A matter of time: Evaluating the storage of fecal samples for steroid analysis

The extraction and immunoassay of fecal steroids is an increasingly common technique, used in both captive and field studies to provide an approximation of an animal's circulating concentration of hormones through non-invasive methods. Storage of fecal samples is of critical concern because fecal bacteria metabolize fecal steroids within hours after deposit. Ethanol is often used as a preservative for fecal samples stored for several hours at room temperature. We examined the stability of fecal estrogen (fE) and glucocorticoid (fGC) metabolites from baboon (Papio cynocephalus) samples in a 95% ethanol solution at ambient temperature and at -20°C over the course of six months, to determine the effect of storage on steroid concentrations. As measured by radioimmunoassay, fE metabolite concentrations increased by 122% at 90 days and fGC metabolite concentrations increased by 92% at 120 days. After peaking, both hormones declined to near initial concentrations by 180 days in ambient temperature samples. In samples stored at sub-zero temperatures, fGC metabolite concentrations showed a similar but dampened pattern, while fE metabolite concentrations exhibited small and variable changes with no consistent trend. We discuss explanations for the dynamic pattern of changing fecal metabolite concentrations and offer practical and analytical guidance to field workers for situations in which ideal conditions for stabilizing hormones are not available.