Dialysis addition of trehalose/glycerol cryoprotectant allows recovery of cryopreserved mouse spermatozoa with satisfactory fertilizing ability as assessed by yield of live young

Mouse sperm cryopreservation provides a means for storing the genetic infor mation in genetically modified mice (mutants, transgenics, and "knockouts") in a cost- and space-effective manner. Sperm from this species are highly sensitive to cryodamage, which has impeded their cryopreservation in the pa st. The cryoprotectant used in this study was 6% glycerol (0.65 M) plus 7.5 % trehalose (0.22 M), which was added to a concentrated suspension of sperm from B6SJLF1/J mice in bicarbonate-free buffer by dialysis to minimize osm otic stress on the cells. Sperm suspensions were frozen in 0.25 mL straws a nd stored in liquid N-2. Eggs were obtained from B6SJLF1/J superovulated fe males. For in vitro fertilization (IVF), 15-25 muL of sperm suspension post -thaw from one straw was added directly to each of three 1.5 mL drops of fe rtilization medium containing 30 eggs each, for 3 replicates per experiment . The fertilized eggs were scored for blastocyst formation, after which 12 blastocysts from each drop were implanted into pseudopregnant CD-1 females. The number of live pups were then scored at birth. Ten experiments yielded 21.7 +/- 1.4 (SD) blastocysts per 30 eggs inseminated (72%) and 7.3 +/- 0. 4 (SD) live pups per 12 blastocysts implanted (61%). The overall yield of l ive pups was 44 per 100 eggs inseminated (44%). This yield should be satisf actory for maintaining a mouse strain through sperm cryostorage, with resta rt of the strain through IVF and embryo transfer. The method should also pr ovide improvement in human sperm cryopreservation, as human sperm are less sensitive to cryodamage than are mouse sperm.