Technique.

Vaginal cytology is not a commonly used procedure in queens. This is primarily because the queen enters an obvious estrus abruptly and because the patterns seen on exfoliative vaginal cytology are not always clearly demarcated. Vaginal exfoliative cytology smears from the queen can be obtained as described for the bitch (see Chapter 19). The assistant usually grasps the queen by the scruff of her neck and holds her down on her elbows. The tail is held in the assistant’s other hand, to raise the rear quarters slightly and allow visualization of the vulva. The veterinarian separates the lips of the vulva and can simultaneously raise the perineum slightly. A sterile cotton swab, moistened with saline, is placed into the vaginal vestibule and guided into the vaginal vault along the dorsal surface of the tract. The swab is gently rotated in both directions and smoothly withdrawn.

The entire procedure usually takes seconds and is rarely painful. Other methods have been described, as in the bitch, but the moistened cotton swab method is simple, quick, reliable, and inexpensive. The veterinarian must remember that the queen is an induced ovulator (i.e., coital contact induces ovulation). Therefore any method used in obtaining a vaginal smear may induce ovulation.

Once removed from the vaginal vault, the cotton swab is gently rolled across a glass slide two or three times and the slide is stained. Slides are usually air-dried or fixed with 90% methanol followed by air-drying. The slides can be stained with various solutions, as described for the bitch. Diff-Quik Stain (Harleco, Gibbstown, NJ) is recommended because it is reliable and easy to use.

Terminology.

The stained slide is studied microscopically at low power to obtain an impression of the degree of “clearing” on the slide. Clearing is defined as the absence of noncellular debris and of eosinophilic or basophilic strands of mucus, as well as a lack of coalescence of vaginal cells into sheetlike aggregates. Estrogen “liquefies” vaginal mucus in cats. Vaginal epithelial cells can be closely examined at higher magnification. These cells can be classified using the categories described in Chapter 19. These categories include parabasal, intermediate, superficial (cornified), and anuclear superficial cells.

Interpretation.

It has been suggested that clearing of the background on a vaginal smear may be the most sensitive and consistent indicator of estrogen activity in the queen (Shille and Sojka, 1995) (Table 33-1). The vaginal epithelial cells become easier to visualize in association with the reduction and the absence of debris. Clearing of the vaginal smear is observed 2 days before the onset of the “follicular phase” in approximately 10% of queens. Clearing is observed in one-third of all estrous cycles before estrus behavior is noted and in more than 90% of estrous cycles during the follicular phase. If clearing is seen on vaginal cytology prior to the onset of estrus behavior, it is consistent with the occurrence of a brief proestrus period.

TABLE 33-1 VAGINAL CYTOLOGY DURING FOLLICULAR PHASE AND BEHAVIORAL ESTRUS IN A SERIES OF CATS^*

Cytologically and clinically, proestrus can be defined as starting with evidence of clearing on vaginal cytology smears and ending when the queen allows a tom (male cat) to mount and breed her. Progressive changes in the morphology of vaginal epithelial cells correspond with increasing follicular estrogen secretion rates. The proportion of anuclear superficial cells consistently increases above 10% on the first day of the follicular phase. The proportion of nucleated superficial cells remains relatively constant whereas intermediate and parabasal cell numbers decline (see Table 33-1). Erythrocytes and leukocytes are not often seen in vaginal smears, the former occurring during the early follicular phase and the latter seen occasionally at the end of the follicular phase (Shille et al, 1979).

Average.

The average duration of estrus behavior in the queen is approximately 7 days. There is a wide range of duration, however, and healthy fertile queens may exhibit estrus for as little as 1 day to as long as 21 days. Queens experiencing coitus (whether or not they subsequently ovulate) are in estrus for approximately 8.5 days. Queens that do not have coital contact are in estrus for only 6 days on average (Shille et al, 1979).

Effect of coital contact on the duration of estrus.

It has been hypothesized that the factors which initiate the ovulatory release of luteinizing hormone (LH) may also influence the centers that control sexual behavior. Vaginal stimulation is transmitted via a spinal afferent nervous pathway to the hypothalamus, where it is converted to a hormonal (gonadotropin-releasing hormone [GnRH]) signal. Release of GnRH following coitus may potentiate or extend sexual receptivity. Coital contact certainly does not immediately bring the queen out of estrus, nor does it shorten the period of receptivity, even if the coital contact induces ovulation.

Effect of personality on exhibition of estrus.

Absence of behavioral estrus or extreme prolongation of estrus behavior has been and is observed during regularly recurring “normal” follicular phases in healthy cats. Failure to manifest estrus behavior at expected times, predicted by plasma hormone (estrogen) concentrations, has been seen in a few queens thought to be timid and at the lower end of the “social scale” (Shille et al, 1979). However, this personality trait is not always obvious in a queen with active follicular phases but no behavioral estrus. It is difficult to simply label these queens abnormal. Many queens failing to exhibit behavioral estrus during one season may appear normal in subsequent seasons.

Prolonged estrus.

Prolonged estrus behavior is another trait occasionally seen in queens that may later be shown to be fertile and otherwise normal. In some cases, prolonged estrus is the result of overlapping waves of maturing follicles and persistent exposure to increased plasma estrogen concentrations (see Fig. 33-1). However, prolonged or continuous estrus behavior is also seen in cats experiencing distinct, repeated follicular phases. These cats have estrogen concentrations indistinguishable from those observed in queens exhibiting the more typical, repeated sexual receptivity patterns. The reason for the lack of coordination between sexual behavior and plasma estrogen concentrations in a small percentage of queens remains, for the present, unexplained.

Interpretation.

Two major findings are observed in vaginal smears from queens in estrus. The first change is the background clearing, and the second is reapportionment of the percentage of each type of vaginal epithelial cell. Background clearing is defined as the absence of noncellular debris and eosinophilic or basophilic strands of mucus, allowing the vaginal epithelial cells to be easily visualized. This process is due, in part, to vaginal mucus being liquefied by estrogen. Clearing begins before the onset of the follicular phase in approximately 10% of queens, and one-third of the females studied had cytologic clearing prior to their showing any evidence of estrus behavior. During the follicular phase, at least 90% of the vaginal smears have clear backgrounds. This clearing of the vaginal smear continues in some queens after the follicular phase ends, with 20% of smears continuing to show clearing 5 days later (Shille et al, 1979). Vaginal clearing is easily recognized on the smear and is one of the most consistent alterations, other than blood hormone (estrogen) concentrations, that allow identification of an actively cycling queen.

Progressive changes are seen in the relative proportion of the types of vaginal epithelial cells during estrus (see Table 33-1). The anuclear superficial cells increase to 10% of the total on the first day of the follicular phase and continue to increase in number, reaching approximately 40% of the total from the 4th through 7th days, before slowly returning to less than 10% by the 12th to 13th day (5th to 6th day after the end of the follicular phase). Intermediate cells decrease in number, from approximately 40% to 50% of the total during the interestrous period to less than 10% during the 4th through 7th days of the follicular phase. Parabasal cells disappear during the follicular phase, returning to 5% to 10% of the total 5 days after the follicular phase has ended. The nucleated superficial cell maintains its 40% to 60% proportion of the vaginal epithelial cell population throughout the follicular phase (see Table 33-1) (Shille et al, 1979).

Clinical uses.

Occasionally, the veterinarian is consulted regarding a queen that is failing to cycle. A number of these cats experience repeated follicular phases and appropriate fluctuations in plasma estrogen concentration. However, the shy queen or one housed with others and at the bottom of the “pecking order” may not display behavioral estrus. Vaginal smears obtained and reviewed once or twice weekly for 1 to 2 months, especially during February and March, reveal the cyclic nature of the queen’s hormonal status.

One can teach owners how to obtain and make smears. They can then bring the air-dried slides to their veterinarian for staining and analysis. This becomes a simple, cost-effective method for studying an apparently healthy but noncycling queen. Vaginal cytology is not commonly used in feline practice, but recognition of subclinical cycles is one important use for this tool. Queens that are shipped for breeding can also be studied in this manner, especially if they fail to exhibit estrus behavior after being placed in a new environment.

Vaginal smears can be obtained after coital contact, following the after-reaction, to determine whether the male ejaculated and whether he is fertile. One can simply look for sperm. However, even when sperm are present, it is not possible to know whether or not the breeding induced an LH surge and thus ovulation.

Effect of plasma estrogen concentration.

The interestrous period follows cessation of follicular function, which is characterized by an abrupt decline of plasma estrogen concentration to levels below 20 pg/ml. The estrogen concentration remains at the low, or basal, level for the duration of the interestrous period, which averages 8 days and ranges from 2 to 19 days (Shille and Sojka, 1995).

Effect of coital contact.

The duration of the interestrous period is probably not affected by coital contact that fails to induce ovulation. Interestrous periods in queens experiencing coitus but not ovulation were shorter in duration than those in animals that had not experienced coitus (8 days versus 10 days, respectively).

Estrus spanning two or more follicular phases.

Occasionally, queens appear to miss an interestrous interval (i.e., estrus activity spans two or more follicular phases despite typical cyclic follicular function; see Fig. 33-1). These queens do experience a hormonal interestrous period but not the behavioral interval one would expect. In some cats, the estrus behavior spanned interestrous intervals with typical basal plasma estrogen concentrations below 20 pg/ml. The estrogen surges during their follicular phases were not, as a rule, different from those seen in the average cat in behavioral interestrous. In other queens, however, cyclic estrogen fluctuations were observed in which the lowest estrogen concentrations remained above the 20 pg/ml plateau. In this latter situation, persistent estrus behavior, without an interestrous interval, is easier to comprehend because the queen is constantly under the influence of follicular estrogen secretion.

The interestrous period is much longer than the 8-day average if ovulation is induced (pseudopregnancy or pregnancy). The ovulation results in development of corpora lutea that secrete progesterone. Because sexual phases of progesterone dominance are referred to as “diestrus” phases, these are discussed in the subsequent section on diestrus.

Female behavior.

Mating is initiated by the female. A point is reached during proestrus when the queen progresses into an “estrus posture.” This lordosis posture (with or without other possible factors) is quickly recognized by the male. The queen in estrus allows the male to grasp her neck from the side (Table 33-2; see Fig. 33-6). Once held, most females elevate the pelvis, deviate the tail, and tread with the rear legs. If this behavior is not elicited, it still may occur following mounting, the alternating stepping movements of the male’s hind feet, or the stroking of the female’s thorax by the front feet of the male. Treading of the female’s back feet may stimulate pelvic thrusting and intromission by the male, but lordosis and treading by the queen are not always seen (Voith, 1980).

TABLE 33-2 DURATION AND CHARACTERISTICS OF THE PREMATING, MATING, AND POSTMATING PERIODS