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: Glucocorticoids (GCs; i.e. cortisol/corticosterone) are a central component of the stress response and thus their measurement is frequently used to evaluate the impact of stressful situations. Their metabolites from faeces of various animal species are more and more taken as a non-invasive aid to assess GC release and thus adrenocortical activity. The current literature review includes an extensive collection (1327 papers) and evaluation (see also Supplementary Tables) of the literature on faecal cortisol/corticosterone metabolite (FCM) analysis published to date. It aims at giving reference for researchers interested in implementing FCM analysis into their study or seeking to improve such methods by providing background knowledge on GC metabolism and excretion, conveying insights into methodological issues and stating caveats of FCM analysis and by highlighting prerequisites for and some examples of a successful application of such methods. Collecting faecal samples and analysing FCMs may appear simple and straightforward, but researchers have to select and apply methods correctly. They also need to be aware of the many pitfalls and potentially confounding factors and, last but not least, have to carefully interpret results. Applied properly, measurement of FCMs is a powerful non-invasive tool in a variety of research areas, such as (stress) biology, ethology, ecology, animal conservation and welfare, but also biomedicine.

Abstract: Non-invasive faecal oestrogen and progesterone metabolite evaluations are well established approaches for monitoring reproductive function in a variety of mammalian species. The route of excretion of steroid hormone metabolites varies considerably among species, and also between steroids within the same species. Steroid concentrations in faeces exhibit a similar pattern to those in plasma, but have a lag time, which depending upon the species, can be from 12 h to more than 2 days. Faecal steroid metabolites in mammals are mainly unconjugated compounds. Faecal oestrogens consist predominantly of oestrone and/or oestradiol-17α or -17β. Therefore, specific oestrogen antibodies or antibodies against total oestrogens can be used for their determination. Progesterone is metabolised to several 5α- or 5β-reduced pregnanediones and hydroxylated pregnanes prior to its faecal excretion. Therefore, relevant antibodies for their determination show considerable cross-reactivities with several pregnane metabolites, whereas specific progesterone antibodies are less suitable. Faecal oestrogen evaluations have been used as reliable indicators of pregnancy in several ungulate and some primate species. They have also been used to determine the preovulatory period in carnivores, corpus luteum activity in New World primates, and to diagnose cryptorchidism in horses. Faecal progesterone metabolite analysis has been successfully used for monitoring corpus luteum function and pregnancy, abortion, seasonality and treatment therapies in an ever expanding list of species.

Abstract: The aim of this comparative study was to gain more information about the excretion of steroid hormones in farm animals. This should help to establish or improve non-invasive steroid monitoring procedures, especially in zoo and wildlife animals. Over a period of 4 h the 14C-steroid hormones (3.7 MBq) progesterone (three females), testosterone (three males), cortisol and oestrone (two males, two females) were infused intravenously in sheep, ponies and pigs. Faeces were collected immediately after defecation. Urine was sampled via a permanent catheter in females and after spontaneous urination in males. A total of 88 +/- 10% (mean +/- SD) of the administered radioactivity was recovered. Considerable interspecies differences were measured both in the amounts of steroid metabolites excreted via faeces or urine and the time course of excretion. Progesterone and oestrone in ewes, and progesterone in mares were excreted mainly in the faeces (over 75%). The primary route of excretion of all other 14C-steroids was via the urine but to a different extent. In general, sheep showed the highest degree of faecal excretion and pigs the least. The highest radioactivity in urine (per mmol creatinine) was observed during the infusion or in one of the next two samples thereafter, whereas in faeces it was measured about 12 h (sheep), 24 h (ponies) or 48 h (pigs) after the end of the infusion. Thereafter the radioactivity declined and reached background levels within 2-3 weeks. In faeces, steroid metabolites were present mainly in an unconjugated form, but in blood and urine as conjugates. Mean retention time of faecal radioactivity suggested that the passage rate of digesta (duodenum to rectum) played an important role in the time course of the excretion of steroids. The information derived from this investigation could improve the precision of sampling as well as the extraction of steroids from the faeces. Furthermore, the study demonstrates that it should be possible to establish methods for measuring faecal androgen and cortisol metabolites for assessing male reproductive endocrinology and stress in animals.

Abstract: The ability of horses to learn and remember new tasks is fundamentally important for their use by humans. Fearfulness may, however, interfere with learning, because stimuli in the environment can overshadow signals from the rider or handler. In addition, prolonged high levels of stress hormones can affect neurons within the hippocampus; a brain region central to learning and memory. In a series of experiments, we aimed to investigate the link between performance in two learning tests, the baseline level of stress hormones, measured as faecal cortisol metabolites (FCM), fearfulness, and social rank. Twenty-five geldings (2 or 3 years old) pastured in one group were included in the study. The learning tests were performed by professional trainers and included a number of predefined stages during which the horses were gradually trained to perform exercises, using either negative (NR) or positive reinforcement (PR). Each of the learning tests lasted 3 days; 7min/horse/day. The NR test was repeated in a novel environment. Performance, measured as final stage in the training programme, and heart rate (HR) were recorded. Faeces were collected on four separate days where the horses had been undisturbed at pasture for 48h. Social rank was determined through observations of social interactions during feeding. The fear test was a novel object test during which behaviour and HR were recorded. Performance in the NR and PR learning tests did not correlate. In the NR test, there was a significant, negative correlation between performance and HR in the novel environment (rS=-0.66, P<0.001, i.e. nervous horses had reduced performance), whereas there was no such correlation in the home environment (both NR and PR). Behavioural reactions in the fear test correlated significantly with performance in the NR test in the novel environment (e.g. object alertness and final stage: rS=-0.43, P=0.04), suggesting that performance under unfamiliar, stressful conditions may be predicted by behavioural responses in a fear test. There was a negative correlation between social rank and baseline stress hormones (rS=-0.43, P=0.04), i.e. high rank corresponded to low FCM concentrations, whereas neither rank nor FCM correlated with fearfulness or learning performance. We conclude that performance under stressful conditions is affected by activation of the sympathetic nervous system during training and related to behavioural responses in a standardised fear test. Learning performance in the home environment, however, appears unrelated to fearfulness, social rank and baseline FCM levels.