Validation and use of hair cortisol as a measure of chronic stress in eastern chipmunks (Tamias striatus)

Abstract

Stress levels of individuals are documented using glucocorticoid concentrations (including cortisol) in blood, saliva, urine or faeces, which provide information about stress hormones during a short period of time (minutes to days). In mammals, use of hair cortisol analysis allows for the assessment of prolonged stress over weeks and months and provides information on chronic stress levels without bias associated with handling. Here, we validate hair cortisol analysis in wild rodents using exogenous adrenocorticotrophic hormone (ACTH challenge) and apply the technique to evaluate stress in eastern chipmunks inhabiting logged and natural sites. Chipmunks were subjected to a mark-recapture study and injected weekly with ACTH (Synacthen Depot) or saline, with hair being collected at the conclusion of the challenge. Subsequently, faecal and hair samples were collected from chipmunks occupying logged and natural sites to assess the utility of hair cortisol in comparison with faecal cortisol metabolites. Following extraction, cortisol concentrations were quantified in hair and faecal extracts by enzyme immunoassay. Hair cortisol concentrations were significantly elevated in samples from ACTH-injected chipmunks compared with saline-injected control animals (five times higher). Chipmunks inhabiting logged sites had increased faecal cortisol metabolite concentrations compared with those in natural sites, but no differences were observed in hair cortisol concentrations. Faecal cortisol metabolite levels were positively correlated with hair cortisol levels in chipmunks. Hair cortisol levels reflect changes in circulating cortisol levels and can be used to evaluate the adrenal stress response, and thus stress, in natural populations. Nonetheless, because of the differences in the temporal scale of stress that hair and faeces represent, we caution the use of hair cortisol for detecting differences in physiological stress when comparing individuals within populations and suggest that it is best suited to examining population-level differences.

Keywords: ACTH challenge; faecal cortisol; logging.

Figure 1:

Cortisol assay validation. (a) Recovery of exogenous cortisol from pooled hair extracts. (b) Parallelism for serial dilutions of pooled hair extracts against the cortisol standard curve. Symbol key: filled diamonds, standard curve; and filled squares, pooled hair extract dilutions.

Figure 2:

Hair cortisol concentrations of saline- and adrenocorticotrophic hormone (ACTH)-injected eastern chipmunks. (a) Initial hair cortisol concentrations were similar for animals in both control (saline) and ACTH treatment groups (P = 0.76). (b) The ACTH-injected chipmunks had significantly higher hair cortisol concentrations than control chipmunks (P < 0.001). The mean, standard error (box) and standard deviation (whiskers) are presented. Data points from the ACTH treatment represent two chipmunks that did not respond to the ACTH injection.

Figure 3:

Hair and average faecal cortisol levels (expressed as nanograms per gram of substrate) were positively correlated in wild-caught chipmunks. The filled circles highlight two individuals with high levels of hair cortisol but low levels of faecal cortisol. Results are presented in the text with these points included (P = 0.055) and excluded (P < 0.001). Cortisol levels were natural-log transformed.

Figure 4:

Chipmunks caught in natural sites had lower levels of faecal cortisol (expressed as nanograms per gram of faeces; mean ± SEM) than those trapped in logged sites. The non-transformed data are shown, with average faecal values plotted on the left and hair values on the right.