Spermatozoa Buck semen was collected with an artificial vagina two to three times per week from the nine males. Im- mediately after semen collection, any gel plug was removed using Pasteur pipettes and forceps. Ejacu- lated semen with more than 60% motile spermatozoa was used for sperm cryopreservation in the present
study. Sperm motility was evaluated by placing a small portion of it into an examination chamber (FA255,Fujihira Inc., Tokyo, Japan) on a micro-warm plate (Kitazato, Shizuoka, Japan) at 37°C, and examined by light microscopy at 100×. Sperm motility was defined as the percentage of spermatozoa that showed any sperm head movement. Forward progressive motility was the percentage of spermatozoa that moved linearly. The number of spermatozoa in samples was determined I duplicate by direct microscopic examination with ahemocytometer (a Thoma chamber).