Oestrogen Conjugate

The predominant urinary oestrogen in giant panda urine is oestrone conjugated (or attached) to a sulphate molecule (oestrone-3-sulphate; Czekala et al, 2003). Urinary oestrogen conjugates were quantified using a single antibody, charcoal separation RIA previously validated for this species (Monfort et al., 1989; Czekala et al., 2003). Oestrone-3-glucuronide antiserum (0.1 ml, 1:5000; D. Collins, University of Kentucky, Lexington, KY) and tritiated oestrone-3-sulphate (0.1 ml, 10 000 counts per minute; New England Nuclear, Dupont, Boston, MA) were added to unprocessed panda urine (0.05-0.002 ml depending on reproductive state) or oestrone-3-sulphate standards (39-5000 pg per tube) in duplicate. After a one-hour incubation (4°C), bound and free fractions were separated by adding a charcoal dextran solution (0.25 ml), incubating for 30 minutes and centrifuging for 15 minutes (1500 g). Supernatant was counted for radioactivity in a beta radiation counter (Wallac 1409, Perkin Elmer, Gaithersburg, MD). Intra- and interassay coefficients of variation were 8% (n = 10) and 14.8% (n = 39), respectively. Interassay variation for female SB 374 and SB 14 was 7.0% and 14.5% (n = 10), respectively.

PREGNANEDIOL-3-GLUCURONIDE (PDG) RIA

Urinary progestins were quantified by RIA (Hodges et al, 1984; Chaudhuri et al., 1988; Monfort et al., 1989; Mainka et al., 1990). Briefly,

0.1 ml of urine was added to 0.9 ml of pure ethanol, vortexed and centrifuged for 10 minutes at 1500 g. Supernatants were poured off, dried down and reconstituted in 0.5 ml of phosphate buffer saline (PBS). Urine samples (0.01 ml) and standards (19.5-5000 pg per tube), in duplicate, were added to 0.1 ml of PdG antiserum (Courtauld Institute of Biochemistry, London) and tritiated PdG (7000 counts per minute) and incubated overnight at 4°C. Bound and free fractions were separated by adding charcoal dextran (0.3 ml) and incubating for 45 minutes. After centrifugation, supernatant was poured off and counted. Intra- and interassay variation was <5% (n = 10) and 6.2% and 7.3%, respectively.

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