Abstract
Enzyme immunoassays (EIA) that measure faecal testosterone metabolites (fTM) are useful tools to monitor gonadal activity. The aim of this study was to validate an “in-house” epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of 3H-testosterone. High-performance liquid chromatography (HPLC) analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using β-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because β-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used β-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that β-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM levels in response to a testosterone injection within 16 h, (2) no biological responsiveness to an adrenocorticotropic hormone (ACTH) injection and (3) significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.
Highlights
In mammals, androgens are involved in shaping phenotypic and life history traits[1, 2]
High-performance liquid chromatography (HPLC) profiles of radiolabelled testosterone metabolites in non-hydrolysed and hydrolysed spotted hyena faeces are shown in Fig 2A&2B, respectively
In this study we demonstrated that an enzyme immunoassays (EIA) based on an antibody against epi-A is a useful tool for monitoring gonadal activity in spotted hyenas
Summary
Androgens are involved in shaping phenotypic and life history traits[1, 2]. Contest competition among males for access to mating partners can be associated with elevate androgen concentrations Blood samples are used to measure circulating hormone concentrations to evaluate an animal’s endocrine status. Restricted access to animals may limit blood sampling, in wildlife species [12]. Handling procedures including capture, restraint and anaesthesia, which are necessary for blood sampling, may alter circulating hormone concentrations, which is especially problematic when an individual is handled repeatedly to obtain multiple samples [13,14,15]. The measurement of faecal hormone metabolite concentrations using enzyme immunoassays (EIA) provides a non-invasive alternative method to the use of blood samples. Faecal hormone metabolite concentrations are integrated over a period of time and are less affected by the pulsatory hormone secretion pattern present in blood samples. The application of EIAs has many advantages—but their application presents several challenges
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