Establishment and in vitro validation of a sheep-specific, low-cost TLR7/8 agonist R848-mediated X/Y sperm separation system.

Sex control is crucial for maximizing the economic benefits of sheep husbandry: male lambs increase farm income by 30-40% due to superior growth and feed efficiency, while female lambs are essential for fine-wool production and population expansion. However, the only commercialized method (flow cytometry) has critical sheep-specific limitations, including 25-30% flow channel blockage, sperm DNA oxidative damage, and prohibitive cost for large-scale breeding. TLR7/8 agonist R848 has been validated for sperm separation in mice, cattle and goats, but no sheep-specific optimized system has been established due to unique biological characteristics of ovine sperm. In this study, we systematically optimized key parameters of R848-mediated sheep sperm separation, comprehensively evaluated its effects on sperm biological characteristics, and verified separation efficiency at the embryo level via IVF combined with nested PCR sex identification. The optimal R848 concentration was 1 µM: the upper layer was enriched with 90.30% ± 5.21% TLR8-negative Y sperm (validated by 80.20% ± 3.70% male embryos), and the lower layer was enriched with 80.50% ± 4.96% TLR8-positive X sperm (validated by 87.56% ± 9.51% female embryos). R848 specifically inhibited X-sperm motility by reducing ATP production (24.82% decrease, P = 0.006) and mitochondrial membrane potential (29.75% decrease, P = 0.023), without damaging acrosome or plasma membrane integrity. The optimal supporting conditions were determined as 1000 µL elution twice, Y-sperm storage at 17 °C for up to 120 h with stable in vitro performance, and X-sperm use within 24 h after sorting. This study provides a low-cost, sheep-specific sperm separation system with comprehensive in vitro validation, which has significant potential for application in precision sheep breeding after further in vivo validation.