Chapter 19 OVARIAN CYCLE AND VAGINAL CYTOLOGY
THE OVARIAN CYCLE
Onset of Puberty
Breed has a significant effect on the timing of a dog’s first estrus. Generally, bitches exhibit their first cycle several months after they achieve adult height and body weight. There is, however, considerable variation within a breed as well as between different breeds. The Beagle, for example, usually exhibits her first proestrus between 7 and 10 months of age. Even in a controlled laboratory environment, the first proestrus may occur as early as 6 months of age or as late as 13 months (Concannon, 1987). Thus it is reasonable to advise owners that some small breeds enter their first heat between 6 and 10 months of age. Although a large-breed bitch may also begin her first proestrus before 1 year of age, some normal large-breed dogs may not begin to cycle until 18 to 24 months of age. A good deal of individual and breed variation is reported. This natural variability, coupled with cycles referred to as silent heats, adds to a veterinarian’s or owner’s inability to predict the timing of a first season (estrus) (Concannon, 1980; Evans and White, 1988; Johnston, et al, 2001).
Silent heat is simply one that goes unnoticed by the owner because little vulvar swelling, bleeding, attraction of males, or behavioral change may be associated with estrus in a young bitch. The owner’s experience, hair length of the dog (vulvar bleeding is easier to see in a short-haired dog), cleanliness of the dog (one that licks a great deal is more apt to hide bleeding), and presence of a male dog in the home are just a few of the variables that determine the ease with which an owner may notice estrus (Concannon, 1980). We tend not to begin to evaluate a female dog clinically for failure to experience an ovarian cycle until she is at least 2 years of age. (See Infertility, Chapter 24.) The ideal age for breeding is between 2 and 6 years. First breeding is recommended during the second or third estrus, after an owner has witnessed at least one complete normal ovarian cycle.
Intervals Between Ovarian Cycles
The “average” bitch begins proestrus approximately every 7 months. Thus, an ovarian cycle would begin at least once in every month of the year during a bitch’s life if this schedule were maintained. There is a tendency to vary somewhat. The common interestrus interval (the period from the end of standing heat to the beginning of the following proestrus) is 5 to 11 months, with the normal interestrus interval as short as 3.5 months and as long as 13 months (Concannon, 1987). Interestrus intervals more frequent than every 4 months, however, are often associated with infertility, and those greater than every 12 months may be associated with sufertility or infertility. Breed and individual variability can be striking. The German Shepherd dog, for example, is one breed that often cycles every 4 to 4.5 months and yet maintains fertility. Rather than relying on knowledge concerning individual breeds, we use the 5- to 11-month interval as normal. It also appears that most dogs 2 to 6 years of age are relatively consistent in cycle length as well as in the duration of each phase. The obvious exceptions to the 7-month average normal interestrus interval are the African dog breeds such as the Basenji. These dogs cycle once yearly. Litters of this breed are typically whelped in December, with January and November following as likely months of births.
Phases of the Ovarian Cycle
There are four phases of the canine ovarian cycle.
Proestrus
DURATION.
Proestrus is usually, and most reliably, defined as beginning when vaginal bleeding is first seen and ending when the bitch allows a male dog to mount and breed. Additional criteria used in defining the onset and completion of proestrus include changes in the appearance of the vaginal mucosa viewed endoscopically (Fig. 19-1) or changes seen on exfoliative cytology of vaginal epithelial cells (Fig. 19-2). Less reliable criteria used in describing the onset of proestrus include enlargement of the vulva, attraction of males, and changes in behavior toward males (Fig. 19-3).
The length of time from the onset of proestrus to the time of first breeding is usually 6 to 11 days, with an average of 9 days. However, variations within “normal” can be extreme (i.e., as brief as 2 or 3 days to as prolonged as 25 days) (Fig. 19-4). These extremes in normal duration of proestrus can be confusing, and their significance is discussed in greater detail in association with the evaluation of female infertility (Chapter 24).
CLINICAL SIGNS.
Bitches that keep themselves clean through licking may present a difficult challenge in detecting proestrus. Short-haired dogs without tails (e.g., German Short-Haired Pointers) can be contrasted with long-haired dogs with flowing tails (e.g., Newfoundlands). Obviously, vaginal bleeding associated with proestrus is easier to detect in some breeds than in others. Occasionally, a grayish mucoid-like vaginal discharge is observed before actual bleeding or swelling of the vulva. Classically, dogs cease vaginal bleeding as proestrus proceeds into estrus. In these bitches the bloody discharge fades and becomes transparent to straw-colored. However, changes in color of the vaginal discharge are inconsistent, with some bitches showing bloody discharge throughout proestrus, estrus, and into diestrus, whereas others bleed little or only at the beginning of proestrus.
HORMONAL CHANGES AND OVARIAN ANATOMY.
The bitch in proestrus is under the influence of estrogen. This estrogen is synthesized and secreted by developing ovarian follicles. Proestrus is the phase of estrogen dominance in the bitch (see Fig. 19-2). FSH and LH pulses are released in concordance in late anestrus, and progression from anestrus into proestrus is associated with an increase in the secretion of FSH with a concomitant rise in LH secretion (Kooistra and Okkens, 2000). Follicles that develop at a time coinciding with gonadotropin stimulation mature and attain the capacity for estrogen synthesis and secretion. The secreted estrogen results in vaginal discharge, attraction of males, and uterine preparation for pregnancy. The potent effects of estrogen can be demonstrated in the ovariohysterectomized female, which can easily be brought into a classic proestrus state (without vaginal bleeding) by administration of estrogens (Concannon, 1987). Estrogen alone usually does not result in breeding activity. Breeding in the dog is typically associated with a serum combination of decreasing estrogen and increasing progesterone concentrations. Estrogen alone usually does not cause behavioral estrus (Leedy, 1988).
The appearance of the ovaries has been monitored during proestrus with ultrasonography. Ovaries of bitches in anestrus can be visualized as structures with an echogenicity equal to or slightly greater than that of the renal cortex. Follicles appear as focal hypoechoic to anechoic rounded structures. Ovaries are easier to identify as follicular development progresses. Ovarian size increases throughout proestrus (England and Allen, 1989a, 1989b; Wallace et al, 1992).
Decreasing plasma estradiol concentrations stimulate the onset of estrus. During the subsequent 5 to 20 days, plasma estradiol concentrations taper progressively to basal levels. Therefore, proestrus is a phase of rising and then stable serum estrogen concentrations, and estrus is a phase of decreasing serum estrogen concentrations. “Basal” serum estrogen concentrations are observed in anestrus and in diestrus, although variations within individual dogs may be observed (Olson et al, 1982). Concentrations of testosterone increase in the serum of bitches during late proestrus, reaching concentrations of 0.3 to 1.0 ng/ml at the time of the LH surge that occurs in early estrus (Olson et al, 1984).
ANATOMY OF THE UTERUS, OVIDUCTS, AND MAMMARY GLANDS.
Several “estrogen-target” tissues are affected by increases in serum estrogen concentrations during proestrus. These include growth of mammary gland ducts and tubules, proliferation of oviductal fimbria, thickening of oviducts, elongation of uterine horns, thickening of endometrium, increased myometrial sensitivity, enlargement of the cervix, elongation and edema of the vagina, proliferation of the vaginal wall, and synthesis of hepatic steroid-metabolizing enzymes (Concannon and DiGregorio, 1986; Concannon, 1987). The preparation for implantation in the endometrium includes a remarkable increase in wall thickness and glandular activity, changes that are associated with bleeding. This uterine hemorrhage is the primary source of vaginal bleeding associated with proestrus and, in some bitches, estrus.
VAGINAL ANATOMY AND ENDOSCOPY.
Thickening in the vaginal mucosa, easily demonstrated with cytology, may also be detected with vaginoscopy. The procedure is simple and well tolerated by nonsedated bitches that have normal vaginovestibular anatomy, but does require some practice and experience. Before inserting any vaginoscope, one should first perform a digital examination of the vaginal vault to confirm that no stricture is present (see Chapter 25). The mid- to anterior vagina can be visualized using a rigid pediatric proctoscope or flexible endoscope. The less expensive proctoscope is an excellent tool for this purpose. Otoscopes are completely inadequate for viewing anything but the “vestibule” of the vagina. The vaginal mucosa is flat in anestrus because relatively few cell layers are present (see Fig. 19-1).
During proestrus, the mucosa appears markedly rounded, edematous, smooth, and shiny (Jeffcoate, 1988). Vaginal folds are pink and billow out into the lumen as a result of fluid retention, resulting in an inability to visualize the lumen (Goodman, 2001). The decreasing estrogen and increasing progesterone concentrations associated with the final 1 to 3 days of proestrus (or the first days of estrus in some bitches) cause edema in the vaginal mucosa to subside, and the stretched luminal surface becomes progressively wrinkled (see Fig. 19-1). This is referred to as crenulation. Initial vaginal crenulation, observed as this subtle wrinkling of the mucosa, appears within 24 hours of the preovulatory LH surge (Lindsay and Concannon, 1986; Concannon, 1987; Jeffcoate, 1998).
The LH surge, in turn, precedes the onset of ovulation by approximately 24 to 48 hours. Breeding should begin at this time. The mucosa becomes progressively more crenulated, the lumen more obvious, and the vaginal folds more flattened as the edema diminishes. This wrinkling is most obvious during the fertile period 4 to 7 days after the LH surge. By diestrus the mucosa is flat and variegated. Further, because the protective thickened layers of epithelium have disappeared, the mucosa once again becomes fragile and superficial hemorrhage may be associated with vaginoscopy (Goodman, 2001). With practice and observation of individual variation in this process, vaginoscopy becomes a useful adjunct when attempting to best time a breeding.
Classification of Vaginal Cells
SUPERFICIAL-INTERMEDIATE CELLS.
These are vaginal cells that appear to have relatively healthy vesiculated nuclei but also have the angular, sharp, flat cytoplasmic border typical of superficial cells (Olson et al, 1984b; Olson, 1989) (Fig. 19-8). The superficial-intermediate cell provides evidence for potent estrogen effect on the vaginal lining, but not quite the full effect. Full estrogen effect is associated with superficial cells and anuclear squames. Vaginal exfoliative cytology may not progress beyond the presence of superficial-intermediate cells at the peak of estrogen effect. No study has demonstrated associations between failure to develop superficial cells on vaginal cytology and subsequent breeding or fertility problems.
ANUCLEAR SQUAMES.
These large, dead, irregular vaginal cells with no nuclei represent the end of a process that began with healthy round parabasal cells. This cell death is caused by the thickened vaginal lining. As the vaginal wall thickens in response to increases in serum estrogen concentrations, from several cell layers to 20 to 30 cell layers, those cells lining the lumen become far removed from their blood supply and their death is inevitable (see Fig. 19-2). These cells are also called anuclear superficial cells. They are large cells with flat, angular borders (Fig. 19-9). These are the cells that have also been called “fully cornified” or “fully keratinized” cells.
Vaginal Cytology in Proestrus
EARLY PROESTRUS.
The vaginal smear from a bitch in early proestrus is similar to that from a bitch in anestrus, with one difference: the presence of blood within the vagina derived primarily from a rapidly developing endometrium (see Fig. 19-2). In addition to varying numbers of red blood cells, the vaginal smear usually contains numerous parabasal, small, and large intermediate vaginal epithelial cells. Neutrophils are common in varying numbers, and bacteria may be present in small to large numbers (Fig. 19-11; Table 19-1). The background of these smears is often granular or “dirty” in appearance, owing to the presence of viscous cervical and vaginal secretions that appear to take a slight amount of stain.
MID-PROESTRUS.
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