Immunomagnetic Sex-Sorting of Landrace Boar Semen Using a Porcine Y-Specific scFv Antibody: Efficiency and Post-Sorting Sperm Quality

This study evaluated the efficiency of an scFv antibody-based sex-sorting method using HL magnetic microbeads for the separation of X- and Y-chromosome-bearing sperm in Landrace boar semen, as well as its effects on sperm quality parameters. A dose-dependent plateau in H4L4 antibody coupling efficiency was observed, with 2 mg identified as the optimal concentration, consistently achieving approximately 50% sperm-binding efficiency across boars. Sex ratio analysis confirmed effective discrimination between X- and Y-bearing sperm, with the X-enriched fraction showing a high proportion of X-sperm (77–81%) and the Y-enriched fraction exhibiting high Y-sperm purity (>83%). CASA revealed a significant effect of the sex-sorting process on sperm kinematics (p < 0.001). X-enriched sperm maintained physiological motility, with total and progressive motility comparable to CON, whereas the Y-enriched fraction showed a marked decline in kinematic performance. Flow cytometric analysis demonstrated that membrane integrity remained high in CON and X-enriched semen (>81%), while a significant reduction in viability was observed in the Y-enriched fraction (50.85%; p < 0.001). Consistently, mitochondrial membrane potential analysis indicated pronounced physiological stress in Y-enriched sperm, with significantly reduced mitochondrial activity compared to CON and X-enriched fractions. No significant differences were detected among individual boars (p > 0.05). In conclusion, this study demonstrates that HL magnetic bead-based sperm sexing effectively separates X- and Y-sperm in chilled Landrace boar semen, while preserving the quality of X-enriched sperm.