2.3. Freezing Protocol
After collection, the ejaculates were diluted 1:1 with
INRA96 extender, divided into aliquots according to the
number of freezing extenders to be tested, and centrifuged
at 1000 g for 7 minutes. The supernatant was removed,
and the sperm pellets were resuspended with a specific
freezing extender (INRA plus 1.25% soybean lecithin þ 2%
CEYþ 2%G(INRA2), INRA plus 1.25% soybean lecithin þ 4%
CEYþ2% G [INRA4], INRA plus 1.25% soybean lecithinþ2%G
[INRA0]) to obtain 200 106 sperm/ml. Each tube of diluted
semen was cooled to 4C over 75 minutes. Cooled semen
was loaded into 0.5-ml polyvinyl chloride straws (IMV
Technologies, France) that were then sealed with polyvinyl
alcohol sealing powder. The samples were packaged into
0.5-ml straws, incubated at 5C for 20 minutes, and frozen at
4 cm above the level of liquid nitrogen for 12 minutes before
being plunged into liquid nitrogen for storage.