Fecal Glucocorticoid Metabolites as Biomarkers in Equids: Assay Choice Matters

From Wiley via Jisc Publications RouterHistory: received 2020-05-06, rev-recd 2021-03-29, accepted 2021-04-09, pub-electronic 2021-06-01Article version: VoRPublication status: PublishedFunder: Royal Society; Id: http://dx.doi.org/10.13039/501100000288; Grant(s): UF110641Funder: Chester Zoo; Id: http://dx.doi.org/10.13039/501100005359; Grant(s): Conservation FellowshipABSTRACT: Free ranging animals are exposed to environmental, demographic, and ecological challenges over time, which can affect their health and fitness. Non‐invasive biomarkers can provide insight into how animals cope with these challenges and assess the effectiveness of conservation management strategies. We evaluated how free ranging ponies (Equus ferus caballus) on the Carneddau Mountain range, North Wales respond to 2 stimuli: an acute stressor of an annual roundup event in November 2014, and spatial and temporal variation in ecological factors in 2018. We evaluated fecal glucocorticoid metabolites using 2 enzyme immunoassays (EIAs): an 11‐oxoetiocholanolone EIA (measuring 11,17‐dioxoandrostanes [11,17‐DOAs]) and a corticosterone EIA. The former assay has been validated in equids, whereas there is limited evidence for the suitability of the latter. We used an additional parent testosterone EIA to measure fecal androgen metabolites in response to the ecological challenges. Following the roundup, the metabolite concentrations measured by the 2 glucocorticoid EIAs were not correlated. The 11,17‐DOAs were elevated from the second day following the roundup and then slowly returned to pre‐round levels over the next 2 weeks. In contrast, the metabolites measured by the corticosterone assay showed no response to the roundup. For the ecological data, all 3 assays detected a positive correlation between metabolites and social group size in males but not in females. The metabolite concentrations measured by the testosterone and corticosterone assays were highly correlated and were temporally associated with the onset of the breeding season, whereas the 11,17‐DOAs were not. The co‐variance of metabolites measured by the corticosterone and testosterone assays, and the lack of an acute response in the corticosterone assay to the roundup, suggests that metabolites detected by the corticosterone assay were not primarily associated with increased glucocorticoid production. We recommend using well‐validated fecal biomarker assays of hypothalamus‐pituitary‐adrenal axis activity to evaluate and compare the effect of different management interventions and environmental change. © 2021 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society

Hybride Systeme zur Objekterkennung

Sagerer G. Hybride Systeme zur Objekterkennung. In: Dreschler-Fischer L, Pribbenow S, eds. KI-95 Activities: Workshop, Posters, Demos. Gesellschaft für Informatik e.V. Bonn; 1995: 146-147

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

[This corrects the article DOI: 10.1371/journal.pone.0128706.]

HPLC profiles of immunoreactive testosterone metabolites in captive hyenas.

<p>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).</p

Changes in fTM concentrations in response to a testosterone challenge in a female spotted hyena.

<p>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 <i>Helix pomatia</i>. The arrow represents the time of testosterone injection; the dashed line indicates the baseline level. The * indicates peaks (values exceeding mean + 2SD).</p

HPLC profiles of steroid standards.

<p>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.</p

Comparison of faecal cortisol and testosterone immunoreactivity.

<p>Changes in fGM and fTM concentrations were determined in faecal samples from the ACTH challenge following hydrolysis with β-glucuronidase from <i>Helix pomatia</i> 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.</p

HPLC profiles of immunoreactive testosterone metabolites in free-ranging hyenas.

<p>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).</p

Comparison of testosterone immunoreactivity following hydrolysis with β-glucuronidase from <i>Helix pomatia</i> and from <i>Escherichia coli</i>.

<p>FTM concentrations were determined in faecal samples from the testosterone challenge in the epiandrosterone EIA following hydrolysis with β-glucuronidase from <i>Helix pomatia</i> and β-glucuronidase from <i>Escherichia coli</i>, respectively. The linear regression indicates that both hydrolysis methods are congruent, as the regression explains a large segment of the variance (r^<b>2</b> = 0.84) and the intercept does not significantly differ from zero (see text for details).</p