Combination medium of cryoprotective agents containing L-glutamine and methyl-{beta}-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-beta-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.
Takeo
T
T
Center for Animal Resources and Development, Kumamoto University, Japan.
Nakagata
N
N
D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D003470:Culture Media / Q000737:chemistry*; D005307:Fertilization in Vitro / Q000187:drug effects* / Q000379:methods*; D005973:Glutamine / Q000494:pharmacology; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects*; D047392:beta-Cyclodextrins / Q000494:pharmacology
D003451:Cryoprotective Agents; D003470:Culture Media; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D005973:Glutamine