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.


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

How important are foundational, novel and review papers?

By Leila Lemos, PhD Student

As I wrote in my last blog post, I am in the process of studying for my preliminary exams that will happen in late March (written exams) and late April (oral exam).

My committee members provided me with reading lists of material they thought was important for me to know in order for me to become a PhD candidate. This will serve as the basis for my dissertation research, and provides the framework for how my contribution will advance the field. In the last month, I have been reading many, many articles, book chapters, theses, etc. to build this foundation.

One of the first steps was to organize all of the readings for my prelims on
a big board that would help me visualize what has been done and
what is still missing for each of the committee members


The material I am reading is a mixture of foundational and novel material, which are equally important. Foundational articles tell us about the origin of a specific field or theme, and help me to understand fundamental concepts and theories. It is really interesting to see what the pioneer researchers in the field first thought and how they tested their hypotheses many years ago. It is also remarkable to read novel papers and see how these foundational ideas have evolved and developed into new hypotheses, leading to new studies and experiments which push the boundaries of what we already know.

Review papers can also give a sense of this timeline by compiling studies on a particular topic. By assembling all of the available findings in my field, it becomes clear what questions remain unanswered, justifying the goals of my research, and establishing the project’s theoretical and methodological framework.

In my PhD project we are attempting to address some of the unanswered questions related to stress responses in baleen whales. Reading about other studies, their results, and the diverse techniques that have been applied to other taxa makes me really excited about what I can still incorporate in the project.

Source: http://binapatel.me/2017/05/25/literature-review-citation-


At the end of my PhD, if we are able to answer our proposed questions, we will have contributed to advancing the field of knowledge, and we will be able to apply our results to the conservation and management of baleen whales in nearshore coastal ecosystems.

The more I read the content proposed by my committee members, the more I find connections between my PhD project, its aims, and the title I proposed for myself as being a “Conservation Physiologist”. Being a Conservation Physiologist is exactly what I want to be, during my PhD, and in the future.




Who am I?

By Leila Lemos, PhD Student
(hopefully PhD candidate soon)


Here I am with the first GEMM Lab blog post of 2018.

Many people begin a New Year thinking about the future and planning goals to achieve in the following year, and that’s exactly how I am starting my year. After two and a half years of my PhD program, my classes and thesis project are nearing the end. However, a large hurdle stands between me and my finish line: my preliminary exams (as opposed to final exams that happen when I defend my thesis).

Oregon State University requires two sets of preliminary examinations (a.k.a. “prelims”) in order to become a PhD candidate. Thus, planning my next steps is essential in order to accomplish my main objective: a successful completion of these two exams.

The first set of exams comprises written comprehensive examinations to be taken over the course of a week (Monday to Friday), where each day belongs to a different member of my committee. The second type of exam is an oral preliminary examination, conducted by my doctoral committee. The written and oral prelims may cover any part of my proposed research topic as described in the proposal I submitted during my first PhD year.

In order to better understand this entire process, I met with Dr. Carl Schreck, a Fisheries and Wildlife Department professor and one of the members of my committee. He has been through this prelim process many times with other students and had good advice for me regarding preparation. He told me to meet with all of my committee members individually to discuss study material and topics. However, he said that I should first define and introduce myself with a title to each committee member, so they know how to base and frame exam questions. But, how do I define myself?

How do you define yourself?
Source: www.johngarvens.com/wpcontent/uploads/2013/02/how_do_you_ define_yourself.jpeg


As part of my PhD committee, Dr. Schreck is familiar with my project and what I am studying, so he suggested the title “Conservation Physiologist”. But, do I see myself as a Conservation Physiologist? Will this set-up have implications for my future, such as the type of job I am prepared for and able to get?

I can see it is important to get this title right, as it will influence my exam process as well as my scientific career. However, it can be hard and somewhat tricky when trying to determine what is comprised by your work and what are the directions you want to take in your future. I believe that defining the terms conservationist and physiologist, and what they encompass, is a good first step.

To me, a conservation specialist works for the protection of the species, their habitats, and its natural resources from extinction and biodiversity loss, by identifying and mitigating the possible threats. A conservation specialist’s work can help in establishing new regulations, conservation actions, and management interventions. As for an animal physiology specialist, their research may focus on how animals respond to internal and external elements. This specialist often studies an animal’s vital functions like reproduction, movement, growth, metabolism and nutrition.

According to Cooke et al. (2013), conservation specialists focus on population characteristics (e.g., abundance and structure) and indicators of responses to environmental perturbations and human activities. Thus, merging conservation and physiology disciplines enables fundamental understanding of the animal response mechanisms to such threats. Using animal physiology as a tool is valuable for developing cause-and-effect relationships, identifying stressor thresholds, and improving ecological model predictions of animal responses. Thus, conservation physiology is an inter-disciplinary field that provides physiological evidence to promote advances in conservation and resource management.

My PhD project is multidisciplinary, where the overall aim is to understand how gray whales are physiologically responding to variability in ambient noise, and how their hormone levels vary across individual, time, body condition, location, and noise levels. I enjoy many aspects of the project, but what I find myself most excited about is linking information about sex, age, body condition, and cortisol levels to specific individuals we observe multiple times in the field. As we monitor their change in body condition and hormones, I am highly motivated to build these whale ‘life-history stories’ in order to better understand patterns and drivers of variability. Although we have not yet tied the noise data into our analyses of whale health, I am very interested to see how this piece of the puzzle fits into these whale ‘life-history stories’.

In this study, animal physiology facilitates our stories. Scientific understanding is the root of all good conservation, so I believe that this project is an important step toward improved conservation of baleen whales. Once we are able to understand how gray whales respond physiologically to impacts of ocean noise, we can promote management actions that will enhance species conservation.

Thus, I can confidently say, I am a Conservation Physiologist.

Me, in Newport, OR, during fieldwork in 2017.
Source: Sharon Nieukirk, 2017.


Over the next three months I will be meeting with my committee members and studying for my prelims. I hope that this process will prepare me to become a PhD candidate by the time my exams come around in March. Then, I will have accomplished my first goal of 2018, so I can go on to plan for the next ones!



Cooke SJ, Sack L, Franklin CE, Farrell AP, Beardall J, Wikelski M, and Chown SL. What is conservation physiology? Perspectives on an increasingly integrated and essential science. Conserv Physiol. 2013; 1(1): cot001. Published online 2013 Mar 13. doi:  10.1093/conphys/cot001.