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Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).

Pribbenow S, East ML, Ganswindt A, Tordiffe AS, Hofer H, Dehnhard M - PLoS ONE (2015)

Bottom Line: Our results showed that β-glucuronidases from both sources produced similar results from spotted hyena faeces.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.

View Article: PubMed Central - PubMed

Affiliation: Department Reproduction Biology and Evolutionary Ecology, Leibniz Institute of Zoo and Wildlife Research, Forschungsverbund Berlin e.V., Berlin, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

Comparison of faecal cortisol and testosterone immunoreactivity.Changes in fGM and fTM concentrations were determined in faecal samples from the ACTH challenge following hydrolysis with β-glucuronidase from Helix pomatia in the cortisol-3CMO, epiandrosterone and testosterone-11-HS EIAs, respectively. Levels of fGM and fTM are shown as percentage increase over pre-injection levels.
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pone.0128706.g007: Comparison of faecal cortisol and testosterone immunoreactivity.Changes in fGM and fTM concentrations were determined in faecal samples from the ACTH challenge following hydrolysis with β-glucuronidase from Helix pomatia in the cortisol-3CMO, epiandrosterone and testosterone-11-HS EIAs, respectively. Levels of fGM and fTM are shown as percentage increase over pre-injection levels.

Mentions: To exclude the possibility that the epi-A EIA tracks androgenic steroids from cortisol metabolism, faecal samples from an ACTH challenge experiment were assayed for fGM in hyenas with antibodies directed against cortisol-3-CMO and epi-A EIA (Fig 7), respectively. The cortisol EIA detected a large percentage increase in fGM in response to ACTH, with a maximum concentration 16-fold above pre-treatment levels. No similarly large percentage corresponding increase was obtained when applying the epi-A EIA. This result suggests that androgenic glucocorticoid metabolites do not contribute to the measurement of immunoreactive testosterone metabolites present in faeces of spotted hyenas. In contrast, the use of an EIA with an antibody directed against testosterone-11-HS-BSA revealed that this assay was responsible for substantial cross-reactivities with fGM in faeces from spotted hyenas.


Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).

Pribbenow S, East ML, Ganswindt A, Tordiffe AS, Hofer H, Dehnhard M - PLoS ONE (2015)

Comparison of faecal cortisol and testosterone immunoreactivity.Changes in fGM and fTM concentrations were determined in faecal samples from the ACTH challenge following hydrolysis with β-glucuronidase from Helix pomatia in the cortisol-3CMO, epiandrosterone and testosterone-11-HS EIAs, respectively. Levels of fGM and fTM are shown as percentage increase over pre-injection levels.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481319&req=5

pone.0128706.g007: Comparison of faecal cortisol and testosterone immunoreactivity.Changes in fGM and fTM concentrations were determined in faecal samples from the ACTH challenge following hydrolysis with β-glucuronidase from Helix pomatia in the cortisol-3CMO, epiandrosterone and testosterone-11-HS EIAs, respectively. Levels of fGM and fTM are shown as percentage increase over pre-injection levels.
Mentions: To exclude the possibility that the epi-A EIA tracks androgenic steroids from cortisol metabolism, faecal samples from an ACTH challenge experiment were assayed for fGM in hyenas with antibodies directed against cortisol-3-CMO and epi-A EIA (Fig 7), respectively. The cortisol EIA detected a large percentage increase in fGM in response to ACTH, with a maximum concentration 16-fold above pre-treatment levels. No similarly large percentage corresponding increase was obtained when applying the epi-A EIA. This result suggests that androgenic glucocorticoid metabolites do not contribute to the measurement of immunoreactive testosterone metabolites present in faeces of spotted hyenas. In contrast, the use of an EIA with an antibody directed against testosterone-11-HS-BSA revealed that this assay was responsible for substantial cross-reactivities with fGM in faeces from spotted hyenas.

Bottom Line: Our results showed that β-glucuronidases from both sources produced similar results from spotted hyena faeces.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.

View Article: PubMed Central - PubMed

Affiliation: Department Reproduction Biology and Evolutionary Ecology, Leibniz Institute of Zoo and Wildlife Research, Forschungsverbund Berlin e.V., Berlin, Germany.

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.

No MeSH data available.


Related in: MedlinePlus