Assessing chronic stress in wild mammals using claw-derived cortisol: a validation using European badgers (Meles meles)

Abstract

Measuring stress experienced by wild mammals is increasingly important in the context of human-induced rapid environmental change and initiatives to mitigate human-wildlife conflicts. Glucocorticoids (GC), such as cortisol, mediate responses by promoting physiological adjustments during environmental perturbations. Measuring cortisol is a popular technique; however, this often reveals only recent short-term stress such as that incurred by restraining the animal to sample blood, corrupting the veracity of this approach. Here we present a protocol using claw cortisol, compared with hair cortisol, as a long-term stress bio-indicator, which circumvents this constraint, where claw tissue archives the individual's GC concentration over preceding weeks. We then correlate our findings against detailed knowledge of European badger life history stressors. Based on a solid-phase extraction method, we assessed how claw cortisol concentrations related to season and badger sex, age and body-condition using a combination of generalized linear mixed models (GLMM) (n = 668 samples from 273 unique individuals) followed by finer scale mixed models for repeated measures (MMRM) (n = 152 re-captured individuals). Claw and hair cortisol assays achieved high accuracy, precision and repeatability, with similar sensitivity. The top GLMM model for claw cortisol included age, sex, season and the sex*season interaction. Overall, claw cortisol levels were significantly higher among males than females, but strongly influenced by season, where females had higher levels than males in autumn. The top fine scale MMRM model included sex, age and body condition, with claw cortisol significantly higher in males, older and thinner individuals. Hair cortisol was more variable than claw; nevertheless, there was a positive correlation after removing 34 outliers. We discuss strong support for these stress-related claw cortisol patterns from previous studies of badger biology. Given the potential of this technique, we conclude that it has broad application in conservation biology.

Keywords: glucocorticoids; steroids; stress.

Figure 1

Comparisons of cortisol assays for claw and hair from European badgers. (A) Serial dilution of claw (y = 33.24–0.421log(x)), but not hair samples (y = 43.205–0.529log(x)) were not significantly different from the cortisol assay standard curve (y = 2810.662–0.453log(x)). (B) Cortisol amount recovered from both pooled claw and nail samples increased curvilinearly with mass of the sample. (C) Increased recovery of known (“spiked”) cortisol concentrations in claw (y = 0.935x + 0.71) compared to hair (y = 1.351 + 0.43) samples.

Figure 2

Box plots for the coarse analysis indicating claw cortisol concentrations in male and female European badgers (M. meles) sampled during two-week trapping efforts in Wytham Woods, England between Sept 2017 and Nov 2019. Seasons correspond to Spring (late May—early June); Summer (early September) and Autumn (mid to late November). The top and bottom of each box represents the 25% and 75% quartiles (Q1 and Q3), with the solid and dashed inside line representing the median and mean, respectively. The bottom and top error bars indicate the 5% and 95% range and the points represent the 1% to 99% range of values. Numbers within the box indicate sample sizes.

Figure 3

Bar plot indicates significant differences in claw cortisol concentrations between male and female European Badgers (M. meles) sampled from Wytham Woods, England as part of the fine-scale analysis (see Methods for details).

Figure 4

Scatterplot for the fine-scale analysis indicating claw cortisol concentrations in European badgers (M. meles) of varying ages. Age was based on mark-recapture data and/or estimation based on tooth wear (see Methods).

Figure 5

Relationship between claw cortisol concentrations and body condition index (BCI) scores in European badgers (M. meles). BCI is derived from standardized residuals of a least squares’ regression of body mass onto total body length (see Methods).

Figure 6

Relationship between claw cortisol concentrations and social group size in both male and female European badgers (M. meles). Size of badger social groups was estimated using a novel minimum number alive estimate (see Methods). Claw cortisol concentrations are positively correlated with group sizes in males (solid line: r = 0.30, P = 0.046), but not females (dashed line: r = 0.12, P = 0.366).

Figure 7

Pearson’s correlation of cortisol concentrations extracted from both claw and hair samples from European badgers (M. meles). Symbols indicate samples collected in different seasons. Trendline is significant at r = 0.772 with n = 431 samples and p < 0.001.