Measurement of fecal cortisol metabolites to evaluate adrenocortical function in Bottlenose dolphins (Tursiops truncatus)

The detection of hormones in fecal samples has been used during the last years in many different domestic and exotic species. However little information is available concerning metabolism and excretion of cortisol in bottlenose dolphins. Several studies have indeed focused on the detection and quantification of the hormone in blood or in feces, without identifying metabolites and their amounts with respect to cortisol. The aim of this work was to develop an analytical method to assess adrenal hormone and metabolites in the fecal sample of bottlenose dolphins (Tursiops truncatus), and determine the feasibility of its use in wild cetaceans in order to establish a non invasive method for the evaluation of stressful conditions. In the present work we developed a method for the extraction of cortisol and its metabolites (namely corticosterone, cortisone, dihydrocortisol and tetrahydrocortisol) from feces and their HPLC quantification as an alternative to the commonly used immunoassays. For the present study, 100 samples collected from 10 bottlenose dolphins were used. Age, reproductive status and physical characteristics of the animals were registered. All the dolphins used for the present study were trained for fecal sample collection as part of the normal routine of the clinical examination of the animals. Fecal samples (10/animal) were collected by voluntary behavior. After collection fecal samples were placed in a plastic container and kept frozen at minus 23 °C until analysis. First step of the method was fecal samples extraction, which was performed with a simple method implying the use of methanol, which was added to the samples in a 8:1 ratio. Extraction was performed by agitation for 30 minute and methanol was then liquid:liquid extracted with ethylic ether. Obtained extract were then dried under gentle nitrogen stream and resuspended in the mobile phase to be used for HPLC analysis. This was performed with a dC18 column (Atlantis, Water S.p.A.) following Bonilla-Jaime et al. (2006). Obtained data were compared with those previously obtained with petroleum or ethylic ether extraction and RIA detection. No differences were noticed in the cortisol and its metabolites levels in comparison with different physiological status in females. Considering group when male and females were kept together rise in fecal cortisol levels were observed during the warm season, when animals were showing more frequently sexual behaviors and were busier with shows and interactive activities. The difference observed in fecal cortisol is possibly due to a different metabolism between sexes, as excretion in females can occur earlier than in males. The method allowed us to identify the excretion profile of cortisol and its metabolites with feces and to underline all changes occurring in hormones excretion during altered health status. Present results allows us to conclude that fecal cortisol and its metabolites can be a useful tool for adrenal gland activity monitoring in bottlenose dolphins and that HPLC can be reliably used for the detection of over-stimulation of adrenal glands and for quantification of hormone metabolites in one single analysis.