Abstract
Background: The cryopreservation of bovine semen is associated with several challenges, including cellular stress, cold shock, and the potential toxicity of cryoprotective agents. These limitations highlight the need for improved cryopreservation strategies that enhance sperm survival after thawing. Aim: The present study investigated the effect of different combinations of glycerol and dimethylformamide (DMF), supplemented with either chicken egg yolk (CEY), or quail egg yolk (QEY) on the post-thaw quality of bull spermatozoa. Methods: Semen samples were diluted in a Tris-based extender containing either CEY or QEY combined with different cryoprotectant treatments: 7% glycerol, 5% DMF, or a combination of 4% glycerol and 2% DMF. After gradual cooling to 5°C and equilibration for 2 hours, the semen was packaged into mini-straws and cryopreserved in liquid nitrogen. Post-thaw sperm quality was assessed by evaluating progressive motility, viability, acrosomal integrity, plasma membrane functionality using the hypo-osmotic swelling test, mitochondrial activity, and DNA fragmentation. Results: The results demonstrated that the extender containing a combination of 4% glycerol and 2% DMF, supplemented with QEY, significantly improved the freezability of bull spermatozoa compared with other treatments. Furthermore, the combination of glycerol 4% and DMF 2% with either QEY or CEY increased the proportion of spermatozoa with intact DNA following thawing. Conclusion: The combined use of DMF and glycerol, particularly when supplemented with QEY, provides enhanced protection to bovine spermatozoa during cryopreservation and may represent a promising alternative strategy for improving post-thaw semen quality.