Urinary corticosteroids were measured using a single antibody RIA. Cortisol standards (7.8-1000 pg per tube; ICN Biomedicals) and 0.010.002 ml of unprocessed panda urine (depending on appropriate binding) were combined in duplicate with cortisol antiserum (0.1 ml, 1:3000; ICN Biomedicals) and tritiated hydrocortisone (10 000 counts per minute per 0.1 ml; New England Nuclear). Assays were incubated overnight (4°C), and bound and free ligands were separated by adding charcoal dextran (0.25 ml) for 30 minutes (4°C). Samples were centri-fuged (1500 g, 15 minutes), and the supernatant was counted for radioactivity. Intra-assay variation was 8% (n = 20), and interassay variation was 20% (n = 5); for females SB 374 and SB 414, interassay variation was 8% and 5% (n = 11), respectively.

Enzyme immunoassays

Oestrogen conjugate EIA

Urinary and faecal oestrogen conjugates were measured by single antibody EIA (Stabenfeldt et al., 1991) with minor modifications. Unprocessed urine (0.4-5.0 ml) or reconstituted faecal extract (1.6-5.0 ml) or oestrone-3-sulphate standards (1.95-500 pg per well) were added in duplicate to 96-well microtitre plates (Maxisorp, Nalge Nunc International, Rochester, NY) coated with the R583 oestrogen metabolite antibody (C. Munro, University of California, Davis, CA). An enzyme conjugate (0.05 ml) then was added to all wells and, after a two-hour incubation at room temperature (25°C), 0.1 ml of substrate solution (ABTS, in citrate buffer; Sigma Chemical Co.) was added. After a 30-to 60-minute incubation (25°C), depending on how fast the reaction/ colour change occurred, optical density readings (405 nm, reference 540 nm) were taken using a microplate reader (MRX, Dynex Technologies, Chantilly, VA). Intra- and interassay variation was <10% and 22% (n = 15), respectively.

Progestin EIA

Urinary progestins were measured using an established EIA (McGeehan et al., 2002). Unprocessed urine (0.01-0.001 ml) and PdG standards (range 1.98-625 pg per well) were added, in duplicate, to 96-well microtitre plates (Immulon 1, Dynex Technologies) coated with a pregnan-ediol metabolite specific antibody (C. Munro). An enzyme conjugate (0.05-ml) was added to all wells and, after a two-hour incubation (25°C), a 0.1-ml substrate solution (tetramethylbenzidine in phosphate citrate buffer; Sigma Chemical Co.) was added to all wells. After a 30-minute incubation (25°C), stop solution (0.4 N H2SO4, 0.05 ml) was added to all wells, and optical density readings (450 nm, reference 620 nm) were taken (as above). Intra- and interassay variation was 5% (n = 16) and 18% (n = 49), respectively. Interassay variation for female SB 374 and SB 414 was 8% (n = 16).

Faecal progestins were measured by a different broad spectrum progestin EIA (Cl 425; C. Munro). Extracted faeces (2.5-0.31 ml) or progesterone standards (0.78-200 pg per well; Sigma Chemical Co.) were added to 96-well microtitre plates coated with the Cl 425 progestin antibody followed by adding an enzyme conjugate (0.05 ml) and incubation at 25°C for two hours. Then an ABTS substrate solution was added to all wells (0.1 ml) and incubated (25°C) for 30 to 60 minutes; and plates were read (as above). Inter- and intra-assay variation was 9% and 5%, respectively.

Data analysis

Weekly mean hormone concentrations were expressed as the mean ± SEM (standard error of the mean) of urine samples grouped in one-week increments. Values were aligned to the Julian calendar by month, week or day from oestrogen peak or by week from natural mating. Statistical differences were measured using a Student's t-test with equal variance. For females SB 374 and SB 414, all data were aligned to the day of peak oestrogen excretion (Day 0). Baseline oestrogen concentrations were defined for each individual female as the mean oestrogen concentration outside breeding and pregnancy/pseudopregnancy intervals. Beginning in February of each year, oestrogen concentrations were determined weekly for each female. When hormone concentrations increased twofold above an individual's own baseline, frequency of endocrine monitoring was increased to one to three times daily, depending on if hormonal data were being used to schedule a timed mating or AI. A greater than six-fold increase in urinary oestrogen above basal, followed by a rapid decline to baseline concentrations within one to four days was considered presumptive evidence of ovulation. During the post-ovulatory interval, progestins were monitored bi-weekly until a female's mean progestin concentrations increased two-fold above concentrations observed during the previous week. This inflection point generally occurred four to five months post-ovulation (40 to 50 days pre-partum), at which time daily progestin assays were conducted to identify a sharp and steady decline in hormone concentrations that could alert animal care and veterinary staff that parturition was imminent.

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