Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol

When mouse epididymal spermatozoa were rapidly frozen in two steps (37 to -70 degrees C for solid CO2 and -70 to -196 degrees 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.