Assessment of Percoll density gradient centrifugation for sperm sexing in Indonesian local bulls

This study provides a preliminary assessment of Percoll density gradient centrifugation (PDGC) in Indonesian local bulls by evaluating post-thaw sperm quality and the proportions of X- and Y-chromosome-bearing spermatozoa. Ninety frozen semen straws from six ejaculates of Bali (Bos javanicus), Madura (Bos indicus), and Peranakan Ongole (PO; Bos indicus) bulls were analyzed. Fresh semen was divided into a non-sexed (NS) group and an aliquot processed using PDGC to produce putative X-enriched (SX) and Y-enriched (SY) sperm fractions. After thawing, sperm motility, kinematics, capacitation status, acrosome reaction (AR), and sperm DNA fragmentation (SDF) were evaluated, together with the proportions of X- and Y-chromosome-bearing spermatozoa using imaging flow cytometry. Progressive motility (PM) showed the most consistent decline following PDGC, whereas other motility changes varied among bulls and fractions. Imaging flow cytometry revealed an approximately 50:50 X ratio across bulls and treatments. Although PDGC maintained low SDF, relatively low capacitation and AR levels, and post-thaw PM above 40%, it did not produce biologically meaningful X- or Y-sperm enrichment. These findings indicate that PDGC effectiveness may be influenced by biological and technical variability, warranting further standardization and validation to establish its reproducibility and practical applicability for sperm sexing.