Fig 1.
HPLC profiles of steroid standards.
Elution positions of authentic cortisol, corticosterone, testosterone, epiandrosterone and dihydrotestosterone (fractions 12, 23, 36, 40, and 41 respectively) obtained by applying the corresponding steroid hormone specific assays. For comparison results are presented as percentage of overall eluted steroid concentration.
Fig 2.
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.
Fig 3.
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).
Fig 4.
HPLC profiles of immunoreactive testosterone metabolites in free-ranging hyenas.
Testosterone immunoreactivity was analysed in faecal extracts of one free-ranging adult male (A) and one free-ranging adult female (B) hyena. Immunoreactivity was determined in the epiandrosterone EIA and is presented in 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).
Fig 5.
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).
Fig 6.
Comparison of testosterone immunoreactivity following hydrolysis with β-glucuronidase from Helix pomatia and from Escherichia coli.
FTM concentrations were determined in faecal samples from the testosterone challenge in the epiandrosterone EIA following hydrolysis with β-glucuronidase from Helix pomatia and β-glucuronidase from Escherichia coli, respectively. The linear regression indicates that both hydrolysis methods are congruent, as the regression explains a large segment of the variance (r2 = 0.84) and the intercept does not significantly differ from zero (see text for details).
Fig 7.
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.
Fig 8.
Hormonal data of juvenile and adult immigrant male hyenas.
FTM concentrations (ng/g faeces) in juvenile male (n = 15) and adult male (n = 15) spotted hyenas determined in the epiandrosterone EIA following hydrolysis with β-glucuronidase from Helix pomatia. The * indicates a significant difference between both categories.