Development of a quality assessment method for early-stage embryos derived from freeze₋dried spermatozoa.

Freeze-dried (FD) sperm preservation enables safe, long-term storage, even during disasters, but its use is limited by lower offspring rates than with fresh sperm. In this study, we aimed to improve birth outcomes by identifying high-quality embryos early and selectively transferring competent ones. Using live-cell imaging, we compared the average developmental kinetics between embryos derived from FD and fresh sperm and identified the greatest delay at pronuclear fusion. Embryos were therefore categorized at this stage as Prefusion, During fusion, or Postfusion. Analyses of chromosomal segregation errors and developmental potential revealed that Postfusion embryos consistently demonstrated significantly improved outcomes across all mouse strains, with birth rates approximately fourfold higher than those of prefusion embryos. Because pronuclear fusion can be easily assessed by bright-field microscopy, this simple classification provides a practical criterion for selecting embryos and could markedly enhance the efficiency and broader application of FD sperm preservation.