ENO1 as an Age-related Molecular Biomarker of Post-thawed Semen Quality in Bali Bulls (Bos sondaicus)

Artificial insemination (AI) efficiency in cattle is highly dependent on the quality of frozen semen, particularly sperm motility. Bull age is an important biological factor influencing semen quality, yet its molecular implications remain insufficiently understood, especially in indigenous breeds such as Bali cattle (Bos sondaicus). This study aimed to evaluate the effect of bull age on post-thawed semen quality and enolase 1 (ENO1) gene expression, and to determine the relationship between ENO1 expression and sperm motility in Bali bulls. Frozen semen samples were obtained from clinically healthy Bali bulls and classified into two age groups: 11 years. Post-thawed semen quality (abnormalities, acrosome integrity, and DNA fragmentation) was assessed by cell staining methods. ENO1 gene expression was quantified using qRT-PCR. The results demonstrated that total and progressive motility, acrosome integrity, and ENO1 expression were significantly higher in bulls aged 11 years (p<0.05), while non-motile sperm and secondary abnormalities were increased in older bulls. No significant differences were observed in sperm kinetic parameters or DFI between age groups (P>0.05). ENO1 expression showed positive correlations with total motility (r = 0.943, P<0.01), progressive motility (r = 0.943, P<0.01), and acrosome integrity (r = 0.886, P<0.05), and a negative correlation with DFI (r = −0.829, P<0.05). These findings showed that age-related decline in post-thawed sperm motility is associated with reduced ENO1 expression, reflecting impaired glycolytic energy metabolism in older bulls. In conclusion, ENO1 represents a promising age-related molecular biomarker for cryotolerance semen and fertility assessment in Bali bulls.