Cryopreservation of Mouse Spermatozoa

Abstract

A procedure to cryopreserve mouse spermatozoa has been derived to bank the genetics of valuable strains of mice in a practical way. The primary objective of this study was to apply the cryopreservation method developed for spermatozoa of strain B6D2F1 to those of strains 129/J and C57BL6/J. Using the capability of spermatozoa to fertilize oocytesin vitroas the criterion of survival, we found differences in survival after cryopreservation among the three strains. Blastocysts were obtained afterin vitrofertilization of oocytes with frozen spermatozoa from B6D2F1 (51%) and 129/J (12%); none was obtained from C57BL6/J. Transfer of embryos into recipients resulted in the birth of 69 live pups from 164 embryos produced with frozen B6D2F1 spermatozoa and 11 pups from 35 embryos produced with 129/J spermatozoa. To seek an explanation of these differences among the three strains, spermatozoa were exposed to anisotonic solutions ranging from 5 to 3200 mOsm; viability of spermatozoa was assessed by a double stain using flow cytometry. Mouse spermatozoa tolerated exposure to solutions of osmolalities between 200 and 400 mOsm, but were damaged when exposed to solutions exceeding this range. Spermatozoa from C57BL6/J were the most sensitive: 20, 35, and 40% of C57BL6/J, 129/J, and B6D2F1 spermatozoa survived exposure to an 800 mOsm solution, respectively. This study suggests that there is a genetic basis for sensitivity of mouse spermatozoa to osmotic shock and freezing injury. Nevertheless, the birth of live pups produced with frozen spermatozoa from 129/J as well as with spermatozoa from B6D2F1 mice indicates that cryopreservation of spermatozoa can be used to preserve the genetics of valuable strains of mice.