Stress and reproductive events detected in North Atlantic right whale blubber using a simplified hormone extraction protocol

As studies quantifying steroid hormones in marine mammal blubber progress, methodological refinements may improve the utility and consistency of blubber hormone measurements. This study advances blubber extraction methodologies by testing a simplified extraction protocol that reduces time and complexity compared to a protocol widely used in cetacean blubber studies. Using blubber samples archived from remote biopsy (n = 21 live whales) and necropsy collection (n = 7 dead whales) of North Atlantic right whales (NARW; Eubalaena glacialis) of known life history states, we performed analytical and biological validations to assess the feasibility of measuring reproductive (testosterone, progesterone) and glucocorticoid (cortisol) hormones in blubber via enzyme immunoassay following the simplified extraction. Analytical validations (parallelism, accuracy, extraction efficiency, repeatability) showed the simplified extraction produced similar results to the extended protocol, offering a more efficient and consistent technique. In live, apparently healthy whales, blubber testosterone concentrations (mean ± SE) were significantly higher in males (2.02 ± 0.36 ng/g) compared to females (0.81 ± 0.15 ng/g). Blubber progesterone was highest in a confirmed pregnant female (60.3 ng/g), which was 12-fold greater than the mean concentration of non-pregnant females (4.56 ± 0.88 ng/g). Blubber cortisol concentrations in whales that died from anthropogenic causes averaged 5.31 ± 2.28 ng/g, whereas most live, healthy whales had cortisol values below 1 ng/g. Among living whales, a whale actively entangled in fishing gear had the highest blubber cortisol measurement (3.51 ng/g), exhibiting levels similar to whales that died from acute entanglement (2.88 ± 0.42 ng/g). Overall, the highest blubber cortisol concentration (18.0 ng/g) was measured in a dead whale with a severe chronic entanglement, approximately 30-fold greater than mean blubber cortisol of apparently healthy whales (0.58 ± 0.11 ng/g). The methodological approach presented here provides a reference for researchers interested in an alternative, streamlined technique for hormone extraction of cetacean blubber and contributes to the diverse tool set for stress and reproductive assessments of endangered NARWs.