• Proteomics reveal glycolysis-biased Y sperm and OXPHOS-biased X sperm in boars. • Metabolic polarized diluent enriches Y sperm from 50.4% to 74.3% with swim up step. • Enriched semen preserves motility and oxidative stability during 17°C storage. • Artificial insemination with enriched semen yield 69% male piglets. Sex sorting is a crucial agricultural technology in livestock production, enhancing productivity, reducing costs, and accelerating genetic progress. However, an efficient and non-damaging method for Y sperm sorting in swine production is still lacking. This study aimed to develop a physiology-based method for sex sorting in pigs by exploiting metabolic differences between X- and Y-bearing spermatozoa. Quantitative proteomic profiling of highly purified sperm populations revealed distinct metabolic patterns, with Y sperm enriched in glycolytic enzymes and X sperm showing higher abundance of oxidative phosphorylation (OXPHOS)–related proteins. Based on these findings, a metabolically polarized diluent (MPD) was formulated to enhance glycolysis-dependent motility in Y sperm while reducing OXPHOS in X sperm and combined with an upstream selection step. MPD treatment increased the proportion of Y-bearing sperm from 50.4% to 74.3% without compromising total motility, viability, acrosome integrity, or plasma membrane function. During 17 °C storage for up to 7 days, MPD-sorted semen exhibited comparable kinematic parameters, structural integrity, and redox stability (ROS, MDA, T-AOC, SOD, GPX, CAT) to unsorted controls. Artificial insemination using MPD-enriched semen produced a male-biased sex ratio of 69%, with no adverse effects on pregnancy rate, litter size, neonatal survival, or birth weight. These results demonstrate that metabolic polarization enables effective Y-sperm enrichment while maintaining sperm quality, providing a cost-efficient alternative to flow cytometry sexing approaches in swine reproduction.
Cao et al. (Wed,) studied this question.