Faecal cortisol metabolites to assess stress in wildlife: evaluation of a field method in free‐ranging chamois

Abstract

1. Non-invasive faecal cortisol metabolite (FCM) analysis is a well-established tool to quantify stress in captive and free-ranging species. While the method has great potential, its suitability in field studies might be limited when faecal samples from unknown individuals are used. Possible factors affecting final results and thus jeopardizing correct data interpretation are individual and sex-specific variation, storage conditions and uneven distribution of metabolites in the faeces. 2. We tested these factors on a population of free-ranging Alpine chamois Rupicapra rupicapra rupicapra in the Austrian Alps. Faecal samples (n = 183) were analysed with an established enzyme immunoassay (EIA). To further validate the assay for FCM in chamois, a high-performance liquid chromatography (HPLC) was performed. Sex-specific differences in metabolite excretion were evaluated. Effects of storage length and temperature on FCM were tested with two experiments. The distribution of metabolites in the faeces was determined by the analysis of subsamples of single faecal samples. Potential individual effects on FCM levels and individually variable reactions to stressful events were evaluated with a simulation experiment. 3. Patterns of immunoreactive peaks after HPLC separation were similar for different faecal samples, except in one sample of a male. In the stability tests, storage time at ambient temperature prior to freezing and the individual were the most important variables in modulating FCM. Concentrations within single samples varied significantly between pellets. Analysis of faecal samples collected from June to October showed a highly significant seasonal trend (P < 0Á001) and a considerable variance of FCM levels within the population. Simulations confirmed that individual reactions to stressors in terms of varying gradients and FCM levels can explain the observed FCM patterns. 4. Using FCM to assess adrenocortical function requires measuring extensively metabolized products of glucocorticoids, whose excretion and detection in faeces depend on several environmental, endogenous and methodological factors. In free-ranging wildlife, these factors and the intrinsic individual differences in FCM excretion generate systemic noise and substantially distort final results. Therefore, sampling of unknown individuals inevitably jeopardizes meaningful interpretation of data, if the above named factors are not taken into consideration.

The importance of individual heterogeneity for interpreting faecal glucocorticoid metabolite levels in wildlife studies

“…Previous studies showed that many endogenous and exogenous factors can affect the concentration of faecal corticosteroid metabolites (hereafter referred to as FCM levels) (Hadinger, Haymerle, Knauer, Schwarzenberger, & Walzer, ). In free‐living animals, FCM levels can be affected by food availability (Jenni‐Eiermann, Glaus, Grübler, Schabl, & Jenni, ; Schoech, Bowman, Bridge, & Boughton, ), with increased food availability being associated with lower FCM levels (Jenni‐Eiermann et al., ).…”

“…Despite this variety of drivers and associated sources of variance, most studies focus only on a small number of factors to assess their hypothesized effect on FCM levels. Inter‐ and intra‐individual differences are often neglected (Goymann, ; Hadinger et al., ; Rehnus & Palme, ). Especially when non‐invasive sampling methods are used, it is often unknown how many individual animals of a population have been sampled and possible pseudo‐replication cannot be excluded (Rehnus & Palme, ).…”

HPLC‐QTOF method for quantifying 11‐ketoetiocholanolone, a cortisol metabolite, in ruminants' feces: Optimization and validation

“…Other factors such as circadian patterns can also seriously influence plasma GC concentrations (Sheriff et al., ). Noninvasive stress monitoring of wildlife using fecal glucocorticoid metabolites (FGCM) has proved to be a powerful tool and is currently being used increasingly (Hadinger et al., ; Keay et al., ; Millspaugh & Washburn, ; Morrow, Kolver, Verkerk, & Matthews, ; Mostl et al., ). Such measurements preclude some of the above‐mentioned problems as feces can be collected easily without disturbing the animal, allowing repeated sampling for monitoring purposes.…”

“…Other factors such as circadian patterns can also seriously influence plasma GC concentrations (Sheriff et al, 2011). Noninvasive stress monitoring of wildlife using fecal glucocorticoid metabolites (FGCM) has proved to be a powerful tool and is currently being used increasingly (Hadinger et al, 2015;Keay et al, 2006;Millspaugh & Washburn, 2004;Morrow, Kolver, Verkerk, & Matthews, 2002;.…”

“…Compared with other chromatographic methods, highperformance liquid chromatography (HPLC) provides good resolution and it has been frequently used for FGCM determination (Hadinger et al, 2015;Touma, Sachser, Mostl, & Palme, 2003;Turner, Tolson, & Hamad, 2002;Young et al, 2004).…”