Abstract: Objective To establish a high-efficiency sperm cryopreservation and in vitro fertilization (IVF) method on C57BL/6J background gene-modified mice. Methods This study compared the effects of different methods on sperm cryoprotectant, sperm concentration, treatment after thawing and IVF medium on 2-cell cleavage rate, and then used technologies developed to cryopreserve and recover sperm of knockout mouse lines on inbred C57BL/6J backgrounds. Results Sperm were frozen with R18S3, which containing 18% gossylose and 3% skimmed milk powder, then thawed for IVF in HTF medium and their fertility (two-cell cleavage) was 8.4%. Sperm were frozen with M-R18S3, a cryoprotective solution that containing 477 µmol/L monothioglycerol (MTG) in R18S3 and then IVF in HTF medium, their fertility (two-cell cleavage) was 20.6%. Sperm cryopreserved in M-R18S3, IVF in HTF with 1mmol/L reduced glutathione (GSH), have better IVF rate than when cryopreserved in R18S3 alone (43.5% vs 25%). High concentration sperm was cryopreserved in M-R18S3, then thawed for IVF in HTF medium with 1 mmol/L GSH and their fertility (two-cell cleavage) was higher than low concentration sperm (64.2% vs 43.5%). Sperm from 3 knockout and 1 transgenic mouse lines on C57BL/6J backgrounds cryopreserved using this techniques were all thawed successfully to obtain IVF fertility (two-cell cleavage) between 34% and 90%, and after embryo transplantation to get genotypically confirmed offspring. Conclusion High concentration sperm was frozen in M-R18S3, screened in IVF drop after thawing, and then IVF in HTF with 1 mmol/LGSH, can be used as an effective method for sperm preservation and recovery of C57BL/6J background genome-modified mice.

Key words: Sperm cryopreservation, In vitro fertilization (IVF), Monothioglycerol (MTG)