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Validation of 8 Assays in 8 Species of Whales

Even with significant protections in place, whale populations continue to be low relative to apparent historic norms.   Unfortunately, the reasons for low population and slow population growth have proven to be elusive.  Wild whales are difficult subjects for study.  Samples are hard to obtain, collection stressful for the whales, and getting repeated samples from the same individual over time is extremely rare.   It is therefore incredibly valuable to develop and validate procedures using available, non-invasive sample types to provide insight into the lives and health of wild whale populations.

Studies have demonstrated progesterone and cortisol can be recovered and measured in samples of powdered baleen from Bowhead and North Atlantic right whales and that highest levels of the hormones could be correlated to known observed pregnancies.   Baleen grows continuously and slowly from a root region in the upper jaw of the whale.  Years of growth for a baleen sample can be determined by counting annual cycles in stable isotope levels deposited as the plate grows spanning anywhere from 1.3-10 years on average depending upon the species of whale.  This means samples from a single plate have the potential to follow the hormone levels of an individual over time.  Baleen plates can be easily collected post mortem; they are dry and highly stable, with steroid hormone levels having been shown to be stable in room temperature baleen for at least a decade.

A recent study by K. Hunt et. al seeking to expand the potential of baleen as a sample type for hormone studies, sought to determine if the techniques initially developed may be suitable for other whale species and additional hormones.  In this study baleen samples from eight species of whales (right, bowhead, blue, sei, minke, fin, humpback and grey) were assayed for eight different hormones of interest (progesterone, testosterone, estradiol, aldosterone, corticosterone, cortisol, T3 and T4) using Arbor Assays colorimetric EIA kits.   Because baleen sampling involves the destruction of a portions of the baleen plate, initial validation was done using just a single baleen plate from a single individual in each species limiting the amount of sample available for testing.

To prepare samples, 500 mg of powdered baleen was collected from each plate to be sampled.  Hormones were then extracted by mixing a 100 mg aliquot of weighed powder with 6.00 mL of HPLC-grade methanol, vortexed for 2 hours, and then centrifuged for 15 minutes at 3000g.  Supernatant was removed and dried under vacuum at 35°C for 5 hours.  At the time of assay, dried samples were reconstituted with 500 mL of assay buffer, sonicated for 1 minute and vortexed for 5 minutes.   Samples were then assessed for parallelism in each of the hormone assay kits.   Parallelism was tested by assaying eight serial dilutions of the baleen extract for each species alongside known concentration hormone standards.    Because of the limited sample volume available and the value of the samples being tested, accuracy testing was only carried out for the progesterone, testosterone and cortisol assays.

Target hormones were detectable in baleen powder for all of the species tested in all eight hormone assays.  In some cases the hormone concentration was quite low, especially for cortisol in the blue, fin and grey whale samples, but parallelism was demonstrated for all species in all the assays.  Accuracy was also demonstrated for all species in the testosterone, progesterone and cortisol assays with linear relationships of observed vs expected concentration and a slope within the desired range of 0.7-1.3.   Overall, hormone level patterns were consistent with relative concentrations typically seen in mammalian plasma.

Somewhat unexpectedly, corticosterone was found to be present in baleen at higher concentrations than cortisol in all samples except the minke whale.  Historically, cortisol has been assumed to be the more abundant of the two glucocorticoids in whale plasma.  However, it is important to note that this assumption was based on just a few cases of plasma glucocorticoid measurement in mysticete whales, all of which involved acute stress situations such as stranding or hunting.    Data from the current study suggests corticosterone may prove to be the better target for those interested in assessing HPA stress levels through baleen samples especially if sample amounts are limited.

Although these results represent only an initial validation, good data from the immunoassays for a variety of steroid hormones across a wide range of whale species suggests strong potential for steroid assessment in baleen as a tool for investigating large whale physiology including pregnancy and inter-calving intervals, estrous and testosterone cycles, metabolic rate, and long term responses to acute and chronic stress.   The availability of hormone data from baleen could have huge impact on the conservation and management of wild whales, especially given the availability of historic archives of baleen – which could potentially provide new and very valuable information on how the health of today’s whales compares to those of the past.

 

Arbor Assays Kits Used in This Study

Progesterone EIA Kit, K025-H Testosterone EIA Kit, K032-H
Estradiol EIA Kit, K030-H Aldosterone EIA Kit, K052-H
Cortisol EIA Kit, K003-H Corticosterone EIA Kit, K014-H
T3 EIA Kit, K056-H T4 EIA Kit, K050-H

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