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.
