Alaskan seals are found in remote and sometimes inaccessible locations, making it difficult to collect time‐series information. This study explores a novel method to examine temporal changes in diet ...and physiological status of ringed (Pusa hispida), spotted (Phoca largha), and harbor (Phoca vitulina) seals using cortisol concentrations and δ15N and δ13C stable isotopes (SIs) measured in serial sections of whiskers. As whiskers grow, whisker tissue is deposited sequentially making these measurements temporally aligned. Whisker cortisol presented in a distinct pattern with elevated concentrations at the root section followed by a curvilinear decline moving toward the tip of most whiskers. Comparing SIs at the root to the rest of the whiskers, δ13C values were slightly lower in ringed and harbor seal whiskers and δ15N values were slightly higher in harbor seal whiskers. The data were modeled controlling for the observed trends in cortisol concentrations and further associations between cortisol concentrations and SIs were detected in spotted and harbor seal whiskers. Additional research examining the source and stability of whisker cortisol is warranted. However, the methods presented here demonstrate that whiskers could prove valuable to gather long‐term and naturally aligned dietary and physiological information.
To determine the potential influence of hair follicles on cortisol levels, we conducted an experiment comparing cortisol concentrations in paired subsamples of brown bear hair with and without ...follicles. We found significantly greater cortisol values in subsamples that included follicles, suggesting that the presence of follicles may influence cortisol concentrations measured in hair. Our findings underscore the importance of standardizing hair collection and preparation methods to enable comparisons across studies.
Abstract
Cortisol concentrations in hair are used increasingly as a biomarker of long-term stress in free-ranging wildlife. Cortisol is believed to be integrated into hair primarily during its active growth phase, typically occurring over weeks to months or longer periods, depending on latitude. Cortisol concentrations in hair thus reflect the activity of the hypothalamic–pituitary–adrenal axis over this time. However, local, independent cortisol secretion within the skin, which includes hair follicles, may also contribute to cortisol levels in growing hair. Methodological differences between studies include the measurement of cortisol in only the hair shaft (i.e. follicle absent, as with shaved hair) versus the whole hair (i.e. follicle present, as with plucked hair). If the concentration of cortisol in the follicle is high enough to influence the overall hair cortisol concentration (HCC), this could confound comparisons between studies using different types of hair samples (hair shafts vs. whole hair) and collection methods. Here, we test the hypothesis that cortisol present in follicles influences HCC. We compared HCC in paired subsamples of hair with and without follicles from 30 free-ranging Scandinavian brown bears (Ursus arctos) and observed significantly greater HCC in samples with follicles present. The effect of follicles remained significant also with sex and age of sampled bears taken into account in a linear mixed model. Finally, we provide an overview of collection methods and types of hair samples used for HCC analysis in 77 studies dealing with stress in wild mammal species. Our findings highlight the need to unify methods of hair collection and preparation to allow for valid comparisons, and to optimize labour input in ecophysiological studies.
Hair cortisol concentration (HCC) is being used increasingly to evaluate long‐term stress in many mammalian species. Most of the cortisol is assumed to passively diffuse from circulating blood into ...hair follicles and gradually accumulate in growing hair. However, our research with free‐ranging grizzly bears (Ursus arctos) suggests HCC increases significantly within several hours following capture, a time too brief to be explained by this mechanism alone. In this study with captive grizzly bears, we sought to determine if a brief spike in blood cortisol concentration, thus mimicking a single stressful event, would cause an increase in HCC over a 7‐day period. To do this, we administered a single intravenous dose (5 μg/kg) of cosyntropin to three captive unanaesthetised adult female grizzly bears on two occasions, during April when hair growth was arrested and during August when hair was growing. In both trials, the cosyntropin caused a two‐fold or greater increase in serum cortisol levels within 1 hr but did not appear to influence HCC at 1, 48, and 168 hr following cosyntropin administration. We conclude the cosyntropin‐induced cortisol spike was likely insignificant when compared to the adrenocortical response that occurs in free‐ranging bears when captured. We suggest further study with a larger sample of captive bears to evaluate the combined effects of anaesthesia and multiple doses of cosyntropin administered over several hours would better simulate the adrenocortical response of free‐ranging grizzly bears during capture.
Administration of cosyntropin to captive grizzly bears caused increased serum cortisol levels within 1 hr. However, cosyntropin did not influence hair cortisol concentration at 1, 48, and 168 hr following administration. Cosyntropin‐induced hair cortisol levels were insignificant compared to free‐ranging bears during capture.
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Methodological differences among laboratories are recognized as significant sources of variation in quantification of hair cortisol concentration (HCC). An important step in ...processing hair, particularly when collected from wildlife, is the choice of solvent used to remove or “wash” external hair shaft cortisol prior to quantification of HCC. The present study systematically compared methanol and isopropanol as wash solvents for their efficiency at removing external cortisol without extracting internal hair shaft cortisol in samples collected from free-ranging grizzly bears and polar bears. Cortisol concentrations in solvents and hair were determined in each of one to eight washes of hair with each solvent independently.
•There were no significant decreases in internal hair shaft cortisol among all eight washes for either solvent, although methanol removed detectable hair surface cortisol after one wash in grizzly bear hair whereas hair surface cortisol was detected in all eight isopropanol washes.•There were no significant differences in polar bear HCC washed one to eight times with either solvent, but grizzly bear HCC was significantly greater in hair washed with isopropanol compared to methanol.•There were significant differences in HCC quantified using different commercial ELISA kits commonly used for HCC determinations.
Environmental contaminants like arsenic (As), cadmium (Cd), mercury (Hg) or lead (Pb) may disrupt hypothalamic–pituitary–adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes due to their ...endocrine toxicity potential. Resulting long-term physiological stress or adverse effects on wildlife reproduction and ontogeny may cause detrimental effects at the individual and population levels. However, data on environmental metal(loid)sʼ impact on reproductive and stress hormones in wildlife, especially large terrestrial carnivores, are scarce. Hair cortisol, progesterone and testosterone concentrations were quantified and modelled with hair As, Cd, total Hg, Pb, biological, environmental and sampling factors to test for potential effects in free-ranging brown bears (Ursus arctos) from Croatia (N = 46) and Poland (N = 27). Testosterone in males (N = 48) and females (N = 25) showed positive associations with Hg and an interaction between Cd and Pb, but a negative association with interaction between age and Pb. Higher testosterone was found in hair during its growth phase compared to quiescent phase. Body condition index was negatively associated with hair cortisol and positively associated with hair progesterone. Year and conditions of sampling were important for cortisol variation, while maturity stage for progesterone variation (lower concentrations in cubs and yearlings compared to subadult and adult bears). These findings suggest that environmental levels of Cd, Hg and Pb might influence the HPG axis in brown bears. Hair was shown to be a reliable non-invasive sample for investigating hormonal fluctuations in wildlife while addressing individual and sampling specificities.
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•First effect study on hormonal association with metal (loid)s in European brown bear.•Hair testosterone was associated with potentially toxic metal (loid) levels.•Year and conditions of sampling important for cortisol variation in hair.•Maturity stage important for progesterone variation (lowest in cubs & yearlings).
The deer ked (
), a hematophagous ectoparasite of cervids, is currently spreading in Scandinavia, and the moose (
) is its main host. However, little is known about the impact of deer keds on moose. ...We analyzed the hair cortisol concentration (HCC) from 262 moose harvested in the fall in relation to age class, sex, body mass (BM), and deer ked infestation intensity, and BM in relation to age class, sex, and infestation intensity. We found that HCC decreased with increasing deer ked intensity at low ked intensities, but for the higher levels of ked intensities, there was a positive relationship between HCC and ked intensity. The HCC was higher in males than in females and lower in yearlings than in calves and adults. Our failure to find any association between BM and deer ked intensity suggested a negligible impact of deer ked infestation on moose foraging and metabolism at the level of infestation observed early in the infestation, but did not exclude an effect later in winter. Our findings suggested that moose generally tolerated moderate parasitism by keds. However, the increase in HCC at higher ked intensities suggested that the tolerance strategy could be disrupted with further increases in intensities and consequently may negatively affect animal health and welfare.
The scraping and counting technique (SCT), with sensitivity values close to 100 %, has been the protocol recommended by global regulatory bodies for the extraction of
Echinococcus
cestodes from the ...intestines of wild carnivores. The proposed scraping, filtration and counting technique (SFCT) maintained the sensitivity (
p
= 0.801,
α
= 0.05) and increased the efficiency of sample processing. SCT had sensitivity and negative predictive value of 91 and 97 %, respectively, when compared to SFCT. The SFCT significantly decreased processing time (
p
= 0.0001,
α
= 0.05) for each sample. The SFCT took an average of 68.5 min less to quantify than SCT, as the SFCT samples consistently contained less debris. The SFCT is therefore appealing for general post-mortem surveillance, to determine if prevalence and intensity of infection are changing in an established region, or if these important parasitic zoonoses are newly established in a region or host species.
We evaluated if steroid hormone concentration profiles in hair collected from brown bears could be used to discriminate between immature and adult bears. Classification accuracy was excellent for ...male age classes. We also had good success with accurately classifying adult females, but poor accuracy with classifying immature females.
Abstract
Although combining genetic and endocrine data from non-invasively collected hair samples has potential to improve the conservation of threatened mammals, few studies have evaluated this opportunity. In this study, we determined if steroid hormone (testosterone, progesterone, estradiol and cortisol) concentration profiles in 169 hair samples collected from free-ranging brown bears (Ursus arctos) could be used to accurately discriminate between immature and adult bears within each sex. Because hair samples were acquired opportunistically, we also needed to establish if interactions between hormones and several non-hormone factors (ordinal day, year, contact method, study area) were associated with age class. For each sex, we first compared a suite of candidate models by Akaike Information Criteria model selection, using different adult-age thresholds (3, 4 and 5 years), to determine the most supported adult age. Because hair hormone levels better reflect the endocrine state at an earlier time, possibly during the previous year, then at the time of sampling, we re-analysed the data, excluding the records for bears at the adult-age threshold, to establish if classification accuracy improved. For both sexes, candidate models were most supported based on a 3-year-old adult-age threshold. Classification accuracy did not improve with the 3-year-old bear data excluded. Male age class was predicted with a high degree of accuracy (88.4%) based on the concomitant concentrations of all four hormones. Female age class was predicted with less accuracy (77.1%) based only on testosterone and cortisol. Accuracy was reduced for females, primarily because we had poor success in correctly classifying immature bears (60%) whereas classification success for adult females was similar to that for males (84.5%). Given the small and unbalanced sample used in this study, our findings should be viewed as preliminary, but they should also provide a basis for more comprehensive future studies.
We used enzyme immunoassays to measure testosterone, progesterone, estradiol and cortisol concentrations in serial hair samples collected from captive adult brown bears. Reproductive hormone levels ...changed throughout the year in step with different reproductive events underscoring the strong potential for these measurement to be used to augment non-invasive genetic sampling.
Abstract
Recognizing the potential value of steroid hormone measurements to augment non-invasive genetic sampling, we developed procedures based on enzyme-linked immunoassays to quantify reproductive steroid hormone concentrations in brown bear (Ursus arctos) hair. Then, using 94 hair samples collected from eight captive adult bears over a 2-year period, we evaluated (i) associations between hair concentrations of testosterone, progesterone, estradiol and cortisol; (ii) the effect of collecting by shaving vs. plucking; and (iii) the utility of reproductive hormone profiles to differentiate sex and reproductive state. Sample requirements (125 mg of guard hair) to assay all hormones exceeded amounts typically obtained by non-invasive sampling. Thus, broad application of this approach will require modification of non-invasive techniques to collect larger samples, use of mixed (guard and undercoat) hair samples and/or application of more sensitive laboratory procedures. Concentrations of hormones were highly correlated suggesting their sequestration in hair reflects underlying physiological processes. Marked changes in hair hormone levels during the quiescent phase of the hair cycle, coupled with the finding that progesterone concentrations, and their association with testosterone levels, differed markedly between plucked and shaved hair samples, suggests steroids sequestered in hair were likely derived from various sources, including skin. Changes in hair hormone concentrations over time, and in conjunction with key reproductive events, were similar to what has been reported concerning hormonal changes in the blood serum of brown bears. Thus, potential for the measurement of hair reproductive hormone levels to augment non-invasive genetic sampling appears compelling. Nonetheless, we are conducting additional validation studies on hair collected from free-ranging bears, representative of all sex, age and reproductive classes, to fully evaluate the utility of this approach for brown bear conservation and research.
Abstract
Stress hormone levels and stable isotope ratios in hair or feathers represent an integrated picture of hormone physiology and foraging ecology of an animal. We evaluate the compatibility of ...laboratory preparation procedures used in ecophysiology with the intention of optimizing the efficiency of future research.
Abstract
The measurement of naturally occurring glucocorticoids and stable isotopes of several elements has gained importance in wildlife studies in recent decades and opened a myriad of ecological applications. Cortisol and stable isotopes equilibrate in animal tissues over periods of integration related to the growth rate of the tissue, providing information reflecting systemic cortisol secretion and dietary intake. Sample preparation shares the common step of first cleaning the sample of external contamination. However, it is not well understood how different solvents used in sample preparation affect isotopic and cortisol values, and whether it is safe to follow the same procedures for both measures to optimize analyses of the same sample. We conducted an experiment to compare different preparation protocols for the analysis of cortisol concentrations and stable carbon (δ13C) and nitrogen (δ15N) isotope ratios in hair. Hair samples from 12 brown bears (Ursus arctos) were each divided into five aliquots; two aliquots were rinsed with a 2:1 chloroform:methanol (v/v) mixture with one aliquot ground prior to cortisol analysis and the other left intact for stable isotope analyses; two aliquots were washed with methanol with one aliquot ground prior to cortisol analysis and the other left intact for stable isotope analyses; and one aliquot washed with methanol and ground prior to stable isotope analyses. The cortisol, δ13C and δ15N values remained consistent following all treatments. Our results indicate that hair samples rinsed with a 2:1 chloroform:methanol mixture or washed with methanol can be used for both types of analyses. Further, hair that has been ground in a standard hair cortisol procedure can also be used for stable isotope analysis. This information is important for improving laboratory efficiency and compatibility of procedures used for wildlife physiological ecology studies where concurrent measurements of cortisol and stable isotopes in hair are required.
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