Abstract

The physiological stress response involves activation of the hypothalamic-pituitary-adrenal axis with the release of catecholamines and glucocorticoids such as cortisol in the blood stream. It has been shown that catecholamines and glucocorticoids can alter immune function, impacting health status. Monitoring cortisol in blood is an important part of conservation medicine and stress biology, yet the process of capturing an animal and drawing a blood sample can be a stressor in and of itself. This project aimed to validate methodology for measuring cortisol in exhaled breath condensate (blow) in beluga whales as a non-invasive means of monitoring stress. To make such a method cost-effective and easily reproducible in laboratories without specialized equipment such as a mass spectrometer, five commercially available cortisol EIA kits were tested for parallelism and linearity. Based on these results, further validation was carried out on a single kit. Blow samples were collected from four beluga whales at the Mystic Aquarium that were trained to exhale on signal. In addition blow samples were collected from wild belugas in Bristol Bay, AK during capture/release health assessments. Fifty ml conical tubes, 250 ml Nalgene bottles and petri dishes were tested as collection devices, while cotton, tulle, nylon stocking and nylon membrane were tested as collection materials. Two, 4 and 8 exhales were targeted and compared for volume of condensate recovered. To recover condensate, membranes were removed and centrifuged for 30 min at 3500 rpm, 10°C in 50 ml conical tubes. Nylon membrane over the bottom of a petri dish provided the best collection material. The number of exhales required to collect at least 50 μl varied between individuals based on depth of breath and length of exhale. Blow samples were pooled between females and males. A dilution series from 1 (undiluted) to 1:64 of each pool was run to test parallelism and linearity. Pools were spiked with three cortisol standards (640 pg/ml, 256 pg/ml, 102.4 pg/ml) in order to test for interfering substances in the blow matrix. A comparison of slopes for cortisol values vs. % bound showed no significant difference between standard curve and each pool. A significant relationship was observed between expected cortisol values and measured values for each pool, with slopes approximating 1. In addition, 256 pg/ml and 102.4 pg/ml standards were spiked onto membrane and showed recovery of 100 ± 10% of hormone.

Results indicate that blow is a possible matrix for measuring changes in cortisol as a non-invasive means to monitor stress in beluga whales. Further work is being conducted to perfect this methodology, adapt collection methods to free ranging whales, and quantify the relationship between blood and blow cortisol levels.

Acknowledgements

The authors would like to acknowledge the arctic coast husbandry staff at Mystic Aquarium for training blow collection behaviors, as well as Mandy Keogh and Suzin Webb for their help and guidance with sample collection and assay validation. Thank you also to Carrie Goertz, Rod Hobbs and the Bristol Bay Field team for the opportunity to participate in beluga health assessments and obtain samples from wild animals.

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+ Student presenter
^ Deceased