Characterisation and validation of an enzyme-immunoassay for the non-invasive assessment of faecal glucocorticoid metabolites in cheetahs (Acinonyx jubatus)

The non-invasive measurement of adrenocortical function in cheetahs is an important tool to assess stress in captive and free-ranging individuals, because stress has been suggested to be one of the causes of poor reproductive performance of captive cheetahs. We tested four enzyme immunoassays (EIA) in two captive cheetahs in Germany using adrenocorticotropic hormone (ACTH) challenges and identified the corticosterone-3-CMO EIA to be most sensitive to the increase in faecal glucocorticoid metabolite (fGCM) concentrations after the ACTH challenge. This EIA performed also well in five captive cheetahs in South Africa. The fGCM concentrations across all seven cheetahs increased within 24 h by 681% compared to the baseline levels prior to ACTH. Storage of faecal samples at 0–4 °C did not strongly affect fGCM concentrations within 24 h, simplifying sample collection when immediate storage at −20 °C is not feasible. The two cheetahs in Germany also received an injection of [3H]cortisol to characterise fGCMs in faecal extracts using high-performance liquid chromatography (HPLC) immunograms. HPLC fractions were measured for their radioactivity and immunoreactive fGCM concentrations with the corticosterone-3-CMO EIA, respectively. The results revealed a polar peak of radiolabelled cortisol metabolites co-eluting with the major peak of immunoreactive fGCMs. Thus, our EIA measured substantial amounts of fGCMs corresponding to the radioactive peaks. The peaks were of higher polarity than native cortisol and corticosterone, suggesting that the metabolites were conjugated, which was confirmed by solvolysis of the HPLC fractions. Our results show that the corticosterone-3-CMO EIA is a reliable tool to assess fGCMs in cheetahs.

We developed an enzyme immunoassay (EIA) based on an antibody directed against corticosterone-3-CMO which is a reliable tool to assess faecal lucocorticoid metabolites (fGCM) in cheetahs. Faecal GCM concentrations measured with the corticosterone-3-CMO assay before and after ACTH administration in three female and four male cheetahs. Concentrations are expressed as % (mean ± SEM) from the pre-challenge baseline.Figure optionsDownload as PowerPoint slideHighlights
► We tested four enzyme immunoassays (EIA) in two captive cheetahs to monitor adrenocortical activity.
► The assay comparison was carried out based on two ACTH challenges.
► The corticosterone-3-CMO EIA can be used to reliably assess faecal glucocorticoid (fGM) concentrations in cheetahs.
► A radiometabolism study used to identify fGM revealed a polar peak of radiolabelled cortisol metabolites.
► We suggest that these metabolites were conjugated, because solvolysis released free, unpolar metabolites.