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Journal of Andrology, Vol 22, Issue 2 339-344, Copyright © 2001 by The American Society of Andrology

JOURNAL ARTICLE

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

K. A. Thompson, J. Richa, S. A. Liebhaber and B. T. Storey
Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical Center, Philadelphia 19104-6142, USA.

Mouse sperm cryopreservation provides a means for storing the genetic information 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 past. 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 osmotic stress on the cells. Sperm suspensions were frozen in 0.25 mL straws and stored in liquid N2. Eggs were obtained from B6SJLF1/J superovulated females. For in vitro fertilization (IVF), 15-25 microL of sperm suspension post-thaw from one straw was added directly to each of three 1.5 mL drops of fertilization 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 live pups was 44 per 100 eggs inseminated (44%). This yield should be satisfactory for maintaining a mouse strain through sperm cryostorage, with restart of the strain through IVF and embryo transfer. The method should also provide improvement in human sperm cryopreservation, as human sperm are less sensitive to cryodamage than are mouse sperm.


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