Abstract
<title>Abstract</title> <p>This study re-evaluated the effectiveness of Percoll density gradient centrifugation (PDGC) for separating X- and Y-chromosome-bearing spermatozoa in Holstein–Friesian (HF) cattle. Although PDGC has long been used in Indonesia, previous evaluations mainly relied on sperm morphometry, which assumes size differences between X- and Y-bearing spermatozoa. Non-sexed (NS) semen and PDGC-derived putative X- and Y-enriched fractions (SX and SY) were produced from contemporaneous ejaculates collected from two HF bulls, with eight ejaculates per bull. Post-thaw sperm quality was evaluated using computer-assisted semen analysis (CASA), chlortetracycline (CTC) staining, the Halomax assay, imaging flow cytometry, and real-time PCR (RT-qPCR) targeting X- and Y-linked markers. A field trial involving 1,649 HF cattle inseminated with NS or SX semen was conducted to assess pregnancy rate (PR), calving rate (CvR), and female calf proportion. PDGC processing reduced progressive motility and increased immotile spermatozoa compared with NS semen. In the absence of a sham-processed control, these effects likely reflected the PDGC workflow rather than chromosomal enrichment. Capacitation status and sperm DNA fragmentation were not significantly affected by treatment, whereas acrosome reaction showed a significant overall treatment effect. Imaging flow cytometry detected statistically significant but biologically small X:Y shifts, whereas RT-qPCR showed only modest, non-significant differences; both results remained close to the physiological 50:50 ratio. Field results showed no significant differences in PR or female calf proportion between NS and SX semen, although CvR was lower in the SX group. The evaluated PDGC protocol did not achieve meaningful chromosomal enrichment or enhance female calf production.</p>