Birth of live mice resulting from oocytes fertilized in vitro with cryopreserved spermatozoa

The objective of this study was to develop a reliable method to cryopreserve mouse spermatozoa, using in vitro fertilization (IVF) of oocytes, development of resultant zygotes into blastocysts, and the birth of live young after embryo transfer as criteria of sperm survival. Epididymal sperm were frozen in a raffinose-glycerol solution containing either egg yolk (EY) or BSA. Sperm frozen in the presence of EY yielded higher survivals than those frozen in the presence of BSA; when used for IVF, the former resulted in more blastocysts (26% vs. 5%). Comparison between one-step and step-wise dilution of the cryoprotectant revealed no differences in sperm viability as assayed by a vital stain. Nevertheless, IVF using frozen sperm recovered by one-step or stepwise dilution resulted in 33% and 17% blastocyst formation, respectively. When embryos produced with cryopreserved sperm were transferred into recipients, pregnancies (12 of 15 recipients) and the birth of live young (57 pups from 167 embryos transferred) resulted. These results were comparable to those obtained from transferral of embryos produced by IVF with fresh sperm (17 pregnancies from 19 recipients, 87 live young from 197 embryos). Thus, this study has resulted in the development of a simple and efficient cryopreservation procedure for mouse sperm.