LIVE MICE FROM CRYOPRESERVED EMBRYOS DERIVED IN-VITRO WITH CRYOPRESERVED EJACULATED SPERMATOZOA

This study was undertaken to try to reduce the number of animals requi red to maintain mouse strains by banking of embryos or spermatozoa. Th e principal objective was to cryopreserve ejaculated mouse spermatozoa , using a method recently developed fbr epididymal spermatozoa. Within 30 min after mating, ejaculated spermatozoa were flushed from the ute rus of mated females; shortly afterwards, epididymal spermatozoa were also collected from the same males that had mated with the females. Th e average values for spermatozoal motility and viability of ejaculated specimens of nine males were 43 and 46%, respectively, and for epidid ymal specimens, the corresponding values were 60 and 52%, In experimen t 1, ejaculated or epididymal spermatozoa were incubated with oocytes for 0.5 to 4 h, As evidenced by development into two-cell embryos with in 24 h, kinetics of fertilization of the two spermatozoa types were s imilar. In experiment 2, ejaculated and epididymal spermatozoa of thre e 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 in to liquid nitrogen for storage. When cryopreserved epididymal or ejacu lated 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 p roduced with cryopreserved spermatozoa were transferred into recipient s, 18 and 22 live pups were obtained from 62 and 54 embryos resulting from ejaculated or epididymal spermatozoa, respectively. This study do cumented the feasibility of cryopreserving ejaculated spermatozoa as a n effective alternative to preserving germ plasm from genetically valu able mice.