The extenders were prepared by using 300 mM glucose and 10% DMSO supplemented with different
concentrations of CLC (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg per 120 106 spermatozoa) and without
CLC (control). The pooled semen was diluted separately at a ratio of 1:3 (v/v) by using CLC extenders.
Diluted semen placed into 0.25 ml straws were equilibrated at 4 C for 15 min and frozen in liquid nitrogen
vapor.