Abstract

Few opportunities exist to collect blood from wild cetaceans, principally because most species cannot be safely captured and restrained.¹ Thus, the collection of non-blood samples, such as blubber, feces, and respiratory vapor (‘blow’), has been the preferred approach for these species.^1,2 These samples have frequently been used in endocrine studies, primarily to diagnose pregnancy,^3-5 evaluate male reproductive activity,^6,7 and identify severe adrenal stress;^8-10 however, they have only been validated for these purposes in a few species. Further, the extent to which sample treatment (e.g., collection materials, storage methods) influences hormone levels, in these samples, remains largely unknown.

The objective of this research was to evaluate the use of blow and blubber steroid hormone monitoring for reproductive and adrenal studies of cetaceans, using captive bottlenose dolphins (Tursiops truncatus and T. aduncus) as model species. Cortisol, progesterone, testosterone, and estradiol concentrations were measured in blow and blubber using enzyme-immunoassays. Concentrations were compared to patterns in serum hormone levels, and examined in relation to age class, sex, reproductive status, season, time of day (morning or afternoon), and collection method (in- or out-of-water sampling). Control samples were also prepared to identify if our collection, storage, and extraction methods interfered with hormone measurements. Hormones were not detected in any blubber sampling controls. In contrast, blow hormone levels increased with the mass of nylon sampling material used and with the amount of seawater contamination. Concentrations were further affected by variability in blow sample volumes and respiratory water dilution. To combat these issues, we trialed the use of two alternative units of measure to absolute blow hormone concentrations: (1) relative hormone concentrations (hormone ratios), and (2) normalized hormone levels (using urea nitrogen concentration as a potential endogenous standard). Neither of these metrics performed significantly better than absolute levels. Nevertheless, some biologically relevant trends that were present in blubber were also detected in blow samples. For instance, pregnant females possessed significantly higher progesterone levels (blow: 0.60±0.2 ng/mL; blubber: 13.012±0.7 ng/g, p<0.001) than non-pregnant females (blow: 0.17±0.01 ng/mL; blubber: 1.17±0.1 ng/g), and estradiol levels were highest in females sampled during an estrous cycle (blow: 0.69±0.2 ng/mL; blubber: 5.044±1.4 ng/g). There were also significant differences in testosterone levels between demographic groups but only for blubber samples. As anticipated, concentrations were higher in adult males (6.59±0.8 ng/g) than in juvenile males (1.15±0.5 ng/g, p<0.001) or females (1.76±0.2 ng/g; p=0.0032). Overall, blubber hormone monitoring appears suitable for detecting pregnancies, identifying sexually mature males, and possibly detecting estrous cycles in bottlenose dolphins. In contrast, our blow sampling methods are unsuitable for evaluating the reproductive and adrenal condition of wild cetaceans.

Acknowledgements

This project was funded by the Holsworth Wildlife Research Endowment and by the School of Veterinary Science, The University of Queensland. The authors would like to thank SeaWorld Australia, in particular Trevor Long, Mitchell Leroy, Andrew Mulville, Benjamin Markham, Christina Holsheimer, Shari Bryden, Deon Griffin, and Cathrine Atkin for continuous support in sample collection, and for use of their facilities. Thanks to Professor Jonathan Hill for additional discussion and comments.

* Presenting author
+ Student presenter

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