Margaret V. Root Kustritz, DVM, PhD
Diplomate, American College of Theriogenologists
C311 VMC; 612-624-7290; email@example.com; 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
- 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
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.
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.
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
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.
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
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
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 non-cornified to completely cornified. Cornification will be complete about 2 days before estrogen peaks, about 4 days before standing heat begins.
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.
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.
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
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
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
The etiologic agent is Brucella canis.
- 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.
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.
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 CYCLE||DEFINITION YOU SHOULD KNOW|
|Proestrus||Increasing percentage cornified cells over this stage of the cycle, PMNs present early|
|Estrus (standing heat)||100% cornified cells with at least 50% being anuclear 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.
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.
A) Prostate diagnostics
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)
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).