Biological Differences in Sperm and the Future Potential of Animal Husbandry

Hanwoo is a native Korean cattle breed known for its highly marbled 1++ grade beef, forming a premium market with prices exceeding 100,000 KRW per kg. In Hanwoo production systems, females are essential for herd expansion and genetic improvement, whereas males mainly contribute to revenue through beef production. Accordingly, X and Y sperm separation technology enables sex ratio control at fertilization to optimize the production of females for breeding and males for beef, improving overall efficiency and profitability. This review examines the biological differences between X and Y sperm and separation technologies that exploit these differences, along with recent research trends. X and Y sperm differ in membrane protein composition, metabolic activity, and epigenetic characteristics, providing a basis for selective separation strategies. Through label-free proteomics and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, specific proteins such as CLRN3, ADAM2, and ATP11C in X sperm, and SCAMP1 in Y sperm, have been identified, proposed as targets for immunological separation or aptamer development. In addition, studies using the toll-like receptor 7/8 (TLR7/8) agonist R848 combined with the swim-up method have demonstrated selective regulation of X sperm motility. Whole-genome bisulfite sequencing (WGBS) has further identified 12,175 differentially methylated regions related to genes involved in energy metabolism and sperm function. Current sex selection technologies include flow cytometry, proteomics-based surface protein analysis, TLR7/8-mediated separation, aptamer-based enrichment, and WGBS-based methylation analysis. This review evaluates their principles and limitations and discusses future research directions applicable to Hanwoo and other livestock species such as pigs and sheep.