Immediately after milting, the sperm sample was subjected to a low temperature (15oC) and
was diluted with the prepared extenders (0.8%NaCl, HBSS, HBSS-CF and Kurokura). Samples of the
extended sperm were then placed in cryovials at different volumes (0.5, 1.0, 2.0 and 4.0 ml). All
samples were placed in a cryochamber and plunged into the cryobath with liquid nitrogen at 10-cm
depth. A computer program (Cryogenesis V5, Australia) was then run to attain a constant cooling rate
of 10oC per minute until the target temperature of -80oC was achieved. The samples were then kept in
a liquid nitrogen tank.