Abstract

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 samples 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, placed into plastic containers, and stored at minus 23°C until analysis. The first step of the extraction involved adding methanol to the samples in an 8:1 ratio, followed by agitation for 30 minutes, and then the methanol was liquid:liquid extracted with ethylic ether. The extracts 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.¹ 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. Difference between males and females were noticed for fecal cortisol (P< 0.05). Considering five animals housed at the same facility, fecal cortisol was examined considering two social situations: first when the five animals were separated in two groups and second when the five animals were kept together in the same pool. Results showed that when the animals were all together the fecal cortisol levels did not vary significantly (P>0.05) even though mean fecal cortisol levels were higher when animals were kept separated. On the contrary serum cortisol was higher (P< 0.001) when the five animals were together.

This can be due to the fact that in both groups there were females and males and the difference in fecal cortisol is not evident because of the homogeneity of the groups and to the different metabolism between sexes. Hunt et al² reported that in Steller sea lions excretion in females can occur earlier than in males. It was noticed that serum cortisol was higher when the animals were all together and it usually happened during the winter season. The increase of cortisol during those months could have been because of the lower temperature³ or because animals were fighting to establish hierarchic positions.

The method allowed us to identify the excretion profile of cortisol and its metabolites with feces and to underline all changes occurring in hormone excretion during altered health status. Present results allow us to conclude that fecal cortisol and its metabolites can be useful tools 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.

References

1.  Bonilla-Jaime H, Vázquez-Palacios G, Arteaga-Silva M, Retana-Márquez S. 2006. Hormonal responses to different sexually related conditions in male rats. Hormones and Behavior 49: 376-382.

2.  Hunt KE, Trites AW, Wasser SK. 2004. Validation of a fecal glucocorticoid assay for Steller sea lions (Eumetopias jubatus). Physiology & Behavior 80: 595-601.

3.  Suzuki M, Uchida S, Ueda K, Tobayama T, Katsumata E, Yoshioka M, Aida K. 2003. Diurnal and annual changes in serum cortisol concentrations in Indo-Pacific bottlenose dolphins (Tursiops aduncus) and killer whales (Orcinus orca). General and Comparative Endocrinology 132: 427-433.