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 2) 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 decreased by identifying 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 evaluating 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 when they take place before the fertile window. Breeding dates do not correlate with whelping dates closely as actual ovulation does.
Pearls & Pitfalls
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Sound knowledge of the canine reproductive cycle is essential. (See also Reproductive Diseases of the Female Small Animal.) Normal female dogs may vary from the average, be presented at variable times during their estrous cycle for evaluation, or 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 endocrinological 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 (O-shaped like doughnuts). Little or no estrogen influence.
Intermediate cells: Medium oval cells with more cytoplasm and smaller nuclei (shaped like fried eggs), indicating early estrogen influence.
Superficial cells: Large cells with angular cytoplasmic edges and pyknotic to absent nuclei (shaped like corn flakes), indicating peak estrogen concentrations have been reached and are characteristic of the fertile period, which is better defined by serial progesterone with or without luteinizing hormone (LH) testing. This hormone testing should begin when vaginal cytology is > 70% superficial cells.
Vaginal mucosal cells during the different stages of the estrous cycle can be remembered by comparing them to different breakfast foods (see vaginal epithelial cells image).
Courtesy of Dr. Autumn Davidson.
Vaginal cytology can also be performed in the queen; cell transitions are similar, except that superficial cells remain nucleated. Vaginal cytology samples in the queen should be acquired by saline lavage of the vagina as described for semen harvest, not using a swab as in female dogs, to avoid stimulating ovulation inadvertently.
The Estrous Cycle in Breeding Management of Bitches and Queens
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 physiological controls terminating anestrus are not well understood; however, 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 GnRH. Hypothalamic GnRH secretion is itself pulsatile; its intermittent secretion is a physiological requirement of gonadotropin release. Mean FSH concentration is moderately increased, and LH concentration slightly increased, during anestrus. At late anestrus, the pulsatile release of LH increases, causing the proestrous folliculogenesis. Estrogen concentration is basal (2–10 pg/mL) and progesterone concentration 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 concentrations are low during most of proestrus, rising during the preovulatory surge. Under the influence of rising estrogen concentrations, the number of layers composing the vaginal epithelium increases dramatically, presumably to provide protection to the vagina during copulation. Estrogen rises from basal anestrous concentrations (2–10 pg/mL) to peak concentrations (50–100 pg/mL) at late proestrus, whereas progesterone remains at basal concentrations (< 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 concentrations and increasing progesterone concentrations. 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 concentrations decrease markedly after the LH peak to variable concentrations, while progesterone concentrations 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 occurs 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 concentrations are variably low, and progesterone concentrations 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. Glandular proliferation of the endometrium and quiescence of the myometrium develop under the influence of increased progesterone concentrations, as well as closure of the cervix.
Diestrus usually lasts 2–3 months in the absence of pregnancy. Parturition terminates pregnancy 64–66 days after the LH peak. Prolactin, a luteotroph, increases as progesterone concentration decreases at the termination of diestrus or gestation, reaching much higher concentrations in the pregnant state. Mammary ductal and glandular tissues increase in response to prolactin concentrations.
Vaginal cytological evaluation can confirm when bitches are in heat and indicate when serial hormone (LH with or without progesterone) testing should begin. Following cytological results to the first day of diestrus (the diestrual shift) enables calculation of the due dates (56–58 days later) with accuracy.
Luteinizing Hormone in Breeding Management of Bitches
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 concentrations and increase in progesterone concentrations. The LH surge triggers ovulation, making it the central endocrinological 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 in bitches. Affordable semiquantitative in-house kits are available to measure serum LH concentrations in bitches 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 bitches. 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 in Breeding Management of Bitches
Progesterone concentrations begin to rise at approximately the time of the LH surge (before ovulation). Rising progesterone acts synergistically with declining estrogen to decrease edema of the vulva and vagina, which can be observed as crenulation during vaginoscopic examination. Other observable clinical signs are minimal.
Serial blood samples taken every 2 days may identify the initial rise in progesterone concentration (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. Whichever methodology is chosen (commercial laboratory versus in-house testing), sampling methodology must remain consistent throughout measurements because results are not comparable across methodologies.
No single absolute value of progesterone concentration correlates to any particular stage of the cycle. Progesterone concentration varies throughout the cycle: 0.8–3 ng/mL at the point of the LH surge, 1–8 ng/mL at ovulation, and 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 concentration above the baseline concentration occurs. Although this is not as accurate as actual identification of the LH surge by assay, estimation by progesterone concentrations is very useful and is often more widely available, more convenient, and less expensive.
Progesterone concentration will not rise in queens until after the copulation-induced LH surge; it is only valuable in confirming that event retrospectively.
Use of Hormonal Evaluation to Time Breeding in Bitches
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 their observation of vaginal discharge or vulvar swelling and 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 concentration (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 concentration > 1.5 ng/mL is identified. The day of the initial rise in progesterone concentration (to > 1.5–2.5 ng/mL) is identified as “day zero.” Breedings are then advised between days 3 and 6. Progesterone should be checked again in 2–3 days to verify that concentrations 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 concentration is determined, serum from the same date can be tested for the LH surge (> 1 IU/L), confirming day zero. Vaginal cytological evaluation 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 artificial insemination can be performed between days 3 and 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 (see vaginoscopy during proestrus and estrus images). 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 cytological evaluation, vaginoscopy, and progesterone and LH tests).
Courtesy of Dr. Autumn Davidson
Courtesy of Dr. Autumn Davidson
Artificial Insemination in Breeding Management of Bitches
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 using a rigid insemination pipette of appropriate length, or into the uterus via transcervical catheterization (see transcervical insemination image).
Access to the uterus via laparoscopy or laparotomy is less desirable because of invasiveness and need for anesthesia.
Courtesy of Dr. Autumn Davidson.
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 long-term 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.
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