Fecal and salivary cortisol determinations have been used in many species to monitor adrenal activity and the stress response. The present study examines the feasibility of measuring cortisol in saliva and feces of belugas as an alternative to blood and utilization of these measurements as indicators of acute vs. chronic stress. Multiple methods have been used to measure cortisol in the blood, saliva and feces. An Enzyme Immunoassay (EIA) was tested and utilized based on the expense and impracticality of utilizing radioactivity at our facility. We predict that a cortisol EIA kit designed for human serum will cross-react and measure cortisol in beluga serum, feces, and saliva. We hypothesize that cortisol levels in saliva reflect real-time blood levels of cortisol and can be used to detect acute stress whereas cortisol levels in feces reflect levels over a longer time frame and may be indicative of chronic stress.
Blood, saliva, and feces were collected from July 2008-March 2009 from three beluga whales housed at Mystic Aquarium & Institute for Exploration (MAIFE) as approved by Mystic Aquarium's Institutional Animal Care and Use Committee. At the end of August 2008, seven beluga whales were introduced in the MAIFE pool. We considered the introduction of the new animals and the following alteration of the social structure of the group as a possible stressor. Both feces and saliva were collected on the same day of a blood draw whenever possible. In addition, fecal samples were collected from wild beluga whales.
After collection, saliva and feces were immediately frozen at -20°C, while serum was frozen at -80°C. Diethyl ether was used to extract cortisol from 100 mg of feces (N = 81 from captive belugas and N = 6 from wild belugas), 0.2 ml of saliva (N = 19), and 0.2 ml serum (N = 16). The protocol provided by the EIA kit manufacturer (Cayman Chemical, Ann Arbor, MI) was carried out, including a series of dilutions to test for interfering substances remaining after the ether extraction process. In addition, serum samples were sent to an external laboratory (Cornell University) to validate results obtained with the EIA kit.
Each sample was assayed in triplicate. The intra-assay coefficients of variation were 1.95% for serum, 2.26% for saliva and 2.75% for feces. Fecal cortisol in captive belugas ranged between 0.27 ng/g and 28.60 ng/g, while in wild belugas fecal cortisol ranged from 1.12 ng/g to 7.04 ng/g. Salivary cortisol in captive whales ranged from 0.02 ng/ml to 0.45 ng/ml and serum cortisol ranged from 4.34 to 24.49 ng/ml. Cortisol levels in saliva and serum collected at the same time showed a positive correlation (r2 = 0.63, N = 11). As predicted, cortisol levels in feces did not correlate with cortisol levels in serum or saliva collected on the same day.
Although further investigation is needed to better characterize the time course of cortisol in the feces after a stressor and to identify the relative abundance of fecal glucocorticoid metabolites, our preliminary results suggest that the tested EIA can be used to measure cortisol levels in beluga serum, feces, and saliva, that salivary cortisol levels may be used as a means to monitor acute stress responses, and that fecal cortisol levels may reflect an average stress response over a longer time frame.