Variations, validations, degradations, and noninvasive determination of pregnancy using fecal steroid metabolites in free-ranging pronghorn

•Fecal progesterone metabolite a determinant of pregnancy in free-ranging pronghorn.•Timing of noninvasive fecal sampling crucial for accurate pregnancy estimates.•Temporal variations exist in fecal progesterone and glucocorticoids metabolites.•Free-range settings showcase natural hormone variations.•Fresh samples required for accurate fecal hormone concentrations. Pregnancy status is a key parameter used to assess reproductive performance of a species as it represents a starting point for measuring vital rates. Vital rates allow managers to determine trends in populations such as neonate survival and recruitment; two important factors in ungulate population growth rates. Techniques to determine pregnancy have generally involved capture and restraint of the animal to obtain blood samples for determining serum hormone levels. Non-invasive pregnancy assessment, via feces, eliminates any hazards between handler and animal, as well as removes handling-induced physiological biases. Using noninvasive fecal sampling, we conducted hormone validations, investigated pregnancy rates, and determined hormone degradation rates across five subpopulations of pronghorn (Antilocapra americana) in Idaho. Samples were collected during April-May of 2018 and 2019 from adult pronghorn of known sex and age class. Metabolites of testosterone, cortisol, 17β-estradiol, and progesterone were measured in fecal samples, and concentrations of estradiol and progesterone were examined for pregnancy determination. Average fecal progesterone metabolite (FPM) levels of pregnant females were more than double compared to levels of nonpregnant females. Fecal estrogen metabolite (FEM) levels did not differ during concurrent sampling. The largest difference in FPM levels between pregnant and nonpregnant females began on 28 April. Pregnancy determination sampling showed average FPM levels for all five subpopulations were significantly different than the nonpregnant female validation group. Nonetheless, pregnancy rates for some subpopulations lacked conclusive estimates due to early fecal sampling. Fecal glucocorticoid metabolites (FGM) levels significantly differed between pregnant females and male pronghorn, but did not differ from nonpregnant females. Degradation rates of FPM and FGM differed across days, with values for FPM from Day 1 being significantly different from all subsequent days, and after Day 9 for FGM, demonstrating the requirement of fresh samples to accurately measure hormone conc