Get the most out of blow hormones: validation of sampling materials, field storage and extraction techniques for whale respiratory vapour samples

Studies are progressively showing that vital physiological data may be contained in the respiratory vapour (blow) of cetaceans. Nonetheless, fundamental methodological issues need to be addressed before hormone analysis of blow can become a reliable technique. In this study, we performed controlled experiments in a laboratory setting, using known doses of pure parent hormones, to validate several technical factors that may play a crucial role in hormone analyses. We evaluated the following factors: (i) practical field storage of samples on small boats during daylong trips; (ii) efficiency of hormone extraction methods; and (iii) assay interference of different sampler types (i.e. veil nylon, nitex nylon mesh and polystyrene dish). Sampling materials were dosed with mock blow samples of known mixed hormone concentrations (progesterone, 17 beta-estradiol, testosterone, cortisol, aldosterone and triiodothyronine), designed to mimic endocrine profiles characteristic of pregnant females, adult males, an adrenal glucocorticoid response or a zero-hormone control (distilled H2O). Results showed that storage of samples in a cooler on ice preserved hormone integrity for at least 6 h (P = 0.18). All sampling materials and extraction methods yielded the correct relative patterns for all six hormones. However, veil and nitex mesh produced detectable assay interference (mean 0.22 +/- 0.04 and 0.18 +/- 0.03 ng/ml, respectively), possibly caused by some nylon-based component affecting antibody binding. Polystyrene dishes were the most efficacious sampler for accuracy and precision (P < 0.001), but required an ethanol rinse for improved progesterone recovery (increased 81%; P < 0.001). Awareness of assay interference from exogenous materials is crucial to future studies. This study establishes critical groundwork to help ensure that hormones can be measured accurately in samples obtained from field collections of whale blow.