Summary. When mouse epididymal spermatozoa were rapidly frozen in two steps (37 to −70°C for solid CO2 and −70 to −196°C for liquid nitrogen) as pellets, 18% raffinose provided the greatest protection to ICR mouse spermatozoa against cold-shock; sperm motility and fertilizing ability were 43% and 22·4%, respectively. A small proportion of spermatozoa frozen with 10% sucrose was motile but incapable of fertilizing ovulated oocytes. Glycerol and dimethylsulphoxide were less effective at any concentration examined. However, the fertilizing ability of frozen–thawed ICR spermatozoa was significantly improved (35·5%) by addition of glycerol (1·75% final concentration) to medium containing 18% raffinose. Spermatozoa from one outbred (ddY) and 5 inbred (C57BL/6N, C3H/HeN, DBA/2N, BALB/c and kk) strains of mice were successfully frozen in the presence of 18% raffinose and 1·75% glycerol, although the fertilization rates of frozen–thawed spermatozoa varied among strains (13% for C57BL/6N to 64% for DBA/2N). A small fraction of mouse eggs resulting from fertilization by frozen–thawed spermatozoa developed normally in vitro (37% in C57BL/6N to 71% in ICR) to the blastocyst stage and in vivo (19% for C57BL/6N spermatozoa and ddY oocytes) to Day 18 of gestation.
Keywords: mouse; spermatozoa; cryopreservation; pellet method; cryoprotectant; raffinose