Integrative functional and molecular characterization of Bali bull semen and its relationship with reproductive performance

Background and Aim: Bali cattle (Bos javanicus) are an important indigenous genetic resource in Indonesia, characterized by high adaptability and reproductive efficiency. However, conventional semen evaluation based on motility and morphology often fails to accurately predict bull fertility. Integrating functional sperm analysis with molecular quality indicators may provide a more reliable assessment of reproductive potential. This study aimed to perform an integrative evaluation of Bali bull semen by combining computer-assisted sperm analysis (CASA) parameters with molecular sperm quality indicators, including acrosome integrity, DNA integrity, and protamine status, and to determine their multivariate relationships using canonical correlation analysis (CCA). Materials and Methods: Frozen–thawed semen samples were collected from ten Bali bulls maintained at a Regional Artificial Insemination Center. Functional sperm parameters were evaluated using CASA, including total motility, progressive motility, velocity parameters (velocity average path, velocity curved line, and velocity straight line), linearity, straightness, wobble, amplitude of lateral head displacement, and beat cross frequency. Acrosome integrity was assessed using fluorescein isothiocyanate-conjugated peanut agglutinin staining, DNA integrity was evaluated using acridine orange staining, and protamine integrity was determined using chromomycin A3 staining. Data were analyzed using one-way analysis of variance followed by Tukey’s test, and multivariate relationships between functional and molecular parameters were examined using CCA. Results: CASA evaluation demonstrated inter-individual variation in semen quality, with total motility ranging from 49.80% to 60.48%. Acrosome integrity remained high across all bulls (85.03–95.59%), while DNA integrity (96.41%–98.55%) and protamine status (90.35%–97.04%) indicated well-preserved chromatin structure. The first canonical function showed a strong correlation between functional and molecular variables (r = 0.906), although the association was not statistically significant (p = 0.134). The canonical plot revealed a consistent positive trend, suggesting coordinated variation between sperm kinematics and chromatin integrity markers. Conclusion: The integrative evaluation of CASA-derived kinematic parameters with molecular sperm quality indicators provides a more comprehensive assessment of Bali bull fertility than conventional semen analysis alone. The strong but non-significant canonical correlation suggests a biologically plausible relationship between sperm motility and chromatin stability. This study provides preliminary evidence supporting the use of combined functional and molecular semen evaluation to improve bull selection, enhance artificial insemination efficiency, and support genetic conservation of Bali cattle. Further studies with larger sample sizes and fertility-validated outcomes are required to confirm these findings. Keywords: acrosome integrity, Bali cattle, canonical correlation analysis, chromatin integrity, computer-assisted sperm analysis, DNA integrity, protamine integrity, semen quality.