Classification of Vaginal Epithelial Cells

A majority of cells observed in a normal vaginal smear are, not surprisingly, vaginal
epithelial cells. In addition, varying numbers of leukocytes, erythrocytes and bacteria are
usually evident, and small numbers of other contaminating cells and microorganisms are
sometimes observed.
Analyzing a vaginal smear is largely an exercise in classifying the epithelial cells into
one of three fundamental types: parabasal, intermediate or superficial cells. Keep in
mind, however, that the epithelial cells reflect a developmental continuum; some of the
cells you observe will not fit perfectly into these rigidly-defined categories.
All of the figures on this page are presented at the same magnification and are from a
single dog.

Parabasal Cells
Parabasal cells are the smallest epithelial cells seen on a typical vaginal smear. They are
round or nearly round and have a high nuclear to cytoplasmic ratio.

Parabasal cells are prevalent on smears taken during diestrus and anestrus, and not
uncommon during early proestrus. Parabasal cells are conspicuously absent during
estrus.

Intermediate Cells
Intermediate cells vary in size and shape, but typically have a diameter two to three
times that of parabasal cells. Many cytologists subclassify these cells into:

small intermediates: nearly round or oval shape with large, prominent nuclei

large intermediates: polygonal shape with a small nuclear/cytoplasmic ratio

In the figures below, all of the cells are typical intermediates except for the one cell in
the middle panel (arrow), which might be classified as a superficial cell (small, dark
nucleus). Note that the intermediate cells vary in size and that some have rounded
outlines (small intermediates), while others have a polygonal shape (large
intermediates).

Intermediate cells are prevalent during all stages of the cycle except estrus.

Superficial Cells
Superficial cells are the largest cells seen on a vaginal smear. The are polygonal in shape
and distinctly flat, sometimes having the appearance of being rolled up. Their nuclei are
either absent or pyknotic (very small and dark). Superficial cells without nuclei are often
referred to as being "fully cornified".

Superfical cells are often seen in large sheets or strings, as seen below with fully
cornified cells.

Superficial cells are not normally seen during anestrus and increase in prevalence during
proestrus. The presence of large numbers of superficial cells or only superficial cells is
the defining characteristic of cytologic estrus, and their abrupt and precipitous decline
marks the onset of diestrus.

Other Cells
Aside from the epithelial cells described above, a number of other cells are seen on
vaginal smears.

Erythrocytes are usually observed in large numbers during proestrus. In some

bitches, they are seen through estrus and even into early diestrus.

Neutrophils are often abundant in smears taken during early diestrus, and are not
uncommon at other stages, though rare during estrus. Moderate numbers of
neutrophils are a common, though not consistent feature of normal canine
vaginal smears and not by themselves indicative of vaginitis.

"Foam cells" is a term given to non-descript epithelial cells containing numerous

vacuoles that are typically seen on smears prepared during anestrus.

The following figure, of a proestrus smear, shows a group of intermediate cells

associated with neutrophils and red blood cells.

Finally, bacteria are often seen on vaginal smears in huge numbers, covering cells and
spilling onto the background. The minute dark specks covering the superfical cells in the
image below are bacteria.

Cytologic Changes Through the Canine Estrous

Cycle

Stages of the canine estrous cycle can be defined by sexual behavior, physical signs
(vulvar swelling, vaginal bleeding) or by vaginal cytology. The period of receptivity to a
male varies considerably among bitches; some bitches are receptive well before and after
the period of potential fertility. Similarly, signs such as "proestrus bleeding" are often
unreliable indicators; some bitches bleed very little and other show bleeding through
estrus and into diestrus.

Since cytologic changes reflect the underlying endocrine events of the cycle, they are
almost always a better predictor of the "fertile time" and gestation length than are
behavioral or physical signs.
Cytologic changes through the canine estrous cycle reflect changes in blood
concentrations of estrogen. As depicted below, and described in more detail in the section
on Canine Reproduction, estrogen levels rise prior to and during proestrus and fall in
conjunction with the preovulatory surge of luteinizing hormone. Rising levels of estrogen
induce the "cornification" that is characteristic of smears examined during estrus.
Ovulation occurs two days after the LH surge.

The sections below describe the cytologic picture typical of different stages of the canine
estrous cycle. Examination of a single smear can sometimes provide useful information,
but can also be quite misleading. For example, it is often difficult to differentiate
proestrus and diestrus from an isolated smear. It is therefore highly recommended that
multiple smears be evaluated.

Anestrus
Intermediate and parabasal cells predominate in smears taken during anestrus. Superficial
cells are absent or found in very small numbers. Neutrophils may also be present or
absent.

Proestrus

Serum concentrations of estrogen rise during proestrus, leading to capillary breakage and
leakage of red blood cells through uterine epithelium, as well as proliferation of the
vaginal epithelium.
Examination of vaginal smears from early to late proestrus will reveal a gradual shift
from intermediate and parabasal cells to superficial cells. Typically, red blood cells are
present in large numbers and neutrophils are commonly observed. Large numbers of
bacteria are also often present.

In some bitches, proestrus can persist for two to three weeks. In such cases, prolonged
lack of receptivity may suggest the need to artificially inseminate or force-breed the
animal. Examining vaginal smears in such cases will alleviate such concerns - certainly,
if more than a very small percentage of cells are parabasals and small intermediates,
breeding is a waste of time.

Estrus
The defining characteristic of cytologic estrus is the predominance of superficial cells.
Most, but not all, bitches will undergo full cornification, and the smear will reveal a
monotonous pattern composed almost exclusively of anucleate superficial cells.

If the bitch has been bred within a day of preparing a vaginal smear, it is quite likely that
sperm will be observed among the epithelial cells. Indeed, careful examination for sperm
in a smear taken within a few hours of an alleged breeding is a fairly reliable means of
confirming or denying such an incident. In the image below, an intact sperm (left panel)
and a sperm head (right panel) are present next to superficial cells.

Diestrus
The onset of diestrus is marked by a precipitous decline in the number of superficial cells
and reappearance of intermediate and parabasal cells. Most commonly, the cellular
profile changes within a single day from essentially 100% superficial cells to less than
20% superficial cells. However, it is best to confirm the onset of diestrus by examining a
smear prepared on diestrus day 2.

The significance of identifying the onset of diestrus is that it is a considerably more

accurate predictor of the time of ovulation, and hence gestation length, than sexual
behavior.
Dogs ovulate 5-7 days prior to the onset of diestrus (7-9 days after the preovulatory LH
surge), and hence, gestation length is usually 57 + 1 day from the onset of diestrus day 1.
The period of behavioral estrus is variable, and often extends up to several days before
and/or after cytologic estrus. Gestation lengths calculated from the onset or cessation of
receptivity are correspondingly inaccurate. The onset of diestrus also correlates well with
loss of fertility, and breedings after the diestrus shift are rarely fertile.
Diagnostic Techniques in Reproduction

Margaret V. Root Kustritz, DVM, PhD

Diplomate, American College of Theriogenologists
C311 VMC; 612-624-7290; rootk001@tc.umn.edu; http://www.tc.umn.edu/~rootk001
All veterinary students require some training in clinical theriogenology in all species, to permit you to pass
NAVLE and so you can function most effectively as a veterinary practitioner or scientist. A survey of
veterinarians in the United States yielded a clear description of what knowledge and skills in which procedures
were of value in practice (Root Kustritz MV, Chenoweth PJ, Tibary A. Efficacy of training in theriogenology as
determined by a survey of veterinarians, J Amer Vet Med Assoc 2006;229:514-521). Small animal information
presented in this course reflects data from that reference.
No information about cats will be presented due to time constraints. References available include one with
tremendous detail (Canine and feline theriogenology, ISBN 0-7216-5607-2); one intended to be a concise guide
for practitioners (Clinical canine and feline reproduction, ISBN 978-0-8138-1584-8); and one for breeders with
much less detail and good color images (The dog breeder's guide to successful breeding and health
management, ISBN 1-4160-3139-1).
Learning objectives: By completion of this module, the student will be able to:
- Describe reproductive anatomy of the female and male dog
- Explain performance and interpretation of vaginal cytology

- Describe the canine estrous cycle

- Order and interpret appropriate tests for canine brucellosis
- Work through a decision making scheme for canine dystocia
- Explain performance and interpretation of semen collection and evaluation
Reproductive examination of the female dog
I) Anatomy - Because the uterus of the dog is inaccessible for practical purposes, diagnostic
capabilities are limited.

A) Ovaries - Not routinely palpable or visible by ultrasound in normal bitches.

B) Uterine tubes
C) Uterus - Bicornuate - Not routinely palpable or visible by ultrasound in normal non-pregnant bitches.
D) Cervix - Abdominal - Tightly closed except during estrus, the peripartum period, or in the presence
of uterine disease.
E) Vagina - Extremely long - The dorsal median postcervical fold obscures the external cervical os. Formed
from the paramesonephric (Mullerian) ducts.
F) Vestibule - Forms junction with the vagina just cranial to the urethral papilla. Forms from the urogenital
sinus. The ventral portion is the clitoral fossa.
II) Techniques
A) Vaginal culture
Direct uterine culture is only possible via laparotomy with hysterotomy. The vaginal discharge present during
proestrus and estrus originates in the uterus, so indirect uterine culture can be performed by anterior vaginal
culture during proestrus or estrus. Use a long guarded culture instrument. Insert as for collection of a vagina
cytology specimen. Be aware that there is a large population of normal flora present in the vagina.
B) Vaginoscopy
Instruments used = endoscope, vaginoscope, anoscope, otoscope. Insert as for collection of a vaginal cytology
specimen. Vaginoscopy allows visualization of the vaginal mucosa, and assessment for source of discharges,
presence of masses, foreign objects, vaginal anomalies, etc.
C) Hormone assay
1) Estradiol
Estradiol is not routinely measured in serum. It is present in very low concentrations (pg/ml) and the assay
sensitivity is often above this level. The levels of serum estradiol vary greatly even within one animal. It is
generally better to use vaginal cornification as a bioassay, as described below.
2) Progesterone
Progesterone can be measured by enzyme-linked immunoassay (ELISA) or radioimmunoassay (RIA). ELISA
tests can be run in-house, but the accuracy is not good. RIAs must be sent out but the accuracy is excellent.
RIAs can be done at any commercial or hospital laboratory.
D) Vaginal cytology
1) Technique
Vaginal epithelial cells mature to keratinized squamous epithelium under the influence of estrogen. Estrogen
levels rise through proestrus and peak just prior to the onset of standing heat. Cornification (=keratinization) of

the vaginal epithelium develops gradually, paralleling estrogen levels. You can interpret the population of
vaginal epithelial cells, WBCs and bacteria collected on a swab of the dorsal vaginal surface during the estrous
cycle to try to predict reproductive events, and can do vaginal cytology at any time to assess for reproductive
tract pathology and as a bioassay for estrogen.
Moisten a cotton-tipped swab with water or saline (this is not a sterile procedure). You may use a standard
length swab since changes in cytology are the same throughout the vagina. Insert at the dorsal commissure of
the vulva, advance craniodorsally till the swab goes over the ischial arch, then advance cranially. Roll the swab
against the dorsal vaginal surface, pull it straight out, roll onto a glass slide, allow to air dry, and stain with new
methylene blue or DiffQuik.
Interpretation - Four cell types exist. Parabasal and intermediate cells are the two non-cornified cell types, and
superficial cells and anuclear squames are the two cornified cell types. There is a gradual increase in percent
cornification as the dog progresses from proestrus to estrus, and an abrupt return to complete non-cornification
at the onset of diestrus. Swabs from early proestrus and diestrus look exactly alike.

2) Estrous cycle
a) Proestrus
Endocrinology = This is the follicular stage of the cycle. Estrogen levels rise during proestrus and peak at the
end of this stage. Serum progesterone and luteinizing hormone (LH) levels are low. Vaginal cytology = RBCs
may be present throughout the stage. PMNs are present early in this stage but disappear as estrus nears and the
vaginal epithelium thickens. The vaginal epithelial cell population gradually changes from completely noncornified to completely cornified. Cornification will be complete about 2 days before estrogen peaks, about 4
days before standing heat begins.
b) Estrus
Endocrinology = Estrogen levels fall at the beginning of estrus. This decrease in estrogen, along with a
preovulatory rise in progesterone, is necessary for onset of breeding behaviors in the bitch, and presumably
elicits the LH surge. A surge of LH is released from the pituitary on or about the first day of estrus, and causes
ovulation of a primary oocyte 2 days later. Great variation in time of ovulation exists in the normal dog. After
ovulation. CLs form and progesterone production begins. Measurement of serum LH is not routinely performed.
However, measurement of serum progesterone concentration can easily be performed and this data used to
optimize breeding management. Vaginal cytology = The vaginal epithelial cell population will be completely
cornified, with greater than 50% of the cells anuclear squames. No PMNs or debris are present. Intra- and
extracellular bacteria are commonly present. RBCs may or may not be present. Vaginal cytology cannot be used
to predict ovulation time prospectively; however, vaginal cytology changes abruptly as the bitch enters the next
stage of the cycle, diestrus, with this change consistently occurring 6 days after ovulation.
c) Diestrus
Endocrinology = Bitches maintain the CL for about 60 days whether they were bred or not at that cycle.
Progesterone levels will be high throughout. Diestrus ends with a decline in serum progesterone to less than 2
ng/ml. This is associated with whelping if the bitch is pregnant, false pregnancy or not outward signs if she is
not. The CL is the sole source of progesterone during pregnancy in the bitch. Vaginal cytology = On the first
day of diestrus, the vaginal epithelial cell population abruptly shifts to complete non-cornification. There may

be a large number of PMNs present, and metestrum cells (non-cornified cells containing leukocytes) and/or
foam cells (non-cornified cells containing vacuoles) may be present.
d) Anestrus
This is a period of reproductive quiescence with no characteristic physical, behavioral or endocrinologic
changes. Vaginal cytology reveals only scant numbers of non-cornified epithelial cells at this stage.
III) Dystocia management
DYSTOCIA FLOW-CHART
SHOULD THE BITCH BE SEEN BY THE VETERINARIAN?
Dsytocia most likely is occurring if any of the following are present. The bitch should be seen if there is:
" Obvious malpresentation of a pup
" First stage labor (panting, restlessness, inappetance, vomiting) for more than 12 hours
" Second stage labor with weak and intermittent contractions for more than 4 hours before birth of the first pup
" Second stage labor with weak and intermittent contractions for more than 2 hours between pups
" Second stage labor with hard or continuous contractions for more than 30 minutes before the birth of the first
pup or between pups
" Green vulvar discharge before the birth of the first pup
" Purulent or frankly hemorrhagic vulvar discharge
" A history of decline in rectal temperature more than 24 hours ago
" Clinical evidence of systemic illness in the bitch
" A history suggesting high risk pregnancy (previous pelvic trauma or dystocia)
SHOULD THE BITCH BE TREATED MEDICALLY OR SURGICALLY?
If elective Cesarean section has been requested by the client and okayed by the therio clinician, this scheme
should not be followed and Cesarean section should be performed if onset of labor is confirmed.
This scheme should be abandoned and Cesarean section performed if green vulvar discharge is evident prior to
the birth of any pups, if fetal heart rate (by ultrasound) is less than 150 bpm, or if the bitch appears systemically
ill.
Key for dystocia management:
1. The puppy is present in the birth canal and can be manipulated for delivery ----- 2
1'. The puppy is not present in the birth canal or cannot be manipulated for delivery ------ 3
2. Attempt delivery with lubrication and gentle traction. After that pup is passed or if other pups are present in
utero ------ 3
2'. Attempt delivery with lubrication and gentle traction. If the pup cannot be delivered ----- 4
3. Fetal heart rate is less than 150 beats per minute ----- 4
3'. Fetal heart rate is 150 beats per minute or more ------ 5

4. Perform Cesarean section.

5. Abdominal radiographs have been taken ------ 6
5'. Abdominal radiographs have not been taken ----- 7
6. Pups are too large to pass or are malpositioned ----- 4
6'. Pups are not too large to pass and are not malpositioned ----- 8
7. Take abdominal radiographs and go to ----- 6
8. Four or fewer pups are present ----- 9
8'. More than four pups are present ----- 4
9. Oxytocin therapy may be attempted as follows: Give 2-5 IU IM, watch for effect for 20 minutes. If no effect
is seen, give 2-5 IU oxytocin IM plus a 5 ml bolus of 10% calcium gluconate SQ and watch for effect for 20
minutes. If no effect seen ----- 4
IV) Serology
A) Brucellosis
The etiologic agent is Brucella canis.
Diagnosis:
- Serologic tests - Dog must be off antibiotics for 4 weeks prior to testing. Chronically infected animals are
intermittently bacteremic, and show intermittent decrease in titres. Chronically infected animals may therefore
test negative falsely with any test. If you are unsure, 3 negative tests at monthly intervals are required to call a
suspicious animal truly negative.
* Rapid slide agglutination test (RSAT) - This test is sensitive but not specific. False positives are due to
cross reaction with Bordatella, Pseudomonas, Staph sp., etc. The RSAT becomes positive earliest of all tests in a
true infection. All positives should be rechecked!
* Tube agglutination test (TAT) - Gives a titre instead of just a yes/no answer. A titre of 1:200 or greater is
considered indicative of active infection. The same problem with false positives exists as described above.
* Agarose gel immunodiffusion test (AGID) - This test is performed at Cornell University. It identifies
antibodies to cytoplasmic antigens (very specific) and cell wall antigens (less specific). This test remains
positive longer after the animal becomes abacteremic.
* ELISA and PCR tests are described in the literature. PCR tests appear to have the greatest sensitivity and
specificity of all testing modalities evaluated to date. PCR tests for canine brucellosis are commercially
available in the United States as of this writing but are not yet considered the gold standard by the CDC,
suggesting more evidence of accuracy is required.
- Culture of canine brucellosis is definitive as a diagnostic test, but difficult. Consider blood, lymph node
aspirates, and obvious samples such as aborted tissues and vaginal or preputial discharge for culture. If you
submit blood cultures, submit at least three samples, in sodium citrate or heparin tubes.
Vaginal cytology cheat sheet

This handout is to ensure you're all clear on terminology and to help you better understand vaginal cytology as
you move toward clinics and the board exam.
TERMINOLOGY
Non-cornified vaginal epithelial cells = those always present, lining the vaginal vault. They are round and do
not cluster on top of each other. Two varieties exist; parabasal cells (very small, often with less cytoplasm than
nucleus) and intermediate cells (larger, still round, about equal proportions of cytoplasm and nucleus).
For an image of non-cornified canine vaginal epithelial cells, click here.
Cornified cells = dying or dead cells present after stimulation of the vaginal epithelium to divide. This stimulus
usually is estrogen, secreted from ovarian follicles during proestrus and estrus. Two varieties exist; superficial
cells (angular, sharp-edged, clumped, pyknotic nucleus) and anuclear squames (morphology as superficial cells
but with no visible nucleus). Some people call all cornified cells superficial cells and do not differentiate
anuclear squames as a distinct cell type. Some authors call cornified cells superficial intermediate cells.
For an image of cornified canine vaginal epithelial cells, click here.
WHAT YOU NEED TO KNOW TO FUNCTION IN CLINICS AND TO PASS BOARDS
1) How to identify non-cornified cells and cornified cells
2) Which cytology is associated with which stage of the estrous cycle
STAGE OF THE
DEFINITION YOU SHOULD KNOW
CYCLE
Proestrus

Increasing percentage cornified cells over this stage of

the cycle, PMNs present early

Estrus
100% cornified cells with at least 50% being anuclear
(standing heat) squames
Diestrus

Abrupt return to predominantly non-cornified cell

population, PMNs present early

Anestrus

Few cells present, all are non-cornified, scattered PMNs

may be present

3) When ovulation occurs in bitches - Bitches are different from all other species in that ovulation dose not
occur reliably relative to standing heat. Ovulation may occur anywhere from late proestrus well into estrus. The
average bitch ovulates two days after standing heat begins but there are many, many normal bitches that are not
average. The most consistent cytologic measure of ovulation is onset of diestrus, which occurs quite
consistently 6 days after ovulation. If you really need to know when ovulation occurred, measure progesterone
in serum.

Here is the URL for an overview of vaginal cytology and some self-test modules from Colorado State for those
of you with an interest - we'll also cover this in more detail in Small Animal Therio for those of you taking that
course later this semester.
http://www.vin.com/WebLink.plx?URL=http://arbl.cvmbs.colostate.edu/hbooks/pathphys/reprod/vc/
Reproductive examination of the male dog

I) Anatomy
A) The two testicles should be completely descended by 6 months of age.
B) The prostate is the only accessory sex gland.
C) The penis of canids contains a bone, the os penis.
II) Techniques
A) Prostate diagnostics
1) Palpation
The normal prostate is palpable on rectal examination as a bilobed symmetrical organ 2-3 cm caudal to the
pelvic brim. As the animal ages, the prostate enlarges and may be pulled cranially to the point where it may be
palpable per abdomen. The prostate secretes fluid constitutively; this prostatic fluid normally drips down into
the bladder and out the penile urethra.
2) Prostatic massage
Prostatic massage is used for collection of prostatic fluid from dogs that cannot or will not ejaculate. The dog is
sedated if necessary, and placed in lateral recumbency. A sterile polypropylene urinary catheter is used to empty
the urinary bladder which is then flushed with 3-4 ml sterile saline. A gloved finger is inserted into the rectum
and the urinary catheter withdrawn until its tip is palpable in the post-prostatic urethra. The catheter is then
advanced until it is judged to be within the prostatic urethra. A volume of 1-2 ml sterile saline is flushed in
while the prostate is massaged vigorously for 1 minute. Fluid and cells are aspirated and submitted for cytology
and culture.
3) Prostatic radiography and ultrasound
Flat films are generally unrewarding, as prostatomegaly will only be evident by cranial dislocation of the
urinary bladder. Reflux of contrast medium into the prostatic parenchyma during retrograde cystourethrography
may be used as an indicator of degree of prostate damage. Cysts and abscesses, and mineralization may be
visualized within the prostate by ultrasound.
4) Prostate biopsy
Blind biopsy per rectum can be performed. Ultrasound-guided biopsy generally yields more useful information
as it allows for biopsy of obviously abnormal tissue.
B) Semen collection and evaluation
1) Semen collection
a) Equipment - Teaser bitch (may increase number of sperm in ejaculate), collection vessel (AV,
syringe case, cup or plastic bag)
b) Technique
The dog is manually stimulated through the prepuce. As erection begins, the prepuce is pushed caudal to the
bulbus glandis and the artificial vagina (AV) introduced. The fingers encircle the penis caudal to the bulbus

glandis tightly, stimulating contraction of the constrictor vestibulae muscles during the copulatory lock (= tie).
Three fractions of semen are ejaculated; the clear pre-sperm, cloudy sperm-rich (thrusting behavior) and clear
prostatic fluid fractions (rhythmic anal contractions and urethral pulsations). When you are done collecting
semen, release the grip caudal to the bulbus and gently peel off the AV. Ensure detumescence and replacement
of the penis within the prepuce prior to kenneling the dog.
2) Semen evaluation
a) Color - Normal = milky, red or brown = blood, yellow = urine, green = pus
b) Volume (ml/ejaculate) - Normal = 1 - 30 ml, extremely variable
c) pH - prostatic fraction only. Normal 6.5 - 7.0 - pH may be used to direct antibiotic therapy in
prostatic disease
d) Progressive motility - Look at one drop of semen on a warmed glass slide with or without extender.
Normal = > 70%
e) Concentration (sperm/ml) - Hemacytometer and WBC Unopette system. Count center square to get
number of million sperm per milliliter. The normal number is variable as it is dependent on the volume of
prostatic fluid collected.
f) Total sperm number (sperm/ejaculate) = volume x concentration. Normal = 300 - 2000 million.
Larger dogs make more sperm as they possess a larger mass of spermatogenic tissue.
g) Morphology - Stain with eosin-nigrosin (SFT Morphology stain) or DiffQuik stains. Normal = >
80% morphologically normal sperm. Examine at least 100 individual sperm under the oil immersion objective.
Correlation of defects with fertility is unknown in the dog.
h) Cytology - Examine the sample for abnormal cells, bacteria, and/or inflammatory cells.
i) Microbial culture - Aerobes, anaerobes, Mycoplasma. Perform a quantitative culture with > 10,000
bacteria (CFU) per ml indicative of significant overgrowth of an organism. The correlation between
inflammatory cytology and presence of infection is not 100%. Perform a culture even if the cytology is normal
if you are suspicious of reproductive tract infection (e.g. recurrent prostatitis or cystitis, infertility).

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