Effects of heat and chemical treatments on fecal glucocorticoid measurements: Implications for sample transport

The United States Department of Agriculture requires that fecal material shipped to the United States from foreign countries be treated with heat or chemicals to kill potential pathogens and prevent disease transmission. However, these treatments could affect fecal steroid metabolite measures. We evaluated whether treating feces of white-tailed deer (Odocoileus virginianus) and elk (Cervus elaphus) with 10% formalin, 2% acetic acid, 90% ethanol, 2% sodium hydroxide or autoclaving and shipping influenced fecal glucocorticoid concentrations. We applied treatments to feces collected fresh and frozen (n=20 individual deer samples, n=20 individual elk samples), including a shipping period of 6 days. We also shipped untreated samples. A control sample was retained frozen in the lab (no treatment and no shipping). We used a radioimmunoassay procedure previously validated for deer and elk to quantify fecal glucocorticoid metabolites. Upon returning to our lab, samples had partially thawed, although the innermost portion of the sample remained frozen. Deer and elk fecal glucocorticoid concentrations were significantly altered by heat and chemical treatments (F_6,114=6.85, P<0.0001; F_6,114=28.08, P<0.0001, respectively), although treatments were not consistent by species. Fecal glucocorticoid metabolites in shipped autoclaved deer feces were significantly higher than the control (F_1,19=58.2, P<0.0001). In contrast, the 10% formalin treatment significantly decreased fecal glucocorticoid concentrations (F_1,19=5.38, P=0.03). The shipped and untreated deer samples were nearly 20% higher than the control; however, this difference was not significant (F_1,19=3.38, P=0.08). For elk samples, 2% sodium hydroxide (F_1,19=48.73, P<0.0001) and formalin (F_1,19=20.21, P=0.0002) significantly reduced fecal glucocorticoid levels compared with the control. Also, ethanol (F_1,19=4.84, P=0.04) and autoclaving elk feces (F_1,19=13.08, P=0.002) significantly increased fecal steroid concentrations. Fecal glucocorticoid concentrations in shipped and untreated elk samples were significantly less than the control (F_1,19=7.48, P=0.01). Immersing deer and elk samples in a 2% acetic acid treatment had the least impact on fecal glucocorticoid metabolite measures; on average these samples were <2% different from the control for deer and <6% for elk. We recommend a 2% acetic acid solution when treatment is necessary (i.e., international shipping) or thawing is possible. Irrespective of the treatment used, samples should remain frozen during shipment to prevent microbial metabolism of fecal material.