ARTICLE IN PRESS Available online at www.sciencedirect.conn SCIENCE /^) DIRECT? CE^I ELSEVIER General and Comparative Endocrinology xxx (2004) xxx-xxx GENERAL AND COMPARATIVE ENDOCRINOLOGY www.elsevier.com/locate/ygcen Noninvasive monitoring of adrenocortical activity in carnivores by fecal glucocorticoid analyses K.M. Young,^ S.L. Walker,^ C. Lanthier,^ W.T. Waddell,'^ S.L. Monfort,^ and J.L. Brown^* '^ Conservation and Research Center, National Zoological Park, Smithsonian Institution, Front Royal, VA, USA Le Zoo de Granhy, Granby, Quebec, Canada " Point Defiance Zoo and Aquarium, Tacoma, WA, USA Received 4 December 2003; revised 25 February 2004; accepted 26 February 2004 Abstract Measurement of glucocorticoid metabolites in feces has become an accepted method for the noninvasive evaluation of adre- nocortical activity. The objective of this study was to determine if a simple cortisol enzyme immunoassay (EIA) was suitable for monitoring adrenocortical activity in a variety of carnivore species. Performance of the cortisol EIA was gauged by comparison to a corticosterone radioimmunoassay (RIA) that has been used for measuring glucocorticoid metabohtes in feces of numerous species. Tests for parallelism and extraction eflSciency were used to compare the cortisol EIA and corticosterone RIA across eight species of carnivores (Himalayan black bear, sloth bear, domestic cat, cheetah, clouded leopard, black-footed ferret, slender-tailed meerkat, and red wolf). The biological relevance of immunoreactive glucocorticoid metabolites in feces was established for at least one species of each Carn?vora family studied with an adrenocorticotropic hormone (ACTH) challenge. High performance liquid chromatog- raphy (HPLC) analysis of fecal extracts for each species revealed (1) the presence of multiple immunoreactive glucocorticoid me- tabohtes in feces, but (2) the two immunoassays measured different metabolites, and (3) there were differences across species in the number and polarities of metabolites identified between assay systems. ACTH chaUenge studies revealed increases in fecal me- tabohte concentrations measured by the cortisol EIA and corticosterone RIA of ~228-1145% and ----231^150% above pre-treat- ment baseline, respectively, within 1-2 days of injection. Concentrations of fecal glucocorticoid metabolites measured by the cortisol EIA and corticosterone RIA during longitudinal evaluation (i.e., >50 days) of several species were significantly correlated (P < 0.0025, correlation coefficient range 0.383-0.975). Adrenocortical responses to physical and psychological Stressors during longitudinal evaluations varied with the type of stimulus, between episodes of the same stimulus, and among species. Significant elevations of glucocorticoid metabolites were observed following some potentially stressful situations [anesthesia (2 of 3 subjects), restraint and saline injection (2 of 2 subjects), restraint and blood samphng (2 of 6 episodes), medical treatment (1 of 1 subject)], but not in aU cases [e.g., gonadotropin injection (n = 4), physical restraint only (n = 1), mate introduction/breeding (n = 1), social tension {n = 1), construction (n = 2) or relocation (n = 1)]. Results reinforced the importance of an adequate baseline period of fecal sampling and frequent collections to assess adrenocortical status. The corticosterone RIA detected greater adrenocortical responses to exogenous ACTH and stressful exogenous stimuli in the Himalayan black bear, domestic cat (female), cheetah, clouded leopard, slender-tailed meerkat, and red wolf, whereas the cortisol EIA proved superior to resolving adrenocortical responses in the black- footed ferret and domestic cat (male). Overall results suggest the cortisol EIA tested in this study offers a practical method for laboratories restricted in the usage of radioisotopes (e.g., zoological institutions and field facilities) to integrate noninvasive mon- itoring of adrenocortical activity into studies of carnivore behavior and physiology. ? 2004 Published by Elsevier Inc. Keywords: Fecal glucocorticoids; Cortisol; Corticosterone; Enzyme immunoassay; Radioimmunoassay; HPLC; ACTH; Stress; Carnivores 1. Introduction Corresponding author. Fax: 1-540-635-6506. E-mail address: jbrown@crc.si.edu (J.L. Brown). 0016-6480/$ - see front matter ? 2004 Published by Elsevier Inc. doi:10.1016/j.ygcen.2004.02.016 The mammalian order Carn?vora represents a diverse group of ~274 extant species that vary considerably in ARTICLE IN PRESS K. M. Young et al. I General and Comparative Endocrinology xxx (2004) xxx?xxx their behavior, ecology, morphology, and physiology (Gittleman, 1989; Macdonald, 1992). Although some debate continues regarding the phylogeny of carnivores, one of the accepted taxonomic arrangements divides the order into 10 families: Canidae (dogs, jackals, foxes, and wolves); Ursidae (bears); Procyonidae (raccoons, coatis, and kinkajou); Mustehdae (badgers, skunks, otters, and weasels); Viverridae (civets, genets); Herpestidae (mon- gooses, meerkats); Hyaenidae (hyenas, aardwolf); Feli- dae (cats), Otariidae (walrus, sea Hons, and fur seals), and Phocidae (earless seals) (Wozencroft, 1989a,b). In a recent report, the World Conservation Union identified 76 carnivore taxa threatened with extinction globally and four taxa lost to extinction since the mid-nineteenth century (lUCN, 2002). Captive management of carnivores has afforded conservationists the opportunity to gather detailed sci- entific information concerning the unique biology of these species. For example, our considerable knowledge of wild felid reproductive endocrinology and gamete biology has been gained from in-depth studies only possible with frequent access to zoo-maintained animals (Brown et al., 1994, 2001; Wildt and Roth, 1997; Wildt et al., 1984, 1986a,b, 1988). However, inappropriate captive environments are known to have deleterious effects on the behavior and physiology of mammals (Carlstead, 1996; Estep and Dewsbury, 1996). Adverse physical, physiologic, and psychogenic stimuli evoke a series of biological responses (behavioral and/or hor- monal changes) that help animals cope with stressful stimuH (Moberg, 1985, 1987, 2000), including those imposed by captivity. In mammals, stress modulates the activities of the hypothalamic-pituitary-adrenal (HPA) axis and sym- pathoadrenal axis, releasing a variety of hormones to counter aversive stimuli (Axelrod and Reisine, 1984; Breazile, 1987; Melmed and Kleinberg, 2003; Stewart, 2003). Activation of the HPA axis during stress en- hances the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary, stimulating the synthesis and secretion of glucocorticoids from the ad- renal cortices (Axelrod and Reisine, 1984; Melmed and Kleinberg, 2003; Stewart, 2003). Chronic stress and heightened levels of HPA axis hormones (i.e., gluco- corticoids, ACTH, and corticotropin releasing hor- mone) can have detrimental effects, including inhibition of normal reproductive function (deCatanzaro and MacNiven, 1992; Dobson and Smith, 1995; Liptrap, 1993; Rivier and Rivest, 1991), suppression of the im- mune system and atrophy of tissues (Munck et al., 1984; Stewart, 2003). Elevated levels of glucocorticoids re- sulting from chronic stress may also cause depression, hypertension, gastrointestinal ulc?ration, electrolyte imbalances, calcium loss and bone mass reduction, and inhibition of growth (Breazile, 1987; Stewart, 2003). Thus, improving the health and general well-being of carnivores in captivity requires identifying stressful en- vironmental conditions or management practices and developing mitigating strategies. While blood glucocorticoid concentrations are ac- cepted indices of stress (Broom and Johnson, 1993), their usefulness in long-term studies with intractable wildlife species is limited due to the circadian rhythm and pulsatile nature of glucocorticoid secretion (Fox et al., 1994; Monfort et al., 1993; Stewart, 2003; Thun et al., 1981) and the possible induction of a stress response during samphng procedures (Cook et al., 2000; Kenagy and Place, 2000; Reinhardt et al., 1990, 1991). Con- versely, the excretion of metabolized blood steroids into feces (Macdonald et al., 1983; Taylor, 1971) permits the monitoring of physiological functions without distur- bance to animal subjects. Analysis of fecal steroid me- tabolites also provides a more representative measure of adrenocortical activity over time because the pooling of metabolites during excretion dampens the episodic se- cretion of blood glucocorticoids. Measurement of fecal glucocorticoids has been used to investigate adrenocortical activity during exposure to stressful stimuli, such as novel environment, transpor- tation, social tension and aggression, human distur- bances, and exposure to predators, in a diverse number of species, including fehds (Terio et al., 1999; Wieleb- nowski et al., 2002), canids (Creel et al., 1996, 1997), hyenas (Goymann et al., 1999, 2001), primates (Wallner et al., 1999), lagomorphs (Teskey-Gerstl et al., 2000), and cervids and bovids (Dehnhard et al., 2001; Millsp- augh et al., 2001; Morrow et al., 2002; Palme et al., 2000; Schwarzenberger et al., 1998). Most of the studies in carnivores have successfully evaluated adrenocortical responses to an ACTH challenge by measuring gluco- corticoid metabolites in feces using a commercially available corticosterone radioimmunoassay (RIA) (Graham and Brown, 1996; Monfort et al., 1998; Terio et al., 1999; Wasser et al., 2000; Wielebnowski et al., 2002). By contrast, attempts to use antibodies specific to Cortisol have produced inconsistent results. Jurke et al. (1997) found differences in basal fecal glucocorticoid metabolite concentrations for cheetahs classified as 'nervous' or 'calm' by zookeepers using a cortisol en- zyme immunoassay (EIA), while others were unable to monitor adrenal activity in felids using a commercial RIA specific for cortisol (Graham and Brown, 1996; Terio et al., 1999). Following ACTH stimulation and dexamethasone suppression of the adrenal gland, Schatz and Palme (2001) found adrenocortical activity could be monitored in the domestic dog using a cortisol EIA. They further determined an antibody produced against 11-oxoetiocholanolone and measuring 11,17-dioxoand- rostanes was effective for monitoring adrenal function in the domestic cat. However, this antibody was not ef- fective for use in the spotted hyena (Goymann et al., 1999). ARTICLE IN PRESS K. M. Young et al. I General and Comparative Endocrinology xxx (2004) xxx-xxx Recently, a cortisol EIA has proved useful for non- invasively monitoring adrenocortical activity in the black-footed ferret (Young et al., 2001) and red wolf (Walker, 1999). Based on these findings, a study was conducted to determine if the cortisol EIA was suitable for monitoring adrenocortical activity in a variety of carnivore species and how it performed when compared with the corticosterone RIA. The objectives were to: (1) validate each immunoassay system for measuring glu- cocorticoid metabolites in feces of an array of carnivore species, (2) determine the correspondence in glucocor- ticoid data generated by the two immunoassays, (3) in- vestigate the potential for fecal glucocorticoid metabolite analysis to detect adrenocortical responses to stressful stimuli, and (4) determine the suitability of each immunoassay for use as a standardized technique for noninvasive monitoring of adrenocortical activity in carnivore species. 2. Materials and methods 2.1. Study animals Adult animals from five Carn?vora families (Canidae, Felidae, Herpestidae, Mustelidae, and Ursidae) were evaluated in this study. Study subjects were housed under comparable conditions at the following zoological facilities across North America: Himalayan black bear (? = 1, Le Zoo de Granby, Granby, QC; ? = 1, Little Rock Zoo, Little Rock, AK); sloth bear (? = 1, Little Rock Zoo, Little Rock, AK); domestic cat (? = 2, Conservation and Research Center, Front Royal, VA); cheetah (? = 2, White Oak Conservation Center, Yulee, PL); clouded leopard (? = 3, Conservation and Re- search Center, Front Royal, VA); black-footed ferret (? = 2, Toronto Zoo, Toronto, ON), slender-tailed meerkat {n = 2, Saint Louis Zoo, Saint Louis, MO); and red wolf (n = 1, Point Defiance Zoo and Aquarium, Tacoma, WA; ? = 1, Red Wolf Breeding Facility, Graham, WA). The level of visual, auditory, and ol- factory exposure to conspecifics and heterospecifics varied for each study subject. In general, study subjects were fed high-quality commercial diets that, depending on the species, were frequently supplemented with other items, including whole animals (e.g., rabbits, mice, mealworms, and crickets), animal parts (e.g., chicken), vegetables (e.g., carrots, potatoes, and yams), and/or fruit (e.g., grapes, apples, oranges, and raisins). All an- imals had ad libitum access to fresh water. 2.2. ACTH challenges To determine the biological relevance of glucocorti- coid metabolites excreted in feces, an ACTH challenge was conducted in at least one species from each Car- n?vora family. The species, number and sex of subjects, ACTH dosages, and methods used to facilitate ACTH administration are presented in Table 1. Most species received a single intramuscular (i.m.) injection of a slow- release ACTH gel (Wedgewood Pharmacy, Sewell, NJ or Medicine Shop, Front Royal, VA). Slender-tailed meerkats received two separate i.m. injections of ACTH gel (10 lU each; 20 lU total) with 2 h between injections. The domestic cats received a series of five 0.125 mg in- travenous infusions of ACTH liquid (Cortrosyn, ~62.5IU total; Organon, West Orange, NJ) into the jugular vein at 1.5 h intervals over 6h. To facilitate the administration of ACTH, animals were physically re- strained (squeeze cage, catchpole or manually) or chemically immobilized. Prior to the ACTH challenge, a surgical plane of anesthesia was induced in the domestic cat (ketamine hydrochloride, 20mg/kg body weight, i.m.; Vetalar, Parke-Davis, Morris Plains, NJ; and ace- promazine mal?ate, 0.2 mg/kg body weight, i.m.; Ayerst Laboratories, Rouses Point, NY) and cheetah (tileta- mine hydrochloride and zolazepam hydrochloride, Telazol, 3.5mg/kg body weight, i.m.; Fort Dodge Lab- oratories, Fort Dodge, lA). Anesthesia was maintained during the procedures for both species with isoflurane gas (Aerane, 1-2%; Anaquest, Madison, WI). Animals were fasted for 12-24 h prior to anesthesia. Feces were collected from each species during their daily husbandry routine. Most animals defecated once per day, but occasionally no defecations or multiple scats were found. Generally, sample collections com- menced 1-10 days before ACTH injection (day 0) and continued for 5-10 days following the adrenocortical stimulation. Fecal material was stored frozen (-20 ?C) in plastic bags or tubes until processing and analysis. 2.3. Longitudinal evaluations and exogenous Stressors To assess the ability of fecal glucocorticoid analysis to detect acute changes in adrenocortical activity, sev- eral species were evaluated over extended periods of time (i.e., >50 days) that included exposure to different types of potentially stressful stimuli. Longitudinal monitoring of fecal glucocorticoids also provided sufn- cient data for comparisons between concentrations of steroid metabolites measured by the two immunoassay systems. The longitudinal assessment periods for three felid species encompassed single episodes of chemical immo- bilization. A female cheetah was immobilized with a tiletamine-zolazepam combination (Telazol, 3.5mg/kg body weight, i.m.) for a routine physical examination. During an assisted reproduction study, domestic cat (? = 1) and clouded leopard (? = 1) females were re- strained for injection of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) to induce follicular development and ovulation, respec- ARTICLE IN PRESS K. M. Young et al. I General and Comparative Endocrinology xxx (2004) xxx?xxx Table 1 ACTH challenges in species from 5 families of carnivores Species Sex ACTH Dose Restraint Glucocorticoid MetaboUte concentration Rise (%) (lU) method metabolite (ng/g feces) Baseline (?SEM) Peak - Himalayan black bear M 955 Squeeze cage Cortisol 107.9 ?8.0 432.6 401 Corticosterone 34.5 ?3.9 197.9 573 Domestic cat M 62.5 Anesthesia Cortisol 181.0?40.4 1673.2 925 Corticosterone 348.1 ?70.6 2015.6 579 F 62.5 Anesthesia Cortisol 745.1 2068.5 278 Corticosterone 746.2 2314.6 310 Cheetah F 400 Anesthesia Cortisol 628.4 ?95.3 7193.2 1145 Corticosterone 436.6 ?107.7 5941.1 1361 Clouded leopard M 400 Squeeze cage Cortisol 223.1 ?14.8 569.5 255 Corticosterone 579.2 ?68.5 3225.9 557 M 400 Squeeze cage Cortisol 176.2 ?30.5 1350.1 766 Corticosterone 85.3 ?10.5 3541.1 4150 F 400 Squeeze cage Cortisol 183.6 ?46.9 431.8 235 Corticosterone 555.4 ?190.3 5664.7 1020 Black-footed ferret F 2 Squeeze cage Cortisol 220.2 ?19.3 548.6 249 Corticosterone 114.5?8.8 264.3 231 Slender-tailed meerkat M 20 Manual Cortisol 110.2?17.5 614.1 557 Corticosterone 175.0 ?32.4 2672.8 1527 M 20 Manual Cortisol 169.5 ?14.4 386.9 228 Corticosterone 173.5 ?5.3 554.4 320 Red wolf M 140 Catchpole/ Cortisol 525.6 ?94.6 2130.7 405 manual Corticosterone 162.1 ?22.6 1875.0 1160 With two exceptions, ACTH was administered to subjects by a single intramuscular injection. For the domestic cats, ACTH was administered via five intravenous infusions (12.5 lU each) into the jugular vein over a period of 6h. Slender-tailed meerkats received two separate ACTH injections (10 lU each) with 2 h between injections. Restraint method refers to the techniques used to facilitate the injection of exogenous ACTH. Manual restraint was defined as the use of hands to restrain the animal. Glucocorticoid metabohtes were measured in extracts prepared from fecal samples with the Cortisol EIA and corticosterone RIA. Baseline represents the average glucocorticoid metabolite concentrations for all pre-treatment samples. Peak refers to the ACTH-induced peak in metabolites. Rise represents the peak expressed as a percentage of the pre-treatment baseline. lively. To permit laparoscopic examination of the ovary and confirmation of ovulation, the domestic cat was immobilized with a tiletamine-zolazepam combination (Telazol, 5mg/kg body weight, i.m.) and the clouded leopard was anesthetized using ketamine hydrochloride (Vetalar, 12mg/kg body weight, i.m.). Cats were main- tained in a surgical plane of anesthesia by delivering isoflurane gas (Aerane, 1-2%; Anaquest, Madison, WI) following intubation. Only the clouded leopard ovulated and was subsequently artificially inseminated. Feces were collected regularly during the range of days (53-56 days) examined, providing a total of 51 samples for the cheetah and 50 samples each for the domestic cat and clouded leopard. A black-footed ferret and two red wolves were eval- uated during periods of physical restraint. Fecal samples were collected from a female (? = 70 samples) and male (n = 60 samples) red wolf during the natural reproduc- tive season. The female was physically restrained with a catchpole on six occasions to collect blood for deter- mination of reproductive status and timing of artificial insemination. The male red wolf experienced several potentially stressful stimuli: (1) restraint with a catch- pole to facilitate the administration of a sahne injection (1.75 ml, i.m.); (2) construction in the adjacent enclosure on two occasions; and (3) relocation to an adjacent en- closure within the zoological facihty. Fecal samples (? = 53) also were evaluated over a 54-day period from a female black-footed ferret that experienced single ep- isodes of cage restraint and cage restraint combined with saline injection (0.2 ml, i.m.). Fecal samples (? = 100) were analyzed from a fe- male sloth bear during a 241-day period that included the introduction of a male and subsequent breeding. This subject also suffered from a chronic nematode (ascarids) infection and frequently defecated abnormal stools. Although treated periodically with deworming medication (mebendazole, Telmin; Pitman-Moore, Mundelein, IL), the nematode infection persisted throughout the study period. Fecal samples (? = 65) were evaluated over a 151-day period from a female Himalayan black bear that was housed with a male. An episode of social tension uncharacteristic for the pair of Himalayan black bears occurred on a single day during the study period. During this episode, the bears exhibited a series of aggressive displays, in- ARTICLE IN PRESS K. M. Young et al. I General and Comparative Endocrinology xxx (2004) xxx-xxx eluding snarling, mouthing, and pounding of the ground. 2.4. Extraction of steroids from feces Frozen fecal samples were dried using a Savant Instruments Speedvac Rotary Evaporator and then pulverized to a fine powder. Hormones were extracted from dried fecal samples using established methods for extraction of ovarian (Brown et al., 1994, 1996) and adrenocortical (Graham and Brown, 1996) ste- roids. Briefly, ~0.2g well-mixed powdered feces was boiled in 5 ml of 90% ethanol for 20 min. After cen- trifugation (500g, 20 min), the supernatant was trans- ferred into a glass tube and the pellet was resuspended in an additional 5 ml of 90% ethanol, vortexed for 1 min, and recentrifuged for 20 min at 500g. Com- bined ethanol supernatants were dried under air and resuspended in 1 ml of 100% methanol. Methanol extractants were vortexed (Imin), sonicated (15 min) and revortexed (30 s) prior to decanting into a plastic tube for storage at -20 ?C until assayed. The efliciency of steroid extraction from feces of each species was evaluated by adding radiolabeled glucocorticoid (^H- cortisol or ^H-corticosterone; 4000-8000 dpm) to a subset of fecal samples prior to boiling extraction. The mean recoveries of ^H-cortisol from fecal extracts were: domestic cat, 80.8% (? = 50 samples); cheetah, 85.0% (? = 71); clouded leopard, 87.1% (? = 97) and black-footed ferret 81.5% (? = 58). The mean recov- eries of ^H-corticosterone from fecal extracts were: Himalayan black bear, 85.5% (? = 21); black-footed ferret, 85.8% (? = 58); slender-tailed meerkat, 75.3% (? = 46); sloth bear, 85.8% (? = 30) and red wolf, 73.7% (? = 71). 2.5. Fecal glucocorticoid metabolite analyses 2.5.1. Cortisol enzyme immunoassay A Cortisol EIA was used to analyze fecal extracts by a modification of methods described by Munro and Lasley (1988). The assay employed a cortisol-horse- radish peroxidase ligand and antiserum (No. R4866; C.J. Munro, University of California, Davis, CA) and Cortisol standards (hydrocortisone; Sigma-Aldrich, St. Louis, MO). The polyclonal antiserum was raised in rabbits against cortisol-3-carboxymethyloxime linked to bovine serum albumin and cross-reacts with cortisol 100%, prednisolone 9.9%, prednisone 6.3%, cortisone 5% and <1% with corticosterone, desoxycorticoster- one, 21-desoxycortisone, testosterone, androstenedione, androsterone, and 11-desoxycortisol (C.J. Munro, pers. comm.). The EIA was performed in 96-well microtiter plates (Nunc-Immuno, Maxisorp Surface; Fisher Sci- entific, Pittsburgh, PA) coated 14?18 h previously with cortisol antiserum (50 |.il/well; diluted 1:20,000 in coating bufi"er; 0.05 M NaHCOs, pH 9.6). Fecal ex- tracts evaporated to dryness and diluted (bears 1:10- 1:30, felids 1:30-1:115, ferrets 1:20, meerkats 1:15, and wolves 1:60-1:100) in steroid buffer (0.1 M NaP04, 0.149 M NaCl, pH 7.0) were assayed in duplicate. Cortisol standards (50^1, range 3.9-1000 pg/well, di- luted in assay buffer, 0.1 M NaP04, 0.149 M NaCl, 0.1% bovine serum albumin, pH 7.0) and sample (50|il) were combined with cortisol-horseradish per- oxidase (50^1, 1:8500 dilution in assay buffer). Fol- lowing incubation at room temperature for 1 h, plates were washed five times before 100(il substrate buffer [0.4 mM 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt, 1.6 mM H2O2, 0.05 M citrate, pH 4.0] was added to each well. After incubation on a shaker for 10-15 min, the absorbance was measured at 405 nm. Parallel displacement curves were obtained for each species by comparing serial dilutions of pooled fecal extracts and the cortisol standard preparation. Intra- and interassay coefficients of variation were 6.4% (? = 26 replicates of a single sample) and 11.0% (? = 57 assays), respectively. Assay sensitivity was 3.9 pg/well at maximum binding. Cortisol metabolite concentrations are expressed as nanograms per gram dry fecal matter (ng/g). 2.5.2. Corticosterone radioimmunassay Fecal extracts were also analyzed using a double-an- tibody '25i RIA (ICN Biomedicals, Costa Mesa, CA) for corticosterone according to the manufacturer's instruc- tions, except all reagent volumes were halved. The polyclonal antiserum was raised in rabbits against cor- ticosterone-3-carboxymethyloxime coupled to bovine serum albumin and cross-reacts with corticosterone 100%, desoxycorticosterone 0.34%, testosterone 0.1%, cortisol 0.05%, aldosterone 0.03%, progesterone 0.02%, androstenedione 0.01%, 5a-dihydrotestosterone 0.01%, and <0.01% with afl other steroids tested (manufac- turer's data). For each species, serial dilution of pooled fecal extracts produced a displacement curve parallel to the corticosterone standard preparation. Samples were diluted in steroid buffer (bears 1:10-1:15, felids 1:30- 1:1000, ferrets 1:40, meerkats 1:50-1:500, and wolves 1:60-1:200) and analyzed in duplicate. Intra- and in- terassay coefficients of variation were 5.0% (? = 20 rephcates of a single sample) and 14.4% (? = 33 assays), respectively. Sensitivity of the assay at maximum bind- ing was 12.5ng/ml. Corticosterone metabolite concen- trations are expressed as nanograms per gram dry fecal matter (ng/g). 2.6. High-performance liquid chromatography The number and relative proportions of immunore- active glucocorticoid metabolites in feces were deter- mined by reverse-phase high-performance liquid ARTICLE IN PRESS K. M. Young et al. I General and Comparative Endocrinology xxx (2004) xxx?xxx chromatography (HPLC) as previously described by Monfort et al. (1991). For each species, 4-6 extracts were prepared from the fecal sample containing the peak value of glucocorticoid metabolites following injection of exogenous ACTH. Fecal extracts were pooled, evaporated to dryness, and reconstituted in 0.5 ml phosphate-buffered saline (0.01 M NaP04, 0.14M NaCl, 0.5% bovine serum albumin, pH 5.0) before loading the total volume on a pre-conditioned C-18 matrix cartridge (Spice Cartridge; Analtech, Newark, DE). The cartridge was washed with 5 ml distilled water and the total ste- roids eluted with 5 ml of 100% methanol, evaporated to dryness, then reconstituted in 300^1 of 100% methanol containing ^H-cortisol, ^H-corticosterone and ^H-des- oxycorticosterone (~4000-8000 dpm for each radiola- beled glucocorticoid). Filtered fecal extracts (55 |il) were separated on a Microsorb C-18 column (Reverse Phase Microsorb MV 100 Cl8, 5 (im diameter particle size; Varian, Woburn, MA) using a linear gradient of 20- 100% methanol in water over 80 min (1 ml/min flow rate, 1ml fractions). A subsample of each fraction (100 (il) was assayed for radioactivity to determine the retention times for the radiolabeled reference tracers. The re- mainder of each fraction (900 (il) was evaporated to dryness, reconstituted in 125 \? steroid buffer and an aliquot (50^1) assayed singly in each immunoassay as described above. 2.7. Analysis of data For the ACTH challenges, individual pre-treatment baselines were calculated as the average of fecal samples collected on all days before the ACTH in- jection, including the day of treatment (day 0). Adrenocortical responses to stimulation with ACTH are expressed as a percentage of the pre-treatment baseline, with the baseline value being equivalent to 100%. Several measures were calculated to summarize fecal hormone values during the longitudinal steroid evalua- tion of each animal: (1) an overall mean of all samples for the collection period; (2) a mean baseline that ex- cludes all values greater than the overall mean plus 1.5 standard deviations (SD); and (3) a peak mean that in- cluded all values greater than the overall mean plus 1.5 SD. An increase in glucocorticoid metabolites following exposure to an exogenous Stressor was considered sig- nificant if the value exceeded the mean baseline plus 3 SD (Goymann et al., 1999). Responses to stressful stimuH (i.e., post-stressor rise in metaboHtes) are pre- sented as a percentage of the mean baseline, which was designated as 100%. Data from the longitudinal evaluations were cor- rected for a nonnormal distribution by performing a common logarithm (logio) transformation. Pearson product moment correlation analysis was used to de- termine the relationship between fecal metabolites (logio transformed data) measured by the cortisol EIA and corticosterone RIA. Average data are presented as mean ? standard error (SEM). The level of significance defined for statistical tests was P < 0.05. 3. Results 3.1. ACTH challenges In all species, fecal glucocorticoid metabolites in- creased sharply to peak concentrations 1-2 days fol- lowing ACTH administration and then declined rapidly. Cortisol and corticosterone immunoassays generated temporally similar fecal glucocorticoid me- tabolite excretion profiles for most species, but mea- sured differing levels of immunoreactivity (Fig. 1). Concentrations of glucocorticoid metabolites during the pre-treatment baseline and at the ACTH-induced peak are shown in Table 1. The corticosterone RIA detected a greater ACTH-induced rise from pre-treat- ment baseline glucocorticoid metabolite concentrations in the Himalayan black bear, cheetah, clouded leop- ard, slender-tailed meerkat, red wolf, and female do- mestic cat, whereas the cortisol EIA measured a greater rise in metabolite concentrations for the black- footed ferret and male domestic cat (see Table 1 for comparison). 3.2. Longitudinal evaluations and exogenous Stressors Figs. 2-A depict longitudinal profiles of cortisol and corticosterone metabolites excreted in feces of seven different carnivore species. Mean overall, mean baseline, and mean peak concentrations of glucocorticoid me- tabolites are presented in Table 2. Significant correla- tions were found between glucocorticoid metabolites quantified by the cortisol EIA and corticosterone RIA in feces of all carnivores (Table 3). 3.2.1. Physical restraint, nonanesthetic injections, and blood sampling Considerable intra- and interindividual variation was observed in adrenocortical responses to manipu- lations involving physical restraint (Figs. 2 and 3). Cage restraint of a black-footed ferret (Fig. 2A), gonadotropin injection of a domestic cat (Fig. 3A), and clouded leopard (Fig. 3B), and most episodes of blood sampling for a female red wolf (Fig. 2B) did not elevate fecal glucocorticoid concentrations above the set level of significance (mean baseline + 3 SD). In contrast, saline injection combined with physical re- straint increased fecal glucocorticoid metabolite con- centrations above mean baseUne in the black-footed ARTICLE IN PRESS K. M. Young et al. I General and Comparative Endocrinology xxx (2004) xxx-xxx A 500' 400' 300' 200' 100 0 Himalayan black bear cfsai^y. B 2500 2000 1500- 1000- 500- 0 I'l'I'I'I'T'T'T'I'l?! 10-8 -6 -4 -2 0 2 4 6 8 10 Domestic cat I ' I ' I I r I I ' I ' I ' I ' I ' I I I ' 10-8 -6 -4 -2 0 2 4 6 8 10 D 4000-1 3000 2000 1000 H 0 Clouded leopard I ' I ? I ? I ? I ??I ' I ' I ? I '?I ' I ' 10-8 -6 -4 -2 0 2 4 6 8 10 3000- 2500- 2000- 1500 1000- 500- 0 Slender-tailed Meerkat i I ' I ' I ' I ' I 10-8 -6 -4 -2 0 2 4 6 8 10 - Cortisol ELA. ? Corticosterone RIA C 8000 6000 4000- 2000- 0 Cheetah I ' I ' I ' I ' I ' I ' I ?" I ? I ' I 1 I " 10 -8 -6 -4 -2 0 2 4 6 8 10 Black-footed ferret 600 ? 400- 20O G 0 2500 2000 1500 1000 500 0 I ' I ' I ' I ? I ' I ' I ' I ' I ' I ? 1 10-8 -6 -4 -2 0 2 4 6 8 10 Red wolf I ' I ' I ' I ' I ' I ? I ' I ' I ? I I I 10 -8 -6 -4 -2 0 2 4 6 8 10 Days From ACTH Administration Fig. 1. Glucocorticoid metabolites in feces of a Himalayan black bear (A), domestic cat (B), cheetah (C), clouded leopard (D), black-footed ferret (E), slender-tailed meerkat (F), and red wolf (G) before and after administration of exogenous ACTH (day 0). The dosages of ACTH administered to each species are presented in Table 1. Immunoreactive metabolites in extracts prepared from fecal samples were measured with a cortisol EIA (open circles) and corticosterone RIA (closed circles). ferret (Fig. 2A) and potentially in the male red wolf (Fig. 2C). One day following the saline injection in the black-footed ferret, fecal cortisol and corticosterone metabohte concentrations were 199% (338.0 ng/g) and 189% (192.8 ng/g) above mean baseline, respectively. Three and four days after saline injection in the male red wolf, fecal cortisol metabolites were 264% (936.0 ng/g) and 304% (1078.8 ng/g) above baseline, respectively. Restraint of the female red wolf for blood sampling on day 23 was followed by a significant in- crease in cortisol metabohtes (301% above mean baseline; 657.0 ng/g) on day 25, whereas blood sam- pling on day 30 was followed by a significant increase in cortisol (298% above mean baseline; 805.2 ng/g) and corticosterone metabolites (409% above mean baseline; 896.3 ng/g) on day 31. ARTICLE IN PRESS K. M. Young et al. I General and Comparative Endocrinology xxx (2004) xxx?xxx 500 400 300 200 100 0 Cortisol EIA Corticosterone RIA Black-footed ferret (female) Restraint Restraint & saline injection ? e o S 'S u 1 ? 0 5 10 15 20 25 B 1500 1 30 35 40 45 50 55 1250 1000 750 500 H 250 Red wolf (female) Multiple restraints & blood sampling I I i , i I O I" "I" "I" "l""l" '?l""l""l""l "" I "" l'""l '?" l""l "" !?" ' I" "l""l" "|i ' "I O 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 C 2000 1600 1200 800 400 Red wolf (male) Construction Restraint & saline injection 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 Days Fig. 2. Longitudinal profiles of fecal cortisol (open circles) and corticosterone (closed circles) metabolites for a female black-footed ferret (A), female red wolf (B), and male red wolf (C). All three animals experienced procedures that involved physical restraint. The male red wolf also experienced minor construction in an adjacent enclosure and relocation to an adjacent enclosure. 3.2.2. Chemical immobilization Fecal glucocorticoid metabolite concentrations in the clouded leopard and cheetah, but not the domestic cat, increased after chemical immobilization (Fig. 3). In the clouded leopard, fecal glucocorticoid concen- trations increased 472% (cortisol metabolites) and 666% (corticosterone metabolites) in response to an- esthesia combined with laparoscopic artificial insemi- nation, reaching maximum values 3 days (1175.1 ng/g) and 2 days (2013.5 ng/g) after anesthesia, respectively. Fecal cortisol and corticosterone metabolite concen- trations 2 days (1026.4 ng/g feces; 412% above mean baseline) and 3 days (1230.0 ng/g; 407% above mean baseline) following anesthesia, respectively, were also significantly elevated. Two days after chemical immo- bilization of the cheetah, fecal metabolites of cortisol and corticosterone increased 390% (2434.3 ng/g) and 1540% (3578.0ng/g) above baseline, respectively. Be- cause all fehds were fasted for 12-24 h prior to anes- thesia, the animals failed to defecate either on the day of (domestic cat) or following (clouded leopard, cheetah) immobilization. Therefore, fecal samples col- lected from nondomestic felids on days 2 and 3 fol- lowing anesthesia represent the first and second defecations after the immobilization event, respec- tively. 3.2.3. Environmental disturbances For the male red wolf, fecal glucocorticoid metabo- lites were not elevated or did not surpass the set level of significance (mean baseline + 3 SD) during construction or following relocation (Fig. 2C). ARTICLE IN PRESS K. M. Young et al I General and Comparative Endocrinology xxx (2004) xxx-xxx Cortisol EIA Corticosterone RIA A 800 600 400 200 Domestic cat liCG Anesthesia ' I ' ? ' ' t I ? I ' I ' ' ' ' I ? ' ? ' ' ' ' ' ' I I ? ' ? I ? ' ? ' I I 10 15 20 25 30 35 40 45 50 55 J5X1 ?530 B o a 'S u o u e O 2500 2000 1500 1000 500 0 Clouded leopard Anesthesia ? I ? ' ' ' I ' ' ? ' 1 ' ' ' ' I ' ' ' ? I ' ' 5 10 15 20 25 30 35 40 45 50 55 4000 ? Cheetah 3000 2000 1000 Fig. 3. Longitudinal profiles of fecal cortisol (open circles) metabolites and corticosterone (closed circles) metabolites for three felid species subjected to anesthesia. A domestic cat (A) and clouded leopard (B) were treated with gonadotropins before laparoscopic examination of the ovaries for ev- idence of ovulation. Only the clouded leopard was artificially inseminated. A cheetah (C) was anesthetized to faciUtate a routine physical examination. 3.2.4. Social tension Concentrations of fecal glucocorticoid metabolites were not elevated in the female Himalayan black bear following a series of aggressive displays between this study subject and a co-housed male (Fig. 4A). 3.2.5. Mate introduction Fecal glucocorticoid metabolite excretion did not increase in the female sloth bear following mate intro- duction or breeding. Instead, significantly elevated concentrations were associated with abnormal defeca- tions and treatment with deworming medication (Fig. 4B). Peaks in fecal glucocorticoid metabolite con- centrations following day 210 coincide with the rise and sustained elevation in fecal progestins during pregnancy (data not shown). 3.3. HPLC 3.3.1. Felidae The majority of cortisol and corticosterone immu- noreactivity in eluates from the domestic cat was asso- ciated with polar peaks eluting in fractions 7-23 (86% of total immunoreactivity) and fractions 12-26 (89%), re- spectively (Fig. 5A). A minor portion of the immuno- reactivity (<4%) detected by the cortisol EIA eluted in fractions slightly more polar (37^0) than the ^H-corti- sol reference tracer. Analysis of HPLC-purified fecal eluates from the cheetah revealed that 47% and 89% of the cortisol EIA and corticosterone RIA immunoreactivity, respectively, corresponded to single polar peaks (fractions 8-18 for the EIA and 9-18 for the RIA) (Fig. 5B). Both immu- ARTICLE IN PRESS 10 K. M. Young et al. I General and Comparative Endocrinology xxx (2004) xxx?xxx ?--^ Cortisol EIA ??? Corticosterone RIA Himalayan black bear ?Si 2 o 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 0 20 40 60 80 100 120 140 160 180 200 220 240 Days Fig. 4. Longitudinal profiles of fecal cortisol (open circles) and corticosterone (closed circles) for a female Himalayan black bear (A) and sloth bear (B) during the presence of conspecific males. A period of social tension involving aggressive displays between the female and male Himalayan black bears was observed on a single day (A). Introduction of a male to the female sloth bear was followed by breeding activity (B). The female sloth bear had a persistent nematode infection during the period of evaluation. Asterisks indicate abnormal defecations and treatments with deworming medication. The horizontal bar denotes the rise and sustained elevation of fecal progestins during pregnancy (B). Table 2 Mean overall, baseline, and peak concentrations of fecal glucocorticoid metabolites for individuals of several carnivore species examined by lon- gitudinal steroid evaluation Species Sex Samples (n) Glucocorticoid metabolite Mean overall (?SEM) Mean baseline (?SEM) Mean Peak (?SEM) Himalayan black bear Sloth bear F F Domestic cat F Cheetah F Clouded leopard F Black-footed ferret Red wolf F M F 65 100 50 51 50 53 60 78 Cortisol Corticosterone Cortisol Corticosterone Cortisol Corticosterone Cortisol Corticosterone Cortisol Corticosterone Cortisol Corticosterone Cortisol Corticosterone Cortisol Corticosterone 107.3? 12.8 60.6 ?7.5 76.9 ?23.8 127.7?24.0 234.1?11.1 377.4?13.2 751.1 ?66.8 298.5 ?67.5 282.9 ?27.8 355.0 ?47.1 184.7 ?8.3 110.2?4.5 462.1 ?42.8 245.3 ?21.5 309.6 ?19.6 264.9 ?18.1 80.7 ?7.1 45.1 ?4.3 48.7 ?3.1 64.1 ?6.5 213.6?8.8 354.7 ?11.2 618.5?49.5 232.9 ?16.4 248.8 ?14.7 302.2 ?28.1 170.1 ?6.4 101.9?3.6 354.8 ?26.5 212.7 ?13.0 270.3 ?12.6 218.9?9.4 369.6 ?42.3 212.8 ?23.0 1428.8 666.5 ?76.6 384.6 ?7.8 544.5 ?7.7 1850.3 ?202.6 3578.0 1100.7 ?74.3 1621.8?391.7 324.6 ?6.5 174.7 ?4.6 1159.4?47.6 702.5 ?133.6 780.3 ?44.8 667.1 ?42.1 Glucocorticoid metabolite refers to the immunoassay, cortisol EIA or corticosterone RIA, used to measure steroids in fecal samples. Mean overall represents the average concentration of metabolites (ng/g feces) in all fecal samples (n) from the collection period. Mean baseline represents the average metabolite concentrations for all samples that fall below the overall mean-i-1.5 SD. Mean peak represents the average metaboUte concentration for all samples that are greater than the overall mean-i-1.5 standard deviations. All values are expressed as means ? standard error unless there was only one value in the category. ARTICLE IN PRESS K. M. Young et al I General and Comparative Endocrinology xxx (2004) xxx-xxx 11 Table 3 Correlation matrix describing the relationship between glucocorticoid metabolites measured with the cortisol enzyme immunoassay and cortico- sterone radioimmunoassay in feces of seven different carnivore species Species Sex Samples (n) Correlation coefficient (r) P value Himalayan black bear F Sloth bear F Domestic cat F Cheetah F Clouded leopard F Black-footed ferret F Red wolf M F 65 100 50 51 50 53 60 78 0.975 0.660 0.786 0.728 0.794 0.830 0.383 0.652 -Cortisol EIA ? Corticosterone RIA <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0025 <0.0001 Pearson product moment correlation analyses were performed on data transformed with a common logarithm (logio). 30 - 20 - 10 Domestic cat t?' if c<>c. 300 - 200 150 100 50 0 50 60 70 80 eassiid 0 10 20 30 '?C-^?X/?S?i?Ss?i - 5 - 4 3 2 1 0 40 50 Fraction 70 80 O o n 250 I s- o s Fig. 6. Reverse-phase HPLC separation of immunoreactive glucocorticoid metabolites in feces of the red wolf (A), slender-tailed meerkat (B), and Himalayan black bear (C). Immunoreactivity in each fraction was measured with a cortisol EIA (open circles) and corticosterone RIA (closed circles). Retention times for co-eluted tritiated reference tracers are indicated by arrows. ARTICLE IN PRESS K. M. Young et al I General and Comparative Endocrinology xxx (2004) xxx-xxx 13 3.3.2. Canidae The corticosterone RIA measured two immunoreac- tive substances in HPLC-separated red wolf fecal ex- tracts, which eluted in fractions 46^8 (10% of total immunoreactivity) and 49-51 (59%) (Fig. 6A). The majority of cortisol El A immunoreactivity (56%o of total immunoreactivity) corresponded to a peak (fractions 36-41) slightly more polar than ^H-cortisol (Fig. 6A). Additional cortisol EIA immunoreactivity was associ- ated with metabolites more (fractions 43^5; 8% of total immunoreactivity) and less (fraction 47-50; 1 Wo) polar than ^H-corticosterone. 3.3.3. Herpestidae Cortisol EIA immunoreactivity corresponded to multiple immunoreactive substances in fecal extracts from the slender-tailed meerkat (Fig. 6B). At least five metabohtes more polar than ^H-cortisol were found to elute in fractions 4-9 (5%) of total immunoreac- tivity), 13-15 (12%), 16-23 (46%), 25-27 (10%), and 29-30 (4%). One metabolite (fractions 45^7; 7% of total immunoreactivity) also detected with the cortisol EIA was less polar than ^H-corticosterone. The cor- ticosterone RIA measured two immunoreactive me- tabohtes more polar than ^H-cortisol in HPLC- separated fecal extracts, which eluted in fractions 13- 17 (22%) of total immunoreactivity) and 25-27 (74%) (Fig. 6B). 3.3.4. Ursidae Analysis of HPLC-purified fecal eluates from the Himalayan black bear determined that 28%) and 15% of cortisol EIA and corticosterone RIA immunoreac- tivity, respectively, co-eluted with ^H-cortisol (frac- tions 38^1) (Fig. 6C). Additional cortisol EIA immunoreactivity was associated with peaks at frac- tions 42-47 (23%) of total immunoreactivity), 49-52 (11%)) and 56-58 (3%o). The majority of corticosterone RIA immunoreactivity was associated with a metab- olite that eluted in fractions 25-27 (74%o of total im- munoreactivity). 4. Discussion In recent years there has been growing concern for the welfare of animals in captivity and increasing de- mand for the development of noninvasive methods to measure the stress associated with management prac- tices and environmental conditions. The aim of this study was to determine if a cortisol EIA was suitable for noninvasively monitoring adrenocortical activity in a variety of carnivore species. The performance of this EIA was gauged by comparison with a corticosterone RIA, an assay currently favored for monitoring fecal corticoids in wildlife species. Standardized procedures (i.e., tests for parallehsm and mass recovery) were used to validate the cortisol EIA and corticosterone RIA for measurement of glucocorticoids in feces of each species. HPLC analyses revealed that the majority of glucocorticoid immuno- reactivity in fecal extracts corresponded with two or more peaks, indicating the presence of multiple me- tabolites. The two assay systems appeared to be mea- suring different glucocorticoid metabolites between the different Carn?vora suborders. Major immunoreactive metabolites in felids and meerkats were more polar than those identified in the red wolf and Himalayan black bear, and previously reported for the African wild dog (Monfort et al., 1998) and black-footed ferret (Young et al., 2001). In contrast, similarities between HPLC profiles for domestic and nondomestic cats suggest that like ovarian steroids (Brown et al., 1994), glucocorticoid metabolism may be conserved across felid species. For most species, only a minor portion of the im- munoreactivity detected in HPLC-separated fecal ex- tracts co-eluted with radiolabeled cortisol or corticosterone. These results are consistent with radi- ometabolism studies that demonstrated the near absence of authentic radiolabeled cortisol and corticosterone from feces of carnivores (Graham and Brown, 1996; Schatz and Palme, 2001), lagomorphs (Teskey-Gerstl et al., 2000), domestic livestock (Palme and M?stl, 1997; M?stl et al., 1999) and primates (Bahr et al., 2000). By contrast, immunoreactive substances in feces of the Himalayan black bear and clouded leopard co-eluted with ^H-cortisol, suggesting these species may excrete native cortisol in variable amounts. Although the identity of most glucocorticoid me- tabolites in HPLC-purified fecal eluates remains un- known, their biological relevance as indices of adrenocortical activity in carnivores was demonstrated by a transient increase in excretion following adminis- tration of exogenous ACTH as determined by both the cortisol EIA and corticosterone RIA. In the present study, ACTH induced a ~228-1145% and ~231^150% rise above pre-treatment baseline concentrations of cortisol and corticosterone metabolites across all spe- cies of carnivores, respectively. These results were similar to other studies utilizing the corticosterone RIA (or its antibody) in carnivores, such as the cheetah (range ~690^294% above baseline, 400 lU ACTH, Terio et al., 1999), clouded leopard (mean ~1400%o, 500 lU ACTH, Wielebnowski et al., 2002), African wild dog (range ~1000-3000%, 400 lU, Monfort et al., 1998), spotted hyena (range ~ 1300-4500%) as calculated from data, 200 lU ACTH, Goymann et al., 1999), black-footed ferret (mean ~208%) as calculated from data, 2IU ACTH, Young et al., 2001), and Malaysian sun bear (~300%) as calculated from graph, 88 lU, Wasser et al, 2000). ARTICLE IN PRESS 14 K. M. Young et al. I General and Comparative Endocrinology xxx (2004) xxx?xxx The adrenal cortex rapidly synthesizes and secretes glucocorticoids in response to ACTH. Although serum Cortisol concentrations in mammals rise within 10- 30 min (Brown et al., 1995; Carlstead et al., 1992; Gra- ham and Brown, 1996; Terio et al., 1999), the appear- ance of elevated concentrations of glucocorticoid metabolites in feces following ACTH injection is much slower. The delay between circulating steroid hormones and detection of their metabolites in feces is approxi- mately equivalent to the time required for digesta to pass from the duodenum to the rectum (Palme et al., 1996). In the present study, the delays between ACTH administration and the appearance of peak glucocorti- coid metabolite concentrations in feces (1-2 days post- ACTH treatment) were within the range previously re- ported for other carnivores, including the domestic cat (24-28 h, Graham and Brown, 1996; 24-49 h, Schatz and Palme, 2001), domestic dog (8-71 h, Schatz and Palme, 2001), African wild dog (24-30 h, Monfort et al., 1998), black-footed ferret (~20-44h. Young et al., 2001), and spotted hyena (16-50 h, Goymann et al., 1999). Capture and physical restraint elevate plasma gluco- corticoid concentrations in wildlife species (Kenagy and Place, 2000; Morton et al., 1995; Wesson et al., 1979). Handling and mild restraint in unfamiliar surroundings for serial blood sampling also increases circulating Cor- tisol levels in animals more habituated to these proce- dures and/or the presence of human investigators (Fox et al., 1994; Reinhardt et al., 1990, 1991). In domesti- cated and nondomesticated mammalian species, acute elevations in fecal glucocorticoid metabolites also have been observed following restraint in a squeeze cage (Jurke et al., 1997; Terio et al., 1999), brief handling and blood sampling (Harper and Austad, 2000), anesthesia (Terio et al., 1999; Whitten et al., 1998), relocation within an institution (Terio et al., 1999), translocation between facilities (Goymann et al., 1999; Morrow et al., 2002; Palme et al., 2000; Schwarzenberger et al., 1998), mate introduction and breeding (Terio et al., 1999), exposure to a novel environment (Morrow et al., 2002), and aggressive social interactions (Goymann et al., 1999). Despite monitoring biologically relevant metab- olites in feces, our ability to detect adrenocortical re- sponses to acute physical and psychological Stressors using the described immunoassay methods varied with the type of stimulus, between episodes of the same stimulus, and among species. A stress response was elicited in 2 of 3 felid species experiencing anesthesia. Significantly elevated concentrations of fecal glucocor- ticoid metabolites also were observed following saline injections in the black-footed ferret and red wolf, but not after gonadotropin injections in the domestic cat and clouded leopard. In the red wolf, only 2 of 6 epi- sodes of blood sampling were followed by a significant rise in excreted glucocorticoid metabolites. Surgery and anesthesia are known to increase plasma or serum Cor- tisol concentrations in dogs (Church et al., 1994; Fox et al., 1994), cats (Smith et al., 1996, 1999), horses (Taylor, 1989), rhesus monkeys (Puri et al, 1981), and humans (Bozkurt et al., 2000; Oyama and Wakayama, 1988). The dramatic rise in fecal glucocorticoid metabolites following procedures involving anesthesia in the cheetah and clouded leopard were consistent with previous re- ports for the cheetah (mean ~475% rise above baseline, Terio et al., 1999) and chimpanzee (mean ~300% rise above baseline, Whitten et al., 1998). Although no increases of fecal glucocorticoid me- tabolites were associated with courtship or breeding activity in the female sloth bear, there was a close as- sociation between enhanced excretion of metabolites and medicinal treatment/abnormal defecations. Because the female sulfered from a persistent nematode infection, the data suggest that factors associated with this medical condition may have induced an adrenal response. Higher plasma levels of cortisol are found in dogs (Church et al., 1994) and horses (Santschi et al., 1991) requiring surgery for abdominal illnesses. In horses, concentrations of fecal 11,17-dioxoandrostanes have been used as indices of pain experienced during colic and after castration (Merl et al., 2000). Alternatively, the deworming process and diarrhea could have increased steroid excretion independent of the stress associated with the nematode infestation. In some instances, it would appear that accurate identification of a true adrenocortical response to an exogenous stimulus may have been hindered by the high day-to-day variation in concentrations of fecal metab- olites. For example, metabolite concentrations following saline injections of the black-footed ferret and male red wolf, and after several episodes of blood sampling in the female red wolf exceeded values required for significance (i.e., mean basehne + 3 SD), suggesting that these pro- cedures were stressful. However, it was diificult to de- termine if these elevations were true stress responses because a number of unexplained peaks in metabolites during the period of longitudinal evaluation also reached the threshold value. Because our behavioral and environmental eifect data were collected opportunisti- cally and based primarily on keeper observations, it is possible that many situations were not adequately doc- umented. Other studies have also had varied success in detecting adrenocortical responses to acute exogenous Stressors. M?stl et al. (2002) measured increased excre- tion of fecal cortisol metabolites in cows following transportation to a novel environment, but not after use for practicing invasive sampling techniques for blood and rumen contents. The absence of significant elevations in glucocorticoid metabolites following relocation, construction, social tension/minor aggression, mate introduction/breeding, and some procedures involving restraint suggests that ARTICLE IN PRESS K. M. Young et al. I General and Comparative Endocrinology xxx (2004) xxx-xxx 15 these stimuli may not have been stressful for the indi- viduals examined in this study. However, adrenocortical responses are known to reflect interanimal variation in the perception of a stimulus, and the biological re- sponses evoked to cope with a Stressor depend on fac- tors such as previous experience, genetics, age, and physiological state (Moberg, 1985, 2000). Alternately, it is plausible that the stimuli were stressful, but the acute adrenocortical rise was undetectable. Brief or small in- creases in circulating levels of glucocorticoids are likely to be masked by the pooling of metabolites in bile and feces. Also, fecal samples containing stressor-induced peaks in metabolites may have been missed due to in- frequent sample collection (e.g., Himalayan black bear and sloth bear, ~3 fecal collections per week) or failure to collect all scats defecated each day. Rather than studying brief events that enhance adrenocortical activity, future efforts should focus on the more physiologically detrimental state of chronic stress by diagnosing its associated persistent elevation of fecal glucocorticoids and identifying possible causative factors. Recently, Wielebnowski et al. (2002) determined that higher mean concentrations of fecal glucocorticoids were present in clouded leopards on public display and in close proximity to potential predators. In addition, a positive association was identified between glucocorti- coid levels and detrimental behaviors (e.g., fur-plucking, tail chewing, excessive pacing, and hiding). Steroids circulating in the blood are catabolized in the liver before excretion in urine and bile (Brownie, 1992; Macdonald et al., 1983; Taylor, 1971). Further changes to steroid metabolites are facilitated by the enzymatic activities of bacterial flora during transit through the intestinal tract (Macdonald et al., 1983; Taylor, 1971). Because steroid hormones are exten- sively metabolized prior to excretion, it is not surpris- ing that immunoassays featuring antibodies specific to blood glucocorticoids may not be suitable for quanti- fying fecal glucocorticoid metabolites (Goymann et al., 1999; Graham and Brown, 1996; Palme and M?stl, 1997; Schatz and Palme, 2001; Terio et al, 1999). As such, group-specific antibodies cross-reacting with a family of metabolites derived from a single parent steroid are preferred over more specific antibodies when using fecal steroid analysis to characterize phys- iological functions such as reproductive cycles and status or adrenocortical activation (Brown et al., 2001; Palme and M?stl, 1997; Palme et al., 1997; Schwar- zenberger et al., 1996). Although the corticosterone RIA was specifically developed to measure corticosterone levels in serum or plasma of rodents, its antibody cross-reacts well with glucocorticoid metabolites in feces and has proved use- ful for evaluating adrenal function in numerous species, including carnivores (domestic cat, spotted hyena, Af- rican wild dog, clouded leopard, cheetah, Alaskan sea otter, and Malayan sun bear), primates (yellow ba- boon), birds (northern spotted owl, chicken), herbivores (African elephant, black rhinoceros, gerenuk, elk, scimitar-horned oryx, and dairy cattle), and rodents (house mouse, deer mouse, and red-back voles) (Dehn- hard et al., 2003; Goymann et al., 1999, 2001; Graham and Brown, 1996; Harper and Austad, 2000; Millspaugh et al., 2001; Monfort et al., 1998; Morrow et al., 2002; Terio et al, 1999; Wasser et al., 1997, 2000; Wieleb- nowski et al., 2002). In the spotted hyena, an EIA using the same corticosterone antibody outperformed three other El As (cortisol, corticosterone, and 11-oxoetio- cholanolone) when resolving changes in adrenocortical activity after an ACTH challenge (Goymann et al., 1999). The aflinity of the corticosterone antibody for fecal glucocorticoid metabolites in such a diverse array of species thus supports the assertion by Wasser et al. (2000) that the antibody is group-specific. In the present study, a cortisol EIA generated im- munoreactive fecal metabolite profiles in carnivores that were temporally similar to the corticosterone RIA. Al- though the polyclonal cortisol antibody was developed to measure serum or plasma cortisol, results from our study suggest that it may also behave as a group-specific antibody. HPLC analyses further confirm that the cor- tisol antibody cross-reacts with a number of glucocor- ticoid metabolites in feces of a variety of carnivores. Still, there were examples where the two assays did not agree, especially in several of the longitudinal profiles. It remains to be determined whether combining the two assays may be of some benefit, since it might quantity a greater majority of the biologically significant metabo- lites. In conclusion, the present study suggests that both a cortisol EIA and corticosterone RIA measure immu- noreactive substances in feces reflective of adrenocorti- cal activation in carnivores. Enzyme immunoassays often are preferred by laboratories at zoological insti- tutions and field facilities because they lack the restric- tive radioisotope licensing associated with RIAs. The described cortisol EIA offers a practical alternative for investigators wishing to noninvasively monitor adreno- cortical activity for improving the health and well-being of carnivores. Acknowledgments We are grateful for the assistance provided by animal care and veterinary staff at the Conservation and Re- search Center, Granby Zoo, Little Rock Zoo, Point Defiance Zoo and Aquarium, Red Wolf Breeding Fa- cility, Saint Louis Zoo, Toronto Zoo, and White Oak Conservation Center. Excellent technical assistance was provided by Dessa Dal Porto. We thank Carolyn Wil- son and Dr. Malcolm Griffin (Queen's University, ARTICLE IN PRESS 16 K. M. Young et al I General and Comparative Endocrinology xxx (2004) xxx?xxx Kingston, ON) for providing helpful comments during the preparation of the manuscript. References Axelrod, J., Reisine, T.D., 1984. Stress hormones: their interaction and regulation. Science 224, 452^59. Bahr, N.I., Palme, R., M?hle, U., Hodges, J.K., Heistermann, M., 2000. Comparative aspects of the metabolism and excretion of Cortisol in three individual nonhuman primates. Gen. Comp. Endocrinol. 117,427-438. Breazile, J.E., 1987. Physiologic basis and consequences of distress in animals. J. Am. Vet. Med. Assoc. 191, 1212-1215. Broom, D.M., Johnson, K.G., 1993. Stress and Animal Welfare. Chapman & Hall, London. Brown, J.L., Graham, L.H., Wielebnowksi, N., Swanson, W.F., Wildt, D.E., Howard, J.G., 2001. Understanding the basic reproductive biology of wild felids by monitoring of faecal steroids. J. Reprod. F?rtil. Suppl. 57, 71-82. Brown, J.L., Wasser, S.K., Wildt, D.E., Graham, L.H., 1994. Comparative aspects of steroid hormone metabohsm and ovarian activity in felids, measured noninvasively in feces. Biol. Reprod. 51, 776-786. Brown, J.L., Wemmer, CM., Lehnhardt, J., 1995. Urinary cortisol analysis for monitoring adrenal activity in elephants. Zoo Biol. 14, 533-542. Brown, J.L., Wildt, D.E., Wielebnowski, N., Goodrowe, K.L., Graham, L.H., Wells, S., Howard, J.G., 1996. Reproductive activity in captive female cheetahs (Acinonyx juhatus) assessed by faecal steroids. J. Reprod. F?rtil. 106, 337-346. Brownie, A.C., 1992. The metabolism of adrenal cortical steroids. In: James, V.H.T. (Ed.), The Adrenal Gland, second ed. Raven Press, New York, pp. 209-224. Bozkurt, P., Kaya, G., Altintas, F., Yeker, Y., Hacibekiroglu, M., Emir, H., Sarimurat, N., Tekant, G., Erdogan, E., 2000. Systemic stress response during operations for acute abdominal pain performed via laparoscopy or laparotomy in children. Anaesthesia 55, 5-9. Carlstead, K., 1996. Effects of captivity on the behavior of wild mammals. In: Kleiman, D.G., Allen, M.E., Thompson, K.V., Lumpkin, S. (Eds.), Wild Mammals in Captivity. University of Chicago Press, Chicago, pp. 317-333. Carlstead, K., Brown, J.L., Monfort, S.L., Killens, R., Wildt, D.E., 1992. Urinary monitoring of adrenal responses to psychological Stressors in domestic and nondomestic felids. Zoo Biol. 11, 165- 176. Church, D.B., Nicholson, A.I., Ilkiw, J.E., Emslie, D.R., 1994. Effect of non-adrenal illness, anaesthesia and surgery on plasma cortisol concentrations in dogs. Res. Vet. Sei. 56, 129-131. Cook, C.J., Mellor, D.J., Harris, P.J., Ingram, J.R., Matthews, L.R., 2000. Hands-on and hands-off measurement of stress. In: Moberg, G.P., Mench, J.A. (Eds.), The Biology of Animal Stress. CABI Publishing, New York, pp. 123-146. Creel, S., Creel, N.M., Mills, M.G.L., Monfort, S.L., 1997. Rank and reproduction in cooperatively breeding African wild dogs: behav- ioral and endocrine correlates. Behav. Ecol. 8, 298-306. Creel, S., Creel, N.M., Monfort, S.L., 1996. Social stress and dominance. Nature 379, 212. deCatanzaro, D., MacNiven, E., 1992. Psychogenic pregnancy disrup- tions in mammals. Neurosci. Biobehav. Rev. 16, 43-53. Dehnhard, M., Clauss, M., Lechner-Doll, M., Meyer, H.H.D., Palme, R., 2001. Noninvasive monitoring of adrenocortical activity in Roe deer (Capreolus capreolus) by measurement of fecal cortisol metabolites. Gen. Comp. Endocrinol. 123, 111-120. Dehnhard, M., Schreer, A., Krone, O., Jewgenow, K., Krause, M., Grossman, R., 2003. Measurement of plasma corticosterone and fecal glucocorticoid metabolites in the chicken (Gallus domesticus), the great cormorant (Phalacrocorax carho), and the goshawk (Accipiter gentilis). Gen. Comp. Endocriol. 131, 345-352. Dobson, H., Smith, R.F., 1995. Stress and reproduction in farm animals. J. Reprod. F?rtil. Suppl. 49, 451-461. Estep, D.Q., Dewsbury, D.A., 1996. Mammalian reproductive behav- ior. In: Kleiman, D.G., Allen, M.E., Thompson, K.V., Lumpkin, S. (Eds.), Wild Mammals in Captivity. University of Chicago Press, Chicago, pp. 379-389. Fox, S.M., Mellor, D.J., Firth, E.C., Hodge, H., Lawoko, C.R.O., 1994. Changes in plasma cortisol concentrations before, during and after analgesia, anaesthesia and anaesthesia plus ovariohysterec- tomy in bitches. Res. Vet. Sei. 57, 110-118. Gittleman, J.L. (Ed.), 1989. Carnivore Behavior, Ecology and Evolu- tion. Chapman & Hall, London. Graham, L.H., Brown, J.L., 1996. Cortisol metabolism in the domestic cat and implications for non-invasive monitoring of adrenocortical function in endangered felids. Zoo Biol. 15, 71-82. Goymann, W., East, M.L., W?chter, B., H?ner, O.P., M?s?, E., Van't Hof, T.J., Hofer, H., 2001. Social, state-dependent and environ- mental modulation of faecal corticosteroid levels in free-ranging female spotted hyenas. Proc. R. Soc. Lond. B 268, 2453-2459. Goymann, W., M?stl, E., Van't Hof, T., East, M.L., Hofer, H., 1999. Noninvasive fecal monitoring of glucocorticoids in spotted hyenas, Crocuta crocuta. Gen. Comp. Endocrinol. 114, 340-348. Harper, J.M., Austad, S.N., 2000. Fecal glucocorticoids: a noninvasive method of measuring adrenal activity in wild and captive rodents. Physiol. Biochem. Zool. 73, 12-22. lUCN, 2002. 2002 lUCN Red List of Threatened Species. Available from http://www.redlist.org. Downloaded on 14 June 2002. Jurke, M.H., Czekala, N.M., Lindburg, D.G., Millard, S.E., 1997. Fecal corticoid metabolite measurement in the cheetah (Acinonyx juhatus). Zoo Biol. 16, 133-147. Kenagy, G.J., Place, N.J., 2000. Seasonal changes in plasma gluco- corticosteroids of free-living female yellow-pine chipmunks: effects of reproduction and capture and handling. Gen. Comp. Endocri- nol. 117, 189-199. Liptrap, R.M., 1993. Stress and reproduction in domestic animals. Ann. N.Y. Acad. Sei. 697, 275-284. Macdonald, D., 1992. The Velvet Claw: A Natural History of the Carnivores. BBC Books, London. Macdonald, I.A., Bokkenheuser, V.D., Winter, J., McLernon, A.M., Mosbach, E.H., 1983. Degradation of steroids in the human gut. J. Lipid Res. 24, 675-700. Melmed, S., Kleinberg, D., 2003. Anterior pituitary. In: Larsen, P.R., Kronenberg, H.M., Melmed, S., Polonsky, K.S. (Eds.), Williams Textbook of Endocrinology, tenth ed. Saunders, Philadelphia, pp. 177-279. Merl, S., Scherzer, S., Palme, R., M?stl, E., 2000. Pain causes increased concentrations of glucocorticoid metabolites in horse feces. J. Equine Vet. Sei. 20, 586-590. Millspaugh, J.J., Woods, R.J., Hunt, K.E., Raedeke, K.J., Brundige, G.C., Washburn, B.E., Wasser, S.K., 2001. Fecal glucocorticoid assays and the physiological stress response in elk. Wildl. Soc. Bull. 29, 899-907. Moberg, G., 1985. Biological response to stress: key to assessment of animal well-being. In: Moberg, G.P. (Ed.), Animal Stress. Amer- ican Physiological Society, Bethesda, Maryland, pp. 27^9. Moberg, G.P., 1987. Problems in defining stress and distress in animals. J. Am. Vet. Med. Assoc. 191, 1207-1211. Moberg, G.P., 2000. Biological response to stress: implications for animal welfare. In: Moberg, G.P., Mench, J.A. (Eds.), The Biology of Animal Stress. CABI Publishing, New York, pp. 1-21. Monfort, S.L., Arthur, N.P., Wildt, D.E., 1991. Monitoring ovarian function and pregnancy by evaluating excretion of urinary oestro- ARTICLE IN PRESS K. M. Young et al. I General and Comparative Endocrinology xxx (2004) xxx-xxx 17 gen conjugates in semi-free-ranging Przewalski's iiorses (Equus przewalskii). J. Reprod. F?rtil. 91, 155-164. Monfort, S.L, Brown, J.L., Wildt, D.E., 1993. Episodic and seasonal rhythms of Cortisol secretion in male Eld's deer (Cervus eldi thamin). J. Endocrinol. 138, 41^9. Monfort, S.L., Mashburn, K.L., Brewer, B.A., Creel, S.R., 1998. Evaluating adrenal activity in African wild dogs (Lycaon pictus) by fecal corticosteroid analysis. J. Zoo Wildl. Med. 29, 129-133. Morrow, C.J., Kolver, E.S., Verkerk, G.A., Matthews, L.R., 2002. Fecal glucocorticoid metabolites as a measure of adrenal activity in dairy cattle. Gen. Comp. Endocrinol. 126, 229-241. Morton, D.J., Anderson, E., Foggin, CM., Kock, M.D., Tiran, E.P., 1995. Plasma cortisol as an indicator of stress due to capture and translocation in wildhfe species. Vet. Rec. 136, 60-63. M?s?, E., Maggs, J.L., Schr?tter, G., Besenfelder, U., Palme, R., 2002. Measurement of cortisol metabolites in faeces of ruminants. Vet. Res. Commun. 26, 127-139. M?stl, E., Messmann, S., Bagu, E., Robia, C, Palme, R., 1999. Measurement of glucocorticoid metabolite concentrations in faeces of domestic livestock. J. Vet. Med. A 46, 621-631. Munck, A., Guyre, P.M., Holbrook, N.J., 1984. Physiological func- tions of glucocorticoids in stress and their relation to pharmaco- logical actions. Endocr. Rev. 5, 25^4. Munro, C.J., Lasley, B.L., 1988. Non-radiometric methods for immunoassay of steroid hormones. In: Albertson, B.D., Haseltine, F.P. (Eds.), Non-radiometric Assays: Technology and Application in Polypeptide and Steroid Hormone Detection. Alan R. Liss, New York, pp. 289-329. Oyama, T., Wakayama, S., 1988. The endocrine responses to general anesthesia. Int. Anesthesiol. Clin. 26, 176-181. Palme, R., Fischer, P., Schildorfer, H., Ismail, M.N., 1996. Excretion of infused '''C-steroid hormones via faeces and urine in domestic livestock. Anim. Reprod. Sei. 43, 43-63. Palme, R., M?stl, E., 1997. Measurement of cortisol metabohtes in faeces of sheep as a parameter of cortisol concentration in blood. Int. J. Mamm. Biol. 62 (Suppl. II), 192-197. Palme, R., M?stl, E., Brem, G., Schellander, K., Bamberg, E., 1997. Faecal metabolites of infused '''C-progesterone in domestic live- stock. Reprod. Dom. Anim. 32, 199-206. Palme, R., Robia, C, Baumgartner, W., M?stl, E., 2000. Transport stress in cattle as reflected by an increase in faecal cortisol metabolite concentrations. Vet. Rec. 146, 108-109. Puri, C.P., Puri, V., Anand Kumar, T.C., 1981. Serum levels of testosterone, cortisol, prolactin and bioactive luteinizing hormone in adult male rhesus monkeys following cage-restraint or anaes- thetizing with ketamine hydrochloride. Acta Endocrinol. 97, 118- 124. Reinhardt, V., Cowley, D., Eisele, S., Scheflfler, J., 1991. Avoiding undue cortisol responses to venipuncture in adult male rhesus macaques. Anim. Technol. 42, 83-86. Reinhardt, V., Cowley, D., Sche?ler, J., Vertein, R., Wegner, F., 1990. Cortisol response of female rhesus monkeys to venipuncture in homecage versus venipuncture in restraint apparatus. J. Med. Primatol. 19, 601-606. Rivier, C, Rivest, S., 1991. Effect of stress on the activity of the hypothalamic-pituitary-gonadal axis: peripheral and central mech- anisms. Biol. Reprod. 45, 523-532. Santschi, E.M., LeBlanc, M.M., Weston, P.G., 1991. Progestagen, oestrone sulphate and cortisol concentrations in pregnant mares during medical and surgical disease. J. Reprod. F?rtil. Suppl. 44, 627-634. Schatz, S., Palme, R., 2001. Measurement of faecal cortisol metabolites in cats and dogs: a non-invasive method for evaluating adreno- cortical function. Vet. Res. Commun. 25, 271-287. Schwarzenberger, F., Kolter, L., Zimmerman, W., Rietschel, W., Matern, B., Birher, P., Leus, K., 1998. Faecal cortisol metabolite measurement in the okapi (Okapi johnstoni). Adv. Ethol. 33, 28. Schwarzenberger, F., M?stl, E., Palme, R., Bamberg, E., 1996. Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild and zoo animals. Anim. Reprod. Sei. 42, 515- 526. Smith, J.D., Allen, S.W., Quandt, J.E., 1999. Changes in cortisol concentration in response to stress and postoperative pain in client- owned cats and correlation with objective clinical variables. Am. J. Vet. Res. 60, 432^36. Smith, J.D., Allen, S.W., Quandt, J.E., Tackett, R.L., 1996. Indicators of postoperative pain in cats and correlation with clinical criteria. Am. J. Vet. Res. 57, 1674-1678. Stewart, P.M., 2003. The adrenal cortex. In: Larsen, P.R., Kronen- berg, H.M., Melmed, S., Polonsky, K.S. (Eds.), Williams Textbook of Endocrinology, tenth ed. Saunders, Philadelphia, pp. 491-551. Taylor, W., 1971. The excretion of steroid hormone metabolites in bile and feces. Vitam. Horm. 29, 201-285. Taylor, P.M., 1989. Equine stress responses to anaestheisa. Br. J. Anaesth. 63, 702-709. Terio, K.A., Citino, S.B., Brown, J.L, 1999. Fecal cortisol metabolite analysis for noninvasive monitoring of adrenocortical function in the cheetah {Acinonyxjuhatus). J. Zoo Wildl. Med. 30, 484^91. Teskey-Gerstl, A., Bamberg, E., Steineck, T., Palme, R., 2000. Excretion of corticosteroids in urine and faeces of hares (Lepus europaeus). J. Comp. Physiol. B 170, 163-168. Thun, R., Effenberger, E., Zerobin, K., L?scher, T., Vetter, W., 1981. Twenty-four-hour secretory pattern of cortisol in the bull: evidence of episodic secretion and circadian rhythm. Endocrinology 109, 2208-2212. Walker, S.W., 1999. Reproductive endocrinology of the red wolf (Canis rufus). MSc thesis. Department of Biom?dical Sciences, University of Guelph, Guelph, Ontario, Canada. Wallner, B., M?stl, E., Dittami, J., Prossinger, H., 1999. Fecal glucocorticoids document stress in female Barbary macaques (Macaca sylvanus). Gen. Comp. Endocrinol. 113, 80-86. Wasser, S.K., Bevis, K., King, G., Hanson, E., 1997. Noninvasive physiological measures of disturbance in the northern spotted owl. Conserv. Biol. 11, 1019-1022. Wasser, S.K., Hunt, K.E., Brown, J.L., Cooper, K., Crockett, CM., Bechert, U., Millspaugh, J.J., Larson, S., Monfort, S.L., 2000. A generalized fecal glucocorticoid assay for use in a diverse array of nondomestic mammalian and avian species. Gen. Comp. Endocri- nol. 120, 260-275. Wesson, J.A., Scanlon, P.F., Kirkpatrick, R.L., Mosby, H.S., Butcher, R.L., 1979. Influence of chemical immobilization and physical restraint on steroid hormone levels in blood of white-tailed deer. Can. J. Zool. 57, 768-776. Whitten, P.L., Stavisky, R., Aureli, F., Russell, E., 1998. Response of fecal cortisol to stress in captive chimpanzees (Pan troglodytes). Am. J. Primatol. 44, 57-69. Wielebnowski, N., Fletchall, N., Carlstead, K., Busso, J.M., Brown, J.L., 2002. Noninvasive assessment of adrenal activity associated with husbandry and behavioral factors in the North American clouded leopard population. Zoo Biol. 21, 77-98. Wildt, D.E., Roth, T.R., 1997. Assisted reproduction for managing and conserving endangered felids. Int. Zoo Yrbk. 35, 164-172. Wildt, D.E., Howard, J.G., Chakraborty, P.K., Bush, M., 1986a. Reproductive physiology of the clouded leopard: II. A circannual analysis of adrenal-pituitary-testicular relationships during elec- troejaculation or after an adrenocorticotropin hormone challenge. Biol. Reprod. 34, 949-959. Wildt, D.E., Howard, J.G., Hall, L.L., Bush, M., 1986b. Reproductive physiology of the clouded leopard I. Electroejaculates contain high proportions of pleiomorphic spermatozoa throughout the year. Biol. Reprod. 34, 937-947. Wildt, D.E., Meltzer, D., Chakraborty, P.K., Bush, M., 1984. Adrenal-testicular-pituitary relationships in the cheetah subjected to anesthesia/electroejaculation. Biol. Reprod. 30, 665-672. ARTICLE IN PRESS 18 K. M. Young et al. I General and Comparative Endocrinology xxx (2004) xxx?xxx Wildt, D.E., Phillips, L.G., Simmons, L.G., Chakraborty, P.K., Brown, J.L, Howard, J.G., Teare, A., Bush, M., 1988. A comparative analysis of ejaculate and hormonal characteristics of the captive male cheetah, tiger, leopard and puma. Biol. Reprod. 38, 245-255. Wozencroft, W.C., 1989a. The phylogeny of the recent carn?vora. In: Gittleman, J.L. (Ed.), Carnivore Behavior, Ecology, and Evolu- tion. Chapman & Hall, London, pp. 495-535. Wozencroft, W.C., 1989b. Appendix: classification of the recent carn?vora. In: Gittleman, J.L. (Ed.), Carnivore Behavior, Ecology, and Evolution. Chapman & Hall, London, pp. 569- 593. Young, K.M., Brown, J.L., Goodrowe, K.L., 2001. Characterization of reproductive cycles and adrenal activity in the black-footed ferret (Mustela nigripes) by fecal hormone analysis. Zoo Biol. 20, 517-536.