A field-friendly method of measuring faecal glucocorticoid metabolite concentration as a simple stress checker in snow leopards

Abstract

The hormonal analysis of excrement has been employed to non-invasively assess the physiological conditions of domestic, experimental, zoo and wild animals to. However, conventional hormone analysis techniques require frozen and refrigerated reagents, and laboratory equipment; therefore, it is almost impossible to obtain the results on-site. This study attempted to establish a method for the simple and rapid quantitative on-site analysis of faecal glucocorticoid metabolite (fGM) concentrations; this method involved using hand-shaking for faecal hormone extraction, and immunochromatography using test strips and a smartphone application. This study focused specifically on snow leopards and developed a new, simple fGM measurement method to evaluate their stress levels on-site. First, the effects of ultraviolet exposure and bacterial activity on fGM concentration, including sample age, were evaluated using faeces from a captive snow leopard. Second, a field-friendly hormone extraction method was evaluated; this method does not require storage of faeces in the field because the hormones are extracted by hand-shaking of faeces in ethanol at the time of collection. In addition, to measure hormone concentrations on-site, a simple immunochromatography measurement kit was used. The concentrations were measured by quantifying the colour reactions using a smartphone application. There was no consistent increase or decrease in fGM concentration with ultraviolet irradiation time. In addition, a high correlation was observed between concentrations extracted using the conventional method (using a vortex mixer and methanol extraction) and those from the field-friendly method (r = 0.812, p < 0.05). Changes in fGM concentration measured using immunochromatography were also highly consistent with those measured using the conventional method (enzyme immunoassay; r = 0.825, p < 0.05). The analytical validation of immunochromatography also showed high accuracy, that is parallelism (ANCOVA: F = 0.597, df = 1, p = 0.445) and recovery tests (98.2% ± 5.1% and 107.5% ± 18.7%). Further, for biological validation, the change in fGM concentration was examined before and after the transportation of the snow leopards to another zoo. This field-friendly hormone analysis method is expected to contribute as a simple and rapid stress evaluation tool to manage conservation and animal welfare by monitoring the stress levels.