Validation of a Fecal Glucocorticoid Assay to Assess Adrenocortical Activity in Meerkats Using Physiological and Biological Stimuli

Abstract

In mammals, glucocorticoid (i.e. GC) levels have been associated with specific life-history stages and transitions, reproductive strategies, and a plethora of behaviors. Assessment of adrenocortical activity via measurement of glucocorticoid metabolites in feces (FGCM) has greatly facilitated data collection from wild animals, due to its non-invasive nature, and thus has become an established tool in behavioral ecology and conservation biology. The aim of our study was to validate a fecal glucocorticoid assay for assessing adrenocortical activity in meerkats (Suricata suricatta), by comparing the suitability of three GC enzyme immunoassays (corticosterone, 11β-hydroxyetiocholanolone and 11oxo-etiocholanolone) in detecting FGCM increases in adult males and females following a pharmacological challenge with adrenocorticotropic hormone (ACTH) and biological stimuli. In addition, we investigated the time course characterizing FGCM excretion, the effect of age, sex and time of day on FGCM levels and assessed the potential effects of soil contamination (sand) on FGCM patterns. Our results show that the group specific 11β-hydroxyetiocholanolone assay was most sensitive to FGCM alterations, detecting significant and most distinctive elevations in FGCM levels around 25 h after ACTH administration. We found no age and sex differences in basal FGCM or on peak response levels to ACTH, but a marked diurnal pattern, with FGCM levels being substantially higher in the morning than later during the day. Soil contamination did not significantly affect FGCM patterns. Our results emphasize the importance of conducting assay validations to characterize species-specific endocrine excretion patterns, a crucial step to all animal endocrinology studies using a non-invasive approach.

Fig 1. Representative profiles of immunoreactive 11β-hydroxyetiocholanolone (black circles) and CCST (white triangles) in two female (top) and male (bottom) meerkats after administration of ACTH at time 0.

Data points before time 0 represent median ± SE concentrations (μg/g) of pre-treatment samples collected 5–8 days before ACTH injection.

Fig 2. Percentage of response in immunoreactive fecal 11β-hydroxyetiocholanolone and CCST levels to ACTH administration in meerkats.

Data points represent mean ± SE values calculated for 8-12-h intervals across the 14 individuals that responded to the ACTH challenge. Percentages were calculated in relation to pre-treatment baseline values (pre = 100%).

Fig 3. HPLC profiles of immunoreactivity detected with the CCST (top) and 11β-hydroxyetiocholanolone EIA (bottom) for a male and a female meerkat.

Samples tested were those that showed peak GC metabolite concentrations after the ACTH challenge. Arrows and numbers show the elution positions of associate reference standards: 1) cortisol (fractions 14–15), 2) corticosterone (22), 3) 11β-hydroxyetiocholanolone (25), 4) 11-oxoetiocholanolone (30), 5) 5β-androstane-3,11,17-trione (36), 6) testosterone (43), 7) androstendione, dehydroepiandrosterone (55–56), 8) epiandrosterone, 5β-DHT, 5b-androstane-3β-ol-17-one (72), 9) 5β-androstane- 3α-ol-17-one (82).

Fig 4. Representative FGCM (11β-hydroxyetiocholanolone) profiles of a female (top) and male (bottom) meerkat after administration of ACTH in samples uncorrected for soil contamination (with soil) and those corrected for soil content (without soil).

Data points before time 0 indicate median ± SE concentrations of pre-treatment samples collected 5–8 days before ACTH injection. Profiles for CCST showed a similar degree of correspondence between the two conditions (see text).

Fig 5. Magnitude of 11β-hydroxyetiocholanolone and CCST elevation to ACTH (peak to baseline ratio; graphs on the left) and variability in 11β-hydroxyetiocholanolone and CCST (pre-treatment) baseline values (coefficients of variation (CV) of baseline variability; graphs on the right) in samples uncorrected for soil contamination (with soil) and those corrected for soil content (without soil) in 10 meerkats.