Three extenders with different glucose concentrations were prepared; they consisted of 10% methanol and 0.1, 0.2, and 0.3 M glucose, respectively. We did not use a variant without glucose, because an external cryoprotectant is needed for successful cryopreservation. Cryopreservation was performed using a 1:3 semen-to-extender dilution ratio. Therefore, the final concentrations of glucose were 0.075, 0.15, and 0.25 M, respectively, and the final concentration of methanol was 7.5% for all variants. Sperm motility was measured for fresh-diluted, and frozen-thawed semen (n = 9 different males) using CASA (see 2.9). Mean sperm concentration and osmolality were 9.87 ± 2.74 × 109 spermatozoa and 235 ± 63 mOsm/kg, respectively. Based on the motility results shown in Fig. 1, the extender (producing final concentrations of 0.15 M glucose and 7.5% methanol) was used in the following experiments. Analyses were run in duplicates for all of the experiments in the present study.