Live mice from cryopreserved embryos derived in vitro with cryopreserved ejaculated spermatozoa

This study was undertaken to try to reduce the number of animals required to maintain mouse strains by banking of embryos or spermatozoa. The principal objective was to cryopreserve ejaculated mouse spermatozoa, using a method recently developed for epididymal spermatozoa. Within 30 min after mating, ejaculated spermatozoa were flushed from the uterus of mated females; shortly afterwards, epididymal spermatozoa were also collected from the same males that had mated with the females. The average values for spermatozoal motility and viability of ejaculated specimens of nine males were 43 and 46%, respectively, and for epididymal specimens, the corresponding values were 60 and 52%. In experiment 1, ejaculated or epididymal spermatozoa were incubated with oocytes for 0.5 to 4 h. As evidenced by development into two-cell embryos within 24 h, kinetics of fertilization of the two spermatozoa types were similar. In experiment 2, ejaculated and epididymal spermatozoa of three males were separately cryopreserved in medium containing raffinose, glycerol, and egg yolk. Samples were cooled and seeded at -4 degrees C, cooled to -70 degrees C at 20 degrees C/min, and then were placed into liquid nitrogen for storage. When cryopreserved epididymal or ejaculated spermatozoa were thawed at > 1,000 degrees C/min and used for in vitro fertilization, > 60% of oocytes cleaved, and approximately 95% of cleaved embryos developed into morulae or blastocysts. When embryos produced with cryopreserved spermatozoa were transferred into recipients, 18 and 22 live pups were obtained from 62 and 54 embryos resulting from ejaculated or epididymal spermatozoa, respectively. This study documented the feasibility of cryopreserving ejaculated spermatozoa as an effective alternative to preserving germ plasm from genetically valuable mice.