Within-sample variation of fecal glucocorticoid measurements

Fecal glucocorticoid metabolite analysis is a useful tool for monitoring adrenocortical activity in captive and free-ranging wildlife. Glucocorticoid metabolites may not be evenly distributed within fecal samples and this variability could affect the interpretation of glucocorticoid metabolite measurements. Furthermore, the precision (i.e., repeatability of measurements) of fecal glucocorticoid measurements from well-mixed samples is unknown. We collected fresh white-tailed deer (Odocoileus virginianus) feces at various times pre- and post-adrenocorticotropin injection to provide samples with low (<75 ng/g), medium (75-90 ng/g), and high (>90 ng/g) glucocorticoid concentrations in case variability differs in samples of dissimilar hormone metabolite concentrations. We compared two sampling methods (selection of three pellet groups [one from each end of the fecal mass and one from the center] versus sampling three small portions of the thoroughly mixed fecal mass) to estimate within-sample variation of glucocorticoid metabolites. Glucocorticoid metabolite measures from pellet groups were higher than fecal glucocorticoid measures from mixed samples in the low (F=3.10; df = 1,56; P=0.08) and medium concentration (F=7.28; df = 1,50; P=0.01) groups. Fecal glucocorticoid metabolite estimates from mixed samples were less variable than glucocorticoid metabolite measures using pellet groups from the same fecal mass, although these differences were not statistically significant (low group: F=0.59; df = 1,38; P=0.45; medium group: F=0.13; df = 1,34; P=0.72; high group: F=2.30; df = 1,28; P=0.14). The mean coefficient of variation was <10% across all treatment groups and sampling methods. However, a power analysis indicated the mixed sub-sample technique requires fewer samples to detect statistically significant differences than pellet groups. Our results suggest that glucocorticoid metabolites may not be evenly distributed in white-tailed deer feces. Consequently, using only a few pellets from a fecal mass may bias assay interpretation. We suggest researchers homogenize the entire fecal mass before removing a sub-sample of fecal material for analysis. Also, other sources of variation must be considered when interpreting results of fecal glucocorticoid studies.