Limits...
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

Changes in fTM concentrations in response to a testosterone challenge in a female spotted hyena.Faecal samples were collected from 6 days prior to injection until 8 days post-injection and were analysed with an epiandrosterone EIA following hydrolysis with β-glucuronidase from Helix pomatia. The arrow represents the time of testosterone injection; the dashed line indicates the baseline level. The * indicates peaks (values exceeding mean + 2SD).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4481319&req=5

pone.0128706.g005: Changes in fTM concentrations in response to a testosterone challenge in a female spotted hyena.Faecal samples were collected from 6 days prior to injection until 8 days post-injection and were analysed with an epiandrosterone EIA following hydrolysis with β-glucuronidase from Helix pomatia. The arrow represents the time of testosterone injection; the dashed line indicates the baseline level. The * indicates peaks (values exceeding mean + 2SD).

Mentions: Administration of testosterone results in a rapid artificial induced increase in plasma testosterone levels and should be clearly reflected in the concentrations of fTM after a species-specific time lag. In the female spotted hyena an increase in concentrations of plasma testosterone from 11 ng/g to 238 ng/g was detected 15 min after injection. To validate the ability of the epi-A EIA to detect the expected increase in fTM caused by the testosterone challenge, we analysed the faecal extracts following hydrolysis with Hp-enzymes. A significant increase in fTM levels (Fig 5) was detected 15.5 h post-injection, with a maximum concentration 4-fold above baseline (0.72 ± 0.24μg/g wet faeces). FTM returned to baseline levels within 24 h following injection. This course is congruent with the course of total amounts of radiolabel excreted with faeces. However, a peak of almost a similar intensity was detected 3 days before testosterone treatment. Ec-hydrolysed extracts revealed a similar profile of fTM concentrations in response to the testosterone challenge (Fig 6). A linear regression of Ec-hydrolysis fTM levels on Hp-hydrolysis fTM levels produced a high degree of congruence (r2 = 0.83; F = 81.36; df = 1, 16; p < 0.0001, with the regression equation being fTM concentration (Hp) = 0.06 + 1.32 x fTM concentration (Ec), and the intercept not being significantly different from 0, t = 0.792, p = 0.44). Therefore, all further analyses were carried out in Hp-hydrolysed faecal extracts.


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)

Changes in fTM concentrations in response to a testosterone challenge in a female spotted hyena.Faecal samples were collected from 6 days prior to injection until 8 days post-injection and were analysed with an epiandrosterone EIA following hydrolysis with β-glucuronidase from Helix pomatia. The arrow represents the time of testosterone injection; the dashed line indicates the baseline level. The * indicates peaks (values exceeding mean + 2SD).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128706.g005: Changes in fTM concentrations in response to a testosterone challenge in a female spotted hyena.Faecal samples were collected from 6 days prior to injection until 8 days post-injection and were analysed with an epiandrosterone EIA following hydrolysis with β-glucuronidase from Helix pomatia. The arrow represents the time of testosterone injection; the dashed line indicates the baseline level. The * indicates peaks (values exceeding mean + 2SD).
Mentions: Administration of testosterone results in a rapid artificial induced increase in plasma testosterone levels and should be clearly reflected in the concentrations of fTM after a species-specific time lag. In the female spotted hyena an increase in concentrations of plasma testosterone from 11 ng/g to 238 ng/g was detected 15 min after injection. To validate the ability of the epi-A EIA to detect the expected increase in fTM caused by the testosterone challenge, we analysed the faecal extracts following hydrolysis with Hp-enzymes. A significant increase in fTM levels (Fig 5) was detected 15.5 h post-injection, with a maximum concentration 4-fold above baseline (0.72 ± 0.24μg/g wet faeces). FTM returned to baseline levels within 24 h following injection. This course is congruent with the course of total amounts of radiolabel excreted with faeces. However, a peak of almost a similar intensity was detected 3 days before testosterone treatment. Ec-hydrolysed extracts revealed a similar profile of fTM concentrations in response to the testosterone challenge (Fig 6). A linear regression of Ec-hydrolysis fTM levels on Hp-hydrolysis fTM levels produced a high degree of congruence (r2 = 0.83; F = 81.36; df = 1, 16; p < 0.0001, with the regression equation being fTM concentration (Hp) = 0.06 + 1.32 x fTM concentration (Ec), and the intercept not being significantly different from 0, t = 0.792, p = 0.44). Therefore, all further analyses were carried out in Hp-hydrolysed faecal extracts.

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