Abstract: Adult male elephants periodically show the phenomenon of musth, a condition associated with increased aggressiveness, restlessness, significant weight reduction and markedly elevated androgen levels. It has been suggested that musth-related behaviours are costly and that therefore musth may represent a form of physiological stress. In order to provide data on this largely unanswered question, the first aim of this study was to evaluate different assays for non-invasive assessment of adrenocortical function in the male African elephant by (i) characterizing the metabolism and excretion of [3H]cortisol (3H-C) and [14C]testosterone (14C-T) and (ii) using this information to evaluate the specificity of four antibodies for determination of excreted cortisol metabolites, particularly with respect to possible cross-reactions with androgen metabolites, and to assess their biological validity using an ACTH challenge test. Based on the methodology established, the second objective was to provide data on fecal cortisol metabolite concentrations in bulls during the musth and non-musth condition. 3H-C (1 mCi) and 14C-T (100 microCi) were injected simultaneously into a 16 year old male and all urine and feces collected for 30 and 86 h, respectively. The majority (82%) of cortisol metabolites was excreted into the urine, whereas testosterone metabolites were mainly (57%) excreted into the feces. Almost all radioactive metabolites recovered from urine were conjugated (86% 3H-C and 97% 14C-T). In contrast, 86% and >99% of the 3H-C and 14C-T metabolites recovered from feces consisted of unconjugated forms. HPLC separations indicated the presence of various metabolites of cortisol in both urine and feces, with cortisol being abundant in hydrolysed urine, but virtually absent in feces. Although all antibodies measured substantial amounts of immunoreactivity after HPLC separation of peak radioactive samples and detected an increase in glucocorticoid output following the ACTH challenge, only two (in feces against 3alpha,11-oxo-cortisol metabolites, measured by an 11-oxo-etiocholanolone-EIA and in urine against cortisol, measured by a cortisol-EIA) did not show substantial cross-reactivity with excreted 14C-T metabolites and could provide an acceptable degree of specificity for reliable assessment of glucocorticoid output from urine and feces. Based on these findings, concentrations of immunoreactive 3alpha,11-oxo-cortisol metabolites were determined in weekly fecal samples collected from four adult bulls over periods of 11-20 months to examine whether musth is associated with increased adrenal activity. Results showed that in each male levels of these cortisol metabolites were not elevated during periods of musth, suggesting that in the African elephant musth is generally not associated with marked elevations in glucocorticoid output. Given the complex nature of musth and the variety of factors that are likely to influence its manifestation, it is clear, however, that further studies, particularly on free-ranging animals, are needed before a possible relationship between musth and adrenal function can be resolved. This study also clearly illustrates the potential problems associated with cross-reacting metabolites of gonadal steroids in EIAs measuring glucocorticoid metabolites. This has to be taken into account when selecting assays and interpreting results of glucocorticoid metabolite analysis, not only for studies in the elephant but also in other species.

Abstract: REASONS FOR PERFORMING STUDY: It is recognised that the amount of psychological stress that an animal encounters determines the degree of response of the hypothalamic-pituitary-adrenal (HPA) axis. In human athletes, the added emotive stress of competition is an important element in the adrenal response. The aim of this study was to examine the effect of show-jumping as well as dressage on stress levels by comparing horses' stress response at a horse show compared to their familiar home. METHODS: Fifty-one horses involved in competition were used. EDTA blood samples were collected before exercise, upon arrived to the schooling area (control), and k over a jump or dressage course. After sampling, plasma was separated and stored at -80 degrees C until determinations of cortisol and ACTH were performed. Fourteen healthy horses not involved in competition were used as control group. RESULTS: Competition induced a significant increase in cortisol and ACTH responses in both, jumping and dressage horses and this effect was more apparent in dressage horses. When horses were most experienced, cortisol and ACTH responses were much lower. CONCLUSION: This study shows that competition elicits a classic physiological stress response in horses and that different training programmes induce different responses. It suggests that horses involved in competition can provide a good model to study the exercise-induced stress response.

Abstract: Reactions to stress vary between individuals, and physiological and behavioral responses tend to be associated in distinct suites of correlated traits, often termed stress-coping styles. In mammals, individuals exhibiting divergent stress-coping styles also appear to exhibit intrinsic differences in cognitive processing. A connection between physiology, behavior, and cognition was also recently demonstrated in strains of rainbow trout (Oncorhynchus mykiss) selected for consistently high or low cortisol responses to stress. The low-responsive (LR) strain display longer retention of a conditioned response, and tend to show proactive behaviors such as enhanced aggression, social dominance, and rapid resumption of feed intake after stress. Differences in brain monoamine neurochemistry have also been reported in these lines. In comparative studies, experiments with the lizard Anolis carolinensis reveal connections between monoaminergic activity in limbic structures, proactive behavior in novel environments, and the establishment of social status via agonistic behavior. Together these observations suggest that within-species diversity of physiological, behavioral and cognitive correlates of stress responsiveness is maintained by natural selection throughout the vertebrate sub-phylum.

Abstract: Conservation medicine is a discipline in which researchers and conservationists study and respond to the dynamic interplay between animals, humans, and the environment. From a wildlife perspective, animal species are encountering stressors from numerous sources. With the rapidly increasing human population, a corresponding increased demand for food, fuel, and shelter; habitat destruction; and increased competition for natural resources, the health and well-being of wild animal populations is increasingly at risk of disease and endangerment. Scientific data are needed to measure the impact that human encroachment is having on wildlife. Nonbiased biometric data provide a means to measure the amount of stress being imposed on animals from humans, the environment, and other animals. The stress response in animals functions via glucocorticoid metabolism and is regulated by the hypothalamic-pituitary-adrenal axis. Fecal glucocorticoids, in particular, may be an extremely useful biometric test, since sample collection is noninvasive to subjects and, therefore, does not introduce other variables that may alter assay results. For this reason, many researchers and conservationists have begun to use fecal glucocorticoids as a means to measure stress in various animal species. This review article summarizes the literature on many studies in which fecal glucocorticoids and their metabolites have been used to assess stress levels in various mammalian species. Variations between studies are the main focus of this review. Collection methods, storage conditions, shipping procedures, and laboratory techniques utilized by different researchers are discussed.