Rachel M. Santymire

Validation of a cortisol enzyme immunoassay and characterization of salivary cortisol circadian rhythm in chimpanzees (Pan troglodytes).

Monitoring concentrations of stress hormones is an important tool for behavioral research and conservation for animals both in the wild and captivity. Glucocorticoids can be measured in mammals as an indicator of stress by analyzing blood, feces, urine, hair, feathers, or saliva. The advantages of using saliva for measuring cortisol concentrations are three‐fold: it is minimally invasive, multiple samples can be collected from the same individual in a short timeframe, and cortisol has a relatively short response time in saliva as compared with other materials. The purpose of this study was to: (1) conduct an adrenocorticotropic hormone (ACTH) challenge as a physiological validation for an enzyme immunoassay to measure salivary cortisol in chimpanzees and (2) characterize the circadian rhythm of salivary cortisol in chimpanzees. We determined that salivary cortisol concentrations peaked 45 min following the ACTH challenge, which is similar to humans. Also, salivary cortisol concentrations peaked early in the morning and decreased throughout the day. We recommend that saliva collection may be the most effective method of measuring stress reactivity and has the potential to complement behavioral, cognitive, physiological, and welfare studies. Am. J. Primatol. 73:903–908, 2011.

Development of a field-friendly technique for fecal steroid extraction and storage using the African wild dog (Lycaon pictus).

Hormonal analysis provides information about wildlife populations, but is difficult to conduct in the field. Our goal was to develop a rapid and effective field method for fecal steroid analysis by comparing: (1) three extraction methods (laboratory (LAB), homogenize (HO) and handshake (HS)) and (2) two storage methods (solid-phase extraction (SPE) tubes vs. plastic tubes (PT)). Samples (n=23) from captive African wild dogs (Lycaon pictus) were thoroughly mixed, three aliquots of each were weighed ( approximately 0.5 g) and 5 ml of 90% ethanol was added. For LAB, samples were agitated (mixer setting 60; 30 min), centrifuged (1,500 rpm; 20 min) and poured into glass tubes. Or aliquots were HO (1 min) or HS (1 min) and poured through filter paper into glass tubes. Samples were split, analyzed for corticosterone (C) and testosterone (T) metabolites using enzyme immunoassays or stored in SPE or PT. Samples were stored (room temperature) for 30, 60 or 180 days, reconstituted in buffer and analyzed. Mean C and T recoveries of HO were greater (P=0.03) than HS compared with LAB, which was similar to HO (P>0.05). After 30 days <21% of C and T was recovered from SPE, but approximately 100% of each was recovered from HO-PT and HS-PT. Similarly, after 60 and 180 days, approximately 100% of C and T was recovered from HO-PT and HS-PT. Results demonstrated that, for C and T, HO was more comparable (P<0.001) to LAB than HS and PT storage was more efficient than SPE (P<0.001).

Validation of a field technique and characterization of fecal glucocorticoid metabolite analysis in wild chimpanzees (Pan troglodytes).

Monitoring adrenocortical activity in wild primate populations is critical, given the well‐documented relationship between stress, health, and reproduction. Although many primate studies have quantified fecal glucocorticoid metabolite (FGM) concentrations, it is imperative that researchers validate their method for each species. Here, we describe and validate a technique for field extraction and storage of FGMs in wild chimpanzees (Pan troglodytes). Our method circumvents many of the logistical challenges associated with field studies while yielding similar results to a commonly used laboratory method. We further validate that our method accurately reflects stress physiology using an adrenocorticotropic hormone challenge in a captive chimpanzee and an FGM peak at parturition in a wild subject. Finally, we quantify circadian patterns for FGMs for the first time in this species. Understanding these patterns may allow researchers to directly link specific events with the stress response. Am. J. Primatol. 75:57‐64, 2013.

Using fecal hormonal and behavioral analyses to evaluate the introduction of two sable antelope at Lincoln Park Zoo.

Introductions of sable antelope (Hippotragus niger) can be difficult due to the potential ensuing aggression compounded by their large horns. The goal was to use hormonal assays and behavioral analyses to evaluate the success of an introduction of 2 adult females at Lincoln Park Zoo. The objectives were to (a) document behavioral and hormonal changes in 2 female sable antelope during the introduction, (b) compare fecal glucocorticoid metabolites (FGM) in each individual during the introduction stages, (c) measure fecal androgen metabolites (FAM) during introduction and compare with dominance rank and observed aggression, and (d) monitor estrous cycle synchronization. Results demonstrate that FGM were higher before than during and after the introduction. Behavioral observations indicated limited aggression between females, although the keeper survey results revealed that the new female was more dominant and had higher mean FGM and FAM than the resident. Both sable antelope were reproductively active throughout the year. Results indicate that fecal hormone analysis can provide zoo management with valuable information to minimize the risk of aggression, injury, and stress during introductions of nonhuman animals.

International Society for Wildlife Endocrinology: the future of endocrine measures for reproductive science, animal welfare and conservation biology

Hormone analysis is a precise and widely accepted tool for monitoring reproductive function and responses to stressors. Although hormones are present and can be measured in various biological matrices, non-invasive methods have gained popularity over the past 30 years as a more practical approach for assessing ovarian, testicular and, more recently, adrenocortical activity in intractable wildlife species. Non-invasive hormone monitoring also has been key to understanding biological mechanisms related to observed behaviours of captive and free-ranging animals. Despite the increasing popularity of this research field, wildlife endocrinologists have not had a specific forum for sharing and discussing their latest findings, technical developments and common challenges. To provide such a communication platform, the International Society for Wildlife Endocrinology (ISWE) was established in 2010, followed by an international meeting held on 3–4 November 2011 at the Toronto Zoo, Canada. Over several sessions, keynote speakers and participants discussed recent developments of new and innovative methods for hormone monitoring, as well as the latest advances in basic endocrinology as applied to adrenal function, reproductive physiology, animal health, ecology and evolution. Here, we introduce ISWE to the scientific community and discuss how this new society will serve as a resource for wildlife endocrinologists worldwide.

Environment influences morphology and development for in situ and ex situ populations of the black-footed ferret (Mustela nigripes)

For selected species, conservation breeding has become integrated into recovery plans, most often through the production of offspring for reintroduction into nature. As these programs increase in size and scope, it is imperative that conservation managers retain the biological integrity of the species. This study investigated the causes of morphological changes that are known to occur in black-footed ferrets (Mustela nigripes) maintained ex situ. In a previous study, ferrets maintained in captivity were 5–10% smaller in body size than pre-captive, in situ animals. In the present study, the authors compared nine morphological characters among ex situ animals and their in situ descendants. Within the ex situ population, cage types were compared to determine whether housing influenced morphometry. Black-footed ferrets born to reintroduced individuals quickly returned to their pre-captive size suggesting that a diminutive morphology ex situ did not have a genetic basis. Furthermore, cage type affected overall body size and shape; ulnas and tibias were as much as 9% shorter for ex situ animals. The authors hypothesise that small cage size and environmental homogeneity inhibit the mechanical stimuli necessary for long bone development. These findings have ramifications for ex situ managers who need to create artificial captive settings that promote natural physical development. In the absence of such an environment, ‘unnatural’ morphologies can result that may contribute to poor fitness or perhaps even domestication.

Environmental enrichment affects adrenocortical stress responses in the endangered black-footed ferret

Potential stressors of wildlife living in captivity, such as artificial living conditions and frequent human contact, may lead to a higher occurrence of disease and reduced reproductive function. One successful method used by wildlife managers to improve general well-being is the provision of environmental enrichment, which is the practice of providing animals under managed care with environmental stimuli. The black-footed ferret (Mustela nigripes) is a highly-endangered carnivore species that was rescued from extinction by removal of the last remaining individuals from the wild to begin an ex situ breeding program. Our goal was to examine the effect of environmental enrichment on adrenocortical activity in ferrets by monitoring fecal glucocorticoid metabolites (FGM). Results demonstrated that enrichment lowered FGM in juvenile male ferrets, while increasing it in adult females; enrichment had no effect on FGM in juvenile females and adult males. These results correspond with our findings that juvenile males interacted more with the enrichment items than did adult females. However, we did not detect an impact of FGM on the incidence of disease or on the ability of ferrets to become reproductive during the following breeding season. We conclude that an environmental enrichment program could benefit captive juvenile male ferrets by reducing adrenocortical activity.

Hormonal Correlates of Paternal Care Differences in the Hylobatidae

Only one of the 15 species of monogamous hylobatids, the siamang (Symphalangus syndactylus), demonstrates direct paternal care in the form of infant‐carrying, providing a unique model for examining hormonal correlates of paternal care differences between siamangs and gibbons. We used behavioral data and fecal hormone analysis to investigate (1) differences in monthly percent father–infant proximity in relation to monthly fecal androgen metabolite concentrations from infant birth to the late postpartum period between siamangs and gibbons, (2) the pattern of change in fecal androgen and fecal estrogen metabolite concentrations during the 8‐week peripartum period between siamangs and gibbons, and (3) the change in mean fecal glucocorticoid metabolite concentrations at 1‐month postpartum from individual baseline between siamangs and gibbons. Father–infant proximity increased as androgen concentrations decreased over the postpartum period in siamangs but not in gibbons. Androgen concentrations increased around birth in siamangs during the 8‐week peripartum period, but exhibited a decreasing trend around birth in gibbons. Estrogen concentrations increased from pre‐ to postpartum in siamangs during the 8‐week peripartum period, but exhibited a decreasing trend from pre‐ to postpartum in gibbons. The difference in mean glucocorticoid metabolite concentrations from baseline was greater in siamangs than gibbons. Our data suggest a relationship between specific steroid hormone patterns and differences in paternal care among the hylobatids, warranting further investigation of such proximate mechanisms.

Reproductive gonadal steroidogenic activity in the fishing cat (Prionailurus viverrinus) assessed by fecal steroid analyses

Non-invasive fecal steroid analyses were used to characterize gonadal activity in the fishing cat (Prionailurus viverrinus). Estrogen, progestagen and androgen metabolites were quantified in fecal samples collected for 12 months from four males and 10 females housed at seven North American zoological institutions. Male reproductive hormone concentrations did not vary (P>0.05) among season, and estrogen cycles were observed year-round in females and averaged (±SEM) 19.9±1.0 days. Mean peak estrogen concentration during estrus (460.0±72.6ng/g feces) was five-fold higher than baseline (87.3±14.0ng/g feces). Five of seven females (71.4%) housed alone or with another female demonstrated spontaneous luteal activity (apparent ovulation without copulation), with mean progestagen concentration (20.3±4.7μg/g feces), increasing nearly five-fold above baseline (4.1±0.8μg/g feces). The non-pregnant luteal phase averaged 32.9±2.5 days (n=13). One female delivered kittens 70 days after natural mating with fecal progestagen concentrations averaging 51.2±5.2μg/g feces. Two additional females were administered exogenous gonadotropins (150IU eCG; 100IU hCG), which caused hyper-elevated concentrations of fecal estrogen and progestagen (plus ovulation). Results indicate that: (1) male and female fishing cats managed in North American zoos are reproductively active year round; (2) 71.4% of females experienced spontaneous ovulation; and (3) females are responsive to exogenous gonadotropins for ovulation induction, but a regimen that produces a normative ovarian steroidogenic response needs to be identified.

A Road Map for 21st Century Genetic Restoration: Gene Pool Enrichment of the Black-Footed Ferret

Interspecies somatic cell nuclear transfer (iSCNT) could benefit recovery programs of critically endangered species but must be weighed with the risks of failure. To weigh the risks and benefits, a decision-making process that evaluates progress is needed. Experiments that evaluate the efficiency and efficacy of blastocyst, fetal, and post-parturition development are necessary to determine the success or failure or species-specific iSCNT programs. Here, we use the black-footed ferret (Mustela nigripes) as a case study for evaluating this emerging biomedical technology as a tool for genetic restoration. The black-footed ferret has depleted genetic variation yet genome resource banks contain genetic material of individuals not currently represented in the extant lineage. Thus, genetic restoration of the species is in theory possible and could help reduce the persistent erosion of genetic diversity from drift. Extensive genetic, genomic, and reproductive science tools have previously been developed in black-footed ferrets and would aid in the process of developing an iSCNT protocol for this species. Nonetheless, developing reproductive cloning will require years of experiments and a coordinated effort among recovery partners. The information gained from a well-planned research effort with the goal of genetic restoration via reproductive cloning could establish a 21st century model for evaluating and implementing conservation breeding that would be applicable to other genetically impoverished species.