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

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 < 0·001) 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.

1. 无创粪便皮质醇代谢物（faecal cortisol metabolite, FCM）分析是一项成熟的研究工具，可用于量化圈养及野生自由活动物种的应激水平。尽管该方法具备极大应用潜力，但当使用来源未知个体的粪便样本开展野外研究时，其适用性可能受到限制。可能影响最终结果、进而干扰数据正确解读的因素包括：个体与性别特异性差异、样本保存条件，以及代谢物在粪便中的分布不均一性。
2. 我们针对奥地利阿尔卑斯山区的野生阿尔卑斯岩羊（Rupicapra rupicapra rupicapra）种群开展了上述因素的相关测试。采用已成熟的酶联免疫吸附实验（enzyme immunoassay, EIA）对183份粪便样本进行检测。为进一步验证该检测方法适用于岩羊FCM分析的有效性，我们还开展了高效液相色谱（high-performance liquid chromatography, HPLC）分析。评估了代谢物排泄的性别特异性差异；通过两组实验测试了保存时长与温度对FCM水平的影响；通过对单份粪便样本的子样本进行分析，确定了代谢物在粪便中的分布情况；通过模拟实验评估了个体因素对FCM水平的潜在影响，以及个体对应激事件的差异化反应。
3. 高效液相色谱分离后的免疫反应峰模式在多数粪便样本中保持一致，仅1份雄性样本例外。在稳定性测试中，冷冻前室温保存时长与个体差异是调控FCM水平的最关键变量。单份样本内不同粪粒的FCM浓度存在显著差异。对6月至10月采集的粪便样本的分析显示，FCM水平存在极显著的季节趋势（P < 0·001），且种群内部FCM水平存在较大变异。模拟实验证实，个体对应激源的反应差异（表现为反应梯度与FCM水平的变化）可解释观测到的FCM模式。
4. 利用FCM评估肾上腺皮质功能时，需要检测糖皮质激素的大量代谢产物，而这些产物在粪便中的排泄与检测依赖于多种环境、内源性及方法学因素。在野生自由活动的野生动物种群中，上述因素以及个体间FCM排泄的固有差异会产生系统性噪声，显著干扰最终结果的准确性。因此，若未考虑上述提及的各类因素，对未知个体的粪便样本进行检测，将不可避免地影响数据解读的有效性。