Toward Practical Embryo Genomic Selection in Cattle: An Optimized Workflow Enabling High-Accuracy Breeding Value...

The implementation of embryo genomic selection (EGS) is constrained by the lack of a standardized workflow that balances high genotyping accuracy with the preservation of embryo viability, particularly given embryonic heterogeneity. To address this, we systematically optimized the critical steps of trophectoderm biopsy and whole-genome amplification, establishing a diameter-stratified strategy that links embryo diameter to a biopsy area window designed to protect embryo recovery while ensuring sufficient DNA input for reliable genotyping. Our results demonstrated high concordance (> 98%) between embryo and calf genomic estimated breeding values (GEBVs), supporting the reliability of predictions. Crucially, we defined a diameter-based biopsy framework: for embryos < 150 μm, biopsy areas < 1000 μm² supported viability, whereas larger embryos (≥ 150 μm) tolerated up to 1500 μm², with a minimum threshold of 840 μm² guaranteeing > 90% genotype call rates. By preventing oversampling in small embryos and undersampling across embryo sizes, this diameter-guided control improves both embryo survival and GEBV prediction accuracy. This study provides a validated technical pathway for EGS that addresses the major practical bottleneck in livestock breeding. By ensuring reliable selection without compromising developmental potential, our work facilitates the rapid introgression of superior genetics and marks a significant step toward making EGS scalable, accelerating genetic progress in cattle and other species.