ObjectiveTo optimize the technical parameters of mouse embryo transfer (including oviduct opening method, pseudopregnant recipient body weight, number of embryos transferred per recipient, and unilateral vs. bilateral oviduct transfer), improve the efficiency of embryo transfer (the pregnancy rate of recipient female mice after embryo transfer is used as the evaluation index), and improve the utilization rate of high-quality embryo germplasm resources (the birth rate of mice after embryo transfer is used as the evaluation index). Methods560 SPF-grade ICR mice and 440 C57BL/6J mice were used as experimental animals. 2-cell embryos were obtained through in vitro fertilization. A single-factor controlled variable method was used to systematically investigate the effects of four factors, including oviduct opening method, pseudopregnant recipient body weight, number of embryos transferred per recipient, and unilateral vs. bilateral oviduct transfer, on the pregnancy rate and birth rate of mouse embryo transfer. ResultsThe pregnancy rate of recipients with an opening made in the oviduct using a 26G syringe needle reached 90%, which was significantly higher than the 43% in the group with oviduct openings cut by Vannas scissors (P<0.05). Pseudopregnant recipients weighing 29-35 g exhibited optimal embryo transfer outcomes, with a pregnancy rate of 90% and a birth rate of 44%, and no pups were crushed by the recipient mothers. The highest birth rate of 42% was observed in the group receiving 16-25 embryos per recipient. Transferring 26-35 embryos per recipient achieved a 90% pregnancy rate and maximized embryo utilization efficiency. However, transfer efficiency decreased significantly when more than 35 embryos were transferred. Transfer of 24-26 embryos per recipient balanced relatively high pregnancy and birth rates. Bilateral oviduct transfer of mouse embryos yielded slightly higher pregnancy and birth rates than unilateral transfer, but the difference was not statistically significant (P>0.05). ConclusionSelecting pseudopregnant recipient female mice weighing 29-35 g, using a 26G syringe needle to make an opening in the oviduct, and controlling the number of transferred embryos at 24-26 per recipient can effectively improve the success rate of embryo transfer and achieve efficient utilization of high-quality embryo germplasm resources.