Most "infertile" dogs are healthy and fertile. Breeding practices should be reviewed before engaging in diagnostic testing for the less common causes of canine infertility. A basic understanding of the reproductive cycle of the bitch is essential to diagnose "infertility" problems related to breeding at the incorrect time, artificial insemination, etc. Most bitches cycle twice a year, with an interestrous interval of at least 4 mo. Bitches with <4 mo between two cycles usually do not get pregnant. Some large breeds (eg, Great Danes) may cycle every 9–12 mo with normal fertility.
The bitch is a spontaneous ovulator, ie, ovulation occurs without any breeding stimulus. The bitch starts cycling with the influence of hormones released from the hypothalamus and pituitary glands. Gonadotropin-releasing hormone (GnRH), released from the hypothalamus, stimulates the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH has a primary responsibility for growth of follicles on the ovaries, whereas LH causes ovulation of the follicles.
The estrous cycle in the bitch is unique and different from that of other domestic animals. It starts with proestrus, which most breeders refer to as the start of the "season," when spotting or bleeding from the vagina is noticed. During proestrus, the bitch is attractive to males but does not allow breeding. Estrogen produced by ovarian follicles causes changes in the epithelial cells of the vaginal mucosa, which can be examined by vaginal cytology (see Vaginal Cytology). Estrus or time of receptivity follows proestrus, when the bitch accepts the male for breeding. The duration of proestrus and estrus is traditionally considered to be 9 days each, on average, but each phase can range from 2 or 3 to 21 days. During proestrus, the vulvar lips of the bitch are turgid or firm and become soft and "wrinkly" as the bitch nears the time of receptivity. In most bitches, the bloody discharge of proestrus also changes to a straw color as estrus nears, but some may bleed all the way through estrus.
The production of progesterone is also unique during the estrous cycle of the bitch. The early rise of progesterone during estrus is from the luteinized follicles of the ovaries and can be used in breeding management. The bitch ovulates ~2–3 days after the LH peak. After ovulation, the follicles are replaced by corpora lutea (CLs), which produce progesterone. The production of progesterone continues throughout diestrus, the phase of the estrous cycle after estrus, regardless of whether the bitch is pregnant. The duration of diestrus is the same as that of pregnancy, ~62–63 days from the LH peak. Unlike in other domestic species, in the bitch there is no production of prostaglandin F2α from the endometrium to cause luteolysis (CL regression). During diestrus, many bitches go through pseudopregnancy in which they may gain weight and have an enlarged abdomen; other overt signs may be seen, including mammary gland enlargement, as well as prewhelping behavior such as nesting or "adopting" toys and shoes. The pseudopregnancy (or diestrus) is considered a normal occurrence and may not need any treatment.
The ova released during ovulation in the bitch are at the primary oocyte stage, ie, the first polar body has not come out of the ovum and sperm are unable to penetrate the ovum. It takes ~3 days for the ovum to become a secondary oocyte, which can be penetrated by sperm. Sperm can survive as long as 10 days in the bitch’s reproductive tract. This can become a diagnostic challenge when a bitch’s owner requests a cesarean section 62 days after breeding, because theoretically the gestation period may be only 52 days. A fairly reliable predictor of whelping is hypothermia or a decrease in rectal temperature of 2°–3°F, caused by a progesterone decrease 12–36 hr before. Vaginal cytology can also be used to predict whelping by determination of day 1 of diestrus.
Successful vaginal swabbing technique requires knowledge of the unique reproductive anatomy of the bitch. The vagina of the bitch is very long, ~20 cm (9 in.) in a medium-sized bitch. The cervix is located in the abdominal cavity, whereas it is located in the pelvic inlet in other domestic animals (eg, cow, mare). Therefore, the cervix in the bitch cannot be visualized by an endoscope or speculum; a flexible endoscope with a light source is required. This is important to deposit semen transcervical in the uterus for artificial insemination (AI). The size of the uterus and ovaries vary considerably with the breed.
A clean, cotton tip swab is commonly used to swab the vagina to obtain a cytology sample. An endoscope cone or vaginal speculum can be used to guide the swab. A right-handed person can hold the bitch’s vulva with a gloved left hand, opening the vulvar lips with the thumb and middle finger, while using the index finger behind the vulva to support it. The swab is moistened with warm tap water and inserted almost vertically into the vagina, avoiding the clitoral area, up and over the brim of the pelvis. It may be helpful to forward the swab, especially in small size bitches and bitches without serosanguineous discharge. If the swab feels "stuck" in the vaginal folds, it may be pulled back slightly and redirected before proceeding. Once the swab is inserted to a depth of at least 6–10 cm (2–4 in.), it is rolled a few times in one direction (if rotated back and forth, the cotton may unroll and drop in the vagina) and withdrawn. Inserting the swab as deep as possible to reach the cranial part of the vagina (versus the caudal part) minimizes collection of extraneous debris. Getting in a habit of reaching the cranial vagina is also helpful to take samples for vaginal culture and for AI. The swab is rolled on a clean microscope slide, air-dried, and stained; two slides are recommended. Stains that can be used include Wright-Giemsa, Romanowsky, methylene blue, eosin-nigrosin, Gram stain, etc. The slides with smears are dipped 5–7 times in each jar, rinsed with tap water, air-dried, and examined under the microscope. Viewing under 200X magnification first is helpful to get an overall impression of the types and distribution of cells before examining more closely under 400X magnification.
Various theriogenologists, clinical pathologists, and other practitioners have interpreted vaginal cytology of the bitch a little differently. One method is described here. The vaginal mucosa is responsive primarily to estrogens, and so vaginal cytology is useful only during the estrogenic phase of the cycle. Parabasal cells are small, round cells with large and distinct nuclei. The total area of the cytoplasm part of the cell is smaller than the nucleus. These cells (along with RBCs) are present during proestrus. Superficial intermediate cells are larger than the parabasal cells, with small nuclei and irregular/folded borders. Large numbers of these cells are seen during late proestrus to early estrus. Superficial cells, also called cornified or anucleated, are the largest of the epithelial cells present during estrus. Under a simple microscope, the nuclei of these cells appear faded or absent. The cells also appear "light in weight," multi-layered, and with folded borders. The appearance of 80%–90% of these cells in the smear is used to indicate that breeding can start. A series of vaginal cytology samples is needed to observe the progressive change in epithelial cells; only one smear is unreliable. Epithelial cells may remain the same for many days in some bitches, whereas they may change within 24 hr in others. In addition, many bitches reach 80%–90% cornification during estrus, but in others cornification never goes above 70%. In these bitches, waiting for 80%–90% cornification will result in loss of breeding during that cycle. Another breeding management tool is the progesterone assay, discussed below. The appearance of neutrophils on the vaginal cytology sample indicates the first day of diestrus, and whelping will occur 57–58 days later. Daily vaginal cytology samples are recommended because in some bitches, the cells change from cornified to superficial intermediate and neutrophils appear within 24 hours.
Progesterone is secreted not only from the CLs but also from the early luteinized follicles of the ovaries. The early rise in progesterone can be used to determine ovulation in the bitch, which occurs ~3 days after the LH peak. The LH peak is very short. LH kits are available, and LH should be checked every day during the expected ovulation time. Progesterone assays are commonly used for canine breeding management. These ELISA-based qualitative test kits are based on a color change that corresponds to a range of progesterone values. Many clinicians prefer to send the blood samples to an endocrinology laboratory for progesterone determination by radioimmunoassay. Progesterone is the same hormone in all species, including people; therefore, blood samples can be analyzed for progesterone in veterinary or human diagnostic laboratories.
The progesterone assay is an excellent tool to determine ovulation time for appropriate breeding. This becomes critical when using chilled transported or frozen-thawed semen for AI. As in vaginal cytology, progesterone concentration is monitored starting a few days after the start of proestrus, projecting ahead using progesterone concentrations of 2–10 ng/mL, with 2–2.9 ng/mL indicative of ovulation in approximately 2 days, 3–3.9 ng/mL in 1 day, and 4–10 ng/mL indicative of ovulation day.