Female dogs can be bred naturally, or be artificially inseminated using fresh, chilled and shipped, or frozen-thawed semen. The practice of ovulation timing has become increasingly desirable to breeders. Owners of popular stud dogs commonly permit a limited number of breedings (usually two) and may need to prioritize females based on their ovulation timing. Owners of female dogs wish to minimize travel time to the stud dog facility. Boarding of females in season can be reduced with identification of their 4-day optimal fertile period. The use of extended and chilled semen and frozen semen as well as subfertile stud dogs necessitates ovulation timing for optimal conception.
Proper ovulation timing permits accurate evaluation of gestational length (important for scheduling elective cesarean sections) and is essential in the evaluation of apparent infertility in female dogs. In addition, litter size is optimized with properly timed breedings. Fresh canine semen can live in the vagina for 9+ days, explaining why breedings can result in conception even if outside the fertile window. Breeding dates do not correlate with gestational length closely for this reason as well (58–72 days after copulation).
Sound knowledge of the canine reproductive cycle is essential. (Also see Reproductive Diseases of the Female Small Animal.) Normal female dogs may vary from the average, may be presented at variable times during their estrous cycle for evaluation, or may have actual abnormalities of their cycles. Each of these scenarios requires veterinary interpretation. The normal canine reproductive cycle can be divided into four phases, each having characteristic behavioral, physical, and endocrinologic patterns, although considerable variation exists. Females 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 female can be crucial to successful breeding management. Evaluation of the estrous cycle for true abnormalities is an important part of the evaluation of an apparently infertile female dog, many of which have not had accurate timing performed or have been bred with poor husbandry or to a subfertile male.
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.
Vaginal cytology is a powerful tool for the practitioner performing ovulation timing. Three main types of vaginal epithelial cells are informative because the vaginal wall responds to estrogen:
Parabasal cells: Small round cells with large nuclei. Little or no estrogen influence. Characterized as "Cheerios."
Intermediate cells: Medium oval cells with more cytoplasm and smaller nuclei, indicating early estrogen influence. Characterized as "fried eggs."
Superficial cells: Large cells with angular cytoplasmic edges and pyknotic to absent nuclei, indicating peak estrogen levels have been reached and are characteristic of the fertile period, which is better defined by serial progesterone +/- LH testing. This hormone testing should begin when vaginal cytology is >70% superficial cells.
Vaginal cytology can also be performed in the queen; cell transitions are similar with the exception that superficial cells remain nucleated. Vaginal cytology samples in the queen should be acquired by saline lavage of the vagina as described above for semen harvest, not using a swab as in female dogs, to avoid stimulating ovulation inadvertently.
The interestrous interval is normally 4–13 months, with 7 months the average. The anestrus phase of the estrous cycle is marked by ovarian inactivity, uterine involution and endometrial repair. An anestrous female 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 extracellular 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 increased, and those of LH slightly increased, during anestrus. At late anestrus, the pulsatile release of LH increases, causing the proestrous folliculogenesis. Estrogen levels are basal (2–10 pg/mL) and progesterone levels at nadir (<1 ng/mL) in late anestrus. Anestrus normally lasts 1–6 months.
During proestrus, females attract male dogs but are still not receptive to breeding, although they 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 parabasal cells to small and large intermediate cells, superficial-intermediate cells, and finally superficial epithelial cells, reflecting the degree of estrogen influence. RBCs are usually, but not invariably, present. Endoscopically, the vaginal mucosal folds appear edematous, pink, and round.
FSH and LH levels are low during most of proestrus, rising during the preovulatory surge. Under the influence of rising estrogen levels, the number of layers composing the vaginal epithelium increases dramatically, presumably to provide protection to the vagina during copulation. Estrogen rises from basal anestrous levels (2–10 pg/mL) to peak levels (50–100 pg/mL) at late proestrus, whereas progesterone remains at basal levels (<1 ng/mL) until rising with the LH surge (2–4 ng/mL). Proestrus lasts from 3 days to 3 weeks, with 9 days average. The follicular phase of the ovarian cycle coincides with proestrus and very early estrus.
During estrus, healthy female dogs display 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 predominately superficial cells; RBCs 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 weeks, 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 2–3 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,healthy female dogs become 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, known as the diestrual shift. 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 increased progesterone levels.
Diestrus usually lasts 2–3 months 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.
Vaginal cytology can confirm when females are in heat and indicate when serial hormone (LH +/- progesterone) testing should begin. Following cytology to the first day of diestrus (diestrual shift) enables calculation of the due dates (56–58 days later) with accuracy.
At the end of the follicular phase of the estrous cycle, a marked increase in LH over usual baseline values develops over 24–48 hours, 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 female dogs.
Daily serial measurement of LH to identify the exact date of the LH surge is an accurate diagnostic tool to time breedings. Affordable semiquantitative in-house kits are available to measure serum LH levels in dogs and to identify the preovulatory LH surge and thus the time of ovulation and the true fertile period. Blood samples must be drawn daily (at approximately the same time) for LH testing, because the LH surge may last only 24 hours in many dogs. The kits can be subject to variable interpretation, so the same person should run the tests if possible. Progesterone testing should always be performed concurrently in case the LH surge is missed.
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 as crenulation during vaginoscopic examination. Other observable clinical signs are minimal. Serial blood samples performed every 2 days may identify the initial rise in progesterone (usually 1.5–2.5 ng/mL), which correlates with the LH surge. Chemiluminescent progesterone testing is considered the gold standard. 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 ng/mL at the point of the LH surge, from 1–8 ng/mL at ovulation, and from 4–20+ 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 above the baseline level is seen. Although this is not as accurate as actual identification of the LH surge by assay, estimation by progesterone levels is very useful and is often more widely available, convenient, and less expensive.
Owners of breeding animals should be advised to notify their veterinarian when they first notice that a female dog 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 2–3 days until >70% superficial cells are present. At that point, serial hormonal assays should begin. For routine breedings, progesterone testing may be done every other day until a rise in progesterone >1.5 ng/mL is identified. The day of the initial rise in progesterone >1.5–2.5 ng/mL is identified as “day 0.” Breedings are then advised between days 3 through 6. An additional progesterone should be performed in 2–3 days to check that levels are >5 ng/mL, confirming ovulation.
When increased accuracy of ovulation timing is necessary (eg, frozen or chilled semen breedings, infertility cases, breedings with subfertile stud dogs), LH testing is recommended. Blood can be drawn daily, processed, and stored while progesterone testing occurs every 48 hours. Once the initial rise in progesterone is determined, serum from the same date can be tested for the LH surge (>1 IU/L) , confirming "day zero." Vaginal cytology may be continued until the diestrual shift is identified, which gives a retrospective evaluation of the breeding just completed. The LH surge should have been 7–9 days previously.
Natural breedings or fresh semen AI can be performed between days 3–6; generally, two breedings are advised. Insemination with extended, chilled semen should be done on days 4 and 6, or 3 and 5. 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. Vaginal mucosa changes from edematous to crenulated (flat, wrinkled appearance) during the fertile period. Behavior and other observations (interest of the stud dog) should also be made. Ovulation timing is most accurate when information from several tests is pooled (vaginal cytologies, vaginoscopy, and progesterone and 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, it can be deposited in the cranial vagina of the female 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 because of invasiveness and need for anesthesia.
Semen (the second fraction) may be diluted with extenders and chilled for later or distant use (within 48 hours), 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. A drop of chilled semen should be warmed for evaluation before use. Frozen semen should be thawed as directed by the cryopreservation center, evaluated, and immediately inseminated. Dogs should be screened for Brucella canis when semen is collected for cryopreservation.
The queen should be taken to the tom when showing signs of estrus, as early as the second day. 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 2–3 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 21–30 days after breeding by abdominal palpation and ultrasonography.