Glycerol is commonly used as a cryoprotectant in bovine sperm cryopreservation; however, its cytotoxic effects can negatively impact sperm quality. This study aimed to reduce glycerol concentration in semen extenders by supplementing with natural antioxidants (taxifolin and fetuin) and the disaccharide trehalose. Twelve ejaculates were collected from three bulls and initially evaluated, then diluted in a tris-egg yolk (TEY) extender containing either 7 % or 4 % glycerol. The control group received 7 % glycerol (TEY), while experimental groups included G7F (TEY + fetuin), G7T (TEY + taxifolin), G7M (TEY + fetuin + taxifolin), and 4 % glycerol groups supplemented with 50 mM trehalose: G4 (TEY + trehalose), G4F (TEY + fetuin), G4T (TEY + taxifolin), and G4M (TEY + fetuin + taxifolin). Post-thaw evaluations included assessments of sperm motility, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), DNA fragmentation, and oxidative stress markers. The G4F and G4T groups exhibited significantly higher total and progressive motility, viability, plasma membrane integrity, and MMP compared to other groups (p < 0.05). Furthermore, these groups showed significantly lower malondialdehyde (MDA) levels, indicating reduced lipid peroxidation and oxidative stress (p < 0.05). In conclusion, co-supplementation of 50 mM trehalose with 50 μM taxifolin and 5 mg/mL fetuin in a cryopreservation extender containing 4 % glycerol may enhance the post-thaw quality of bull spermatozoa.