Optimizing hormone extraction protocols for whale baleen

By Alejandro Fernández Ajó, Postdoc, OSU Department of Fisheries, Wildlife, and Conservation Science, Geospatial Ecology of Marine Megafauna Lab

Large whale conservation is challenged by our limited understanding of the impacts of natural and anthropogenic disturbances on the whale´s health and its population level consequences. To better mitigate human-wildlife conflicts, we need to improve our ability to predict multi-scale responses of whales to disturbances, describe and identify disease dynamics, and understand the reproductive biology of whales (Madliger, 2020; McCormick and Romero, 2017). Conservation physiology and conservation endocrinology can provide tools to illuminate the underlying physiological mechanisms whales use to cope with changing environments and different stressors, thus filling information gaps to guide management and conservation actions.

In brief, conservation physiology is a multidisciplinary field wherein a broad suite of tools and concepts are used to understand how organisms and ecosystems respond to both environmental and anthropogenic change and stressors (Madliger et al., 2020). Conservation endocrinology is a subdiscipline within conservation physiology, which relies on endocrine measurements (hormone quantifications). However, monitoring the physiology of free ranging animals in wild populations presents many technical challenges and it is particularly difficult when studying whales. Traditionally, conservation endocrinology relied on laboratory analyses of plasma samples (derived from blood). Yet implementing this techniques for monitoring the physiology of mysticetes (baleen whales) is currently impossible, as there are no feasible, non- (or minimally) invasive, methods to obtain a blood sample from living large whales (Hunt et al., 2013).

Therefore, we are interested in the development and further validation of alternative sample types from whales to obtain endocrine data. During my Ph.D. dissertation I worked to develop and ground truth the endocrine analyses of whale baleen as a novel sample type that can be used for retrospective assessments of the whale´s physiology. Baleen, the filter-feeding apparatus of the mysticete whales (Figure 1), consists of long fringed plates of stratified, keratinized tissue that grow continuously and slowly downward from the whale´s upper jaw (Hunt et al., 2014). Baleen plates are readily accessible at necropsy and routinely collected from carcasses of stranded whales.

Like hair, nails, feathers, spines, or horns of other animals, baleen is a keratinized tissue that can store steroid and thyroid hormones in detectable and relevant concentrations to provide an integrated measure of hormonal plasma levels over the period that the structure was growing. Thus, baleen contains a progressive time-series that captures months and often years of an individual’s endocrine history with sufficient temporal resolution to determine seasonal endocrine patterns allowing to explore questions that have historically been difficult to address in large whales, including pregnancy and inter-calving interval, age of sexual maturation, timing and duration of seasonal reproductive cycles, adrenal physiology, and metabolic rate. Additionally, their robust and stable keratin matrix allows baleen samples to be stored for years to decades, enabling the analysis and comparison of endocrine patterns from past and modern populations. Therefore, keratinized sample matrices are valuable tools to investigate reproductive and stress physiology in whales and other vertebrates.

However, due to its novelty, the extraction and analysis of hormones from baleen and other keratinized tissues requires both biological and analytical validations to ensure the method fulfills the requirements for its intended use. Baleen hormone analyses has already passed several essential assay validations, including parallelism and accuracy of immunoassays (Hunt et al., 2017b), and numerous biological validations, such as the study of animals with known physiological status (i.e., pregnancy, and known stress events such as entanglement in fishing gear or presence of lesions) to assess the degree to which the endocrine data reflect the physiology of the individual (Fernández Ajó et al., 2020, 2018; Hunt et al., 2018, 2017a; Lysiak et al., 2018; Palme, 2019). Yet, other questions essential for technical validation remain unknown, including choice and volume of extraction solvent, the effect of solvent-to-sample ratio (solvent:sample) on extraction yield, and the amount of sample (e.g., mg) needed for analysis to obtain reliable hormonal data.

In our recent contribution, Optimizing hormone extraction protocols for whale baleen: Tackling questions of solvent:sample ratio and variation, we aimed to tackle two of these important questions: “1) what is the minimum sample mass of baleen powder required to reliably quantify hormone content of baleen samples analyzed using commercially available enzyme immune assays (EIAs); and 2) what is the optimal ratio of solvent volume to sample mass for steroids extracted from baleen, i.e., the ratio that yields the maximum amount of hormone with high accuracy and low variability between replicates.”

We performed the extraction with methanol and tested a variety of sample masses with the objective to provide methodological guidance regarding optimizing sample mass and solvent volume for steroid hormone extraction from powdered baleen. Our results suggest that the optimal sample mass for methanol extraction of steroid hormones from baleen samples is 20 mg, and that larger sample masses did not produce either better yield or less variation in the apparent hormone per g of baleen sample (Figure 2). In addition, when the extraction was performed keeping the volume of solvent proportional to the sample mass (namely, a solvent:sample ratio of 80:1), masses as small as 10 mg yielded reliable hormone measurement (Figure 2).


Our results indicate how baleen hormone analytic techniques can be more widely employed on small sample masses from rare specimens (i.e., less sample than is currently employed, which is typically 75 mg or 100 mg in most studies to date), such as from natural history museums and stranding archives. Thus, we demonstrate that greater use of this valuable technique to reconstruct the endocrine and physiological history of individual whales over time can be achieved with reduced sample size (so reduced damage to the sample). I hope these findings encourage researchers to apply these methods more broadly to analyze historical archives of baleen plates that can date back to the era of commercial whaling, and modern archives of baleen collected from stranded animals to help continue further developing techniques that can make headway in gaining conservation-relevant physiological knowledge of this particularly challenging taxon.

Bibliography:

Fernández Ajó, A., Hunt, K.E., Dillon, D., Uhart, M., Sironi, M., Rowntree, V., Loren Buck, C., 2021. Optimizing hormone extraction protocols for whale baleen: Tackling questions of solvent:sample ratio and variation. Gen. Comp. Endocrinol. 113828. https://doi.org/10.1016/j.ygcen.2021.113828

Fernández Ajó, A.A., Hunt, K.E., Giese, A.C., Sironi, M., Uhart, M., Rowntree, V.J., Marón, C.F., Dillon, D., DiMartino, M., Buck, C.L., 2020. Retrospective analysis of the lifetime endocrine response of southern right whale calves to gull wounding and harassment: A baleen hormone approach. Gen. Comp. Endocrinol. 296, 113536. https://doi.org/10.1016/j.ygcen.2020.113536

Fernández Ajó, A.A., Hunt, K.E., Uhart, M., Rowntree, V., Sironi, M., Marón, C.F., Di Martino, M., Buck, C.L., 2018. Lifetime glucocorticoid profiles in baleen of right whale calves: potential relationships to chronic stress of repeated wounding by Kelp Gulls. Conserv. Physiol. 6, 1–12. https://doi.org/10.1093/conphys/coy045

Hunt, K.E., Lysiak, N.S., Moore, M., Rolland, R.M., 2017a. Multi-year longitudinal profiles of cortisol and corticosterone recovered from baleen of North Atlantic right whales (Eubalaena glacialis). Gen. Comp. Endocrinol. 254, 50–59. https://doi.org/10.1016/j.ygcen.2017.09.009

Hunt, K.E., Lysiak, N.S., Robbins, J., Moore, M.J., Seton, R.E., Torres, L., Loren Buck, C., Buck, C.L., 2017b. Multiple steroid and thyroid hormones detected in baleen from eight whale species. Conserv. Physiol. 5. https://doi.org/10.1093/conphys/cox061

Hunt, K.E., Lysiak, N.S.J., Matthews, C.J.D., Lowe, C., Fernández Ajó, A., Dillon, D., Willing, C., Heide-Jørgensen, M.P., Ferguson, S.H., Moore, M.J., Buck, C.L., 2018. Multi-year patterns in testosterone, cortisol and corticosterone in baleen from adult males of three whale species. Conserv. Physiol. 6, 1–16. https://doi.org/10.1093/conphys/coy049

Hunt, K.E., Moore, M.J., Rolland, R.M., Kellar, N.M., Hall, A.J., Kershaw, J., Raverty, S.A., Davis, C.E., Yeates, L.C., Fauquier, D.A., Rowles, T.K., Kraus, S.D., 2013. Overcoming the challenges of studying conservation physiology in large whales: a review of available methods. Conserv. Physiol. 1, cot006–cot006. https://doi.org/10.1093/conphys/cot006

Hunt, K.E., Stimmelmayr, R., George, C., Hanns, C., Suydam, R., Brower, H., Rolland, R.M., 2014. Baleen hormones: a novel tool for retrospective assessment of stress and reproduction in bowhead whales (Balaena mysticetus). Conserv. Physiol. 2, cou030–cou030. https://doi.org/10.1093/conphys/cou030

Lysiak, N.S.J., Trumble, S.J., Knowlton, A.R., Moore, M.J., 2018. Characterizing the Duration and Severity of Fishing Gear Entanglement on a North Atlantic Right Whale (Eubalaena glacialis) Using Stable Isotopes, Steroid and Thyroid Hormones in Baleen. Front. Mar. Sci. 5, 1–13. https://doi.org/10.3389/fmars.2018.00168

Madliger, C. L., Franklin, C. E., Love, O. P., & Cooke, S.J. (Ed.), 2020. Conservation Physiology: Applications for Wildlife Conservation and Management., 1st ed. Oxford University Press. https://doi.org/10.1093/oso/ 978019883610.001.0001

McCormick, S.D., Romero, L.M., 2017. Conservation Endocrinology. Bioscience 67, 429–442. https://doi.org/10.1093/biosci/bix026

Palme, R., 2019. Non-invasive measurement of glucocorticoids: Advances and problems. Physiol. Behav. 199, 229–243. https://doi.org/10.1016/j.physbeh.2018.11.021

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