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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

HPLC profiles of 3H-testosterone metabolites.3H-testosterone metabolites were analysed in non-hydrolysed (black circles) and hydrolysed (white circles) faecal extracts of one captive male (A) and one captive female (B) spotted hyena. Extracts were separated by RP-HPLC and then radioactivity of each fraction was analysed. Radioactivity is presented as a percentage of the overall eluted activity. The arrows represent the elution positions of reference standards cortisol (1), corticosterone (2), testosterone (3), epiandrosterone (4) and dihydrotestosterone (5), as detailed in Fig 1.
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pone.0128706.g002: HPLC profiles of 3H-testosterone metabolites.3H-testosterone metabolites were analysed in non-hydrolysed (black circles) and hydrolysed (white circles) faecal extracts of one captive male (A) and one captive female (B) spotted hyena. Extracts were separated by RP-HPLC and then radioactivity of each fraction was analysed. Radioactivity is presented as a percentage of the overall eluted activity. The arrows represent the elution positions of reference standards cortisol (1), corticosterone (2), testosterone (3), epiandrosterone (4) and dihydrotestosterone (5), as detailed in Fig 1.

Mentions: HPLC elution positions of the reference steroid standards cortisol, corticosterone, testosterone, dihydrotestosterone and epiandrosterone obtained when applying the corresponding steroid hormone specific assays are shown in Fig 1. HPLC profiles of radiolabelled testosterone metabolites in non-hydrolysed and hydrolysed spotted hyena faeces are shown in Fig 2A&2B, respectively. Using reversed-phase HPLC, metabolites were separated according to their polarity and the more polar metabolites were eluted first. In non-hydrolysed extracts of one captive adult male (Fig 2A) and captive adult female (Fig 2B) hyena, only one polar peak was detected in fractions 1–5, probably indicating a cluster of conjugated radiolabelled metabolites. Minor variations in the composition of radiolabelled metabolites in both sexes might result of individual or sex-specific differences in testosterone metabolism. Enzymatic hydrolysis of faecal extracts changed the elution patterns of polar radiolabelled metabolites in extracts from both sexes. In the male (Fig 2A) as well as in the female (Fig 2B) polar radiolabelled conjugates disappeared and were substituted by a major radiolabelled peak in fractions 39–41, co-eluting with the epi-A standard. In the male an additional minor proportion of radiolabelled metabolites occurred in fractions 44–46. In both sexes no radiolabelled metabolites were detectable at the elution position corresponding to the testosterone standard, thus the circulating hormone itself is not present in faeces. Additionally, no radiolabelled metabolites were detected at the elution positions of authentic cortisol, corticosterone and DHT.


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)

HPLC profiles of 3H-testosterone metabolites.3H-testosterone metabolites were analysed in non-hydrolysed (black circles) and hydrolysed (white circles) faecal extracts of one captive male (A) and one captive female (B) spotted hyena. Extracts were separated by RP-HPLC and then radioactivity of each fraction was analysed. Radioactivity is presented as a percentage of the overall eluted activity. The arrows represent the elution positions of reference standards cortisol (1), corticosterone (2), testosterone (3), epiandrosterone (4) and dihydrotestosterone (5), as detailed in Fig 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128706.g002: HPLC profiles of 3H-testosterone metabolites.3H-testosterone metabolites were analysed in non-hydrolysed (black circles) and hydrolysed (white circles) faecal extracts of one captive male (A) and one captive female (B) spotted hyena. Extracts were separated by RP-HPLC and then radioactivity of each fraction was analysed. Radioactivity is presented as a percentage of the overall eluted activity. The arrows represent the elution positions of reference standards cortisol (1), corticosterone (2), testosterone (3), epiandrosterone (4) and dihydrotestosterone (5), as detailed in Fig 1.
Mentions: HPLC elution positions of the reference steroid standards cortisol, corticosterone, testosterone, dihydrotestosterone and epiandrosterone obtained when applying the corresponding steroid hormone specific assays are shown in Fig 1. HPLC profiles of radiolabelled testosterone metabolites in non-hydrolysed and hydrolysed spotted hyena faeces are shown in Fig 2A&2B, respectively. Using reversed-phase HPLC, metabolites were separated according to their polarity and the more polar metabolites were eluted first. In non-hydrolysed extracts of one captive adult male (Fig 2A) and captive adult female (Fig 2B) hyena, only one polar peak was detected in fractions 1–5, probably indicating a cluster of conjugated radiolabelled metabolites. Minor variations in the composition of radiolabelled metabolites in both sexes might result of individual or sex-specific differences in testosterone metabolism. Enzymatic hydrolysis of faecal extracts changed the elution patterns of polar radiolabelled metabolites in extracts from both sexes. In the male (Fig 2A) as well as in the female (Fig 2B) polar radiolabelled conjugates disappeared and were substituted by a major radiolabelled peak in fractions 39–41, co-eluting with the epi-A standard. In the male an additional minor proportion of radiolabelled metabolites occurred in fractions 44–46. In both sexes no radiolabelled metabolites were detectable at the elution position corresponding to the testosterone standard, thus the circulating hormone itself is not present in faeces. Additionally, no radiolabelled metabolites were detected at the elution positions of authentic cortisol, corticosterone and DHT.

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