Results

Morphology and staining traits

The morphology and staining characteristics of vaginal cells are illustrated in Figure 9.3. Basophilic cells (see Fig. 9.3a) stained blue, regardless of morphology. This classification included cells traditionally classified as basophils (small, round cells) as well as intermediates (larger, more angular cells). Acidophilic cells (see Fig. 9.3b) included all pink-staining cells regardless of morphology. The cells pictured in Figures 9.3a,b are not morphologically distinct; if a monochrome stain had been used, the cells would have been incorrectly classified together as the same type. We have categorised the sudden shift from blue to pink cells (without morphological transformation) as the 'first chromic shift'. A 'second chromic shift' also occurs when cells abruptly change from pink to yellow. A keratinised cell (see Fig. 9.3c) stains orange, yellow or tan and only occasionally contains a visible nucleus. This distinction often results in higher numbers of keratinised cells than superficial cells.

Relationship of vaginal cytology patterns to urinary oestrogen profiles

Figure 9.4 aligns vaginal cytology with a typical urinary oestrone conjugate (EjC) profile. Vaginal cytology data (see Fig. 9.4a) represent average values for eight cycles from the four evaluated giant pandas. Endocrine data (see Fig. 9.4b) represent five EaC cyclic patterns of two of the females (SB 371 and 291) and are a subset of the vaginal cytology cycles. Superficial cells began to rise sharply eight days (Day —8) prior to ovulation (Day 0, as defined above as the first day of significant E1C decline post-peak concentration). At Day —8, the proportion of acidophilic cells surpassed the proportion of basophilic cells in the first chromic shift (arrow 1 in Fig. 9.4a). The increase in acidophilic cells was not closely aligned with the increase in superficial cells. In most cycles, the percentages of acidophilic cells exceeded the proportion of superficial cells until Day —5. Superficial cells then climbed steadily from Day —4 until Day 0. On Day —2, the second chromic shift occurred (arrow 2 in Fig. 9.4a), which was characterised as a decline in acidophilic

-11 -10 -9 -e -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 b Day from E,C fall

Figure 9.4. (a) Relative proportions of vaginal cell types for eight giant panda cycles (mean ± SEM; four females; basophilic; x, acidophilic; o, kerantinised; —, superficial. (b) correlated with urinary oestrone sulphate conjugate (EjC) patterns (mean ± SEM; for a subset of two females and five cycles). Data are standardised to Day 0, defined as ovulation on the basis of the first significant EjC decline post-peak concentration. Arrows 1 and 2 in panel 'a' indicate days of the first and second chromic shifts, respectively. Vaginal swabs were not obtained on Days +3 and +6.

cells with a concomitant rise in keratinised cells such that the latter predominated. While superficial cells remained elevated, the relative percentages of acidophilic and keratinised cells reversed again on Day +2 as keratinised cells declined rapidly. By Day +5, superficial and keratinised cells had returned to baseline, acidophilic cells were decreasing and basophilic cells were rising.

Concentrations of E1C in urine rose above 25 ng mg-1 Cr on Day —12, gradually climbing until Day —3 when the rate of increase accelerated (see Fig. 9.4b). Peak levels were recorded on Day —1, and the rapid decline on Day 0 was considered the day of ovulation.

Case studies and representative profiles

Figure 9.5 illustrates a representative vaginal cytology profile from an individual giant panda, SB 371, in 1998. The first chromic shift occurred eight days before the second shift. The significance of the chromic shifts had not yet been recognised. However, in this case, the female was artificially inseminated with fresh semen on the day before, and the day of, the second shift, and no pregnancy resulted. Hormonal data (not shown) indicated that E1C concentrations peaked two days after the second chromic shift. In retrospect, conception failure may be attributed to premature insemination (at least two days before ovulation).

The following year, 1999, AI of this same female with fresh semen resulted in the birth of a single female offspring 135 days later. Figure 9.6 depicts a false (unsustained) second chromic shift on 2 April, seven days after the first shift. On this date, urinary E1C concentrations were steadily rising to ~150 ng mg—1 Cr (data not shown). Another false second chromic shift occurred two days later on 4 April. Urinary E1C dipped for a single day on 5 April, corresponding to the decrease in keratinised cells, which indicated the false nature of this second chromic shift. The authentic, sustained second shift occurred two days later on 6 April and initiated a four-day peak in keratinised cells, at which time superficial cells peaked. On 8 April, two days after the authentic second chromic shift, urinary E1C concentrations both peaked at >800 ng mg—1 Cr and then declined precipitously to <100 ng mg—1 Cr. Artificial insemination was performed for three consecutive days beginning on 9 April. False second shifts were not observed in vaginal cytology profiles for other individual pandas or cycles.

In 2002, SB 291 experienced a first chromic shift on 20 February, but no second shift occurred (Fig. 9.7). Keratinised cells never exceeded

Figure 9.5. Relative proportions of vaginal cell types for SB 371 during a normal oestrus in 1998. Two artificial inseminations were performed (arrows labelled AI) but no cub was born. Days of the two chromic shifts are also indicated with arrows labelled 1 and 2, respectively. Based on the second chromic shift, ovulation was predicted to occur on 10 April. Note that after 8 April vaginal swabs were not obtained daily. •, basophilic; x, acidophilic; o, kerantinised; —, superficial.

Figure 9.5. Relative proportions of vaginal cell types for SB 371 during a normal oestrus in 1998. Two artificial inseminations were performed (arrows labelled AI) but no cub was born. Days of the two chromic shifts are also indicated with arrows labelled 1 and 2, respectively. Based on the second chromic shift, ovulation was predicted to occur on 10 April. Note that after 8 April vaginal swabs were not obtained daily. •, basophilic; x, acidophilic; o, kerantinised; —, superficial.

42%, and superficial cell counts did not rise above 63%. Urinary E1C concentrations rose slightly, but failed to peak (data not shown), mimicking the vaginal cytology profile. This anomalous vaginal cytology and E1C profile may have reflected an irregular or anovulatory oestrus, perhaps related to this nulliparous female's advanced age (17 years old in 2002).

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