Age-Associated Alterations in Sperm Quality and DNA Integrity in Cryopreserved Bali Bulls (Bos javanicus) Semen

Age-related reproductive changes in breeding bulls may influence semen quality and long-term efficiency of artificial insemination (AI) programs. However, molecular indicators of aging, particularly in indigenous tropical cattle such as Bali cattle (Bos javanicus), remain insufficiently characterized. This study aimed to evaluate age-associated alterations in post-thaw sperm kinematics, DNA integrity, protamine integrity, and testosterone concentrations in Bali bulls. Frozen semen from 10 bulls was categorized into two age groups (< 8 years and > 10 years; n = 5 per group). Sperm motility parameters were evaluated using computer-assisted sperm analysis (CASA), DNA integrity was assessed by sperm-bos-halomax, protamine integrity using chromomycin A3 staining, and testosterone concentrations were measured using ELISA. Total and progressive motility did not differ significantly between age < 8 years and > 10 years (p > 0.05). However, bulls > 10 years exhibited significantly higher VSL, DCL, and BCF values, while sperm DNA integrity was significantly reduced. Notably, DNA integrity was significantly reduced in older bulls (91.97 ± 1.32%) compared with younger bulls (97.82 ± 0.48%; p = 0.01), whereas protamine integrity and testosterone concentrations showed no significant age-related differences. Correlation analysis revealed a strong positive association between progressive motility and DNA integrity in younger bulls (r = 0.84), whereas in older bulls, progressive motility was strongly negatively correlated with testosterone concentration (r = -0.91). These findings indicate that reproductive aging in Bali bulls is primarily reflected by impaired genomic integrity rather than alterations in conventional semen quality or endocrine status. Although total motility, progressive motility, protamine integrity, and testosterone concentrations did not differ significantly between bulls aged < 8 years and > 10 years, sperm DNA integrity was significantly lower in bulls aged > 10 years than in bulls aged < 8 years. These findings suggest that sperm DNA integrity may serve as a more sensitive biomarker of reproductive aging than conventional semen characteristics and may improve the accuracy of breeding soundness evaluation in AI programs.