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Monitoring reproductive physiology in wildlife can be a useful tool for assessing population dynamics for conservation and management purposes. Utilizing non-invasive approaches for this, such as quantifying reproductive hormone metabolites from faeces, can be challenging when defaecation events are not observed, or when cryptic species like African clawless otters (Aonyx capensis) are involved. Additionally, test systems for quantifying hormone metabolites in a species for the first time must first be reliably validated prior to use. Our results indicate that Epiandrosterone and Progesterone EIAs are most suitable for determining fAM and fPM concentrations in African clawless otter spraints. The fAM:fPM ratio and respective thresholds are more reliable in sex identification compared to the separate use of individual hormone classes. Sex-related hormone metabolite concentrations remained comparable for up to 12hrs post-defaecation in both sexes. • We screened two androgen and two progestagen enzyme-immunoassays (EIAs) for suitability and reliable quantification of faecal androgen metabolites (fAM) and faecal progestagen metabolites (fPM) in African clawless otters. • We assessed whether the ratio of fAM:fPM concentrations can be used to assign sex to faecal samples from unknown individuals. • We tested the stability of fAM and fPM concentrations post-defaecation to determine the effects of environmental exposure and bacterial metabolism.

Hormone monitoring in keratinous tissues has become increasingly popular. The insoluble keratin materials are generally pulverised before hormone extraction; however, this is difficult for thicker keratin structures like baleen plates or hooves. A new method, involving the use of keratinase, allows enzymatic digestion of keratin and hormone analysis in the resulting suspension. Pangolins are unique mammals covered in keratinous scales, which are one of the reasons these animals are extensively trafficked. This study aimed to investigate the suitability of Temminck's pangolin scales as hormone matrix for quantifying reproductive steroids. A protocol was developed to digest scales with a keratinase before measuring hormone concentrations. This method can be used to investigate the reproductive endocrinology of Temminck's pangolins but may also be extended to the other extant pangolin species. • Keratinase digests Temminck's pangolin scales and reproductive steroid metabolite concentrations are measurable in the resulting suspension. • Isopropanol is an ideal washing solvent for scales to remove surface contaminants and scale sample mass should be standardised to allow comparisons. • Any section of a scale and scales from any pangolin body region can be used as samples for hormone quantification.