Validating enzyme immunoassays for non-invasive reproductive hormone monitoring in Temminck's pangolin

Gonadal hormones play a central role in reproductive function and success. As such, quantifying reproductive hormones non-invasively in threatened, vulnerable and endangered wildlife species offers an ideal tool for assessing general and individual reproductive patterns in situ. Whilst the use of faeces as a hormone matrix is often preferred in these cases, the required enzyme immunoassays (EIAs) for measuring faecal androgen (fAM), oestrogen (fEM) and progestagen metabolite (fPM) concentrations must first be validated if a species gets investigated for the first time to ensure biologically relevant patterns can be observed. In this study we aimed to biologically validate the EIAs for monitoring fAM, fEM and fPM concentrations in Temminck's pangolin, Smutsia temminckii. Hormone metabolite concentrations derived from each EIA tested were compared between different age and sex classes. An epiandrosterone EIA effectively measured androgen levels in males, distinguishing between adult and juvenile individuals, as well as both female age classes. Similarly, the tested oestrogen EIA successfully distinguished between adult and juvenile female fEM concentrations, and both tested progestagen EIAs demonstrated adequate differences between fPM concentrations of adult and juvenile females. The now-validated EIAs offer robust tools for a non-invasive monitoring of reproductive activity in Temminck's pangolin. The development of such techniques will allow researchers to assess reproductive hormone patterns of the species in situ, whilst also paving the way for further studies in this field. Despite the small sample size due to the species' conservation status, the study provides a foundation for future research using a robust, validated, non-invasive monitoring tool. The latter can now be implemented in long-term monitoring with larger sample sizes to yield more comprehensive data, aiding in the conservation of Temminck's pangolin.