Simple Summary Testosterone may be the primary reproductive hormone in man vertebrates. to assess fecal TMs can be widely used and will benefit various research disciplines. Abstract Testosterone is the main reproductive hormone in male vertebrates and conventional methods to measure testosterone rely on invasive blood sampling procedures. Here, we aimed to establish a noninvasive alternative by assessing testosterone metabolites (TMs) in fecal and urinary samples in mice. We performed a radiometabolism study to determine the effects of daytime and sex on the metabolism and excretion pattern of radiolabeled TMs. We performed physiological and biological validations of the applied EIA to measure TMs and assessed diurnal fluctuations in TM excretions in male and female mice and across strains. We found that males excreted significantly more radiolabeled TMs via the feces (59%) compared to females (49.5%). TM excretion patterns differed significantly between urinary and fecal samples and were affected by the daytime of 3H-testosterone injection. Overall, TM excretion occurred faster in urinary than fecal samples. Peak excretion of fecal TMs occurred after 8 h when animals received the 3H-testosterone in the morning, or after 4 h when they received the 3H-testosterone injection in the evening. Daytime had no effect on the formed TMs; however, males and females formed different types of TMs. As expected, males showed higher fecal TM levels than females. Males also showed diurnal fluctuations in their TM levels but we found no differences in the TM levels of C57BL/6J and B6D2F1 hybrid males. Finally, we successfully validated our applied EIA (measuring 17-hydroxyandrostane) by showing that hCG (human chorionic gonadotropin) administration increased TM levels, whereas castration reduced them. In conclusion, our EIA proved suitable for measuring fecal TMs in mice. Our non-invasive method to assess fecal TMs can be Prazosin HCl NEK3 used in various research disciplines like animal behavior broadly, reproduction, pet welfare, ecology, conservation, and biomedicine. = Prazosin HCl 0.94) or between sexes (men: 56.4 6.3%, females: 57.3 6.3% MannCWhitney U check: U = 145.5, N = 32, = 0.52). Though Interestingly, we discovered that men secreted a lot more radiolabeled TMs via the feces than females (men: 59.0 7.3%, females: 49.5 5.5%; MannCWhitney U check: U = 39, N = 32, < 0.001), whereas females showed higher proportions of radioactivity in the urine in comparison to men (men: 41.0 3.7%, females: 50.3 5.5%; Prazosin HCl < 0.001). General, animal defecation prices were significantly reliant on daytime (Combined = ?35.95, < 0.001): Experimental mice produced normally 8.02 g feces through the 12 h collection intervals from 9 p.m. to 9 a.m. in comparison to 3.22 g feces through the 12 h collection intervals from 9 a.m. to 9 p.m. The excretion of radiolabeled TMs assorted significantly as time passes and demonstrated different patterns in urinary and fecal examples (LMM feces: F15, 464 = 39.33, < 0.001; LMM urine: F15, 464 = 57.65, < 0.001). Within both, fecal and urinary samples, we discovered a significant impact of enough time of shot for the excretion design (LMM feces: F1, 30 = 48.21, < 0.001; LMM urine: F1, 30 = 19.07, < 0.001): Prazosin HCl In fecal examples, pets that received the radiolabeled testosterone at night showed a faster rise in excretion prices and excreted the radiolabeled testosterone quicker compared to pets that received the radiolabeled testosterone each day (Figure 1b). In urinary examples, pets from both Prazosin HCl treatment organizations showed an instantaneous rise in excretion prices, but pets from the night group excreted the radiolabeled testosterone quicker compared to pets from the morning hours group (Shape 1a). Within both test types no sex-specific variations in the excretion patterns had been recognized (LMM feces: F1, 29 = 1.64, = 0.21; LMM urine: F1, 29 = 1.55, = 0.22) and almost all radiolabeled metabolites in feces (97.3 1.5 %) and urine (84.6 12.2%) was excreted inside the 1st 24 h. Open up in another window Figure one time course (1st 48 h) of 3H-testosterone excretion in (a) urinary and (b) fecal examples of male and feminine C57BL/6J mice. White colored boxplots (n = 16) represent data from mice that received the 3H-testosterone each day (1 hour after the starting point from the light stage) and gray boxplots (n = 16) represent data.