12 GENERATION OF RAT OFFSPRING DERIVED FROM CRYOPRESERVED SPERMATOZOA IN JAPANESE NATIONAL BIORESOURCES

Abstract

The purpose of the National BioResource project is to facilitate the availability of genetically and phenotypically standardized rat strains for life sciences. The BioResource is available to scientists worldwide. To bank genetic resources efficiently in the rat, cryopreservation of both sperm and embryos is a very important technology. The objective of the present study was to confirm the ability of banked and transported rat spermatozoa to fertilize oocytes through intrauterine insemination and for the embryos to develop to term, with the ultimate aim of developing a system for banking rat genetic resources. The epidydimal spermatozoa from the KLM rat, whose body size is small because the Prkg2 gene is partially defective, were frozen with egg yolk medium supplemented with 0.7% Equex Stm (Nakatsukasa et al. 2001 Reproduction 122, 463–467) and banked in the Institute of Laboratory Animals, Kyoto University. The cryopreserved sperm in 0.25-mL straws were transported to the laboratory at Azabu University, Kanagawa. Two straws from different males were thawed in a 37°C water bath for 15 s. Thawed semen was diluted with 1.0 mL of mR1ECM (Miyoshi et al. 1997 Biol. Reprod. 56, 180–185) with 0.4% (w/v) bovine serum albumin (BSA, fraction V; Sigma-Aldrich Japan K.K., Tokyo, Japan) at 37°C and then incubated at 37°C in 5% CO₂ in humidified air until insemination. The percentage of motile spermatozoa was assessed visibly and determined by direct observation at 37°C under a light microscope at 100×. The thawed semen (50 µL, 3–4 × 10⁵ sperm cells) was then inseminated into the top of both uterine horns of recipient females that were mated with a vasectomized male. The post-thaw motility of frozen spermatozoa was 10%. Seven of 15 inseminated females became pregnant and 13 live pups were born. It is thought that the low number of pups born in spite of the relatively high pregnancy rate was caused by sperm damage during the freezing and thawing procedure. The results of the present study show that rat spermatozoa cryopreserved in the BioResource have the ability to revive genetic resources through intrauterine insemination.