Measuring fecal metabolites of endogenous steroids using ESI-MS/MS Spectra in Taiwanese Pangolin, (Order Pholidota, Family Manidae, Genus: Manis): A non-invasive method for endangered species

Pangolins are 'keystone species' driven towards extinction due to a lack of profound awareness and illegal trade. The drivers urge for immediate development in the understanding of demographics and reproductive dynamics of this species. In this study, we developed and validated a quantitative method to measure pangolin fecal extracts using the electrospray (ESI-MS/MS) interface in positive ionization mode. The method aids in the measurement of hormones from the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axis, making it a possibly appropriate technique to understand the cross-talk between the axes. The study aims to measure the relative abundance of adrenal and gonadal hormones such as corticosterone, cortisol, estrone, estradiol-17β, progesterone, testosterone, and a number of its metabolites. From the dried fecal extract, the principal metabolite identified from the estrogen family was estradiol-17β, whereas the gestagen family revealed that the pregnane series is predominated in 5α-configuration. On the other hand, epiandrosterone was seen as the dominant form in the male fecal extracts. Additionally, the glucocorticoids are excreted majorly as corticosterone, but traces of cortisol are also present in both the male and female fecal samples. The physiological validation confirmed that the ESI-MS/MS technique is suitable to determine physiologically caused differences in the fecal steroid concentrations. Physiologically, the age structure in pangolin is not responsible for causing differences within gender. However, the results revealed that glucocorticoids might vary between the sexes, i.e., males have a higher relative abundance of glucocorticoids over females. Other steroids such as androstane, androstenedione, estriol, pregnanediol-3-glucuronide, and cortisone were also detected in the fecal extracts but were not reliably measurable due to steroidal restrictions. Therefore, our studies show that some of the main adrenal and gonadal metabolites can be predicted by exploiting MS/MS, which can steer research to potentially assess the reproductive status of captive and free-ranging pangolin species.

Keywords: Taiwanese pangolin; adrenal metabolites; electrospray MS/MS; gonadal metabolites.