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Figure 9.1. Female SB 371 moderately restrained in a squeeze cage. The upright sitting position was maintained with positive food reward during the collection of vaginal cells.

(Studbook, SB, 371) was also trained to allow swabbing while in dorsal recumbency (Fig. 9.2).

A phosphate buffer saline-moistened sterile cotton swab (Fisher Scientific, Tustin, CA) was inserted 2 to 3 cm into the vagina, rotated

Figure 9.2. Female SB 371 in the 'down' position for vaginal swabbing. Dorsal recumbency was maintained with positive food reward.

and removed. Care was taken not to scrape cells from the vaginal epithelium. Each swab was rolled gently onto a clean microscope slide, which was immediately sprayed with cell fixative (1:1, methanol:ether) and allowed to air dry. Cells were stained with modified trichrome Papanicolaou (Fisher Scientific) (Papanicolaou, 1942) as described by Durrant et al. (2002). In brief, this involved increasing the haematoxylin immersion from two to five minutes and reducing subsequent rehydration and clearing times. Coverslipped slides were evaluated immediately, then cured for three to four days in 37°C dry heat before archiving. A minimum of 200 cells was evaluated per slide (usually at x40 magnification) and classified as basophilic (stained blue), acido-philic (stained pink), keratinised (stained yellow to orange), in addition to the traditional categories of basophil, intermediate and superficial (Fig. 9.3; Plate X) (Shutte, 1967; Feldman & Nelson, 1987). A predominance of basophils in the vaginal smear is correlated with low circulating oestrogen. Intermediate cells reflect increasing oestrogen concentrations as the female approaches oestrus, and superficial cells are indicative of

b

Figure 9.3. Exfoliated vaginal epithelial cells of the giant panda stained with modified trichrome Papanicolaou. (a) Nucleated basophilic cells (x60); (b) nucleated acidophilic cells (x40); and (c) anucleated, keratinised cells (x40). (See also Plate X.)

peak oestrogen associated with oestrus. Cells were examined twice, the first time to record colour only and the second to record morphology regardless of colour.

Swabs were obtained daily during the breeding season beginning at the first behavioural or hormonal signs of pro-oestrus and continuing until behavioural oestrus was complete, or the last AI had been conducted. Behavioural indications of impending oestrus included increased vocalisation, water play and/or scent marking (see Chapter 11). Urinary oestrone sulphate conjugate concentrations (E1C) were assayed daily by enzyme immunoassay or radioimmunoassay procedures (see Chapter 8). An increase in EaC above 25 ng mg-1 creatinine (Cr) was considered evidence of approaching oestrus. The day oestrogen metabolites fell significantly from peak value was designated the day of ovulation (Day 0).

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