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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 immunoreactive testosterone metabolites in captive hyenas.Testosterone immunoreactivity of faecal extracts were analysed in faecal extracts of one captive adult male (A) and one captive adult female (B) spotted hyena. Immunoreactivity was determined in the epiandrosterone EIA and is presented as a percentage of overall eluted activity. Lines with black circles represent immunoreactivity in each fraction. Lines with white circles show immunoreactivity in the fractions of the same extract after hydrolysis. The arrows represent the elution positions of reference standards cortisol (1), corticosterone (2), testosterone (3), epiandrosterone (4) and dihydrotestosterone (5).
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pone.0128706.g003: HPLC profiles of immunoreactive testosterone metabolites in captive hyenas.Testosterone immunoreactivity of faecal extracts were analysed in faecal extracts of one captive adult male (A) and one captive adult female (B) spotted hyena. Immunoreactivity was determined in the epiandrosterone EIA and is presented as a percentage of overall eluted activity. Lines with black circles represent immunoreactivity in each fraction. Lines with white circles show immunoreactivity in the fractions of the same extract after hydrolysis. The arrows represent the elution positions of reference standards cortisol (1), corticosterone (2), testosterone (3), epiandrosterone (4) and dihydrotestosterone (5).

Mentions: Before hydrolysis, HPLC profiles revealed polar radiolabelled testosterone metabolites (Fig 2) that were not apparent in the HPLC immunograms of the captive male (Fig 3A), the free-ranging adult male (Fig 4A) and the free-ranging adult female (Fig 4B), probably due to the inability of the polar radiolabelled metabolites to cross-react with the antibody. In contrast, before hydrolysis the polar radiolabelled metabolites from the captive female (Fig 2B) fitted with corresponding polar immunoreactivities (Fig 3B). Nevertheless, hydrolysis with the Hp-enzymes standardizes the patterns of immunoreactivities in both the captive male and female (Fig 3), liberating a major metabolite from its conjugate corresponding to the elution position of epi-A in fraction 40 and a minor one at fraction 25 (Fig 3). Similarly, in the hydrolysed extracts from an adult free-ranging male (Fig 4A) and an adult free-ranging female (Fig 4B), the epi-A EIA demonstrated a major peak of immunoreactive metabolites in fractions 40, co-eluting with the epi-A standard, and two minor peak in fractions 25 and 43, respectively. As a consequence of these results, all further analyses were carried out in hydrolysed extracts. To exclude crossreactivities of the epiandrosterone antibody with other possible faecal steroid metabolites, we analysed the HPLC elution fractions from the captive female with a cortisol-21, corticosterone-21, testosterone and dihydrotestosterone (DHT) EIA, respectively. In comparison to the epi-A EIA, none of the EIAs detected significant amounts of immunoreactivities (S1 Fig). This suggests that additional steroid metabolite levels did not falsify the measurement of immunoreactive epiandrosterone metabolites.


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 immunoreactive testosterone metabolites in captive hyenas.Testosterone immunoreactivity of faecal extracts were analysed in faecal extracts of one captive adult male (A) and one captive adult female (B) spotted hyena. Immunoreactivity was determined in the epiandrosterone EIA and is presented as a percentage of overall eluted activity. Lines with black circles represent immunoreactivity in each fraction. Lines with white circles show immunoreactivity in the fractions of the same extract after hydrolysis. The arrows represent the elution positions of reference standards cortisol (1), corticosterone (2), testosterone (3), epiandrosterone (4) and dihydrotestosterone (5).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128706.g003: HPLC profiles of immunoreactive testosterone metabolites in captive hyenas.Testosterone immunoreactivity of faecal extracts were analysed in faecal extracts of one captive adult male (A) and one captive adult female (B) spotted hyena. Immunoreactivity was determined in the epiandrosterone EIA and is presented as a percentage of overall eluted activity. Lines with black circles represent immunoreactivity in each fraction. Lines with white circles show immunoreactivity in the fractions of the same extract after hydrolysis. The arrows represent the elution positions of reference standards cortisol (1), corticosterone (2), testosterone (3), epiandrosterone (4) and dihydrotestosterone (5).
Mentions: Before hydrolysis, HPLC profiles revealed polar radiolabelled testosterone metabolites (Fig 2) that were not apparent in the HPLC immunograms of the captive male (Fig 3A), the free-ranging adult male (Fig 4A) and the free-ranging adult female (Fig 4B), probably due to the inability of the polar radiolabelled metabolites to cross-react with the antibody. In contrast, before hydrolysis the polar radiolabelled metabolites from the captive female (Fig 2B) fitted with corresponding polar immunoreactivities (Fig 3B). Nevertheless, hydrolysis with the Hp-enzymes standardizes the patterns of immunoreactivities in both the captive male and female (Fig 3), liberating a major metabolite from its conjugate corresponding to the elution position of epi-A in fraction 40 and a minor one at fraction 25 (Fig 3). Similarly, in the hydrolysed extracts from an adult free-ranging male (Fig 4A) and an adult free-ranging female (Fig 4B), the epi-A EIA demonstrated a major peak of immunoreactive metabolites in fractions 40, co-eluting with the epi-A standard, and two minor peak in fractions 25 and 43, respectively. As a consequence of these results, all further analyses were carried out in hydrolysed extracts. To exclude crossreactivities of the epiandrosterone antibody with other possible faecal steroid metabolites, we analysed the HPLC elution fractions from the captive female with a cortisol-21, corticosterone-21, testosterone and dihydrotestosterone (DHT) EIA, respectively. In comparison to the epi-A EIA, none of the EIAs detected significant amounts of immunoreactivities (S1 Fig). This suggests that additional steroid metabolite levels did not falsify the measurement of immunoreactive epiandrosterone metabolites.

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