Rapid freezing without cooling equilibration in canine sperm

The aim of this study was to develop a rapid method of canine semen freezing without cooling equilibration using treatment with different cryoprotectant agents (CPAs) and freezing in liquid nitrogen (LN2) vapor in a 0.5-mL straw via modifying vitrification. Ejaculates from eight beagle dogs were frozen with different CPAs (CPA-free, 5% glycerol, 5% ethylene glycol, and 10% ethylene glycol) and freezing times (direct plunging into LN2 or freezing for 1, 2, 3, or 10 min in LN2 vapor before plunging into LN2). Frozen–thawed sperm were evaluated for motility, viability, normal morphology, and plasma- and acrosome-membrane integrities. The 5% glycerol treatment resulted in improved sperm motility, plasma-membrane integrity and acrosome-membrane integrity (P < 0.05). Freezing in LN2 vapor showed improved sperm motility, viability, and plasma membrane integrity (P < 0.05), and freezing for more than 2 min in LN2 vapor increased acrosome-membrane integrity compared with direct plunging into LN2 (P < 0.05). The direct plunging into LN2 showed no motile sperm. However, freezing for more than 2 min in LN2 vapor increased the total abnormalities compared to direct plunging into LN2 (P < 0.05). In conclusion, use of 5% glycerol and freezing in LN2 vapor were essential for the rapid freezing of canine sperm without cooling equilibration. In particular, holding for 2 min in LN2 vapor was sufficient to yield successful rapid freezing. This rapid freezing method is simple and effective in canine sperm and would be helpful to offer information for trial of vitrification in large volumes of canine sperm.