In vitro fertilization (IVF) of eggs by frozen and thawed C57BL/6J mouse sperm is inhibited by dead sperm and enhanced by preincubation of the sperm in calcium-free medium. In other species, the presence of sperm killed by freezing and thawing has been associated with the generation of hydrogen peroxide.

The proportion of eggs fertilized by cryopreserved C57BL/6J mouse sperm was increased significantly by increasing the volume of fertilization medium in which sperm and eggs were coincubated. Enhanced fertilization occurred even though the concentration of potentially fertile sperm was decreased fivefold. This suggested that if a putative soluble factor was inhibiting fertilization, dilution of that factor, but not the sperm, should increase the fertilization rate. This was achieved by coincubation of the gametes in cell culture inserts (Transwells^®) that during incubation were transferred progressively to wells containing fresh fertilization medium. Fertilization rates using inserts were high (66.6±2.4% versus 27.3%±2.8% in wells alone). On the assumption that the soluble factor could be H₂O₂, reduced glutathione was added to the fertilization medium. This enhanced fertilization rate significantly (76.6%±2.0% versus 21.2%±1.9%), while addition of oxidized glutathione did not (82.7%±6.5% with reduced glutathione; 44.5±8.8% with oxidized glutathione; 47.8%±12.1% with no glutathione). Positive effects of reduced glutathione on IVF were also seen with frozen 129S1, FVB, and C3H sperm, and sperm from two lines of genetically modified C57BL/6J mice.

IVF in cell culture inserts and addition of glutathione to fertilization medium significantly increased the proportion of eggs fertilized by cryopreserved mouse sperm from four inbred strains, suggesting that reactive oxygen species generated during fertilization inhibit fertilization. The modified IVF techniques developed here enhance the feasibility and efficiency of using cryopreserved sperm from genetically modified lines of inbred mice.