Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India

pp 89-98

Measures to reduce equine infertility G.N.Purohit

Department of Animal Reproduction Gynaecology and Obstetrics College of Veterinary and Animal Science, Rajasthan Agricultural Univesity Bikaner (Rajasthan).
Mare infertility can be frustrating because time and money restrictions are frequently imposed on the veterinarian. The approach of she wont get pregnant unless she is bred (repeatedly) may not always be true, because surgical correction of an anatomical defect, a period of sexual rest to resolve uterine inflammation, or better semen quality may be what is needed for the mare to conceive. Information on these issues can often be gleaned from the mares reproductive history. In some cases, a physical and reproductive examination (perineal-conformation examination, rectal and ultrasonographic exam, vaginal and cervical evaluation) is all that is needed to make a diagnosis. In other cases, the cause of the infertility might not be determined with a single examination. Reproduction is a dynamic process, and subtle abnormalities may only be identified during specific times of the estrous cycle. Guidelines for conducting a breeding soundness exam, treatment protocols, and management of specific problems are provided for reference. Basic Reproductive Examination The basic reproductive examination described (LeBlanc, 2006) for an infertile mare includes obtaining a history, conducting a physical examination, evaluating the perineal conformation, performing a rectal/ultrasonographic exam of the reproductive tract, performing a vaginal speculum examination, and performing a digital examination of the cervix. Ancillary aids include uterine cytology and culture, endometrial biopsy, and endoscopic examination of the reproductive tract. The latter diagnostics are chosen on a caseby-case basis. The importance of obtaining the mares performance and reproductive history (as completely as possible) cannot be overstated. Often times, the repeatability of a specific clinical finding gives clues to the primary cause of the infertility. If the case is a referral, discussing previous diagnostics and treatments with the referring veterinarian and obtaining his or her insight provides valuable information on a possible direction to pursue. It is imperative that all findings are recorded. They may provide clues as to what to do next if the mare returns a second time, because she did not become pregnant after the first examination. A one-page check-off sheet that includes space after each category 1

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 (history, perineal conformation, physical exam, etc.) to write ones findings will ensure that a complete and thorough examination is performed each time. Before examination of the reproductive tract, the general physical condition of the mare and farm management should be appraised. Systemic problems, poor body condition, laminitis, or pain may adversely affect fertility. Mares with laminitis may accumulate fluid within the uterine lumen because they have limited mobility. Abnormal estrous-cycle length or lack of estrous cycles during the mares natural cyclic season may be associated with pain, systemic endocrine abnormalities such as Cushings disease, or uterine infection. Feeding or management changes, dental care, or parasite control may be indicated. Race mares that are brought from the track to the breeding farm in late winter may not adapt to the group dynamics of a broodmare band because they were stalled separately during their racing career. Placing them in a small paddock with another mare may improve their ability to cope. Mares housed in northern climates require more calories in January, February, and March, because they expend more energy in the cold. Mares that lose weight in the early spring tend not to cycle properly even when placed under 14.5 h of artificial light in December. Most uterine infections are caused by bacteria, or yeast ascen-ding through the vagina. Conformational characteristics that correlate with high fertility include a long sloping hip, a sacral iliac joint located dorsal to the tail setting, and a vulva that is 10 off of the vertical plane. Mares that pool fluid after breeding often are flat over their croup with a tail setting level with the sacral iliac joint and a sunken anus. The perineum is best evaluated during estrus when relaxation and elongation of the vulvar lips are greatest. The integrity of the vulvar lips, the angulation of the vulva, and the location of the dorsal commissure of the vulva in relation to the pelvis need to be evaluated. The vulvar lips should meet evenly and appear full and firm. They function as a seal against external contamination of the uterus. The vulvar lips should lie vertically with a cranial to caudal slope of 10 from the vertical plane. The dorsal commissure of the vulva should be 4 cm dorsal to the pelvic floor. If it is 4 cm, the vulva is pre-disposed to cranialventral rotation, which can lead to pneumovagina and contamination. The lips of the vulva should be parted to determine the integrity of the vestibulo-vaginal sphincter. An intact sphincter is present in a mare when the labia can be spread slightly without air entering the cranial vagina. By parting the labia, the color and moisture of the vestibular walls can be assessed. Estrus produces a glistening pink to slightly red mucosa. Anestrus generally is reflected by a pale, dry mucosa; dark red or muddy color suggests inflammation. A white, tacky mucosa indicates progesterone dominance. The perineal body may be defective in older, pluriparous mares. The defect most likely occurs

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 from repeated foalings and poor reproductive conformation, and it results in a sunken anus and lack of tissue between the rectum and the vagina. The integrity of the perineal body can be assessed by placing one finger into the rectum (usually the second finger) and the thumb into the vestibule. A vestibuloplasty or deep caslicks is helpful in decreasing contamination of the anterior reproductive tract, especially if the vestibule-vaginal seal is lost. Rectal/Ultrasonographic Examination of the Reproductive Tract Repeated ultrasonographic evaluation of the reproductive tract during the estrus cycle is routine in many reproductive practices. Correlations between uterine edema, cervical dilation, and follicular dynamics can be made, and inconsistencies such as excessive edema early in estrus (lack of cervical dilation or endometritis) or free intra-luminal fluid (Severe uterine-drainage problem or endometritis) can be identified. With the advent of ultrasonography, veterinarians can more closely time breeding with ovulation, can determine ovulation within a matter of hours, and can identify pathologies such as anovulatory follicles, granulosa cell tumors, intra-luminal fluid, and uterine cysts. However, ultrasonography does not take the place of good manual skills. The tone and consistency of the uterus and follicles and the length and width of the cervix is identified by a veterinarian skilled in rectal palpation. Inconsistencies such as a turgid, firm uterus during estrus or a narrow, long cervix in a mare on the day of ovulation indicate problems such as endometritis and post-breeding endometritis, respectively. A grading system for uterine edema, fluid accumulation, and follicular dynamics should be developed in each practice. By doing so, veterinarians can follow up on each others cases, if needed. Reviewing the ultrasonographic findings over a number of cycles in an infertile mare can provide valuable information on possible causes of the infertility. Vaginal-Speculum and Digital-Cervix Examination Vaginal-speculum examination is an integral part of the breedingsoundness examination. Findings include, but are not limited to, determining the integrity of the vestibulo-vaginal sphincter, identifying the presence of abnormal fluids in the vaginal vault such as pus or urine, and finding a discrepancy between cervical relaxation and stage of estrous cycle. Mares with endometritis or maiden mares may have a closed cervix that is located high on the vaginal wall during estrus. Adhesions of the external os of the cervix to the vaginal fornix may be identified, especially if the examination is conducted during diestrus. The cervix will be covered below. It must be examined visually and manually in infertile mares, because it is frequently compromised and the cause of the problem. Ancillary Aids Many detailed texts are available on the proper techniques of obtaining and interpreting uterine cytology, culture, and biopsies (Pycock, 2000; 3

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 Blanchard et al., 2003; LeBlanc et al., 2003). In addition, the correlation between uterine cytology, uterine culture, and 28-day pregnancies rates can be reviewed in last years proceedings (Riddle et al., 2005) It is recommended (LeBlanc, 2006) that both a uterine cytology and culture be taken in all mares that are infertile, because neither diagnostic technique alone identifies all mares with endometritis. Obtaining a uterine swab from the uterine body during estrus may not be the most accurate method for evaluating the bacterial status of the uterus, because the swab only comes in contact with a small area of endometrium. Performing a small volume uterine lavage seems to identify a higher number of sub-clinical infections, most likely because the fluid infused into the uterus comes in contact with the majority of the endometrium. Nowadays, the technique is suggested to be used in all mares presented for infertility (LeBlanc, 2006). Mares with potential problems are identified at stall side, because efflux collected from these mares commonly is cloudy or has strains of articulate matter. The technique is simple. Sixty milliliters of saline is infused into the uterus through a sterile Bivona catheter that has been placed aseptically into the uterus. The veterinarian then manipulates the uterus rectally to move the fluid from horn to horn. The efflux is drained in a sterile 50-ml conical tube that an assistant is holding. Either the pellet at the bottom of the tube is cultured after it is allowed to settle for 12 h or the fluid is centrifuged for 10 min and the pellet cultured. Endometrial biopsies provide valuable information on the integrity of the endometrium; however, endometrial biopsies have lost favor as a diagnostic technique, because the histological interpretation provided rarely changes the management strategy. The problem, as seen LeBlanc, (2006), is in the classification system used, which is 28 yr old (Keeney, 1978). Additional histological lesions such as angeosis, lymphatic lacunae, severe edema, mitotic figures in endometrial glands, and mucus on the endometrial surface need to be incorporated into the classification. The importance of these lesions in reproductive function has only come to the forefront in the last 510 yr and therefore, is not addressed by the current classification system used in the United States. Endoscopy is a helpful tool in identifying mares with uterine adhesions, foreign material in the uterine lumen, endometrial cysts, fetal remnants, and fungal plaques. It is reserved for select cases.

Causes of Infertility (Subfertility)

The basic reproductive failures in mares have been considered to be of three types a) a mare does not evidence estrus cyclicity b) a mare cycles normally estrus but does not conceive and c) a mare cycles normally and conceive but then suffers from early or late embryonic deaths. Infertility has been described in detail in classical texts (Ricketts et al., 2006; Samper et al., 2007) but, for the ease of 4

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 understanding the causes of infertility mentioned previously (Purohit, 1997) are classified as follows: 1. Problems because of the transition period 2. Irregularities of the estrous cycle 3. Genital Infections and abnormalities 4. Problems of management Management of the Transition Period The physiologic breeding season of horses extends from April to October in the Northern Hemisphere. Mares are polyestrous in that they exhibit repeated estrous cycles during the breeding season (Sharp and Davis, 1993). In the winter months, 80% or more of mares enter a prolonged anovulatory phase referred to as winter anestrus. The onset of the transition period may be defined as the first significant increase in follicular diameter greater than that noted during deep anestrus. From a practical standpoint, transition begins in horse mares when a follicle first reaches 2025 mm in diameter. In pony mares, the onset of transition occurs when the diameter of the largest follicle reaches 21 mm in anovulatory mares (Donasdeu and Ginther, 2002). The term late transition may be used when one or more follicles are 30 mm in diameter in horse mares. Mares may exhibit multiple waves of follicular growth and regression during the transition period (Donadeu and Ginther, 2002; Davis et al, 1987). The total number of follicles present on each ovary increases during the transition, and ultrasound examination classically reveals a grapelike cluster of small- to medium-sized follicles. During transition mares show irregular periods of sexual receptivity, prolonged heats of 10-20 days, split heats, and heats without ovulation. The transition period may last for 5070 days or more prior to the first ovulation of the season. Once a mare has ovulated, she will generally continue to cycle at regular intervals. Behaviorally, transitional mares may display prolonged or irregular periods of estrus. This is likely caused by an extreme sensitivity to the low levels of estrogens being produced by developing follicles. The mean calendar date of the first spontaneous ovulation of the year in anestrous mares maintained under ambient light conditions in North America ranges from the middle of March to early May. Exposure of mares in deep anestrus to a stimulatory artificial photoperiod has been used for decades to advance the first ovulation of the year. Mares maintained under lights still go through a transition period of relatively normal length, but the transition begins earlier in the year. In most instances, the duration from onset of adequate light exposure to ovulation is 6070 days(Guillaume et al., 2000). General recommendations for light treatment mentioned recently (McCue, 2006) are: 1. a fixed-length period of light exposure for 14.516 h, allowing 7.59 h of darkness. It is not necessary to increase duration of light exposure incrementally. 5

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 2. Alternatively, adding 2.56 h of additional light exposure after dusk is effective. Light treatment should be initiated during the first week of December if it is desirable to begin breeding mares in February (Guillaume et al., 2000). Minimum light intensity of 10 foot-candles should be used (100 lux = ~ 10 foot-candles). A 100- to 200-W incandescent bulb in a 12 X12-ft box stall should be sufficient. A number of hormone therapies have been used in attempts to stimulate follicular development and advance the first ovulation of the year in seasonally anestrous mares. These include gonadotropin-releasing hormone (GnRH) agonists, gonadotropins, progestins, and dopamine antagonists. Administration of low doses of native GnRH and GnRH agonists (i.e., buserelin and deslorelin) and equine follicle-stimulating hormone (eFSH) have been used successfully to induce follicular development in transitional mares. Higher doses of GnRH agonists (i.e., deslorelin) and human chorionic gonadotropin will induce ovulation of large follicles (>35 mm in diameter) in late transition. Dopamine antagonists, such as domperidone and sulpiride, may not be effective under all management conditions. Success rate is likely to be higher in transitional mares and mares maintained indoors under a stimulatory artificial photoperiod. The most current proposed mechanism (Duchamp and Daels, 2002) by which dopamine antagonists may stimulate follicular development is as follows: 1. Dopamine antagonist treatment results in increased plasma prolactin levels. 2. Prolactin acts directly on the ovary to stimulate expression of gonadotropin receptors. 3. Pituitary gonadotropin (follicle-stimulating hormone [FSH] and luteinizing hormone [LH]) secretion is not altered by dopamine antagonist therapy, but treated mares are more sensitive to endogenous gonadotropins because of increased numbers of gonadotropin receptors. Conflicting reports exist as to the efficacy of progesterone or synthetic progestins in advancing the first ovulation of the year. However, a majority of controlled research studies indicate that progesterone therapy (Sharp and Davis, 1993) will not consistently advance the first ovulation when administered to mares in deep seasonal anestrus or early transition, (Donadeu and Ginther, 2002) may synchronize the return to estrus and advance the first ovulation in late transitional mares, and (Davis et al, 1987) is effective

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 in suppressing the prolonged and

(Table 1 replicated from McCue, 2006)

often, irregular periods of estrus in transitional mares (Allen et al., 1980; Turner and Irvine, 1993). Initiation of any treatment to mares in deep winter anestrus is less likely to be successful and is associated with a higher risk of return to anestrus after conclusion of therapy than treatment when mares are in transition. Dosage regimens described by McCue, (2006) are listed in Table 1. Some of the other procedures described recently ( Samper et al., 2007) document the use of progestagen vaginal implants. This can be done by the use of progestagen devices like PRID, CIDR-B and Cu-Mate. These are applied intravaginally aand kept for 14-20 days. A mare resumes estrus shortly after withdrawing the device and ovulates within 4-8 days of removal of the device.

Irregularities of the estrous cycle

The normal estrous cycle in the mare is 21 to 22 days long, as defined by the intervals between ovulation. The duration of estrus, however can vary, so the most consistent period in the estrous cycle is the length of diestrus. Diestrus has a fairly consistent 14 day (hormonal) or 15 day (behavioral) duration. Estrous behavior in the mare is a display to a stallion. Without a stallion, assessing behavioral estrus is difficult, if not impossible. Regular exposure allows the mare to familiarize herself with the stallion, and the observer to better read the individual mare. Monday, Wednesday, and Friday provides a convenient teasing schedule for most situations. Sometimes, it may be necessary to tease every day. In the absence of a stallion, mares may rarely show estrus to geldings or exceptionally to people. In all cases, regular observation and recording is essential. During summer, the modal duration of the estrous cycle is 21 to 22 days, but it can vary within a range of 19 to 24 days. In donkeys (E. asinus) the estrous cycle is usually about 24 days long, mainly because their luteal phases are about 3 days longer than in mares.

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 Estrus usually lasts for 5 or 6 days in both mares and Jennies. Prolonged Estrus: As already mentioned earlier prolonged periods of estrus are charachteristic findings during the transition period when mares exhibit prolonged estrus or diestrus. Many a times mares evidence estrus without ovulation during the transition period(Fig1)

Fig.1 The progesterone profiles in mares during the transition periods

Prolonged Diestrus (Psuedopregnancy) The two terms had been used in the past the two terms had been used synonymously but more recently Bergfelt and Adams( 2007) mention that the term psuedopregnancy should be used in mares that have a prolonged interestus interval, a tense uterine tone at palpation and a confirmed embryonic loss. Idiopathic persistence of the corpus luteum may be used to describe prolonged diestrus. The therapy for both the conditions eseentially is the same. Administration of a prostaglandin is all that is required (Ricketts et al., 2006). Silent Heat Mares that on examination appear to be in physiologic estrus, but do not respond with any positive signs of heat when teased and reject natural mating are said to be in silent heat. Gynaecologic examination reveals a pink moist, relaxed cervix; palpation and sonography reveals a mature ovarian follicle. The problem appears to be purely a psychologic one and one must try different approaches such as changing the surroundings, teaser or the stallion. (Newcombe, 2007). Other authors suggest Tranquillizaation, the administration of exogenous estradiol benzoate or artificial insemination (if appropriate) (Ricketts et al., 2006). Shortened disestrus Endometritis causes premature endogenous endometrial prostaglandin secretion resulting in premature luteolysis, shortened diestrus (< 12 days) and premature return to estrus. gynaecologic examinations reveal an inflamed, moist, relaxed cervix, sometimes but not always with purulent discharge. After confirmation of uterine infection with cytologic 8

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 and bacteriologic examinations, uterine antibiotic therapy or uterine lavage and correction of predisposing factors like pnuemovagina will usually be followed by normal diestrus length. Ovulation Failure Failure of ovulation during the breeding season represents a significant cause of reproductive inefficiency in the mare and may be responsible for significant economic loss. The incidence of ovulation failure during the physiologic breeding season has been reported to range from 3.1% to 8.2 %.( McCue and Squires, 2002). Post-partum mares and Jennies (Dadarwal et al., 2004) may exhibit physiologic ovulation failure. A majority of mares develop follicles and ovulate early in the post-partum period (foal heat), and they continue to cycle if they do not become pregnant at a foal-heat breeding. Alternatively, a foal-heat ovulation may be followed by a variable period of anestrus or anovulation until the mare resumes normal cyclic activity. Finally, some mares may have no significant follicular development or may exhibit moderate to substantial follicular development without ovulation during the postpartum period. Mares in the latter groups may remain anestrus or anovulatory for weeks or months before cyclic ovarian activity is initiated. A majority of mares that do not cycle after giving birth are mares that foal early in the year. Consequently, it may be difficult to distinguish between post-partum anestrus caused by a short ambient photoperiod and anestrus caused by the effects of lactation. In general, failure to ovulate post-partum is more likely to be attributed to seasonal affects than lactation affects (Loy, 1980; Palmer and Driancourt, 1983). However, some mares do not develop follicles in the post-partum period or become anestrus following a foalheat ovulation, and they will exhibit rapid follicular development and estrus as soon as the foal is weaned (Ginther, 1992; Nagy et al., 1998). The incidence of lactationassociated anestrus in mares has been reported to be 2174% (Palmer and Driancourt, 1993). In contrast, other investigators have reported that suckling had no effect on postpartum ovarian activity (Nagy et al., 1998). Poor body condition in late gestation and the early post-partum period may also contribute to poor reproductive performance. The effects of inadequate nutrition and poor body condition may be manifested in delayed return to reproductive cyclicity post-partum, reduced pregnancy rates, and increased embryo loss rates (Henneke et al., 1984). The phenomenon of lactational anestrus may, in fact, represent the combined effects of season, body condition, and lactation (Ginther, 1992). Maintenance of late-term pregnant mares due to foal between January and March (Northern Hemisphere) under a stimulatory artificial photoperiod for the last 23 mo of pregnancy may be beneficial. Pregnant mares housed under lights have been reported to foal 10 days earlier than mares not maintained under lights (Hodge et al., 1982). They are also more likely to have a foal-heat ovulation, have continued estrous cycles, and ovulate earlier in the post-partum period (Koskinen et al., 1991). Ovulation failure occasionally occurs during the physiologic breeding season. Anovulatory follicles may be quite large (515 cm in diameter), persist for up to 2 mo, and result in abnormal estrous behavior and prolonged interovulatory intervals (Meyers, 1995) Specific causes of ovulation failure in the mare are not known; however, it may be caused by pituitary gonadotropin stimulation insufficient to induce ovulation (McKinnon, 1997), insufficient estrogen production from the follicle itself, or hemorrhage into the lumen of the pre-

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 ovulatory follicle. Mares may develop anovulatory follicles without prior exposure to exogenous hormones (Loy, 1980). Anovulatory follicles were reported in a recent study to occur in 8.2% of equine estrous cycles (McCue, 2002). The incidence of anovulatory follicles increases with age. Mares 1620 yrs old were noted to form anovulatory follicles during 13.1% of estrous cycles during the physiologic breeding season. A high percentage (43.5%) of mares that developed anovulatory follicles experienced subsequent estrous cycles with anovulatory follicle formation during the same breeding season. The interval between actual ovulations for mares that developed anovulatory follicles was noted to be 38.5 days. It is often difficult to determine beforehand if a dominant follicle in an estrual mare will fail to ovulate. Formation of an anovulatory follicle is usually preceded by development of normal endometrial folds or edema. Initial growth patterns of follicles destined to become anovulatory are usually within normal limits, and the first indication of a problem is typically detection of echogenic particles within the follicular fluid during ultrasound examination. Anovulatory follicles may contain blood and have, consequently, also been called hemorrhagic anovulatory follicles. Hemorrhage can be detected ultrasonically as scattered free-floating echogenic spots within the follicular fluid. The follicular fluid may form a gelatinous, hemorrhagic mass within the follicular lumen. Ultrasonographically, hemorrhagic follicles may contain echogenic fibrous bands or strands traversing the follicular lumen. A progression from echogenic particles and strands to complete infiltration of the follicular lumen with echogenic material is commonly observed. In other instances, the only ultrasonographic sign observed during development of an anovulatory follicle is a thickening of the follicular wall. A majority of anovulatory follicles eventually become luteinized (85.7%), while some remain as follicular (non-luteal) structures (14.3%). Progesterone levels may be used to determine the luteal status of anovulatory follicles (Loy, 1980). Mares with anovulatory follicles containing a highly echogenic lumen invariably have elevated progesterone levels. Administration of prostaglandins will result in the destruction of the luteal cells in mares with luteinized anovulatory follicles, a rapid decline in serum progesterone levels, and a return to estrus. Prostaglandin treatment has no apparent effect on nonluteinized anovulatory follicles. Fortunately, a majority of non-luteinized anovulatory follicles will spontaneously regress in 14 wks. Administration of human chorionic gonadotropin or the GnRH agonist deslorelin is generally not effective in inducing ovulation or luteinization of a follicular-type anovulatory follicle. Recently the human C-terminal decepeptide kisspeptin-10 (10 mg dissolved in saline given i.v when the follicle was 33 mm) has been described to be effective in inducing ovulation in Welsh pony mares (Briant et al., 2006). Likewise, recombinant equine LH at a dose of 750 g was found to be as effective as hCG in inducing ovulation in mares and prevented the problem of antibody formation, common with the repeated use of hCG (Niswender et al., 2006).

Genital Infections and Abnormalities

Genital infections and abnormalities constitute one of the major shares for infertility in the equine species. However, Ricketts and Troedsson (2007) consider that true infertility and epidemic subfertility (e.g., epidemic venereal infections or viral pregnancy failures) are uncommon in mares. It is beyond the scope of this manuscript to describe all the genital infections or abnormalities and the reader can refer standard texts. However, the more common and problematic ones are listed below Congenital abnormalities 10

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 The most chromosome abnormalities seen in phenotypic females are the 63XO or 64XY genotype, or a variety of combination of mosaicism. The most common clinical presentations are persistent anestrus, non-cyclic estrus or conception failure. Gynaecologic examinations reveal persistently small and non functional ovaries(less than 1 cm) and a small uterus. The diagnosis is confirmed by karyotyping and affected mares are permanently sterile. Ovarian and Oviductal Abnormalities Diagnosis of disorders involving the oviduct, alongwith associated fimbrial disorders should be one of the last procedures employed to diagnose infertility in the mare and involves the use of specialized techniques like hysteroscopy. The equine oviduct has a well developed muscularis layer which is a sphinchter like structure that prevents the entry of instruments from the utero-tubal junction. Oviductal disorders are more prevalent in older mares (Samper et. al., 2007). Three dimensional internal structure microscopy has been recently described for visualization of internal ovarian structures during the estrous of mares (Hirano et al., 2006). Likewise the inflammatory lesions in the mares oviduct have been reported recently (Fiala et al., 2006). Age related ovarian dysfunction is common in the mare and currently no treatment options are available. Exogenous administration of anabolic steroids in low doses can result in stallion like behaviour and higher doses can result into inhibition of inhibition of ovarian activity, and hence they are contraindicated in mares and fillies intended for breeding. Ovarian cysts are uncommon and difficult to be described for the mare. Many large anovulatory follicles may ovulate at later time. Ovulation fossa inclusion cysts sometimes occur in older mares where multiple small unovulated follicles block the progression and ovulation of other follicles into and through the ovulation fossa. Equine cushings disease (ECD): This is a disease shown in mares with hypertrophy, hyperplasia or adenoma formation in the pars intermedia of the pituitary. The problem is more prevalent in mares above 20 years of age and mares evidence abnormal and reduced estrus activity and reduced fertility. Other signs include hirsutism and abnormal hair coat, polyuria, polydipsia. Diagnosis involves measurement of cortisol and ACTH which are elevated. The therapy involves the administration of pergolide mesylate 0.5 to 2.0 mg every 24h or cyproheptadine 0.25 mg/Kg every 24h given in the morning (Samper et al., 2007). Ovarian Tumors The most common ovarian tumor is Granulosa Cell Tumor (GCT). GCTs are almost always unilateral, slow growing, and benign. An examination of the affected ovary by transrectal ultrasonography often reveals a multicystic or honeycombed structure, but the tumor may also present as a solid mass or as a single large cyst (Hinrichs and Hunt, 1990). The contralateral ovary is usually small and inactive, although Mares with a GCT on one ovary and a functional contralateral ovary have been reported (McCue et al., 1991). Behavioral abnormalities such as prolonged anestrus, aggressive or stallion-like behavior, and persistent estrus or nymphomania may be expressed in affected mares. GCTs are hormonally active, and clinical diagnostic assays for the detection of a GCT include the measurement of inhibin, testosterone, and progesterone.

11

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 Inhibin is elevated in >90% of the mares with a GCT (McCue, 1992). GCTs are usually surgically removed if the tumor affects follicular development on the contralateral ovary, causes behavioral abnormalities, or is a source of colic. Surgical approaches for tumor removal include colpotomy, flank and ventral midline laparotomy, paramedian laparotomy, and laparoscopy. Ovulation from the remaining ovary will occur after 68 months after tumor removal (Samper et al., 2007). Other tumors described include Dysgerminoma which is metastatic, teratoma or serous cysadenoma. Uterine Infections and abnormalities Venereal disease: The commonly occurring venereal infections in mares include dourine (Trypanosoma equiperdum), equine coital exanthema (Equine Herpes virus EHV3), contagious equine metritis CEM (due to Tayllorella equigenitalis), and certain venereal infections caused by some strains of Psuedomonas and Kleibsiella. The diagnosis of these diseases depends upon the culture of endometrial swabs and swabs from medial clitoral sinuses and the presence of specific lesions as shown in dourine and EHV3. Therapies for these conditions involve local and parentral therapy described elsewhere (Ricketts et al., 2006). Endometritis is by far the commonest clinical entity affecting fertility of broodmares. Other problems include uterine cycts, adhesions and fetal mummification (Samper et al., 2007). Endometritis could be sexually transmitted like CEM, chronic infectious (because of microbes like Streptococcus zoopidemicus, Salmonella, Psuedomonas, Klebsiella, Proteus, E Coli, Shigella and fungi like Candida, Aspergillus), chronic degenerative (endometriosis) or mating induced (commonly known as Persistent Mating Induced Endometritis; PMIE, and is a common cause of uterine fluid accumulation post breeding and infertility). The cause of chronic endometritis often lies not in the uterus but elsewhere (poor vulvar or perineal conformation or a cervical problem) their therapy therefore should begin with repair reconstruction of these conditions and not directly with antibiotic infusions in the uterus. Repeated and prolonged indiscriminate use of intrauterine antibiotics may in fact result in development of chronic endometritis. The diagnosis may involve speculum examination and a uterine cytology. A endometrial culture when taken properly may be helpful in deciding the type of antibiotic to be used intra-uterine. The specific benefits and prerequisites for the use of intrauterine placement of antibiotics has been suggested recently (Causey, 2007) and is mentioned in Table 2. Table 2 Antibiotics for treatment of Bacterial uterine infections in the mare. Drug Amikacin sulfate Ampicillin Dose 2g 3g Comments Buffer with equal volume of 7.5% sodium carbonate Use soluble preparation 12

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 Carbenicillin Ceftiofur Gentamicin sulfate Kanamycin sulfate Neomycin Penicillin G Polymyxin B Ticarcillin Ticarcillin/clvula nic acid 6g 1g 1-2 g 1-2 g 4g 5 million units 1 million units 6g 6g/200mg Some Psuedomonas are sensitive Broad spectrum probable including the anaerobe B. fragilis Gram negative spectrum; buffer with equal volume of 7.5% sodium carbonate Most E.Coli are sensitive E.Coli and some Klebsiella are sensitive Use sodium or potassium salts (soluble). Preferred for Streptococci Lack of clinical response often seen Broad spectrum Broad spectrum

Dilute povidone-iodine (0.05%) mixed with saline can be used but higher concentrations of iodine can be hazardous to the equine uterus. Large volume uterine lavage using isotonic saline or ringers lactate is suggested in some cases to clear the uterus. The lavage should be followed by administration of 10-15 IU of intravenous ocytocin to clear the uterus. Fungal endometritis is common in older mares and mares treated repeatedly for bacterial endometritis with antibiotics. Such endometritis resolves with great difficulty and some medications like Clotrimazole 500 to 700 mg, Amphotericin B; 100 to 200 mg, Nystatin 0.5 to 2.5 million units or Fluconazole 100 mg are suggested for intrauterine infusion for 7-12 days (Dascanio, 2007). Persistent Mating Induced Endometritis A transient inflammatory response in the endometrium is an inevitable consequence of mating by either natural service or by artificial insemination, and it is caused primarily by the exposure of the uterus to spermatozoa. (Troedsson, 1995). The inflammatory response is characterized by an influx of neutrophils, immunoglobulins, complement, and other factors into the uterine lumen. Neutrophil numbers are highest 812 h post-mating, and the majority of reproductively normal mares are capable of eliminating the inflammation within 2436 h. The intensity of the inflammatory response may be increased in mares inseminated with frozen-thawed spermatozoa. This may be due, in part, to the limited amount of seminal plasma present in frozen semen. A persistent mating-induced endometritis (PMIE) may develop in some mares. Mares prone to PMIE are usually older (i.e., >12 yr of age), pluriparous, and may have poor perineal conformation. The primary cause of PMIE is impaired or

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Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 delayed uterine clearance due to a defect in uterine muscular contractility. Inadequate cervical relaxation, poor perineal conformation, impaired uterine lymphatic drainage, and endometrial vascular degeneration may also contribute to the problem. Management of mating-induced endometritis is aimed at limiting the severity and duration of the inflammatory response and clearing the uterus of fluid, inflammatory by-products, and bacteria. Treatment strategies may include: 1. limiting the number of breedings or inseminations (i.e., one mating, if possible). 2. Using artificial insemination (if permitted by breed regulations) to reduce the amount of bacteria and debris introduced into the uterus. 3. Lavaging the uterine lumen post-mating to eliminate dead spermatozoa, inflammatory cells, fluid, and debris. An initial lavage may be performed 48 h after mating without adversely affecting pregnancy rates. A total of 14 L (or more) of sterile saline or lactated Ringers solution may be required. The lavage procedure is repeated until the uterine effluent is clear. Early treatment is aimed at interrupting and/or limiting the inflammatory response. Delaying lavage treatment until after ovulation may allow time for the inflammatory response to reach peak levels. 4. Administering oxytocin (1020 IU) intravenously, subcutaneously, or intramuscularly beginning 48 hrs after mating to stimulate uterine contractions and promote physical clearance of fluid and inflammatory by-products. Oxytocin does not appear to adversely affect the transport and function of gametes within the oviducts and does not adversely affect fertility. Oxytocin causes uterine contractions for 3050 mins, and therapy may need to be repeated multiple times per day for several days in mares with a history of fluid retention. Prostaglandins (i.e., cloprostenol, 250 g, IM) may be administered instead of oxytocin to promote uterine contractions if given before ovulation. Uterine contractions are stimulated for 45 hrs after prostaglandin administration. Administration of prostaglandins after ovulation may adversely affect formation and function of the corpus luteum. Low doses of dinoprost (2.5 mg; 0.5 ml) have been described for luteolysis in the mare with equal efficacy (Barker et al., 2006) 5. Infusing antibiotics into the uterine lumen may be indicated in conjunction with uterine lavage and oxytocin treatment if a bacterial infection is suspected. Systemic antibiotics are not routinely administered for uterine infections, but they are occasionally recommended for refractory or recurrent infections. 6. Using the Caslicks procedure for mares with poor perineal conformation or poor muscular tone to the vulva prevents aspiration of air and fecal material.

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Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 Pyometra There are several possible etiologies for pyometra in the mare. Cervical adhesions or malfunction with failure to drain contents are often implicated. Reduced endometrial resistance or other unknown factors may also be involved. A variety of organisms can be involved and in some cases a negative culture may be obtained. There is usually no sign of systemic illness. In some cases there may be a discharge. On rectal exam, a fluid filled uterus is found which must be differentiated from pregnancy. The majority of affected mares cycle normally. In some cases, with severe endometrial damage, PGF release may not occur so that luteal function is prolonged. In other cases, the mare may cycle at shortened intervals due to endometritis and PGF release. In all cases, however, the prognosis for future fertility is poor. The damage to the uterus is most severe in long standing cases or those with a closed cervix. In these cases the endoetrium may be replaced by granulation tissue. In less severe cases endometritis with atrophy and fibrosis of the endometrium is found. If the pyometra is cleared up, it will likely reoccur. Endometriosis and Uterine Cysts Endometriosis refers to wide range of degenerative changes seen in the endometrium of mares on histopathologic examination. The condition is degenerative and lacks the typical featues of active inflammation. Endometriosis is a feature of uterine wear and tear that develops during a mares advancing reproductive lifetime.. The major changes observed are fibrosis, lymphatic stasis, uterine sacculation, transluminal adhesions and glandular cysts (Hoffmann et al., 2009). Reliable and specific treatments have not been described that can permanently alter or reverse the course of endometriosis. Uterine cysts are smaller (5-10mm) or sometimes several centimeters in diameter. They are diagnosed by rectal palpation when they are large or multiple or by hysteroscopy. Such cysts may prevent a pregnancy or result into abortion when their number is large or size greater. Therapies of such cysts involve mechanical curettage (Ricketts, 1985), manual rupture per vaginum, fine needle aspiration (Brook and Frankel, 1987) and endoscopically guided photoablation using laser technology (Ley et al., 2002). However, the treatments may or may not offer attaining optimal fertility post therapy. Cervical Problems Proper cervical function is needed for a successful pregnancy. Trauma such as a cervical tear, fibrotic changes to the cervical body, or adhesions to the vaginal fornix renders many mares infertile. Afflicted mares frequently accumulate intra-uterine fluid and have microorganisms isolated from their uterus over subsequent cycles, even after repeated treatment with uterine lavage, intrauterine antibiotic, or antimycotic drugs. Some mares clear the infection during winter anestrus only to have the infection flair in the spring when the mare starts to exhibit estrous behavior. Cervical defects or adhesions between 4 and 8 oclock are the most difficult to identify, because vaginal exams are most commonly conducted during estrus when the cervix is lying on the vaginal floor. They tend to be associated with uterine infections, because the cervix is pulled open

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Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 ventrally during diestrus. This allows bacteria that reside in the anterior vagina to gain access into the cervical canal and possibly seed the uterus. If a cervical defect is suspected during estrus, the cervix should be reexamined when the mare is under the influence of progesterone. In addition to a vaginal-speculum examination of the cervix, a digital exam of the external os and the entire length of the cervical canal should be conducted. Fibrotic lesions associated with a previous foaling injury may be identified during rectal palpation of the cervix and reproductive tract. The cervical canal may be bent laterally to the right or left, which is likely the result of tearing and subsequent scarring of the muscle on one side of the cervical canal during foaling. Mares with this type of defect have a history of delivering a large foal, needing assistance during the foaling, or having a dystocia or abortion in late gestation. Whether or not lesions will require surgical intervention is determined by clinical history (repeated uterine infections or pregnancy loss) and the clinical experience of the veterinarian. Cervical tears that affect more than one-third of the length of the canal usually need to be repaired surgically. Treatment for cervical adhesions (adhesion of the external os to the vaginal fornix) must be determined on an individual basis, because their removal may result in additional adhesion formation. Cervical tears that are not identified for 1 yr after they occur require a longer convalescence between repair and successful breeding than tears that are repaired within months of their occurrence. Long-standing cervical lesions are commonly associated with chronic bacterial or fungal endometritis. The infection may flair after surgical repair, because air and blood may enter the uterus during the procedure. The blood is a medium for growth of microorganisms, and the air irritates the uterine lining. It has been recommended (LeBlanc, 2006) to observe 60 days of sexual rest after repair of a cervical lesion that is more than 1 yr old, but mares with a cervical lesion that is <90 days old may be bred successfully in as little time as 3 wk after repair. Cervixes that have torn and are repaired surgically may lose their pliability. They may not open completely, which results in a physical drainage problem. The uterus should be treated after breeding with uterine lavage, ecbolics, and possibly, intrauterine antibiotics. Mares mated by natural service should be reenforced with semen after the horse dismounts. A surgically repaired cervix will likely tear again when the mare foals; therefore, a cesarean section is recommended. Incomplete dilation of the cervical canal during estrus is associated with chronic endometritis, fluid retention in the uterine body after breeding, and lack of pregnancy after repeated breeding by natural service. Mares with this type of cervical incompetence may have fibrosis of the cervical canal, an elongated cervix during estrus with a narrow canal diameter, or a tight internal cervical os in the presence of a soft, edematous, open external os. Fibrotic cervixes are not pliable or soft when examined digitally during estrus. They have a meaty texture, and the canal is narrow and, in some cases, seems elongated. Defects may be 16

Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 identified on rectal palpation during diestrus, because the cervix may have a 2045 bend in the canal. The reproductive history may include delivery of a large foal with or without assistance. There is minimal histological data on damaged cervixes; the tissue is difficult to cut for histological assessment, because it is thick and tough. Mares with cervical fibrosis may develop uterine infections that require prolonged intra-uterine and systemic treatment, especially if the lesion is >1 yr old and the mare had been bred repeatedly before the defect was diagnosed. After the infection is cleared, sexual rest for 30 days is recommended so that the chronic uterine inflammation can resolve. Aggressive post-breeding management may be required to assist with physical uterine clearance. Uterine lavage between 4 and 8 h after breeding and repeated use of ecbolics and intra-uterine antibiotics in the first 48 h after breeding has been suggested (LeBlanc, 2006). Systemic antibiotics around the time of breeding and a single dose of dexamethasone (10 mg, IV) 24 h after breeding to reduce uterine inflammation have also been used. The number of cases with cervical fibrosis and chronic endometritis that we have identified has been low; however, those treated aggressively have carried a foal to term. Some of these mares tend to be every other year mares, because cervical involution is prolonged after foaling. An inability of the cervix to dilate properly during estrus is a common cause of infertility in old, maiden mares (>10 yr of age) and in some young, maiden mares (Thoroughbred mares). If mares are bred by natural service, there is the additional problem of decreased semen deposition within the uterus. Prostaglandins F2 (cloprostenol), prostaglandin E1 or E2, and estradiol 17 have been used in an attempt to dilate the cervix during estrus. There are no scientific reports on the ability of any of these hormones to dilate the cervixes of mares during estrus. Estradiol 17 has been used for >40 yr as a means of improving estrous behavior of young Thoroughbred mares that are mated naturally and dilating their cervixes. A dose of 10 mg given 1 and 2 days before breeding has been advocated. Clinicians have noted that estrous behavior is improved after treatment and that the cervix is more edematous and open on vaginal-speculum examination. Repeated doses of cloprostenol (250 g, IM) before breeding have also been advocated. Cloprostenol is given every 12 h beginning on day 2 of estrus, and treatment is continued until 12 h before breeding. Treatment with cloprostenol is resumed between 8 and 12 h after breeding in an attempt to clear the uterus of fluids associated with breeding. It should be discontinued 1224 h after ovulation. However, repeated doses of cloprostenol in mares with cervical incompetence, has been associated with diarrhea. Intracervical PGE2 or PGE1 have been used in mares for cervical ripening before induction of abortion or parturition (Rigby et al., 1998). Although successful in inducing abortion, their use has been

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Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 associated with complications such as retained dead fetuses, retained placentas, and a partially prolapsed uterus after repeated use. Intracervical PGE2 or PGE1, either in tablet form or as a compounded cream, have been used to dilate the cervix during estrus. Local application of tablets include either 2 mg of prostaglandin E2 (mixed in 24 ml of lubricating jelly and deposited in the cervical canal) or 200 g of misoprostole (PGE1 analog) tablets (softened in a small volume of sterile saline or lubricating jelly and inserted into the external cervical os) one time daily. The use of a compounded PGE1 cream (misoprostol; 2000 g in 3 g of cream) has been suggested (LeBlanc, 2006) in mares whose cervixes do not dilate properly during estrus. A small amount of cream is rubbed on the external os and within the cervical canal preferable 23 h before breeding. Clinical impression is that the cervix remains dilated for 8 h after treatment. It is possible that cervical dilation is prolonged by the presence of semen within the uterus, because the cervical response can be dramatic in some maiden Thoroughbred mares naturally mated. Repeated use of the cream may induce cervicitis, and therefore, only one treatment during estrus is recommended. Mares with an incompetent cervix during estrus should be treated for postmating-induced endometritis and fluid retention. The use of hormones to dilate the cervix of mares with fibrotic changes does not seem to be successful. Vulvar and perineal conformation Vulvar conformation is one of the most important and well known contributor of mare infertility. A vulvometer has been designed to measure the length and angulation of the vulva (Pascoe, 2007). Likewise the Caslick index can be determined to decide whether or not to perform a Caslicks operation on a mare. Other operations described include vestibuloplasty and perineal body transection, but these operatives procedures require sufficient exposure of the surgeons.

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Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 Breeding accidents Whenever bleeding is observed after natural mating, it is important to determine the origin of the hemorrhage. It may be due to vaginal perforation. If so, the location and extent of the tear must be determined. Vaginal tears may be peritoneal vs. retroperitoneal depending on their location. Treatment will depend on their location (retroperitoneal -antibiotics; peritoneal -antibiotics, surgery or cross tie the mare). Prevention should be practiced if any problems are anticipated (large stallion with small mare, stallion with previous history) as they can result into cervical tears (Dadarwal et al., 2002). AI would certainly avoid the problem but if natural service is required, the use of a stallion roll will improve safety. Varicose veins in the vagina may rupture during breeding but are not of any serious consequence. Rectal breeding can cause a rectal tear. If a stallion penetrates the rectum during breeding, do a rectal exam to check for tears. If present, treat as previously discussed. Early (Resorption) and late pregnancy losses in mares Early pregnancy loss (Early Embryonic Deaths EED) has been for long times referred to as Resorption by clinicians and stud owners. A clinician often finds the disappearance of a pregnancy once confirmed by his examination. Ultrasonography is considered the best way to detect EED. The causes for early pregnancy loss could be multiple and are often associated with uterine, oviductal or cervical pathology or stress. The therapies suggested include the administration of progesterone but the results are inconspicuous. The hormonal profiles and treatments in the late pregnant mare have been extensively reviewed recently (Ousey, 2006).

Management Factors affecting equine fertility

The management of equine breeding is an art collectively excercised by the stud owners and managers and the veterinarians. An effective teasing program, a good nourishing diet, regular exercise, hygienic breeding and foaling, and regular and sometimes prompt veterinary advice are essential to attain high fertility at the stud farm. The management philosophy for the problem mares have been recently reviewed (McCue, 2008).

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Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98

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Compendium Training Course on Equine Health and Production Management January 20-Feb 2 2010 NRC on Equines Hissar India pp 89-98 Pascoe RR. (2007). Vulvar conformation. In: Samper JC, Pycock JF, McKinnon AO. Eds. Current Therapy in Equine Reproduction. Saunders, 140-45. Purohit GN.(1997). Causes of Infertility in equids. In: Training Manual Short course on equine reproduction NRC on Equines EPC Bikaner. 32-40. Pycock JF. (2000) Breeding management of the problem mare. In: Samper JC, ed. Equine breeding management and artificial insemination. Philadelphia: W.B. Saunders, 195228. Ricketts S, Troedsson MHT. (2007). Fertility expectations and management for optimal fertility. In: Samper JC, Pycock JF, McKinnon AO. Eds. Current Therapy in Equine Reproduction. Saunders, 53-69. Ricketts SW, Barrelet A, Barrelet FE, Stoneham SJ. (2006). The stallion and mare reproductive system. In: Higgins AJ, Synder JR. eds. The Equine Manual Saunders, USA. 727-744. Ricketts SW. (1985).Endometrial curettage in the mare. Equine Vet J. 17:324-28. Riddle TW, LeBlanc MM, Pierce SW, et al.(2005). Relationships between pregnancy rates, uterine cytology, and culture results in a Thoroughbred practice in central Kentucky, in Proceedings. 51st Annual American Association of Equine Practitioners Convention, 198201. Rigby S, Love C, Carpenter K, et al. (1998). Use of prostaglandin E2 to ripen the cervix of the mare prior to induction of parturition. Theriogeneology. 50:897904. Samper JC, Pycock JF, McKinnon AO. (2007) Eds. Current Therapy in Equine Reproduction. Saunders, Elsevier USA. Sharp DC, Davis SD. (1993). Vernal transition. In: McKinnonAO, Voss JL, eds. Equine reproduction. Baltimore: Williams & Wilkins,133143. Troedsson MHT(1995). Uterine response to semen deposition in the mare, in Proceedings. Annual Meeting of the Society for Theriogenology, 130135. Turner JE, Irvine CHG. (1991). The effect of various gonadotrophinreleasing hormone regimens on gonadotrophins, follicular growth and ovulation in deeply anoestrous mares. J Reprod Fertil. 44(Suppl):213225.

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