DOI: 10.5061/dryad.kkwh70s1c
title: Faecal cortisol metabolites and reactive oxygen species are biomarkers of acute and chronic stress in Carneddau mountain ponies

description: 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 (&lt;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.
description: Study species and sample collection We sampled individuals from a free-ranging population of ca 300 recorded individual Carneddau ponies in Snowdonia National Park in North Wales (53.22°N, 3.95°W). They are not provisioned or subject to veterinary intervention. However, once a year they are rounded up by the farmers who own and manage the population for health checks and potentially remove individuals. The round-up thus provides an ideal opportunity to collect samples from individuals known to be exposed to an acute stressor. Ponies were observed and identified from a distance of 10-20 m. Faecal samples were collected as soon as possible (no more than one hour following defecation). The sample was mixed thoroughly by hand and a subsample was removed for storage. Samples were then frozen at -18 °C for 7-10 months prior to extraction. The time of defecation and the day of sampling were recorded. Annual round-up Faecal samples were collected from October to early December 2014. Baseline samples were collected over a four-week period before the annual round-up, for each of eight focal individuals (seven mares and one stallion). The time sequence of the round-up event was as follows: ponies were rounded up and driven onto farmland (the drift) and kept on a fenced paddock overnight. The next day they were put through a livestock crush and had their tails trimmed. The following day they were moved back onto the commons land of Snowdonia National Park. Individuals were left for 24 hours to return to their social group and home range, after which faecal samples were collected opportunistically from the following day onwards from seven adult mares and one stallion from two focal groups (ca 72 hours post drift and 48 hours post handling, which we term day 2 in the analyses). Mare faecal samples were collected on days 2, 3, 4, 6, 8, 11, 15 and 19 such that each mare was sampled at least six times over this period. For stallions, we only had regular post samples for one stallion. In total we had 70 mare samples and ten stallion samples. There was one day prior to the round-up where the two focal bands were in close spatial proximity (inter-band distance &lt;150 m). When we did exploratory data analyses to evaluate variation in assays between days, this day stood out as a clear outlier as all sampled individuals had marked elevated hormone concentrations (see SI). We excluded these samples from the main analysis, but present these data in the SI as a potential case of social conflict (Fig S2). Although, we do not have observational data for the days preceding sample collection, these encounters often involve stallions harassing mares and aggression between the herd stallions. Seasonal data collection We focussed on easily recognisable and accessible social groups in order to collect repeated samples from the same individuals. We selected groups that would represent the lower elevation band (higher quality habitat in terms of water and food availability, more favourable weather conditions) and upper elevation band (lower quality habitat, harsher living conditions). Samples were collected from February to May 2018, where the same route across the National Park was taken which typically results in encountering ten focal groups across upper and lower bands (Fig 1). When a group was encountered, a maximum of one hour was spent with the group to collect samples from known individuals. All collections took place from 08:00 am to 12:30 pm and the ponies tend to defecate on an hourly basis in synchrony. During sampling, there was a noticeable shift in seasons reflected in the weather conditions, starting with cold weather and a heavy snowfall in February (the “Beast from the East” storm), a wet and windy March, followed by occasional sunny spells in April and a heatwave at the end of that month. Therefore, we collected faeces that we expected to reflect low, medium or high levels of ‘stress hormones’ to allow us to determine the influence of varying environmental conditions on initial concentrations of stress hormone metabolites. Online available climate data for the Carneddau area (53.18oN, 4.00oW; www.meteoblue.com) was accessed to estimate temperature, wind speed and humidity for each sample day. Weather parameters were taken 24 hours prior to sampling collection, given the time lag for metabolites be excreted. We also took an average of the previous seven days to evaluate whether there was a signal for a cumulative, or chronic, stress response. We collected social data on foraging group size and inter-individual interactions during each sampling period and used the maximum observed group size over all days.