Faecal Glucocorticoid Metabolites in African Penguin: Biological validation of an enzyme immunoassay

Animals in zoos and aquaria are subject to a variety of physical, ecological, and social limitations. Physiological responses to stress in captive animals can be used to evaluate their welfare. Previous studies demonstrated that exposure to stress results in an increased secretion of glucocorticoids (GCs) from the adrenal cortex. In particular, corticosterone is the main avian gluticorticoid. Measurement of GCs metabolites in faeces has become a well established method for the non-invasive evaluation of the adrenocortical activity. The biological validation is a crucial step to demonstrate that a method can detect changes in adrenocortical activity and thus providing reliable results. The aim of this study was to validate a method for non-invasively measuring glucocorticoid in faeces of the African penguin (Spheniscus demersus). Samples were collected from a colony in a zoological institution (ZOOM Torino, Italy) accredited by the EAZA (European Association of Zoos and Aquaria). To perform the biological validation, we used a known stressful event: the capture and immobilisation of the animals for veterinary treatments. Faecal samples were collected from four adult males and five adult females before and after the stress. All samples were stored at -20°C for later analyses. After the extraction of metabolites with methanol, samples were analysed by an expressly developed enzyme immunoassay (EIA) based on the use of antibodies against corticosterone. Experimental results showed that the peak of secretion of GC metabolites occurred 7 to 10 hours after the stressful event, both in males and females. To verify the reliability of our methodology, we compared the results obtained with those from an EIA for tetrahydrocorticosterone methodology already validated to measure CG metabolites in faeces of the Adelia penguin (Pygoscelis adeliae) and the Wilson's storm petrel (Oceanites oceanicus). Our results concerning the biological validation suggest that the developed EIA for corticosterone can be a useful tool for non-invasively measuring GC metabolites in faeces of the African penguin. Moreover, our results are in agreement with those obtained through the EIA for tetrahydrocorticosterone method. Therefore, we propose to exploit this methodology to evaluate sources of stress such as abundance of visitors, noise level, and the environmental constrains. Moreover the ability to non-invasively monitor adrenocortical activity in African penguin is important for welfare management strategies since prolonged periods of elevated GC concentrations interfere with numerous physiological processes, including immune and reproductive function.