Abstract

The use of blow collection to gather physiological and biological information from cetaceans, has increased in popularity as evidenced through application to new species, and through the development of new sampling techniques. Previous work within our lab has validated the use of blow sampling in belugas to monitor cortisol, testosterone, and progesterone.^1,2 However; questions remain as to the feasibility of blow collection in free-swimming small cetaceans and the influence of dilution on sample measures. This dilution may be due to variation in condensation within airways, force of breath, or environment (e.g., water splashing). The goal of this study is to determine a suitable marker of dilution in blow, which can be used to normalize hormone data from samples with different contributions of water content. For this work, cortisol was measured in blow samples using the previously validated Cayman Chemical Cortisol EIA.¹ Validation of two commercially available assays were carried out for each of two potential dilution markers: urea (Arbor Assays BUN Colorimetric Detection Kit and Quantichrome™ Urea Assay Kit) and total protein (Bradford Assay and Nanodrop).

We aimed to:

1.  Investigate the relationship between blow sample volume and cortisol, urea, and total protein measures

2.  Compare the relationship between cortisol values in blood and blow, with and without urea and total protein correction

3.  Test how dilute a blow sample can be for urea and total protein to provide accurate correction

4.  Test urea and total protein correction on samples collected from free swimming belugas

Blow samples were collected on 150 x 15 mm petri dishes with, and without, nylon membrane, held inverted over the blowhole. For aims 1, 2, and 3, blow samples were collected from three belugas using an approach that minimizes environmental water. Paired blood and blow samples were collected once per month, with additional monthly blow sampling occurring for aim 3. For aim 4, blow samples were collected from a single beluga during ‘free swim’ sessions, utilizing a collection pole with attached petri dish, to simulate collection in the wild. Complete analysis of data is forthcoming. However, large variation in the volume of blow collected for both monthly and free swim behaviors has been noted. Preliminary analyses suggest both urea (Quantichrome™) and total protein (Nanodrop) may be used to correct cortisol values, but that free swim samples may be extremely dilute and require extraction and concentration in order to measure cortisol and the target dilution markers. It is likely that there is a limit to how dilute samples can be while still providing reliable hormone databased on this approach.

Acknowledgements

The authors would like to thank the Arctic Coast Husbandry staff at Mystic Aquarium for training and maintaining breath behaviors for blow collection, as well as free-swim behaviors. We also thank the Veterinary team at Mystic Aquarium for monthly blood samples. This work was funded by the Office of Naval Research (ONR) awards N00012-18-1-2779 and N00014-21-1-2568.

*Presenting author

Literature Cited

1.  Thompson LA, Spoon TR, Goertz CE, Hobbs RC, Romano TA. 2014. Blow collection as a non-invasive method for measuring cortisol in the beluga (Delphinapterus leucas). PLoS One. 9(12):e114062.

2.  Richard JT, Robeck TR, Osborn SD, Naples L, McDermott A, LaForge R, Romano TA, Sartini BL. 2017. Testosterone and progesterone concentrations in blow samples are biologically relevant in belugas (Delphinapterus leucas). Gen Comp Endocrinol. 246:183–193.