Potential sex-sorting of Hampshire boar sperm with ScFv-antibody-coated magnetic beads

The practical application of sex-sorted semen in pig production remains limited because conventional flow cytometric sorting requires specialized equipment and relatively low throughput. The H4:L4 single-chain variable fragment (scFv) is an antibody developed in a previous study that preferentially recognizes an epitope on the plasma membrane of Y-chromosome-bearing sperm from Duroc boars, thereby providing a knowledge base for evaluating its broader utility in other pig breeds. This study evaluated H4:L4 single-chain variable fragment (scFv)-coated magnetic microbeads as an immunomagnetic platform for boar sperm sex sorting. Antibody binding was assessed in different pig breeds, followed by evaluation of antibody concentration, sperm separation efficiency, sex-ratio enrichment, and post-sorting sperm quality. Breed-associated binding response in H4:L4 scFv were observed, with the strongest response in Duroc sperm and detectable binding in Landrace, Large White, Hampshire, and Native Black pigs. ELISA confirmed concentration-associated binding, and 4 mg/mL was selected as the optimal coating concentration because it achieved sex-ratio enrichment comparable to 8 mg/mL while reducing antibody use. Following immunomagnetic separation, the supernatant fraction was enriched with X-chromosome bearing sperm, whereas the eluted fraction was enriched with Y-chromosome-bearing sperm. Imaging flow cytometry showed 75.9-77.0% X chromosome-bearing sperm in the supernatant and 76.3-78.6% Y-bearing sperm in the eluted fraction. qPCR supported these findings, showing increased BMP15 expression in the supernatant and increased UTY expression in the eluted fraction. Sperm quality was preserved in the X-enriched fraction, with motility, membrane integrity, mitochondrial membrane potential, and oxidative status comparable to conventional semen, whereas reduced sperm quality was observed in the Y-enriched fraction. These findings provide in vitro proof of concept that H4:L4 scFv-coated magnetic microbeads can produce X- and Y-chromosome-enriched fractions from Hampshire boar semen. However, the study did not evaluate fertilization, artificial insemination, pregnancy, farrowing outcomes, or offspring sex ratio. Consequently, the observed sperm enrichment cannot be extrapolated directly to fertility or piglet sex ratio, and controlled biological validation is required before practical application can be considered.