Catharine J. Wheaton

As part of its mission to advance the field of wildlife endocrinology, the International Society of Wildlife Endocrinology aims to develop cost-effective antibodies and enzyme immunoassay kits that support research across a diverse range of species and sample matrices. To provide additional options for the quantification of faecal glucocorticoid metabolites (fGCMs), an antibody against 11-oxoetiocholanolone-17-carboxymethyl oxime (CMO) was generated in rabbits, and an enzyme immunoassay incorporating a horseradish peroxidase-conjugated label and 11-oxoetiocholanolone standard has been developed, designed for use with anti-rabbit IgG secondary antibody coated plates. This mini-kit was used to quantify glucocorticoid metabolites with a 5β-3α-ol-11-one structure in faecal extracts from 23 species: African and Asian elephants, Alpine chamois, American bison, Bengal tiger, blue wildebeest, blue-and-yellow macaw, brushtail possum, cape buffalo, fat-tailed dunnart, Florida manatee, ghost bat, giraffe, golden langur, Gould’s wattled bat, hippopotamus, Leadbeater’s possum, mandrill, okapi, roan antelope, samango monkey, short-beaked echidna, and western lowland gorilla. Pharmacological (adrenocorticotropic hormone challenge) and biological (inter-zoo translocation, wild capture, social disruption, illness/injury and veterinary intervention) challenges resulted in expected increases in fGCM concentrations, and in a subset of species, closely paralleled results from a previously established immunoassay against 11-oxoetiocholanolone-17-CMO. Two additional species tested, Krefft’s glider, which showed contradictory results on this assay compared to a previously validated enzyme immunoassay (EIA) and Ankole cow, where the magnitude increase post-event did not quite reach the 2-fold change criteria, highlight that differences in excreted faecal metabolites across species mean that no EIA will be suitable for all species. This assay provides a valuable new option for assessing adrenal activity across taxa using a group-specific antibody. Future studies should put similar emphasis on validation to determine optimal assay choice for measuring fGCMs in a variety of species.

Background: Leopard sharks (Triakis semifasciata) are abundant, coastal, eastern Pacific, mesopredatory sharks and are frequently managed in aquariums and zoos. Medical examinations are a routine part of good husbandry practices, but the handling protocols vary greatly between facilities. In this study, we compared the physiological stress responses of T. semifasciata associated with manual restraint and sedated handling under parallel holding and handling procedures in a 10 min interval. Methods: Blood was collected and analyzed for the hematocrit (Hct), lactate, glucose, beta-hydroxybutyrate (β-HB), pH, blood gasses, 1α-hydroxycorticosterone (1α-OHB), and corticosterone (B). Results: Overall, a minimal physiological stress response was observed in both groups. However, statistically significant increases in the PCO₂, β-HB and lactate occurred, while the pH_TC, SO₂, and BE decreased over time in the manual-restraint group. In the sedated group, the B, PCO₂, and lactate increased while the pH_TC, SO₂, and base excess (BE) decreased over time. When comparing treatments, the sedated group had a higher B and PCO_{2 TC} and a lower pH_TC, BE, and SO₂ than the manual-restraint group at the second time point. Conclusions: This study indicates that manual restraint is comparable to the described sedation in T. semifasciata for minimally invasive procedures. The overall results show that this species had low physiological stress responses to both methods.

Though lion‐tailed macaques are managed ex situ as an assurance population, little information is available on the relationship between behavior and reproductive physiology to ensure successful management and evaluate welfare. To facilitate social and reproductive management in a group of one male and three female lion‐tailed macaques at Disney's Animal Kingdom®, we observed behaviors associated with dominance and reproduction, rated genital swellings, recorded menses, tracked ovarian cycles as determined by measurement of fecal immunoreactive progesterone (P4) and estradiol (E2), and monitored fecal cortisol (CORT). We analyzed the dominance hierarchy of females, compared hormone patterns of ovarian cycles with genital swellings and copulations relative to menses, and tested for correlations between hormones and behavior. The strength of the dominance hierarchy indicated moderate linearity, but relaxed after the dominant female received anxiolytic medications to reduce intraspecific aggression. Fecal P4 and E2 patterns revealed ovarian cycle lengths averaging 29.7 ± 0.8 days. The timing of single‐day E2 peaks varied between and within females' cycles. However, on average, E2 peaks occurred 6–10 days after the first day of menses, with maximum genital swellings and copulations occurring slightly thereafter in uncontracepted females. Female‐to‐female aggression and sexual behaviors with the male were related to ovarian cycle phase, but correlations between behaviors and hormones varied between females. Initiated aggression was positively correlated with E2 in the dominant and mid‐ranked females, while aggression received was positively correlated with P4 or negatively correlated with E2 in the mid‐ranked and subordinate females. In one uncontracepted female, sexual behaviors with the male were negatively correlated to P4. CORT was positively correlated to aggression in the dominant and mid‐ranked females and negatively correlated to grooming in the subordinate female. Results indicate observations of visual and behavioral cues can be used to track ovarian cycles and evaluate social dynamics and welfare in zoo‐housed lion‐tailed macaques.

Seasonal availability of resources can influence the timing and success of reproduction in primates. This study examines the annual pattern of conceptions, mid‐term (13 weeks) and full‐term (26 weeks) pregnancy losses, births, and infant survivorship of the Critically Endangered cotton‐top tamarin (Saguinus oedipus) at Proyecto Tití's long‐term field site in Santa Catalina, Colombia. Using 18 years of behavior, physical exam, and fecal steroid data (N = 51 females, 168 conceptions, 121 liters), fecal estrone conjugate (E₁C) and pregnanediol‐3‐glucuronide (PdG) or progesterone (P4) profiles were examined to investigate the relationships between female reproductive patterns and reproductive success with feeding and climatic data. Cotton‐top tamarins demonstrated seasonal patterns of reproduction and pregnancy losses. We found positive correlations between peak conception and nectarivory, births and rainfall, and mid‐lactation and frugivory. High body mass mothers reproducing during the peak season were more likely to have infants survive. Overall, these results suggest that tamarins use a “relaxed income‐breeder” strategy. Cotton‐top tamarins appear to maximize reproductive output at the time of ovulation, and can reduce their investment in the current reproductive effort at many stages. Changes in food availability due to habitat loss and climate change will require the Critically Endangered cotton‐top tamarins to alter their reproductive strategies to survive.

OBJECTIVE: To characterize physical examination, plasma biochemical, and ultrasonographic findings in aquarium-housed, managed semiwild, and wild southern stingrays (Hypanus americanus) with and without reproductive disease. ANIMALS: Southern stingrays from aquarium (n = 48), lagoon (managed semiwild; 34), and wild (12) habitats. PROCEDURES: Limited, opportunistic prosections were performed of presumed anatomically normal wild southern stingrays and compared with findings for aquarium-housed stingrays with reproductive disease. Ultrasonographic video data from both groups were used to assign a score (1 to 5) indicating increasing severity of ovarian and uterine reproductive disease. Plasma total 17β-estradiol, estrone, progesterone, and testosterone concentrations were measured with enzyme immunoassays validated for use in southern stingrays. RESULTS: Ultrasonographic ovarian scores were significantly correlated with uterine scores. No reproductive disease was detected in semiwild or wild stingrays, but 65% (31/48) of aquarium-housed stingrays had developing or advanced reproductive disease (ie, ultrasonographic ovarian or uterine score of 4 or 5). Significant correlations were identified between ovarian and uterine disease status and plasma concentrations of all steroid hormones except testosterone. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that ultrasonography and plasma hormone concentrations may be useful in the identification of reproductive disease and determination of disease severity in southern stingrays.

Stress is a major factor in determining success when releasing endangered species into the wild but is often overlooked. Mandrills (Mandrills sphinx) are vulnerable to extinction due to habitat loss and demand for bush meat and the pet trade. To help bolster in situ populations, rehabilitated rescued mandrills recently were released into a protected area in the Republic of Congo. The goal of this study was to validate the use of faecal glucocorticoid metabolite enzyme immunoassays (EIAs) in mandrills and test field-friendly faecal hormone extraction techniques that can subsequently be used to monitor the stress physiology and welfare of mandrills throughout the release process. Using faecal samples collected from ex situ mandrills, we tested cortisol, corticosterone, 11β-hydroxyetiocholanolone (69a), and 11-oxoetiocholanolone EIAs. Absolute concentrations, hormone profiles following medical procedures or translocation, and high-performance liquid chromatography fraction immunoreactivity showed that the 69a assay was the best choice to monitor the stress response in this species. Samples with delayed extraction or drying times had 40–80% lower 69a concentrations than samples extracted immediately post-collection and frozen. The 69a EIA is an appropriate assay for monitoring welfare in this species in situ or ex situ, and results indicated that consistent extraction methods are important for accurate comparisons.

Large flying foxes (Pteropus vampyrus) are a socially complex species. In situ colonies typically comprise thousands of individuals in small harems of one male to many females. In ex situ environments, all‐male colonies are becoming more common due to a surplus of males in the population. There is limited information describing the hormonal and behavioral patterns of all‐male colonies during the breeding season. We assessed seasonal changes in hormones and behavior in an all‐male colony of 12 large flying foxes at Disney's Animal Kingdom^®. We validated hormone assays using morning urine and fecal samples to assess seasonal changes in excreted immunoreactive testosterone and glucocorticoid metabolites. We collected behavior data using an all‐occurrence method, recording agonistic behaviors related to territorial defense (hooking, biting, wing flexing, vocalizing, and wrestling), and sexual behavior (mounting and frontal grabbing). Results indicated that (i) we could reliably measure testosterone and glucocorticoid metabolites concentrations from fecal and urine samples collected from individual bats; (ii) there were distinct relationships between changes in levels of agonism and hormone concentrations throughout the year; and (iii) three agonistic behaviors (chasing, wrestling, and open‐mouth threat) peaked prior to the increase in testosterone and glucocorticoid hormones measured during the breeding season. These three behaviors could potentially be used as early indicators to signal the onset of the breeding season and allow time to implement ex situ management changes to reduce the incidence of agonism between individuals.

Differences in reported testosterone concentrations in male sea turtle blood samples are common in the veterinary literature, but may be accounted for by differences in sample handling and processing prior to assay. Therefore, our study was performed to determine best practices for testosterone analysis in male sea turtles (Caretta caretta and Chelonia mydas). Blood samples were collected into 5 collection tube types, and assay validation and measured testosterone concentrations were compared across different sample storage (fresh, refrigerated 1 week, or frozen), extraction (unextracted or ether-extracted), and processing treatment (untreated, homogenized, or dissociation reagent) conditions. Ether-extracted and dissociation reagent–treated samples validated in all conditions tested and are recommended for use, as unextracted samples validated only if assayed fresh. Dissociation reagent treatment was simpler to perform than ether extraction and resulted in total testosterone concentrations ~2.7–3.5 times greater than free testosterone measured in ether-extracted samples. Sample homogenization did not affect measured testosterone concentrations, and could be used to increase volume in gelled samples. An annual seasonal testosterone increase was observed in both species when ether extraction or dissociation reagent treatment was used. Annual deslorelin implant treatments in a Chelonia mydas male resulted in suppression of seasonal testosterone following the fourth treatment. Seasonal testosterone patterns resumed following discontinuation of deslorelin. Comparison of in-house and commercially available enzyme immunoassay kits revealed similar patterns of seasonal testosterone increases and deslorelin-induced suppression. Our study highlights the importance of methodological validation and provides laboratorians with best practices for testosterone enzyme immunoassay in sea turtles.

Amphibian biology is intricate, and there are many inter‐related factors that need to be understood before establishing successful Conservation Breeding Programs (CBPs). Nutritional needs of amphibians are highly integrated with disease and their husbandry needs, and the diversity of developmental stages, natural habitats, and feeding strategies result in many different recommendations for proper care and feeding. This review identifies several areas where there is substantial room for improvement in maintaining healthy ex situ amphibian populations specifically in the areas of obtaining and utilizing natural history data for both amphibians and their dietary items, achieving more appropriate environmental parameters, understanding stress and hormone production, and promoting better physical and population health. Using a scientific or research framework to answer questions about disease, nutrition, husbandry, genetics, and endocrinology of ex situ amphibians will improve specialists' understanding of the needs of these species. In general, there is a lack of baseline data and comparative information for most basic aspects of amphibian biology as well as standardized laboratory approaches. Instituting a formalized research approach in multiple scientific disciplines will be beneficial not only to the management of current ex situ populations, but also in moving forward with future conservation and reintroduction projects. This overview of gaps in knowledge concerning ex situ amphibian care should serve as a foundation for much needed future research in these areas. Zoo Biol. 33:485–501, 2014. © 2014 Wiley Periodicals Inc.