Margaret V. Root Kustritz, DVM, PhD
Diplomate, American College of Theriogenologists
435F AnSc/VM 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 (The
practical veterinarian: Small animal theriogenology. ISBN 1-7506-7408-3); 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).
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 non-cornified 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.
* 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. 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.
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. 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.
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).
Dr. Peggy Root Kustritz
C333 VTH
Office: 612-624-7290
Home: 651-457-4102
E-mail: rootk001@umn.edu