Efficient bovine sex selection via Toll-like receptors 7/8 agonist 24e through the GSK3α/β-hexokinase pathway

Sex selection is pivotal in animal husbandry and reproductive biotechnology, critical for preferential production of male beef cattle and female dairy herds. Current bovine sex selection via flow cytometry is limited by high costs, low efficiency, and sperm motility damage. This study revealed X sperm-specific Toll-like receptors 7/8 (TLR7/8) expression. Pharmacological activation by 24e induced GSK3α/β and NF-κB phosphorylation in X sperm, causing reduced motility, mitochondrial dysfunction, and ATP depletion. Optimized conditions (0.27 μM 24e, 60 min in HTF medium, 6 × 10⁷ sperm/mL) achieved effective separation: upper layer contained 84.07 % ± 0.95 % Y sperm, lower layer contained 82.80 % ± 1.31 % X sperm, without compromising sperm viability or function. IVF trials demonstrated precise sex ratio control, yielding 77.0 % ± 2.08 % male embryos from Y sperm and 85.32 % ± 1.45 % female embryos from X sperm. Mechanistically, 24e suppressed X sperm motility via the GSK3α/β-NF-κB/hexokinase axis. These findings suggest that 24e can effectively achieve sperm sex sorting by inhibiting the motility of X sperm.