Whiskers as a Novel Tissue for Assessing Reproductive Histories in Phocid and Otariid Species: Laboratory and Biological Validations
Several populations of North Pacific pinnipeds are currently listed as depleted, endangered or with unknown status, highlighting the need for new methods to assess the reproductive rates of these populations. Keratinized tissues including baleen and whiskers have already proven an ideal tissue for acquiring a temporal record of marine mammal physiological parameters, such as stable isotope signatures for diet. ADF&G and our collaborators previously demonstrated the utility of whiskers as tissue matrix for quantifying steroid hormones (cortisol and progesterone) in phocid whiskers. Unlike other tissues currently used for determining reproductive status (e.g., serum, reproductive tracts), whiskers do not require special storage or handling and whiskers have been routinely collected from both live-captured pinnipeds and bio-sampled carcasses, providing archived tissues necessary for this proposed study. More importantly, unlike current sampling methods which provide a snap shot of reproductive status, reproductive hormone signatures are laid down sequentially along the length of the whiskers, allowing for measurement of hormone concentrations longitudinally over the course of time the whiskers are retained: one year in phocids and multiple years for otariids. In order to understand how different reproductive strategies impact the use of whiskers as a tool to assess reproductive histories, we have targeted four North Pacific pinniped species: 2 phocids (harbor and ringed seal) and 2 otariids (Steller sea lion and Northern fur seal).
In the present study, whiskers were sectioned with a hand chisel (5–15 mm) and sections were processed as previously described.1 Ground whisker sections were stored in 1.5 ml plastic vials at room temperature in the dark until extraction of steroid hormones following the methods previously described.1,2 Briefly, 100% methanol was combined with powdered hair samples, vortexed and then allowed to slowly spin on a rotator for 24–96 h at room temperature, following which the samples was centrifuged and snap frozen after which the supernatant was collected and frozen. Subsamples of the methanol with extracted steroid hormones were dried under forced air and reconstituted in assay buffer. Pools of extracted hormones from females for each species were used to validate commercially available progesterone enzyme immunoassay kits. Standard methods including recovery of added mass, parallelism and dilution linearity were used for the laboratory validations of ELISA kits for each adrenal hormone.2,3 Progesterone concentrations were determined along the length of whiskers collected from females with known reproductive events to determine progesterone concentrations during pregnancy with pup produced, diapause or pseudo-pregnancy, and non-reproductive years.
This work was supported in by NPRB # 1528. We thank Drs. Russ Andrews, Jason Waite and Lori Polasek from ASLC and UAF, Dr. Tom Gelatt from the NMFS/Alaska Fisheries Science Center, and Lori Quakenbush with ADF&G for whiskers and life history information.
* Presenting author
1. Macbeth B, Cattet M, Stenhouse G, Gibeau M, Janz D. Hair cortisol concentration as a noninvasive measure of long-term stress in free-ranging grizzly bears (Ursus arctos): considerations with implications for other wildlife. Can J Zool. 2010;88:935–949.
2. Hunt KE, Stimmelmayr R, George C, Hanns C, Suydam R, Brower H, Rolland RM. Baleen hormones: a novel tool for retrospective assessment of stress and reproduction in bowhead whales (Balaena mysticetus). Conserv Physiol. 2014;2:cou030.
3. Keogh MJ, Atkinson S, Maniscalco JM. Body condition and endocrine profiles of Steller sea lion (Eumetopias jubatus) pups during the early postnatal period. Gen Comp Endocrinol. 2013;184:42–50.