Enhancing cryopreservation performance in semen quality of Bali bulls (Bos sondaicus) using tris-nano-liposome...

Objectives: Post-thaw semen quality in Bali bulls is largely determined by the extender used during cryopreservation. Conventional tris egg yolk-based extenders (TEY) are widely used in bovine semen freezing; however, their effectiveness may be limited by variability in biochemical composition and potential biosecurity concerns. Tris-nano-liposome-based extenders (TNL) have recently emerged as a promising alternative due to their membrane-stabilizing properties and improved cryoprotective efficiency. This study aimed to evaluate the effectiveness of TEY and TNL extenders in maintaining post-thaw semen quality in Bali bulls. Materials and Methods: Semen samples from nine Bali bulls were assessed for macroscopic characteristics, microscopic parameters, and kinematic traits using a computer-assisted sperm analysis (CASA) system. Correlation analysis, principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA) were performed to explore parameter relationships and distinguish responses between extenders. Results: Both extenders maintained satisfactory post-thaw sperm quality, with no significant differences (p > 0.05) observed in total motility, viability, membrane integrity, acrosome integrity, and DNA fragmentation. The kinematic parameters remained within optimal ranges, indicating preserved progressive and curvilinear movement patterns. Multivariate analyses identified motility and membrane integrity as the primary contributors to variation in semen quality. Furthermore, the TNL extender produced more stable and homogeneous responses than TEY. Conclusions: The TNL extender demonstrated cryoprotective performance comparable to that of the conventional TEY extender while offering advantages in clarity, biosecurity, and compositional consistency. These findings indicate that TNL represents a promising alternative extender for bovine semen cryopreservation.