Abstract: In recent years, the noninvasive monitoring of steroid hormone metabolites in feces of mammals and droppings of birds has become an increasingly popular technique. It offers several advantages and has been applied to a variety of species under various settings. However, using this technique to reliably assess an animal's adrenocortical activity is not that simple and straightforward to apply. Because clear differences regarding the metabolism and excretion of glucocorticoid metabolites (GCMs) exist, a careful validation for each species and sex investigated is obligatory. In this review, general analytical issues regarding sample storage, extraction procedures, and immunoassays are briefly discussed, but the main focus lies on experiments and recommendations addressing the validation of fecal GCM measurements in mammals and birds. The crucial importance of scrutinizing the physiological and biological validity of fecal GCM analyses in a given species is stressed. In particular, the relevance of the technique to detect biologically meaningful alterations in adrenocortical activity must be shown. Furthermore, significant effects of the animals' sex, the time of day, season, and different life history stages are discussed, bringing about the necessity to seriously consider possible sex differences as well as diurnal and seasonal variations. Thus, comprehensive information on the animals' biology and stress physiology should be carefully taken into account. Together with an extensive physiological and biological validation, this will ensure that the measurement of fecal GCMs can be used as a powerful tool to assess adrenocortical activity in diverse investigations on laboratory, companion, farm, zoo, and wild animals.

Abstract: The continuous development of tourism and related leisure activities is exerting an increasingly intense pressure on wildlife. In this study, a novel noninvasive method for measuring stress in the black grouse, an endangered, emblematic species of European ecosystems that is currently declining in several parts of its European range, is tested and physiologically validated. A radiometabolism study and an ACTH challenge test were performed on four captive black grouse (two of each sex) in order to get basic information about the metabolism and excretion of corticosterone and to find an appropriate enzyme-immunoassay (EIA) to measure its metabolites in the feces. Peak radioactivity in the droppings was detected within 1 to 2 hours. Injected (3)H-corticosterone was excreted as polar metabolites and by itself was almost absent. A cortisone-EIA was chosen from among seven tested EIAs for different groups of glucocorticoid metabolites, because it cross-reacted with some of the formed metabolites and best reflected the increase of excreted corticosterone metabolites, after the ACTH challenge test. Concentrations of the metabolites from fecal samples collected from snow burrows of free-ranging black grouse were within the same range as in captive birds. The noninvasive method described may be appropriate for evaluating the stress faced by free-living black grouse populations in the wild, particularly in mountain ecosystems where human disturbance, especially by winter sports, is of increasing conservation concern.

Abstract: Fecal steroid analyses are becoming increasingly popular among both field and laboratory scientists. The benefits associated with sampling procedures that do not require restraint, anesthesia, and blood collection include less risk to subject and investigator, as well as the potential to obtain endocrine profiles that are not influenced by the sampling procedure itself. In the feces, a species-specific pattern of metabolites is present, because glucocorticoids are extensively metabolized. Therefore, selection of adequate extraction procedures and immunoassays for measuring the relevant metabolites is a serious issue. In this review, emphasis is placed on the establishment and analytical validation of methods to measure glucocorticoid metabolites for a noninvasive evaluation of adrenocortical activity in droppings of birds.

Abstract: IgA antibodies reflecting airways or intestinal mucosal immune responses can be found in saliva and feces, respectively, and IgG antibodies reflecting serum antibodies can be found in saliva. In this study, antibodies were detected in samples of saliva and feces which had been air-dried at room temperature (+20°C) or +37°C, and stored at these temperatures for up to 6 months. In saliva the antibody levels increased, while the antibodies in feces decreased upon storage. The individual IgA antibody concentrations which were adjusted by using the ratios of specific IgA/total IgA were relatively stable in both saliva and feces, and correlated with corresponding antibody levels in samples which had been stored at -20°C. The results indicate that air-dried saliva and feces can be used for semiquantitative measurements of mucosal antibodies, even after prolonged storage at high temperatures and lack of refrigeration.

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.