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As fecal steroid methods increasingly are used by researchers to monitor the physiology of captive and wild populations, we need to expand our validation protocols to test the effects of procedural variation and to identify contamination by exogenous sources of steroid hormones. Mammalian carnivore feces often contain large amounts of hair from the prey they consume, which itself may contain high concentrations of hormones. In this study, we report first a validation of two steroid hormone antibodies, corticosterone and progesterone, to determine fecal concentrations of these hormones in wild spotted hyenas (Crocuta crocuta). Next, we expand on these standard validation protocols to test two additional metrics: (i) whether hair from consumed prey or (ii) the specific drying method (oven incubation vs. lyophilization) affect steroid hormone concentrations in feces. In the first biological validation for the progesterone antibody in this species, progesterone concentrations met our expectations: (i) concentrations of plasma and fecal progesterone were lowest in immature females, higher in lactating females, and highest in pregnant females; (ii) across pregnant females, fecal progesterone concentrations were highest during late pregnancy; and (iii) among lactating females, fecal progesterone concentrations were highest after parturition. Our additional validation experiments indicated that contamination with prey hair and drying method are hormone-specific. Although prey hair did not release hormones into samples during storage or extraction for either hormone, its presence appeared to “dilute” progesterone (but not corticosterone) measures indirectly by increasing the dry weight of samples. In addition, fecal progesterone, but not corticosterone, values were lower for lyophilized than for incubated samples. Therefore, in addition to the standard analytical and biological validation steps, additional methodological variables need to be tested whenever we measure fecal hormone concentrations, particularly from predatory mammals.

 

Abstract Studies in rodents and captive primates suggest that the early-life social environment affects future phenotype, potentially through alterations to DNA methylation. Little is known of these associations in wild animals. In a wild population of spotted hyenas, we test the hypothesis that maternal care during the first year of life and social connectedness during two periods of early development leads to differences in DNA methylation and fecal glucocorticoid metabolites (fGCMs) later in life. Here we report that although maternal care and social connectedness during the den-dependent life stage are not associated with fGCMs, greater social connectedness during the subadult den-independent life stage is associated with lower adult fGCMs. Additionally, more maternal care and social connectedness after den independence correspond with higher global (%CCGG) DNA methylation. We also note differential DNA methylation near 5 genes involved in inflammation, immune response, and aging that may link maternal care with stress phenotype.