Assisted reproductive technologies (ARTs) in mice were recently advanced when two long-existing technical barriers were overcome. The first barrier was the limited number of mature oocytes after conventional superovulation, especially in inbred strains of mice. A combination of estrous cycle synchronization and antiinhibin serum treatments increased the number of collected oocytes from female mice by approximately 3–4 times in many strains. The second barrier was the low fertilization rate after in vitro fertilization (IVF) using frozen-thawed spermatozoa. The addition of reduced glutathione in the fertilization medium dramatically increased the IVF yields, even in cryopreserved/warmed spermatozoa from the C57BL/6J strain, which is one of the strains most sensitive to cryoinjury. This result encouraged the use of cryopreserved spermatozoa in mouse strains worldwide for the preservation and transportation of their genetic characteristics. The final yield to produce offspring from one female was increased from 9 to 30. In IVF with cryopreserved spermatozoa from the C57BL/6J strain, the final yield using these technological innovations was estimated to be ninefold higher than previously. Following this improvement, the efficiency of ARTs in mice was increased dramatically and the decrease in the number of euthanized animals contributes to animal welfare and reduces labor and expense.