Abstract

Steroid hormones have been measured in the blow (respiratory vapor) of several species of cetaceans.^1-3 This method may provide a minimally invasive way to assess the reproductive status of a free-swimming cetacean. While testosterone has previously been measured in cetacean blow, the resulting concentrations are difficult to interpret because the relationship between testosterone concentrations in blow and blood are unknown. Utilizing samples collected from trained belugas, the aims of this project were to validate an assay for testosterone in beluga blow, determine if testosterone concentrations in blow correlated with those in matching blood samples, and to evaluate the ability to assess the reproductive seasonality and status of the beluga using blow testosterone concentrations.

Using pooled male blow samples, a commercially available enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) was validated for testosterone in beluga blow samples. A serially diluted pool had binding parallel to the standard curve (p = 0.45). The recovery of testosterone from spiked blow sample pools (n = 5, range 15–250 pg) was 102 ± 7% (y = 1.04x–2.04, R² = 0.99). Matched blood and blow samples (n = 38) collected from 4 male belugas (3 adults and 1 juvenile) demonstrated a significant positive relationship between testosterone concentration in blood and blow (F_{1, 37} = 24.41, p < 0.001). Seasonality of testosterone secretion in blow was demonstrated (p < 0.001) in two adult males sampled for one full year, with peak concentrations occurring during the breeding season (February–April). A comparison between 4 adult males from February–April (n = 29) and May–January (n = 62), 4 juvenile males (n = 12), and 10 female belugas (n = 20) demonstrated a significant difference in blow testosterone concentrations (Bonferroni adjusted ANOVA, p < 0.017) between each group except juvenile males and females. Although there is a high degree of overlap between out-of-season adult males, juvenile males, and females at low testosterone concentrations, a threshold value of 85 pg/ml would allow the detection of an adult male. Of the samples that exceeded this threshold, 57/59 (97%) were collected from adult males, which accounts for 63% of all adult male samples (n = 91). From February through April, 28/29 (97%) of the adult male samples exceeded this threshold. This method can be used to detect maturity or monitor seasonality in male belugas in aquaria. Testosterone analysis in blow is also applicable to temporarily stranded wild belugas in areas such as the Cook Inlet, Alaska, where temporary mass strandings occasionally occur, providing a minimally invasive way to identify males in breeding condition.

Acknowledgements

This research is made possible by the beluga training and veterinary staffs at the Mystic Aquarium (especially Kristine Magao, Lindsey Nelson, and Kate McElroy), Georgia Aquarium (especially Cara Field and Tonya Clauss), SeaWorld San Antonio (especially Todd Robeck, Shannon Bond, Steve Aibel, and Steve Osborn), and Shedd Aquarium (especially Lisa Naples, Lisa Takaki, Maris Muzzy, and Frank Oliaro). Special thanks to Renee Bakker for laboratory assistance and to Mandy Keogh and Laura Thompson for their guidance. This project is supported by the National Science Foundation Graduate Research Fellowship and Marine Mammal Commission Research Grant awarded to Justin Richard.

* Presenting author
⁺ Student presenter

Literature Cited

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2.  Hunt KE, Rolland RM, Kraus SD. Detection of steroid and thyroid hormones via immunoassay of North Atlantic right whale (Eubalaena glacialis) respiratory vapor. Mar Mamm Sci. 2014;30:796–809.

3.  Thompson LA, Spoon TR, Goertz CE, Hobbs RC, Romano TA. Blow collection as a non-invasive method for measuring cortisol in the beluga (Delphinapterus leucas). PLoS ONE. 2014;9:e114062.