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" DOB = date of birth; h WC = wild caught; c male/female access, oestrus/mating and offspring data are confined to the study period; A Oe, oestrus; e M, mating; ^ Al, artificial insemination; g EEJ, Electroejaculation; h San Diego Zoo, San Diego, CA; ' Smithsonian's National Zoological Park, Washington, DC;J Zoo Atlanta, Atlanta, GA; k Adventure World, Wakayama, Shirahama, Japan;1 China Conservation and Research Centre for the Giant Panda (Wolong Nature Reserve), China; Zoológico de Chapultepec, Mexico City, Mexico.

Adventure World (Wakayama, Japan)

Urinary androgens were measured in male SB 390 under two different conditions - when housed singly (2000) and when he was paired with a female conspecific (2001).

Zoológico de Chapultepec (Mexico City, Mexico)

Female SB 332 was housed in visual contact with two other females (a sibling and niece). Assessments of urinary ovarian and adrenal steroids commenced in 1998.

China Conservation and Research Centre for the Giant Panda (Wolong Nature Reserve, China)

Two case studies at this location are presented illustrating the usefulness of urinary hormone monitoring for evaluating hormonal therapy. Because SB 414 had never exhibited strong behavioural oestrus or mated, twice daily intramuscular (i.m.) injections of follicle stimulating hormone (FSH, 75 international units, IU, Metrodin HP, Serono, Rockland, MA) were administered for seven days starting on 17 March 1999. The dose was doubled on 24 March (to 150 IU), and twice-daily injections continued up to 29 March. Although mating occurred on 30 March, human chorionic gonadotrophin (hCG; 10 000 IU, Profasi 500, Serono) was administered (i.m.) on this day, and artificial insemination (AI) was performed 48 hours later (1 April); no pregnancy resulted. In 2000, SB 414 exhibited strong oestrous behaviours, mated and later gave birth.

The other case involved SB 374 who consistently displayed strong behavioural oestrus, naturally mated and also was artificially inseminated, but never produced offspring. This animal was given hCG (10 000 IU) on 25 March 1999 during her usual robust oestrus. She mated the next day (26 March), was artificially inseminated on 28 and 29 March, was given additional hCG (10 000 IU) on 30 March, but failed to give birth. In 2000, SB 374 exhibited strong behavioural oestrus again, mated and gave birth.

Urine and faeces collection

At each institution, urine and/or faeces were collected four to seven times per week. Typically, specimen collection was year-round, with the exception of female SB 414 and 374 for which collections occurred from March to May (1998 to 2000). Each urine sample (2-5 ml) was aspirated from the enclosure floor with a plastic syringe and stored in a 12 x 75-mm plastic tube or 2-ml plastic cryovials (—20°C); any sample containing faecal matter was discarded. Generally, each urine sample was collected in the morning (0700 to 0900 hours) and within one to two hours post-urination. If freshly excreted urine was unavailable, samples voided during the night were collected and marked as 'overnight' specimens. Freshly voided faeces (10 to 20 g) were collected by one to two hours post-defaecation and each stored in a sealed plastic bag (—20°C). All specimen containers were marked with the animal's identification number and date, and samples were either processed immediately or stored frozen for up to one year before analysis.

The value of urinary creatinine

Creatinine (Cr; a by-product of muscle degradation, which occurs normally in all mammals) is excreted in urine at a constant rate under homeostatic conditions. Thus Cr concentration, which can be determined colourimetrically (Taussky, 1954), serves as an indirect index of an animal's day-to-day fluid balance. If a urine specimen is 'dilute', because an animal is over-hydrated or because the urine is water contaminated (e.g. with water used to clean an enclosure floor), Cr concentration is low. Conversely, animal dehydration elevates Cr. The concentration of other excreted by-products, including hormones, rise and fall in parallel with Cr. Thus, dividing urinary hormone content (mass hormone per ml of urine excreted) by Cr concentration (mg Cr per ml urine excreted) 'indexes' the hormone concentration, which then is expressed as mass of hormone excreted per mg of Cr excreted (e.g. x ng hormone per mg Cr).

For Cr determinations in the giant panda, 0.1 ml urine (diluted 1:100 in distilled water) was added in duplicate to 96-well, flat-bottomed microtitre plates (Costar, Cambridge, MA), combined with 0.05 ml of 0.4 N picric acid and 0.05 ml of 0.75 N NaOH and incubated at room temperature (25°C) for 15 minutes. Optical density was measured at 490 nm (reference 620 nm) using a microplate reader (Benchmark, Biorad, Hercules, CA), and urine samples were compared to a single reference Cr standard (0.001 mg ml—1, Sigma Chemical Co., St Louis, MO) assayed in quadruplicate. Urine samples with Cr concentrations

<0.1 mg Cr per ml were considered too dilute (probably from water contamination). This criterion generally excluded about 7% of all samples.

Faecal extraction

Steroids must be transferred or 'extracted' from the solid faeces into a supernatant (or liquid medium) to allow measuring the hormone concentration. Numerous methods are available for processing, extracting and accurately quantifying faecal steroid metabolites (Wasser et al, 2000) but typically employ an aqueous-based, alcohol-containing (10100%) solvent, with variation, depending on whether samples are extracted wet or dry, if procedural losses are documented and if heat is used during processing (Monfort, 2003). For the giant panda, a fixed mass of dried (lyophilised; 0.09-0.1 g) or wet (0.45-0.55 g) faeces was placed in a 16 x 125-mm glass tube, combined with 80% ethanol (5 ml) in water and boiled for 20 minutes (90°C). During and after boiling, ethanol (80%) was added to maintain the pre-boil volume (5 ml). Extraction tubes were centrifuged (1500g, 20 minutes), and the supernatant was decanted into a clean glass tube. Faeces were resuspended in 80% ethanol (5 ml) and recentrifuged; both extractants were then combined and evaporated to dryness under a stream ofair in a 37°C water-bath. To concentrate dried extractant and rinse particulates adhering to the vessel wall, tubes were successively rinsed with 4, 3 and 1 ml of ethanol, allowing evaporation under a stream of air after each alcohol addition. Finally, extractants were resuspended in 1 ml of dilution buffer (195 ml of 0.2 M NaH2PO4; 305 ml of 0.2M Na2HPO4; 500 ml distilled water; 8.7 g NaCl, pH 7.0), vortexed and sonicated until dissolved. Hormonal data were expressed as mass of steroid per gram of faeces excreted.

Immunoassays

Minute hormone concentrations can be quantified in biological fluids using antibody-based immunoassays that employ radioactive (i.e. radioimmunoassay or RIA) or non-radioactive (i.e. enzyme immunoassay or EIA) reagents. RIA requires expensive equipment capable of quantifying radioisotopes whereas EIA is colourimetric, relying on less expensive, portable instruments. While both provide equivalent results, each has its advantages and disadvantages (Table 8.2).

Endocrinology of the giant panda 205 Table 8.2. Comparison of radioimmunoassay (RIA) versus enzyme immunoassay (EIA)

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