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: Stress responses play an important role in allowing animals to cope with challenges. Glucocorticoids, key elements in the neuroendocrine stress axis, are traditionally measured as a parameter for welfare assessment. As blood sample collection itself disturbs an animal, non-invasive or minimal invasive methods have gained importance for assessing stress. In horses saliva and faeces are most frequently used. Faecal samples offer the advantage that they can be collected easily and stress-free. In faecal samples circulating hormone levels are integrated over a certain period of time. As a consequence faecal glucocorticoid metabolites represent the cumulative secretion and they are less affected by short episodic fluctuations of hormone secretion.
However, in order to gain reliable information about an animal’s adrenocortical activity, certain criteria have to be met: Depending whether the impact of acute or chronic stressors is assessed, frequent sampling might be necessary whereas in other cases, single samples will suffice. Background knowledge regarding the metabolism and excretion of glucocorticoids is essential and a careful validation is obligatory. In addition, this presentation will address analytical issues regarding sample storage, extraction procedures, and immunoassays and various examples of a successful application in equids will be given. Applied properly, non-invasive techniques to monitor stress hormones are a useful tool for animal welfare assessment.

Abstract: Non-invasive methods enable stress evaluation through measuring fecal glucocorticoid metabolites (FGMs), and immunoglobulin A (IgA) in the feces avoiding stressful blood drawing or stressful restraining of animals in the field. However, FGMs and IgA are mostly analysed in freshly frozen samples, which is difficult when fresh samples cannot be frozen immediately or frozen samples cannot be stored or transported. Good results were also derived from air-dried fecal samples, which are hampered by unstable air humidity in the field. These difficulties may be overcome, when drying of samples could be induced with colorless silica gel (SiO2) granules in a secure set-up, such as an air tight tube. We determined the speed of drying 1.5 g of a fresh fecal sample from six horses on air and on silica gel. Furthermore, FGMs and IgA were analysed in differently stored subsamples from 12 horses: in frozen fecal samples, in air- or silica gel-dried samples stored for 1 day and for 7 days, and in wet fecal samples kept in a tube at room temperature for 7 days. FGM levels remained stable in feces dried on air or on silica gel for 7 days, whereas IgA quantities showed a significant loss. Under field conditions, when freezing or transporting the frozen samples is not possible and humidity hampers air drying, drying samples on silica gel in air tight tubes appears to be very helpful and reliable for analysing FGMs.

Abstract: In this study, we investigated endocrine factors and behaviour in free-living Common hamsters (Cricetus cricetus) during reproductive and non-reproductive periods of the annual cycle. We applied a non-invasive method to gain information on seasonal changes in adrenocortical activity in male and female hamsters by analysing faecal glucocorticoid metabolite concentrations (FCM). In addition, plasma progesterone concentrations were monitored in females throughout the non-hibernation season. The animals were live-trapped from spring emergence until the onset of hibernation in autumn. Reproductive status was determined at capture and blood and faecal samples were collected. During behavioural observations, agonistic and sexual interactions were recorded. FCM concentrations were significantly higher in males than in females during the reproductive period. In males, a pronounced increase in FCM during the reproductive period coincided with high frequencies of intrasexual aggression. In females, FCM levels remained relatively constant. Aggressive behaviour in females increased during the reproductive period, but was much less frequent than in males. Females, which successfully raised a second litter after a postpartum oestrus and concurrent lactation and gestation had lower FCM levels than individuals, which lost their second litter after parturition. As expected, plasma progesterone concentrations were low before and after the reproductive period. During gestation, levels peaked and remained elevated during lactation. The results of this field study provide insight in critical periods associated with reproduction in male and female Common hamsters.

Abstract: Faecal steroid hormone metabolites are becoming increasingly popular as parameters for reproductive functions and stress. Theextraction of the steroids from the faecal matrix represents the initial step before quantification can be performed. The steroid metabolites present in the faecal matrix are of varying polarity and composition, so selection of a proper extraction procedure is essential. There have been some studies to address this complex but often neglected point. Radiolabelled
steroids (e.g. cortisol or progesterone) have frequently been added to faecal samples to estimate the efficiency of the extraction procedures used. However, native, unmetabolized steroids are normally not present in the faeces and therefore the results are artificial and do not accurately reflect the actual recoveries of the substances of interest. In this respect, recovery experiments based on faecal samples from radiometabolism studies are more informative. In these samples, the metabolite content accurately reflects the mixture of metabolites present in the given species. As a result, it is possible to evaluate different extraction methods for use with faecal samples. We present studies on sheep, horses, pigs, hares and dogs that utilized samples containing naturally metabolized, 14C-labelled steroids.