title: Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).
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.
authors.affiliation: Department Reproduction Biology and Evolutionary Ecology, Leibniz Institute of Zoo and Wildlife Research, Forschungsverbund Berlin e.V., Berlin, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Pribbenow
authors.affiliation: Department Reproduction Biology and Evolutionary Ecology, Leibniz Institute of Zoo and Wildlife Research, Forschungsverbund Berlin e.V., Berlin, Germany.
authors.forename: Marion L
authors.initials: ML
authors.lastname: East
authors.affiliation: Endocrine Research Laboratory, Department of Anatomy and Physiology, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, Republic of South Africa; National Zoological Gardens of South Africa, Pretoria, Republic of South Africa.
authors.forename: Andre
authors.initials: A
authors.lastname: Ganswindt
authors.affiliation: National Zoological Gardens of South Africa, Pretoria, Republic of South Africa; Department of Companion Animal Clinical Studies, Faculty of Veterinarian Science, University of Pretoria, Onderstepoort, Republic of South Africa.
authors.forename: Adrian S W
authors.initials: AS
authors.lastname: Tordiffe
authors.affiliation: Department Reproduction Biology and Evolutionary Ecology, Leibniz Institute of Zoo and Wildlife Research, Forschungsverbund Berlin e.V., Berlin, Germany.
authors.forename: Heribert
authors.initials: H
authors.lastname: Hofer
authors.affiliation: Department Reproduction Biology and Evolutionary Ecology, Leibniz Institute of Zoo and Wildlife Research, Forschungsverbund Berlin e.V., Berlin, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Dehnhard
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000008:administration & dosage; D000738:Androsterone / Q000096:biosynthesis / Q000097:blood / Q000302:isolation & purification*; D000818:Animals; D001711:Biotransformation; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D005243:Feces / Q000737:chemistry*; D005260:Female; D005966:Glucuronidase / Q000737:chemistry; D006372:Helix, Snails / Q000737:chemistry / Q000201:enzymology; D045968:Hyaenidae; D007124:Immunoenzyme Techniques; D007273:Injections, Intramuscular; D008297:Male; D010053:Ovary / Q000187:drug effects* / Q000502:physiology; D012098:Reproduction / Q000502:physiology*; D013737:Testis / Q000187:drug effects* / Q000502:physiology; D013739:Testosterone / Q000008:administration & dosage / Q000378:metabolism*; D014316:Tritium
chemical_list: D014316:Tritium; D013739:Testosterone; D000324:Adrenocorticotropic Hormone; D000738:Androsterone; D005966:Glucuronidase