Faecal cortisol metabolites and reactive oxygen species are biomarkers of acute and chronic stress in Carneddau mountain ponies

Free ranging animals are exposed to environmental, demographic and ecological challenges over time, which impact their health and fitness. Non-invasive biomarkers can provide insight into how animals cope with these challenges. Here, we evaluate how free ranging Carneddau ponies (Equus ferus caballus) respond to two ‘challenges’: (i) an acute stressor of an annual round-up event, and (ii) spatial and temporal variation in abiotic and social factors across the population using three stress biomarker assays: two enzyme immunoassays (EIAs) of faecal glucocorticoid metabolites (an 11-oxoaetiocholanolone EIA measuring 11,17-dioxoandrostanes – 11,17-DOAs - and a corticosterone EIA), and a novel application of faecal reactive oxygen species (H2O2). With the second set of samples, we also used a faecal testosterone metabolite (TM) assay to assess seasonal changes in breeding condition. The two “glucocorticoid” EIAs showed very different responses to the round-up. Levels of 11,17-DOAs were significantly elevated from day two after the round-up and then slowly returned towards baseline levels. Metabolite concentrations measured by the corticosterone assay were not significantly elevated following the round-up. In contrast, the third ‘stress’ marker, H202,showed a pronounced, but delayed, increase from day eight following the round-up. For the longitudinal data across all ponies, H2O2was elevated during periods of cold weather and was higher in young ponies (<1 year old). 11,17-DOA levels were positively correlated with social group size in stallions but not in mares or immature individuals. The metabolite concentrations measured by the androgen and corticosterone assays were strongly correlated. Across all ponies, both assays failed to detect any drivers of physiological responses. However, there was a strong temporal signature in that levels of both assays were high during the pre-breeding season, but then declined in stallions but continued to rise in mares to the breeding commencement of the breeding season. The co-variance of metabolites measured by the corticosterone and testosterone assays, coupled with the lack of an acute response in the corticosterone assay to the round-up, suggests there are likely to be issues with cross-reactivity. We suggest the corticosterone assay is detecting metabolites that are not specifically associated with an upregulation of glucocorticoid production from the hypothalamic pituitary adrenal (HPA) axis. We demonstrate that validated faecal biomarkers of HPA activity can indicate acute stress and, for the first time, that reactive oxygen species may indicate chronic stress with more long-lasting physiological consequences. Together, they can be used as a powerful tool for monitoring animal health, documenting the physiological impacts from management interventions, and for evidencing causal mechanisms underlying poor performance and population decline in the face of future environmental changes.