377 SPERMATOZOA RETRIEVED FROM MALE MICE FROZEN FOR 15 YEARS CAN PRODUCE NORMAL OFFSPRING

Abstract

Cryopreservation of male germ cells is a strategy to conserve animal species and strains of animals valuable to biomedical research. However, to minimize damage that may occur during freezing and thawing, complex cryopreservation protocols that have been optimized for the stage and species of male germ cells are usually employed. Recently, we have found that mouse male germ cells can be cryopreserved at -80�C by freezing the whole epididymides and testes without cryoprotectant for at least one year (Ogonuki et al. 2006 Reprod. Fertil. Dev. 18, 286 abst). This study was undertaken to determine whether mouse male germ cells retrieved from the bodies of mice frozen at -20�C for 15 years could produce normal offspring by microinsemination. Mature males of BALB/c-nude and C3H/He (8 weeks of age) were euthanized by overdose of pentobarbital on February 20 and March 8, 1991, respectively, and kept in a -20�C freezer. The frozen body was thawed about 15 years after freezing (February 2006) by putting it in a water bath until the outer surface of the body was softened. The body was then removed from the water, and the testes were isolated through an abdominal incision. Testicular spermatozoa were collected from the testes and microinseminated into B6D2F1 oocytes. Within 24 h after sperm injection, over 80% of oocytes developed into 2-cell embryos. Apparently normal pups were born after embryo transfer in both strains of mice at rates of 21% (17/81) and 12% (12/97) per transfer, respectively. Two pups from the BALB/c-nude group died shortly after Caesarian section due to respiratory failure, but others grew normally and were proven to be fertile when they matured (at least 19 mice out of 20 mice tested). We further mated these F1 offspring and confirmed that the nude gene was safely propagated. The present study demonstrates that spermatozoa can retain their fertilizing ability in frozen whole bodies for longer than we anticipated. If spermatozoa of extinct mammalian species (e.g. woolly mammoth) can be retrieved from animal bodies that were kept frozen in permanent frost, live animals might be restored by injecting them into oocytes from females of closely related species.