Age-related differences in sperm chromatin packaging and protein profiles based on SDS-PAGE in Bali bulls (Bos javanicus).

Objectives: This study evaluated the effect of advancing age on sperm chromatin integrity and protamine status and also evaluated sperm protein profiles based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in Bali bulls (Bos javanicus). The study also examined the relationship between chromatin integrity parameters and sperm motility to provide insights into molecular determinants of sperm quality supporting sustainable cattle production and food security. Materials and Methods: Eight Bali bulls maintained at an artificial insemination center were divided into two groups: bulls younger than 10 years (n = 4) and bulls older than 10 years (n = 4). Sperm motility parameters were evaluated using computer-assisted sperm analysis (CASA). Chromatin integrity was assessed through acrosome integrity, DNA fragmentation, and protamine status using microscopic staining techniques, including chromomycin A3 (CMA3). Sperm protein profiles were analyzed using SDS-PAGE followed by densitometric analysis with ImageJ. Statistical analysis was performed using independent sample t-tests and Pearson correlation. Results: Bulls older than 10 years showed significantly higher progressive motility (p = 0.04) and protamine status (p = 0.03) than younger bulls, while acrosome integrity and DNA fragmentation were not significantly different. A strong positive relationship was observed between protamine status and progressive motility (R2 = 0.72, p = 0.007). SDS-PAGE revealed several protein bands, with the ~10-11 kDa band showing significantly higher intensity in older bulls (p = 0.007). Conclusions: Advancing age in Bali bulls was associated with improved protamine-mediated chromatin packaging and higher progressive sperm motility. Integrating chromatin integrity assessment with sperm protein profiling may support more effective bull selection strategies and contribute to improved cattle reproduction and food security.