Immunoreactive testosterone, progesterone and cortisol were quantified in blubber extracts using commercially available enzyme immunoassay systems (catalogue #ISWE001, ISWE003, ISWE002, respectively; Arbor Assays, Ann Arbor, MI), following the manufacturer’s protocols. These bulk-reagent immunoassay kits were developed specifically for measuring hormones and their metabolites in alternative sample matrices from diverse wildlife species. All samples, standards and controls were assayed in duplicate, with the coefficient of variation (CV%) between all duplicates < 10%. Quality control samples of high (~30%) and low (~70%) binding were included on each plate, with resulting inter-assay CVs of 1.6% and 3.6% for testosterone (n = 7 assays); 6.2% and 11.4% for progesterone (n = 7 assays); and 1.8% and 5.3% for cortisol (n = 7 assays). Final results were reported as nanograms of immunoreactive hormone and metabolites per gram of blubber tissue (ng/g), subsequently referred to simply as blubber testosterone, progesterone and cortisol. Antibody cross-reactivity, assay sensitivity and lower limit of detection values are available on the manufacturer’s website:

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