The objectives of the present work were 3-fold. First, a new method for estimating daily sperm production was validated. This method, in turn, was used to evaluate testis output as well as deferent duct throughput. Next, this analytical approach was evaluated in 2 experiments. The first experiment compared left and right reproductive tracts within roosters. The second experiment compared reproductive tract throughput in roosters from low and high sperm mobility lines. Standard curves were constructed from which unknown concentrations of sperm cells and sperm nuclei could be predicted from observed absorbance. In each case, the independent variable was based upon hemacytometer counts, and absorbance was a linear function of concentration. Reproductive tracts were excised, semen recovered from each duct, and the extragonadal sperm reserve determined by multiplying volume by sperm cell concentration. Testicular sperm nuclei were procured by homogenization of a whole testis, overlaying a 20-mL volume of homogenate upon 15% (wt/vol) Accudenz (Accurate Chemical and Scientific Corporation, Westbury, NY), and then washing nuclei by centrifugation through the Accudenz layer. Daily sperm production was determined by dividing the predicted number of sperm nuclei within the homogenate by 4.5 d (i.e., the time sperm with elongated nuclei spend within the testis). Sperm transit through the deferent duct was estimated by dividing the extragonadal reserve by daily sperm production. Neither the efficiency of sperm production (sperm per gram of testicular parenchyma per day) nor deferent duct transit differed between left and right reproductive tracts (P > 0.05). Whereas efficiency of sperm production did not differ (P > 0.05) between low and high sperm mobility lines, deferent duct transit differed between lines (P < 0.001). On average, this process required 2.2 and 1.0 d for low and high lines, respectively. In summary, we developed and then tested a method for quantifying male reproductive tract throughput. This method makes the study of semen production amenable to systems biology.