Dogs

• Must determine ovulation day so that breeding(s) occur(s) at the proper time.
• Fresh, chilled, or frozen semen-usually limited to one or two inseminations; insemination must be timed relative to ovulation for maximum fertility.
• Ovulation may vary relative to onset of standing heat (estrus), vaginal cytology.
• Luteinizing hormone-controls ovulation; peaks on same day or after full cornification is observed; ovulation occurs approximately 2 days after the peak; 2–3 days (54–72hours) more required for oocyte maturation; mature oocytes viable for a minimum 2–3 days; thus fertile period is 4–8 days after the LH peak, and maximum fertility is 5–6 days after the LH peak.
• Physical signs alone-unreliable for precise determination of fertile period.
• Onset of estrus-usually associated with a change in the vaginal discharge from sanguineous to barely red and decreased vulvar edema; sanguineous discharge may continue during estrus and cease only at the onset of diestrus (fertile period has passed; bitch no longer receptive; cervix closed).
• Receptivity-may be detected by touching the perineum near the vulva; if receptive, female will “flag” by elevating the tail to one side.
• Vaginal cytologic examination-better but imprecise indicator of fertile period; cornification of the vaginal epithelium controlled by estrogen; full cornification with a clear background usually coincides with sexual receptivity.
• Serum progesterone-increase closely associated with the LH peak; useful for estimating ovulation, and thus the fertile period; concentration <1 ng/mL (3.18 nmol/L) before the LH peak, 1.5–4 ng/mL (4.8–12.7nmol/L) at LH peak, 4–10 ng/mL (12.7–31.8nmol/L) at ovulation; continues to rise during diestrus/pregnancy. Commercial laboratories use various methods of progesterone concentration measurement so values indicative of LH and ovulation vary among labs. Documenting a rapid rise in progesterone subsequent to the initial rise is a more reliable indicator of ovulation than is the single measurement of the LH peak or the initial rise in progesterone.

Cats

• Ovulation-usually induced; timing of breeding is not as critical as with dogs; depends on adequate GnRH and then LH release triggered by vaginal stimulation.
• Adequate stimulation-characterized by a copulatory cry and a post-coital reaction; frequency of coital stimuli important in determining adequacy of coital contact.
• LH-peak concentration and duration of the elevation determine ovulation; higher concentration with multiple copulations; response to copulation depends on the day of estrus (greater release on estrus day 3than on estrus day 1); release partially depends on duration of exposure to estrogen.

General Comments

Historical Findings

Physical Examination Findings

Dogs

• Limited number of breedings
• Female unreceptive to male
• Artificial insemination (fresh, chilled, or frozen semen)

Cats

• Coitus-too early or too late in estrus; too few times
• Artificial insemination

Dogs

• In-house semiquantitative progesterone ELISA-as adjunct to vaginal cytologic examination
• Quantitative progesterone testing preferred when breeding with frozen semen; especially useful in bitch or male with reduced fertility.
• LH testing-must sample daily to observe the LH peak, can use serum progesterone to signal when to start testing or as a surrogate for the LH peak.

Cats

Progesterone testing to verify ovulation.

Dogs

• Behavior-flagging, male very interested, females mounting one another.
• Vaginal discharge much less hemorrhagic, vulva softer, less swollen.
• Palpation of vagina per digitum may be resented by bitch in proestrus improving throughout proestrus until estrus.
• May feel edematous mass on floor of caudal vagina, just cranial to urethral os that should shrink as optimal breeding period approaches.
• Vaginal cytologic examination-proestrus (in breeder terms, “day 1” at first sign of hemorrhagic vaginal discharge), most epithelial cells are non-cornified; percentage of cornified cells (cells with angular cytoplasm and pyknotic nuclei or nuclei that fail to take up stain) increases during proestrus, often reaching 90% or more by estrus (individuals vary). (Breeders refer to this stage by day, rather than using the term “estrus”; this is usually about day 10–18 in breeder terms.)
• Background of slide is free of debris during estrus.
• Diestrus, an abrupt decline in the percentage of cornified cells (20–50%) occurs in a single day-day 1 of diestrus (D1); normal to see neutrophils on days 1–4 of diestrus.
• Vaginoscopy-edematous vaginal folds until the LH peak, then vagina pale with slight wrinkling (crenulation) as edema decreases with estradiol decline, by optimum breeding period obvious wrinkles until diestrus when folds are very flat and edema disappears.
• Serologic test for B. canis (dogs)-rapid slide agglutination test used as a screen (D-TEC^® CB; Zoetis, (888)963-8471) sensitive but not specific, looks at antibodies so need 4to 8 weeks post-exposure for a positive test; confirm by agar gel immunodiffusion test (Cornell University Diagnostic Laboratory, (607)253-3900) or bacterial culture of whole blood or lymph node aspirate.

Dogs

• Fresh semen: multiple breedings; inseminate q48h after the initial rise in progesterone is observed until D1.
• Two vaginal breedings fresh semen: inseminate either on days 3and 5or on days 4and 6after the LH peak or initial rise in progesterone; use standard AI pipettes or modified Foley catheters (several sizes available); vaginal insemination on or after day 5may be associated with reduced pregnancy rates and litter sizes due to beginning of cervical closure.
• Frozen or chilled semen: frozen semen is less viable than chilled, thus timing is more critical; one or two intrauterine inseminations most common: inseminate on day 5or 6after the LH peak or initial rise in progesterone (day 0) or 3 days after progesterone 5 ng/mL (16 nmol/L); intrauterine insemination (via transcervical endoscopy (TCI) or “Norwegian” catheter or surgical insemination).
• Progesterone on day of insemination should be 12 ng/mL (38nmol/L).
• Timing of insemination based on progesterone improves chance of conception and increased litter size.
• Blood collection and vaginal examination q48h are adequate in most cases.

Cats

• Increase the likelihood of ovulation and litter size by maximizing the number of matings; breed on successive days.
• Breed at least four times daily at least 2–3hours apart on days 2and 3 of estrus to maximize LH release.
• May induce ovulation by administration of exogenous hormones-GnRH or hCG after mating.

Dogs

• Vaginal cytologic examinations-compare D1with the prospective estimation based on progesterone; if the estimates differ, pregnancy rates are reduced.
• Semiquantitative progesterone kits-must come to room temperature before use; false high values common when using a cold kit.
• Serum progesterone-allow blood to clot at room temperature; separate cells from serum 20minutes after collection; false low values occur when using serum mixed with RBCs (progesterone binds to RBCs).
• A hemolyzed or lipemic specimen-may give a false low progesterone value.
• Quantitative (chemiluminescence, fluorescence, enzyme immunoassay) progesterone assay-more accurate when performed by a commercial laboratory than semiquantitative kits; in house analyzers are available; turn-around times should be less than 24h.
• Do not use serum separator tubes-false elevation if progesterone measured by chemiluminescent assay; type of anticoagulant may affect reported value (serum > heparin plasma > EDTA plasma).