Use of faecal samples to monitor the oestrous cycle, reproductive status and adrenal gland activity in the bottlenose dolphin (Tursiops truncatus)

The detection of hormones in faecal samples has been used during the last years on many different domestic and exotic species, including humans. The information gained through the to application of non-invasive techniques has become an essential tool to investigate endocrinological and physiological mechanisms of several wild animal species, for which traditional methods of study would be difficult to perform. The scope of this thesis was to develop analytical methods to assess adrenal and reproductive hormones in bottlenose dolphin (Tursiops truncatus) faecal samples and determine the feasibility of its use with wild cetaceans. Due to the lack of information regarding the levels of hormones present in the faeces of bottlenose dolphins, I compare the results obtained from faecal samples with those obtained from serum and blood samples, vaginal cytology, ultrasounds and behaviour observations. After a general introduction to the production and role of steroids hormones and the non invasive techniques applied to study reproduction and endocrinological aspects of wildlife terrestrial and marine animals, in the second chapter I present the methodology applied to collect the samples from bottlenose dolphins used in the present study. All the samples were collected from the ten dolphins by voluntary behaviour. The first step was to set up a radio immuno assay (RIA) to determine progesterone, estrogens, testosterone and cortisol concentrations in faeces of bottlenose dolphins and each test was validated and parallelism test and recovery test were performed. In the fourth chapter I evaluate the presence of progesterone and estradiolo in faecal samples of eight females of different age and under different physiological conditions (Pre puberty, puberty, cycling, resting and pregnant). The results highlight as a major difference that in pregnant animals, progesterone never drop below 10 pmol/g of faeces, while in resting females it was always below that value. The results showed as well a correspondence between faecal levels and results observed with ultrasound and vagynal cytology. Two males were studied and results are reported in the fifth chapter. A baseline level for testosterone in juvenile animals has been established at 30pmol/g faeces. The results obtained for reproductive hormones suggest that the RIA can be applied to investigate the reproductive physiology in bottlenose dolphins. In chapter six I investigate the cortisol levels in faecal samples and comparing the results obtained with those values obtained from serum samples, the technique seems feasible to monitor chronic stress, while, due to the lag time between the moment the hormone is produced from the adrenal gland, until gets excreted in the faeces, it does not seems feasible to detect state of acute stress. On the contrary to results reported in literature for other marine mammals, no differences were noticed in the cortisol levels in comparison with different physiological status in females, while observing the group when male and females were kept together, rise in faecal cortisol levels were observed during the warm season, when animals were showing more frequently sexual behaviours and were more busy with shows and interactive activities. Important observation was done comparing the sexual hormonal levels in peri-pubertal females and the male sharing the pool with those: a rise of progesterone level was noticed to follow the rise of testosterone in the male. This suggest the possible "male effect" on the hormones production and cycle in females. It was further concluded that the radio immuno assay is effective to detect hormonal levels in faeces of bottlenose dolphins, but further steps are required to investigate the faecal presence of hormonal metabolites through the application of high-performance liquid chromatography and mass spectrometry (HPLC-MS)