Non-invasive monitoring of gonadal activity in two carnivores, the spotted hyena (Crocuta crocuta) and the cheetah (Acinonyx jubatus)

The development of enzyme immunoassays (EIAs) to measure faecal testosterone metabolites (fTM) facilitates a non-invasive study design to objectify an animal’s gonadal status without taking blood samples. Despite the wide use of these techniques, EIAs need to be carefully validated for each species and hormone. This dissertation was designed to establish and validate EIAs to monitor gonadal activity non-invasively based on faecal hormone analyses in two carnivores, the spotted hyena (Crocuta crocuta) and the cheetah (Acinonyx jubatus). In Article 1, I detail the development and validation of an epiandrosterone EIA for the measurement of fTM in spotted hyenas. We performed radiometabolism studies to characterise fTM excreted in a captive male and female spotted hyena, respectively and demonstrated that the epiandrosterone EIA detected immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations after a testosterone challenge test, (2) a lack of crossreactivities with faecal glucocorticoid metabolites (fGM), and (3) significant differences in fTM levels in juvenile males and adult males of a free-ranging population. In Article 2, we validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible crossreactivities with fGM. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations after a testosterone injection, (2) a significant increase in fTM concentrations after a gonadotropin-releasing hormone (GnRH) injection, and (3) significant differences in fTM concentrations in adult male, adult female and juvenile male cheetahs of a free-ranging population. InArticle3, we highlight the challenge of undesired cross-reactivities of testosterone EIAs to unknown fGM. For this purpose, we compared the ability of four different testosterone EIAs to monitor gonadal activity in spotted hyenas non-invasively based on fTM analyses. Our study demonstrates that a careful methodological and biological validation is requested to ensure that fTM concentrations quantitatively reflect gonadal activity. Even for an EIA passed the biological validation based on a challenge experiment, it is highly recommended to ensure that stress-related increases in fGM are not monitored whenapplying an assay for fTM quantification. Our studies describe the first EIAs for the non-invasive monitoring of testosterone and its underlying endocrine mechanisms, thereby providing the necessary tools for long-term investigations of hormone-behaviour relationships in both carnivore species.