variety of infections is more common and injury to the cervical mucous
membrane can occasionally be serious in nature.
 In cervicitis the mucosa as seen at the external os is usually congested and a dark red to
purple color; the os is generally edematous, pendulous, and dilated.
 In rare cases the cervical glands become cystic; deep suppuration and abscess formation may
occur.
 Fibrosis of the cervix is usually not as pronounced as it may appear clinically, nor as severe as
in the cow with advanced cervicitis.
 The inflammatory process involves mainly the mucosa of the cervix. In rare cases a
membranous or solid, thick adhesion may completely obstruct the cervical lumen and since
uterine infection is usually present, pyometra is likely to develop.
 Severe lacerations, scarring, and malformation of the cervix may occasionally occur in the
mare at the time of parturition and produce a chronic cervicitis or cause a failure of the
normal closure of the cervix by the cervical seal, resulting in repeated early embryonic deaths
due to infection and permanent sterility.

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 SPECIFIC INFECTIOUS DISEASES CAUSING INFERTILITY

COW

BACTERIAL AGENTS

1. CAMPHYLOBACTERIOSIS

 Also known as Vibriosis

 Causative organism is Campylobacter fetus
 The use of artificial insemination has reduced the incidence of the disease because of bull
screening at artificial insemination centers and the use of antibiotics in semen extenders.
 Where natural service is used its venereal route of transmission means that
campylobacteriosis must always be considered as a potential cause of infertility.
 About 90% of infertility due to C.fetus is due to the subspecies venerealis however, the
subspecies fetus can also be involved.

Clinical Signs and Course of Disease

 The bull carries the infection for life and acts simply as a mechanical carrier, transmitting the
infection at service to the female.
o Organism is confined to the glans penis, prepuce and distal urethra, with no lesions.
 The sites of infection in the cow are the vagina, cervix, uterus and uterine tubes.
o No lesions of the vagina, but the organisms can persist in that site for some time.
o Infection causes mild endometritis characterized by peri glandular accumulations of
lymphocytes and the collection of exudates in the uterine lumen
o Fertilization occurs but nidation of the embryo is affected and is followed by early
embryonic death.
 Infection into a susceptible herd results in dramatic decrease in pregnancy rate.
o Embryonic deaths that occur before the maternal recognition of pregnancy causes the
cow to return to estrus within 3 weeks after service.
o Embryonic deaths that occur after recognition of pregnancy result in later, irregular
return to estrus, often between 25 and 35 days after service.
 A small proportion of susceptible cows and heifers may conceive to first service by an
infected bull and may carry their calves to full term.
o Immunity to the organism slowly develops and, as it does so, cows conceive and
remain pregnant.
o After an average of five services, the majority of cows become safely pregnant and
carry their calves to term.
o Most cows which have had normal gestations after breeding by an infected bull will
be free of infection at the time of next service.
 Following a serious infertility spell for about 6 mounts, the affected herd will gradually
become immune
 Maiden heifers, if they are bred to an infected bull, may show low conception rates and
irregular returns to estrus
 Purchased animals, show the effects of the disease during their first season in the herd.
 The majority of the abortions due to C. fetus occur between the fourth and seventh months of
gestation.
o The placenta is often autolysed with lesions are very similar to but less severe than,
those caused by Brucella abortus.
o There is necrosis, with yellowish brown discoloration of the fetal cotyledons and
leather-like thickening or edema of the inter cotyledonary spaces.
o Lesions in the fetus are not specific.

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Diagnosis

 History of regular or irregular return to service, especially if the infertility coincides with the
introduction of a new bull.
 The possibility of defective semen of the newly introduced bull should first be eliminated
 Diagnostic tests used to diagnose C. fetus infection include
o Identification of the organism in preputial washings.
o Direct smears, culture and fluorescent antibody tests.
o Serological tests
o Vaginal mucus agglutination

Identification of the Organism in Preputial Washings

 Preputial washings or scrapings of the penile or preputial mucosa can be examined for the
presence of organisms.
 Phosphate buffered saline will maintain the viability of organisms or a selective enriched
transport medium could be used as an alternative.
 Antibiotics, such as polymyxin B, inhibit the growth of contaminants, which obviates the
need for refrigeration. Even after a delay of 2-5 days, such media can result in good recovery
of the organism.

Direct Culture or Fluorescent Antibody Techniques

 A bull can be declared non-infected after four consecutive negative fluorescent antibody tests.
 Tissues from an aborted fetus and abomasal fluid should be removed aseptically and
maintained at 4° C until they reach the laboratory.
 Direct smears of abomasal contents can be examined using phase contrast or dark field
microscopy.
 Positive cultures are diagnostic, although the fastidiousness of the organism means that
negative results should be interpreted with caution.

Serological Tests

 Serological tests are of little or no value, since genital campylobacteriosis does not produce
measurable serum antibody levels.

Vaginal Mucus Agglutination Test

 Mucus can be collected by a variety of different methods.

 The copious mucus of estrus should be avoided as the agglutinin will be diluted
 The simplest and most effective method in cows, as opposed to heifers, is to insert a clean,
gloved hand into the vagina and to scoop mucus into the palm of the hand from the ventral
fornix which is can be transferred to a wide-mouth collecting bottle.
 The vaginal mucus agglutination test should be used for herd diagnosis rather than for
individual cows. False positives can be obtained if the mucus is contaminated with blood.
 One positive reaction is sufficient to establish a herd infection; for this reason, confirmation
of an infected bull can be made by allowing test mating of two virgin heifers and performing
a mucus agglutination test 60-80 days later.
 A piece of Whatman filter paper is placed on the lateral wall of the vagina cranial to the
urethral opening until it is saturated; secretory immunoglobulin is then detected using enzyme
linked immunosorbent assay.

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Treatment and Control

Control is based on three epidemiological facts:

 Transmission is venereal.
 Bulls remain permanently infected.
 Infected cows overcome the infection, or become immune, in a period of 3-6 months from
service.
o ‘Self-cure’ of the cows will occur if natural service by infected bulls is replaced by
artificial insemination.
o Removal of bulls from the herd
o Regular testing of AI bulls
o Addition of antibiotics to semen diluents
o Using a clean bull on the virgin heifers.
o Breeding the heifers and any non-exposed cows artificially or to a clean bull and to
continue service by the herd bull on the infected group.
o Dihydrostreptomycin at a dose rate of 22 mg/kg subcutaneously, together with the
local application of the same antibiotic to the penis and prepuce, is effective, although
it must be remembered that the bulls will be susceptible to reinfection.
o A combination of neomycin and erythromycin, in a waxy base, is effective in
eliminating C. fetus from bulls in which streptomycin has been ineffective.
o Antibiotics have no beneficial effect in the cow, whether administered locally or
parentally.
o Vaccination should preferably be carried out 30-90 days before breeding commences
and, since the immunity wanes annual revaccination is recommended for optimum
protection as close to the time of service as possible.
o Vaccination has also been used to cure infected bulls. Two doses of vaccine at a
month’s interval, together with annual vaccination programmes, greatly reduces the
incidence of genital vibriosis.

2. BRUCELLOSIS

 Commonly caused by Brucella abortus.

 Brucella melitensis, which occurs in sheep and goats, can also be transmitted to cattle.
 Results in abortion in the second half of pregnancy, together with metritis and retained fetal
membranes.
 In bulls, it causes orchitis, epididymitis, seminal vesiculitis or infection of the ampullae.

Epidemiology

 Infection can be through

o ingestion of B. abortus from contaminated pasture, food or water.
o licking an aborted fetus, infected afterbirth or genital exudates from a recently
aborted or recently calved cow.
o teat by infected milk of another cow
o vagina by infected semen.

 The organism colonises the udder and supramammary lymph nodes of non-pregnant animals
and infected cows often shed the organism in the milk, thereby endangering public health.
 In pregnant animals, production of erythritol within the placenta allows rapid multiplication
of the bacteria, leading to
o endometritis, infection of cotyledons and placentitis
o abortions within 48 -72 hours after death of fetus, by which time a degree of autolysis
has occurred.

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 o retention of fetal membranes.
 Outside the animal body B. abortus may live for months in aborted fetuses or fetal
membranes, but when exposed to drying and sunshine it is soon killed.
 Calves that derive milk-borne infection throw off infection from the lymph glands of the
gastrointestinal tract in 50-80 days.

Clinical Signs

 The disease causes serious economic loss, primarily due to abortion in the second half of
gestation, although earlier abortions occur at the beginning of an outbreak.
 Calves may be born alive but may be weak and unthrifty.
 Infected cows usually abort once and seldom more than twice, although in subsequent
pregnancies the uterus may be re infected from the udder even though the cow carries the
fetus to term.
 Retained fetal membranes, delayed involution of the uterus leading to puerperal metritis.

Diagnosis

 Identification of organisms in stained smears

 Fluorescent antibody technique for direct identification of the organism
 Cultures from fresh afterbirth, or uterine exudates. Technique is time consuming and
expensive.
 A colony blot ELISA using monoclonal antibodies provides a rapid, inexpensive and reliable
method of identifying B. abortus.
 Serological tests such as agglutination test, complement fixation test, antiglobulin test,
fluorescent antibody test and immunodiffusion or electroimmunodiffusion tests using a wide
range of biological materials such as milk, whey, serum, vaginal mucus and semen can be
used to diagnose brucellosis.
 The Rose bengal plate test can be used as an initial screening test of serum samples. All
positive samples are re-examined using the serum agglutination test or complement fixation
test. Rose bengal negative samples are not normally retested.
 A Serum Agglutination Test (SAT) is very widely used but detects non-specific antibodies as
well as specific antibodies from Brucella infection and vaccination.
 The Compliment Fixation Test is a more definitive test than the SAT, especially in
differentiating titres arising from infection from vaccination.
 The Milk Ring Test (MRT), which detects Brucella antibodies in milk, is very useful in
screening the presence of brucellosis in herds by collecting bulk milk samples or in individual
animals. Positive results can then be followed up by using other diagnostic tests on individual
animals.
 The vaginal mucus agglutination test can be used on samples from individual cows but is not
very reliable.

Control

 Brucellosis is not only a cause of abortion in cattle, but it also causes a serious disease,
undulant fever, in man. Hence, control of the disease has to be directed at both its animal
health and its public health aspects.
 From the animal health viewpoint, abortions can be prevented in herds by calfhood
vaccination, using the B. abortus S19 live antigen. But, since this vaccination programme
does not eliminate the infection from cattle, such a method is unsatisfactory from the public
health perspective as there is an on-going risk of undulant fever in those who consume the
raw milk.

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Vaccination

 Vaccinations are being done with Strain 19 a smooth variant of a strain of B. abortus, of
reduced virulence but of high antigenic quality.
 Calves have to be vaccinated between 2 and 10 months of age.
 Vaccination of calves causes a febrile reaction and rapid sero conversion, with titers declining
over the next 12 months in 90% of animals.
 In self-contained herds, calfhood vaccination is sufficient for life, but where adult cattle are
brought in, or in the presence of active infection cows should be revaccinated after their first
calving.
 When infection is introduced into an unvaccinated herd, all adult female stock as well as
calves and cows pregnant up to 4 months, should be vaccinated. The S19 vaccine gives a
better immunity when used on cows rather than calves, but in sexually mature cattle, higher
and more persistent agglutinating titres are produced. Vaccinal titres occurring in adult cows
may be confused with natural infection, but they seldom rise above 1:200.
 It is not usual to vaccinate bull calves, mainly because brucellosis of bulls is uncommon and
also because a vaccinal titre might throw suspicion on the bull and would preclude its
purchase for artificial insemination or for export. In addition, it has been reported that S 19
may produce permanent infection in bulls which is similar to the natural disease, and thus
should not be used.

Eradication

Eradication can be undertaken by a programme of testing and slaughter of seropositive animals.

The brucellosis eradication scheme consists of;

 Positive identification of cows and their calves

 Tracing movement of cattle, so that potential carriers and in-contact animals can be found.
 Securing the boundaries to individual farms or to eradication areas so that uncontrolled
movements of animals are prevented.
 Regular testing of all cows, followed by immediate slaughter of reactors for dairy cows.
 Isolation and testing of any cows that abort or have premature calving.
 In practice, the method of control depends upon the prevalence of the disease. Thus, in
positive herds with no recent history of abortion, repeated herd blood samples should be
taken, and if these disclose inactive infection with a small proportion of reacting animals, it is
advisable to sell the reactors. Further herd blood samplings are undertaken with a view to
obtaining a certificate of freedom from the disease. If there are too many reactors for
immediate disposal to be an economical proposition, the disease is controlled as far as
possible, on the farm; reactors and are strictly isolated when they calve or if they abort.
Rigorous cleaning, disinfection and disposal of infective material is practiced. The complete
isolation of the reactor from 4 days before calving or abortion to 14 days afterwards is the key
to successful reduction in incidence of the disease on the farm. Calfhood vaccination should
be performed in these infected herds. When the incidence of infection is sufficiently reduced,
the reactors may be slaughtered.
 Lastly, in heavily infected herds with current abortion, the spread of infection must be
controlled by
o Isolation of all parturient or aborting animals from 4 days before to 14 days after
parturition.
o Disposal of infected material, thorough cleansing an disinfection after an abortion and
segregation of reactors are practiced.
o Cows in controlled herds should be served only by non-reacting bulls, or inseminated
with semen from Brucella-free bulls.

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 3. BOVINE TUBERCULOSIS

 Bovine tuberculosis infection may reach the tract either by spread from the peritoneum via the
uterine tubes, or by penetration of the serosa, or by bloodstream invasion, in which case the
endometrium may be involved in the absence of serous or tubal lesions.
 Occasionally, primary uterine infections may arise from contaminated instruments or hands
during gynaecological or obstetrical interferences.

Clinical Signs

 Uterine tuberculosis is of three clinical types - peritoneal, glandular and epithelial.

Peritoneal

 The outstanding feature is extensive adhesions of the uterine horns to themselves, the parietal
peritoneum and adjacent organs. The adhesions often contain multiple abscesses, which may
attain several centimeters in diameter.

Glandular

 Glandular layer of the mucous membrane is mainly involved and is characterized by marked
hypertrophy of a diffuse or nodular nature. Caseous or purulent foci of variable size are found
throughout.
 The presence of a vulval discharge varies, depending on the degree to which the mucous
membrane is involved.

Epithelial

 This type generally originates in the bloodstream and the lesions take the form of multiple
pinhead sized granulomata. Often there is no appreciable enlargement of the uterus, but a
vulval discharge, from which acid-fast organisms can readily be isolated. The discharge may
be sero sanguineous or frankly purulent.
 Tuberculosis of the uterus is not an inevitable barrier to reproduction, for quite frequently a
calf is born from a grossly infected uterus. It is probable in such cases that the uterine
infection was acquired or, at least, rapidly developed during pregnancy. The epithelial form is
especially liable to develop after parturition.
 The uterine tubes are frequently involved in tuberculosis of the genital tract. They become
progressively thickened, often attaining a diameter of 1 cm, and may contain local abscesses.
These are generally adhesions of the bursa to the ovary. An ovary itself may be the site of
tuberculous abscesses. The cervix is rarely affected.

Diagnosis

 Tuberculosis of the uterus is not an inevitable barrier to reproduction

 The uterine tubes become progressively thickened, often attaining a diameter of 1 cm, and
may contain local abscesses.
 Adhesions of the bursa to the ovary occurs. An ovary itself may be the site of tuberculous
abscesses. The cervix is rarely affected.
 Diagnosis by rectal examination and the detection of thickened, tortuous tubes is diagnostic.
In advanced cases, diffuse or nodular enlargement of the uterus will be readily detected.

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 4. LEPTOSPIROSIS

 Leptospirosis is an important zoonotic disease of cattle

 Caused by pathogenic spirochaetes of the species Leptospira interrogans.
 The main serovar of L. interrogans, whose maintenance host is cattle, is hardjo. However,
serovars like pomona, canicola, copenhageni, grippotyphosa, icterohaemorrhagiae and
traassovi whose maintenance hosts are species other than cattle are also regularly
encountered.
 Leptospirosis is also of considerable public health importance, as it causes a zoonotic disease
in man

Mode of Transmission

 Infection can enter via skin abrasions or through the mucous membranes of the eye, mouth or
nose.
 Transmitted in semen after natural service or AI.
 Organisms get localized in tissues that are inaccessible to antibodies, notably the kidney
tubules, cotyledons and fetus.
 Leptospires are excreted in urine over a variable period of time, providing a source of
environmental contamination and of direct infection both of other cows and of humans.
Urinary excretion normally occurs for several weeks and it can be for the animal’s lifetime.
 Renal damage can be severe, which is more serious in non-maintenance hosts than in
 Dairy heifers usually become infected at 2-3 years of age, either from older cows or an
infected bull; sometimes they become infected when they are introduced into the main herd
after calving.

Clinical Signs

 The clinical signs of leptospirosis depend upon the infecting organism, the route and dose of
organisms and the immune status of the cow. The role of the bull in the transmission of the
disease is questionable.
 Leptospires can be present in puerperal discharges for up to 8 days and can persist in the
pregnant and non-pregnant uterus for up to 142 and 97 days after infection, respectively.
 An acute febrile disease, characterized by temperatures of 40°C or more, together with
haemoglobinuria, icterus and anorexia is seen.
 Leptospiral mastitis caused by strains such as pomona, canicola, icterohaemorrhagiae and
grippotyphosa may also be present.
 Deaths may occur, especially in calves, and there may be abortions.
 In some herds, abortions have occurred after a ‘leptospiral mastitis’ or agalactia has been
observed during the previous 3 months. Infection causes a bacteraemia with or without
concurrent pyrexia.
 There is a precipitous fall in milk yield, especially in cows that are in early lactation.
 Milk from all four quarters is thick and colostrum like with clots, and is frequently blood-
tinged known as "Gargetty milk".
 The udder is soft and flaccid.
 Agalactia in cows near the end of their lactation may occur without any recovery in milk
production.

Diagnosis

 There are no lesions that are specific for leptospirosis; thus diagnosis of leptospirosis as a
cause of abortion is based almost entirely upon
o demonstrating specific antibodies in fetal sera
o by demonstrating leptospires in fetal organs, particularly lungs, kidneys and adrenal
glands
~ 191 ~
 o by culture or immunofluorescence.
 The MAT is used extensively in the diagnosis of leptospirosis, using serum from animals that
have aborted or are suspected of being infected. Its value is limited in individual animals, but
a herd screening test it is useful for both serovars pomona and hardjo, particularly in herds
where the infection is endemic without clinical signs of the disease, and where certain groups
might be at risk, i.e. heifers, newly purchased animals and farm staff.
 The various categories within the herd, i.e. heifers, dry cows, cows in milk, should be
sampled proportionately. When a partial or herd test reveals hardjo seropositive animals, then
if the titres are below 1:400 and are confined to older animals in the herd which have mixed
freely in the herd, then the infection can probably be considered to be historical rather than
active.
 Where more than 20% of the herd are seropositive or if titres are over 1:1600, then an active
infection is present and further spread of the disease is possible.
 Single samples from individual cows are of little value and it is impossible to separate
infected from vaccinated animals. However, a high titre in a cow at the time of abortion is
generally proof of infection; unfortunately, low titres <1:100 can occur in infected animals.
Paired samples from individual animals are of no value, since there is usually an interval of 6-
12 weeks between infection of the dam and fetal expulsion, by which time the dam’s antibody
titre is either falling, static or not detectable.

Treatment and Control

 General control measure is related to good hygiene, thus minimizing the risk of infection with
leptospires from other host species, should be implemented. These include the strict
segregation of cattle from pigs, rodent control and the draining or fencing off of contaminated
water sources. The role of sheep in the epidemiology of serovar hardjo is still not clear;
however, since they have been shown to excrete the organism in their urine, it seems prudent
not to graze them together.
 There are two methods of specific treatment and control: the use of a vaccine or parenteral
streptomycin/dihydrostreptomycin, or a combination of both. The antibiotic should be used at
a does rate of 25 mg/kg by intramuscular injection with no greater a volume than 20 ml at any
one site. Milk should be withdrawn for 7 days and meat for 28 days. Repeated doses may be
necessary. Streptomycin is effective in clearing pomona from the urine of infected cattle and
treatment with antibiotic plus vaccination has been effective in arresting the progress of an
abortion storm. Dihydrostreptomycin is less effective in treating hardjo, for which other
antibiotics may be preferable.
 In closed herds, vaccination of all members of the herd should be done annually. In open
herds, the frequency should be increased to 6-monthly intervals; this is particularly important
for heifers between 6 months and 3 years of age. Vaccines are based upon bacterins, which
produce relatively low antibody titres, but which confer protection for about 12 months. There
is little or no cross-protection between the main serovars that affect cattle, so the use of
bivalent vaccine hardjo and pomona or trivalent vaccines is common. In situations where the
losses due to leptospirosis are low, vaccination may not be cost-effective. However, the
zoonotic risk of the disease is such that, even when losses are not great, public health
authorities may exert considerable pressure to ensure that susceptible cattle are vaccinated.

5. SALMONELLOSIS

 Salmonellosis induced abortion has been reported from many countries. S. typhimurium is
endemic in cattle throughout the world, but is not a major cause of reproductive failure. S.
typhimurium is endemic in cattle throughout the world, but is not a major cause of
reproductive failure. S. Newport is probably the most common of the ‘exotic’ salmonellae to
infect cattle, but a wide variety of other species are isolated during individual outbreaks.

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Pathogenesis

 Following experimental infection of pregnant heifers with S. Dublin, the organism rapidly
spreads to the liver, spleen, lungs and adjacent lymph nodes of the dam; this is associated
with pyrexia. Six to eight days later it spreads to the placentomes, causing a second bout of
pyrexia. The placentome is damaged, probably by endotoxin, causing necrosis, placental
failure, fetal death and abortion.

Clinical Signs

 The disease is contracted following the grazing of pasture possibly contaminated with slurry
from animal units, human sewage or infected river water. The classical signs of salmonellosis
in adult cattle include a marked pyrexia severe diarrhoea and dysentery, which may be
associated with abortion. More frequently, salmonella abortions occur in late pregnancy in the
absence of any other clinical signs, although malaise, pyrexia and inappetance have also been
recorded.RFM is a common sequel, although there is no adverse effect upon fertility.

Diagnosis

 A definite diagnosis depends upon the isolation of the organism from fetal tissues and
membranes, uterine discharges or vaginal mucus. Serological tests can be used, especially the
SAT, although agglutinins fall to low titres fairly soon after the event.

Control

 Cows that have aborted only excrete the organism for a very short period of time, unlike the
continuous or intermittent excretors that occur following enteric infection. Potential excretors
need to be isolated until vaginal discharge ceases; fetuses and fetal membranes together with
contaminated bedding should be disposed of safely. Adequate cleansing and disinfection of
premises should be performed.
 Vaccination has been used to control salmonellosis. S. Dublin can be controlled by
vaccination with the Strain 51 live vaccine when its use is combined with a closed-herd policy
and effective hygiene measures. Killed vaccines and bacterins have also been used, largely
against S. typhimurium, but their effectiveness has been a matter of debate.

6. LISTERIOSIS

 Listeria monocytogenes is primarily a pathogen of the central nervous system in sheep and
cattle, in which it causes encephalitis. It is consistently, if not frequently, isolated from bovine
abortuses, and is also a cause of abortion in sheep and goats.

Pathogenesis

 The organism gains entry by ingestion or by penetration of mucous membranes of the

respiratory system or conjunctiva, as well as the central nervous system. L. monocytogenes
has a predilection for the placenta, causing placentitis, and affects the fetus to cause abortion.
A latent infection can occur with abortion occurring after a time lag and triggered by stress.

Transmission

 L. monocytogenes is ubiquitous in the environment, being present in the soil, sewage effluent,
bedding and foodstuffs; it persists as it is particularly resistant to the effects of drying,

~ 193 ~
 sunlight and extreme temperature. There is good evidence that there is an association between
listeriosis and the feeding of poor-quality silage of higher-than-normal pH. Cross-infection
between sheep and cattle is possible.

Clinical Signs

 Usually abortions are sporadic, occurring towards the end of gestation. However, there are
rare reports of serious outbreaks, or abortion storms in some herds. In some individuals, there
may be pyrexia before, at the time of or after abortions have occurred. The aborted fetus
frequently has characteristic multiple yellow or grey necrotic foci in the liver and cotyledons,
similar to those described for sheep.

Diagnosis

 This is dependent upon the identification of the organism in the abomasum and liver of the
fetus, and in the placenta and vaginal discharges by a direct smear or by immunofluorescence.
Culture of the organism is not easy, although a series of sub cultures following refrigeration
has proved to be successful. Serological tests are not used in its diagnosis.

Treatment and Control

 The possibility of preventing further abortions occurring in a herd might be considered by

using oxytetracycline or penicillin; however, this is rarely practicable. If silage is being fed
this must be considered to be a potential source of infection and, if possible, withheld from
pregnant cows. There is evidence that some individuals become symptomless carriers,
excreting the organism in faeces and milk.

Bacillus abortion

 It is only in the last decade that abortion due to bacillus spp., in particular B. licheniformis,
has been demonstrated.

Clinical Signs

 Sporadic cases occur in late gestation although there are reports of small outbreaks in two
consecutive years. Sometimes live calves can be born with some evidence of placental
lesions. The placentitis due to B. licheniformis is similar to that following mycotic infection.
The allantochorion is dry, leathery and yellow or yellowish brown in colour. There is often
edema of the allantochorion, especially around the cotyledons, which appears almost as if
there are vesicles present. The cotyledons are haemorrhagic and necrotic. The fetus may be
infected and, if so, there will usually be evidence of a fibrous pleurisy, pericarditis and
peritonitis. There are no systemic signs of disease in the cow.

Diagnosis

 This depends upon the appearance of the placenta and the culture of the Bacillus in placenta
and vaginal swab.

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Transmission and Pathogenesis

 B. licheniformis is ubiquitous; however a common source of infection is silage, especially

when water, other foodstuffs and bedding are contaminated with silage effluent. Wet, spoilt
hay can also be a source.

Control

 Infected silage or hay should not be fed.

Eschericha coli

 Sporadic abortions due to E. coli have been reported. It is suggested that, following stress, the
organism reaches the fetus and placenta via haematogenous spread or ascending the genital
tract.

Mycoplasma

 M. bovigenitalium is found in the vaginal mucus of normal and repeat breeder cows, which
has led to speculation concerning its role as a pathogen. It has, however, been found in cows
of low fertility in which no other cause of infertility could be found. The organism may also
cause granular vulvovaginitis although the evidence for its role in natural occurrences of the
disease is not unequivocal.
 M. bovis causes mastitis in adult cattle and polyarthritis in calves. It is a successful pathogen
of the uterus, causing extensive lesions of the uterus, uterine tubes and even peritonitits.
 Other Mycoplasma species have been isolated occasionally from abortuses, but for these, as
well as for M. bovigenitalium, the evidence for being the initiating cause of abortion is not
clear-cut, since mycoplasmas have frequently been isolated from spontaneously aborted
fetuses.

Ureaplasma diversum

 Ureaplasma diversum is a common inhabitant of the genital tract of the cow. It persists only
briefly in the uterus and uterine tubes, but is most commonly found in the vagina and
vestibule. Differences in virulence of strains probably account for the presence of the
organisms in normal reproductive tracts.
 One of the conditions attributed to U. diversum infection is granular vulvovaginitis. Acute
infection produces granules around the clitoral region and on the lateral walls of the vagina,
which are accompanied by hyperemia of the vulva and a profuse, mucopurulent vaginal
discharge. Large, purulent lesions may also be present, which resemble those of IPV. These
may give way to less obviously inflamed, chronic lesions.
 U. divrsum can also produce endometritis and salphingitis. These lesions have been
associated with high levels of embryonic death and returns to estrus, which are accompanied
by a mucopurulent vaginal discharge. Abortions may also occur, but Ureaplasma may often
be isolated as an incidental finding from calves that have been aborted for other reasons.
Hence, unless there are histological lesions in the abortus that are characteristic of
ureaplasmosis or the presence of a virulent strain is demonstrated, Ureaplasma isolations
should be interpreted with a degree of caution.
 U. diversum can infect the penis and prepuce of the bull and has occasionally been isolated
from all parts of the male tract. It is generally regarded as non-pathogenic in the male,
although some have attributed low-grade lymphoid granulomas on the penile integument to
the presence of the organism.
 The main means of transmission of the infection is by the venereal route. Infected semen used
in AI seems of particular importance, since its deposition into the uterus allows the
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 development of chronic endometritis, rather than of acute vulvovaginitis. However, infection
of virgin females and males has been described and it has been suggested that direct
transmission between females or even transmission by dogs sniffing the vulvas of cows may
occur. Whether it is transmitted between bulls is uncertain.

Acholeoplasma

 Three species of Acholeplasma have been isolated from cattle. A. modicum, A. laidlawii and
A. axanthum. Of these, a laidlawii has been isolated most often, largely from the bull. It is
possible that Acholeplasma infection of cows may cause pathological changes in the genital
tract, but the case is far from proven. It is often isolated from aborted calves, but as described
above, may not be the cause organism. It probably causes no pathological lesions of the bull.

Transmission

 Spread of the organism from infected bulls and resultant infertility have also been
demonstrated. M. bovigenitalium also inhabits many parts of the reproductive tract of the
bull. It has been suggested that the prepuce and urethral orifice are the primary locations of
the organism but it has also been recovered from virtually every part of the male tract. When
it infects the testes or epididymides, M. bovigenitalium may cause detrimental changes to
semen quality, especially after cryopreservation.
 M. bovis has been shown to cause abortion in both natural and experimental infections. Since,
it is seldom found in the reproductive tract of normal cows, isolation of the organism from the
placenta or aborted fetus can be considered significant. M. bovis is found in bovine semen
less often than M. bovigenitalium and its pathogenicity for the bull has not been established.

Diagnosis

 Most bovine mycoplasmas are easily recovered in conventional mycoplasma media, although
some may require special supplements or condition for optimum growth .
 The development of ELISA and other diagnostic tests is likely in the near future.

Treatment and Control

 Natural service, if used, should be suspended and semen should be collected and cultured for
the presence of mycoplasmas. Instead, animals should be inseminated with semen that is
known to be free of contaminant organisms. Infected bulls should be rested for 3 months and
treated systemically for 5 days with tetracyclines, together with sheath irrigation.
 A number of antibiotics have been incorporated in semen for the control of these organisms.
A combination of lincomycin, spectinomycin, tylosin and gentamycin, spectinomycin, added
to raw semen, and non-glycerolated whole milk or egg yolk-based extenders has been shown
to control M. bovis, M. bovigenitalium and Ureaplasma spp. If artificial insemination is used,
the standard Cassou pipette should be protected by a disposable polythene sheath to prevent
vulval or vaginal contamination before it is introduced through the cervix. The uterus can be
infused with a solution containing 1 g of tetracycline or spectinomycin 1 day after
insemination, a treatment that has been shown to improve pregnancy rates.
 Stress, associated with intensive management systems, is said to predispose to the disease;
thus transfer to pasture of affected animals should be considered. This may reduce spread by
direct contagion.

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PROTOZOAL

1. TRICHOMONIASIS

 Trichomoniasis is a classic venereal disease that is transmitted to cows from asymptomatic

carrier bulls during coitus. The causal organism is a flagellate protozoan.

Transmission

 Bulls become infected by serving an infected cow. The infection rate from cows to bulls is
high. Bulls can remain infected for life, remaining asymptomatic throughout. The organism
lives within the crypts and folds of the penile integument. Control of trichomoniasis through
AI can only be achieved if the stud bulls are free of the disease, since trichomoniasis can also
be spread from bull to bull via contaminated artificial vaginas and T. fetus survives
cryopreservation quite well.
 Although the number of trichomonads needed to establish an infection in the cow is large
transmission rates are high. Under conditions of heavy work, the number of trichomonads
present in the preputial area of the bull is reduced, so transmission may be less than 100%, but
under normal conditions, it is common for virtually every cow that is mated by an infected
bull to become infected. In addition to natural service, cows can be infected via insemination
with contaminated semen. Rarely, infection can occur following the use of contaminated
instruments such as vaginal specula.

Clinical Signs

 In the cow, T. fetus colonizes the uterus, cervix and vagina, but it survives poorly in the
vulva. Within the uterus, the organism produces a catarrhal endometritis and vaginitis, with
edema of vulva, perivaginal tissue and uterine wall. It does not generally invade through the
epithelial surface. Affected animals show an intermittent vulval discharge. Manipulation of
the uterus often provokes a discharge from the vulva in which motile trichomonads can
generally be demonstrated. The disease does not prevent fertilization, but causes embryonic
death at an early stage of gestation. Typically, embryonic death occurs after the maternal
recognition of pregnancy causing an irregularly extended return to estrus, although some
animals exhibit normal, or even short, returns to estrus. Many pregnancies fail at between 30
and 50 days of gestation. Embryonic death is not infrequently accompanied by the
development of pyometra, in which the uterus is filled with enormous quantities of
trichomonad filled, thinnish pus. Vaginal discharge with pus is common.
 Many cows experience a series of embryonic deaths before they become pregnant and carry
the calf to term. The return to fertility is dependent upon the development of immunity to the
parasite. However, immunity is slow to develop, for even if the cow is only served once by an
infected bull, subsequent services will not result in successful pregnancies until the
trichomonads have been eliminated from the uterus. Antibody-mediated immunity develops
over several months although antigens to some components of the protozoan are present much
sooner. However, infected cows will conceive to both infected and non-infected services and
eventually carry to term once immunity has developed. Nevertheless, although cows are free
of parasites after a normal gestation, immunity has been lost by the end of gestation, so that
cows again become susceptible to infection from an infected carrier bull.
 Some abortions occur between the second and fourth months of gestation, but very few occur
after the fourth month. In later-term abortions, trichomonads can be found in the chorion, fetal
lung and fetal gut. The fetus is smaller than that appropriate to the period of gestation, due to
growth retardation. In such abortion cases, the fetus, which is grey in colour, is generally
expelled complete in its membranes. There are no signs of putrefaction and T. fetus can
readily be demonstrated in fetal fluids. Parasites quickly disappear from the vaginal
discharges after abortion. Following an abortion the cows
o become pregnant and carry to term without clinical signs of infection developing.

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 o return to multiple services, but show no obvious signs of infection; estrous cycles
may be regular or irregular.
o fail to become pregnant and develop an edematous condition of the endometrium
with a mucoflocculent discharge.
o become pregnant, but abort at 2-4 months of gestation.
o develop pyometra and become acyclic.

Diagnosis

Diagnostic samples

 Diagnosis in the female cow is best achieved by demonstrating the presence of trichomonads
in uterine pus, vaginal discharges, cervical mucus or abortus material. The best source of
material is the fetal membranes or the organs of an aborted fetus. Failure to demonstrate the
presence of the organism does not necessarily imply its earlier absence. The organism also
degenerates very rapidly after death, so unless samples are handled properly, the organisms
may be absent by the time the samples are examined. Materials contaminated with faeces
should be discarded, because non-pathogenic trichomonad-like organisms may be present.
 In the bull, diagnosis is made by the collection of preputial scrapes or preputial washes.
Vigorous scraping of the preputial mucosa was needed to diagnose the presence of
trichomonads. The bull should be allowed a period 5-10 days of sexual rest before sampling
so that the number of trichomonads can increase. Alternatively the presence of the infection in
a bull can be demonstrated using a test mating with a virgin heifer. Cervical mucus should be
collected 10-20 days later to demonstrate the presence of T. fetus by direct examination or
culture.

Demonstration of the organism

 Whatever the source of the material which might contain trichomonads it should be examined
as soon as possible after collection. Preputial washings are centrifuged in order to concentrate
the organisms.
 Various media can be used for culture, including.
o trypticase-yeast extract-maltose
o diamond’s medium for this method, an incubation period of up to 9 days is required
o in-pouch system.

Treatment

 As a general principle, carrier bulls should be culled since, unlike the infection in the female,
it persists indefinitely. However, in a valuable animal whose blood line it is desirous to
maintain, treatment may be considered.
 Treatment of the bull can be attempted by the use of topical substance infused into the
prepuce or applied to the penis. The original method used by Abelein and Swangard involved
the withdrawal of the penis under epidural anaesthesia, bilateral internal pudendal nerve block
or with the aid of a tranquillizing drug, followed by thorough manual application to the penis
and prepuce of an ointment which contained trypaflavine and a protozoacidal agent. Iodine-
based compounds, acriflavine and imidazoles have all been used. Success rates are variable,
elimination of infection is not reliable and the application of such substances is anything but
straightforward.
 Systemic treatment was first attempted by Bartlett who used sodium iodide at a dosage of
5g/45kg body weight in 500 ml water, by intravenous injection on five occasions at 2-day
intervals. More recently, treatment with imidazoles has been reported as both feasible and
effective. Dimetridazole can be given orally but has unpleasant side-effects of rumen stasis,
inappetance and digestive disorders. When given intravenously, different side-effects occur,

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 including respiratory distress, ataxia, short-term recumbency and weakness. In either route of
administration it is however, effective. Metronidazole is also fairly effective. Resistance to the
entire group of imidazoles is easily induced by the use of sub therapeutic doses. A new
antibiotic, trichostatin, has been found to be effective against T. fetus in vitro and in vivo.
 Even when treatment of individual animals is effective, it has no impact upon the presence of
disease in the herd unless other steps are taken to ensure its eradication.
 Treatment of cows is largely unnecessary, as the disease is self-limiting and, indeed, there is
no evidence that the treatment of cows or heifers hastens the time to self-cure. Cows with
pyometra may be induced into estrus with prostaglandin F2a. Intrauterine administration of
Lugol’s iodine, antiseptics or acriflavine at regular intervals has been advocated for many
years as a means of treating trichomoniasis, but is probably useless. Maybe treatment with
imidazoles is occasionally indicated.

Control

 Control can be attempted by:

o eliminating bulls and replacing natural service by AI.
o ‘active’ management of groups of cows and used of bulls
o treatment and/or vaccination of cows and bulls

Artificial insemination

 Control through artificial insemination is based upon the assumption that recovery in the
female is spontaneous, and that infection of healthy animals cannot occur if natural service is
replaced by AI with semen from non-infected bulls. Of all of the available methods, the
elimination of bulls from the herd and AI with uncontaminated semen is by far the most
effective and efficient means of control. The method does require that cows should be bred
exclusively by AI throughout at least one and, preferably, two seasons. Pregnancy rates to AI
are likely to be poor during the initial period of its introduction, since many of the cows may
still be infected.
 It should be noted that elimination of bulls does mean exactly that, all potentially infected
bulls being slaughtered or, if exceptionally valuable, vigorously treated and repeatedly
sampled to ensure that they no longer harbor the parasite. Simply putting bulls away into a
remote paddock for a couple of years does no good at all. They will still be infected at the end
of the period.

Group management

 Many different ideas have been suggested as ways of managing trichomonad- infected herds
without resorting to the total use of AI.
 In principle, when it is established that T. fetus infection exists in a herd, the females should
be grouped as follows:
o Group I (Clean group): Those which are definitively known not to have been exposed
to infection. This group will comprise maiden heifers and any recently calved cows
that have not been served since the introduction of an infected bull.
o Group II: All other cows whose disease-free status is not definitively established.
o Group III: All bulls on the farm should be regarded as being infected, unless
individuals’ disease-free status is beyond debate.
 The ‘clean’ group is bred to known uninfected bulls. The other group can be bred to any bull
until they conceive, after which those bulls are eliminated. After a full-term pregnancy the
Group 2 cows should be free of infection. Absolute separation is a prerequisite for such
scheme to work, although the apparent simplicity of the programme belies the huge practical
difficulties of its implementation.
 An alternative strategy relies upon the limitation of effects of the disease by only using young
bulls for breeding. It is argued that, since 2-year-old bulls are relatively resistant to infection,
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 their use in breeding will result in less spread of the disease than occurs with older bulls. This
may be true, but herd fertility remains below normal. Nevertheless, whilst relying upon the
resistance of young bulls is unlikely to result in elimination of infection, their use may well
help to reduce the level of infection that is present.

Vaccination

 Many attempts have been made to develop a vaccine against T. fetus. Initial work used killed
trichomonads in a mineral oil adjuvant which helped eliminate infection from bulls. However,
most development has been based upon fragmented cells or isolated membrane fractions,
which stimulate a significant antibody response. These too have helped prevent and/or
eliminate infection in cows and bulls.Efficacy of trichomonas vaccines is estimated to be, at
best, 60%. Hence, as the vaccine does not completely protect, it can only be used as an
adjunct to other control or prevention methods.

2. NEOSPORA CANINUM

 Neospora caninum was first discovered as a protozoan parasite which causes

encephalomyelitis of dogs. Neosporosis is now recognized as a significant cause of bovine
abortion in most of the major cattle-producing regions of the world. Infected dams can
produce calves which are apparently normal, but are congenitally infected or which are born
alive with neurologic limb defects. Abortions due to neosporosis can be sporadic, but abortion
storms, in which up to 30% of calves are lost, are also common. Cows can abort in successive
pregnancies.
 The dog is both the definitive host and an intermediate host for the parasite although oocysts
have only been found in the feaces of experimentally infected dogs. Tachyzoites are in neural
and vascular cells, together with a number of other tissues of the body. Tachyzoites are also
found in bovine placental and neural tissue In some outbreaks, vertical transmission has
proved to be the main route by which cattle became infected. However, although the means
by which horizontal transmission could take place are poorly understood, epidemiological
evidence from abortion storms suggests that a point source of infection was implicated.
Routes of horizontal infection could include colostrum, fetal membranes and fluids from
infected cows or oocyst-contaminated feed. None of these routes of infection has been
convincingly proved. However, there is a clear association between the presence of abortion
in dairy cows. On the other hand, post natal sero conversion is uncommon and abortion
storms could originate from previously infected animals which become synchronously
immune suppressed by, for example, BVD infection.
 In consequence of this poor understanding of the mode of transmission of N. caninum, it has
been difficult to devise effective control strategies for the disease. Culling is probably not a
viable option under most circumstances, due to the high prevalence of the disease in some
herds. Prevention of access by dogs to fetal membranes and abortuses may help reduce
horizontal spread, as may prevention of soiling of feed stores by dog faeces. However, there
is little or no epidemiological evidence to show whether such methods are effective. Since
cows that are apparently immune to the disease can still undergo repeat abortions, the
prospect for control of neosporosis is not promising.
 There are several tests which can be used to detect the disease in dairy herds. Immuno-
fluorescent antibody and ELISA tests can detect serological responses against N. caninum.
However, because of the widespread prevalence of seropositive cows, a positive result does
not necessarily indicate infection at the time of testing. Abortion diagnosis should therefore
be made by a combination of serology, with immunohistochemistry and histopathology of
aborted fetuses.

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VIRAL

1. BOVINE VIRAL DIARRHOEA

 BVD was initially recognized as a cause of diarrhoea during the 1940s. Although it was
originally considered to be a simple virus-induced diarrhoea, more recent understanding of
the infection has shown that it also causes infertility. The BVD virus is a pestivirus, which is
related to the viruses of Border disease of sheep and classical swine fever. There are two main
biotypes; a cytopathic and a non-cytopathic strain.

Transmission

 Infection with the non-cytopathic strain in utero between about days 30 and 125 of gestation
leads to the birth of a calf that is persistently infected with the virus. Such calves are immune
tolerant and, if they are subsequently infected with the cytopathic strain of BVD, they may
develop mucosal disease. Persistently infected animals shed the virus throughout life. The
incidence of persistently infected calves is about 1 per 100 - 1000 calves born, but such
animals are a major source of infection and are important in maintaining the BVD virus in
nature Persistently infected cows can transmit the disease vertically through trans placental
infection to their calves, although the majority of persistently infected calves are born to
normal cows that were susceptible to infection during the first 4 months of gestation. Animals
that are persistently affected, or have acute infections, shed large amounts of virus in occulo
nasal discharges, saliva, urine and faeces.
 In countries where vaccination has been used, contaminated modified-virus vaccines or
poorly attenuated modified-live BVD virus vaccines have been responsible for the
introduction of the infectious agent on to a farm. In addition, where virus –contaminated fetal
calf serum has been used in embryo transfer techniques there is also a possibility of disease
transmission.

Pathogenesis

 Infection of cows at other stages of pregnancy causes early embryonic death and abortion,
with aborted fetuses exhibiting abnormalities of the central nervous and ocular systems.
Infection in the last third of pregnancy does not cause immune tolerance, but results in the
birth of a calf that is immune to the disease.
 Infection of susceptible adult animals that are not immune tolerant produces a transient
disease, which is characterized by a period of pyrexia plus a leucopenial viraemia that persists
for up to 15 days. In susceptible herds, there will be diarrhoea, with a high morbidity but low
mortality rate, occulonasal discharge and mouth ulcers. There is usually a drop in milk yield
in dairy cows. The virus has a profound immunosuppressive effect, which can increase the
susceptibility of the host to other diseases. Most adult animals, however, seroconvert without
showing any overt signs of illness. it is the mild clinical form of the disease that is likely to
have the greatest effect upon reproductive function, since the mild pyrexia and modest
mucosal lesions generally go undetected.
 Bulls have been shown to excrete the virus in their semen following spontaneous, persistent
and chronic infection and also following experimental infection. Mucosal disease is usually
seen in younger animals. The disease is characterized by pyrexia, anorexia, watery diarrhoea,
nasal discharge, buccal ulceration and lameness. The morbidity rate is generally low, but,
amongst affected animals, the mortality rate is high.

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Clinical Signs

 The effect of the BVD virus on reproduction depends upon the stage of pregnancy at which
the cow becomes infected. Acute infection, with either biotype, can severely affect the
embryo or fetus. During the first month of gestation, infection results in the death and
resorption of a high proportion of embryos. The only signs of reproductive disease that such
affected cows or heifers exhibit is returning to estrus at normal or extended intervals.
Pregnancy rates are therefore reduced in affected animals.
 Low pregnancy rates also result from the insemination of semen that is contaminated with
BVD virus, whether by AI or natural service and can give rise to the birth of persistently
infected calves. BVD can also be transmitted through virus contaminated embryos.
 From the second to the fourth month of gestation, infection may be followed by abortion,
death with mummification, growth retardation, developmental abnormalities of the central
nervous system and alopecia; some infected cows or heifers will carry calves to term, but
these may well become persistently infected.
 From the fifth and sixth months of gestation, there can be abortion or the birth of calves with
congenital abnormalities of the central nervous system and eyes. Typically, there is a time
interval of between several days and 2 months between infection with BVD virus and
abortion.
 Irrespective of the biotype, infection of the fetus late in pregnancy will lead to the birth of an
immune calf, since the fetus can develop a measurable antibody response to the organism by
5-6 months of gestation However, fetal infection can also be followed by the birth of normal
premature live, stillborn or weakly calves, as well as those with congenital abnormalities.

Diagnosis

 The recent introduction of a persistent infected cow or heifer into a susceptible herd should be
viewed with concern. There may be a history of the overt disease. However, since in most
cases there may only be slight pyrexia, inappetance and respiratory distress which may go
undetected, the first signs are likely to be abortions and birth of congenitally deformed calves.
The fetuses may be fresh, autolysed or mummified.
 The virus can be isolated from the fetus, particularly lymphoid tissue such as the spleen.
Immuno cytochemical identification of kidney, lung or lymphoid tissue, can sometimes be
detected, even though the virus cannot be demonstrated. A substantial rise in neutralizing
antibodies in herds experiencing abortions and the presence of antibodies in the serum of
newborn calves or the thoracic fluids of abortuses is diagnostic of infection. In the case of live
calves, serum must be obtained before colostrums is ingested.

Treatment and Control

 This can be expensive and may not be cost-effective if it requires extensive culling of
persistently infected animals. The basic principles are that farms do not breed from
persistently infected cows; that only immune animals are introduced to the breeding herd –
this can be achieved by deliberate exposure to persistently infected cattle before breeding; and
that any purchased animals introduced into the herd should be screened beforehand. Since
there is some suggestion that cross-infection can occur between cattle and sheep and goats the
species should be separated.
 The absence of antibody titres is generally assumed to indicate the absence of infection. With
BVD this is not the case; a seropositive animal would be a safe purchase but a seronegative
one requires to be free of virus to assure freedom from risk.
 Vaccines are used in many countries as a control measure. Killed-virus vaccines can be used
in pregnant cows; modified-live-virus vaccines cannot. Concern at the use of the latter has
been expressed.

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 2. IBRT

 Infectious bovine rhinotracheitis virus is present world – wide and causes an acute respiratory
disease of cattle with conjunctivitis.It also causes a disease of the genital organs of the bull
and cow, a syndrome that has been recognized for many years, long before the respiratory
form of the disease was described or the causal organism identified; the disease of the genital
system has been variously called infectious pustular vulvovaginitis, vesicular venereal disease
and coital vesicular exanthema. BHV-1 causes both the respiratory and genital forms of the
disease, although the two forms usually occur independently. BHV-1 also causes abortion,
more commonly after the respiratory, rather than the genital, form of the disease. BHV-1
infection is also associated with infertility in cows and heifers.

Transmission

 The genital form of the disease is readily transmitted venerally, but this is not the only route,
since it can occur via contaminated bedding and the mutual licking and sniffing of the vulva
and perineum of infected and non-infected animals.
 Also, it can be transmitted by virus contaminated semen. Once it has gained entry, it is
transported haematogenously in leucocytes.

Pathogenesis

 Some animals can become lifelong latent carriers of the virus, despite the formation of
specific antibodies. The infection enters a latent phase in the ganglion cells of the nervous
system.
 Under certain circumstances, such as stress, calving, transportation, vaccination or
corticosteroid therapy, the latent infection can be reactivated so that the virus migrates along
nerves to the periphery, where it multiplies and is excreted. These animals represent a
reservoir of the virus.

Clinical Signs

Infectious pustular vulvovaginitis

 The onset of vulvovaginitis is sudden and acute. Signs appear 24-48 hour after veneral
transmission; heifers tend to be more severely affected than cows. The vulval labia become
swollen and tender and, in light-skinned animals, deeply congested. This is quickly followed
by the development of numerous red vesicles on the mucosa. These may rapidly rupture or
develop into pustules which give rise to haemorrhagic ulcers, 3 mm or so in diameter.
 The quantity of vulval discharge is variable, ranging from small quantities of exudates, which
adhere to the vulval and tail hairs, to a copious mucopurulent discharge. A speculum is useful
to examine the vaginal mucosa but, because of the pain and discomfort, caudal epidural
anaesthesia is worthwhile. The lesions are obviously painful since affected animals are
restless, with swishing of the tail, frequent urination and straining. There may be transient
pyrexia and reduced milk yield, but the systemic effects are variable depending upon the
presence of respiratory problems. The acute phase of the disease will subside in about 10-14
days, but a few animals will display a persistent vulval discharge for several weeks.
 When females show signs of IPV, the bull must be examined for the presence of lesions,
since, unlike the situation with most veneral diseases of cattle, the signs in the bull are
dramatic

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Infertility

 Opinions have varied over the role of BHV-1 as a cause of infertility. Artificial insemination
of contaminated semen is undoubtedly associated with embryonic death; however, the
evidence for such an effect of natural service by an infected bull is less clear-cut.
 The virus can affect a number of other aspects of reproduction. It can cause a bilateral
necrotizing oophoritis, to which the corpus luteum appears particularly susceptible, especially
during the first few days after ovulation. This damage to the developing corpus luteum may
directly affect its function, perhaps resulting in lower than normal progesterone production. In
consequence, the survival of the embryo would be compromised. The virus can also directly
cause embryonic death, by direct invasion of cells. The consequence is embryonic death, with
the cow returning to estrus at a normal interval after insemination after infection of heifers at
the time of breeding.

Abortion

 Abortion is a common sequel to infection, with or without previous respiratory tract signs of
disease, and also following vaccination with a modified live vaccine. The age of gestation at
the time of infection appears to be critical, since cows that are 51/2 months pregnant, or less,
do not abort, whilst those older than this have a 25% probability of aborting. In beef herds,
abortion ‘storms’ occur, with between 5 and 60% of cow aborting.
 Abortions occur from 4 months of gestation to term. Some calves are stillborn, and a few may
be born alive, but succumb subsequently. The time interval from infection to abortion varies
from a few days to 100 days. in the latter case the fetus is extensively autolysed and may be
reported as being too decomposed for diagnostic work-up. However, even in such cases,
diagnostic lesions are generally present in the fetal liver and adrenal, if a careful search is
made.

Epivag

 ‘Epivag’ is a specific bovine venereal disease causing epididymitis and vaginitis in cattle. It
causes diffuse infection of the vagina, but not the presence of distinct lesion as occur with
IPV. A severe mucopurulent vaginal discharge may be present during the earlier stages of the
disease. Most infected cows fail to conceive to service, and subsequently develop adhesions,
hydrosalpinx and ovarian and bursal adhesions. Likewise, some cows develop parametritis as
a result of Epivag infection and adhesions may be widespread throughout the pelvis and even
extend into the abdomen.

 Most bulls have a mild balanoposthitis after infection, although, since this is far less severe
than IPV infection, it may not be observed. Subsequently, most bulls develop an induration of
the epididymis, particularly of its tail. Orchitis may occasionally occur.

Diagnosis

 The genital tract lesions of IPV are fairly characteristic of the disease, but must be
differentiated from granular vulvovaginitis due to Ureaplasma sp and catarrhal
vaginocervicitis.
 Some investigators consider that a severely autolysed fetus strongly suggests BHV-1
infection. There is frequently a liquefactive necrosis of the whole of the kidney cortex with
peri-renal haemorrhagic edema. Histologically, there is always focal necrosis of the liver and
in many cases there are necrotic lesions in the brain, lungs, spleen, adrenal cortex and lymph
nodes. There are characteristic virus inclusion bodies at the periphery of these necrotic lesions
in fresh experimental cases but, because of autolysis, they are not always demonstrable in

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 field cases of abortion. The virus has been found in all fetal tissue and is concentrated in the
cotyledons.
 Paired serum samples should be taken from the dam at the time of abortion and a second set
of samples 2-4 weeks later. However, since cows may have been infected up to 4 months
before abortion occurs, a significant rise in antibody titres is unlikely to be demonstrated.
Serological examination of paired serum samples from at least 10 cows in the herd should
reveal sero conversion or a four-fold increase in titres if IBR infection is active in the herd.
 For subsequent fluorescent antibody tests, pieces of fetal tissue, particularly kidney and
adrenal gland, should be taken together with a piece of placenta. Such tests that demonstrate
specific focal fluorescence are diagnostic of the disease. Virus isolation is not particularly
reliable but should be used if only placental tissue is available.
 Following the presence of genital lesions, vaginal swabs, preputial washings and semen
should be placed in virus transport medium. Paired serum sample should be taken from the
affected cows.

Treatment and Control

Treatment

 Spontaneous recovery of the genital lesions will occur and therefore treatment is not really
necessary; however, the administration of emollient creams to the vulva, vagina and penis
may be useful. Vulval stenosis and penile/preputial adhesions and phimosis can occur during
the healing phase.

Control

 Infected animals should be isolated and natural service suspended. Vaccination is the most
effective way of controlling the disease; a number of live, attenuated vaccines are available,
often combined with a bovine para influenza virus vaccine. Heifers should be vaccinated after
6 months of age and before their first service; thereafter, annual vaccination is preferable.
Pregnant animals should only be vaccinated with a killed vaccine. Both the intranasal and
intramuscular routes can be used. Vaccination of bull is of questionable value since on they
will be seropositive blood testing and may be rejected for sale as being infected. Routine
examination of semen for the presence of the virus is preferable as a method of control.

3. BLUE TONGUE

 Blue tongue is mainly a disease of sheep and deer, but cattle and wild ruminants are important
reservoir hosts for the virus. The virus is primarily transmitted by insect bites. Culicoides
species are the main vectors and there may be some transmission by ticks, keds and
mosquitoes. Bulls that are infected by blue tongue virus can transmit the virus in their semen.
 In cattle, clinical disease is rarely caused by blue tongue virus but it does have a number of
effects upon bovine reproduction. Infection of susceptible cattle causes a viraemia, during
which the virus can cross the placenta. Infection of the post-hatching embryo can result in its
death and, if susceptible herds are bred during the season of maximal infection with the virus,
seasonal infertility can result. Infection later in pregnancy can lead to abortion or
mummification of the fetus. The neruropathogenicity of the virus produces hydroencephaly
an abnormal contractures of extremities. Calves may be born alive, which are weak and ataxic
or which are persistent carriers of the infection.
 In the aborted fetus, diagnosis of blue tongue can be made by demonstration of central
nervous lesions or by virus isolation from fetal blood, spleen, lung or brain. Serology can be
used to diagnose maternal infection, although the presence of antibody-negative, viraemic
animals during an epizootic outbreak can confuse diagnosis.

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Catarrhal Vaginocavitis

 This contagious, mainly venereally transmitted, disease was first described in South Africa
since then it has been reported in many countries. It is caused by an enterovirus from the
enteric cytopathic bovine orphan (ECBO) group.
 Transmission and Pathogenesis
o Although the disease is transmitted venereally, it can also be spread by faecal
contamination of the vulva, or by animals licking the perineum of infected and non
infected individuals; hence the disease can occur in virgin heifers.
 Clinical Signs
o Affected animals have a profuse, postcoital non-odorous, yellow, mucoid vulval
discharge. The cervix and vagina are inflamed but there are no pustules, such as occur
in IPV infection, and no fever. The typical yellow gelatinous exudate frequently
accumulates in the vagina, varying in quantity from a few to several hundred
milliliters. The disease persists for a few days to a few weeks. Only a few animals
show clinical signs of the disease at any one time. As a consequence, pregnancy rates
are reduced and there are prolonged, irregular returns to estrus, presumably due to
late embryonic death. In some herds, fetal mummification, abortion and stillbirth
have been reported as being a problem.
o Bulls may or may not become clinically infected. The penis and prepuce do not show
the lesions that occur following BHV-1 infection.
 Diagnosis
o The most reliable method of diagnosis is serological examination of paired blood
samples, collected at least 15 days apart, for evidence of rising antibody titres; the
first sample should be collected as soon as possible after the disease is suspected.
o The virus can be isolated from vaginal mucus, but the recovery rate is frequently low.
 Treatment and Control
o There is no specific treatment or vaccine. Infected bulls should not be used for service
for several months, even after clinical signs of disease have disappeared. Potentially
infected animals should be isolated after purchase and, in closed herds; serological
examination of potential additions to the herd might be contemplated.

Parinfluenza3(PI3) Virus Abortion

 This widely distributed virus has been recovered from aborted fetuses in which it caused a
septicaemic disease. Experimentally, it can cause fetal death and abortion after interafetal
inoculation, but after introduction into the maternal respiratory system.
 Vaccines to PI 3 virus are available commercially, often combined with IBR vaccines.
Vaccination can be done during calf hood or in adult cattle to give lifelong protection.

Transmissible Genital Fibropapillomas

 Wart-like tumours commonly occur on the penis of young bull and occasionally similar
growths occur on the vulva, perineum and vestibulovaginal epithelium of heifers. They are
caused by a virus of the papovavirus group and are transmitted by contact with infected
animals.
 These fibropapillomata regress spontaneously in 2-6 months; the speed of regression may be
expedited by the use of a wart vaccine Except in so far as the larger tumours might interfere
mechanically with coitus, they do not cause infertility in female animals.

FUNGAL

 Fungal invasion of the placenta and fetus is a frequent and consistent cause of abortion In
cattle. Abortions are normally sporadic, although in some herds the incidence may be as high

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 as 5-10%.The fungi that are most frequently isolated following abortion are Absidia sp.,
Rhizopus sp and Aspergillus sp.
 Other fungi such as Mortiella wolfii and Petriellidium boydii have also been implicated. In
the northern hemisphere, A fumigatus is the most common cause of abortion, while in the
southern hemisphere, M. wolfii is the most important organism.

Transmission and Pathogenesis

 Many of the species of fungi are ubiquitous in the air and environment in which cattle live;
however, there is good evidence that mouldy hay and straw and other food such as silage and
sugar beet pulp are important sources of infection. Mycotic abortion is most prevalent in the
winter months when cattle are housed. There is still speculation about how the organism
reaches the uterus and infects the placenta and fetus. It is generally agreed that there is
haematogenous spread following entry into the vascular system from the respiratory or
alimentary tracts. There is some evidence that fungal-contaminated semen can cause uterine
lesions although this route is unlikely to be important.
 The fetus and placenta are much more susceptible to mycotic invasion than maternal tissue;
this may be due to growth enhancement of fungi by the products of conception. Once the
fungus has colonized the uterus it probably spreads has colonized the uterus it probably
spreads in two ways: after initial infection of a few placentomes it spreads slowly throughout
the placenta until sufficient is affected to cause abortion, at the same time the mycelium will
invade the fetus and, after initial infection, there is rapid invasion of the placenta with
abortion occurring before the fetus is affected.

Clinical Signs

 Infection does not always cause abortion, since infected live calves can be born. When
abortion occurs, it is usually sporadic in nature, with abortions occurring between 4 and 9
months. The appearance of the lesions on the placenta and the calf are fairly characteristic of
mycotic infection. The whole or part of the placenta usually appears discolored when shed,
and is either grey, yellow or reddish-brown; the intercotyledonary areas of the allantochorion
are thickened, wrinkled or leathery. Those cotyledons that have attached portions of the
corresponding caruncle after the placenta has been shed appear thickened and have a cup-like
or coffee bean appearance. Characteristic fetal skin lesions include circumscribed, grayish-
white thickened patches similar in appearance to skin ringworm in calves and young cattle.
 There are no other clinical signs of disease in the dam associated with abortion due to A.
fumigates. Conversely, although abortion due to M. wolfii is not accompanied by immediate
clinical signs in the dam, a common sequel of abortion is a fatal mycotic pneumonia in the
dam after she has aborted.

Diagnosis

 The appearance of the placenta is fairly typical in fungal abortion, although some bacteria can
produce similar lesions. The fetal skin lesions are almost pathognomonic.
 Laboratory confirmation requires submission of placental tissue, preferably the whole organ
Culture from placental tissue is of no value since the placenta is usually contaminated after it
has been expelled. Culture from fetal lungs and abomasums is more reliable but
contamination can occur.
 The reliable and traditional method of diagnosis is the identification of fungal cells in
histological sections of the placenta. Since, fungal infections are frequently localized,
resulting in focal lesions. Selection of suitable material is important. Another technique is the
potassium hydroxide ‘crush’ mount of non-fixed tissue.
 Conclusive diagnosis of mycotic placentitis can be made if:

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 o the characteristic lesions of placentitis are present in association with the presence of
mycotic elements.
o the characteristic lesions of fetal dermatomycosis are present in association with the
presence of mycotic elements.
o there is a fetal bronchopneumonia associated with mycotic elements.
o Serological tests are at present, unreliable and cannot be used for routine diagnosis.

Control

 The feeding of mouldy forage or the use of mouldy bedding should be avoided.

BOVINE CHLAMYDIAL ABORTION

 C. psittaci is a pathogen of both the male and female bovine genital tract.
 In the bull it affects the testes, epididymides ad other accessory glands. Chlamydial infection
also affects fertility in the cow.

Transmission and Pathogenesis

 It causes orchitis possibly in association with Mycoplasma species. However, it particularly

affects the vesicular glands, where it is believed to be involved in the seminal vesiculitis
syndrome.
 The organism is sometimes excreted in the semen of affected bulls, although it has also been
isolated from bulls that were clinically normal. Chlamydial infection also affects fertility in
the cow. If contaminated semen is used then, after fertilization has occurred, there will be
embryonic death either due to a direct effect upon the embryo or, more likely, via its effect
upon the endometrium. C. psittaci also causes abortion around 7-9 months of gestation
without any other clinical signs.

Clinical Signs

 The lesions following abortion are fairly characteristic. The inter cotyledonary areas of the
placenta are more frequently affected, being thickened and leathery in appearance with a
reddish-white opaque discoloration edema is quite common.
 In the aborted fetus, the liver is enlarged and a mottled reddish-yellow colour. The organism
can be cultured from aborted fetuses and discharges following the use of transport media.
 Giemsa-stained smears for the identification of elementary bodies or inclusions are also
useful. Serological tests such as the CFT have been used but are generally too insensitive. It is
likely that the ELISA tests, used to detect the infection in sheep, will be developed for use in
cattle.

Treatment and Control

 Tetracyclines could be used to treat pregnant cows that have been exposed to infection, but
this it is not really practicable because it requires knowing that the secondary chlamydaemia
has not occurred, and animals must be treated until normal calving.
 Pregnant animals should be segregated from potential sources of infection. Vaccines are
available for use in sheep but none has yet been developed for use in cattle. Following
abortion there should be a natural immunity.

EPIZOOTIC BOVINE ABORTION

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  This disease was first identified in the mid-1950s in California. It is characterized by a high
abortion rate of 30-40% during the last trimester of gestation in cows and heifers newly
introduced to beef herds in particular areas of the states of California, Oregon and Nevada
The dam shows no clinical signs other than abortion.
 Abortions are confined to the habitat of the argasid tick Ornithodoros coriaceus. Hence, it
seems that this tick is the vector for the disease. The causal organism has, however, not been
definitively identified. Early studies suggested that the disease was due to chlamydia psittaci;
however, there is considerable debate about the authenticity of the isolation of the organism
and its role in the pathogenesis. Spirochaetes have also been implicated. It should, however,
be noted that enzootic abortion is a separate disease entity from bovine chlamydial abortion.
 Abortions are seasonal, occurring 100 days or more after exposure to ticks. Once abortions
have occurred, animals are immune, so the cattle which are at greatest risk are those calving
for the first time and animals which have been moved into a tick-infested region. Infection
late in pregnancy can give rise to the birth of live, weak calves. Lesions in aborted fetuses are
characteristic and are used in its diagnosis. Abortuses are not autolvsed, and lymph nodes,
spleen and liver are enlarged, with lymphocytic hyperplasia of most lymphoid organs. Control
is attempted by ensuring that susceptible animals are exposed to ticks before they become
pregnant.

MARE

VIRAL

1. EQUINE HERPES VIRUS

Equine Herpes Virus(EHV)

 EHV is the single most important infectious cause of equine abortion. The disease is caused
by EHV-1 and rarely, EHV-4. EHV-1 is also capable of causing respiratory disease (most
noticeable in foals and yearlings), paralysis, neonatal foal disease and uveitis/hypopyon.
EHV-4 normally causes respiratory disease but occasionally has caused abortion in single
mares. Clinical signs of herpesvirus infection of the respiratory tract are not distinguishable
from those caused by other viruses (and secondary bacterial infection): namely, nasal
discharge, transient pyrexia and depression. The source of the virus is
o Clinically affected animals with nasal secretions
o Aborted fetuses and their membranes
o Infected foals born live at term
o Mares that have aborted, although they only shed virus from the genital tract for a
short period.
o Asymptomatic virus excretors.

 Naturally acquired immunity after EHV-1 infection is short-lived, so that even after only a
few weeks or months, reinfection is possible. Evidence has also been found of latent EHV-1
infection with the virus remaining dormant in the reticuloendothelial cells of clinically normal
animals for an unspecified length of time. Stress can activate the virus.

Clinical Signs

 The majority of EHV-1 abortions occur in the last 4 months of gestation. The mare shows no
signs of impending abortion or clinical disease. The fetus is usually fresh and still enclosed in
its membranes, and typically has excess serosal fluids, minute spots on the liver, jaundice of
the mucous and placental membranes, enlarged spleen, perirenal edema and pulmonary
haemorrhages. Rarely, some foals survive for up to 7 days, but they are weak, jaundiced and
have a marked leucopenia. Histological lesions include foci of necrosis and eosinophilic

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 intranuclear inclusion bodies seen in degenerating hepatocytes and or bronchiolar epithelial
cells.

Diagnosis

 Virus isolation is possible from lung, liver and thymus samples that have been submitted on
viral transport medium. Fluorescent antibody test can be performed on frozen sections of liver
and lung. Unfortunately, there is no test to detect latent carriers.

Control

 Groups of mixed ages and reproductive status are most at risk from virus abortion ; thus
racehorses, hunters, weaned foals and yearlings should be kept away from pregnant mares.
The pregnant mares should be kept in small groups isolated from each other. All abortions
and stillbirths should be investigated and the mare isolated pending the results. Newly arrived
animals should never be mixed with pregnant mares on a farm.
 A killed – virus vaccine and an attenuated live-virus vaccine are available commercially.
However, only the attenuated live-virus vaccine (Duvaxyn EHV 1, 4 ; Fort Dodge Animal
Health) is licensed for the prevention of abortion in mares in the UK, and the manufacturer
advises that it is given during the firth, seventh and ninth months of pregnancy. Results
following vaccination are conflicting. It would seem mostly like that, while vaccination does
not prevent an individual animal aborting, if the stud has a vaccination policy, then the
likelihood of an abortion storm is much reduced.

2. EQUINE VIRAL ARTERITIS

 Equine viral arteritis is a contagious viral disease of the horse first identified in 1993.

Transmission

 The two important routes of EVA transmission are venereal from a stallion with infected
semen, and aerosol via the respiratory secretions of an acutely infected horse. After 7 days of
incubation, EVA is excreted in all bodily secretions, including respiratory secretions and
urine for up to 21 days (possibly longer in urine). The virus may persist indefinitely in the
accessory sex glands in stallions.
 Close or direct contact is required for aerosol transmission to occur. Venereal transmission is
believed to be the major cause of widespread dissemination of the virus. Stallations that
become persistently infected with EVA shed the virus in the semen, which appears to be the
sole route. In breeds that permit the use of artificial insemination, the virus can be transmitted
through the use of fresh, chilled or frozen semen.

Clinical Signs

 The classic clinical signs are an influenza-like illness with pyrexia for 1-5 days, depression, a
nasal discharge, conjunctivitis, anorexia, a focal dermatitis and edema of the limbs, ventral
abdomen, scrotum, prepuce and periorbital regions.
 Abortion may occur during, or shortly after, an acute illness or subclinical infection. Abortion
occurs as a result of myometrial necrosis and oedema leading to placental detachment, and
hence fetal death. Abortions tend to occur in the latter half of pregnancy.

Diagnosis

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  A definitive diagnosis, based on clinical signs alone, is not possible due to their variable
nature. Acute EVA can be confirmed by virus isolation from nasopharyngeal swabs,
heparinised blood samples, and uterine and semen samples. Serological evidence of EVA
exposure can be found by taking an initial serum sample as soon as possible after clinical
onset, followed by a convalescent sample 10-11 days later to detect a rise in the EVA
antibody titre.
 Diagnosis of abortion due to EVA is largely dependent on virus isolation from the placenta or
fetal tissues there are no pathognomonic gross lesions. Mares infected with EVA will usually
abort partially autolysed fetuses, in contrast to fresh fetus aborted by mares infected with
herpesvirus.

Treatment

 At present, there is no effective treatment for a chronically infected stallion. Such animals can
remain persistently infected with the virus in the reproductive tract for variable periods of
time, from several months to a period of years and, in some cases, the lifetime of a particular
statllion.
 Up to now, there is no evidence that mares, geldings or foals that acquire the infection
congenitally become carriers. Since, virtually all acutely infected horses recover uneventfully
after EVA, any treatment is symptomatic.

Control

 A modified live-virus vaccine is available in North America, whereas a killed vaccine

(Artervac, Fort Dodge Animal Health) is available in the UK . It must be remembered that
certain countries will not accept the importation of seropositive animals. If an animal is to be
vaccinated, a blood sample for serology should be taken prior to vaccination.
 A second blood sample should be taken 10 days after the second vaccination to ensure a
serological response to vaccination.

3. EQUINE COITAL EXANTHEMA

 In addition to EHV-1 EHV-4 and equine viral arteritis infection, which cause abortion, EHV-
3 causes a relatively benign venereal disease referred to as coital exanthema; it affects both
sexes. There have been reports of its transfer during gynaecologial examination. The virus can
remain dormant until conditions favour its proliferation with the development of the
characteristic clinical signs.
 Normally, following coitus, they develop after an incubation period of 4-7 days. Multiple
vesicles appear on the vulval mucosa and perineum, resulting in a short period of local
irritation. These rupture leaving small ulcers 3-10 mm in diameter that are painful to touch. In
the absence of infection with opportunist pathogens, healing occurs in 10-14 days, when it
ceases to be contagious. There is permanent loss of pigmentation at the site of the healed
lesions. Pregnancy rates are not reduced. In the stallion, the vesicles develop on the shaft of
the penis and the prepuce; if severe, he may be reluctant to breed. Treatment consists of
immediate sexual rest and the application of an antiseptic powder or spray to prevent
secondary bacterial infection; this allows the ulcers to heal. The disease is controlled by
withholding breeding of all affected stallions and mares and taking hygienic precautions when
handling these animals.

BACTERIAL

 A large number of bacterial species that gain access to the placenta can cause abortion in the
mare. The ascending pathway via the cervix is the primary route of infection, and most
infections occur in early pregnancy. Rarely, bacteria may be in the uterus at the time of
conception or arrive haematogenously. Bacteria that spread rapidly through the allantochorion
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 often infect the fetus, causing acute bacterial septicaemia. More chronic ascending infections
are often localized around the cervical star and cause a focal or local placentitis. The
placentitis often leads to placental insufficiency with abortion of a growth-retarded fetus, or
the birth of a dysmature foal. The placenta is often thickened and covered with exudates and
the fetus septicaemic. Bacteria that cause placentitis are similar to the organisms that cause
endometritis. They are often opportunist pathogens that can be isolated from the caudal
genital tract of normal mares, i.e. Streptococcus spp. and Escherichia coli. Others are
considered to be venereal pathogens, i.e. Pseudomonas spp. and Klebsiella spp.
 Leptospiral abortion is difficult to confirm because there are no clinical signs in the mares in
fetal tissues and the placenta by immunofluorescence, and serology in mares, are needed for a
diagnosis. Treatment of carriers with antibiotics is generally considered useful, but may not
eliminate the shedding of the organism.

FUNGAL

 A large number of bacterial species that gain access to the placenta can cause abortion in the
mare. The ascending pathway via the cervix is the primary route of infection, and most
infections occur in early pregnancy. Rarely, bacteria may be in the uterus at the time of
conception or arrive haematogenously. Bacteria that spread rapidly through the allantochorion
often infect the fetus, causing acute bacterial septicaemia. More chronic ascending infections
are often localized around the cervical star and cause a focal or local placentitis. The
placentitis often leads to placental insufficiency with abortion of a growth-retarded fetus, or
the birth of a dysmature foal. The placenta is often thickened and covered with exudates and
the fetus septicaemic. Bacteria that cause placentitis are similar to the organisms that cause
endometritis. They are often opportunist pathogens that can be isolated from the caudal
genital tract of normal mares, i.e. Streptococcus spp. and Escherichia coli. Others are
considered to be venereal pathogens, i.e. Pseudomonas spp. and Klebsiella spp.
 Leptospiral abortion is difficult to confirm because there are no clinical signs in the mares in
fetal tissues and the placenta by immunofluorescence, and serology in mares, are needed for a
diagnosis. Treatment of carriers with antibiotics is generally considered useful, but may not
eliminate the shedding of the organism.

 PROTOZOAL Trypanosoma equiperdum causes a venereal disease called dourine, which is

currently prevalent in Africa, the Middle East and Central and South America ; it has been
eradicated from Europe and North America.
 The incubation period is 1-4 weeks and the disease has an extremely protracted course that
can extend over a period of weeks of months. It affects horses, mules and donkeys of either
sex.
 The initial sign is a non-painful swelling o the external genitalia of both stallions and mares;
mares show a vaginal discharge and stallions have a paraphimosis. Some weeks later,
depigmented areas and urticuria like raised plaques 2-10 cm in diameter appear over the body
surface. The disease is characterized by a low morbidity, but a high mortality of 50-75%.
 Diagnosis of dourine is made from the clinical signs, particularly the skin plaques, together
with demonstration of the trypanosome in the discharges and in the skin lesions. A
complement fixation test is also available.
 Treatment using quinapyramine sulfate has been attempted, but stallions that recover may
become carriers. Therefore, strict screening using a complement fixation test, with slaughter
of positive and affected animals, as well as institution of quarantine programmes, should be
used to control this disease.

SOW

 An infectious form of infertility can be of great economic significance to a unit. It is vital,

however, to ensure that management and stockmanship are adequate before searching for an
infectious agent in any investigation into infertility. A whole range of management factors

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 (e.g. environment, stress and nutrition) may lower the natural defense mechanisms, rendering
an animal population more susceptible to disease
 The infectious causes of infertility have been classified into three groups.
o Group 1
 Group 1 infections are associated with ubiquitous microorganisms that are
present in the majority of pig populations. Under normal circumstance such
organisms are generally harmless but they may act as opportunist pathogens
when other predisposing factors allow them to gain access to susceptible
reproductive tract. An episode of this type would tend to be sporadic in
nature.
o Group 2
 Group 2 infections result from certain common contagious microorganisms
that are present on a high proportion of pig units, e.g. porcine entero viruses,
and porcine parvovirus (PPV).
o Group 3
 Group 3 infections occur relatively infrequently, but tend to result in severe
reproductive loss, e.g. Leptospirosis and Aujeszky’s disease.

 Group 1 infections are associated with ubiquitous microorganisms that are present in the
majority of pig populations. Under normal circumstance such organisms are generally
harmless but they may act as opportunist pathogens when other predisposing factors allow
them to gain access to susceptible reproductive tract. An episode of this type would tend to be
sporadic in nature. Examples of this type of organism include:

o Escherichia coli
o Erysipelothrix rhusiopathiae
o Listeria sp.
o Mycoplasma sp.
o Salmonella sp
o Klebsiella sp.
o Corynebacterium sp.
o Staphylococcus sp
o Streptococcus sp.
o Campylobacter sp.
 Clinical signs could include conception failure, abortion, stillbirths, perinatal death and
endometritis. Diagnosis and control of Group 1 infections can be difficult due to the
ubiquitous nature of these organisms in normal healthy populations. Control measures must
include removal of all predisposing factors, enhancement of resistance in susceptible animals
and reduction of the weight of infection to exposed individuals. Hygiene in the farrowing
house and at service is particularly important. The boar should not be forgotten as a potential
source of infection.

 Group 2 infections result from certain common contagious microorganisms that are present
on a high proportion of pig units, e.g. porcine entero viruses, and porcine parvovirus (PPV). A
strong immunity to such infection agents is usually developed during early postnatal life.
Such viruses rarely cause clinical disease in adult sows and boars but they are highly
contagious and can spread rapidly through a susceptible population.

 PPV is endemic in most herds and may cause reproductive failures associated with embryonic
death, mummification, stillbirths and subsequent reduction in litter size. The virus has been
recovered from aborted and stillborn piglets, piglets that died soon after birth, and from
vaginal mucus and semen. Infection must occur during the first half of pregnancy in order to
result in disease. Transplacental infection has been demonstrated. Gilts are particularly
susceptible at their first exposure, after which a lifelong immunity will develop. Management
of this disease requires exposure of all gilts to the virus before service by careful integration
into the herd. A more controlled alternative is vaccination. Diagnosis is by serology, and
serological testing also gives an indication of the immune status of the herd.
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  Porcine reproductive respiratory syndrome (PRRS) is a relatively new member of Group2
diseases. It was first recognized in the USA in 1987. The effect on the reproductive
performance of a herd can be devastating. The clinical signs of PRRS are rather variable but
include some or all of the following.

Clinical Signs

Sow

 Inappetance (for 7-10 days), which may appear in waves in herd

 Fever
 Listlessness
 Regular and irregular returns
 Vaginal discharge
 Anestrus
 Abortions (not a major feature)
 Early farrowings
 Stillbirths and mummification
 Poor milking
 Secondary discharges due to cystitis or pyelonephritis
 Sudden death.

Boar

 Lethargy
 Inappetance
 Semen quality – affected for up to 13 weeks, or occasionally on a
 Permanent basis

Piglets

 Weakness
 Puffy eyes
 Lameness
 High pre-and post weaning mortality
 Respiratory signs.
o Gross pathological lesions tend to occur in the respiratory system with a confluent
consolidation of the lungs affecting all lobes. Extensive bronchopneumonia and
occasionally fibrous pleuritis are also features of this disease. The most likely method
of spread of the disease is by the introduction of infected pigs on to the premises,
although local airborne spread between herds has been suspected over distance of up
to 3 km. The virus has been isolated in boar semen, and infection has followed
insemination with infected semen.
o The serious economic impact of this disease results from its devastating effect on
herd productivity in terms of farrowing rate, number of live piglets born, pre-and post
weaning mortality and performance of surviving piglets.

Treatment

 Treatment involves the use of antibiotics. Herds need to adapt their gilt intake programme to
ensure that exposure to the virus occurs at least 4-5 weeks prior to

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 Group 3 infections occur relatively infrequently, but tend to result in severe reproductive
loss, e.g. Leptospirosis and Aujeszky’s disease.

 Reproductive losses from leptospirosis have been reported on a world-wide basis. The
causative agents are a variety of spirochetes belonging to the genus Leptospira. Sero groups
of greatest importance to pig populations are australis, pomona and tarrasovi. Incidental
infections in pigs may also result from canicola, icterohaemorrhagiae, autumnalis, hardjo,
mozdak and muenchen. The epidemiology of the disease is complicated by the fact that some
strains are specifically adapted to the pig, and others to dogs, horses, hedgehogs and other
wildlife.
 The most important route of infection is thought to be via the mucous membranes of the eye,
mouth, nose or vagina. A bacteraemia occurs 1-2 days after infection, may last for a week and
coincides with acute clinical disease.
 Presenting signs include anorexia, pyrexia and listlessness. Primary signs of chronic
Leptospirosis are abortions, stillbirths and birth of weak piglet. Leptospires can localize in the
kidneys, multiply at this site and appear in the urine in varying degrees of intensity and for
different lengths of time. Leptospires can also localize in the uterus of pregnant sows; when
this happens in the last half of gestation abortions and stillbirths often result, occurring 1-4
weeks after infection Usually, diagnosis of leptospirosis is based on serology using the
microscopic agglutination test (MAT), although the presence of seropositive animals in the
herd does not always result in clinical signs. Demonstration of leptospires in the fetus
provides a definitive diagnosis of leptospiral abortion.
 Control of leptospirosis depends upon the combined use of antibiotic therapy and
management. Systemic streptomycin at 25 mg/kg body weight or oral tetracyclines at levels
of 800g per tone of feed have been used to eliminate carriers, although this type of strategy is
not always successful. The main management factor involves prevention of contact between
pig populations and other domestic stock or wildlife, which can be difficult to achieve,
particularly on outdoor units. Vaccination is an option in some parts of the world, although
vaccines are not available in many countries in Western Europe.
 The causative agent of Aujeszky’s disease (or pseudorabies ) is a herpesvirus. Aujeszky’s
infection usually gains access to the pig by inhalation or ingestion of the virus. It may also be
transmitted by coitus although there is some argument as to whether true venereal
transmission occurs. Aujeszky’s disease is characterized by nervous and respiratory signs
associated with a rise in temperature and often death in young piglets. Infection in adults may
result in stillbirths and abortion. In adult boars and sows, the clinical signs of this disease are
seldom severe and usually consist of pyrexia, depression and anorexia that last for up to a
week. Of great significance to the breeding herd is the fact that the virus causes embryonic
death, fetal mummification and stillbirths.
 Brucella suis is a widespread infection of pigs in the USA but has not appeared in Great
Britain. In countries where it does occur, it should always be considered as a cause of herd
infertility or abortion. Pigs of both sexes are much more susceptible to infection after
weaning. Once infection has been introduced into a susceptible herd (usually by pig
movements), it spreads quite rapidly by ingestion or by venereal transmission. An infected
animal suffers an initial generalized bacteraemia similar to undulant fever in humans, which
may last for several weeks or months. Service by an infected boar results in uterine infection,
although establishment and proliferation of the organism do not appear to interfere with
fertilization. Abortion is the most significant effect of venereal infection and can occur at any
stage during pregnancy, although the rate is highest when infection occurs at the time of
breeding. Sows usually abort only once.
 There is also a higher incidence of stillborn and weakly piglets. In sexually mature boars,
infection can localize in the testis, resulting in clinical orchitis with consequent impairment of
spermatogenesis, loss of libido and infertility. Poor reproductive performance of the boars
exacerbates the overall infertility. Herd diagnosis is made by means of a complement fixation
test (CFT) or a serum agglutination test (SAT). B suis may be isolated from aborted fetuses.
 There is no treatment for swine brucellosis, nor is there any means of conferring artificial
immunity. In infected commercial herds, all pigs should be slaughtered as they reach a
suitable marketable weight and the unit left empty for 6 months before restocking. In the case
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 of a valuable breeding herd, depopulation maybe out of the question. In such situations all
pigs are assumed infected, and a clean herd built up by isolating the piglets at birth and
retaining those that pass the agglutination test at weaning age. The public health issues should
be borne in mind as this is an important zoonotic disease.
 Other viruses that interfere with gestation include swine fever (hog cholera), foot and mouth
disease, classical swine influenza, transmissible gastroenteritis, Japanese B encephalitis and
Japanese haemagglutinating virus. Experimental infection with attenuated classical swine
fever virus has caused various effects that vary according to the stage of gestation at which
sows were inoculated. The enteroviruses have been isolated from two serologically distinct
groups (A and B) with an epizootic disease of pigs characterized by stillbirths (S),
mummification (M), embryonic death (ED) and infertility (I) (‘SMEDI viruses’).

EWE

1. ENZOOTIC ABORTION OF EWE

 EAE also known as Ovine enzootic abortion or Kebbing is caused by Chlamydia psittaci
immunotype 1 (recently reclassified as Chlamydophila abortus), which has a predilection for
the pregnant uterus. Chlamydia psittaci immunotype 1 has a highly specialized life-cycle that
involves alternate intra-and extracellular phases that confer advantages for evasion of host
immune responses and facilitates the maintenance of low-grade asymptomatic infection.

Transmission

 The major source of infection, responsible for over 80% of new outbreaks in lean flocks, is
the purchase of infected ewes of any age. Spread may also be by wildlife, e.g. foxes, gulls and
crows. Sheep-to-sheep spread is the commonest route, and lambing time is the greatest time
of risk when infected ewes shed large numbers of infectious particles into the environment.
Susceptible ewes inhale or ingest chlamydiae from infected placentae, uterine discharges,
dead lambs and contaminated bedding. Infective particles (elementary bodies) may survive
for weeks at low environmental temperatures. Ewes infected early in pregnancy usually abort;
otherwise the Chlamydia lie dormant until the next pregnancy. Chlamydia is not transmitted
in the milk of infected ewes. However, lambs may acquire infection from uterine discharge on
the teats.

Clinical Signs

 Chlamydia acquired outwith pregnancy lie dormant. However, they can be reactivated from
their ‘latent’ state during pregnancy. Neither the site of latency nor the precise triggers of
reactivation have been identified. The tonsil and lymphoid tissue of the pharynx has been
shown to be a primary site of infection, with subsequent blood-borne spread to major organs
and lymph nodes. Rapid replication of C. psittaci leads to local necrosis and contagious
spread of infection involving the cotyledonary and intercotyledonary placenta and apposing
endometrium, resulting in abortion that usually occurs in the last 2 weeks of pregnancy. The
macroscopic signs of a placentitis are similar to that following Brucella abortus infection in
cattle. The intercotyledonary allantochorion is edematous, thickened and leathery in
appearance; there is degeneration and necrosis of the fetal cotyledons and a thick yellow
deposit on the chorion.
 Abortion occurs 40-50 days after being infected; however, those ewes infected late in
pregnancy do not abort until the following pregnancy. Infected bought-in sheep may abort in
the first year spreading infection at lambing time to susceptible ewes and lambs, resulting in
an abortion storm the following year.
 Most aborted lambs are well developed, fresh and show no autolytic changes, indicative of
recent death in utero; some infected ewes may produce both dead and live lambs. However,
lambs born alive may be weak, fail to survive and in spite of good nursing contribute to the

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 overall losses from EAE. The disease is extremely rare in hill flocks, unless housed for
lambing in facilities previously used by infected lowland flocks.

 Although rams can become infected and may develop epididymitis, there is no evidence that
they play any significant role in the transmission of EAE.

Diagnosis

Clinical signs

 There are no premonitory sings of impending abortion. Ewes are not ill. However, a few ewes
may show evidence of a vaginal discharge for several days beforehand and possibly
behavioural changes. There may be abortions, premature lambs, weakly live lambs and
normal lambs with infected membranes. Ewes may retain fetal membranes leading to metritis,
but not other clinical signs are seen.

Placental lesions and staining

 The placenta is usually acutely inflamed, thickened and necrosed showing typical signs of a
placentitis. Smears from infected intercotyledonary areas, and the wet skin of the fetus, can be
stained by the modified Ziehl-Neilsen method to detect intracellular inclusion bodies, which
occur as small acid-fast cocci; they may be seen intracellulary as clumps, or singularly
scattered throughout the smear; these may be confused with coxiella burnetti organisms,
which are larger.

Serology

 The demonstration of specific chlamydial antibody in fetal fluids or precolostral lamb serum
with fluorescent antibody test is specific evidence of infection.
 The complement fixation test is the routine diagnostic test used, a titre of over 64 generally
being accepted as positive. Paired samples should be taken, at the time of abortion and 3-4
weeks post aborting; in positive ewes samples show a significant rise in antibody titres.
Vaccinated ewes will have lower titres with no evidence of a rise. An enzyme linked
immunosorbent assay (ELISA) and indirect immunofluorescent antibody test are also
available.

Treatment

 Antibiotics that will reduce rather than eliminate abortions can be used in flocks with
extended lambing seasons. For the best results, treatment should be given as soon after 95-
100 days of gestation as possible, at which time possible cases of placental infection will have
commenced. Although it is expensive, long-acting oxytetracycline, at a dose of 20 mg/kg
repeated every 10-14 days until lambing, has been used. This treatment will reduce the
number of organisms shed, but does not eliminate infection nor can it reverse pathological
changes already present in a heavily infected placenta; hence some abortions will still occur
despite treatment.

Control

 Following diagnosis of EAE

o Isolate, for up to 3 weeks, and mark all ewes that abort.
o Send dead lambs and membranes to a laboratory for diagnosis.
o Reduce risk of spread to other ewes.
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 o Burn or bury dead lambs and membranes not needed for diagnosis.
o Clean lambing area and cover with clean straw.
o Discourage use of ewes to foster lambs, as infection may be picked up from vaginal
discharges and infected fleeces. If lambs are fostered they should not be used for
breeding.
 In following years consider vaccination policy and/or strategic use of oxytetracycline.
 Ewes that have acquired infection do not develop positive titres until they abort; therefore it is
not possible to screen a flock to detect latent infection.

Protection by Vaccination

 Enzovac (Intervet UK), which contain a temperature-sensitive strain of Chlamydia psittacti,

requires a 2-month period after injection to develop ‘protective’ antibody levels. Vaccination
can be used from 5 months of age, and also in older animals between 1 and 4 months pre-
tupping. The vaccine will protect lambs from transplacental infection. High-risk flocks, viz.
with >5% abortions per year, and sheep bought from non-accredited flocks should have
vaccination repeated yearly or biannually. Low-risk flocks, viz. where<5% abortions occur,
and sheep bought in only from accredited flocks need to be vaccinated once only.
 If EAE is not present. Strenuous efforts must be made to prevent the disease gaining entry
using the following management strategies:
o Maintain a closed flock and purchase rams from known sources, or replacements for
EAE-monitored flocks.
o Purchased ewes should be lambed separately from the indigenous flock in the first
year, and all abortions and barren ewes should be investigated.

Zoonotic Risks

 C. psittaci from sheep can be extremely dangerous to pregnant women, growing rapidly in the
unborn baby’s placenta. Initial mild influenza-like symptoms become progressively more
severe, and abortion occurs within a week.
 Disseminate intravascular coagulation may develop in the mother causing critical illness.
Intensive nursing normally results in complete recovery, but regrettably, to date all the babies
have died.

2. TOXOPLASMOSIS

 Toxoplasma gondii infection is the second most common cause of abortion in ewes. Infection
in non-pregnant sheep is typically mild and in apparent, but in pregnancy it is essentially a
disease of the conceptus.
 The causal organism has a complex life cycle, involving an asexual cycle that can occur in
any species of mammal or bird, and a sexual cycle that can only be completed in cats and wild
Felidae. In the cat family, the parasites multiplies within the epithelial cells of the intestine
and, as a consequence, oocysts will be excreted in the faeces for about 8 days, during which
time tens of thousands of oocysts can e shed. These sporulate within a few days and are then
ingested by sheep.

Epidemiology

 The principal vector in the spread of toxoplasmosis is the cat and its related wild species.
Infective toxoplasma oocysts may survive up to 2 years on pasture, feed or bedding they are
passed in the faeces of young cats, who become infected when they first being to hunt.
Although toxoplasms have been demonstrated in the semen of experimentally and naturally
infected rams infection of the ewe at tupping would be unlikely to cause abortion.

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  Toxoplasms can also be passed in milk during acute infections. Lateral spread within a flock
from aborting ewes is likely to be relatively unimportant. However, lambs born alive that
survive from infected ewes can be congenitally infected. As few as 200 oocysts will infect
one ewe, and as many as 1 million may be present in 1 g of cat faeces. Once ingested
sporozoites are released, they penetrate the intestines and are distributed to many organs
where tissue cysts form, a febrile response occurs after 5-12 days in conjunction with a
parasitaemia. Toxoplasma can be detected in the uterine caruncular septa 10 days after oocyst
ingestion, and placental trophoblast cells after 10-15 days; toxoplasma-specific fetal antibody
is present after 30 days.

Transmission and Pathogenesis

 Without doubt, the principal vector in the spread of toxoplasmosis is the cat and its related
wild species. They excrete oocysts in their faeces and contaminate pasture, forage and other
foodstuffs. Young cats and older breeding cats are the main problem. Old neutered cats on a
farm are helpful, not only in reducing levels of vermin, but in keeping feral cats away.
Foodstuffs should be protected from faecal contamination by cats. Wild rodents, especially
mice, are the main source of infection for cats.
 Infection of ewes outside pregnancy produces a good immunity. Replacement ewes should be
exposed to the farm environment as soon as possible after purchase, so that they can ingest
oocysts and acquire immunity before tupping.
 Toxoplasma gondii can affect humans but usually does not cause clinical disease. The
exceptions are women who become infected for the first time during pregnancy. Placental and
fetal infection results in severe damage to the unborn child.

Clinical Signs

 The effect upon reproduction depends upon the stage of pregnancy when infection occurs. If
early in gestation, i.e. before 60-70 days, fetal resorption usually occurs, with ewes returning
to estrus or remaining barren. Unlike EAE, numerous barren ewes, which have not been seen
to abort, may be detected in the flock at scanning or lambing a ram is still present and the
breeding season has not ended, ewes are capable of conceiving, now with a good immunity.
Infection in mid-gestation results in abortion or mummification; in the latter case, only one
member of a set of twins or triplets may be involved. Infection after 120 days usually results
in stillbirth, or weakly or normal lambs.
 The gross appearance of the placenta, particularly the cotyledons, is fairly typical of the
disease. Cotyledons are bright-to dark-red in colour with multiple small white nodules 1-3
mm in diameter. These nodules may be sparse or so numerous that they become confluent;
sometimes normal cotyledons are present. The inter cotyledonary areas of the allantochorion
appear normal (Unlike infection with C. psittaci).

Diagnosis

 The condition is characterized by barren ewes, abortions, stillbirths, and mummified and
weakly lambs. The appearance of the placenta is diagnostic.
 Confirmation can sometimes be made using Giemsa or Leishman-stained smears of those
cotyledons containing the white nodules. Alternatively, histological sections of the cotyledons
may be required to demonstrate the presence of the parasite. Examination of the brain,
especially in those lambs that die soon after birth, may reveal foci of glial cells and
leuconephalomalacia, which are characteristic of the infection. Immunofluorescent staining of
cotyledon sections can also be used.
 A number of satisfactory serological tests on the maternal serum have been used including the
dye test of Sabin and Feldman, the indirect fluorescent antibody (IFA) test,
radioimmunoassay and the ELISA test. The ELISA test has been modified to detect anti-

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 toxoplasma immunoglobulin G (LgG) in body fluids. A single serum sample with an elevated
titre may well only indicate past infection.
 Serology of ewes is difficult to interpret as antibodies remain elevated for years. However,
paired samples may prove beneficial. Those taken at a 14-day interval and showing a rising
titre are indicative of an active infection.
 Infected lambs will have precolostral antibodies, and serology can be performed on pleural,
pericardial, or peritoneal fetal fluids or lamb serum, providing that the lambs have not
received colostrum. If postcolostral samples only are available, it is necessary to demonstrate
IgM and IgG antibody.

Treatment and Control

 Chemoprophylaxis with monensin given in the food at the rate of 15 mg/animal /day during
pregnancy can significantly suppress toxoplasma infection in sheep. Decowuinate, the
anticoccidial drug fed daily at 2 mg/kg body weight, also significantly reduces the effect of T.
gondii oocysts ingested by pregnant sheep. Both these products work best if they are being
fed at the time sheep encounter infection rather than after infection has been established.
 Ewes can treated during the acute phase of the disease with sulfonamides and potentiated
sulfonamides such as trimethoprim. A recent study has demonstrated the efficacy of a
combination of sulfamethazine and pyrimethamine, which is used to treat the disease in
humans.
 Although there is virtually no danger of lateral spread from aborting ewes, isolation in the
early stages of an abortion outbreak should be implemented, since there is a possibility that
other infectious agents may also be present.
 Ewes that have aborted will be immune and should be retained in the flock, and new additions
should be exposed to possible infection with oocysts from contaminated food as early as
possible before the start of the breeding season.

3. CAMPYLOBACTERIOSIS

 Campylobacter is the third most common cause of abortion in the UK and although both C.
fetus fetus and C. jejuni cause abortion in ewes, the former is the main organism isolated.

Epidemiology

 Infected and often symptomless animals excrete these organisms in faeces. C.jejuni is mainly
from a wildlife source and C. fetus from carrier sheep. Unlike in cattle, where the route of
infection is mainly venereal, in sheep it is by ingestion and mainly intestinal. Once abortions
occur, there is lateral spread to other susceptible pregnant ewes. The organism survives well
in cold moist conditions but soon perishes in hot, dry weather. The only clinical signs of the
disease are abortion, usually in the last 6 weeks of gestation; lambs at full term may be born
dead or in weak condition. Apart from some vulval swelling, and the presence of a reddish –
coloured vulval discharge, ewes rarely show any other clinical signs. Metritis may develop
after abortion, some ewes becoming ill and even dying. Aborted material is infective. Ewes
infected <3months into pregnancy are not affected. If ewes are infected >3 months into
pregnancy, a bacteraemia develops with placentitis being the main lesion. In late pregnancy,
abortion occurs between 1 and 3 weeks after infection. Flocks infected for the first time may
have levels of abortion varying from 5% to 50%. There is a strong immunity after infection,
but this is serotype-specific. Symptomless carriers may excrete the organism for up to 18
months.

Diagnosis

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  There are signs of placentitis with edema and necrosis of the fetal cotyledons. However, these
are not pathognomonic. The aborted fetus looks fresh with no specific gross pathology, and in
approximately 25% of the aborted fetuses, there are characteristic necrotic foci of 10-20mm
diameter in the liver.
o Disease is commonest in young ewes, or older ewes that have lost their immunity.
o The organism can be identified in Gram-or modified Ziehi-Neelsen-stained smears
from the placenta and fetal stomach contents and cultured from the placenta, fetal
stomach or liver.
o Serology is not useful.

Treatment and Control

 As soon as campylobacteriosis is suspected, aborting ewes should be isolated from pregnant

ewes. If extensive lateral spread is a possibility, pregnant ewes should be treated with
intramuscular injections of 300 000 IU of penicillin and 1 g of dihydrostreptomycin on 2
consecutive days.
o Dispose of aborted materials as the disease is zoonotic. Avoid infecting sheep over 3
months in lamb, or wildlife that can later act as reservoirs of infection.
o When campylobacteriosis has been confirmed, mix aborted ewes with those already
lambed to stimulate production of a strong immunity. The disease is self-limiting.
And most ewes from an infected flock acquire immunity in the first year, irrespective
of whether or not they have aborted. This acquired immunity lasts about 3 years, in
most circumstances, equal to the expected breeding life of the ewe.
o Turn over feed troughs, preventing birds from feeding at them.
o Try to keep the flock closed. Bought-in ewes should be mixed with the resident sheep
for as long as possible before mid-pregnancy, then separated in late pregnancy and
lambed separately.

4. SALMONELOSIS

 Several serotypes cause ovine abortion including S. abortus ovis, S. typhimurium, S. dublin
and S. montevideo. Occasionally exotic strains are isolated, and are usually associated with
imported foreign protein.

Epidemiology

 Many species of animal, including humans, may act as sources of infection, which, in early
pregnancy, may result in barren ewes and, in late pregnancy, abortions, and dead and weak
lambs. And those that recover may act as symptomless carriers.

Clinical Signs

 These will vary with the serotype of organism and are summarized below.
 S. abortus ovis- This is a host-specific strain, rarely isolated. Few systemic clinical signs
occur in ewes other than abortion, which usually occurs in the last 6 weeks of pregnancy.
Symptom less carriers are often a problem.
 Two distinct clinical pictures may be observed in the lambs
o They may be born weak and die within a few hours of birth.
o They may be born healthy, suddenly become ill and die in the first 10 days of life
 S. montevideo- Apart from abortion, there has been little evidence of systemic illness.
 S. typhimurium- The clinical picture is totally different to those previously described.
Anorexia, pyrexia (up to 106°F) and profuse scour are usual. A foul-smelling vaginal
discharge may be present. Death may occur from septicemia or dehydration in 6-9 days.
Lambs may be born dead, and those that are born alive may show signs of severe illness with

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 diarrhea and a high mortality rate. Severe losses amongst the ewes are not unusual. Numerous
observers have commented that the disease often follows times of stress, e.g. after gathering,
winter shearing, housing and vaccination.
 S. Dublin. The clinical signs are not normally as severe in both ewes and lambs as with S.
typhimurium infection. Death is usually from septicemia or dehydration, and mortality rates
are usually much lower.

Diagnosis

 It is important to stress that Salmonella sp., other than S. abortus ovis, are zoonotic, hence
care should be exercised when dealing with infected material.
 The clinical picture is that of scouring and pyrexic ewes. Before, or in association with,
abortion a foul-smelling vaginal discharge may be present. Lambs born alive may become ill
with fatal septicemia or pneumonia, especially in cases of S. typhimurium and S. Dublin
infection.
 Identification of the organism follows culture of fetal stomach contents, placental tissue or
vaginal discharges. In addition, fluorescent antibody techniques may also be used for the
rapid diagnosis of organisms in the same tissues.
 Serological tests can be used to diagnose infection with S. abortus ovis.

Treatment and Control

 A number of general principles have been suggested for the control of S. Montevideo that are
applicable to other Salmonella species.
o Isolate affected sheep that have aborted, or scoured profusely, and treat with an
antibiotic to which the salmonellae are sensitive, thus limiting excretion of the
organism.
o Keep aborted ewes separate from those yet to lamb.
o If birds are possibly the source of infection, then turn over feed troughs when not in
use, regularly change the feeding area and avoid feeding on the ground.
o Avoid stress situations arising within the flock, such as frequent moving, and make
sure that foodstuffs are freely available with sufficient trough space to avoid
competitiveness.
o Try to prevent sheep drinking from streams and open ditches by using piped fresh
water.

5. LISTERIOSIS

 Listeria have a world-wide distribution in several domestic species. Two strains of Listeria
produce disease in sheep, L. monocytogenes and L. ivanovii, and the disease may be
presented in one or more of the following forms:
o Encephalitis
o Abortion
o Diarrhea and septicemia
o Keratoconjunctivitis and mastitis
o Septicemia and death in young lambs.
 Neurological signs characteristic of Listerial encephalitis are circling with evidence of
unilateral facial paralysis, head tilting, and turning. Abortions in sheep are produced by both
L. moncytogenes and L. ivanovii, and although they may occur at any stage they are most
frequent in late pregnancy.
 Initially, sheep may be pyrexic. There are no distinguishing characteristics to the abortion,
and usually no typical complications following expulsion of the fetus, which may be
autolysed. Weakly lambs are often born, and grey/white focal necrosis may be seen in the
fetal liver (so-called ‘sawdust liver’). The placental villi are necrotic and chorion is covered

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 with brownish red exudates; there is a heavy brown vaginal discharge and death of the ewe
occurs from metritis or septicemia.

 Several forms of the disease may be present in the flock at the same time. In one outbreak,
diarrhoea and septicemia occurred 2 days after the commencement of silage feeding, and
although the silage was removed, encephalitis was seen 4 weeks later followed by abortions.
 These signs were not seen in the same ewes, and it is rare for the nervous disease and
abortions to occur together.

Diagnosis

 Diagnosis is based on isolation of the organism from vaginal swabs, fetal membranes or the
fetus, and fetal liver lesions.
 Fluorescent antibody techniques have been used whilst inoculation of mice with necrotic liver
and the production of keratitis in rabbits are additional tests.

Transmission and Pathogenesis

 L. monocytogenes is ubiquitous, frequently found in soil but also isolated from foodstuffs and
faeces of healthy animals. Soil is the most likely source of infection.
 Especially following the feeding of soil contaminated silage where poor fermentation has
occurred. Sheep are probably frequently exposed to infection, but presumably it requires
some other factors to precipitate clinical Listeriosis.
 Following ingestion in late pregnancy, the organism penetrates the gut mucosa and infects the
fetus, causing a septicemia and placentitis both of which may kill the fetal lamb. As a
consequence, abortion occurs.

Treatment

 The organisms are sensitive to a wide range of antibiotics that may prove beneficial if given
to ewes that have aborted and are discharging.

Control

 All aborted ewes should be isolated and the abortion site cleaned up.
 In Iceland, listeriosis is called ‘silage disease’. And it has been suggested that the feeding of
silage appears to make ewes more susceptible to Listeriosis. Hence, while feeding silage the
following precautions are to taken
o Use of additives to reduce the pH of the silage; make high-quality silage with pH
below 5, avoiding gross soil contamination (where the ash content exceeds 70 mg/kg
dry matter), i.e. avoid mole hills and having forage harvesters set too low.
o Compact and seal silage the same day as made. Making sure that round bales are
securely tied and not punctured.
o Avoid feeding obviously mouldy or poor quality silage that can be smelt or has come
from the top o sides of the pit.
o Remove any silage that has not been eaten by sheep after 48 hours.
o On farms where Listeriosis occurs annually it would be advisable not to graze any
fields with livestock that were intended for silage-making.

6. BORDER DISEASE

 This disease was first recognized in flocks along the English –Welsh border in the 1950s
affecting newborn lambs that showed neurological symptoms such as tremor and a coarse

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 fleece (so-called ‘hairy shakers)’ and were generally weak with a high mortality rate. Since
then, it has been recognized in many other places in the UK and has also been shown to cause
reproductive failure.

Etiology

 The disease is due to infection with a pestivirus similar to that which causes bovine viral
diarrhoea (BVD) in cattle and European swine fever.

Clinical Signs

 In adult ewes, infection results in a mild pyrexia that would probably go undiagnosed.
However, if ewes are pregnant, the virus will affect the fetus causing fetal death with
mummification, abortion or, if early on in fetal life, death with resorption, or the birth of
weakly affected lambs.
 Abortion can occur at any stage of gestation, although it is most common around 90 days with
the voiding of a brown, mummified or swollen anasarca fetus.
 The conceptus is most susceptible to experimental infection between 16 and 80 days of
gestation. Hence, if infection occurs at an early stage, the only clinical sign will be
barrenness.

Diagnosis

 This can be made on clinical signs in lambs, supported by histopathological examination of

the brain and spinal cord, virus isolation from the lamb, or a fluorescent antibody staining
technique serological tests on ewes that abort, or are barren, have to be examined in relation
to antibody levels in other ewes in the flock.

Transmission

 Pestiviruses from other species can also, under experimental conditions, cause border disease
in sheep, so that among domestic animals, cattle in particular, but also goats, represent
potential sources of infection.
 However, the most likely source of infection is from ewe lambs that have recovered from
border disease being introduced into the flock. These individuals remain chronic excretors of
the virus for a long period of time, yet appear healthy.
 Although likely to have reduced fertility, they can give birth to infected progeny that
themselves are a source of infection. Some ram lambs can excrete the virus in their semen.

Treatment and Control

 There is no treatment and, as yet, there is no commercially available vaccine, although one
may become available eventually. The disease is best controlled by ensuring that the flock
remains closed and hence the disease does not gain entry.
 Once it is present in a flock it is important, in the early stages of the outbreak, to attempt to
segregate pregnant ewes from those that have given birth to clinically affected lambs.
 At the same time, to ensure that nonpregnant ewes develop immunity, they should be exposed
to infection, and thereafter, any surviving lambs from the infected flock should not be
retained for breeding and be sent for slaughter, so that symptomless carriers do not remain.

7. LEPTOSPIROSIS

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  The disease occurs in late gestation and the immediate postpartum period, resulting in severe
losses for the farmer. It usually occurs during two short periods of time when sheep are
physiologically immune compromised, viz. in ewes 2 weeks before lambing, and in neonatal
lambs in the first week of life.

Epidemiology and Transmission

 The disease is not seen in traditionally managed hill flocks, but it occurs when they are
bought as replacements for intensively reared and housed lowland flocks. Reproductive
wastage occurs during the first lambing season, but not in subsequent years. There is still
debate over whether or not sheep are a maintenance host for the infection, or whether there is
a requirement for cattle as an established maintenance host to be closely involved.
Nevertheless, it is generally recognized as good practice, in the control of the disease, to
minimize contact between the two species.

Clinical Signs

 The clinical sign in adult ewes is reproductive wastage in the form of late term abortion,
stillbirth and the birth of weak lambs.

Diagnosis

 In cases of abortion, stillbirth or weakly lambs diagnosis is based on the isolation of the
organism from aborted fetuses or fetal membranes, its demonstration, by a fluorescent
antibody technique, in the same tissues or placenta, or the presence of a rising antibody titre
in paired blood samples.

Control

 Control is by vaccination, using a quarter of the cattle dose of L. hardjo vaccine before
tupping with a repeat dose in 2-4 weeks. Those ewes that have not aborted when an abortion
storm occurs can be treated with 25mg/kg of dihydrostreptomycin as a single dose.

8. BRUCELLOSIS

 Sheep can be infected with both Brucella melitensis and B. ovis.

 B. melitensis is primarily a disease of sheep and goats, but can affect other species, including
man (Malta fever). Transmission is by direct ingestion of the products of abortion or drinking
infected milk. It causes abortions, stillbirths or weak lambs in late pregnancy.
 The placental lesions are similar to those identified with B. abortus infection in cattle, and
diagnosis is by direct examination or culture of placental smears, fetal stomach contents or
vaginal discharges. Serological tests, such as the complement fixation test, are used. The
disease can be controlled by using B meliensis or B. abortus S19 vaccines.
 B. ovis is host-specific. Its main effect is upon the ram, where it causes an epididymitis and
subsequent infertility or sterility (see Chapter 30). Following experimental infection of the
ewe, the organism causes a placentitis followed later in gestation by abortion or the birth of
small weak lambs. However, under field conditions, abortion rarely occurs, and according to
Hartley et al. (1954), if it does occur, the incidence is usually low (7-10%). Whilst a ram can
be infected after serving a ewe previously served by an infected ram, the ewes themselves are
not infected venereally. The method of infection of ewes is not fully understood.

9. Q-FEVER

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  This is due to infection with the Rickettsia, Coxiella burnetii. Although of little significance
in sheep farming, it has been associated with a small number of outbreaks of ovine abortion in
the Uk Its main importance is its public health implications, producing influenza-like
symptoms, pneumonia and cardiac lesions in humans, and thus it is important that the agent is
recognized when fetal material is examined to avoid the possibility of human infection.
Microscopically, it may easily be mistaken for B. abortus or Chlamydia.
 Placenta and vaginal discharges are heavily contaminated, allowing spread of infection at
parturition or later by aerosol from the fleece or dust in the lambing are, acting as a source for
human infection.

10. UREAPLASMOSIS

 Ureaplasma sp. have been isolated from normal ewes and from ewes with granular vulvitis.
There is some suggestion that they are common inhabitants of the urogenital tract of sheep.
 Ureaplasmas have been identified as a cause of infertility and abortions in cattle and in other
non-domestic species, and may have a possible role in infertility and abortion in sheep.The
ram has been implicated as a major distributor of infection.

11. TICK BORNE FEVER

 This is limited to those areas where the sheep tick Ixodes ricinus occurs, since almost all ticks
harbor the infective agent Cytoecetes phagoctophilia. The disease is known in the southwest
of England, Scotlan, Scandinavia, the Netherlands and South Africa. Abortion in adult sheep,
previously unexposed to ticks, occurs naturally following tick-borne fever infection in late
pregnancy, usually 2-8 days after the commencement of a fever, which can be as high as 107
°F. A proportion of pregnant ewes may die. Some fetuses may die in utero, become
mummified and be expelled weeks later. Recovery in non-pregnant ewes is generally
uneventful.

Diagnosis

 Diagnosis can be fonfirmed by identifying the organism in the leucocytes of ewes that have
aborted, or during the septicaemic phase.

Treatment

 Oxytetracycline can be used for the rest of the naive flock.

Control

 Infected ewes that survive develop an immunity, whilst the majority of all sheep from tick-
infected areas will have acquired an immunity at an early age. Newly purchased non-
acclimatised sheep should be introduced to farms before tupping, preferably when tick
numbers are at their highest. Ticks can also be controlled by adopting the appropriate dipping
routine.

DOE

BRUCELLOSIS

 Brucella melitensis is the organism most frequently involved and is endemic in many
Mediterranean countries, Africa and Central America; it does not occur in the UK. B. abortus
occasionally causes abortion but B. ovis has not been isolated from goats.

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  B. melitensis causes abortion in late pregnancy, stillbirths or weakly kids; following the first
exposure, abortion may be in the form of a storm. Few abortions occur in other years, and
some does may become sterile from uterine lesions. Kids may become infected from does’
milk. The disease can be diagnosed by bacterial culture from the fetus, fetal membranes or
vaginal discharges, and can be controlled by routine vaccination. It is important to remember
that B. melitensis is of zoonotic importance.

CAMPYLOBACTERIOSIS

 This is uncommon. It is caused by infection with Campylobacter sp., probably C. jejuni and
possibly C. fetus. Does may or may not show evidence of a systemic illness, and may abort in
late gestation or produce stillborn or weakly kids, and have a post –abortion muco-or
sanguineous purulent discharge.
 Multiple necrotic foci upto 2 mm in diameter may be seen in the liver of the aborted kids.
Diagnosis, treatment and control are similar to those described for sheep.

CHLAMYDIAL (ENZOOTIC) ABORTION

 This is an important cause of infertility in goats in many countries, and is the commonest
cause of infectious goat abortion in the USA. Infection is due to Chlamydia psittaci which is
similar to, or identical with, the strain responsible for enzootic abortion in sheep.
 Abortions usually occur in the last 4 weeks of gestation, with levels as high as 25-60% in
does kidding for the first time; stillborn and weakly kids can also occur.
 Diagnosis and treatment are similar to that described for sheep. Does infected in late
pregnancy usually abort during the subsequent pregnancy and can produce infected kids
which, after a latent phase, abort during their first pregnancy. The disease is best controlled
by good hygiene to prevent lateral spread to susceptible animals, especially young does, and
the use of a vaccine, which has been made compulsory in some countries where the disease
has become widespread.

Leptospirosis

 Although it is not a frequently diagnosed cause of abortion there are reports in the literature of
abortion with Leptospira grippotyphosa infection. There is usually systemic illness preceding
the abortion associated with septicemia, but clinical signs of icterus are not often present.
 Diagnosis is based upon identification of the organism and serological tests. The disease can
be treated in the acute phase with streptomycin, but it is doubtful if this will prevent abortion
occurring. Control measures, involving the use of vaccines, might be tried in an outbreak.

Listeriosis

 Encephalitis, due to Listeria monocytogenes infection, is quite common in goats. The same
organism can cause abortion in late gestation or stillbirth. The does may show no signs before
aborting, but may develop a necrotic metritis and vaginal discharge shortly afterwards.
 As in sheep and cattle, poorly fermented, soil contaminated silage is a likely source of
infection. The pathogenesis, diagnosis, treatment and control are similar to those described for
sheep.

Salmonellosis

 There are no host-specific Salmonella species in the goat. However, the ubiquitous
salmonellae have been reported to cause abortion.

Toxoplasmosis
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  Toxoplasma gondii is the commonest cause of infectious goat abortion in the UK. It causes
fetal death with resorption if infection occurs early in gestation, or abortion of kids, which
may be stillborn, alive but weak or normal, depending upon the time in pregnancy that the
doe was exposed to infection. However, unlike in sheep, fetal death is preceded in some cases
by a period of severe illness with pyrexia, anorexia, diarrhoea and muscle weakness. During
an acute infection, toxoplasma may be excreted in the milk and be a source of human
infection if drunk unpasteurized.
 The placental lesions are very similar to those described in sheep. Diagnosis is dependent
upon identification of the organism in placental tissue or serological tests. Aborting does
develop immunity, and should be retained within the herd whilst young, non-pregnant does
should be exposed to infection before they become pregnant.
 Domestic cats wild felidae play a critical role in the spread or the disease as in sheep.
 Treatment and control measures are similar to those described for sheep.
 T. gondii is of zoonotic importance and thus care should be taken in handling possibly
infected material.

Q Fever

 Q fever, caused by Coxiella burnetii, can cause abortion and stillbirth in goats, without
previous clinical signs or following a few day’s illness involving dullness, depression and
anorexia, the abortion rate can be very high in some infected herds. Large numbers of
organisms are expelled into the environment from placental tissue, uterine fluids colostrums
and milk. Ticks have also been implicated in the spread of Q fever, and may be the initial
means of introduction into the herd.
 Diagnosis is made upon identification of the organism in smears of the placenta or the organs
of the abortus, and serological tests demonstrating a rising antibody titre.
 There is no vaccine, and does can remain chronic carriers of the organism. C. burnetii is a
zoonosis and is excreted in milk.

Mycoplasmosis

 A number of Mycoplasma species have been identified as causing abortion.

BITCH

NORMAL VAGINAL BACTERIAL FLORA

 There is a widespread belief among breeders and veterinarians that infertility, vaginitis and
fading puppy syndrome are caused by bacteria that inhabit the reproductive tract of the dog
and bitch.
 Many aerobic and anaerobic bacteria normally inhabit the vestibule and vagina of the healthy
bitch and the bacterial flora is normally mixed. The aerobic bacteria isolated from normal
bitches include Escherichia coli, staphylococci and streptococci whilst the anaerobic bacteria
include Bacteroides sp. and Streptococcus spp. Mycoplasmas have been isolated from
between 30 and 88% of normal bitches Greater numbers of bacteria are found within the
vestibule compared with the vagina; the uterus is normally sterile the stage of the estrus cycle
may influence the bacterial flora, because there is a significant increase in vaginal bacterial
numbers when estrogen concentrations are elevated. The bacterial species cultured from
infertile bitches did not differ significantly from healthy bitches and hence, the result of
microbiological examination of the reproductive tract of the bitch must be treated with
caution because the simple isolation of bacteria from the vagina does not constitute a
diagnosis of reproductive disease.

OPPORTUNIST PATHOGENS

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  The bacterial species found in bitches with from those found in healthy bitches. However,
disease may result if the uterine or vaginal defense mechanisms are depressed, thereby
allowing overgrowth of the normal commensals. Many of the normal vaginal inhabitants may
become pathogens if a breakdown in local immunity occurs.
 Bacteria may enter the uterus during estrus when the cervix is relaxed and could then cause
infertility either directly by interfering with the zygote or by producing spermicidal factors.
Bacteria might persist within the uterus and be associated with the development of pyometra
during the progesterone-dominant phase of the cycle.
 If vaginal microbiological sampling reveals bacteria present in a pure growth or in very large
numbers, then they may be considered significant, although pure growths of bacteria may also
be isolated from normal dogs. Repeated culture after 1 week should be performed to confirm
the diagnosis before attempting treatment. Appropriate antimicrobial therapy, based upon
sensitivity tests, should only be administered after investigation of possible predisposing
causes such as anatomical, neoplastic or mechanical abnormalities of the vagina. Parenteral
and topical administration has been advocated.
 Mycoplasmas and ureaplasmas have been implicated in causing reproductive disease in the
bitch although they are also frequently isolated in clinically normal animals with no evidence
of reproductive tract disease. Mycoplasma colonization of the vagina has recently been
demonstrated following prolonged treatment of bitches with oral ampicillin and potentiated
sulfonamides which suggests that the widespread use of antimicrobial agents in healthy
bitches should be avoided.

Brucella canis

 B. canis is a Gram-positive bacterium that can produce abortion and infertility. It is the only
bacterium known to be a specific cause of infertility in the bitch. Brucella infertility was first
reported in the USA but has subsequently been found in several countries. B. canis can be
transmitted in several ways, including contact with aborted fetal or placental tissue, contact
with the vaginal discharge of infected bitches, venereal transmission and congenital infection.
The most common method of infection is venereal. Abortion occurs most commonly between
days 45 and 55 of pregnancy; however, there may be early fetal resorption, or the birth of
stillborn or more rarely weak pups.
 The isolation of the bacterium from blood or aborted tissue is diagnostic of the disease;
however, there may be prolonged periods when the bitch is not bacteraemic, so that a negative
blood culture does not rule out infection. Fortunately diagnosis, using the plate agglutination
test for screening and tube agglutination for confirmation, is not difficult, Titres of 1:200 or
greater being diagnostic of infection. Treatment of the condition with a combination of
streptomycin and tetracycline is often effective in clinical cases; however, antimicrobial
treatment does not remove the organism from tissues. Since a carrier state can occur and these
animals may be potential sources of infection, they are best neutered to remove them for the
breeding programme.

Toxoplasma gondii

 T. gondii infection causes abortion, premature birth, stillbirth and neonatal death. Surviving
infected pups may carry the infection. The public health consequences of Toxoplasma
infection should be considered whenever it is diagnosed.

Canine herpesvirus

 Canine herpesvirus in adult dogs generally produces a few mild signs limited to the
respiratory or genital tract. However, the virus may cause genital lesions in the bitch that may
be associated with infertility, abortion and stillbirths.
 It appears that infection of the pregnant bitch results in the production of placental lesion and
the infection of the fetuses. The infected placentae are macroscopically underdeveloped, and
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 possess small grayish white foci characterized by focal degeneration, necrosis and the
presence of eosinophilic intranuclear inclusion bodies. Experimental data suggest that
infection during early pregnancy may result in fetal death and subsequent mummification,
whilst infection during mid pregnancy results in abortion and infection during late pregnancy
results in premature birth.
 Virus has also been recovered from vesicular lesions on the genitalia of bitches. Variable-
sized vesicles are commonly observed in the vestibule and frequently these lesions are evident
at the onset of pro-estrus, suggesting that venereal transmission is probably important in adult
dogs. Virus shedding from the vesicular lesions, may be stimulated by the stress of pregnancy
and parturition.
 Pups may become infected at birth, during passage through the vagina, and subsequently die
with characteristic widespread histological necrotizing lesions. Pups that survive the illness
may show persistent neurological disorders. Pups are only at risk whilst in utero and during
the first 3 weeks of life; attempts to produce the generalized disease in older pups have failed.
In the pups, the disease is rapidly fatal and treatment is often unrewarding; symptomatic
therapy is all that is available since specific antiviral agents are not efficacious.

Canine adenovirus

 It is well established that infection with canine adenovirus during pregnancy can result in the
birth of dead or weak pups that die within a few days of parturition.
 In most cases, however, the virus is ingested and causes neonatal mortality. Carrier bitches
may therefore act as a source of infection for pups.

Canine distemper virus

 Experimental exposure of pregnant bitches to canine distemper virus was found to produce
either clinical illness in the bitch with subsequent abortion, or subclinical infection of the
bitch and the birth of clinically affected pups. This provides evidence for transplacental
transmission, although the frequency of this under natural conditions is unknown.

Canine parvovirus

 Canine parvovirus has been implicated by some breeders as a cause of infertility in their
kennels.
 Canine parvovirus may cause an acute generalized infection in pups less than 2 weeks of age,
which can occur as a consequence of uterine infection or as a result of exposure to the virus
soon after birth.

QUEEN

OPPORTUNIST PATHOGEN

 The vestibule and vagina of the queen are normally inhabited by many aerobic and anaerobic
bacteria that enter the uterus at mating, and subsequently cause abortion because the
progesterone-dominated uterine environment allows them to proliferate. It is not clear,
however, whether bacteria isolated from aborted fetuses have caused the abortion, or whether
they have invaded the uterus after dilatation of the cervix at the time of the abortion.
 Bacteria commonly isolated include E. coli, staphylococci, streptococci, salmonellae and
mycobacterium.
 Immediately prior to an abortion, the queen may become pyrexic and lethargic.
 Treatment includes the administration of broad- spectrum antimicrobial agents, fluid therapy
and drugs to stimulate uterine evacuation. Hysterotomy to remove fetal tissue is rarely
necessary, although ovariohysterectomy may be required should a severe metritis develop.

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Feline Leukaemia Virus (FeLV)

 Feline Leukaemia Virus (FeLV) has been implicated in a variety of clinical syndromes
including infertility, embryonic resorption and abortion. FeLV is believed to be the single
most common cause of infertility in the queen. Fetal resorption is seen frequently, although
abortion and the birth of permanently infected kittens also occur. The aetiology of the
reproductive disease is uncertain, and whilst it is known that the virus may cross the placenta,
one possibility is that secondary bacterial infections occur because of FeLV induced
immunosuppression.
 Diagnosis of FeLV may be achieved by virus isolation; owners should be discouraged from
breeding from FeLV- positive queens since all offspring are born persistently infected. These
kittens usually develop a FeLV-related disease soon after birth. Vaccines are now available
that provide protection against FeLV and its related diseases.

Feline Herpesvirus

 Feline herpesvirus I may result in abortion during the 5th or 6th week of gestation. Lesions
may be found within the uterus; however, placental lesions have only been demonstrated
following experimental infection. In the naturally occurring disease, abortions are thought to
be the result of a non-specific reaction to the infection Transmission of the virus occurs via
the respiratory tract, with up to 80% of cats remaining as chronic carriers.
 The diagnosis of herpes virus infection is based upon the clinical signs and the isolation of
virus. Vaccination of queens provides good immunity and should be recommended for all
breeding animals.

Feline Panleucopenia Virus

 Feline panleucopenia virus is transmitted by direct contact with salvia, faeces and urine.
Infection of pregnant queens may result in abortion, stillbirths, neonatal deaths and fetal
cerebellar hypoplasia. These effects are the result of transplacental infection leading to fetal
death and resorption in early pregnancy and cerebellar hypoplasia when infection occurs from
the middle third of pregnancy onwards. Diagnosis may be made on the basis of the clinical
signs, histopathological findings, virus isolation and paired serum samples that demonstrate a
rising antibody titre. There is no treatment for kittens with cerebellar hypoplasia.

Feline Infectious Peritonitis Virus

 Feline infectious peritonitis virus has been implicated as a cause of infertility, stillbirths,
endometritis, resorption and abortion, chronic upper respiratory tract disease and fading kitten
syndrome. Queens are not always ill and may suffer resorption of abortion which is
unnoticed. Abortion generally occurs during the last 2 weeks of pregnancy. Diagnosis is made
by serological and pathological investigation.

Toxoplasmosis

 Toxoplasmosis has been incriminated as a rare cause of abortion and congenital infection of
cats. Serological screening is necessary to demonstrate the role of this protozoan in cases of
abortion.

Chlamydiae

 There is evidence that the feline strain of Chlamydia psittaci causes abortion in the queen.
The mode of transmission has not been elucidated, although the organism has been isolated
~ 231 ~
 from the genital tract of infected cats and there is circumstantial evidence associating
infection with reproductive disease as well as abortion. Diagnosis is possible by
demonstrating high antibody titres. It is difficult to confirm whether the isolation of this
organism indicates its role in an abortion, since it may simply be an opportunistic bacterium.

FERTILITY PARAMETERS

Reproductive traits Goal

Calving interval 365-380 days
Avg. days to 1st observed heat Less than 40 days
% cows in heat by 60 days post calving Greater than 90
Avg. days open to 1st breeding 50-60 days
Avg. days open to conception 85-100 days
Services/conception 1.5-1.7
First service conception rate
A) replacements 65-70%
B) producing females 55-60%
% breeding intervals between 18-24 days Greater than 85%
% cows open greater than 120 days Less than 10%
Dry period length 45-60 days
Avg. age at 1st calving* 24 months
Avg. age at 1st breeding* 15 months
% cows pregnant less than or equal to 3 Al/services 90%
% cows pregnant on examination 80-85%
Abortion rate Less than 5%
Cull rate for infertility Less than 10%

Non return to first estrus

 This is the percentage of cows or heifers, in a particular group, which have not been presented
for a repeat insemination within a specific period of time.
 The periods are usually 30-60 days or 49 days.
 This is used, particularly in artificial insemination centers, to monitor the fertility of bulls and
the performance of inseminators.

Calving interval and calving index

 The calving interval is the interval in days for an individual cow between successive calvings;
the calving index is the mean calving interval of all the cows in a herd at a specific point in
time, calculated retrospectively from their most recent calving date. These two measurements
have been used traditionally as a measure of fertility, since they indicate how closely the
individual cow or herd approximates to the accepted optimum of 365 days.
 The disadvantages of these measurements are that they are historical in that they are
calculated retrospectively; furthermore, the calving index can give an overoptimistic
assessment of fertility when many of the cows that fail to become pregnant are culled.

Calving to conception interval

 The calving interval (or index CI) is the sum of two components, (a) the interval from the last
calving date to the date of conception and (b) the length of gestation thus:
o CI = a + b
 Therefore
o CI = 85 days + 280 days = 365 days

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  The calving to conception interval (CCI) is calculated by counting the number of days from
calving to the service which resulted in pregnancy (effective service); this is usually the last
recorded service date. The CCI is a useful measurement of fertility but requires a positive
diagnosis of pregnancy to be made. It is influenced by two factors; how soon after calving the
cows are served or inseminated and how readily they become pregnant when they have been
served.
 The CCI can be expressed thus:
o Mean CCI = c +d
 Where ‘c’ is the mean calving to first service interval and ‘d’ is the mean first
service to conception interval, therefore
o Mean CCI = 65 days + 20- days = 85 days.
 The mean CCI is a useful measure of fertility, provided that the interval from calving to first
service is stated, since this probably will have the greatest influence upon its length.

Days open

 This is defined as the interval, in days, from calving to the subsequent effective service date
of those cows that conceive, and from calving to culling or death for those cows that did not
conceive.
 Numerically, it will always be greater than the mean CCI unless all cows that are served
conceive, in which case it would be the same.

Days open (%) = (Total service period in days in all lactations /Total herd life in days) x 100

Calving to first service interval

 In the case of a herd that calves all the year round a mean value of 65 days should result in a
mean CCI of 85 days.The factors that influence the calving to first service interval are:
o Breeding policy of the farm. Although cows will return to estrus after calving as early
as 2-3 weeks, they should not be served before 45 days, and in the case of first
calving, high-yielding cows and those that have had dystocia and problems during the
puerperium slightly longer time should elapse. Thus, in a seasonal calving herd, those
that calve early in the season will have their first service delayed and, for those that
calve late, it may be necessary to advance the date of first service thereby tightening
the calving pattern
o Delayed return of cyclical activity after calving, i.e. true anestrus
o Failure to detect estrus in those cows that have resumed normal cyclical activity.
 Factors (2) and (3) can be improved by ensuring that cows have returned to cyclical activity
postpartum. This can be done by regular and routine examination of those cows, per rectum,
that have failed to be seen in estrus by 42 days postpartum and by the use of milk
progesterone assays.
 Detection of estrus depends upon the herdsman knowing the true signs of estrus, having a
regular routine, recording the events and using estrus detection aids.

Overall pregnancy rate

 This (originally called the overall conception rate) is the number of services given to a
defined group of cows or heifers, over a specified period of time which result in a diagnosed
pregnancy not less than 42 days after service; the figure is expressed as a percentage of the
total number of all services and should include culled cows.
 The method of pregnancy diagnosis should be specified. The first service pregnancy rate is
usually calculated separately and obviously refers to first services only. Thus in a 12 month
period, if 100 cows receive 180 services, of which 90 resulted in a confirmed pregnancy, the
overall pregnancy rate would be 50 per cent.
 The pregnancy rate is influenced by:

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 o The correct timing of artificial insemination, which will be dependent particularly on
the accuracy of estrus detection.
o Correct artificial insemination technique, handling and storage of semen.
o Good fertility of the bull if natural service is used, and the absence of venereal
disease.
o Adequate nutritional status of cows and heifers at the time of service and afterwards.
o Complete uterine involution and absence of uterine infection.
 The pregnancy rate to first service and overall pregnancy rate are very useful measures of
fertility; the latter is used to calculate the reproductive efficiency of the herd.
 The rates for the first service are usually slightly higher than those for all services because the
latter group will include those cows that may be sterile and receive many services before they
are culled.
 In order to identify the influence of management changes, particularly nutrition, it is
worthwhile calculating these two parameters on a monthly basis provided that there is a
minimum of 10 services per month.

Estrus detection aid

 Improving the detection of estrus has a much greater influence upon reducing the calving to
conception interval than improving the pregnancy rates; the latter can only be improved up to
a certain level. This enables herdsmen to anticipate the time of a subsequent estrus and thus
improves the detection rate. It also enables the early detection of acyclic cows.
 It is possible to estimate the estrus detection rate but it is important to stress that it is an
estimate and not an accurate measurement. A number of different methods are used and they
all have some measure of inaccuracy. One method is to determine the number of supposed
missed estrous periods. This an interval of 36-48 days suggests that one estrus has been
missed and an interval of 54-72 days suggests that two have been missed, although this latter
range is fairly wide and can lead to errors. The percentage of estrus detection rate (EDR) is
calculated thus:
o Estrus detection rate (%) =(No. of interservice interval recorded /(No. of
interservice interval recorded + No. of missed estrous period) ) x 100
o Efficiency of estrus detection (%) = (No. of estrus detected / Total No. of estruses )
x 100
o Accuracy of estrus detection (%) =(No. of estrus detected / (No. of estrus detected
+ No. of false estrus detection ))x 100
 One simple method of assessing the estrus detection rate at routine sessions of pregnancy
diagnosis will be the number of cows that are assumed by the herdsman to be pregnant, and
thus submitted for examination, but are found to be non-pregnant. Non-pregnant cows should
have returned to estrus since service or artificial insemination, and hence should have been
seen in estrus.
 In many apparently well-managed dairy herds where the calving to first service interval is on
target, there is a failure to detect returns to estrus in non-pregnant cows. This will result in a
large number of inter estrus intervals that are two or three times the normal interval. Poor
estrus detection may be due to:
 Poor accommodation inhibiting cows from exhibiting overt signs of estrus.
 Poor lighting or identification of animals.
 Failure to record signs of approaching estrus and signs of true estrus.
 Inadequate regimen for observing cows for signs of estrus, perhaps due to the
herdsman being overworked.

First service submission rate

 A days in all-the-year-round calving herds, then she should be served or inseminated within
the next 21 or 24 days. However, pregnancy rates will probably not reach their optimum for at
least 90 days postpartum. Furthermore, cows that have suffered dystocia or an abnormal
puerperium should not be served before 60 days postpartum and should be examined

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 routinely before service. It has been shown that there is a good correlation between the
physical state of the uterus, as determined by rectal palpation and the amount of mucopurulent
discharge and the regeneration of the endometrium.
 Heifers, and cows yielding more than 40 litres per day, should not be served before 50 days
postpartum.
 The submission rate is influenced by: (1) the time interval to the resumption of normal
cyclical activity after calving, and (2) the detection of estrus in those cows that have resumed
normal cyclical activity, and their presentation for service or artificial insemination. A good
submission rate is 80per cent In seasonally calving herds it will tend to be higher in those
cows that calve earlier than in the later calving. This is because, with the former, the presence
of more non-pregnant cows will ensure greater interaction when they are in estrus, which
should improve its. A relatively simple method of obtaining a fairly accurate measurement is
to list all cows that are ready for service (at or beyond the earliest service date of 45 days
since calving) at the start of each 21 or 24 day service period. At the end of this period
identify all those that have been served. The percentage submission rate is calculated thus:
 Submission rate (%) = (No. of cows or heifers served within a 21 days period / No. of cows
or heifers that are at or beyond the start of the 21 days period) x 100
 Another method is to list all cows chronologically in order of the calving date. Add 21 days to
the earliest date on or after which they are ready for service, i.e. 45 + 21 (24) = 66 (69) days.
Thus every cow should be served before the target date of 66 or 69 days postpartum. The
submission rate is calculated thus:
o No of cows served on or before the target date / No. of cows that should have been
served on or before the target date
 In a seasonally calved herd, the earliest service date will be selected in relation to when the
cows are required to calve down the following year. Thus, Cows that calve early in the season
will have a longer time interval before they need to be served compared with those that calve
late in the season. The choice of 21 days is based on the assumption that this is the mean inter
estrus interval. However, 24 days can be used, as it is the normal maximum interval. It is
irrelevant which is selected as long as its use is consistent.

Reproductive efficiency

 Attempts have been made to calculate a single index that provides an overall measurement of
fertility and takes into account many different parameters. One such measurement is the
reproductive efficiency (RE) of the herd; it is calculated thus:

RE = (Submission Rate X Overall Pregnancy Rate) / 100

 Thus if the submission rate is high, i.e. 80per cent and the overall pregnancy rate is good, i.e.
55 per cent, then the RE is 44.
 The advantage of this measurement is that an artificially high submission rate, obtained by an
overzealous herdsman presenting cows for artificial insemination when they are not in estrus,
will be compensated by a reduced pregnancy rate. Conversely, an overcautious herdsman may
have a reduced submission rate but although the reasonable RE value, it is not possible to
increase this further.

Culling rate

 One method of achieving a CI around 365 days is by culling those cows that are slow to get in
calf.
 This is rarely cost-effective because it will be necessary to replace the culled cow with a
heifer.
 The purchase price or the cost of rearing such a replacement is much greater than the price
obtained for the cull.
 Overall culling figures for infertility should not exceed 5per cent; thus 95per cent of the cows
that calve and are served should become pregnant again.
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SEXUAL HEALTH CONTROL AND HERD REPRODUCTIVE HEALTH PROGRAMME

HISTORY

 Before performing a detailed gynecological examination it is important to obtain a detailed

and accurate history, particularly the breeding history, of the cow. The following should be
obtained:
o Age
o Parity (there are certain conditions which can be excluded in nulliparous as opposed
to parous individuals).
o Date of last calving, together with information on the occurrence of dystocia, retained
placenta or puerperal infection.
o Dates of observed estrus since calving, if recorded.
o Presence of any abnormal discharge.
o Numbers and dates of services or inseminations.
o Dates of observed estrus since service or artificial insemination.
o Previous fertility records, particularly calving conception intervals and services per
conception.
o Details of feeding management and milk yield; in suckler cows the number of calves
suckled.
o Details of health, i.e. signs of milk fever, mastitis or ketosis.
o Details of fertility of other cows or heifers in the group or herd.

CLINICAL EXAMINATION

Determine whether the cow is pregnant. If there is a suspicion that she might be pregnant, but it is too
early to detect- leave and re-examine at a later date. Cows can conceive as early as 14 days post
partum. If the cow is definitely not pregnant, proceed as follows:

 Determine the general health status and assess body condition.

 Examine the base of the tail for signs of rub marks, and back and flanks for hoof marks,
which might indicate that the individual has been ridden by other cows.
 Examine vulva tail and flanks for evidence of discharge. If abnormal, perform examination of
vagina and cervix manually or with a speculum.

 Rectal palpation of the cervix and uterine horns to determine whether uterine involution is
complete, which should be so by 42 days post partum at the very latest. Assess extent of
uterine tone, and whether the uterine wall feels doughy and edematous; compare the relative
sizes of the uterine horns. Determine the presence or absence of adhesions involving uterine
horns and broad ligaments.
 Palpate both uterine tubes for evidence of thickening or enlargement (these are difficult to
identify if they are normal)
 Palpate the ovarian bursa for evidence of adhesions.
 Palpate ovaries; assess their mobility and absence of adhesions. Determine size, shape and
structures palpable. Is a CL palpable? Are follicles palpable? Is there evidence of a cyst-
fluctuating structure> 2.5 cm in diameter? Are the ovaries small, flattened, smooth and
featureless?
 Transrectal ultrasound scanning can be used if available.

 Collect a milk sample for progesterone assay to confirm ovarian structures

 Other specific tests, such as the PSP dye test, may be considered.

DIAGNOSITIC TEST

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  Most diagnostic tests are of limited value, although single blood or milk progesterone assays
are useful to identify the presence of luteal tissue if concentrations are high (4-6ng/ml in
plasma or 12-18ng/ml in milk).
 Bacterial swabbing and culture and endometrial biopsy are of limited value. The PSP
(phenolsulphonphthalein) test for tubal patency can also be used to demonstrate occluded
uterine tubes.

SUMMARY OF SIGNS OF INFEERTILITY

 The following summary describes a procedure for investigating an infertile animal on the
basis of the clinical history, signs and examination, with an indication of a possible diagnosis
of the cause and its treatment. Animals can be catagorised into those having
o No Observed Estrus
o Regularly Returning to Estrus
o Short Interestrus Intervals
o Anestrum

NO OBSERVED ESRTRUS

Rectal palpation or diagnostic ultrasonography should establish the presence or absence of pregnancy.
If the individual is pregnant it should be recorded; however, if there is any doubt or if it might be an
early pregnancy then a re-examination at a later date is required. If there is no pregnancy then
palpation of the ovaries is the next step.

Absence of Ovaries

Uncommon due to ovarian agenesis or freemartinism and hence will be seen only in a nulliparous
animal. There is no treatment, and thus the animal should be culled.

Small Inactive Ovaries

 If the ovaries are small, narrow and functionless, in a heifer, then this is due to delayed
puberty, ovarian hypoplasia or, possibly, freemartinism.

 There is no treatment; if delayed puberty is suspected, normal cyclic activity should

eventually occur. If the ovaries are flattened, smooth, small and inactive then this is true
anestrus; if there is a need for confirmation then a milk progesterone determination 10 days
later will help.
 The condition may be due to high yield, suckling, inadequate energy intake and severe
postpartum weight loss or trace element deficiency.
 Check feeding and assess bodily condition; improve deficiencies if present. Insert a PRID or
a CIDR for 12 days; estrus should occur 24-48 hrs. after withdrawal. Alternatively, GnRH
analogues can be used with estrus occurring in 1-3 weeks.

Presence of One or More Corpora Lutea

Number of situations are possible;

 The animal may be pregnant; if in doubt re examine later and check records.

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  Non-detected estrus: if this is the case, improve detection with increased frequency of
observation, heat mount detectors or tail paint or induce luteolysis with PGF2α or an
analogue, followed by artificial insemination at observed estrus or at a fixed time.
 Subestrus or ‘silent heat; this is only likely if soon after calving. Treat with PGF2 α or an
analogue as above.
 Persistent corpus luteum: thoroughly palpate the uterus, using retraction forceps if necessary,
to check for the absence of pregnancy. It may be mummified fetus or, rarely, a non-specific
cause. Treat with PGF2 α or an analogue.

Small Active Ovaries

 The palpation of small follicles, and perhaps a regressing corpus luteum or evidence of recent
ovulation associated with good uterine tone, indicates that the animal is coming into estrus, is
in estrus or has been in estrus.
 Careful inspection of the vulva at the time of palpation should reveal clear mucus.
 Re-examination in 10 days should reveal the presence of a corpus luteum.

Ovarian Cysts (Luteal)

 The presence of one or both enlarged ovaries, containing one or more fluid-filled, thick-
walled structures more than 2.5cm in diameter, particularly if confirmed using
ultrasonography, should confirm the diagnosis.
 A repeat examination several days later will confirm their persistence, and a milk or blood
progesterone determination will show the presence of luteal tissue.
 Treat with PGF2 α or an analogue.

Prolonged Interestrus Interval

The ovaries and genital tract should be examined per rectum. If the ovaries are normal, infertility may
be due to:

 Non-detected estrus: if the interval between successive heats is approximately twice the
interestrus interval, i.e. 36-48 days, then this indicates that one estrus has not been observed
or recorded. Irregular intervals that are not the product of the normal interval are likely to be
due to incorrect identification of estrus. If large numbers of animals are reported then this
suggests that the estrus detection rate is poor. If a susceptible corpus luteum is present,
PGF2α can be used to cause luteolysis and estrus in 3-5 days time. Methods of improving
estrus-detection should be implemented.
 Embryonic or fetal death: the interval between successive heats is unlikely to be an
approximate multiple of 21 and thus will be some other interval such as 35 or 46 days. In an
individual cow it is probably of no significance, but if a number of animals are involved,
especially if natural service is used, specific pathogens should be eliminated and other causes
sought.

REGULAR RETURN TO ESTRUS (repeat breeder or cyclic non breeder)

 The ovaries and genital tract should be examined per rectum to determine the presence of
gross abnormalities such as severe adhesion or uterine infection. This condition can occur
only if there is a failure of fertilization or embryonic death before day 12 of the estrous cycle.
 There are a number of possible causes:

Infertile Bull

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  If a number of cows and heifers are involved the bull should be examined. If artificial
insemination is used from an approved center this can be excluded although it must be
stressed that there is considerable variation in the fertility of bulls standing at artificial
insemination studs, although they will be above a minimal level. A bull with a high fertility
should be selected.
o Incorrect timing of service or artificial insemination: this is unlikely to occur
repeatedly unless the time of ovulation is asynchronous. If a number of animals are
involved, advice on the correct time may be worthwhile or else fixed time artificial
insemination after the administration of PGF2α or progestogens would be ideal.
o Nutritional deficiency or excess: check diet.

Occluded Uterine Tubes

 Palpate carefully and use the PSP test to confirm.

Anatomical Defects

 Palpate carefully, if the animal is nulliparous, look for segmental aplasia; if it is a parous,
check for ovarobursal or uterine adhesions.

Endometritis

 If there are clinical signs, diagnosis is simple but sub clinical disease can be diagnosed only
by biopsy.
 If suspected, treat with antibiotics after insemination or by prostaglandin to shorten the luteal
phase preceding insemination.

Delayed Ovulation

 Diagnosis is difficult.
 Treat with GnRH or hCG at the time of insemination or repeat insemination on the
subsequent day.

Anovulation

 Diagnosis depends on ovarian palpation or transrectal ultrasonography 7-10 days after estrus
to demonstrate failure of ovulation by absence of a corpus luteum.
 Treat with GnRH or hCG at the time of insemination.

Luteal Deficiency

 There is no absolute proof that it occurs, but if other causes have been eliminated the use of a
luteotrophic agent, such as hCG might be worthwhile at 2-3 days after subsequent
inseminations to improve corpus luteum formation or at mid-cycle to stimulate accessory
corpus luteum formation.
 Alternatively, GnRH analogue can be administered at day 12 or 13 after insemination.

Short interestrus interval

This condition is usually identified by other signs of nymphomania and palpation or imaging of
ovaries. The cause may be:

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Enlarged Ovaries

 If either one or, more likely, both contain one or more thin-walled, fluid-filled structures this
should confirm the diagnosis of follicular cysts.
 Persistence of the cyst should be confirmed by a second rectal examination performed at
intervals of ten days.
 Treat with GnRH, hCG or a PRID.

Artificial insemination at the wrong time due to incorrect estrus detection

 This is often preceded or followed by an extended interval so that the sum of the two intervals
is 36-48days.
 If large numbers of cows have the same history, estrus detection should be improved.

Abortion

 This is defined as the production of one or more calves between 152 and 270 days of
gestation: they are either born dead or survive for less than 24 hours.
 The cow should be isolated, the fetus and fetal membranes should be retained and the case
treated as a suspected Brucella abortion.
 The physical appearance of the fetus and fetal membranes should be noted, the fetus aged
approximately and this confirmed by the service or insemination date if available.
 Elimination of infection as a cause is made by being unable to demonstrate organisms in the
fetus, fetal membranes vaginal and uterine discharges and/or by the demonstration of specific
antibodies in body fluids.
 Where possible the whole fetus should be submitted to the laboratory for cultural
examination.Possible infectious causes of abortion are:

Infectious Causes Occurance

Brucella abortus 6-9 months

Leptospira spp 6-9 months

Listeria monocytogenes sporadic outbreaks at 6-9 months

Campylobacter fetus (veneralis) 5-7 months

Trichomonas fetus occurs before 5 months

Salmonella spp: especially S. Dublin usually sporadic with no specific time,

although usually about 7 months
Actinomyces (Corynebacterium) pyogenes usually sporadic and occurs at any stage

Mycobacterium tuberculosis occurs at any stage

Mycotic agents, Aspergillus spp;, Absidia occurs from 4 months to term

spp. Mucoralis group
Bacillus lichenkformis sporadic late abortions

Neospora caninum late abortions

Infectious bovine rhinotracheitis-infectious occurs at 4-7 months

pustular vulvovaginitis (IBR-IPV) virus

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 Bovine viral diarrhea (BVD) virus occurs at any stage

Investigation of abortion

 The approach to investigating the cause of abortion will depend upon the frequency. If
sporadic, then a full laboratory investigation is probably unnecessary because many abortions
are not associated with infection.
 However, if it exceeds 3-5 per cent of the herd-and it is important to consider stillbirths and
premature calving (excluding twins) in this calculation-then a thorough investigation should
be implemented.

Investigation of sporadic abortion

 Perform statutory brucellosis investigation.

 Determine if all abortions have been reported and that it is a true sporadic case. If so, proceed
to (3) if not, or if there is any doubt, then follow the procedure for an outbreak investigation
given below.
 Clinical examination of cow.
 Examine placenta for evidence of obvious lesions, particularly fungi
 Submit serum for Leptospira serovar hardjo serology unless it is a vaccinated herd.
 Request culture of vaginal swab for salmonella Dublin.
 Obtain detailed history of changes in husbandry, movement of livestock, purchase of animals,
hiring of bulls, signs of ill health, and age of aborting cows.

Investigation of abortion outbreak

 Repeat steps (1), (2), (3), (4), and (7) given for Investigation of sporadic abortions.
 Ideally, submit one or more fresh whole fetuses and placentas-or
 Several complete fresh cotyledons.
 Fetal stomach contents (2ml) aseptically collected using vacutainer or syringe and needle,
 Fluid from thorax or abdomen (2ml) using methods described in (4).
 About 5g of fresh lung, liver, thymus and salivary gland, All tissues and other samples should
be refrigerated and packed with ice, but not frozen.
 Air-dried, acetone-fixed impression smears from fresh cotyledons lung, liver and kidney.
 Formal-saline-fixed cotyledon, fetal liver, heart and lung.
 Two 7ml vacutainers of clotted blood from all cows that have recently aborted.
 Repeat samples from the same cows as in (9) 2-3 weeks later for possible rising antibody
titres in the serum.

If an infectious cause is not identified using routine diagnostic tests it may be necessary to extend the
investigation in an attempt to confirm the presence of a less common infectious agent. However
abortions can be caused by many other factors: congenital defects due to genetic factors or teratogens;
trauma; allergies; dietary excesses such as high protein pastures, chemicals such as nitrates and
chlorinated naphthalene; and hormones such as prostaglandins. Diagnosis is generally based on
circumstantial evidence, and in some cases the presence of pathgnomonic lesions.

Evaluation of herd fertility

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  The term “Reproductive efficiency” at once looks to be too simple, in theory, but try to put it
into practice, based on actual performance of the animals, nerve shattering problems
immediately surface.
 On the face of it, successful reproduction is the culmination of several individual stages in the
reproductive life of the individual female animal leading to the birth of young one followed
by lactation. Yet, several factors which are extraneous to the individual-like nutrition,
environment, management, performance of the male, pathological conditions etc.
considerably influence the reproductive performance and can be responsible for lowered
reproductive efficiency.
 To ensure the profitability of any dairy industry, several reproductive parameters used to
measure the reproductive efficiency need to be kept in mind.
 Regular, accurate evaluation of the fertility status of the dairy herd is an essential part of a
control programme and should be done at least twice a year. It is also an important
prerequisite when investigating a suspected herd infertility problem.
 In order to evaluate fertility it is necessary first of all to quantify certain reproductive values,
and in order to do this it is necessary to have access to records of reproductive events.
Obviously, the accuracy and value of such calculations will depend upon the quantity and
quality of the information provided, and it will be necessary to modify one’s assessment
accordingly, depending upon clinical judgment, the history of the herd and the primary
complaints of the herdsman or owner.
 The minimum information required is:
o Identity of cow;
o Last calving date;
o First and subsequent service or insemination dates;
o Confirmation of pregnancy.

ROLE OF HORMONES IN FEMALE REPRODUCTION

 Endocrinology
o A science concerned with chemical integration of the body. Integration is a key word
related to the function of the nervous system.
 Endocrine glands
o Endocrine glands are those ductless glands of the body whose secretion goes directly
in to the blood stream.
 Exocrine glands
o Exocrine glands are those glands of the body whose secretion is carried away by a
duct.
 Hormone
o The word hormone originated from a Greek word meaning "I Stir up or
stimulate". A hormone is a chemical substance produced in one part of the body
(restricted area) that diffuses or is transported to another area where it influences
activity and tends to integrate component parts of the organism.
o Hormones regulate (decrease or increase) the rates of specific processes but do not
contribute energy to the process or initiate metabolic reactions. Instead, hormones
influence an existing reaction which is usually one involving enzymes.
 Local Hormone or Para Hormones
o In strict sense are not hormones, but are chemical messengers or regulators. Eg.
Prostaglandins, erythropoietin, and histamine.

HYPOTHALAMUS

The hypothalamus, the key brain center that controlls all our reproductive activities lies at the base of
the brain, bordered anteriorly by the optic chiasma, posteriorly by the mammillary bodies, dorsally by
the thalamus and ventrally by the sphenoid bone.

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  Its size is 1/300 of the entire brain.
 Within the hypothalamus are numerous neurons. Clusters or groups of these neurons are
called hypothalamic nuclei, each of which have a specific name.
 The hypothalamic nuclei of importance are:
o The preoptic nucleii(PON), the anterior hypothalamic area (AHA), suprachiasmatic
nucleii(SCN) that make up the surge center
o The ventromedian nucleii(VMN), the arcuate nucleii(ARC) and the median
eminence(ME) that make up the tonic center, and
o paraventricular nucleus(PVN).

 The medial portion of the hypothalamus known as third ventricle of the brain separates
most of the paired nuclei.
 Neurons in the hypothalamus communicate with the anterior lobe of the pituitary using a
special circulatory modification known as the hypothalamo- hypophyseal portal system.

Pituitary gland

 The pituitary gland lies below the hypothalamus in a bony depression in the sphenoid bone
called the Sella turcica.
 It consists of anterior and posterior lobes.

HYPOTHALAMO – HYPOPHYSESAL PORTAL SYSTEM

The hypothalamus, the key brain center that controlls all our reproductive activities lies at the base of
the brain, bordered anteriorly by the optic chiasma, posteriorly by the mammillary bodies, dorsally by
the thalamus and ventrally by the sphenoid bone.

 Its size is 1/300 of the entire brain.

 Within the hypothalamus are numerous neurons. Clusters or groups of these neurons are
called hypothalamic nuclei, each of which have a specific name.

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  The hypothalamic nuclei of importance are:
o The preoptic nucleii(PON), the anterior hypothalamic area (AHA), suprachiasmatic
nucleii(SCN) that make up the surge center
o The ventromedian nucleii(VMN), the arcuate nucleii(ARC) and the median
eminence(ME) that make up the tonic center, and
o paraventricular nucleus(PVN).

 The medial portion of the hypothalamus known as third ventricle of the brain separates
most of the paired nuclei.
 Neurons in the hypothalamus communicate with the anterior lobe of the pituitary using a
special circulatory modification known as the hypothalamo- hypophyseal portal system.

Pituitary gland

 The pituitary gland lies below the hypothalamus in a bony depression in the sphenoid bone
called the Sella turcica.
 It consists of anterior and posterior lobes.
 The portal system consists of
o The Superior hypophyseal artery(SHA)
o Primary portal plexus(PPP) (where the neurons of the surge center and tonic center
terminate),
o The medial hypophyseal artery (MHA)that supplies part of the anterior lobe of the
pituitary,
o The portal vessels(PV) that transport the blood containing releasing hormones and,
o The secondary portal plexus(SPP) that delivers blood and releasing hormones to the
cells of the anterior lobe.
 The terminal portion of the hypothalamic neurons release neuropeptides that enter specialized
capillary system at the stalk of the pituitary. Blood enters the capillary system from the
superior hypophyseal artery that divides into small arterial capillary forming plexus (primary
portal plexus). The releasing hormones are transferred to the secondary portal plexus in the
anterior lobe of the pituitary where the releasing hormones cause pituitary cells to release
other hormones. The hypothalomo-hypophyseal portal system is important as it allows for
minute quantities of releasing hormones to act directly on the cells of the anterior lobe of the
pituitary before GnRH gets diluted by the circulation.
 The posterior lobe of the pituitary does not have a portal system. Neurons from certain
hypothalamic nuclei (PVN) extend directly into the postetrior lobe of the pituitary where the
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 neurohormone is released into a simple arteriovenous capillary plexus. For eg. Cell bodies in
the Para Ventricular Nucleus (PVN) synthesize oxytocin that is transported down the axon to
the terminals in the posterior lobe. If the neuron is stimulated, oxytocin is released into the
blood.

ENDOCRINE REGULATION OF REPRODUCTION

 In contrast to the neural system, the endocrine system depends on hormones to cause the
responses.
 Hormone in extremely small quantities can bring about dramatic physiologic responses.
 Hormones are classified as having relatively short half lives.
 Hormonal half life is defined as the time required for one half of the quantity of the hormone
to disappear from the blood or from the body.
 Short half lives are important because once the hormone is secreted and released into the
blood and cause a response, it is rapidly degraded so that further or unnecessary responses do
not occur. However, when hormones such as progesterone are produced during pregnancy,
the action brought about by the hormone continues as long as the hormone is present.
 Compared to neural control, hormonal control is slower and has durations of minutes, hours
or even days.

MECHANISM CONTROLLING SECRETION

Positive and Negative Feedback are the major Controllers of Reproductive Hormones.

 Negative feedback: inhibits GnRH neurons. For eg., High progesterone inhibits GnRH
neurons which secrete only basal levels of GnRH. Such basal levels will allow for some
follicular development but only to the extent where insufficient estrogen production is there.
Hence, when progesterone levels are high, animal does not cycle. The tonic center in both and
female is believed to respond mostly to negative feedback.
 Positive feedback: activates the GnRH neurons in the hypothalamus. When estradiol reaches
threshold level, the surge center will be positively stimulated and will release large quantities
of LH that stimulate ovulation.

Hormones of reproduction are classified based on

o Chemical structure
o Glandular origin
o Mode of action

Chemical structure

 Based on their structure, the hormones are classified into

o Proteins
o Steroids
o Fatty acids

Proteins or Polypeptide Hormones

 Proteins or polypeptide hormones have molecular weight of 300 to 70,000 Daltons and are
easily broken down by enzymes. They must be administered by injections.
 They contain an α and β subunit. The α subunit for FSH, LH and TSH are identical but the β
subunit is unique to each species. When two α or two β subunits combine the resulting
molecule will have no activity. If α subunit of one hormone combines with the β subunit of
another hormone, the activity of the molecule will be determined by the β sub unit only.

~ 245 ~
  The amount of carbohydrate present on the surface of the protein determines the duration of
half life of the hormone.
 Glycoprotein hormones can be degraded easily by proteolytic enzymes in the digestive tract.
There fore, they are not effective when given orally.

Steroids

 Steroid hormones have the common cyclopentano perhydro phenantherene ring nucleus.
 They have a molecular weight of 300 to 400 Daltons.
 Precursor is cholesterol
 Natural steroids are not effective by oral administration.
 Synthetic and plant steroids can be administered orally and by injection.
 Steroid hormones are sexual promoters and cause profound changes in both male and female
reproductive tract.

Fatty acids

 Have a molecular weight of approximately 400 Daltons, and

 Can be administered by injection.

Glandular origin

 Reproductive hormones originate from:

o Hypothalamus
o Pituitary
o Gonads
o Uterus, and
o Placenta
 Based on their origin, they are classified as
o Hypothalamic hormones eg. GnRH.
o Pituitary Hormones eg. FSH/LH
o Gonadal Hormones eg. Estrogen, Progesterone
o Uterine hormones eg. PGF2 α
o Placental hormones eg. hCG, eCG.

Mode of action

 Neurohormones : Synthesized by neurons and released directly into the blood and cause
response in a target tissue. eg. Oxytocin. A neurohormone can act on any number of tissues
provided that the tissue has cellular receptors for the neurohormone.
 Releasing hormones: Synthesized by neurons in hypothalamus and cause release of
hormones from pituitary. eg. GnRH.
 Gonadotrophins: Hormones released by the gonadotroph cells of the anterior pituitary and
stimulate gonads. The suffix `tropin’ means having an affinity for. eg. FSH and LH.
 Sexual promoters (Steroids): Produced by the gonads of both male and female to stimulate
the reproductive tract, to regulate function of hypothalamus and anterior pituitary and to
regulate reproductive behaviour.
 Pregnancy maintenance hormones: are responsible for maintenance of pregnancy. (eg.
Progesterone) and in some cases, assist the female in her lactation ability.
 General metabolic hormones: promote metabolic well being. eg. Thyroxin, adrenal
corticoids and somatrophin.
 Luteolytic hormones: cause destruction of the corpus luteum. The suffix `lytic’ is a
derivative of word lysis. eg. PGF 2 α

OXYTOCIN
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  First hormonal peptide identified in the mammals.
 Synthesized in the supraoptic and paraventricular nuclei of the hypothalamus and stored and
released from posterior pituitary.
 Synthesized along with the carrier proteins called neurophysins.
 Transported in small secretory vesicles enclosed by a membrane. Secretory vesicles flow
down the hypothalamic–hypophyseal nerve axons by axoplasmic streaming and are stored at
nerve endings next to capillary beds in the neurohypophysis.
 Also produced by the corpus luteum of cow, ewe and human.
 Therefore, oxytocin has two sites of origin:
o the ovary, and
o the hypothalamus.

Functions of oxytocin

Oxytocin in Greek means 'rapid birth'

 Causes contraction of oviduct and thus involved in transport of male and female gametes in
oviduct.
 Estrogen enhances responsiveness of smooth muscle to oxytocin.
 Causes milk let down.
 Ovarian oxytocin is involved in luteal function by acting on the endometrium of the uterus to
induce PGF2 alpha release which causes lysis of CL.

GnRH

 GnRH is a deca peptide containing 10 amino acids.

 Molecular weight is 1183 daltons.
 Causes release of FSH and LH from the anterior pituitary.
 The C- terminal portion of this molecule is necessary for attaching to receptor while first 3
amino acids are necessary for activating LH and FSH release.
 The hormones of the anterior pituitary, adrenal cortex, thyroid and gonads feedback to inhibit
and in some instances to stimulate the secretion of the hormone.
 Two types of Analogs to LHRH have been synthesized:
o GnRH antagonists - bind to receptor sites on the pituitary but do not induce release
of LH or FSH and block the action of the natural hormone.
o GnRH agonists - that induce release of more LH and FSH than natural GnRH.
Increased biological activity is due to their ability to stay bound to pituitary receptors
longer than natural hormone and their ability to resist enzyme degradation.

Functions

 Controls the release of FSH and LH

PITUITARY GONADOTROPHIN

 The anterior pituitary gland secretes three glycoprotein hormones viz.

o Follicle Stimulating Hormone (FSH)

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 o Leutenizing Hormone (LH)
o Prolactin
 Consist of two non-identical subunits termed a and ß
 The alpha subunit is identical within species for FSH, LH and TSH.
 Molecular weight is 32,000 daltons with each subunit having molecular weight of 16,000
daltons.
 Alpha and beta subunits by themselves have no biological activity.
 Alpha subunit of one hormone (LH alpha) is recombined with beta subunit of another
hormone (FSH beta), the molecule regains FSH biologic activity or activity of the beta
subunit.
 If two alpha subunits or two beta subunits are combined, no biologic activity is noted.

1. FSH

 Alpha subunit contains 92 amino acids with CHO side chains at aa 52 and 78 : the beta
subunit has between 108 to 118 amino acids with 2 CHO side chains at aminoacid 7 and 24.
 Combination of alpha and beta subunit are necessary to provide tertiary structure for
recognition by the FSH receptor in the gonad.
 Six different species of FSH in a single animal has been identified.
 Half life of 2.0–2.5 h.

Functions

 Stimulates growth and maturation of the graafian follicle in the ovary.

 FSH along with LH causes estrogen production from the ovary and testes.

2. LH

 Also called Luteotrophin or Interstitial Cell Stimulating Hormone (ICSH).

 Glycoprotein composed of alpha and beta subunit with a molecular weight of 30,000 daltons.
 Half life of 30 minutes.

Functions

 Tonic or basal levels of LH act in conjunction with FSH to induce estrogen secretion from the
large graafian follicle.
 Preovulatory LH surge causes rupture of follicle and ovulation.
 LH is the major luteotrophic substance (maintains activity of corpus luteum).
 Stimulates interstitial cells (Leydig cells) in male to produce androgens.

CONTROL OF FSH AND LH SECRETIONS

Tonic LH and FSH Release

 Serum LH and FSH are released in a tonic or basal fashion in both male and female.
 Tonic levels are controlled by negative feed back of estrogen and inhibin from gonads.
 The arcuate nucleus, ventromedian nucleus and the median eminence control the tonic release
of LH and FSH.

Preovulatory LH and FSH Release

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  Preovulatory surge of LH and FSH occurs in female prior to ovulation.
 Initiated by increase in estrogen which exerts a positive feed back on the hypothalamus
inducing release of GnRH surge.
 Preoptic and anterior hypothalamic nuclei controls preovulatory surge of LH and FSH.

PROLACTIN

 Polypeptide hormone containing 198 aa and a molecular weight of 24, 000 Daltons.
 Prolactin molecules are similar in structure to growth hormone and in some sp. have similar
biologic properties.

Functions

 Prolactin initiates and maintains lactation.

 It is considered as a gonadotropic hormone because of its leutrotropic properties in bitches
and rodents. However in domestic animals, LH is the major luteotropic hormone.
 Prolactin may mediate the seasonal and lactational effects on reproduction in farm animals.

PROSTAGLANDINS

 The prostaglandins are a group of compounds synthesized in the body from arachadonic acid
and found in many tissues of the body. The prostaglandins have been arranged into 8 major
series according to chemical makeup: A, B, C, D, E, F, G and H. There may be different
prostaglandins within a series.
 Many different physiological and pharmacological actions reportedly affect a number of body
systems:
o Central nervous
o Cardiovascular
o Urinary
o Gastrointestinal
o Respiratory
o Reproductive
o Certain prostaglandins are involved in the inflammatory reaction and allied adverse
reactions of the body to injury.
 Currently the only prostaglandin available commercially is prostaglandin F2 Alpha
(PGF2Alpha).
 Prostaglandin F2 alpha (PGF2α) release is brought about by the activation of endometrial
oxytocin receptors.
 The receptor concentration increased after 10 day exposure to progesterone and is brought
about by estrogen from antral follicles in late luteal phase.
 If the animal becomes pregnant, the early embryo through ectoderm secretes the protein,
bovine trophoblast interferon which prevents PGF2α release, which is the signal for maternal
recognition of pregnancy.

Action

 Reduction in arterial blood supply by vasoconstriction leading to luteolysis.

 Binding with specific binding receptor sites developed on the plasma membrane of luteal
cells. Binding interferes or prevents PGF2 α binding.
 PGF2α binding brings about intracellular changes and affects steroidogenesis.

Function

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  PGF2α is involved in ovulation in ewes and cows. Ovulation is blocked by administration of
indomethacin, an inhibitor of PG synthesis. LH release is not affected. Hence action of PGs
may be only at the level of ovarian follicle.

PGE

 Stimulates contractions of the uterus,

 Dilatation of blood vessels and has no luteolytic action.

PGF2α

 Stimulates contraction of the uterus,

 Aids in sperm transport in the male and female reproductive tracts,
 Causes constriction of blood vessels,
 Has luteolytic properties by venous constriction,
 Helps in parturition and a parallel rise in the level of PGF2α along with estrogen is observed.
Primary effect of PGF2α is the myometrial contraction which favours release of oxytocin.

GONADAL HORMNES

 Primarily secreted by ovary and testes. The adrenals and placenta are other sources.
 They are of 4 types
o Androgens
o Estrogens
o Progesterone
o Relaxin
 The first three are steroid hormones while relaxin is a protein.
 Steroid hormones have a basic or common nucleus called the
cyclopentanoperhydrophenanantherene nucleus. It consists of a three, six member fully
hydrogenated phenantherene rings designated as A,B,C and D.
o An 18 carbon steroid has estrogen activity
o A 19 carbon steroid has androgen activity
o A 21 carbon steroid has progesterone activity
 The secretory activity of steroid hormones is under endocrine control of anterior pituitary.

ESTROGEN

 The estrogens are steroidal hormones synthesized from cholesterol and produced primarily by
the ovaries, placenta and corpus luteum.
 A significant source of estrogens is the testes of stallions and boars, while a minor source is
the adrenal gland.
 The estrogens are metabolized by the liver and excreted in the bile, feces, and the urine
(horses and ruminants).
 Progesterone decreases the effects of the estrogen and FSH and LH may be involved in
estrogen secretion.

Functions

Some important physiological actions of estrogens include:

 Maturation growth and development of the reproductive organs.

 Stimulation of normal physiological processes of the tubular reproductive tract.
o growth of the uterine muscle
o development of the endometrial lining of the uterus
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 oincrease the vascularity of the uterus
 Induction of behavioral estrus
 The production of edema in folds of the mucosa at the utero-tubal junction
 Dilation of the cervix
 Protein anabolism in ruminants
 Under the influence of the estrogens the uterus is less susceptible to infection
 They play a role in the normal health and function of the skin
 They produce contractions of the uterus
 They enhance the effects of oxytocin on uterine motility
 The estrogens inhibit the secretion of FSH and LH via a negative feedback mechanism
 They are required for the development of the secondary sex characteristics of the female
including hair growth, deposition of body fat, mammary gland development, plumage, etc.
 The estrogens are involved in the regression of the corpus luteum.

Applications

 They are used to evacuate the uterus in cases of fetal mummification, fetal maceration and
pyometra.
 They are used to induce abortion in all species
 They can be used in the treatment of postpartum metritis and retained fetal membranes
 Used in the management of misalliance in dogs
 To produce signs of estrus in anestrual animals
 Treating cases of estrogen responsive urinary incontinence
 In virgin heifers and dry cows estrogen can be used to stimulate mammary development and
lactation
 Large doses of estrogen after parturition can be used to inhibit lactation and relieve
congestion of the mammary glands.
 Used as growth promotants in beef cattle
 Used in the management of skin condition in spayed bitches
 In treating male dogs with prostatic hyperplasia
 Can be used to decrease libido in males

Side effects

 Prolonged use or large doses can produce cystic ovaries or ovarian atrophy
 Because of its effect on the ligaments, the estrogens predispose to prolapse of the vagina and
rectum and dislocations and fractures of the pelvic bones.
 In the dog excessive amounts or prolonged administration of estrogens can produce a fatal
anemia - leukopenia and thrombocytopenia.
 In some species the estrogens in combination with progesterone may increase the incidence of
cystic endometrial hyperplasia - pyometra complex.

PROGESTERONE

 These include the naturally occurring steroid progesterone which is synthesized from
cholesterol and produced by the corpus luteum (main source), placenta (especially of the ewe
and mare after the first 1/3 and 1/2 of gestation respectively), the adrenal gland and the testes,
as well as a number of synthetic progestogens which are much more potent and have a longer
half-life than progesterone.

Functions

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  Progesterone causes growth of the glandular system of the endometrium of the uterus, and the
secretions from the endometrial glands (uterine milk) for the nutrition of the ovum and the
attachment of the embryo.

 Progesterone plays a role in the maintenance of pregnancy (and pseudopregnancy) by

providing a favorable environment for survival of the embryo.
 Progesterone causes growth of the alveolar system of the mammary gland.
 Progesterone inhibits the smooth muscle activity of the uterus - renders it less sensitive to
oxytocin.
 Target tissues are relatively insensitive to progesterone unless primed by estrogen - At low
levels progesterone acts with estrogen to stimulate ovulation by promoting LH release.
 At high levels progesterone inhibits the secretion of FSH and LH via a negative feedback.
However enough FSH is released so that follicles may develop during the luteal phase of the
cycle (diestrual ovulation seen in the mare, and also during the gestation period in some
species - the mare and to some extent the cow).
 Progesterone increases the efficiency of nutrient utilization.

Applications

 Prevent or control habitual abortion due to an actual or possible progesterone deficiency.

 Estrum deferment or suppression in the ewe, cow, sow, mare and bitch.
 Synchronization of estrus in the mare, ewe, cow and sow.
 In heifers the progestins are used to promote growth - suppresses heat
 To treat post-partum hemorrhage in the bitch.
 In the treatment of cows with cystic ovaries progesterone withdrawal
 In cats progesterone has been used to treat miliary eczema and eosinophilic granulomas.
 Progesterone has a calming as well as androgenic effect and has been used to manage
antisocial or aggressive behavior in mares, stallions, dogs and cats.

Problems

 If injections are given beyond the recommended length of time, prolongation of the gestation
period and fetal death may occur.

ANDROGENS
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  Androgens are 19-carbon steroids with a hydroxyl or oxygen at positions 3 and 17 and a
double bond at position 4.
 The androgens are called 17-Ketosteroids when qxygen is found at position 17.
 Testosterone is the principal circulating androgen in the male being produced by the
interstitial cells of the testis.
 Other minor sources include the adrenal cortex and ovaries in females.

Functions

 Testosterone plays a role in

o Maturation, growth and development of the repro- ductive organs and secondary sex
characteristics of the male.
o Erythropoesis
o Protein anabolism
o Maintainance of the secretory responses of the accessory sex organs-provide the fluid
component of semen.
o Suppressing the secretion of the pituitary gonado- trophins through negative
feedback.

Applications

 In general,the use of testosterone in large animal is limited to:

o Developing teaser animals
o Induce libido in geldings
 In general, there is no fertility condition in the male due to a hormonal cause that uniformly
respond to endocrine therapy. The androgens have been used in small animals to manage
several problems.

RELAXIN

 Relaxin is a polypeptide hormone containing alpha and beta subunits that are connected by
two disulphide bonds.
 It has amolecular weight of 5700 daltons. Inhibin and insulins are structurally similar, but
their biological actions are similar.
 Relaxin is primarily secreted by the corpus luteum during pregnancy. In some species the
placenta also secrete relaxin.
 In canines, relaxin is a pregnancy specific hormone.
 The main biological action of relaxin is
o Dilatation of cervix and vagina before parturition.
o It also inhibits uterine contractions
o Causes increased growth of the mammary gland if given in conjunction with
estradiol.
o In the Guinea pig, relaxin causes seperation of the pubic symphysis bone within 6 hrs
after injection. Seperation of pubic symphysis normally occurs during parturition in
this species.

BREEDING MANAGEMENT AND ARTIFICIAL INSEMINATION IN DOGS

The most common problem encountered by veterinarians working in canine reproduction is the
“potentially” infertile bitch or stud. Owners bring these dogs to their veterinarian with the major
concern that the dogs are failing to produce puppies. It is important to emphasize that a vast majority
of these dogs are healthy fertile animals whose apparent infertility problems are related to a
misunderstanding of proper breeding management.

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METHODS OF BREEDING

 Methods of breeding include

o Out-crossing
o In-breeding
o Line breeding
 Out-crossing is the mating of two dogs within the same breed that are less closely related
than the breed average.
 In-breeding occurs when closely related dogs are breed such as parents and offspring or
brother and sister.
 Line breeding (which is a form of in-breeding) occurs with the repeated use of 1-2 dogs
(usually males) for breeding to increase a certain trait.
 The problems with in-breeding are that it increases the manifestation of undesirable traits.
Because of this it is advisable to evaluate at least five generations of pedigree on any bitch
bought into the in-breeding program, especially those bitches with relatives that have
demonstrated potentially inherited diseases, and if the bitch is in any way a likely carrier of a
serious genetic disorder breeding from her should be discouraged. Also where significant in-
breeding is present out-crossing should be seriously considered.

GOALS

 The simple goal in any breeding program is to have sufficient sperm numbers present in the
uterus and oviducts to achieve the optimal chance for fertilization of mature eggs.
 Mature occytes are typically fertilized during the 3 to 8 days following the luteinizing
hormone surge, representing a period beginning 24 to 48 hours after ovulation of primary
immature oocytes.
 Using reliable, clinically practical methods for estimating the day of the LH surge can be
quite valuable. These criteria include
o Behavior observation
o Vaginal cytology
o Vaginoscopy
o Hormone assays
 When used together they enhance the chances of a bitch being inseminated at the proper
times.
 Further, normal sperm are known to survive and retain the capacity for fertilizing mature
oocytes within the uterus of the bitch for at least 4 to 6 days, and in some instance for as long
as 11 days.
 Using this information, a breeding program can be offered to a client with reasonable
confidence of success.

ERRORS

 People who own male dogs allow only one or two breedings per cycle. Since, there is so
much of variation in the duration of proestrus and estrus and in the time of ovulation, it is
almost impossible to fix two breeding dates without using any breeding management tool.
 Most breeders and petowners breed their dogs on the 9, 11 or 13th day of the cycle assuming
that all dogs enter into estrus on the 9th day. Such predetermined dates are fine if it works, but
what if the perfectly normal, but not average, bitch is in proestrus for 16 days or proestrus
lasts 4 days and estrus lasts 4 days?
 Some breeders begin breedings when the bloody vaginal discharge of proestrus becomes clear
and /or strawcolored. Some normal bitches may have a bloody vaginal discharge throughout
proestrus, estrus and even into diestrus. Others may discontinue bleeding days before the
onset of estrus.
 Most breeders tend to depend on the male dog to choose the breeding dates. Male response to
a bitch is simply unreliable. Some males always want to breed. Other males never want to

~ 254 ~
 breed because they may be submissive to a bitch, again rendering the male worthless as a
guide to breeding.
 People always assume that the male is always fertile. Any male may quickly become
transiently or premanently infertile. Any time the fertility of the male is questioned a semen
analysis is warranted.

PREBREEDING EXAMINATION

Prior to breeding the bitch ideally should undergo

 A full physical examination to ensure she is in a fit condition to carry a litter of pups
 Verification of her vaccination status also whether she is free from parasites such as worms,
fleas and mites.
 Screening tests for genetic diseases is recommended for certain breeds. For example breeds
that are susceptible to hip dysplasia should be radiographed and hip scored prior to a breeding
occurring (dogs should be over 12 months of age before being radiographed for hip
dysplasia). Bitches that are determined positive to hip dysplasia should not be bred with due
to the nature of the disease.
 Thyroid testing should be considered in breeds that are over presented for hypothyroidism or
in bitches showing signs of thyroid problems.

EXAMINATION AND TESTING OF REPRODUCTIVE SYSTEM

 All bitches presented for breeding need to be examined for abnormal vulvar conformation and
vulvar discharge. Severe conformational problems can contribute to an inability to breed,
persistent inflammation of part of the reproductive trait and an inability to whelp naturally.
 It is advisable to perform a digital vaginal examination in maiden bitches. We look for
strictures that may interfere with matings, foreign bodies, tumours or any other abnormalities
that may prevent a normal mating.
 Vaginascope should be performed in bitches that have a history of being non-receptive to
mating, that have an abnormal discharge from the vulva, or that have signs suggesting
inflammation of the caudal reproductive trait, such as rubbing the vulva on the floor,
excessively licking the vulva and abnormal vulva odours.

BASED ON SEXUAL BEHAVIOUR

 Behavioral estrus is the factor in determining when breeding of the bitch should begin.
Observation of the bitch's response to a male is an inexpensive, straight forward, and reliable
means of determining when to begin and when to end the breeding phase.
 On day 5 or 6 of proestrus, the bitch should be brought into contact with a male dog for
approximately 10 to 20 minutes. This should be repeated every second or third day. Breeding
should begin whenever the bitch is willing regardless of the color of the vaginal discharge, the
vaginal exfoliative cytology interpretation, or the day of the cycle and should continue every
other day until she is no longer willing to breed.
 It is recommended to breed the bitch every 2 to 4 days, beginning with the first day of
acceptance and continuing throughout the acceptance period. Dogs that are in standing heat
for longer than 12 days should be bred no more often than every third or fourth day. Bitches
in standing heat for only 3 or 4 days should be bred every 48 hours.
 It is of paramount importance to recommend to owners that the male continue breeding the
bitch until the bitch refuses to breed or until the first day of diestrus is documented with
vaginal cytology. Fertilization of eggs is most likely occurring in the final 4 or 5 days of
standing heat, regardless of the length of standing heat, or 4 to 5 days before the onset of
diestrus.

BASED ON VAGINAL CYTOLOGY

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  Vaginal cytology is a simple, inexpensive, and reliable means of evaluating the bitch
and provides a good reflection of rising plasma estrogen concentrations. Vaginal smears
should be monitored beginning with the second or third day of proestrus. The day the smear
shows more than 80 % superficial plus cornified cells put together (cytological estus),
breedings should begin.
 Since, Vaginal cytology does not directly predict the time of ovulation it is advised that once
breeding begins, it should be allowed to continue until the bitch refuses to breed.
Recommendations are to breed the dog every second, third or fourth day of estrus.

BASED ON VAGINAL ENDOSCOPY

 Vaginoscopy can be used to aid in timing natural breeding. Vaginal mucosa in proestrus
appears rounded and edematous.
 "Wrinkling" or "Crenulation" of mucosa is associated with LH surge. This is the time to begin
breeding. Breeding should be continued throughout the phase of maximal mucosal
crenulation, seen as angulated folds of vaginal mucosa with sharp profiles.
 Breeding should be discontinued when the vaginal mucosa again becomes flacid and smooth,
with patchy red and white surface which is observed 6 to 10 days following LH surge.

BASED ON HORMONE ASSAY

LH Assay

 Measurement of the peripheral plasma concentration of LH is a reliable and accurate method

for determining the optimum time to mate. In most countries there is no readily available
commercial assay for canine serum LH, and at present measurement requires
radioimmunoassay. This method is time-consuming, expensive and there is frequently a delay
in obtaining the results, because samples are assayed in batches in service laboratories.
Should LH concentration be measured, critical matings or insemination can be planned
between four and six days after the LH surge.
 At least one ELIZA assay kit for measuring LH in canine serum has been marketed for
ovulation timing (Status-LH, Synbiotics). The test is stored at room temperature and has a
relatively short shelf-life. The kit may not be accurate if used beyond the expiration date, or if
the foil packet containing the test device and pipette is opened well before the test is to be run.
The preferred sample is serum. The sample should not be lipemic or hemolyzed. The test
should be run the same day the serum is collected. If that is not possible, the serum should be
refrigerated, not frozen, until the test can be run.
 Because duration of the LH peak averages about one day in bitches, samples must be drawn
daily, at about the same time of day. Because of the tests short shelf-life, necessity of daily
testing, and the requirement of assessing progesterone to verify accuracy, this assay is rarely
used in practice.

Progesterone Assay

 There is a significant preovulatory luteinization in the bitch during and following the LH
surge as there is in many rodent and primate species. Plasma progesterone concentration
begins to increase rapidly from baseline approximately 2 days before ovulation, during the
LH surge.
 Serial monitoring of plasma progesterone concentrations therefore allows anticipation of
ovulation by about 1 to 2 days, and if continued allows confirmation of ovulation and
detection of the fertilization period.
 Since the initial rise in progesterone is progressive, it is only necessary to collect blood
samples every second or even third day, unlike the daily regime required to detect the LH
surge.Daily assays can determine the day of ovulation within 1 or 2 days in most cases, and
should be used for very critical breedings and inseminations.
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  Progesterone may be measured most accurately by radioimmunoassay (RIA), quantitative
enzyme-linked immunosorbent assay (ELISA), or immuno-chemilluminesce assay. Many
veterinary diagnostic laboratories have a turn-around time of 1 day for these accurate assays.
RIA of LH in daily serum samples can pinpoint the day of the LH surge in the majority of
dogs, and be accurate within 1 day in over 90% of cases.
 In-hospital test kits are available for measuring serum concentrations of progesterone as <3,
3-10 ng/ml and greater than 10 ng/ml. Serum should be assessed beginning 3 or 4 days after
onset of proestrus and should be continued every other day. At the time of ovulation the level
of blood progesterone rises dramatically & ovulation takes place when values of progesterone
are between 4 and 10 ng/ml. Matings should be done at this time.
 It is important to know that at the time of ovulation, the ova are primary oocytes and require
48 hours to mature. Hence a second mating should be done 48 hours later to achieve
maximum litter size.

CRITERIA FOR BREEDING THAT ARE NOT RELIABLE

LH Assay

 Measurement of the peripheral plasma concentration of LH is a reliable and accurate method

for determining the optimum time to mate. In most countries there is no readily available
commercial assay for canine serum LH, and at present measurement requires
radioimmunoassay. This method is time-consuming, expensive and there is frequently a delay
in obtaining the results, because samples are assayed in batches in service laboratories.
Should LH concentration be measured, critical matings or insemination can be planned
between four and six days after the LH surge.
 At least one ELIZA assay kit for measuring LH in canine serum has been marketed for
ovulation timing (Status-LH, Synbiotics). The test is stored at room temperature and has a
relatively short shelf-life. The kit may not be accurate if used beyond the expiration date, or if
the foil packet containing the test device and pipette is opened well before the test is to be run.
The preferred sample is serum. The sample should not be lipemic or hemolyzed. The test
should be run the same day the serum is collected. If that is not possible, the serum should be
refrigerated, not frozen, until the test can be run.
 Because duration of the LH peak averages about one day in bitches, samples must be drawn
daily, at about the same time of day. Because of the tests short shelf-life, necessity of daily
testing, and the requirement of assessing progesterone to verify accuracy, this assay is rarely
used in practice.

Progesterone Assay

 There is a significant preovulatory luteinization in the bitch during and following the LH
surge as there is in many rodent and primate species. Plasma progesterone concentration
begins to increase rapidly from baseline approximately 2 days before ovulation, during the
LH surge.
 Serial monitoring of plasma progesterone concentrations therefore allows anticipation of
ovulation by about 1 to 2 days, and if continued allows confirmation of ovulation and
detection of the fertilization period.
 Since the initial rise in progesterone is progressive, it is only necessary to collect blood
samples every second or even third day, unlike the daily regime required to detect the LH
surge.Daily assays can determine the day of ovulation within 1 or 2 days in most cases, and
should be used for very critical breedings and inseminations.
 Progesterone may be measured most accurately by radioimmunoassay (RIA), quantitative
enzyme-linked immunosorbent assay (ELISA), or immuno-chemilluminesce assay. Many
veterinary diagnostic laboratories have a turn-around time of 1 day for these accurate assays.
RIA of LH in daily serum samples can pinpoint the day of the LH surge in the majority of
dogs, and be accurate within 1 day in over 90% of cases.

~ 257 ~
  In-hospital test kits are available for measuring serum concentrations of progesterone as <3,
3-10 ng/ml and greater than 10 ng/ml. Serum should be assessed beginning 3 or 4 days after
onset of proestrus and should be continued every other day. At the time of ovulation the level
of blood progesterone rises dramatically & ovulation takes place when values of progesterone
are between 4 and 10 ng/ml. Matings should be done at this time.
 It is important to know that at the time of ovulation, the ova are primary oocytes and require
48 hours to mature. Hence a second mating should be done 48 hours later to achieve
maximum litter size.

RECOMMENDATONS FOR A BREEDING PROGRAMME

A set of simple guidelines should be available for the conscientious breeder of an apparently healthy
bitch. These are guidelines that cannot harm the animal and usually increase success of any breeding
program.

 Record the first day of vulvar swelling, bloody vaginal discharge, and when males become
obviously interested in the bitch.
 Begin “teasing” the bitch with a male dog on day 5 or 6 of proestrus, and repeat this
procedure every 2 or 3 days to determine the first day of standing heat. In cases of previous
infertility, begin on the first day that proestrus is observed.
 Allow the bitch to be bred, beginning on her first dayof acceptance of the male, and continue
to breed every 2 to 4 days throughout the acceptance period.
 In cases of infertility, as well as bitches with short or prolonged standing heat, teach an owner
how to obtain vaginal smears. Smears should be obtained once daily throughout apparent
proestrus and estrus as well as several days into diestrus. The veterinarian can then stain and
review slides as they are brought in, or the entire series of slides can be reviewed after estrus
has apparently ended. The results of slide interpretation can then be correlated with breeding
dates and conception rate. When possible, a series of serum progesterone measurements with
or without vaginoscopy should be considered.
 Complete records should be kept on the dates of proestrus, breeding, and vaginal smears.
Notes should be made on the presence or absence of ties, the length of each tie, and the
behavior of both the male and female. The success of the male in siring litters with other
bitches should be recorded. Records should also be kept on whelping dates, litter size, health
of puppies, length of parturition, interval between births, and any other valuable information.
This includes the reason for destroying any puppies.

ARTIFICIAL INSEMINATION IN DOGS

 The manual collection and subsequent deposition of semen into the vaginal vault of a bitch in
castrus is a common procedure used by breeders and veterinarians.
 As a result, artificial insemination is frequently requested by the dog owner or handler. Fresh
undiluted semen, semen mixed with an extender, or frozen semen is used in dogs.
 To enhance changes of success, veterinarians must have a good understanding of the estrus
cycle, semen collection and AI techniques, and potential pitfalls that may be encountered.

INDICATIONS

There are several situations in which AI may be used. They are

 inability of the male and female to breed.

o For the bitch, these problems include
 vaginal strictures
 conformational defects
 rear leg weaknes
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  psychological problems
 pain
o for the male
 weakness
 arthritis
 back pain
 premature ejaculation
 conformational defects that prevent intromission or a “tie” .
 AI may also be chosen because of a major size difference between the mates.
 Psychological problems may also result in the need for AI.
 Some owners wish to use AI avoid any possible venereal contact between their dog and its
mate, thereby controlling the spread of potentially infectious diseases. Inseminating semen
into the vagina still provides intimate contact between bitch and stud dog and therefore any
infectious agent that could be transmitted from the dog to the bitch during natural mating also
has the potential to be transmitted during AI. However AI does avoid transmission of
infectious agents from the bitch to the stud dog. All breeding dogs and bitches should be
Brucella-free as determined by appropriate tests.
 Some male dogs experience prostatic bleeding and hemospermia following exposure to a
bitch “in heat”. The bleeding may be associated with Von Willebrand’s disease (VWD) but
has also been observed in dogs not afficted with VWD. Regradless, AI can be performed
without any contact between stud dog and bitch, occasionally avoiding this problem.

Fresh extended and frozen forms of semen are being used with increasing frequency. Because semen
collection is the difficult task, insemination of extended or previously frozen semen remains a
relatively simple procedure. The shipment and use of fresh, extended or frozen semen helps defray the
cost and removes the hazards associated with shipping the female.

SEMEN COLLECTION

 Semen is usually collected in an artificial vagina or in collection cups. One end of a sterilized,
soft rubber, cone-shaped bovine artificial vagina is cut off and to this is attached a 12 to 15
ml plastic tube. The wide-mouth end of the cone is folded over and sealed with rubber cement
to make a smooth, nonabrasive edge. A small amount of nonspermicidal lubricating jelly is
applied to the inner surface of the rubber cone. This is the only surface that make penile
contact.
 The most difficult task in AI is stimulating the male to ejaculate. Once this is accomplished,
the balance of the procedure is quite simple. With most stud dogs, semen can be collected in a
clean, quiet room with a nonslippery floor. A bitch is not often needed. However, for
experienced males accustomed to natural breeding, a bitch in heat makes collecting semen
easier. With the owner holding the stud dog to minimize movement and to protect the
collector, the penis and bulbus glandis are gently massaged within the penile sheath.When the
bulbus glandis begins to enlarge, the sheath is slipped posteriorly and the penis with the
bulbus glandis is exteriorized. Failure to exteriorize both the penis and the bulbus glandis
from the sheath usually results in an incomplete erection and failure to ejaculate or
incomplete ejaculation, presumably due to pain.
 Once the penis and bulbus glandis are extruded from the sheath, the collector firmly holds
onto the the base of the penis, proximal to the bulbus glandis. The thumb and index finger are
used, providing both massaging movements and downward pressure around the base of the
bulbus glandis. During or immediately after achieving an erection, aggressive pelvic thrusting
movements by the stud dog may make it difficult to place the artificial vagina over the penis.
Fortunately, pelvic thrusting is typically short-lived. The initial phase of the ejaculate consists
primarily of sperm-free prostatic fluid, and the sperm-rich second fraction of the ejaculate
usually begins as the pelvic thrusting begins to subside. Immediately after pelvic thrusting
stops, many males try to”step over” the collector’s arm as if dismounting the bitch. The
collector should simply allow this movement by the male, which results in a 180° rotation of
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 the engorged penis such that it is protruding caudally between the rear legs. Digital pressure
should be maintained on the bulbus glandis and the collection continued until the ejaculate
becomes clear.

 Semen is usually collected for a period of 2 to 5 minutes. This represents the typical duration
of the second phase of ejaculation. The clear plastic tube should already have been connected
to the rubber artificial vagina, and the apparatus can be held under the collector’s arm during
the initial stimulation period to provide some warmth. Canine semen is relatively resistant to
cold shock, alleviating the need for warm water baths or incubators for holding semen. The
use of a clear plastic collection tube allows visualization of the semen. One hand is kept over
the plastic to avoid excessive light exposure. As long as the ejaculate is obviously whitish or
creamy and cloudy the ejaculate continues to be collected. When the ejaculate becomes clear
one can discontinue collection. If one is not certain when the male has ceased ejaculating the
sperm-rich fraction, stop the collection after 5 minutes. Continued collection only dilutes the
sperm with the third-phase clear, sperm free prostatic fluid, resulting in cumbersome fluid
volumes.
 The collection of the third fraction of the ejaculate is not necessary for successful
insemination. The semen volume that can be handled easily is 3 to 10 ml. the collection
system containing sperm-rich semen can be exchanged for a clean system if there is any need
to evaluate the prostatic fluid.
 The bitch should be inseminated within 5 to 10 minutes of collecting semen. Prior to
insemination the color and consistency of the semen should be noted and a small drop placed
on a warm glass slide. The semen can be quickly evaluated microscopically to easure that a
normal number of live, progressively motile sperm are present. During this time, the semen is
kept warm by holding the tube in one’s hand, which also minimizes exposure of semen to
potentially harmful ultraviolet rays. Alternatively, a drop of semen can be evaluated by a
technician while the remainder of the semen is being placed into the insemination device. The
presence of abnormal color or consistency, oligospermia, or dead sperm should be identified.

FRESH SEMEN ARTIFICIAL INSEMINATION

Insemination Procedure

 Although there are a variety of “tools” used for insemination, commonly used tools are
o 12 ml syringe
o flexible disposable male urinary catheter or rigid plastic insemination pipette or the
Cassous' AI Sheath
o surgical gloves.
 These items should be sterile.
 After the gloves are put on, the semen sample is drawn into the syringe, the sterile catheter is
attached, and the syringe is then filled with an additional 1 to 3 ml of air.
 A gloved, non lubricated index finger is placed into the vaginal vault, palm up. If a lubricant
is used it must be nonspermicidal.
 The catheter is then slid over the top of the finger and passed into the vaginal vault,avoiding
accidental catheterization of the urethra. Sliding the catheter over the index finger also aids in
avoiding the clitoral fossa.
 The catheter follows the dorsal curvature of the vaginal vault.
 The catheter is inserted until resistance is met. The resistance indicates that the cranial end of
the vagina vault has been reached or the catheter has simply become trapped within vaginal
folds.
 The catheter should be gently advanced as far cranially as possible before the semen is
deposited to ensure deposition of spermatozoa near the cervix and into the uterus.
 Once the syringe containing the semen has been emptied it should be disconnected from the
catheter, filled with a few more milliliters of air, reattached to the catheter, and emptied,
thereby depositing any semen that may have remined in the catheter.

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  Care should be taken to avoid injecting too much air into the vagina, as this may result in loss
of semen out of the vulva once the procedure is completed.
 Once the semen has been deposited, the catheter should be removed and the hindquarters of
the bitch should remain elevated above her head for a minimum of 20 minutes, thereby aiding
the movement of semen anteriorly in the reproductive tract and into the uterus. Insertion of a
gloved finger and the gentle stroking of the dorsal wall of the vagina during this time may
stimulate muscular contractions within the reproductive tract, further enhancing the
movement of spermatozoa toward the ovaries.
 After elevation of the hindquarters has been completed, the bitch should be kept quiet for an
hour or so to minimize loss of semen out of the vagina. In addition,pressure should not be
applied to the abdomen.
 The entire insemination procedure is rarely a problem for the bitch. There should not be any
pain or discomfort associated with the procedure. For these reasons, the procedure is rather
simple and not time consuming.

EXTENDED CHILLED SEMEN AI

 Semen that has been properly extended and chilled can be refrigerated for several days yet
still yield fertile sperm when warmed and inseminated.
 The extender helps keep the spermatozoal membranes from being harmed by changes in
temperature or shaking during transport, while also providing nutrients and stabilizing the pH
of the medium.
 Semen once extended, is gradually cooled to 5 to 15°C over a period of 30 to 60 minutes.
 Rapid changes in temperature must be avoided.
 When prepared properly, chilled extended spermatozoa easily remain viable for 24 hours and,
depending on the technique, may remain viable for as long as 5 days.
 Sort – term preservation of sperm allows overnight air delivery of freshly extended semen
without the costs of frozen semen or shipment of the bitch.
 The ejaculate is extended, packaged in a small container, and shipped in a thermos-type
container. The ejaculater, should remain cold during shipment and should be kept refrigerated
until use.
 The technique for insemination of the bitch with extended semen is as previously described
for insemination with fresh semen.
 Ideally, multiple inseminations beginning 3 to 4 days after the initial rise in blood
progesterone concentration should be completed to maximize conception rate.

FROZEN SEMEN AI

Long –term preservation of semen utilizing deep freezing techniques has been available for several
years.

 The advantages of using frozen semen include

o wider dispersion of desirable genetic traits,
o disease prevention,
o decreased numbers of breeding males in research colony,
o preservation of semen from dogs with diseases that are models of human disorders,
o elimination of the need for shipping bitches.
 The steps involved in freezing semen for AI include
o Semen collection
o Dilution in an extender
o Equilibration under refrigeration
o Freezing in convenient volumes
o Storage
o Thawing and insemination of the bitch during the peak of her fertile period.

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  Extenders such as egg yolk, glycerol, lactose, skim milk and antibiotics are typically added to
the semen prior to freezing. Extenders have
o nutrients as an energy source,
o buffer against harmful changes in pH,
o provide a physiologic osmotic pressure and concentration of electrolytes,
o prevent growth of bacteria,
o protect cells from cold shock during the cooling process,
o have cryoprotectants that reduce sperm cell damage during freezing and subsequent
thawing.
 Canine sperm is often frozen in 0.5 ml, plastic “French straws.” Straws provide a convenient
format for handling, labeling, storage and thawing.
 Alternative formats include bulk semen volumes sealed in ampoules and semen frozen in
small spherical pellets on a block of dry ice.
 Frozen semen has been stored for as long as 9 years with little to no post-thaw decrease in
sperm motility. More than 4 years have been reported between semen collection/storage and
thawing/conception. Thawing instructions usually accompany the semen and should be
followed exactly.
 At the time of insemination, small drop of thawed semen should be evaluated microscopically
for sperm viability and motility.

CONCEPTION RATES

 The success of AI depends upon several variables and include

o the use of a fertile stud dog with normal semen
o proper handling of the semen during collection and insemination
o and the deposition of semen into the anterior vaginal vault of the bitch at the
appropriate time of estrus.
 The importance of the latter cannot be overemphasized. Determining when to perform AI
should always be based on a combination of
o behavior of the bitch
o sequential vaginal cytology
o plasma progesterone concentrations
o vaginal endoscopy. Evaluation of serial vaginal cytology and plasma progesterone
concentrations, in conjunction with changes in behavior of the bitch, are easy,
relatively inexpensive, and accurate methods to determine when the bitch should be
inseminated.
 Multiple inseminations, performed daily or every other day until diestrus as confirmed with
vaginal cytology, should be done once the decision is made to perform AI. Since, there is no
way to predict exactlywhen ovulation has occurred, multiple inseminations increase the
change of conception by ensuring the presence of spermatozoa in the female tract both early
and late in estrus.
 Artificial insemination may be associated with lower conception rates and smaller litter sizes
than would be achieved with natural breeding. This is likely a result, of several factors such as
o During natural breeding, semen is pressure-forced through the cervix into the uterus
and oviducts, whereas in AI, the semen is placed posterior to the cervix.
o The erect penis virtually fills the vaginal vault, compressing the urethral opening, and
the bulbis glandis prevents backward flow of semen. The large volume of ejaculate
produced during the normally prolonged third phase is forced through the cervix
because there is no other place for distribution.
o With natural breeding, uterine contractions may aid in semen transport. This is an
unlikely event in most AI situations.
o Also in natural breeding the duration of the tie may contribute to improving
conception rates, because the male ejaculates during that entire period producing a
volume of semen too large for the vaginal vault.

CONCEPTION RATES FOLLOWING FRESH SEMEN AI

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  Conception rates of 75 to 80% have been reported when AI is performed properly. This
technique places the semen in the cranial vagina.
 Although deposition of semen in the uterus may improve the conception rate, catheterizing
the cervix of a bitch in estrus is not so easy primarily because of the anatomic conformation
of the canine cervix.

CONCEPTION RATES FOLLOWING CHILLED SEMEN AI

 Conception rates for AI using fresh extended semen are variable but have been reported to be
60% or higher.
 In some studies conception rates for AI using fresh extended semen are higher than with
frozen semen.
 With fresh extended semen, no damage to the sperm occurs due to freezing, the cervix is less
of a barrier, larger numbers of sperm are usually inseminated, fresh sperm live longer in the
reproductive tract of the bitch, and timing of inseminations is not as critical as with frozen
semen.

CONCEPTION RATES FOLLOWING FROZEN SEMEN AI

 Most commercial facilities claim a frozen semen fertility rate of 70 to 80% based on litters
per bitch inseminated, or per bitch inseminated at the appropriate time and excluding
mistimed inseminations performed primarily at the insistence of the owner. These high
success rates are in contrast to published success rates of 0 to 60% when intravaginal
insemination was used Properly timed intrauterine inseminations using either surgical or
transcervical deposition, have reported success rates of 46 to 83% .
 The reasons for poorer conception rates with intravaginal than with intra uterine insemination
are not known but are probably related to poor sperm transport through the cervix.
 Insemination after cannulation of the cervix is preferable to deposition of the semen in the
anterior vaginal vault.
 Cannulation of the cervix is difficult because of its relative inaccessibility and its anatomy.
Nevertheless, cannulation can be accomplished with a flexible catheter under direct
visualization with a fiberoptic endoscope or with a rigid cannula and fixation of the cervix by
palpation per abdomen.
 Obtaining successful pregnancies and reasonable litter size with frozen semen depends greatly
upon performing inseminations during the 2 to 3 days in which healthy, fertilizable ova reside
in the oviducts of the bitch.
 Fresh sperm can remain fertile in the bitch for 6 to 7 days. Frozen sperm die rapidly after
being thawed and do not survive the long periods that fresh dog sperm do. As a consequence,
AI with frozen semen should be performed about 2 to 3 days after ovulation, 4 to 5 days after
the preovulatory LH surge, or 5 to 6 days after the first rise in plasma progesterone
concentration. Once started, inseminations should be done every day or every other day.

MISMATCHING

MEDICAL TERMINATION OF PREGNANCY

Unwanted mating or mismating is a common clinical problem in veterinary practice which may arise
because dog owners are not aware that their pet is “in heat” or the owner may be aware of estrus but
underestimated the will of a stud or bitch that wants to encounter the opposite sex. Therefore, even the
best educated and most careful owners will end up with mismating problems in their bitches.

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Ovariohysterectomy would be the best method to prevent birth of puppies following mismating, but
for bitches whose future breeding potential needs to be maintained, medical termination of pregnancy
becomes imperative.

 Pregnancy in the bitch can be divided into three periods.

o Begins at fertilization and ends at implantation (day 20-22 from LH peak).
o Begins at implantation and ends at the time of fetal ossification (40-42 days).
o Begins at fetal ossification and ends at parturition.
 Although methods to induce abortion are available for all three periods, only the second
period is of practical clinical interest.
 The options to prevent birth of pups include those that prevent or interfere with implantation,
those that alter the normal endocrine environment and induce resorption or abortion, and
those, which are directly embryotoxic. The main objective when planning such treatments
should be aimed at inducing abortion only if the bitch is pregnant

METHODS

Abortion induction methods involve

 Modification of estrogen progesterone ratio by administration of estrogen/synthetic estrogen

derivatives / glucocorticoids.
 Induction of functional luteal arrest or luteolysis by substances that act directly
(prostaglandins) or indirectly on the CL by inhibiting luteotrophic support. For eg. dopamine
agonists suppress prolactin secretion whereas GnRH antagonists deplete LH.
 Blockage of progesterone secretion by inhibiting steroidogenesis (epostane).
 Blockage of progesterone action at the receptor levels (anti progesterone (mifipristone /
algepristone).

ABORTION INDUCTION BEFORE IMPLANTATION

ESTEROGEN

 Large doses of estrogens have been used for many years to prevent implantation following
mismating. However, currently estrogens are to be avoided because of their toxic effects.
 Estrogens exert their action by causing closure of the uterotubal junction, alter the transport
time of zygotes and exert direct embryotoxic effect.

Recommended protocols

 Estradiol benzoate @ 0.5 - 3.0 mg every other day for a total of three injections beginning 4 -
10 days after misalliance.
 Estradiol valerate @ 3.0 - 7.0 mg administered once 4 to 10 days after mismating.

Side effects and Toxicity of Estrogens

 Estrogens at high doses cause

o bone marrow suppression leading to thrombocytopenia, leukopenia, severe anaemia
and death
o development of pyometra related to abnormal increase in estrogen: progesterone
ratio, cystic hyperplasia of the uterine glands, abnormal epithelial secretion,
relaxation of the cervix and ascending infection
o induction and prolongation of estrus behaviour. As a result bitches may continue to
attract males for 7 to 10 days
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 o less severe side effects include alopecia, skin hyperpigmentation, mammary gland
and vulval enlargement.

ABORTION INDUCTION AFTER IMPLANTATION

PROSTAGLANDINS

 Prostaglandins when administered to the bitch cause lysis of corpora lutea and decrease in
plasma concentrations of progesterone. Since, corpora lutea of the bitch are highly resistant to
prostaglandins than those of other species, repeated therapy is necessary to achieve complete
regression of the corpus luteum. Prostaglandins also produce contraction of smooth muscle
having an ecbolic effect that may a part of the mechanism of inducing abortion.

Candidates for Prostaglandin Administration

 The bitches must be healthy and less than 7 years of age.

 Pregnancy must be confirmed using ultrasonography

Treatment Protocols

 The half life of PG is only seconds and it remains in circulation for a few minutes following
an intramuscular injection or perhaps a little longer when administered by subcutaneous
injection. Hence repeated administration several times a day is a must for absolute efficacy,
whether using low, moderate, or high doses. Treatment must be continued until the efficacy
is verified by ultrasound or palpation. Partial abortion of litters can occur if treatment is
discontinued prematurely. Whatever may be the dose, a minimum of 5 to 7 or sometimes
even 9 or more days may be required to terminate pregnancies.

Drug Dose Day of pregnancy Duration

Single dose Natural
treatment PGF2α
Cloprostinol
Multi dose Natural
treatment PGF2α
(SC injections)
20 – 30µg/kg BID or TID After day 30 4-7 days longer
100 – 250µg/kg BID After day 30 4-7 days longer
2.5µg/kg every 48 hrs. After day 30 4-7 days longer
Day 1 Day 2-3 Day 4 onward Between day 25 7 days or longer
25 50 µg/kg 50 to 100 µg/kg and 35 of
µg/kg or higher pregnancy

Note

Ultrasound examination is the only reliable means to evaluate the viability of fetus in early and mid
gestation. Radiography to confirm efficacy based on absence of fetal skeletons would not be reliable
until after day 45 of gestation.

Side Effects

 Since prostaglandins act on all smooth muscles of the body, side effects such as panting,
respiratory distress, excess salivation, vomiting, defecation, stranguria and/or urination are
observed within 3 seconds to 3 minutes after injection and persisted for 4 to 55 minutes.
However, bitches adapt to the PGF2α , with side effects diminishing after each subsequent

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 injections. After 6 to 8 injections, side effects were minimal to absent in all dogs, regardless
of the dose protocol.
 Side effects can be reduced by concurrent administration of anticholinergic drugs like
atropine @ 0.5 mg/kg SC or IM, 15 minutes before prostaglandin administration. Walking the
animal for 30 minutes following PGF2α injections would help to reduced intensity of side
effects. One must bear in mind that physical side effects of PG though frightening are only
transient but not life threatening.

DOPAMINE AGONIST

 Prolactin is the major luteotropic hormone throughout the luteal phase in both pregnant and
non pregnant bitches which means the progesterone production from corpora lutea is prolactin
dependent.
 Dopamine agonists like bromocriptine or cabergoline are ergot alkaloids with strong
dopamine D2 receptor agonist activity and suppress prolactin secretion and thereby
progesterone levels.

BROMOCRIPTINE

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  Bromocriptine, at dose of 0.1 mg / kg PO BID for a minimum of 6 days terminated pregnancy
after day 30. Treatment should not be discontinued until all fetuses are expelled (as confirmed
by U/S).
 Bromocriptine has potent dopamine receptor activity and produces side effects including
severe vomiting, anorexia and depression. Peripherally acting emesis inhibitors other than
metaclorpromide (Metaclorpromide and Bromocriptine are antagonistic) could be given prior
to administration to prevent emesis.

CABERGOLINE

 Compared to Bromocriptine, Cabergoline is a more potent dopamine agonist with milder side
effects as it is a more specific D-2 dopamine receptor agonist and is less able to cross the
blood brain barrier and have CNS effects. A dose of 5 µg/kg PO once daily for 5 days after
day 32 of LH surge caused abortion in dogs.
 A combination therapy of oral cabergoline at 5 µg/kg per day and low dose of cloprostenol
(PGF2α analogues) injection of 1 µg/kg SC every other day for upto 9 days terminated
pregnancy in dogs when administered starting around day 28 of pregnancy. The objective of
this combination is to reduce CL function and progesterone release by a double mechanism of
action.
o Direct local effect of PG on CL steroidogenesis
o Indirect effect due to withdrawal of pituitary prolactin support.
 Resorptions with some vaginal discharge usually occurred 5 to 8 days after the beginning of
treatment which could be visualized and confirmed sonographically.

ANTIPROGESTIN

 Antiprogestins are synthetic steroids which bind with great affinity to progesterone receptors
thereby preventing progesterone induced changes in DNA transcription. As a result, the
effects of progesterone are not there.
 Mifepristone (RU486) is one such antiprogesterone which induced direct luteolysis. In
pregnant bitches, Mifepristone was found to be effective if administered after day 32 of
gestation @ 2.5 mg/kg orally twice daily for 4.5 days. Pregnancy was terminated without any
side effects within 3 to 6 days after treatment.
 A combination of low doses of prostaglandin E such as misoprostol and mifepristone is
currently being used to improve the action of mifepristone.
 Another analog of RU-486, Aglepristone (RU-534) can be used to prevent pregnancy when
administered as two injections SC of 10 mg/kg 24 hrs apart. This drug is found to have an
efficacy of 97.3% and does not cause any side effects.

CORTICOSTERIODS

 Dexamethasone when administered beginning at mid gestation can terminate pregnancy in

dogs by activating endogenous mechanisms similar to those involved in parturition.
 Injectable Dexamethasone (5 mg twice daily IM for 10 days) and oral Dexamethasone (0.1 to
0.2 mg twice daily at a decreasing dose for 5 to 10 days).

Dose schedule for Dexamethasone (µg/kg) used in a 9.5 day protocol of twice daily oral
dexamethasone administration for termination of canine pregnancy

Time of 1 2 3 4 5 6 7 8 9 10
Day
Morning 200 200 200 200 200 200 200 120 60 10
Evening 200 200 200 200 200 20 160 80 20 -

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  Some bitches had brownish vaginal discharge at the time of abortion while no signs were
observed in other bitches.
 Side effects like polydipsia and polyurea were observed shortly after start of treatment and
persisted for a few days after end of treatment.
 Restlessness and milk secretion were also observed in some bitches during period of abortion
or resorption.
 Failure of dexamethasone to terminate pregnancy has resulted in birth of live normal pups at
term; delivery of dead pups near term and partial abortions with expulsion or resorption of
few fetuses and then birth of 1 or 2 dead pups at term.

Epostane

 Epostane is a steroid molecule that inhibits steroid synthesis at the level of the hydroxy
steroid – dehydrogenase – isomerase enzyme system, thereby inhibiting the formation of
progesterone from pregnanolone. Epostane terminates pregnancy when given orally at 2.5 to
5 mg/kg for 7 days starting any time after diestrus begins. No adverse side effects have been
observed upto 10 mg/kg; but at higher doses sterile abscess may form at the site of injection.

GnRH Antagonists

 GnRH antagonists are effective after day 20 of diestrus and act by decreasing concentration of
circulating gonadotropins causing luteolysis, decline in serum progesterone concentrations,
and subsequent pregnancy loss. But, currently this preparation is not available in clinical
practice.

PSEUDOPREGNANCY

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  In dogs, the clinical condition of overt pseudopregnancy called simply pseudopregnancy,
pseudocyesis, false pregnancy or nervous lactation is a syndrome observed in non-pregnant
bitches about 6 to 12 weeks after estrus and is characterized by clinical signs such as nesting,
weight gain, mammary enlargement and lactation.
 Since, there is no pregnancy recognition system in dogs; the corpus luteum continues to
remain functional in both pregnant and non pregnant bitches.
 Hence, all non pregnant dogs in diestrus stage of the cycle are referred to as pseudopregnant
(physiological pseudopregnancy).
 However, when extreme behavior or atypical mammary activity are presented as clinical
problems involving changes similar to those seen in late pregnancy or the early post-partum
period, the condition can best be termed "clinical pseudopregnancy" or "overt
pseudopregnancy".

PATHOPHYSIOLOGY

 The occurrence of pseudopregnancy appears to be related to and dependent on a previous

prolonged, and in most cases a very recent, exposure to elevated levels of progesterone.
Further, it has been suggested, based on indirect evidence, that pseudopregnancy may occur
as a result of increased concentrations of prolactin or an increased sensitivity to prolactin
induced by a more rapid than normal decline of progesterone levels in the late luteal phase. In
fact, spaying or ovariectomy during the luteal phase (i.e., during metestrus/diestrus) induces
pseudopregnancy in some bitches.

Probable and Proposed Causes of Clinical Pseudopregnancy in Female Dogs

 Idiopathic occurrence of a more extensive increase in prolactin than occurs in normal diestrus.
 Idiopathic increase in sensitivity to the endocrine changes that normally occur in late diestrus,
including the normal progressive decline in progesterone and modest elevation in prolactin.
 Pseudo-luteal phase induced by administration of exogenous progestins
 Progesterone withdrawal caused by:
a. ovariectomy during diestrus,
b. termination of long-term or short-term progestin therapy
c. idiopathic or prostaglandin-induced abrupt luteolysis
d. antiprogestin therapy
 Idiopathic hyperprolactinemia potentially associated with pituitary microadenomas.
 Physchogenic or reflexive hyper-prolactinemia occurring in response to stimulation by
surrogate neonates or other visual, physical or social stimulation.

CLINICAL SIGNS

 Common signs include

o Prepartum and maternal-like behaviors.
o Nesting, digging, over-affection, over-protectiveness, over-defensiveness, aggression,
licking, mothering of inanimate objects.
o Mammary enlargement and distension. Mammary hypertrophy is usually more
evident in the most caudal pair of glands although the entire mammary chain can be
involved.
o Lactation and milk release. Milk production during pseudopregnancy apparently
results from the development of not only intra-acinar but also intra-canalicular
mammary secretion in predisposed bitches. Lactation is often encouraged by self-
nursing or by adoption of unrelated neonates.
o Weight gain
o Anorexia
o Sometimes abdominal contractions that mimic those of parturition

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  Less common clinical signs include
o Emesis
o Abdominal enlargement
o Abdominal contractions
o Diarrhoea
o Polyuria
o Polydipsia
o Polyphagia
 Pseudopregnancy is a self limiting condition and signs normally cease after 2 to 4 weeks.
 A high recurrence rate in successive estrous cycles has been reported in susceptible bitches.
 Pseudopregnancy and prolactin have been implicated in the pathogenesis of mammary
tumors. An increased risk of mammary neoplasia associated with pseudopregnancy might be
explained by a continuous mechanical distension of, and the accumulation of carcinogenic
products within, the mammary acini caused by the formation and retention of milk.

DIAGNOSIS

 Diagnosis is based on reported clinical signs. Ultrasound or radiography should be used to

differentially diagnose from pregnancy as unscheduled matings may be overlooked by
owners.
 Other conditions of the luteal phase, such as pyometra or recent pregnancy and abortion,
should be ruled out by abdominal ultrasonography or radiography, a complete blood cell
count and additional ancillary testing, including vulval and vaginal examination.

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  It is also important to keep in mind that pseudopregnancy can co-exist with other reproductive
or non-reproductive clinical problems, sometimes making diagnosis more difficult

TREATMENT

 Since, pseudopregnancy is typically a self-limiting state, mild cases usually need no treatment
other than discouraging maternal behavior.
 Treatment for pseudopregnancy may either be
o Conservative Treatment
o Aggressive Treatment

CONSERVATIVE THERAPY

 Placing of Elizabethan collars to prevent licking of the mammary glands is recommended.

 Avoid licking, milking, or use of compresses as they stimulate lactation.
 Withdrawal of water for 5 to 7 consequent nights promotes fluid conservation and also helps
to terminate lactation (normal renal function should be documented beforehand).
 When behavioral signs are significant, light tranquilization with non-phenothiazine drugs can
be useful.
 Phenothiazines are not recommended in pseudopregnant bitches as they stimulate prolactin
secretion.
 Considering the possible relationship between pseudopregnancy and subsequent development
of mammary tumors the recent approach is to treat psuedopregnancy with pharmacological
agents.

AGGRESSIVE THERAPY

 Pharmacological approaches to the treatment of moderate to severe pseudopregnancy have

historically included steroids, including estrogens, progestins and androgens. More recently
the preferred method of treatment is the use of prolactin-suppressing drugs, especially
dopamine agonists, whereever available.

Sex Steroid Therapy

 Steroids such as estrogens, progestins and androgens have been traditionally used to treat
pseudopregnancy but the side effects usually outweigh any benefits of these medications.
High doses appear to exert a negative effect, either by suppressing pituitary prolactin or
decreasing sensitivity to prolactin.
 Estrogens
o Estrogens such as diethylstilbestrol, estradiol benzoate or estradiol cipionate have
been used. They may cause signs of proestrus or estrus, uterine disease such as
pyometra, and anemia due to bone marrow depression. The use of estrogen is not
recommended.
 Androgens
o Androgens including testosterone and synthetic androgens can suppress lactation.
Clitoral hypertrophy, virilization, and epiphora are side effects noticed. The synthetic
androgen mibolerone has been shown to reduce the duration of pseudopregnancy.
 Progestins
o Progestins such as megestrol acetate and medroxyprogesterone acetate, administered
orally, have been used to suppress the symptoms of overt pseudopregnancy probably
by suppressing prolactin secretion or reducing the tissue sensitivity to prolactin.
However, withdrawal of progestin results in recurrence of symptoms including
lactation. Progestin administration can cause cystic endometrial hyperplasia-pyometra
complex and insulin resistance, as well as mammary gland nodules, mammary
tumors, and acromegaly. Administration of progestins is therefore not recommended.

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Prolactin-Suppression Therapy

 Dopamine Agonists
o Secretion of prolactin by the pituitary is mainly under tonic inhibitory control of the
hypothalamus, mediated by a direct action of dopamine, the major prolactin inhibiting
factor (PIF).
o The dopamine agonists bromocriptine and cabergoline are the most common ergot
compounds used clinically to inhibit prolactin secretion.
 Bromocriptine
o Oral administration of Bromocriptine at varying doses ranging from 10 to 100
µg/kg/day for 10 to 14 days has been recommended by various authors. Since, it has a
short half-life of + 4 to 6 h and should be administered at least twice a day for
greatest efficacy.
o Unlike cabergoline, bromocriptine also crosses the blood-brain barrier and can
stimulate other brain centers in addition to the hypothalamus. Emetic effects result
from stimulation of the hypothalamic vomiting center.
o The ED50 for emesis is near the commonly used therapeutic doses and digestive side
effects are frequent and proportional to the dose. Common side effects include
vomiting, anorexia, depression, and other behavioral changes. Side effects tend to
decrease during the course of treatment.
o To prevent or reduce the incidence of emesis, bromocriptine can be administered in
low and then increasing doses, or administered with the food. In addition, vomiting
can be managed by administration of anticholinergic drugs such as atropine.
o Care should be taken, when trying to prevent emesis, not to use central dopamine
blockers of synaptic transmission whose action would oppose that of bromocriptine.
o Bromocriptine is formulated in 2.5 mg tablets for use in humans, and fractionation of
the tablets is necessary to achieve dosages of 10 to 30 µg/kg typically administered to
pseudopregnant bitches. This makes it difficult to administer exact doses, and may
have caused an overestimation of the drug's side effects.
o Preparation of exact dosages is important. Administration of the 10 to 30 µg/kg dose
twice or even 3 times a day is preferable to once a day administration.
 Cabergoline
o Cabergoline has greater bioactivity, superior D2-receptor specificity, and a longer
duration of action compared to bromocriptine.
o It can be effectively administered once a day. Cabergoline crosses the blood brain
barrier only slightly and consequently has much less central emetic effects than some
other dopamine agonists.
o The ED50 for emesis is 4 times the therapeutic dose and gastrointestinal signs are
rare. Cabergoline is used for treating pseudopregnant bitches at a dose of 5 µg/kg/day
for 5 to 10 days, given orally.

Anti-Serotonergic

 Metergoline
o Metergoline, an anti-serotonergic veterinary drug marketed for the treatment of
pseudopregnancy in bitches in Europe and in some Latin American countries
(Contralac®) is an ergot alkaloid which has a dopaminergic effect and thus reduces
prolactin secretion when administered at high doses.
o It has a short half-life and hence has to be administered twice a day.
o The recommended dose is 0.1 mg/kg, orally, twice a day, for 8 to 10 days.
o Anxiety, aggressiveness, hyper excitation and whining are the most frequent side
effects of metergoline, which are due to its central anti-serotonergic effect.

OVARIECTOMY

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  Predisposed bitches not intended for breeding should be spayed. Ovariectomy is the only
permanent preventive measure. This should preferably be done during anestrus.
 Ovariectomy during lactation can lead to an extended pseudopregnancy.
 In bitches with a history of overt pseudopregnancy, spaying during diestrus may provoke an
episode of pseudopregnancy 3 to 7 days after surgery .

TRANSMISSIBLE VENERAL TUMOUR IN DOGS

Transmissible venereal tumour (TVT) also known as infections sarcoma, venereal granuloma,
transmissible lymphosarcoma or stickler tumour is a benign reticuloendothelial tumour of the dog that
mainly affects the external genitalia and occasionally the internal genitalia. Since, it is usually
transmitted during coitus, it mainly occurs in young sexually mature animals. The transmissible
venereal tumour cells contain an abnormal number of chromosomes ranging from 57- 64 in contrast to
the normal 78 of the species.

ETIOLOGY

 TVT arises from allogenic cellular transplants and the abnormal cells of the neoplasm are the
vectors of transmission. Exfoliation and transplantation of neoplastic cells due to physical
contact during mating or licking of affected area is responsible for spread onto genital, oral or
nasal mucosa.
 The implantation of the tumour is facilitated by the presence of any mucosal lesion or by the
loss of mucosal integrity.
 Growth of tumour occurs 15 to 60 days after implantation.
 Metastasis may occur in less than 5-17% of cases.

GROSS AND HP

 Small pink to red, 1 mm to 3 mm diameter nodules can be observed 2 or 3 weeks after

transplantation.
 Initial lesions are superficial dermoepidermal or pedunculated. Multiple nodules then fuse
together forming larger, red, hemorrhagic, cauliflower-like, friable masses of 5 to 7 cm in
diameter which then progress deeper into the mucosa as multilobular subcutaneous lesions
with diameters that can exceed 10 - 15 cm.
 Tumours bleed easily and while becoming larger, normally ulcerate and become
contaminated
 Exfoliative cytology shows discrete cells that are round to oval, with moderately abundant
pale blue cytoplasm, an eccentrically located nucleus, with occasional binucleation and
mitotic figures. Single or multiple nucleoli are often observed. The most characteristic feature
of TVT cells is the presence of numerous discrete clear cytoplasmic vacuoles.

CLINICAL SIGNS

 Clinical signs vary according to localization of the tumours.

 Discharge may be confused with urethritis or cystitis.
 In bitches the tumours are localized in the vestibule and/or caudal vagina, protrude from the
vulva and frequently cause a deformation of the perineal region. Rarely, the tumour masses
interfere with micturition.
 A considerable hemorrhagic vulvar discharge may occur and can cause anaemia if it persists.
 The discharge can attract males and the condition of the bitch can be mistaken for estrus by
the owners. Infrequently, TVTs can localize in the uterus.
 In cases with extra genital localization of the TVT, clinical diagnosis is usually more difficult
because TVTs cause a variety of signs depending on the anatomical localization of the

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 tumour, eg., sneezing, epistaxis, epiphora, halitosis and tooth loss, exophthalmos, skin bumps,
facial or oral deformation along with regional lymph node .

Diagnosis

 Diagnosis is based on
o Physical examination.
o Cytological findings of TVT in exfoliated cells obtained by swabs, fine needle
aspirations or imprints of the tumours.

Click to view an You Tube presentation on TVT in dogs

Treatment

 Several treatments including surgery, radiotherapy, immunotherapy, biotherapy and

chemotherapy have been applied for TVT.
 Surgery has been used extensively for the treatment of small, localized TVTs, although the
recurrence rate can be as high as 50 - 68% in cases of large invasive tumours. Contamination
of the surgical site with TVT cells is also a source of recurrence.
 Transmissible venereal tumours are radiosensitive and orthovoltage as well as cobalt have
been used for this purpose.
 Biotherapy studies have also been reported. The intra tumoural application of Calmette-
Guérin's bacillus (BCG) has been used for three weeks with sporadic success.
 Recurrences have been described after immunotherapy using Staphylococcus protein A, BCG
or a vaccine made from tumoural cells. Biotherapy has unfortunately also resulted in a high
rate of recurrence.
 Chemotherapy has been shown to be the most effective and practical therapy, with vincristine
sulfate being the most frequently used drug. Vincristine sulfate is administered weekly at a
dose of 0.5 to 0.7 mg/m2 of the body surface area or 0.025 mg/kg IV. Complete remission
takes 2-8 injections and occurs in more than 90% of the treated cases. Vincristine can cause
myelosuppression and gastrointestinal effects resulting in luekopenia and vomiting. A
complete WBC count is recommended prior to each administration. When the WBC count is
below 4,000 m3, further administration should be delayed 3 to 4 days and its dose reduced to
25% of the initial dose. Local tissue lesions can be caused by extravasations of the drug
during IV.
 Cyclophosphamide @ 5 mg/kg PO for 10 days as a single drug therapy or in combination
with prednisolone @ 3 mg/kg, for 5 days; vinblastine @ 0.1 mg/kg IV for 4-6 weeks;
methotrexate @ 0.1 mg/kg PO every other day can be given alone or in combination for
treatment of TVT. There is no advantage of combination therapy over vincristine
administration alone.
 In cases that fail to resolve with chemotherapy, electro-cauterization, cryo-cauterization or
radiotherapy has found to yield good results.

INDUCTION OF ESTRUS

Anestrus denotes a state of complete sexual inactivity with no manifestations of estrus. It is not a
disease but a sign of variety of conditions. Although anestrus is observed during certain physiological
states- eg. before puberty, during pregnancy, lactaion and in seasonal breeders- it is more often a sign

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of temporary or permanent depression of ovarian activity (true anestrus) caused by seasonal changes
in the physical environment, nutritional deficiencies, lactation stress and aging.

TYPE OF METHOD OF ADMINISTRATION BIOLOGICAL

HORMONE ACTIVITY
GONADOTROPINS
ECG or PMSG Single injection Mimics FSH and
stimulates follicular
growth, long half-life
FSH Single/multiple injections Stimulates follicular
growth; short half-life
hCG Single injection Mimics LH and induces
ovulation
GONATROPIN RELEASING HORMONE AGONIST
GnRH-Buselerin Single injection Induces release of LH
and FSH from the
anterior pituitary;
recruitment and
selection of new
dominant follicle
PROGESTOGENS
Progesterone Multiple injections Inhibits ovulation by
suppressing LH
secretion; mimics
action of CL
Synthetic Oral, subcutaneous implant, intravaginal Inhibits ovulation by
Progestogens pessary/device suppressing LH
secretion, mimics
action of CL
ESTROGENS
Estradiol Injection, implant Induces premature
Conjugates regression of CL and
enhances response to
progestogens
PROSTAGLANDINS
PGF2 alpha or Single intramuscular injection Induces regression of
synthetic analogues CL during responsive
phases

COW

Extensive studies have been carried out regarding the use of different hormones in treatment of
anestrus. The major hormones used are as follows,

Follicle Stimulating Hormone (FSH)

 As the name indicates the physiological role of this hormone is to induce follicular growth.
 Once follicular growth is induced, the estrogen secreted by the developing follicle induces an
endogenous Luteinizing hormone (LH) surge for ovulation.
 But, in several cases of anestrus, administration of FSH alone would not benefit, as ovulation
is not ensured.
 Administration of LH or hCG (human chorionic gonadotrophin) may be necessary to favor
ovulation.
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  Generally, LH is administered after 48 – 72 h of FSH administration. Follicular growth can be
induced in anestrus animals by exogenous administration of FSH or equine chorionic
gonadotrophin (eCG) also called as pregnant mare serum gonadotrophin (PMSG).
 The latter is having both FSH & LH activity and preferred for treatment of anestrus due to its
long half-life.

Dosage

 FSH – 1000 IU intra muscular

 eCG (Folligon, Trophovet) – 500 – 1000 IU intra muscular
 hCG (Chorulon) – 1500 IU intra muscular

Gonadotrophin Releasing Hormone (GnRH)

 This hormone can be used to treat anestrus animals either as single or double injection; the
latter method gives better results.
 In single injection method, it is advised to palpate the ovary for presence of follicle as this
hormone acts better when follicle is present in the ovary.
 In double injection method, administration of GnRH at 10 days apart induces estrus and
improves the ovulation and conception rates.

Dosage

 Gonadorelin (Fertagyl) – 250 – 500 µg intra muscular

 Buserelin acetate (Receptal) – 8 – 20 µg intra muscular

Progesterone

 Administration of progesterone mimics the presence of corpus luteum and induces follicular
growth and ovulation when withdrawn.
 There are several methods of administration of this hormone viz. oral, intravaginal, injection
and ear implants.
 The hormone should be administered for a minimum of 10 days and estrus is induced with in
3 – 5 days of removal of progesterone.
 Oral feeding of progestational compounds continuously for 14 – 18 days induces estrus within
3 – 5 days of last day of feeding, however, fertility at this estrus is not optimum. When
administered through injection also it has to be administered for 10 – 14 days

Oral progesterone

 MGA - 0.5 – 1mg/day/cow

 DHPA – 120 – 150 mg/day/cow
 MPA – 180 mg/day/cow
 CAP – 5 – 10 mg/day/cow

Injectable progesterone Combination of Hormones

 17 alpha hydroxy progesterone caproate(Duraprogen, Proluton)

 50 mg/day/heifer
 100 mg/day/cow

 Several combinations of above said hormones (eg. progesterone releasing intravaginal device-
PRID; Norgestomet ear implants) are also used to augment fertility in anestrus cattle and
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 buffaloes. Recently, administration of GnRH and PGF2 alpha has been reported to induce estrus
within 3 – 5 days of treatment.

GnRH and PGF2 alpha schedule

 Day 1: GnRH (8 m g intra muscular)

 Day 7: GnRH (8 m g intra muscular)
 Day 17: PGF2 a (25 mg – Dinoprost)

Non-Hormonal Approach

 Supplementation of minerals and some herbal drugs fall in this category. Different workers
have reported different success rates with these therapies.
 Simple utero-ovarian massage is also useful in some anestrus cases. Treatment with different
mineral mixtures, boluses, herbal compounds (Aloes compounds, Prajana, Janova etc) and
were also reported to be effective in treatment of anestrus cases.
 Treatment with Lugol’s iodine was also proved worthy in treating some anestrus cattle and
buffaloes.
 Swabbing of uterin externum with 5% Lugol’s solution causes local irritation and hyperemia
of uterus and ovaries.
 The ovary stimulates the hypophysis reflexly, resulting in release of gonadotrophic hormones.
 Besides this, iodine stimulates the thyroid glands directly or indirectly and iodine deficiency,
if any, is corrected. It was also reported that swabbing of Lugol’s iodine releases uterine
PGF2 alpha acting via the utero-ovarian and utero-pituitary-ovarian pathway.

MARE

The mare is seasonally polyestrous and has several cycles during a particular season cycling during
periods of long daylight thus ensuring that the mare will have foals at the time of the year most
conducive to foal survival, the spring.

Light Year

 The mare is light responsive in that increasing daylight causes cycles to start by decreasing
melatonin while decreasing light turns the mare off. The important days to remember in the
light year are:
o Summer solstice - ~June 21, which is the longest day of the year and the peak of
natural breeding season;
o Fall equinox - ~September 21, when there is equal light and dark and the mares are
turning off in fall transition;
o Winter solstice - ~December 21, which is the shortest day of the year and mares are
in deepest anestrus;
o Spring equinox - ~March 21 when there is equal light and dark and the mares are in
spring transition.
 Temperature may also influence the onset of cyclicity as cyclicity may be in part regulated by
a neurotransmitter which is also involved in prolactin secretion. Therefore, temperature is
important in the control of prolactin (low temp, low prolactin) and may therefore also affect
neurotransmitters.
 Reduction of the opioid inhibition of the gonadal axis may also play a role in triggering the
onset of the breeding season.
 Normal cycles occur around the Summer solstice, which is the natural breeding season.

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Anestrus

 Anestrus in caused by the secretion of melatonin, which is secreted in response to increasing

darkness.
 The melatonin inhibits GnRH, so the FSH and LH are low.
 Anestrus occurs around Winter solstice wherin 80% of mares undergo anestrus.
 Mares in anestrus are passive to the stallion advances.
 On rectal palpation the
o ovaries are small, smooth, and inactive
o the cervix and uterus are flaccid.
 Vaginoscopic exam reveals a cervix that is pale and dry, and the cervix may even be open.
 The hormones are all at very low concentrations.
 If the is nutrition poor, the mare may not cycle back in the spring.

Spring Transition

 Melatonin apparently inhibits the production of GnRH in anestrus.

 As the melatonin decreases, GnRH resumes secretion, and FSH and LH also increase.
 FSH during anestrus is low and irregular, whereas during the cycling season it becomes
bimodal (through pregnancy).
 LH during anestrus is also at basal levels, but increases to a normal pattern in cycling animals.
LH production however, lags behind FSH production.
 With increased FSH, follicles start to grow.
 Most of these follicles are not steroidogenically competent so they do not produce estrogen.
 They also do not ovulate. In fact, an average of 3.7 waves of follicular development occur
before the first ovulation.
 After several waves, an estrogen producing follicle finally develops and ovulates.
 During transition mares show irregular periods of sexual receptivity, prolonged heats of 10-20
days, split heats, and heats without ovulation.
 The uterus may be histologically 1 cycle behind the ovaries. In other words, the uterus may
still look anestrus when the mare starts cycling.
 The ovaries have follicles that grow and regress until one is selected to ovulate.
 This occurs when a follicle becomes steroidogenically competent and starts secreting
estrogen.
 The large follicles may persist on the ovaries because of insufficient LH. The follicles are not
cystic !!!
 There is no treatment for this anovulatory receptivity. LH (or hCG) will not make a
transitional mare ovulate.

Treatment

 Nothing prevents transition.

 Regumate- (altrenogest is a progestogen that has no cross reaction with progesterone. If
given at a dose of 1ml/110 lbs for 14 days orally, it shortens and eases the transitional signs,
but does not eliminate transition !!!! The net result is increased fertility earlier in season.
Regumate may induce LH receptors on the follicles?????
 Progesterone in oil -100 mg/day for 7 days has similar effects as Regumate.

Fall Transition

 Fall transition mirrors spring transition and is characterised by

o prolonged heats,
o irregular cycles,

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 o large 'hung' or 'autumn' anovulatory follicles that become atretic and the mare goes
into anestrus.
o This is caused from the low LH release because melatonin is taking its grip again as
the day length decreases.
 There is no treatment for fall transition

Induction of Cycling

 You must start no later than Dec. 1 (about 2 months before you want cycles to begin)
o 10 foot candles or 1 lux is sufficient. This is equivalent to a 100 watt bulb in a 12X12
box stall or two 40 watt fluorescent bulbs / box stall.
o You need a minimum 14.5 hr. light to start cycles.
o It is imperative that light be added at the end of the day, not the beginning.
 GnRH supplementation
o 100 mcg /hour for eight days at a constant infusion caused most mares to start
cycling.
o 40 mcg every 12 hours 28 days vs. implants (Buserelin implants released 100mcg/day
28 days) showed that 0/15 of the controls ovulated by 28 days, whereas 7/15
injections and 9 /15 implants did. Although the implants group retrospectively had
greater LH before the study began.
 Dopamine D2-antagonists
o Dopamine effect on gonadotropin release is unclear .
o Antagonists may act directly on ovary, rather than by hypothalamic pituitary axis
o GnRH is inhibited by dopaminergic neurons acting directly on GnRH neurons
o if given during anestrus the effect is not as great as if given during transition or if
mares have been exposed to extended photoperiod.
 Domperidone -0.1 mg.kg PO SID
o Transitional mares ovulated 12-22 days after treatment started
o Deep anestrous mares took longer to ovulate (50-60 days) .
 FSH-12.5–mg of purified eFSH intramuscularly twice daily for a maximum of 15 consecutive
days

DOES

 Two non-hormonal methods for altering the breeding of does include the sudden introduction
of the buck and the use of an artificially altered photoperiod.
 In general the sudden introduction of a male to a group of mature females during the period of
transition from anestrus to estrus can be expressed to advance the breeding season by a matter
of weeks, whereas the use of photoperiod alteration can allow for the breeding of does during
the traditional “deep anestrous” time of year.
 It is also possible to combine the two techniques in order to achieve both “out of season”
breeding and synchronization of estrus.

Buck Effect

 The introduction of a normal or teaser male induces estrus and ovulation in mature transition
period does within 5 to 7 days. The induced estrus and ovulation are reasonably synchronized.
 The precise mechanism for this induction is not completely understood, but seems to be
mediated through an induced surge of luteinizing hormone (LH). In any case, a group of does
can be bred earlier (e.g., August in the northern temperate regions) if they are suddenly
introduced to an intact male. If it is desirable to ascertain that the does are responding prior to
breeding, as with in artificial insemination program, then the introduced male can be a
surgically prepared teaser.
 The use of a marking harness, or simply painting the brisket of the teaser, should allow for the
detection of responding does. Since the cycle induced by the buck is occasionally of

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 abnormally short length and can be associated with insufficient luteal function, this practice
will preclude the possibility of breeding induced does at an infertile estrus. In cases studied to
date an induced “short cycle” is usually followed by a cycle of normal length.
 Therefore, once it has been determined that the does are cycling in response to a male, the
teaser can be removed and replaced by a breeding buck or by artificial insemination.

Photoperiod Alteration

 Many studies have shown that the adult ewe is susceptible to induction of estrus by
manipulation of length of daylight. Few controlled studies are available for mature goats, but
it is known that yearling does can be induced into estrus at least 60 to 80 days early by
providing feeding areas of a barn with 19 hours of artificial light per day, beginning in mid to
late winter.
 An inexpensive timer is set to turn on the lights ( a pair of 8-foot, 40-watt fluorescent tube for
each 36 to 40 square meters of pen space) at approximately 0500 and to turn them off 19
hours later.
 Animals should be fed in the evening, to encourage them to expose themselves to the
augmented light. A protocol for lighting and animal management.
 Apparently, the relative decrease in length of day when the artificial light is terminated, is the
stimulus that induces hypothalamic events leading to estrus and fertile ovulation.
 The physiological mechanisms that translate "decreased" length of daylight into estrus and
ovulation are under study.
 When photoperiod-primed yearling does are suddenly exposed to bucks, there is a surge of
LH within about 2 weeks. The first surge apparently does not induce ovulation, but may be
responsible for the very slight rise in progesterone that follows and that precedes estrus by 2
to 4 days. This estrus may be tightly synchronized.
 It would appear , then, that the relative decrease in length of day may condition hypothalamic
responses necessary to allow the “buck effect” in yearling does.
 The success of this method has not been examined in a controlled fashion for lactating does,
but some producers have reported success rates similar to those achieved with yearlings.

GILT AND SOW

Prepubertal Gilt

 The gilt is first able to ovulate in response to exogenous gonadotropins at approximately 100
days of age.
 The most common regimens of 500 to 1000 IU PMSG followed in 48 to 96 hours by
approximately 500 to 750 IU hCG and the injection of 400 IU PMSG in combination with
200 IU hCG.
 The split-dose and combination treatment appear to induce comparable rates of ovulation and
estrus. When PMSG is given without hCG, ovulation rates and the per cent of gilts showing
estrus are reduced relative to treatments utilizing hCG in conjunction with PMSG.
 Exogenous GnRH has been used in lieu of hCG at 48 to 60 hours after PMSG administration
to induce synchronized ovulation; however, a more reliable response appears to occur when
hCG is given after PMSG. The time of ovulation following treatment with the PMSG/hCG
combination is approximately 110 to 120 hours.
 When either hCG or GnRH is given following PMSG, ovulation occurs at approximately 40
to 44 hours following the second injection.
 Although conception rates of 40 to 90 per cent are common following gonadotropin-induced
ovulation, less than 60 per cent of gonadotropin-induced gilts are typically able to maintain
successful pregnancies. It is thought that in some gilts induced corpora lutea (CL) are not able
to retain their viability 20 to 30 days after ovulation.
 If females are mated at the spontaneous estrus that occurs in some gilts at approximately 21
days following induced ovulation, farrowing rates are markedly improved. The presence of

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 boars after induced ovulation enhances the ability of gilts to maintain cyclic, ovarian and
estrus activities.

Delayed Puberty

 Gilts reared in confinement commonly show an onset of puberty that is substantially delayed
relative to pen mates. As discussed in other, articles, these anestrus females may have acyclic,
anovulatory or cyclic, ovulatory ovaries.
 Most gilts that are anovulatory and anestrous are able to respond to exogenous gonadotropins
with both estrus and ovulation.
 Prepubertal females that are anestrous and have cyclic ovaries (behaviourally anestrous) are
not able to respond to increased circulating levels of estradiol and thus cannot respond to
gonadotropin therapy with estrus.
 They are, however, able to ovulate and to form accessory CL in addition to their primary CL.

Lactating Sow

 During lactation, the ovaries of sows show only modest follicular development. There is
gradual but progressive development of follicles in response to exogenous gonadotorpins as
early as 15 days postpartum, but pregnancy rate is improved when treatment is initiated at 25
days or more following farrowing.
 The procedure found to be most effective for inducing ovulation in the lactating sow involves
the administration of 1500 IU of PMSG followed after 96 hours by 1000 IU of hCG. Since
estrus is not consistently observed females should be artificially inseminated at 24 hours and
again at 36 to 42 hours following hCG administration.
 The separation of the sow from her litter for one to three 12-hour periods prior to treatment
appears to improve the response to gonadotropin.

Postweaning Sow

 Following weaning the majority of sows show signs of estrus within 3 to 7 days.
 Exogenous gonadotropins have been used to decrease the weaning-to-estrus interval and to
improve the synchrony of the post weaning estrus. Gonadotropins have also been used
prophylactically to decrease following weaning.
 PMSG can be used alone or in combination with hCG to promote earlier onset of the post
weaning estrus.
 For maximum effectiveness, PMSG should be administered on the day following weaning.

Anestrous Sow

 It is not uncommon in some herds for sows to fail to return to estrus for 30 or more days
following weaning.
 As with delayed puberty, anestrous sows may have acyclic or cyclic ovarian activity.
 Females that have anovulatory ovaries are able to respond to PMSG with a fertile estrus. The
combination of estrogen (1 mg of either estradiol benzoate or estradiol cypionate) and hCG
(1000 IU) has been used to return anestrous sows to productivity.
 However, additional studies are warranted to establish the efficacy of this treatment. Because
of its luteotrophic effects in the pig, exogenous estrogen may induce prolonged diestrus in
anestrous sows with cyclical ovaries.
 Estrogens should be used with caution in the pig.

BITCHES

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  A number of methods to induce estrus in dogs have been reported. Most are probably not
appropriate for application in healthy, normally cycling bitches, despite interest in shortening
and synchronizing cycles for purposes of accommodating owners schedules, the availability
of stud dogs, or the shipments of chilled or frozen semen, or for purposes of increasing the
number, frequency or size of litters in such animals.
 All of the methods reported, when assessed in repeated or large studies have a significant
failure rate and involve one or more of the following drawbacks: smaller than normal litters in
a significant percentage of successful attempts; disruption and possible prolongation of the
normal cycle; and, theoretically a possibly increased risk of reproductive tract disease due to
premature and possibly excessive stimulation of the reproductive tract by the administered
hormones or changes in endogenous hormones provoked by the treatment.
 Nevertheless, interest remains high in the development of methods that may be safe enough,
have fewer drawbacks, and/or have a sufficiently high success rate as to merit clinical
application in the course of breeding management.
 Furthermore, some of the current methods would appear to have significant merit for
application in cases of prolonged anestrus and for enhancing fertility of research bitches in
colonies of dogs maintained as animal-models of heritable or genetically-based diseases of
interest in human or veterinary medicine.

Monitoring the Treated Bitch

 Regardless of the method chosen for estrus induction, vaginal cytology should be obtained on
an alternate-day basis.
 It is recommended that natural breeding or artificial insemination begin when superficial cells
compose 60% or more of the exfoliated vaginal epithelial cells and/or when plasma
progesterone concentrations exceed 1 ng/ml.
 Insemination should continue on an alternate-day basis until diestrus is confirmed..

Drug Availability and Reliability

 Problems arise regarding availability, quality, consistency, and dependability of the various
hormone preparations utilized in the studies completed. This accounts, to some degree, for the
different results seen with similar protocols used by separate research groups.
 Many of the agents used are not commercially available and others vary significantly in
potency depending on where they are purchased and how they are prepared.

Use of PMSG or FSH

 PMSG
o A variety of protocols using pregnant mare serum gonadotropin have been evaluated
in one study, approximately 50% of bitches ovulated following 9 consecutive days of
IM or subcutaneous PMSG, injected at 44 IU/kg /day followed by 500 IU of hCG IM
on day 10. The responding animals exhibited behavioral estrus 10 to 15 days after
initiating treatment, but this included only half of the dogs that ovulated.
o In another study, pregnant mare serum was administered to mature anestrus bitches
for 10 consecutive days at subcutaneous does of 500 IU /day. 250 IU/day, or 20
IU/kg/day. This was followed by a subcutaneous injection of 500IU of human
chorionic gonadotropin on day 10. Abnormal ovulations with shortened luteal phases,
and toxic side effects attributed to the excess estrogen. Those side effects included
thrombocytopenia, uterine disease, and termination of pregnancy
o If the PMSG was administered for only 5 days before hCG administration the serum
estrogen concentrations were more physiologic and the protocol resulted in a 50%
conception rate. This latter protocol was evaluated in another study, resulting in
excess concentrations of serum estrogen Differences in results may be due, in part, to
difference in the potency of the PMSG preparations used.
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  FSH with or without Estrogen Priming
o Protocols using FSH (Schering Corporation, Kenilworth), as the sole stimulus for
induction of estrus have not been as successful as those using PMSG. Pre treating
bitches with an “estrogen priming” regimen of diethylstilbestrol (DES) using 5 mg
daily for 7 or more days to produce signs of proestrus, holds promise. Five days after
induction of proestrus 5 mg of LH IM was administered; 10mg of FSH IM was
administered 9 and 11 days after observing vaginal bleeding. Each bitch so treated
became pregnant.
o A successful modification of the estrogen-FSH protocol included administration of
DES daily for 4 to 10 days. Duration of DES administration continued 3 days beyond
the first day of induced proestrus Counting from the first day that signs of induced
proestrus were observed, 10 mg of FSH-P was given IM on days 5,9, and 11. Estrus
behavior was observed in 70% of the bitches 5 to 10 days after the initial dose of
DES. Subsequently, 46% ovulated and 30% became pregnant, carrying litters to term
chard et al.

Gondadotropin-Releasing Hormone or a GnRH Agonist

 Use of GnRH or an agonist of that hormone induces a fertile ovarian cycle only if the
pituitary-ovarian axis is normal. These drugs stimulate the secretion of pituitary
gonadotropins, which should, in turn, stimulate the ovaries. Follicle development, estrogen
secretion, behavioral estrus and pituitary-stimulated ovulation depend on a normal cascade of
physiologic events following “activation” of the system.
 One protocol used a surgically implanted infusion pump that administered a small dose of
GnRH every 90 minutes for 6 to 12 days. In the bitches treated, proestrus began in 3 to days
and fertile estrus in 7 to 14 days. The protocols were successful at inducing fertile cycles in
37 to 85% of bitches treated. However, the cumbersome and impractical nature of expensive
implanted infusion pumps that need to be removed make this protocol interesting but
unavailable to most practitioners:
 Another protocol used a constant infusion of a GnRH agonist for 14 days. This approach
resulted in rapid induction of proestrus and estrus, with fertility rates of 25% when
administered immediately following lactation and 50% when given to anestrus bitches after a
non pregnant cycle. Although the results were promising, the agonist used is not
commercially available Also, the small, inexpensive osmotic pumps require minor surgery for
their subcutaneous placement and removal.
 A less stringent protocol used subcutaneous injections of a GnRH agonist at a dose of 1 pg/kg
TID for 11 days and then 0.5 pg/mg TID for 3 days. Estrus was observed within 9 to 11 days
of initiating treatment in 80% of the dogs, each of which became pregnant. Despite the
inconvenience of a TID injection protocol, this method may present the best combination of
efficacy and clinical utility among the various approaches involving GnRH.

Dopamine Agonist Administration

 Several studies have reported the use of a dopamine agonist (DA) administered orally at doses
sufficient to lower plasma prolactin as a means to terminate anestrus either prematurely in
normal bitches or therapeutically in cases of prolonged or persistent anestrus.
 The efficacy has been anecdotally estimated to be about 70%, and possibly higher in bitches
with prolonged anestrus; the resulting proestrus, when induced, has occurred after a variable
duration of treatment ranging from 8 to 40 days; the average appears to be about 20 days;
duration appears to be dependent on the stage of anestrus, with longer treatment required in
early anestrus.
 Whether the simultaneous reduction in prolactin is part of the mechanism of action or if the
mechanism involves other or additional dopaminergic effects is not known.
 However, efficacy appears to depend on a dopamine responsiveness sufficient to also cause
suppression of prolactin; bitches that fail to experience suppression of prolactin also fail to
show a clinical proestrus response.
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  Two DA treatments reported to be effective have included bromocriptine at 0.05.or 0.1
mg/kg, p.o., q.d. or bid, and cabergoline at 5 ug/kg, p.o, q.d.; administration is until an
induced proestrus is pronounced for 2 days or until the onset of estrus.

SPECIES TREATMENT END OF TREATMENTOF

ESTRUS

CATTLE AND BUFFALO

Prepubertal or Estrogen on day 1 followed by 7-12 Within 5 days
Postpartum Suckled Cows days of progestogen, eCG given on last
day (optional)

Postpartum Milked Cows GnRH on day 14 postpartum 1 day

2-4 days
GnRH (day 0) and PGF (day 6)

SHEEP AND GOATS

Prepubetral or Seasonal Progestogen for 12-21 days with eCG 2-4 days; eCG required for
Anestrus given near the end of progestogen good response
treatment

SWINE
Prepubertal or eCG alone 3-5 days
Postpartum Anestrus eCG on day 1 with h CG given 48-96 h 3-5 days
later 3-5 days
e CG +h CG given on day 1

HORSES
Seasonal Anestrus Lengthen photoperiod by 4 h per day 4 -6 weeks earlier than normal
Progestogen for 15 days Within 1 week

SYNCHRONIZARTION OF ESTRUS AND OVULATION

A limiting factor for the dairy herd productivity and profitability is reproductive efficiency. The
calving interval of cows in a farm to be economically efficient should be 12-13 months. To maintain
this, cows must be pregnant within 85 to 115 days after calving and a recommended leaving of 35 to
74 day breeding period. The detection of estrus and rate of conception are integral components in
achieving this calving interval.Though causes of low conception may be multifactorial,
synchronization of estrus and ovulation will bring about a drastic increase in the conception rate in
large herds. Under the Indian field conditions major use of synchronization agents lies in the
management of infertility rather than estrus synchronization. These synchronizing agents can be
effectively used to address the major causes of infertility viz.

 Poor detection of heat.

 Untimely insemination.
 Subestrus / silent estrus and
 Anestrus.

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In addition, these agents can be used in individual animals to induce heat and inseminate at fixed time
to improve fertility.

ESTRUS SYNCHRONIZATION

 It is the method of bringing a group of animals into estrum at a predetermined time. In farm
animals this is achieved by progesterone, PGF2 alpha or a combination of both.
 Synchronization of the estrus cycle can only take place in females that are already cycling.

Potential Advantages in Large Dairy Herds

 Better control of calving interval.

 Reduction on dependence on heat detection.
 Reduction of errors in heat detection.
 Increased reproduction with the same number of cows.
 Maintain same production with fewer numbers of cows.
 Increased number of calves per cow.
 Increased economic use of AI.
 Schedule entry of heifers into the milking herd.
 Reduce labour.

HOW IT WORKS?

Synchronization products control the estrous cycle by influencing structures that are present on the
ovary.

Prostaglandins (PG)

 PG regulates a female's estrous cycle by causing "luteolysis" or regression of the CL when it

is present on the ovary.
 A synthetic prostaglandin will mimic natural PG release from the uterus and will cause CL
regression. Because the CL produces progesterone, PG eliminates the "progesterone block"
and allows follicles to grow and subsequent ovulation to occur.
 Females with a CL on their ovary when they receive an injection of PG will usually exhibit
estrus 2 to 5 days later. An injection of PG will only regress a CL that is 6 to 17 days old in
the 21-day estrous cycle.
 After the progesterone block is removed, there is an increase in follicle growth and therefore,
an increase in estrogen production and heat and subsequent ovulation will occur 2 to 5 days
after the PG injection.

Estradiol Benzoate

 Estradiol benzoate, the estrogen used in the Syncro-Mate B protocol, will cause regression of
a CL that is 1 to 5 days old. This is the primary reason for using estradiol instead of a
prostaglandin.

Progesterone, MGA and Norgestomet

 Progesterone, MGA and Norgestomet (in the Syncro-Mate B protocol) "hold" the female
from exhibiting heat. During a normal estrous cycle, the CL on the ovary produces
progesterone.

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  Feeding (MGA) or implanting (Syncro-Mate B) progesterone in females will play the same
role as progesterone being produced by the CL.
 Progestins will not allow the follicle to ovulate or allow the female to exhibit heat until the
synthetic progestin is removed.

Gonadotrophin Releasing Hormone (GnRH)

 Gonadotrophin releasing hormone causes the release of luteinizing hormone (LH) and follicle
stimulating hormone (FSH) from the anterior pituitary.
 The target tissue for LH and FSH is the ovary. The ovary needs these hormones to grow
follicles that produce the egg. In a synchronization program, LH and FSH will grow follicles
and also are a part of the process that causes ovulation (release of the egg from the dominant
follicle). Because LH and FSH are involved in follicle maturation, these two hormones also
support estrogen production from the follicle.
 An elevated blood concentration of estrogen is associated with ovulation and bringing the
animal into standing heat.
 Initially estrus control was attempted with progesterone. Though estrus and ovulation was
controlled, the conception rate in the first service was poor. In the 1960’s, the treatment was
mostly oral treatment with progestogens and it was termed ‘grouping ‘.

EVALUATING ESTRUS RESPONSE

The efficacy of synchronizing treatment is evaluated as two parts:

1. Estrus response, which is the proportion of cattle treated that has come to heat in the peak 24
h.
2. Reproductive performance, which is the conception rate in the controlled estrus.

CRITERIA FOR SUCCESSFULL CONTROLLED BREEDING

Animal requirement

 Animal must be disease free - especially of reproductive tract.

 Heifer must have reached maturity with proper weight >200-250 kgs.
 Nutrition must be adequate with the animal in positive balance.
 Adequate postpartum interval of 45-60 days should be there.
 For prostaglandins it is important that the animals must be cycling.
 The animal should have a normal non-pregnant reproductive tract.

Management requirements

 Proper timing is essential - once the program is initiated it must be carried through all steps in
proper sequence and at proper times.
 Good semen quality is an essential requirement. Under present condition existing in the field,
this is very important.
 Good AI technique must be followed.

PG AND THEIR ANALOGUES

 PG are a group of naturally occurring compounds. The specific prostaglandin is F2 alpha

(PGF2 alpha). Synthetic PG function in the same manner as the naturally occurring PG except
they are administered in doses large enough to cause luteal regression according to the
schedule we desire.
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  When injected at the recommended dosage, these products act by causing rapid regression of
the corpus luteum (CL) on the ovaries of cycling females in Days 6 to 16 of their estrous
cycles. The injection decreases the function of the CL, which allows these females to return to
estrus within 2 to 5 days and synchronizes their estrous cycles. Females in Days 17 to 20 will
be in estrus normally within 1 to 4 days and also will be synchronized. Females in Days 1 to 5
of the cycle and non-cycling females that do not have a mature CL will not respond to the
injection. Only about 75% of the cows cycling in a herd can be synchronized with one
injection. If all cycling females are to be synchronized, two injections are needed.
 The PGF2 alpha analogues are more potent than the natural PGF2 alpha and exert lesser side
effects and have a wider margin of safety.
 When the bovine CL matures there is an increase in the number of prostaglandin receptors in
the CL early in the estrous cycle and twice daily injections would bring about luteolysis. PG
has a short biological half life. Generally fertility is not affected after PG and fixed time AI
results in acceptable conception rates.

Effective Response Time

 PG is not effective in estrus, against developing and regressing CL.

RECOMMENDED TIME FOR PG ADMINIDTRATION

SELECTION OF PROGRAMME

 Options given below are suitable for individual as well as group of animals. Selection of
programme depends on the skill to identify CL and economy.
o Program A - requires only a single injection for each animal.
o Program B - requires double injection for all animals.
o Program C - requires one injection in about 2/3 of the animals and double injection
in 1/3 of the animals. ie. as the diestrus period is the longest, when randomly injected
the animal is more likely to be in the diestrus than non-responsive period.

~ 287 ~
Use one of the following options for breeding

Detect heat and breed (Usually 2 to 5 days after injection).

OR

Fixed time AI at 72 h after injection with no heat detection.

OR

~ 288 ~
 Fixed time AI at 72 and 96 h with no heat detection (better than single insemination).

Animal should be observed for the next estrus 18-22 days after this estrus period and inseminated.

GENERAL COMMENTS

 Prostaglandins are very effective in their function i.e. luteolysis.

 With proper use nearly 100% induction can be achieved.
 They are not fertility drugs.
 Under ideal conditions conception rate of the induced estrus cannot be better than the normal
oestrus.
 Double injection regimen allows for higher synchrony.
 Since PG can be absorbed through the skin, pregnant women and asthmatics should especially
be careful when handling these products.

COMMERCIAL PREPARATIONS

Prostaglandin Commercial Preparations, Dose and Route of Administration

Commercial preparations Content Presentation Dose and Route of administration

Lutalyse Dinoprost 5mg/ml 10 ml vial 25 mg i/m

12.5mg IVSM
Prosolvin Luprostiol 7.5 mg/ml 2, 10, 20 ml 15 mg i/m
Estrumate Cloprostenol 263 mcg/ml 10 ml 500 m g i/m
Dinofertin prostmate Dinoprost 5mg/ml 5 ml 25 mg i/m
Bovilene Fenprostalene 2 ml SC injection
Iliren Tiaprost 0.15mg/ml 10 ml 0.75mg /m, 0.45 mg i/v

Administration

Intra muscular route

 Natural PGF2 alpha - 25 mg.

 Synthetic analog - 0.5 mg.

Intra vulvo sub mucosal route

 Natural PGF2 alpha - 10 mg.

 Synthetic analog - 0.2 mg.

Both routes are effective and have been found to give good conception rate.

PROGESTERONES AND PROGESTOGENS

 The luteal phase of the cycle is artificially prolonged and on withdrawal of progesterone i.e.
after the CL has regressed leads to estrus synchronization.

Estrus Response to Progesterone and Progestogens

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  Majority of the animals are found to come to estrum within 24-48 h. Estrus response to a
regimen is lower in cattle compared to sheep probably due to endocrine differences which
results in a shorter duration of estrus, 18-24 h in cows compared to sheep (36 h) and so there
is difficulty in detecting estrus.
 Exogenous progesterone may lead to development of dominant follicle. It may not be able to
exert the same feed back level on LH as the CL and hence there is increased level of LH
leading to the development of a dominant follicle which would become atretic due to lack of
gonadotrophin and this on ovulation would lead to decreased fertility.

SYNCHROMATE –B

 SMB ear implant is approximately 2 x 18 mm size and contains 3 mg of norgestomet (17

alpha acetoxy 11 beta methyl 19-Nor-Preg 4 ene-3, 20 dione), a progesterone-like compound.
 The SMB injection contains 2 ml oily solution of 5 mg estradiol valerate and 3 mg
Norgestomet

ADVANTAGES

 Increases pregnancy rates

 Increases probability of estrous detection and insemination
 Used to control the estrous cycle and mimic the reproductive hormones found within the
normal cow
 Control follicular wave development
 Promote ovulation in anestrous cows
 Regresses the corpus luteum in cyclic cows
 Synchronizing estrus and (or) ovulation
 The advantages of this program include administration at any point within the cow's estrus
cycle will result in a synchronized estrus and accidental administration to pregnant cows
won't cause abortion.

USES

 Syncromate B is used for estrous synchronization.

 The implant is placed in the ear for nine days. At the end of the nine day period the implant is
removed and the injection is given.
 The cows or heifers will start to show signs of heat about twenty-four hours after implant
removal.

PRINCIPLE

 The norgestomet from the injection immediately blocks the release of hormones that cause
ovulation and prevents females from displaying estrus.
 Release of norgestomet from the implant prevents estrus and ovulation over the next nine
days. The estradiol valerate in the injection causes regression of a mature CL and any new
developing CL.
 Together, the estradiol valerate and norgestomet cause luteolysis and advance all cows to
about day 19 of their estrous cycle and hold them there until the implant is removed. On day
9, when the norgestomet implant (progesterone block) is removed, cycling returns with the
release of hormones which stimulate follicular growth and estrogen secretion, and cows
generally exhibit estrus within the next one to four days.
 After the implant removal, cows are observed for standing estrus and bred 12 h later or time
inseminated at 48-54 h after implant removal. SMB also induces estrous cycles in some
anestrus cows.

ADMINISTRATION

~ 290 ~
  SMB ear implant is placed subcutaneously in the backside of the ear. Before inserting the
implant, it is helpful to clip the hair on the back of the ear, and disinfect the implant site.
 Synchronization is done by administering an injection and an ear implant on day 0 and the
implant removed on day 9.
 At the time the implant is being inserted, the animal is given an intramuscular injection that
contains estradiol valerate and norgestomet.

OPTIONS FOR INSEMINATION

Three options for insemination using the Syncro-Mate B program,

 All females are mass inseminated at a predetermined time. Females should be inseminated
between 48 and 54 h after implant removal without regard to time of estrus.
 Animals are inseminated 12 h after first observation of standing estrus. This result in greater
conception rates because the timing for insemination is more accurate and because non-
responding cows are not inseminated.
 A combination of the above two methods. Inseminate females that show estrus before 48 h by
the AM-PM rule and mass inseminate non-responding females at 48-54 h after implant
removal.

ADVANTAGES FOR PROGESTOGENS

 CL need not be identified.

 All the animals can be administered at the same time.
 Can be given at any time of the cycle.
 Even if the animal is not cycling they bring the animal into estrus.
 All the other conditions with prostaglandins are also applicable to progestogens for success.

CIDR

 The CIDR is designed as a T shaped nylon spine molded with a silicone rubber skin, which
contains 1.9 g progesterone.
 The wings of the CIDR insert have the ability to be folded together in order to insert
intravaginally. Once inserted, the wings return to their original T shape position and apply
pressure to the vaginal walls to hold the insert in place.
 The CIDR insert is removed following the treatment period by pulling the plastic tail that
protrudes from the vulva.
 Current research is looking at modifying a CIDR to reduce the initial and residual
progesterone load, while maintaining intended preformance.
 Progesterone is released from the skin of the insert, causing the animals blood progesterone
concentrations to increase rapidly. Maximum concentrations are reached within an hour after
insertion.
 Progesterone acts according to the same mechanism as a typical steroid hormone.
 Progesterone diffuses through the cell membrane and the nuclear membrane, binding to the
progesterone receptor in the nucleus, thus causing a change in cell physiology

ADVANTAGES

 Higher pregnancy rate

 Estrus synchronization
 Improved value from semen, because of a higher success rate of artificial insemination
 Heifers freshening at a younger age
 Exact breeding and calving dates of cows and heifers
 A more profitable herd or flock

~ 291 ~
HOW IT WORKS

 CIDRs are coated with progesterone. Progesterone is a naturally produced steroid hormone by
the corpus luteum of mammalian ovaries. In vivo, progestrone functions to maintain
pregnancy.
 Progesterone provides a potent suppression of estrus, making it important for estrus
synchronization in herds of animals.
 When the CIDR is removed at the end of a treatment period, a rapid drop in concentration of
systemic progesterone occurs in each animal.
 Thus promoting a synchronized estrus effect within the herd, and allowing for artificial
insemination of the herd to take place

ADMNISTRATION

 CIDRs are coated with progesterone. Progesterone is a naturally produced steroid hormone by
the corpus luteum of mammalian ovaries. In vivo, progestrone functions to maintain
pregnancy.
 Progesterone provides a potent suppression of estrus, making it important for estrus
synchronization in herds of animals.
 When the CIDR is removed at the end of a treatment period, a rapid drop in concentration of
systemic progesterone occurs in each animal.
 Thus promoting a synchronized estrus effect within the herd, and allowing for artificial
insemination of the herd to take place

WARNINGS

Taken from product insert:

 Human Warning: Avoid contact with skin by wearing latex gloves when handling the inserts.
Keep this and all medications out of the reach of children.
 Environmental Warning: Store removed EAZI-BREED CIDR Cattle Inserts in a plastic bag
or other sealable container until they can be properly disposed in accordance with applicable
local, state and Federal regulations.
Residue Warning: Neither a pre-slaughter withdrawal interval nor a milk discard time is
required when this product is used according to label directions.

Do Not Use:

 In beef or dairy heifers of insufficient size or age for breeding or in cattle with abnormal,
immature or infected genital tracts.
 In beef cows that are less than 20 days postpartum or in lactating dairy cows less than 40 days
postpartum. The sponsor has not provided effectiveness and animal safety data for the use of
this product in beef cows that are less than 20 days postpartum or in lactating dairy cows that
are less than 40 days postpartum.
 The EAZI-BREED CIDR Cattle Insert in lactating dairy cows concurrently with
LUTALYSE® Sterile Solution or other prostaglandin products for synchronization of the
return to estrus. The concurrent use with prostaglandin products is not approved in lactating
dairy cows.
 An insert more than once. To prevent the potential transmission of venereal and blood borne
diseases the EAZI-BREED CIDR Cattle Insert should be disposed after a single use.

PRID

It is a stainless steel flat coil coated with an inert silicone rubber incorporating 1.55 g of progesterone
and contains a 10 mg of estradiol benzoate capsule.
~ 292 ~
  The PRID is inserted into the anterior vagina, left for 12 days and then withdrawn.
 Estrus occurs 2-3 days after withdrawal.
 Fixed time AI at 48 and 72 h or only at 56 h after removal is recommended.

COMMERCIAL PREPARATIONS OF PROGESTERONE AND PROGESTOGENS

Preparations and Route of Administration Duration of Treatment (in

days)
Oral - MGA 14
Intravaginal Sponges 14
Progesterone Releasing Intra Vaginal Device - PRID 9 -11
Controlled Internal Drug Releasing Device - CIDR 11
Ear implants 9

 Crestar
 Synchromate B

Injection in oil or other media 14 -18

TECHNIQUES FOR SYNCHRONIZING ESTRUS IN CYCLIC FARM ANIMALS

Species Method Treatment Regimen End of Treatment

to Estrus
Cattle and Buffalo PGF Two injections (11-12 days apart) 3-5 days;AIDE/TAI
Cattle GnRH+PGF Inject GnRH (day 0), PGF (day6) 2-4 days; TAI
GnRH+PGF+GnRH Inject GnRH (day 0), PGF (day7), 2-4 days; TAI
GnRH (day 8 or 9)
Progestogen +estrogen Estrogen injection (day 1), CIDR (days 3-5 days;
1-9) AIDE/TAI
Sheep Progestogen + PGF Progestogen (days 1-7), PGF (day 6) 2-3 days;
AIDE/TAI
Progestogen (pessary) +e CG Progestogen (12-14 days),e CG (day of 2 days; AIDE or
pessary removal) double AI
PGF Two injections (9 days apart)
Goat Progestogen (pessary) +eCG Progestogen (18-21 days), e CG (day of 2-3 days; breed at
pessary removal) estrus or double AI
Two injections (11-12 days apart)
Swine Progestogen in feed Altrenogest (14-18 days) 4-7 days; breed at
estrus
Horse Progestogen in feed Altrenogest (15 days) 4-7 days; breed at
estrus
PGF One dose to mares in diestrus 4-7 days; breed at
estrus
PGF +H CG PGF (day 1), hCG (day 7-8), PGF (day 3-5 days; breed at
15), estrus
hCG (day 21-22) 2-4 days

PROTOCOLS FOR SUPEROVULATION IN FARM ANIMALS

Species Pre Treatment Gonadotropin PGF LH

~ 293 ~
Goat Fit a Progestogen pessary for Inject 20 mg PFSH one Inject 125-250 μg None
17 days day before pessary cloprostenol
removal
sheep Fit a Progestogen pessary for Inject 20 mg PFSH one Inject 1000 μg Cloprostenol
12 days day before pessary
removal
Cattle and buffalo Progestogen for 7-8 days Inject 20-30 mg pFSH Inject 25 mg PGF (Lutalyse) Inject (IM) 25 mg
(day 2) porcine LH (day 5)

Cattle Day-7 Progestogen+GnRH Inject 80 mg FSH (day Inject 25 mg PGF (Lutalyse) Inject (IM) 25 mg
implant 0-4) (day 2) porcine LH (day 5)

Pig Feed Altrenogest for 15 days Inject 1500 IU eCG one None None
day before end of
feeding period

FOLLICULAR DYNAMICS

FOLLICULAR DEVELOPMENT

 In the primordial follicle reserve, formed during fetal life or soon after birth, follicles are
released which continues to grow until ovulation or until the follicle degenerates, which is the
case with majority of the follicle.
 Ovarian follicular development in cows and ewes is a progressive and recurring process with
two or three waves of follicular growth occurring in each cycle. In cows two waves appear to
be more common but three waves are frequent in long cycles.
 Thus even though the follicular phase comprises only about 20% of the estrous cycle, the
process of follicular growth and degeneration (known as follicular dynamics) occurs
continuously throughout the entire estrous cycle.If the ovaries are examined at any point
during the estrous cycle significant number of antral follicles of various sizes will be seen.
These antral follicles have been classified as small, medium and large depending on their
diameter.

DYNAMICS OF ANTRAL FOLLICLE

 Dynamics of Antral follicles consists of

o Recruitment
o Selection
o Dominance
o Atresia

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 Click here to
view an
animation on
relative
gonadotropin,
inhibin and
estrogen
secretion
during
recruitment,
selection and
dominance.

Recruitment

 Recruitment is the phase of follicular development in which a group of small antral follicles
begin to grow and produce estradiol.
 Some of these recruited follicles undergo atresia.
 Following recruitment, groups of growing follicles, which have not undergone atresia, are
selected.

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Selection

 Selection involves the emergence of dominant follicles from the host of previously recruited
antral follicles.
 Selected follicles may become dominant or they may undergo atresia.

Dominance

 As the selected follicle proceeds towards dominance, they continue to produce increasing
amounts of estrogen as well as inhibin.
 In the cows and mare (monotocus sp) these are several selected follicles but only one will
develop into the dominant follicle.
 Increasing inhibin levels and reduced blood supply to other follicles, the dominant follicle
causes inhibition of other follicles.
 Suppressed FSH concentration in blood along with reduced blood supply results in atresia.
 Only those follicles receiving a large blood supply and thus higher levels of gonadotrophin
continue to grow.
o Recruitment = High FSH + Low LH + No Inhibin + No Estradiol
o Selection = Low FSH + Moderate LH + Low Inhibin
o Dominance = Low FSH + High LH + High Inhibin
o Atresia = Degeneration of Follicles

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ENDOCRINOLOGY OF FOLLICULAR GROWTH AND OVULATION

 FSH plays a major role in antrum formation.

 This gonadotrophin stimulates granulose cell mitosis and follicular fluid formation.
 FSH also increases the sensitivity of granulosa cells to LH by increasing LH receptors, which
prepares for ovulation. On the other hand theca cells are stimulated only by LH and LH
receptors are present from the beginning of theca cell formation
 During recruitment FSH and LH begin to increase, thus promoting follicle development.
 As follicles enter the selection phase, inhibin is produced by the follicles and inhibits FSH
released by the anterior pituitary.
 As follicular dominance phase is entered, the large follicle produces more and more estrogen
and thus causes the preovulatory surge center to release LH surge. In addition FSH
concentration is reduced because of inhibin. This causes antral follicles to undergo atresia.
FSH and LH are also essential for steroidogenesis (two cell two hormone theory).

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OVULATION

Ovulation is the release of oocyte from mature graafian follicle. Ovulation in mammalian ovary
occurs on any point in the ovarian surface while ovulation in mares is restricted to ovulatory fossa. In
cow, sheep and horses, ovulation occurs at random irrespective of which ovary contains previous CL.
In some animals ovulation consistently alternates between ovaries and in others (whales) ovulation
may predominate in one ovary.In the rhesus monkey, the CL retards subsequent follicular growth so
that ovulation alternates between ovaries.

TYPES OF OVULATORS

 Spontaneous Ovulators (eg. Cow, mare, sheep goat dog). Ovulation occurs spontaneously
irrespective of whether mating has taken place or not.
 Induced Ovulators or Reflex Ovulators (eg. Cat, mink, ferret). Copulation is a must for
ovulation to take place.

THEORIES

Follicular Pressure Theory

 As the follicle grows the amount of liquor folliculi also increases. This liquor folliculi exerts
pressure on the follicular wall thereby follicles rupture.
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  However, this was not the case with cystic ovaries where there was both an increase in
follicular size and follicular fluid but there was no rupture. This theory was not acceptable.

High Osmotic Pressure Theory

 Liquor folliculi consists of more amounts of electrolytes particularly Na and K. Increased

osmotic pressure leads to rupture of the follicle.
 In cystic ovaries size of the follicle and amount of follicular fluids along with electrolyte
content also increased but still there was no rupture. Hence, This theory was also not
acceptable.

Ischemic Theory

 Increased follicular fluid exerts pressure against follicular wall.

 At one point due to pressure, ischemia occurs and leads to stigma formation and ovulation.
Theory was partly acceptable.

Follicular Wall Thinning and Rupture

 Nearing ovulation, the blood supply to the follicle increases Thinning of the follicular wall
occurs at one point called as stigma. Ovarian contractions and follicular rupture occurs. This
theory was accepted.
 Recent theory is that ovulation is a combination of physiological, biochemical and
biophysical mechanisms.

OVULATORY PROCESS

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  A Surge of LH occurs at the beginning of estrus prior to ovulation when progesterone is at its
minimal levels and estradiol has reached its highest cyclical values.
 Several tissue layers separate the oocyte from the outside of the follicle. These are the surface
epithelium, the tunica albugenia, theca externa, and interna, basement membrane and
granulosa cells. All these tissue layers have to be broken down before ovulation can take
place.
 An increased blood flow near ovulation occurs to all classes of follicles but the follicle
destined to ovulate receives the largest volume of blood and has capillaries more permeable
than those in other follicle.
 As the follicle enlarges it begins to protrude from the surface of the ovaries, the vascularity of
the follicular surface increases except at its centre, which is devoid of blood vessels. This
avascular area is the future point of rupture.
 Meanwhile there is dissociation of cumulus cells which detaches the oocyte from the
membrana granulosa and now the oocyte is surrounded by the radiata cells.
 Resumption of Meiosis (nuclear maturation) occurs 3 h after LH surge and ends 1 h before
ovulation when the first polar body is extruded.
 Cumulus cells actively secrete a viscous mass enclosing the oocyte and its corona. After
follicular rupture the viscous mass spreads at the ovarian surface to facilitate the pickup of
oocytes by the fimbriae.
 The LH surge also causes a temporary shift in steroid secretions by increasing progesterone
secretion. This progesterone stimulates collagenase activity in the follicular wall reading to
dissociation of bundles of collagen fibers (increase in plasmin activity causes an increase in
follicular wall elasticity).
 The LH surge also causes an increase of PGF2 alpha and PGE2 levels. These prostaglandins
play a basic role in follicular rupture, and inhibition of their synthesis prevents ovulation.
o PGE2 stimulates production of plasminogen activator thus increasing plasmin activity
which increases in follicular wall elasticity and is involved in tissues cell migration
and thereby causes mixing of theca and granulosa cells during CL formation.
o PGF2 alpha causes rupture of the epithelial cell lysosomes at the follicular
epithelium. Their hydrolase’s destroy the underlying albugenia cells and then the
theca cells. After lysosomal rupture, epithelial cells scale off. The wall of the
follicular apex becomes thin in a circumscribed area called the stigma. The PGF2
alpha causes contraction of the smooth muscle cells that are present in the ovarian
stroma and theca externa thus leading to ovarian contractions and follicular
contractions. These ovarian contractions cause follicular rupture and follicular
contractions causes expulsion of the oocyte.
 At the time of ovulation the ovum, together with surrounding cells in a gelatinous mass,
protrudes at the ovarian surface and is swept into the ostium of the oviduct by the action of
the motile kinocilia of the fimbriae.

Species Ovulation Duration of estrus Ovulation time

type
Cow Spontaneous 14-18 h 12-18 h after end of estrus
Mare Spontaneous 4-7 days Last 2 days of estrus
Sows Spontaneous 2-3 days Last day of estrus
Ewe Spontaneous 1-2 days Last day of estrus
Bitch Spontaneous 7-9 days First 3 days of estrus
Cat Induced 4 days if copulation 1 day after mating
occurs
no ovulation occurs if copulation does not
9-10 days without take place
copulation

SUPEROVULATION AND EMBRYO TRANSFER

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NON-SURGICAL EMBRYO TRANSFER IN CATTLE

Refers to the non-surgical collection of an embryo from a donor cow and its replacement non-
surgically into the uterus of a recipient cow.

RECORDS

Species Year Authors

Rabbit 1890 Heape
Goat 1932 Warwick & Berry
Rat 1933 Nicholas
Sheep 1933 Warwick et.al.
Mouse 1942 Fekete & Little
Cow 1951 Willet et. al.
Pig 1951 Kvasnickii
Horse 1974 Oguri & Tsutsumi
Human 1978 Steptoe & Edwards

APPLICATIONS

 Genetic improvement
o Genetic variation
o Selection of dams
o Selection intensity
o Generation interval
 Genetic screening
 Disease control
 Import and export
 Circumvention of infertility
 Twinning
 Conservation

SELECTION OF DONOR

Two Major Criteria are involved in Selection of Donors

o Genetic merit
o Reproductive performance
 Good body condition and preferably gaining body weight.
 Disease free
 A minimum of 50-60 days post partum, and
 Cycling regularly

Clinical Examination

 Rectal palpation should be performed to rule out any adhesions or other palpable lesions of
the cervix, uterus and ovaries.
 The patency of cervical canal should be checked using a cervical dilator, especially in heifers.
This will help to overcome the frustration that might arise if unable to negotiate the cervix
after superovulation with expensive hormones.
 Vaccinate against prevailing diseases.
 Single embryos or multiple embryos may be collected from naturally ovulating or
superovulated cows, respectively.

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  Recommended donor-recipient ratio:
o For every flushing program, at least 2-4 donors should be prepared and synchronized
with their recipients.
o Recommended ratio is 8-10 potential recipients per donor.

SUPEROVULATION

 Superovulation is defined as the increased ovulatory response, above the number that would
be expected to occur naturally, generated in an animal by the administration of exogenous
gonadotrophic hormones.

General Considerations

 In the process of embryo transfer, superovulation is the least predictable step with great
variation in response due to age, breed, lactational status, nutritional status, season and stage
of the cycle at which treatment is initiated.

Superovulation with FSH

o Follicle stimulating hormone (FSH) or equine chorionic gonadotropin (eCG),

formerly called pregnant mare serum gonadotropin (PMSG) are used.
o Due the short half-life of FSH twice-daily injections over a period of 4-5 days is
required.
 Initiation of Treatment
o Treatment should be initiated during the mid-luteal phase (day 8 to 12) of the donor’s
cycle, and with the use of prostaglandins (PGF2alpha) to synchronize the cycles of
the donors and the recipients.
o Alternatively, treatment can be initiated on day 16 or 17 (day 0 = estrus) of the
donor’s natural estrous cycle.
 Schedule
o In four day FSH regimen, prostaglandins (PG) (25-35 mg PGF2α or 500 mcg PG
analog IM) are administered at the time of the fifth and sixth FSH injections. Estrus
follows in 2 days and ovulation in 3 days. In superovulated cows, the interval from
PG to the onset of estrus is 12-24 h shorter than in naturally ovulating cows or
heifers. Hence, in recipients PG should be injected 24 h before the donors.
 Response
o The response ranges from zero to 20 or more ovulations, with an average of 8-10.
o No difference in response between a 4-day and a 5-day regimen.
o If a donor fails to respond to an established superovulatory treatment regime, the
second attempt with the same or a similar regimen is also likely to result in failure.
 Preparations
o Folltropin-V (Bioniche): Twice daily injections in decreasing doses are
recommended.
 Comparable Products
o Ovagen (ICP Ltd; Auckland New Zealand).
o Pluset (Serono; Rome, Italy).
o Stimufol (Rhone Merrieux; Lyon France).

Superovulation with Pregnant Mare Serum Gonadotropin (PMSG)

 PMSG is a foreign protein that is antigenic, may lead to a reduced response after repeated use.
 Due to its long half-life, a single injection is sufficient.
 The FSH/LH ratio: 1.9 to 95.5 depending on the stage of gestation at which the serum was
collected.

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  Administration of gonadotropin releasing hormone or LH to donor cows at the onset of estrus
in an attempt to precipitate or to group ovulations does not offer any advantage.
 Stage of administration
o On day 16 or 17 of a normal estrous cycle.
o If used in conjunction with prostaglandin (PG), PMSG is administered between day
8-12 of an estrous cycle followed by PG 48 to 72 h later.
 Dose: 1500-3000 IU.
 Route of administration: SC or IM.

Assessment of Superovulatory Response

 It is difficult to accurately assess the number of ovulations by palpation of the CL per rectum
when the number exceeds 4 to 6 per ovary or when several anovulatory follicles are also
present.

WITH FSH

 Follicle stimulating hormone (FSH) or equine chorionic gonadotropin (eCG), formerly called
pregnant mare serum gonadotropin (PMSG) are used.
 Due the short half-life of FSH twice-daily injections over a period of 4-5 days is required.

Initiation of Treatment

 Treatment is initiated during the mid-luteal phase (day 8 to 12) of the donor’s cycle, and with
the use of prostaglandins (PGF2alpha) to synchronize the cycles of the donors and the
recipients.
 Alternatively, treatment is initiated on day 16 or 17 (day 0 = estrus) of the donor’s natural
estrous cycle.

Schedule

 In four day FSH regimen, prostaglandins (PG) (25-35 mg PGF2α or 500 mcg PG analog IM)
are administered at the time of the fifth and sixth FSH injections. Estrus follows in 2 days and
ovulation in 3 days. In superovulated cows, the interval from PG to the onset of estrus is 12-
24 h shorter than in naturally ovulating cows or heifers. Hence, in recipients PG should be
injected 24 h before the donors.

Response

 The response ranges from zero to 20 or more ovulations, average 8-10.

 No difference in response between a 4-day and a 5-day regimen.
 If a donor fails to respond to an established superovulatory treatment regime, the second
attempt with the same or a similar regimen is also likely to result in failure.

Preparations

 Folltropin-V (Bioniche): Twice daily injections in decreasing doses are recommended.

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Comparable Products

 Ovagen (ICP Ltd; Auckland New Zealand).

 Pluset (Serono; Rome, Italy).
 Stimufol (Rhone Merrieux; Lyon France).

WITH PMSG

 PMSG is a foreign protein that is antigenic, may lead to a reduced response after repeated use.
 Due to its long half-life, a single injection is sufficient.
 The FSH/LH ratio: 1.9 to 95.5 depending on the stage of gestation at which the serum was
collected.
 Administration of gonadotropin releasing hormone or LH to donor cows at the onset of estrus
in an attempt to precipitate or to group ovulations does not offer any advantage.

Stage of Administration

 On day 16 or 17 of a normal estrous cycle.

 If used in conjunction with prostaglandin (PG), PMSG is administered between day 8-12 of
an estrous cycle followed by PG 48 to 72 h later.
o Dose: 1500-3000 IU.
o Route of Administration: SC or IM.

ASSEASMENT OF SUPEROVULATORY RESPONSE

It is difficult to accurately assess the number of ovulations by palpation of the CL per rectum when
the number exceeds 4 to 6 per ovary or when several anovulatory follicles are also present.

ESTRUS DETECTION

 Accurate estrus detection is necessary for

o Timely insemination of the donor, and
o To assess the degree of synchronization of estrus and ovulation between the donor
and recipients.
 The age of the embryo is calculated from the time of onset of estrus.

Methods of Estrus Detection

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  Direct observation of estrus behavior, especially mounting, remains the most reliable method.

Mounting Behaviour Estrual Mucus discharge

 Estrus detection aids are available, including the use of teaser animals, tailhead marking or
painting, heat detector patches, and pedometers.

Click here to know more about estrus detection methods

Monitoring of Estrus and Ovulation

 Frequent rectal palpation of the uterus and ovaries.

 By ultrasonography.
 By frequent milk or plasma progesterone assay.

BREEDING OF DONOR

 Twice AI with a 10-12 h interval beginning 4 -6 h after the onset of estrus, to cover the range
of time over which the ovulations may occur.
 Depending on the quality of the frozen semen, a double inseminating dose may be used at
each insemination especially in cows with a large pendulous uterus.

NON SURGICAL EMBRYO FLUSHING

Optimum Time of Flushing

 Bovine embryos descend into the uterus around day 4.5 (estrus = day 0) and shed their zona
pellucida (“hatch”) between days 8-10.
 Optimum time for nonsurgical flushings is between days 6-8.

TECHNIQUES

Foley Catheter

 A 2-way round tip with a 30 ml inflatable balloon.

 French size #16 to 24.
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  The 2-way catheter has one channel for inflation of the balloon plus a single channel for
alternate inflow and outflow of flushing medium.

Rusch Catheter (Germany)

 Red rubber 68 cm long, 18 French gauge, and balloon catheter.

 It has a self-contained locking stylet and a tip which extends 4.5 cm beyond the balloon and
offers advantage in older cows.
 After the stylet has been withdrawn 3-4 cm the catheter can be directed further into the horn
because the rubber is stiffer than the Foley catheter.
 Advantageous in older animals with long pendulous uteri. The uterine lumen is flushed by
alternating in- and out-flow.

Three-way Collection Device (IMV, France)

 A large and rigid, stainless steel.

 One channel serves to inflate the balloon, a second one, the stainless steel cannula, to
introduce the flushing medium, and the third, a small flexible catheter which can be advanced
into the tip of the horn, to recover the flushing medium.

FLUSHING AND HOLDING MEDIA

 Phosphate - (Dulbecco’s phosphate buffered saline, DPBS): advantage is that they maintain
the pH during exposure to the air.
 Bicarbonate-buffered solutions: need a gas phase of 5% CO2, which means that they require a
closed system.
 The pH of most body fluids is 7.2-7.4 and osmolarity in the range of 270-300 mOsmol.
 1% heat-treated bovine serum (10 ml) or Bovine serum albumin (BSA) 0.04% is added to
each individual 1-liter bottle of flushing medium which has been warmed to a temperature
between 30-37 °C. Serum may act as a protein source for embryo growth and membrane
stabilization, and renders embryos less sticky.
 For flushing, PBS only needs to contain penicillin and 1-2% serum. 10-20% serum is added to
the flushing medium to make a holding medium which can also be used for short-term (less
than 24 h) culture.
 The holding medium is filter sterilized by filtration through a 0.22 µm or 0.45 µm millipore
filter attached to a large disposable syringe.
 Embryos are stored in the same type of medium that was used for flushing. Changing
embryos from a phosphate- to a bicarbonate- buffered medium is undesirable because of the
possible changes in osmolarity, pH and energy substrates.

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  The holding dishes should be covered to minimize contamination and evaporation.
 Embryos must be changed to a fresh dish of holding medium from time to time (every 2 h) to
further minimize the effects of contamination and evaporation.

FLUSHING PROCEDURE

 Restrain the donor in a chute.

 Carefully remove the faeces from the rectum avoiding sucking of air, and assess the number
of ovulations (CL).
 Administered (4-6 ml of 2 % lidocaine) epidurally to prevent defecation and straining.
 Wash the vulva and perineal region thoroughly with plain water and wipe it dry.
 Tie the tail to one side of the animal.
 Use an appropriate cervical dilator properly covered with a sanitary sleeve before they are
introduced into the vagina to expand and straighten the small or tortuous cervical canal.
Perforate this protective cover just before the instrument enters the external os of the cervix.
Exercise extreme caution while using a rigid, relatively sharp-pointed dilator as it can readily
perforate the uterine wall.
 Part the lips of the vulva and the Foley catheter, with the stylet in place, is inserted into the
vagina and on into the lumen of the cervix. Carefully manipulate into the appropriate horn
until the inflatable balloon is situated at the base of the uterine horn. Alternately, the catheter
with the balloon may be fixed just anterior to the internal os of the cervix, in the body of the
uterus so as to flush both horns simultaneously.

Location of the Balloon Balloon Placement Bilateral Uterine Flush

The local distension at the base of The balloon is placed at the base The balloon may be placed in the
the right uterine horn indicates of the horn for a unilateral flush. anterior portion of the cervix
the location of the inflated with the tip of the catheter in the
balloon. uterine body to flush both horns
simultaneously. Inflation of the
balloon in the cervix meets with
more resistance, compared with
placement in the uterus.

 Slowly inflate the balloon with 15-20 cc of air in adult cows and 10-15 cc of air in heifers.
The endometrium can easily be split by over distension, resulting in haemorrhage and escape
of the flushing solution into the mesometrium from which it cannot be recovered.
 Position the catheter and remove the stylet.

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  Connect the catheter via a Y-junction by sterile tubing to a 1 litre bottle or bag of flushing
medium. Connect the remaining arm of the Y-junction to a free piece of tubing.
 Quick-release clamps are used to check the flow of medium in both pieces of tubing. The
flushing medium bottle is suspended one metre above the level of the uterus and when the
outlet tubing is clamped, the flushing solution from flows in to the uterus by gravity flow.
 Extend the horn of the uterus by elevating the tubo-uterine junction and by carrying it
anteriorly. When the inflow stops, clamp the inlet tubing and release the clamp on the outlet
tubing.
 Allow the fluid to directly pass through an embryo filter (75 µm pore size).
 If filters are not used, the effluent may be collected in a 1-liter graduated cylinder. Allow the
embryos to settle for 20-30 min and carefully siphon off the supernatant. Start the siphoning
by aspiration using a small syringe fitted at the other end of the tubing and gently lower a
length of small diameter (e.g. 1 mm) tubing into the cylinder until the end of the tubing
reaches the 75 ml mark. Examine the remaining 75 ml directly under a stereomicroscope.

Cervical Dilator Embryo Filter Graduated Cylinder

Cervical dilator: diameter 6 mm, Embryo filter (EmCon) with a A 500 ml or 1000 ml sterile
50 cm long with a 4 cm tapered stainless graduated cylinder may be used
tip to a end diameter of 2.5 to steel sieve. Diameter of the pores to collect the flushing medium.
3.0 mm. Made out of stainless is 75 micrometers. The white The embryos will settle to the
steel or aluminium alloy. clamp on the drainage tube bottom by gravity. After a 15
Compared with a standard 45 controls the level of minute wait the upper portion of
cm long flushing medium in the filter. The the fluid may be gently siphoned
AI syringe. effluent tube of the flushing off by lowering a small diameter
catheter can be connected directly length of tubing to the 50 mm
to the nipple on the mark. The remaining smaller
lid of the filter. volume may then be poured into
a searching dish and examined
under the microscope for the
presence of embryos. This
method is less expensive than the
use of single-use disposable
embryo filters.

 If the returning fluid is blood tinged, the red cells may be washed directly through the filter by
opening both clamps between the bottle of flushing solution and the filter. Never allow the
filter to run completely dry leaving the embryos on the filter disk exposed to the air. Maintain
a minimum layer of 1 cm fluid by regulation with the clamp on the tubing attached to the
bottom of the filter unit.

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  During the final collection of the flushing solution, administer 50 IU of oxytocin IV, which
sometimes may aid in the recovery of the residual portion of the medium from the uterus.
 Repeat the same flushing procedure for the opposite horn using a separate sterile catheter.

IDENTIFICATION AND HANDLING OF EMBRYO

Identification

 The embryo is spherical and is composed of cells (blastomeres) surrounded by a gelatin-like

shell, and acellular matrix known as the zona pellucida (ZP).
 The zona is spherical and translucent, thus is clearly distinguishable from cellular debris.
 Due to its shape the embryo tends to roll on the bottom of the (searching) dish.
 The overall diameter of the bovine embryo is 150-180 μm including a ZP thickness of 12-15
μm.
 The diameter remains constant until expansion of the blastocele begins.

Important Criteria

 Shape of the embryo

 Presence of a zona pellucida
 Size
 Color, and
 Knowledge of the age of the embryo.

Handling

 As soon as an embryo is identified, it is immediately transfered to a small petri dish

containing fresh, filter sterilized medium.
 The embryos are serially rinsed through at least three different dishes containing fresh sterile
medium using a sterile pipette.

MORPHOLOGICAL CLASSIFICATION OF EMBRYO

Embryos recovered 5-8 days after estrus are classified morphologically into the following groups.

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Morula

 Blastomeres are round in shape and are not tightly connected to each other. Individual
blastomeres are difficult to discern from one another. The cellular mass of the embryo
occupies most of the perivitelline space.

Compact Morula (Tight Morula)

 The shape of a tight morula is similar to a golf ball, in that the outer edge is slightly bumpy
(scalloped) in appearance because of compaction. Individual blastomeres are no longer
distinguishable. Cells on the surface of the mass are polygonal in shape. The embryo mass
occupies 60 to 70% of the perivitelline space.

Early Blastocyst

 A tiny transparent (clear) space is visible which contains fluid. This area is the beginning of
the blastocele. The embryo occupies 70 to 80% of the perivitelline space.

Blastocyst

 The prominent blastocele cavity comprises more than 70% of the volume of the embryo. Two
groups of cells are present and clearly recognizable as the trophoblastic layer beneath the
zona pellucida and the darker inner cell mass occupying one side of the embryo. The
perivitelline space may still be visible but is very small.

Expanding or Expanded Blastocyst

 There is no perivitelline space between the layer of trophoblastic cells and the inside of the
zona. The zona pellucida becomes thinner as the blastocyst expands. A small (well
compacted) inner cell mass positioned on one side of the embryo is observed. The color of the
embryo is pale to clear because of the large amount of fluid present inside.

Hatched Blastocyst

 Ultimately the blastocyst expands to the point of rupture and the embryo escapes from the
disrupted zona. Hatched blastocysts may be spherical with a well-defined blastocele or they
may be collapsed, resembling debris. Identification of embryos at this stage can be difficult
for the inexperienced operator. When zona-free, or hatched, blastocysts are collected, there is
a greater risk of damage due to handling. Furthermore, hatched blastocysts are “sticky” and
may adhere to tubing and glassware. Embryo filters should not be used when there is a
possibility that hatched embryos will be recovered (>day 7.5).
 Excellent, good and fair quality embryos are considered transferable. Excellent and good
quality embryos are freezable.

CODES FOR EMBRYO QUALITY

Code 1: Excellent or Good

 Symmetrical and spherical embryo mass with individual blastomeres (cells) that are uniform
in size, color and density.
 Consistent with its expected stage of development.

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  Irregularities should be relatively minor and at least 85% of the cellular material should be an
intact, viable embryo mass. This judgement should be based on the percentage of embryo
cells represented by the extruded material in the perivitelline space.
 The zona pellucida should be smooth and have no concave or flat surfaces that might cause
the embryo to adhere to a petri dish or a straw.

Code 2: Fair

 Moderate irregularities in overall shape of the embryo mass or size, color and density of the
individual cells.
 At least 50% of the cellular material should be an intact, viable embryo mass.

Code 3: Poor

 Major irregularities in shape of the embryo mass, or size, color and density of individual cells.
 At least 25% of the cellular material should be an intact, viable embryo mass.

Code 4: Dead or Degenerating

 Degenerating embryos, oocytes or 1-cell embryos; non-viable.

EMBRYO SCREENING PROCEDURE

 Embryo searching equipment is prepared before the uterus of the donor is flushed when a
concentrating filter is used.
 One to three 100 x 100 mm square, grid bottom dishes are used to search one filter.
 Each dish is labeled with the donor’s number and the sequence in which it was filled from the
filter.
 Fresh straight PBS is drawn into a 30 or 35 ml syringe with sterile precautions. A 22 gauge 1
inch needle is then attached to the syringe. This syringe is used to rinse the filter. The
presence of serum in the PBS must be avoided during this procedure to prevent foaming.
 The medium, left in the filter from the flush, is swirled and poured into first dish.

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Rinsing the Filter Searching the Dish Searching For Searching Pattern
The filter is rinsed with The square, sterile, Embryos The circle indicates the
plain flushing medium (no searching dish is filled Searching for size of the viewing field at
serum) half full of flushing embryos under the 70 X magnification. A
to keep it from foaming. A medium which contains stereoscope at a systematic search is
25 ga needle is used and 1% fetal calf serum. The magnification of 40 conducted along the top
the contents of the filter holding medium in the to 70 X half of the 1cm squares
are poured into a round holding dish from left to right, and then
searching dish with a contains 10% fetal calf along the bottom half
grid, and marked with the serum. from right to left. The
number of the donor. number of the donor is
written on the bottom part
of the searching dish.

 The filter is held at an angle and rinsed into the dish. More fluid may be needed if the filter
contains mucus. Steps 4 and 5 are repeated using as many dishes as necessary, until the filter
is completely clear (no mucus and/or tissue debris left on the filter). Each dish will safely hold
75 ml.
 Five to seven ml of serum or 0.4 percent BSA are added to the searching dish and stirred in
the medium while clearing bubbles from the edge of the dish.
 Dishes are searched under a stereomicroscope (15X). The dish is systematically moved along
the reference lines to ensure the entire dish is searched. When embryos are identified, they are
immediately transferred into a small petri dish containing sterile, filtered holding medium
(PBS + 10-20 % serum, or 0.4 percent BSA). All dishes should be kept covered between
searches to avoid contamination, and particularly evaporation when placed in an incubator.
 The embryo is drawn from the searching dish into a micropipette attached to a 0.5 ml syringe
while observing under the microscope. Then, the embryo is transferred to the small holding
dish. The number of good and/or bad embryos are tentatively recorded on the lid of the
holding dish. The estimated number of corpora lutea usually serves as a guide to the number
of embryos to be searched for.
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  After all the embryos from one donor are accumulated in one small holding dish, they are
transferred three times, each time to a dish containing freshly filtered PBS plus serum. This
procedure serves to rinse the embryos.
 The embryos are now graded and prepared for the next procedure; culturing, transferring,
freezing, splitting or sexing.
 Grading is facilitated under higher magnification (100 X) with a regular light microscope,
preferably an inverted microscope with adequate working room on the stage.
 Alternatively, when a large 500 to 1000 ml graduated cylinder is used, the embryos are
allowed to settle to the bottom of the cylinder for 20 to 30 minutes. All but the bottom 75 ml
of flushing medium are slowly siphoned off with a small diameter piece of tubing. The final
75 ml are gently swirled and then poured into a searching dish. The cylinder is rinsed 2 to 3
times with small volumes of flushing medium (containing 1 % serum) and emptied into a
searching dish. This is followed by steps 7 through 12 as before.

LOADING THE STRAW

 Load the embryos individually in sterile 0.25 ml French straws just before transfer in to
suitable recipients.

 Aspirate the embryo from the holding dish into the straw with the aid of a 1 ml tuberculin
syringe attached to the plug end of the straw.

 First aspirate a 3 cm column of medium, followed by a 0.5 cm column of air, then a 3 cm

column of medium containing the embryo, followed by another air bubble. Fill the remainder
of the straw with medium until the initial column of medium wets and solidifies the plug.

SELECTION OF RECEIPIENTS

Selection of Cows or Heifers

Select cows and heifers

 with large body frame, disease free and in good body condition.
 with a minimum of 2 normal cycles before inclusion.
 that are not too fat and should preferably gain 0.1 - 0.2 kg / day.
 that are vaccinated against common abortion diseases.

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  An average bovine donor yields 6 to 8 transferable embryos.Therefore, Prepare a minimum of
8 recipients per donor.
 Screen atleast 12 recipients in order to definitely identify 8 with active corpora lutea.
 If 8 are injected with prostaglandins, 6 on the average will be suitably synchronized with the
donor.
 Inject recipients with prostaglandins one day earlier than the donor.
 Due to prior gonadotropin treatment, the donor comes into oestrus 48 h after the
prostaglandins, while the recipients which did not receive any gonadotropin treatment will
come into oestrus 72 h after prostaglandin treatment.
 Since all donors will not respond to the superovulatory treatment, for optimal efficiency, 2-4
donors should be superovulated at the same time to permit sharing of the prepared recipients
and avoid the expensive frustration consequent to single donor preparation.

PROSTOGLANDIN PROCEDURES FOR ESTRUS SYNCHRONIZATION OF DONOR AND

RECEIPIENTS

ecipients can be synchronized to exhibit heat on the same day or just ahead of the donor with:

Method - 1

 In animals with a palpable CL, inject 25 mg PGF2α or 0.5 mg PG-analog lM.

 Estrus may be expected in 2-4 days with a peak on the third day.

Method - 2

 Regardless of the presence or absence of a CL, all recipients may be administered

prostaglandins.
 A second PG is administered 11 days later.
 Estrus will peak on the third day after the second injection.

Prostaglandin Protocol

Day What To Do - DONOR What To Do - RECIPIENTS

0 In heat / No Al PG
10 FSH Nothing
11 FSH PG am

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 12 FSH
Nothing
PG am/pm
13 FSH Nothing
14 Estrus +Al Estrus/No Al
21 Collect embryos Transfer

SYNCHROMATE B

ncro-mate-B (SMB ear implant)  Progestogen containing ear implant.

 Each SMB ear implant contains 6 mg of the synthetic progesterone norgestomet.

MB ear implant insertion  In recipient animals, estrus synchronization is induced by placement of 6 m

Syncromate-B (SMB) ear implant.
 Under aseptic precautions, the SMB ear implant is inserted on the outer surface of t
ear under the skin, avoiding blood vessels, using SMB applicator.
 At the time of implant insertion 0.5 ml of SMB injection (1.5 mg of norgestomet a
2.5 mg of estradiol valerate) is administered intramuscularly.
 On day-10 after the insertion of the implant, the animals receive a single no
superovulatory intramuscular injection of 400 IU of PMSG and 10 mg of PGF2α.

MB ear implant removal  The implant is removed on day-11.

 The scab at the point of insertion is gently teased and removed using a sterile needl
 By gently squeezing the skin over the implant from the top end, the implant
removed.
 Apply antiseptic at the site following implant removal.

Syncromate-B Protocol

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Day What To Do - DONOR What To Do - RECIPIENT

0
SMB Implant +injection SMB implant + injection

7
FSH Nothing

8
FSH PG

9
FSH + PG + implant removal Implant removal

10
FSH Nothing

11
Oestrus + AI Oestrus

12
AI Nothing

18
Collect embryos Transfer

NON SURGICAL EMBRYO TRANSFER

 Performed non-surgically similar to artificial insemination.

 Requires a high degree of dexterity and skill.
 Contamination of the uterus should be minimised because it is more susceptible to infection
during the luteal phase.
 Faeces is evacuated from the rectum and the side of the CL is determined.
 Epidural anesthesia is induced to prevent defecation and to minimize straining.
 The perineal region is washed and the vulva is wiped dry.
 In the laboratory, the embryo is loaded into the 0.25 ml French straw. The straw is
inserted into the transfer or Al gun. The transfer gun is covered with a sterilized sheath and
fixed in place with the 0-ring. The gun is wrapped with a second, sterile, larger (sanitary)
sheath which is closed at the distal end over the first to serve as a protective cover and permit
passage of the gun through the vagina without coming into contact with the vaginal flora.

0.25 cc AI Syringe ET Syringe with Sanitary Sleeve

0.25 cc AI syringe with O ring and split end Comparison of a 53 cm embryo transfer syringe
sheath. A 0.25 cc straw loaded with an embryo in with a standard 45 cm artificial insemination
the center column of medium between to air syringe. The transfer syringe is covered with a
pockets is also shown. sanitary sleeve.

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  The embryo is gently deposited approximately one-third of the way into the uterine horn
ipsilateral to the CL and the gun is slowly withdrawn.
 It is important to bear in mind that trauma to the delicate endometrium can result in bleeding,
and blood (complement in the serum) is embryocidal.
 In Embryo Placement the tip of the transfer instrument can be seen through the incision in
this slaughterhouse specimen. Deeper placement may be ideal, but excessive manipulation
and trauma to the endometrium must be avoided.

CARE OF DONOR ANIMALS

 Immediately after flushing of donor animals, Prostaglandin F2 alpha (25 mg; i/m) is
administered to cause rapid reduction in the size of the ovary, and to prevent the
establishment of pregnancy from an unflushed embryo if left in the uterus.
 Administration of Prostaglandin F2 alpha to donor animals will induce estrus usually within 3-
5 days - Remember not to breed the donor on this induced estrus.
 Any contamination that might have gained entry at the time of the embryo flushing will be
eliminated during this induced estrus.
 Superovulation can be carried out thrice at two months interval without drastically affecting
the response.
 In between the superovulatory treatments, if the animals come in to estrus, they can be
artificially inseminated and single embryo flushing may be performed.

CARE OF RECIPIENT ANIMALS

 Pregnant recipient cows should be maintained similar to that of other pregnant cows.
 Presumptive pregnancy diagnosis can be made based on
o Palpation of the functional CL.
o Indirect estimation of progesterone in the milk or plasma on day 21-24 of their cycle
(16-19 days after transfer on day 5), or
o Identification of conceptus by ultrasonography around day 27.
 Definitive pregnancy examinations: at six weeks.
 Confirmative diagnosis: at three months by rectal examination.

SURGICAL EMBRYOTRANSFER IN GOATS

 There are 502 million goats in the world, approximately 56.5% of which are in Asia. About 6
% of the world’s goats are found in developed countries and 94 % in developing countries.
 Genetic improvement necessitates substitution of genetically superior animals for those of
little genetic merit.
 Embryo transfer is an advanced, but well established, animal breeding technology.
 The procedure of superovulation followed by recovery of embryos and transfer to
synchronized recipients has proved to be an effective means of increasing the contribution of
superior females to the gene pool of the population.
 During the last two decades, tremendous progress has been made in the female germplasm
use through multiple ovulation and embryo transfer.
 The first record of successful embryo transfer in goat was reported by Warwick and Berry
(1949). Hunter et. al., (1955) transferred nineteen, 2-16 cell embryos to 18 recipient ewes,
eight lambs were born. This procedure remains to be the basis of surgical embryo transfer in
sheep and goats today.

What is Surgical Embryo Transfer?

 Surgical embryo transfer is the process whereby embryos are surgically flushed from the
reproductive tract (either from the oviduct or uterus) of a "donor" goat and surgically
transferred in to suitably synchronized "recipient" in order to establish a surrogate pregnancy.
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Donors

 Virgin does 6 months and older.

Recipients

 Multiparous does (age approximately 1-1.5 years). The main purpose of having multiparous
does as recipients is to reduce the possible risk of dystocia that may be seen with primiparous
animals.

HORMONES

Generally, the principles of superovulation in goats are similar as in cattle. The following
gonadotrophin preparations are most commonly used for superovulation in goats

 Pregnant Mare Serum Gonadotrophin (PMSG): Administered as a single subcutaneous or

intramuscular injection given one day prior to the last synchronization treatment.
 Pituitary Follicle Stimulating Hormone (FSH-P): is given at 12h intervals in decreasing doses
for 3-4 days on days 5-16 of the estrous cycle. Prostaglandin F2 alpha (PGF2α)
 Horse Anterior Pituitary Extract (HAP)

SUPEROVULATION PROTOCOL

 There are a number of different superovulation protocols that may be used in the goat. The
following protocol produces optimal embryos
o The timing of oestrus in the donors is synchronized on day-0 with the placement of a
subcutaneous synchromate–B (6 mg of norgestomet), SMB-ear implant.
o At the time of implant insertion 0.5 ml of SMB injection (1.5 mg of norgestomet and
2.5 mg of estradiol valerate) is administered intramuscularly.
o Inj. PGF2α (10 mg) is administered intramuscular on day 11.
o Starting from day-9, after insertion of implant a total of 200 mg of FSH-P is
administered intramuscularly over four days in twice daily injections. A decreasing
dose format starting with 32 mg the first day, ending with 16 mg on the fourth day of
FSH-P treatment.
o The implant is removed on day-11.
o On day-13 the animals are observed for estrus signs and bred in the evening to fertile
males.

ESTRUS SYNCHRONIZATION AGENTS

Syncromate-B Components

SMB ear implant approximately 2 x 18 mm size containing 3 mg of Norgestomet (17

alpha acetoxy 11 beta methyl 19-Nor-Preg 4 ene-3, 20 dione).

SMB injection containing 2 ml of oily solution of 3 mg of Norgestomet and 5 mg of

estradiol valerate.

SMB applicator

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 Chrono-gest Vaginal Sponge

Contains 45 mg of fluorogestone acetate

ESTRUS SIGNS

 Standing estrus lasts approximately 24 h in young does but may last 2-3 days in mature does.
 The doe in estrus walks restlessly along the perimeter of her enclosure, searching for a way to
reach the buck, or remain close to the fence.
 The vulva becomes somewhat swollen and the doe’s tail wags vigorously. This tail wag can
often be observed even in the absence of a buck.
 The doe stands firmly when a buck attempts to mount and may even back up the buck.
 The vaginal discharge at the beginning of estrus is clear and colorless, becomes progressively
white and more opaque towards the end of standing estrus.
 Ovulation typically occurs near the end of standing estrus and approximately 24 h after a
serum peak in Luteinising hormone (LH).

SURGICAL EMBRYO COLLECTION

 Embryos are flushed on the second day following breeding or 72 h following implant
removal.
 Few hours prior to surgical collection embryo flushing medium and culture droplets are
prepared under sterile lab conditions and equilibrated at 38.5-39 °C in a 5 % CO2incubator.
 The animals are prepared for surgical collection and transfer by fasting for 24 h and with
holding water for 12 h prior to surgery.
 The animals are subjected to mid ventral laparatomy and anaesthesia induced by i/m
administration of 0.22 mg/kg.b.wt.
 xylazine followed by 11mg /kg.b.wt. ketamine 10 minutes later. This combination leads to
smooth induction with anaesthesia lasting approximately 45 minutes.

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 The animal is put on dorsal recumbency, held in position with the help of cotton ro
tied to the hind limbs with surgical site aseptically prepared.

A longitudinal midline skin incision approximately 10 cm is made ventral to the pel

cavity avoiding the mammary glands.

The muscles and peritoneum are cut at the same time at the subsequent strokeexpos
the viscera.

 The reproductive tract is exteriorised through the midline incision. Corpora lutea and
unovulated follicles are counted to evaluate the superovulatory response and to predict the
number of embryos that should be collected by oviductal flushing.
 For embryo collection a sterile tygon tube of suitable diameter is inserted through the
fimbriated end of the fallopian tube to a depth of about 1 cm. A sterile sequencer tip attached
to a 10 ml syringe with flushing medium (DPBS with 10% FCS) is gently inserted into the
lumen few cms from the utero-tubal junction and held in position between the thumb and
index finger and about 5–10 ml of medium is used for flushing and collected in a sterile
petridish. This procedure repeated on the opposite side and then the reproductive tract

~ 320 ~
 returned to the abdomen. Ensure that no loss of fluid occurs during collection procedure.
After flushing, the abdominal cavity is closed and sutured with size 1 chromic catgut.

The skin edges are closed with cotton thread.

 The animals are returned to the stall for post-surgical observation and recovery to standing
occurs in approximately 1.5 h. Postoperative care is given for about 7 days with antibiotics
and local dressing. The embryos are collected from the DPBS oviductal flushings on
stereomicroscope and evaluated.

SURGICAL ET IN SURROGATE

 Only morphologically normal and 2-4 cell stage embryos are selected for transfer.
 Under the same surgical procedures as for the donors, the fallopian tube is located and the
embryos in minimum medium are transferred into the lumen to a depth of 2-3 cm using a
pipette with a sequencing tip after assessing the ovarian response.
 Each recipient goat usually receives 2-3 embryos ipsilateral to the ovary containing one or
more corpora lutea.

RECIPIENT MANAGEMENT

 It is advisable that feed intake only be maintained or even reduced for recipients after transfer,
as excess feeding during early pregnancy reduces pregnancy rates.
 Recipient does are first evaluated by ultrasonography approximately day-35 from the first day
of estrus to detect pregnancies.
 A confirmatory ultrasound at day-55 will provide the most reliable indication of viable
pregnancy with fetal number and viability evaluated.
 Pregnant does are monitored daily throughout pregnancy and appropriate pre-kidding
procedures are performed.
 After completion of the gestation period (150 + days), the kids will be born.

ET IN MARES

 From a purely academic point of view, equine embryo transfer can be used for the following:
o To obtain foals from subfertile mares;
o To better manage older, valuable broodmares;
o To circumvent problems with neonatal isoerythrolysis (jaundice);
o To obtain foals from mares engaged in competition;
o To manage mares that chronically abort twins;

~ 321 ~
 o To further the knowledge of the mechanisms of the maternal recognition of
pregnancy;
o To produce multiple offspring
o To advance genetic progress.
 The first successful equine embryo transfers were reported in England a little more than 10
years ago. Acceptance of the technique as an approved method for producing foals by a major
American breed association occurred less than 5 years ago.
 The procedure was initially adopted to produce a single foal per year from barren mares that
could not carry a pregnancy to term; however, more recently, embryo transfer is also being
used to produce pregnancies from maiden fillies that are in show competition. This is
probably most often done in the Arabian breed.
 Irrespective of the reason for performing embryo transfer, the efficacy of the procedure in
horses is confounded by the inability to produce multiple viable embryos via superovulation,
a relatively short in vitro survival of the equine embryo, difficulty in synchronizing ovulation
among donors and recipients and the high incidence of uterine infections in barren donor
mares.
 Moreover, in the United States and in virtually all other countries, the two major racing
breeds, Standardbreds and Thoroughbreds, do not accept foals produced by embryo transfer
in their registries.

Dulbecco’s phosphate-buffered saline (PBS) supplemented with 1 per cent heat-inactivated fetal
bovine serum appears to be most suitable medium for field use. Dulbecco’s PBS is supplemented with
1 per cent fetal bovine serum and 100 IU penicillin plus 100 μg streptomucin/ml to prepare the
embryo recovery medium. Embryo culture and transfer medium is prepared by adding 20 per cent
heat-inactivated fetal bovine serum to a volume of recovery medium. This is then Millipore filtered
through a 0.22 μm disposable Falcon filter.

~ 322 ~
 Preparation of the Donor Placement of the Balloon
of the donor mare for the flushing The donor mare is placed in the stocks and cleaned The balloon catheter is directed into an
e is important. in preparation for flushing of the embryos. the cervix towards the uterine lumen w
guidance per vaginam

Recovary Supplies Collection of the Effluent Preparation of the

e a large sterile balloon catheter, a sterile The effluent is passed through an embryo filter. The synchronized recipient mare is pre
ich is set in a beaker, with tubing After the fluid has passed through the filter it is sterile surgery and transfer of the embryo
The forceps are used to clamp off the collected in a graduated cylinder to monitor the
regulate flow. There is also a tube of amount of fluid recovered versus the amount
acidal sterile lubricant. infused. The embryo is retained in the filter.

Collection of Embryo

 Embryo collections are performed non surgically on days 6 to 9 after ovulation ( day of
ovulation = day 0). The equine embryo does not enter the uterus until day 6 after ovulation.
The diameter of equine embryos over days 6 to 9 after ovulation will range from 0.1 to 4.5
mm. By day 8 the equine embryo is generally 10-fold larger than a bovine embryo of the
same age and are too large to be transferred without breakage.
 The nonsurgical collection procedure is a modification of the one developed for cattle. An
extended 30-French Foley or an 18-French Rusch catheter with a 30-ml inflatable cuff is
inserted through the cervix into the uterine body and is secured in position by inflating the
cuff with 1.5 to 30 ml of sterile water or recovery medium. Once the catheter is properly
positioned, both uterine horns and the uterine body are filled simultaneously with 1 liter of
medium by gravity flow. The medium is then collected by gravity flow into sterile, 1 liter
Erlenmeyer flasks or graduated cylinders. This procedure is repeated three additional times.
Uterine palpation is used to facilitate recovery of medium from the uterus during the last three
flushes.

Embryo Handling

 Embyros are of greater density than the medium and therefore settle to the bottom of the
collection vessel within approximately 20 to 30 minutes following collection. The upper 850
to 900 ml of medium is removed by pouring or siphoning into another sterile container. The
bottom portion of the medium is then poured into a gridded sterile plastic petri dish. Attempts
are first made to identify the embryo macroscopically, and then the dish is searched into a 14-
gauge catheter (Soverign) attached to a 1-ml syringe and is then deposited into a sterile plastic
petri dish containing transfer medium. The embryo is then gently agitated (washed) for

~ 323 ~
 approximately 1 to 2 minutes and then placed in a second Petri dish containing transfer
medium. The embryo is stored in the dish until transfer.
 Results have indicated that equine embryos do not remain viable for more than approximately
3 hours in Dulbecco’s PBS. Therefore, transport of fresh equine embryos over long distances
would seem impossible. Embryo freezing would be an obvious solution to this problem.

TRANSFER

Nonsurgical

 Embryos are aspirated into a Luter Flex 22-inch sterile large animal pipette or similar pipette,
which contains 10,000 IU penicillin plus 10,000 μg streptomycin. The aspiration procedure
has the following sequence: 1 ml antibiotics, 0.25 ml air, 0.5 ml transfer medium, 0.25 air, 0.5
ml transfer medium containing the embryo, 0.25 air and 0.05 ml transfer medium.
 The perineal area of the recipient is scrubbed with a dilute betadine solution and water before
transfer. The infusion pipette containing the embryo is passed through the vagina and into the
cervix following manual dilation of the external cervical os.
 The operator’s hand is covered by a plastic palpation sleeve and a sterile surgical glove.
Before passing the pipette into the uterine body, the operator’s hand is transferred from the
vagina into the rectum to grasp the uterus.
 The uterus is elevated, and the pipette is than passed is then passed into the lumen of the
uterine body. Precaution is taken to keep physical trauma to the endometrium to a minimum.
The contents of the pipette are deposited from the internal cervical and uterine bifurcation.

Surgical

 The most practical method for field use is via a flank in incision. This method is similar to
that used in cattle. Mares are given 250 mg Xylazine IV and 25 mg of acepromazine IM.
 The paralumbar fossa is prepared for aseptic surgery and the incision is infiltrated with 30 to
50 ml of lidocaine. A 15- to 20-cm vertical incision is made, the muscle layers and
peritoneum are bluntly dissected.
 The tip of the uterine horn adjacent to the corpus luteum is exteriorized, and a small puncture
is made into the cutting needle. The embryo is loaded into a 14-gauge large animal Sovereign
catheter or glass pipette in a total volume of approximately 0.5 ml of transfer medium.
 The embryo is usually positioned between two air spaces in the catheter to stabilize its
position.
 The catheter is passed through the uterine puncture, and its contents are deposited into the
uterine lumen. The abdominal wall is then closed in a routine manner.

ET IN SWINES

 The major reason for performing commercial embryo transfer in swine is to prevent and/or
control disease. Although swine producers invariably exploit superior females, this is seldom
the primary reason given for doing embryo transfer in swine, because even the most valuable
donors and embryos are inexpensive compared with costs incurred when some of the common
diseases of swine are introduced into a susceptible herd.
 Export of swine embryos is likely to become another important reason for doing embryo
transfer once long-term storage of swine embryos becomes possible.

Synchronization of Estrus

 There are two methods commonly used to synchronize estrus for embryo transfer purposes.
The first method involves weaning a group of sows on the same day, with estrus occurring 4
to 10 days later. However, if the sows are injected subcutaneously with 500 to 750 IU of

~ 324 ~
 pregnant mare serum gonadotropin (PMSG) at weaning, a high proportion of sows will come
into estrus 4 to 5 days later.
 Another method frequently used to synchronize sows is breeding and then aborting sows
when they are between days 16 and 45 of pregnancy. Sows are aborted with one injection
followed 12 hours later by a second injection of prostaglandin F2α (PGF2α) or one of its
analogues.
 A high proportion of sows come into estrus 4 to 7 days after treatment, and conception rates
are high. Better synchrony can be achieved by injecting 500 to 750 IU of PMSG 12 hours
after the second injection of PGF2α. Often, a group of sows will be synchronized by using
both the weaning and the abortion methods.
 Two other methods are sometimes used to synchronize estrus in swine. Pseudopregnancy may
be induced with daily injections of estrogen preparation on days 11 through 15 of the estrous
cycle. The corpora lutea of pseudopregnancy, which can be maintained for as long as 90 to
120 days, can be induced to regress with PGF2α.
 Most sows return to estrus 4 to 7 days later. Another synchronization method is to inject or to
feed progestogens for about 14 to 16 days. However, most of these progestogens induce
ovarian cysts and are seldom used.

Superovulation

 Sows are usually superovulated with one injection of 1200 to 1500 IU of PMSG at weaning
or 24 hours after the first injection of PGF2α in sows that were synchronized by first being
made pregnant or pesudopregnant.
 For gilts and sows in which embryos are collected on more than two consecutive estrous
cycles, the time of estrus is not controlled, and the animals are not usually superovulated. If
these sows are superovulated, PMSG is given on alternate estrous cycles 4 to 5 days before
the expected onset of estrus.
 As with other species, the superovulatory response is quite variable within and among breeds.
However, the average response for small groups of sows to 1200 to 1500 IU of PMSG ranges
from 30 to 45 ovulations.
 Ovulation rates of 45 or more are not desirable because of the increase in the proportion of
abnormal embryos and the proportion of unfertilized eggs.
 Human chorionic gonadotropin (hCG), which can be used to control the time of ovulation, is
seldom used by embryo transfer specialists. If hCG is used, 500 IU is given 3 to 4 days after
administration of PMSG. Ovulation occurs about 40 to 42 hours after hCG injection.

Insemination

 For optimum conception and fertilization rate, donors should be mated or inseminated every
12 hours throughout estrus. If hCG is used to control the time of ovulation, the most
important inseminations of fresh and frozen semen are those done at 24 and 36 hours,
respectively, after hCG injection.
 The volume of the inseminate should be 50 to 100 ml and contain at least 4 to 5 billion live
spermatozoa.

Embryo collection

Timing

 Swine embryos are usually collected 4 to 6 days after the onset of estrus.
 Four days after the onset of estrus most embryos are at the four-to-eight –cell stage, whereas
on the sixth day after the onset of estrus, most are in the expanded unhatched blastocyst stage.
 Most collections of swine embryos are done 4 days after the onset of estrus because four-to-
eight cell embryo are easily identified and evaluated.

~ 325 ~
  In contrast, morulae and the early blastocysts, which are most frequently collected on day 5,
are more difficult to identify and to distinguish from unfertilized eggs.
 Collection and transfer are seldom done before day 4, not only because embryos which are
usually located in the oviduct of the donor, must be transferred to the oviduct of the recipient,
but also because it is more difficult to deposit embryos in the oviduct than in the uterus.
 The collection of embryos on day 7 or later is not usually done because sows that receive
hatched blastocysts may be less likely to farrow than sows that receive unhatched embryos.

Surgical Collection

 Embryos are collected surgically in a clinic or laboratory. Anesthesia is induced by injecting a

barbiturate into the marginal ear vein and is maintained with halothane using a closed-circuit
anesthesia machine.
 A small mid ventral incision is made to expose one ovary along with the adjacent oviduct and
about 30 cm of the uterine horn. A small incision is made for insertion of a glass cannula on
the antimesometrial side of the uterine horn at about 20 to 25 cm from the uterotubal junction.
 To avoid contaminating the cannula with blood when it is introduced into the uterine horn, it
is important (1) to squeeze the blood vessels on the mesometrial side with thumb and
forefinger while forcing the blunt end of a scalpel handle through the wall of the uterus on the
opposite side and (2) to insert the cannula into the uterine lumen as soon as pressure on the
blood vessels is removed.
 The glass cannula should be about 12 to 15 cm long and 9 to 11 cm in diameter. The end that
goes into the uterine horn should be cut at a 45°angle and flared. The opposite end of the
cannula should have a bend of about 45° located 1 to 2 cm from the end. The glass cannula is
inserted about 2 to 3 cm into the uterine horn and is held in place with a towel clamp.
 To collect the embryos about 40 to 50 ml of the medium warmed to 37° C is placed in a
syringe fitted with a blunt 12 to 14-gauge needle. The needle is inserted into the oviduct, and
the entire medium is flushed into the oviduct, through the uterine horn, out the cannula and
into a Petri dish. After removing the entire flushing medium from the uterine horn, the
incision is closed before repeating the entire procedure in the second uterine horn.
 Following the surgical collection of embryos, swines are especially prone to form adhesions
of the reproductive organs. Therefore, to reduce the possibility of adhesion forming, it is
essential to (1) maintain asepsis throughout the procedure, (2) handle the reproductive organs
gently and only when necessary and (3) keep the exposed reproductive organs moist at all
times with saline or another physiological solution.

Handling, storage and evaluation of embryos

Searching for and Handling Embryos

 The flushings, are examined for embryo with a stereomicroscope. Searches are done at 10 to
20x magnification and the evaluation of embryos at 50x or 70x. Good optics and high
magnification are particularly important for distinguishing morulae and early blastocysts from
unfertilized eggs.
 As embryos are located, they are transferred to culture plates or other dishes that contain fresh
medium warmed to 37ºC. After several rinses in fresh medium, the embryos are stored until
transferred to the recipient.
 Tuberculin syringes fitted with a tom cat catheter or a glass pipette are frequently used to
handle embryos.

Short-term Storage

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  The medium for the flushing procedure can also be used to store embryos in vitro for several
hours. Some of the media used for flushing and storing embryos include Brinster’s solution,
Ham’s F-10 and TCM-199 with bicarbonate.
 Embryos should be stored in fresh medium at 37°C. Although not recommended, it is possible
to obtain acceptable conception rates with embryos stored at room temperature for 2 hours.
 Swine embryos have been cultured for 24 hours without a decrease in embryonic survival
rates after transfer to recipients.

Evaluation of Embryos

 Evaluation of the quality of embryos is done by examining the general morphologic

appearance of the embryo at the time of collection. In general, the cleavage rate of embryos
collected from a donor is quite uniform.
 On days 4, 5 or 6 the stage of development for most normally developing embryos ranges
from four to eight cells, eight cells to morulae and blastocysts, respectively.
 Once embryos pass the eight –cell stage, cells fuse, making it difficult to identify and to count
individual cells. Therefore, considerable experience is required to distinguish 8- to 16-cell
embryos, morulae and early blastocysts from degenerating unfertilized eggs. Because four- to
eight –cell embryos are easily identified and evaluated, most embryo transfer specialists
prefer to collect embryos from donors when the embryos are expected to be at the four-to-
eight-cell stage. However, in a recent study it was shown that sows that received morulae
were more likely to farrow than sows that received four-to-eight –cell embryos.

TRANSFER

 Practical methods for the nonsurgical transfer of swine embryos have not been developed.
Surgical transfers are usually done on the farm rather than in a clinic or a laboratory to reduce
the risk of introducing disease. Anesthesia is induced and maintained by injecting a
barbiturate into the marginal ear vein. The reproductive tract is reached through a mid ventral
incision. Corpora should be examined for appropriate stage of development before embryos
are transferred to the recipient. The uterine horns should also be examined for abnormalities,
especially if gilts are used as recipients.
 Embryo are transferred to the recipient by one of two methods. In one method a fine catheter
or pipette that contains the embryos is passed through a small puncture wound into the lumen
of the uterus. The embryos are deposited wound does not require sutures. Inexperienced
individuals should be especially careful not to deposit the embryos into the endometrium or
the myometrium. Depositing embryos into the wall of the uterus is more easily done in swine
than in bovine or in ovine. However, this complication and hemorrhaging of the puncture
wound, which sometimes occur, can be avoided. This is accomplished by introducing a tom
cat catheter or a piece of rubber tubing, which contains the embryos, into the oviduct. The
distal end of the tubing is held firmly in place while the embryos are flushed into the uterus
with a syringe that is attached to the other end of the tubing.

FACTORS AFFECTING RESULTS

 The day of collection and transfer of embryos may influence results. Best results are obtained
when the donor comes into estrus from 2 days before to 1 day after the recipient. Higher rates
of farrowing may be possible when embryos are collected and transferred within 6 days after
the onset estrus.
 For optimum results, at least 12 embryos of high quality should be transferred to each
recipient. Pregnancy fails to occur if there are too few embryos between days 12 and 17 or if
embryos are not distributed throughout most of the uterine horns.
 As in other species, the time and conditions of in vitro storage also influence results. Until
culture methods improve, swine embryos should be transferred to the recipient as soon as
possible after collection. If embryos must be stored for 4 hours or more before transfer, better
results may be obtained by collecting embryos that are past the eight-cell stage.
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  Lastly, considerable experience with surgical collection of embryos is required to minimize
the possibility of donors forming adhesions of the reproductive tract, which can cause
infertility. Sows are more likely than ewes and cows to form adhesions following the surgical
collection of embryos.

IMMUNOMODULATION FOR ENHANCEMENT OF FECUNDITY

Immunomodulaton in reproductive disorders of livestock is a new area of immunotherapeutics. It is

mainly used to treat inflammatory conditions of the reproductive disorders such as endometritis,
metritis, vaginitis in which the immunity barrier of the host is breached. Immunomodulaton is also
done using plant preparations. The normal uterine defense mechanism is brought about by

The anatomical barriers which include vulva, vestibule and cervix,

The physical barrier which includes the cervical-vaginal mucous.
The contractions of circular and longitudinal uterine muscles that help to propel the microbes
outside.
Chemical barrier which consists epithelial and stromal cells which secrete soluble factors and
cause cell to cell adhesions
Immunological barrier constituted by the cellular immunity comprising of polymorphonuclear
inflammatory cells (PMNs), lymphocytes and humoral immunity by antibodies.

An ideal immunomodulator should meet the following criteria.

 It should be nontoxic for animals even at high doses.

 It should not have teratogenic, carcinogenic or any other side effects.
 It should have a short withdrawal period with low tissue residues. It should not be secreted in
milk or come in eggs.
 It should stimulate both specific as well as paraspecific immune responses in the body.
 It should act as adjuvant when given with vaccines.
 The immunomodulator or its breakdown products should be either inactive or readily
biodegradable in the environment.

Uterine Cellular Immunity

 Polymorphonuclear inflammatory cells (PMNs), blood monocytes and tissue macrophytes are
regarded as ‘professional phagocytes’ in the cellular defenses against pathogenic
microorganisms.
 Phagocytosis involves chemotaxis, adherence and attachment of leucocytes to cell surface
antigens presented by the organism before it is ingested by the phagocyte and finally digested.

Uterine Humoral Immunity

 Immunoglobulin concentrations in uterine secretions reflect both the extent of the endometrial
inflammatory process in the face of microbial challenge.
 Immunoglobulins have been found in bovine uterine secretions and their protective role
against pathogens have been widely reported.

INFLUENCE OF STEROID HORMONES ON UTERINEIMMUNE RESPONSES

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  The cyclical pattern of steroid hormone concentrations, characteristic for different stages of
estrous cycle, regulates potential pathogenicity of micro-organisms that contaminate the
uterus postpartum.
 Steroid hormones influence functional activity of leucocytes migrating into uterus.
o During estrogen phase of the ovarian cycle there is increased mucus production, and
identified PMN activity;
o During the luteal phase, there is reduced endometrial epithelial permeability to
bacteria that delays leukocyte stimulation, and an absence of detoxifying agents in
uterine secretions.
 Disruptions of these mechanisms allow opportunist pathogens, mostly microorganisms found
in posterior gastrointestinal tract and around perineal area.
 Inflammation of endometrium can occur following
o coitus,
o artificial insemination (AI),
o more commonly in cattle after parturition,
o in majority of cattle,1-4 weeks after calving,
 Micro-organisms contaminate the uterine lumen but self-cure usually occurs within 6 weeks
postpartum. In those cows unable to eliminate infection, endometritis may develop
subsequently. Endometritis causes a significant delay in calving to conception intervals.

CAUSATIVE ORGANISMS FOR INFLAMMATION OF BOVINE GENITALIA

 Various Gram-positive and Gram negative aerobes and anaerobes, have been isolated from
the bovine uterus following coitus, Al, or postpartum.
 The bacterium most frequently isolated is Acranobacter pyogenes (formerly known as
Corynebacterium pyogenes). Other bacteria, such as Streptococci spp., Staphlococci spp. and
Escherichia coli have also been cultured and identified with endometritis of varying severity.
 Anaerobes affecting bovine genitalia are Fusobacterium necrophorum and Bacteroides
melaninogenicus.
 Diagnosis is usually done by rectal palpation,vaginal examination, bacterial culture,
endometrial biopsy, endometrial cytological examination and Ultrasound examination of the
genitalia.
 Intrauterine oxygen reductase potential (Eh) can be used to find the degree of bacterial
infection as more Eh potential indicates infection. Moreover, pH in bacterial infection as more
Eh potential indicates infection. Moreover, pH in bacterial infection ranges between 6.9-7.3.
Peripheral blood haptoglobin, which is an acute phase protein synthesized in the liver in
repose to tissue damage has been used as a marker for endometritis , its major function is to
bind free haemoglobin and protect the host from the oxidative activity of haemoglobin.
Another circulating acute phase protein, α1-acid glycoprotein is also screened for detecting
endometritis.

USE OF IMMUNOMODULATORS IN TREATMENT

 Plenty of research asociated with immunomodulators of uterine defence mechanism and their
application as treatments for metritis or endometritis has been carried out in mares.

Hormones

 Hormones commonly used as immunomodulators are

o Prostaglandins
o E.Coli Lipopolysaccharides
o Derivatized Polysaccharides
o Oyster Glycogen
o Leukotriene B4

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Other Agents

 Other agents commonly used as immunomodulators are

o Plant Preparations

Prostaglandin

 Prostaglandin F2α is a naturally occurring luteolytic agent in non-pregnant ruminants.

Oxytocin stimulates uterine secretion of PGF2α which in turn, stimulates oxytocin release
from the corpus luteum; this luteal oxytocin positively stimulates endometrial secretion of
PGF2α. As a result, peripheral blood concentrations of progesterone progressively fall from
day 16 of the estrous cycle until estrus, when they are undetectable; conversely, estradiol
concentrations, rise to reach a peak when the animal shows standing heat. Hence, there are
three reasons for using PFG2α for treatment of endometritis.
 Exogenous prostaglandin therapy administered to cattle presenting a functional corpus luteum
will induce luteolysis and bring the animal into heat, thereby removing the suppressive affect
of progesterone on the uterine defense mechanism or, alternatively, stimulate it through
estrogen causing myometrial contraction which expels debris and micro-organisms that
contaminate the uterine lumen after calving. Moreover, PGF2α may have stimulatory effect
on the phagocytic activity of uterine leucocytes. Hence, the luteolytic action of PGF2α has
been used to treat endometritis in cows where a functional corpus luteum is present.

E.coli Lipopolysaccharides

 E. coli lipopolyaccharides (LPS) are thought to act as a chemo attractant to PMNs. This
increase in numbers of PMNs in the endometrium may help to resolve endometritis in both
cows and mares. A single intrauterine infusion of 100 μg. E. coli LPS at estrus in repeat
breeding cows cleared the bacterial infection from the uterine lumen within one estrous cycle.
Following this treatment, a majority of the repeat breeding cows with turbid vaginal discharge
conceived in a preliminary trial. Similar results have been described following intrauterine
administration of LPS in ewes. When E. coli endotoxin has been similarly administered to
cows 5 days after calving at rates of 5 g/kg body-weight, the toxin was absorbed with
associated transient clinical signs causing enhancement in immune response.
 The contaminating micro-organisms associated with uterine infection postpartum may
themselves secrete endotoxins detectable in peripheral blood, and may interfere with ovarian
function characterized by short cycles in cows. Variation between species in endotoxin
absorption from uterus postpartum may be associated with differences in placentation and the
extent of trophoblast invasion of the endometrium. In species, where there is little clinical or
physiological evidence of endotoxin absorption, an intact endometrium may act as a
cytological barrier to totally or partially exclude the toxin.

Derivatized Polysaccharides

 They are generally high molecular weight dextrans.

 Their action is complex possibly by T or B cell mitogenicity e.g. oyster glycogen.

Oyster Glycogen

 Intrauterine administration of oyster glycogen (OG) caused PMN migration, up to 90% of all
cells identified in uterine secretions being neutrophils.
 Measurable immunoglobin (IgG) concentrations were found in uterine secretions following
administration.

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Leukotriene B4

 They are generally glycoproteins of molecular weight around 20 kDa.

 They up regulate immune response. It is an effective chemo attractant, stimulating
preferential migration of PMNs into the lumen of the bovine uterus.
 A single intrauterine treatment of a 30 mm01/l solution increased the intrauterine leukocyte
count 5-10 times within 24h.
 Granulocyte-macrophage colony stimulating factor (GM-CSF; lymphokines) are highly
effective chemo attractants in mares, but their activity in cattle has not been studied.

Plant Preparation

 The active principles of plants may be carbohydrates, glycosides, tannins, lipids and akaloids.
WHO has also been recommended the promotion of native, practices and conservation and
cultivation of medicinal plants.
 Despite these facts, little attention has been given to describe and explore the traditional
herbal medicines used by the local and tribal communities in specific areas for the control and
treatment of various reproductive disorders in farm animals.

Plants Used in Disorders

 Uterine disorders
o A wide variety of medicinal plants and their preparations are found to be useful in
treatment of reproductive ailments.
o They include Abroma augausta, Aristolochia bracteata, Datura alba, Mytrus
communis, Salvadora species, Saraca indica, and ViI species.
 Anestrum
o Anestrum in cows/buffaloes can be overcome by feeding fenugreek powder. Feeding
of bamboo leaves brings cattle and buffaloes into regular heat. Feeding of leaves of
jute plant (about 2-2.5 kg) brings animal into heat.
o Feeding leaves of Mann tree (approx:15-20 kg) can overcome anestrus condition.
o A mixture of black pepper (10 grains) and Vanghuchi (20-25 gm) is given twice a day
at interval of 6-8 hrs for 1-2 days for treatment of anestrus.
 Decreased Conception Rate
o Feeding 200 gm germinated Bengal gram (Cicer arietnum) soaked overnight to the
animal continuously for one week.
o Along with this pounded leaves and unopened fruit of Yanai (Pedalium murex) may
be given once a day for three days without adding water.
 Retained Placenta
o Bamboo leaves and bark are boiled with paddy husk and fed to cows for expulsion of
placenta.
o Ficus bengalensis is used for treatment of retained placenta. Leaves and twigs of ber
(Zyzphus mauritiana) are collected and burnt. The ash is given to the animal with
water to induce the placenta to drop.
o About 250 g of leaves of jingara used for retention of placenta showed 60% success
rate.
o To prevent abortion farmers feed piece of stem of banana (Musa paradisiacal).After
conception, buffalo is fed 10-15 kg; cow is fed 5-10 kg pieces of stem of banana. It is
fed for five times over a period of 2-3 days. It helps to reduce internal heat and
improves health. This practice has been in use for the last 30-40 years.
 Uterine Infection
o The immunomodulatory property of Aristolochia indica (Ischamur) can be proved an
aid in preventing the uterine infection by augmenting local immune system.
Approximately 100 gm of root or bark of Convolvus micrphyllus (roots) powdered

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 and mixed with 300 ml of water and boiled. This concoction is filtered and then
cooled. This is given to affected animals once a day for 3 days.
 Equiman
o This is an immunomodulating phytopreparation having its affect on bovine immune
cells. Equimen is a fixed combination of Echinaces purpurea, Thuja occidentalis and
elemental phosphorous in different concentration. The preparation reduces the
spontaneously generated reactive oxygen species (ROS) by neutrophils.
o Phytotherapy has been followed in the treatment of animals from thousand of years
since ancient time. Plant based drugs (natural drugs) may be used directly i.e. they
may be collected, dried and used as a therapeutic agents (crude drugs) or their active
principles, separated by various chemical process which are employed as medicines.

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