Measuring Cortisol Metabolites in Fecal Samples

Cortisol metabolite levels were measured using a Cortisol ELISA kit (Enzo Life Sciences^®, ADI-900-071, New York, NY, USA) that has broad cross-reactivity and has previously been validated for faecal samples assessment in other mammal species [37 (link),38 (link)], including primates [39 (link)]. According to the manufacturer, the cross-reactivity was cortisol (100%); prednisolone (122.35%); corticosterone (27.68%); 11-deoxycortisol (4.0%); Progesterone (3.64%); prednisone (0.85%); testosterone (0.12%) and <0.10% with androstenedione, cortisone, and estradiol. The sensitivity of the assay was 56.72 pg/mL (range 156–10,000 pg/mL). Samples were diluted 1:10 with the assay buffer. All faecal samples and standards were assayed in duplicate. Assay data were analysed utilising a 4-parameter logistic (4PL) fitting programme (MyAssays^®, Brighton, UK). Intra-assay coefficients of variation at low, medium, and high concentrations of cortisol were 10.5%, 6.6%, and 7.3%, respectively; inter-assay coefficients at low, medium, and high concentrations of cortisol were: 13.4%, 7.8%, and 8.6%, respectively.