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

Abstract

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.

Keywords: Tursiops truncatus; animal welfare; blood cortisol; bottlenose dolphin; saliva cortisol; stress measurement.

Figure A1

Saliva sampling, Image of the saliva sampling procedure.

Figure A2

Blood cortisol values for five female and five male dolphins who underwent two different treatments: restriction and training. (A) Healthy animals versus animals experiencing medical treatment (referred to as sick). (B) The two different test assays, which had different sensitivities.

Figure A3

Correlation between blood cortisol values and saliva values during restriction. A logarithmic curve was fitted. Saliva samples were taken before (green dots) and after (red dots) the blood samples; see Figure 3 for an exemplarily sampling scheme. Three dolphins were sampled (Jenny = 9 samples; Moby = 1 sample; Anke = 1 sample; Nami = 2 samples before, 3 samples after).

Figure A4

Correlation between blood cortisol values and saliva values during restriction. An exponential curve was fitted. Saliva samples were taken before (green dots) and after (red dots) the blood samples; see Figure 3 for an exemplarily sampling scheme. Three dolphins were sampled (Jenny = 9 samples; Moby = 1 sample; Anke = 1 sample; Nami = 2 samples before, 3 samples after).

Figure 1

Scheme of the outdoor pools, called the lagoon, where the dolphins were housed.

Figure 2

Dilution series for saliva cortisol analyzed at two laboratories (Lab R and Lab D). Saliva was collected over some days, accumulated, and then diluted with different proportions of 0.9% NaCl solution. Saliva samples were analyzed with two different methods LC-MS/MS (LC-MS) and immunoassays (IBL).

Figure 3

Exemplary sampling scheme for the simultaneous blood and saliva sampling.

Figure 4

Fixed saliva sampling schemes used for (A) feeding experiment I and (B) feeding experiment II.

Figure 5

Blood cortisol values for five female and five male dolphins for the treatments restriction, and training.

Figure 6

Correlation between blood cortisol values and saliva values during restriction. A linear curve was fitted (see Figure A3 and Figure A4 for other curves). Saliva samples were taken before (green dots) and after (red dots) the blood samples; see Figure 3 for an exemplarily sampling scheme. Three dolphins were sampled (Jenny = 9 samples; Moby = 1 sample; Anke = 1 sample; Nami = 2 samples before, 3 samples after).

Figure 7

Cortisol values of different fodder fish and cephalopods. We measured samples taken from the thawing water of a 20 kg block of Atlantic herring, a 15 kg block of Baltic herring, a 5 kg block of capelin, a 20 kg block of mackerel, an 8 kg block of sprat and an 8 kg block of squid.

Figure 8

Saliva cortisol values during feeding experiment I. Saliva samples were taken every 2 min over a course of 6 min. At minute 0, dolphins were fed with fish (capelin, Mallotus villosus) and a saliva sample was taken directly afterwards. The experiment was conducted with 3 female dolphins and repeated 10–11 times (for Dolly and Donna, 11 times; for Sunny, 10 times). Some selected significance levels of Tukey’s pairwise post hoc comparisons are indicated as follows: * p < 0.05, *** p < 0.001. For all results, see Table A2.

Figure 9

Saliva cortisol values during feeding experiment II. Saliva samples were taken every two minutes over a course of 10 min. At minute 0, dolphins were fed with fish (capelin, Mallotus villosus) and a saliva sample was taken directly afterwards. The experiment was conducted with 5 female dolphins and repeated 5–7 times (Anke, 5 times; Dolly, Jenny, and Sunny, 6 times; Donna, 7 times). Some selected significance levels of Tukey’s pairwise post hoc comparisons are indicated as follows: * p < 0.05, *** p < 0.001. For all results, see Table A3.