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Combination medium of cryoprotective agents containing l-glutamine and methyl-β-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6J mouse sperm

Recently, a vast number of genetically-engineered mice have been created in various laboratories worldwide, all of which need to be effectively archived. The cryopreservation of mouse sperm provides a simple and economical means of storing the mice in mouse resource facilities. The current protocol for sperm cryopreservation using 18% raffinose pentahydrate and 3% skim milk (R18S3) has been adopted in most laboratories. In general, we can attain relatively high fertilization rates for frozen/thawed sperm in many inbred and F1 hybrid strains. However, the sperm of C57BL/6J mice shows an extremely low fertility rate after freezing and thawing (0–20%). In this study, we attempted to improve the low fertility of frozen/thawed C57BL/6J mouse sperm. Our results showed that a combination of R18S3 containing l-glutamine and methyl-β-cyclodextrin (MBCD) in a preincubation medium dramatically increased the rate of fertilization (69.2 ± 12.2%). Furthermore, the developmental potencies of two-cell embryos produced by frozen/thawed sperm to live young were normal (fresh: 46.0 ± 8.2%, frozen/thawed: 51.5 ± 11.1%). In summary, we conclude that a new method of sperm cryopreservation and in vitro fertilization using modified R18S3 with l-glutamine and MBCD in a preincubation medium yields a high fertilization rate for frozen/thawed C57BL/6J strain sperm. Furthermore, the new method provides a reliable archiving and reproducing system for genetically-engineered mice using sperm cryopreservation.