Bitches may be bred naturally, or artificially inseminated using fresh, chilled, or frozen-thawed semen. The practice of ovulation timing has become increasingly desirable to breeders. Popular stud dogs' owners commonly permit a limited number of breedings (usually 2) and may need to prioritize bitches based on their timing. Owners of bitches wish to minimize travel time to the stud dog facility. Boarding of bitches in season can be reduced with recognition of their fertile period. The use of extended and chilled semen and frozen semen, or subfertile stud dogs, necessitates ovulation timing for optimal conception. Proper ovulation timing permits accurate evaluation of gestational length and is essential in the evaluation of apparent infertility in the bitch. In addition, litter size is optimal with properly timed breedings.
Sound knowledge of the canine reproductive cycle is essential. Individual bitches may vary from normal, be presented at variable times during their estrous cycle for evaluation, and sometimes exhibit pathologic variations in cycles. Each of these scenarios requires veterinary interpretation. The normal canine reproductive cycle can be divided into 4 phases, each having characteristic behavioral, physical, and endocrinologic patterns, although considerable variation exists. Bitches with normal estrous cycles but unexpected patterns must be differentiated from those with true abnormalities. Detection of individual variation within the normal range of events in a fertile bitch can be crucial to breeding management. Evaluation of the estrous cycle for true abnormalities is an important part of the evaluation of an apparently infertile bitch.
The interestrous interval is normally 4–13 mo, with 7 mo the average. The anestrus phase of the estrous cycle is marked by ovarian inactivity, uterine involution, and endometrial repair. An anestrous bitch neither attracts nor is receptive to male dogs. No overt vulvar discharge is present, and the vulva is small. Vaginal cytology is predominated by small parabasal cells, with occasional neutrophils and small numbers of mixed bacteria. The endoscopic appearance of vaginal mucosal folds is flat, thin, and red.
The physiologic controls terminating anestrus are not well understood, but the deterioration of luteal function and the decline of prolactin secretion seem to be prerequisites. The termination of anestrus is marked by an increase in the pulsatile secretion of pituitary gonadotropins, follicle stimulating hormone (FSH), and luteinizing hormone (LH), induced by gonadotropin-releasing hormone (GnRH). Hypothalamic GnRH secretion is itself pulsatile, its intermittent secretion is a physiologic requirement of gonadotropin release. Mean levels of FSH are moderately elevated, and those of LH slightly elevated, during anestrus. At late anestrus, the pulsatile release of LH increases, causing the proestrous folliculo genesis. Estrogen levels are basal (2–10 pg/mL) and progesterone levels at nadir (<1 ng/mL) in late anestrus. Anestrus normally lasts 1–6 mo.
During proestrus, the bitch attracts male dogs but is still not receptive to breeding, although she may become more playful. A serosanguineous to hemorrhagic vulvar discharge of uterine origin is present, and the vulva is mildly enlarged. Vaginal cytology shows a progressive shift from small parabasal cells to small and large intermediate cells, superficial-intermediate cells, and finally superficial (cornified) epithelial cells, reflecting the degree of estrogen influence. RBC are usually, but not invariably, present. The vaginal mucosal folds appear edematous, pink, and round. FSH and LH levels are low during most of proestrus, rising during the preovulatory surge. Estrogen rises from basal anestrous levels (2–10 pg/mL) to peak levels (50–100 pg/mL) at late proestrus, while progesterone remains at basal levels (<1 ng/mL) until rising at the LH surge (2–4 ng/mL). Proestrus lasts from 3 days to 3 wk, with 9 days average. The follicular phase of the ovarian cycle coincides with proestrus and very early estrus.
During estrus, the normal bitch displays receptive or passive behavior, enabling breeding. This behavior correlates with decreasing estrogen levels and increasing progesterone levels. Serosanguineous to hemorrhagic vulvar discharge may diminish to variable degrees. Vulvar edema tends to be maximal. Vaginal cytology remains predominated by superficial cells; RBC tend to decrease but may persist throughout. Vaginal mucosal folds become progressively wrinkled (crenulated) in conjunction with ovulation and oocyte maturation. Estrogen levels decrease markedly after the LH peak to variable levels, while progesterone levels steadily increase (usually 4–10 ng/mL at ovulation), marking the luteal phase of the ovarian cycle. Estrus lasts 3 days to 3 wk, with an average of 9 days. Estrous behavior may precede or follow the LH peak—its duration is variable and may not coincide precisely with the fertile period. Primary oocytes ovulate 2 days after the LH peak, and oocyte maturation is seen 2–3 days later; the life span of secondary oocytes is 2–3 days.
During diestrus, the normal bitch becomes refractory to breeding, with diminishing attraction of male dogs. Vulvar discharge diminishes and edema slowly resolves. Vaginal cytology is abruptly altered by the reappearance of parabasal epithelial cells and frequently neutrophils. The appearance of vaginal mucosal folds becomes flattened and flaccid. Estrogen levels are variably low, and progesterone levels steadily rise to a peak of 15–80 ng/mL before progressively declining in late diestrus. Progesterone secretion depends on both pituitary LH and prolactin secretion. Proliferation of the endometrium and quiescence of the myometrium develop under the influence of elevated progesterone levels.
Diestrus usually lasts 2–3 mo in the absence of pregnancy. Parturition terminates pregnancy 64–66 days after the LH peak. Prolactin levels increase in a reciprocal fashion to falling progesterone levels at the termination of diestrus or gestation, reaching much higher levels in the pregnant state. Mammary ductal and glandular tissues increase in response to prolactin levels.
Estrogen, LH, and progesterone are important in ovulation timing. All may be assessed as part of a reproductive evaluation.
Increased estrogen causes an increased turnover rate of vaginal epithelial cells, resulting in the progressive cornification seen on vaginal cytology. Progressive edema of the vaginal mucosa also develops and can be visualized with endoscopic examination. Estrogen assays are performed by many commercial laboratories; however, the information is of little value for ovulation timing because peak estrogen levels vary from bitch to bitch, and even relative changes do not correlate to ovulation or the fertile period. Estrogen is best assessed by serial vaginal cytologies and vaginoscopy. Estrogen levels do not indicate the fertile period because ovulation is triggered by the LH surge, not an estrogen peak.
Examination of the cells on the surface of the vaginal epithelium can provide information about the stage of the estrous cycle. Proper technique is important so that the cells obtained are representative of the hormonal changes occurring. The sample should be collected from the cranial vagina; cells from the clitoral fossa, vestibule, or caudal vagina are not as indicative of the stage of the cycle. Under the influence of rising estrogen levels, the number of layers composing the vaginal epithelium increases dramatically, presumably to provide protection to the mucosa during copulation. As estrogen rises during proestrus, the maturation rate of the epithelial cells increases, as does the number of keratinized, cornified epithelial cells seen on a vaginal smear. Full cornification continues throughout estrus until the “diestral shift” occurs 7–10 days after the LH surge, signifying the first day of diestrus. The vaginal smear then changes abruptly, with appearance of neutrophils and epithelial cells changing from full cornification to 40–60% immature (parabasal and intermediate) cells over the next 24–36 hr. If vaginal cytology is performed until the diestral shift is observed, the LH surge, ovulation, and the fertile period can be analyzed retrospectively.
At the end of the follicular phase of the estrous cycle, a marked increase in LH over usual baseline values develops over 24–48 hr, followed by a return to baseline values. This surge is thought to occur in response to the decline in estrogen levels and increase in progesterone levels. The LH surge triggers ovulation, making it the central endocrinologic event in the reproductive cycle of the bitch.
Daily serial measurement of LH to identify the exact date of the LH surge is an accurate diagnostic tool for timing breedings. Affordable semiquantitative in-house kits are available for measuring serum LH levels in the dog and for identifying the preovulatory LH surge and thus the time of ovulation and the true fertile period. Blood samples must be drawn daily (at about the same time) for LH testing, because the LH surge may last only 24 hr in many bitches. The kits can be subject to variable interpretation, so the same person should run the tests if possible.
Progesterone levels begin to rise at approximately the time of the LH surge (before ovulation). Rising progesterone acts synergistically with declining estrogen to reduce edema of the vulva and vagina, which can be seen on vaginoscopic exam. Other observable clinical signs are minimal. Serial blood samples performed every 2 days may identify the initial rise in progesterone (usually >2 ng/mL), which indicates that the LH surge has occurred. Progesterone can be assayed by radioimmunoassay at most veterinary commercial laboratories. Several in-house semiquantitative kits are also available.
No single absolute value of progesterone correlates to any particular stage of the cycle. Progesterone varies from 0.8–3.0 ng/mL at the point of the LH surge, from 1.0–8.0 ng/mL at ovulation, and from 4.0–20.0 ng/mL during the fertile period. However, if accurate serial quantitative progesterone assays are obtained, the LH surge may be estimated as the day a distinct increase in progesterone level is seen. While this is not as accurate as actual identification of the LH surge by assay, estimation by progesterone levels is still very useful and is often more widely available and convenient.
When timing breeding using semiquantitative in-clinic progesterone assays, only a range of progesterone is obtained, which makes it difficult to accurately identify the day of the initial rise in progesterone or the true fertile period. Technical problems with these kits have also been seen. Therefore, these assays should be used only for routine breedings in which a wider margin of error is acceptable. A safe rule of thumb is that when progesterone is >2 ng/mL, breeding should begin. Optimal ovulation timing should use quantitative progesterone assays from commercial laboratories—the cost difference is minimal. Regardless of which assay is used, an additional test should always be performed 2–4 days after the first rise is detected to indicate that the cycle has progressed as expected, a functional corpus luteum has been formed, and ovulation has occurred.
Use of Hormonal Evaluation to Time Breeding
Owners of breeding animals should be advised to notify their veterinarian when they first notice that a bitch for which timing is planned is in season, based on vaginal discharge or vulvar swelling/attraction to males. Even the most astute owner may not notice the true onset of proestrus for a few days. Early proestrus should be documented with vaginal cytology (<50% cornification/superficial cells). A baseline progesterone level (usually 0–1 ng/mL) might be informative if the true onset of the cycle is unknown. Vaginal cytology should be performed every 2 days until cornification progresses significantly, usually >70% superficial cells. At that point, serial hormonal assays should begin. For routine breedings, progesterone testing may be done every other day, until a rise in progesterone >2 ng/mL is identified. The day of the initial rise in progesterone >2 ng/mL is identified as “day 0.” Breedings are advised on days 2, 4, and 6.
When increased accuracy of ovulation timing is necessary (eg, frozen or chilled semen breedings, infertility cases, breedings with subfertile stud dogs), daily LH testing is recommended. Once the LH surge is identified, breeding days may be planned. The day of the LH surge is also “day 0.” It is useful to perform vaginal cytology every 2–3 days until cornification is complete (>90% superficial cells). This maximal cornification usually develops before the fertile period and continues until the onset of diestrus, which is usually a few days after the end of the fertile period. Vaginal cytology may be continued until the diestral shift is identified, which gives a retrospective evaluation of the breeding just completed. In addition, at least 1 progesterone assay should be performed after day 0 is identified to document that levels continue to rise. This illustrates sustained corpus luteum function and strongly suggests that an ovulatory cycle has occurred.
Insemination with extended, chilled semen should be done on days 4 and 6, or 3 and 5, after day 0. The days chosen can depend on overnight shipping possibilities and the schedules of all involved parties. Frozen semen breedings should be done on day 5 or 6.
Vaginoscopy may be performed throughout the cycle as an adjunct to vaginal cytology and hormonal assays, especially when evaluating an unusual cycle. Behavior and other observations should also be made. Ovulation timing is most accurate when information from several tests is pooled (vaginal cytologies, vaginoscopy, and progesterone or LH tests).
Artificial insemination is becoming more common in canine reproduction, permitting the use of shipped semen, assistance for geriatric or subfertile males, coverage of dominant females, and advanced reproductive technology such as intrauterine deposition of semen. Insemination may be performed with fresh, chilled, or frozen semen. All instruments should be clean and free of any chemical contamination. After semen has been collected and evaluated (see Management of Reproduction: Small Animals: Semen Evaluation), it can be deposited in the cranial vagina of the bitch using a rigid insemination pipette of appropriate length, or into the uterus via transcervical catheterization. Access to the uterus via laparoscopy or laparotomy is less desirable due to invasiveness.
Semen (the second fraction) may be diluted with extenders and chilled for later or distant use (within 48 hr), or extended and frozen in liquid nitrogen (in straws or pellets) for longterm storage. Phosphate-buffered egg yolk diluent or Tris-buffered diluent is used most often; several commercial extenders are available. Chilled semen should be warmed for evaluation before use. Frozen semen should be thawed as directed by the cryopreservation center, evaluated, and immediately inseminated.
The queen should be taken to the tom when showing signs of estrus. The breeding area should be familiar to the tom, quiet, and have good footing and a minimum of interference, while permitting observation. The courtship should not be interrupted unless there is concern for the safety of either cat. Toms have been known to mate to the point of physical exhaustion, but queens normally go through a period of rolling and grooming after a breeding and may not let the tom remount for some time.
Because ovulation is induced by vaginal-cervical stimulation, multiple breedings over 3 days are advised. Periods of separation between matings prevent exhaustion and diminish the chances of fighting. The stress of transportation may affect reproductive functions in nervous queens. Evaluation for pregnancy can be performed at 21–30 days gestation by abdominal palpation and ultrasonography.
Last full review/revision July 2011 by Autumn P. Davidson, DVM, MS, DACVIM