Saliva and Blood Cortisol Measurement in Bottlenose Dolphins (Tursiops truncatus): Methodology, Application, and...

A central task of zoos and aquaria is the frequent and accurate assessment of their animals' welfare. Recently, important steps have been made, such as the introduction of animal welfare evaluation tools and welfare decision trees. To determine animal welfare, it is not only important to collect life history data, such as longevity and reproductive success, but also for experienced observers or caretakers to conduct behavioral observations on a regular basis to assess animals' emotional state. To physiologically validate welfare observations, glucocorticoid levels are usually assessed, as they are a common indicator of stress. While, for many animals, these levels can be easily determined via fecal or hair samples, for cetaceans, the levels are usually determined via blood samples. As blood samples cannot be taken very frequently and the process may cause stress to the animals (if the samples are not taken following medical training), other techniques, such as the measurement of health biomarkers (especially cortisol, which can be measured in saliva), have become the focus of cetacean stress research. However, there are two problems associated with saliva measurements in cetaceans: saliva might either be diluted with pool water or be contaminated by fodder fish, as frozen fish usually contains high levels of cortisol. In our study, we investigated how saliva cortisol levels are connected to blood cortisol levels and how saliva cortisol can be influenced by fodder fish. We examined saliva and blood samples in eleven bottlenose dolphins (Tursiops truncatus) kept in an outdoor and indoor facility in Germany. Furthermore, we assessed the cortisol levels of different kinds of fodder fish. Our data show that, although saliva cortisol values are elevated under stress and arousal, they seem not to be correlated with blood cortisol values. We also show that, after feeding, saliva cortisol values are increased up to 100-fold. Our results suggest that saliva cortisol measurements in dolphins have to be conducted and considered with care, as they can easily be contaminated. Moreover, it is important to use the right laboratory method in order to specifically detect cortisol; in our study, we conducted reliable tests, using LC-MS/MS.