Concentration-dependent effects of caffeine (1,3,7-trimethylxanthine) in Tris-based egg yolk extender on the post-thaw quality of cryopreserved bull semen

Cryopreservation-induced damage to bull spermatozoa remains a limiting factor in artificial insemination programs. Caffeine (1,3,7-trimethylxanthine), a phosphodiesterase inhibitor, has attracted attention as a semen extender additive. However, dose-dependent effects in Tris-based egg yolk extenders at varying incubation times are poorly characterized for cattle. The study evaluated the effects of caffeine supplementation concentrations (0, 3, 5, and 8 mM) in a Tris-based egg yolk extender and post-thaw incubation time (0, 1, 2, 3, and 4 hours) on the quality of cryopreserved Nguni bull semen. Eighteen ejaculates were collected from three Nguni bulls using an electro-ejaculator and cryopreserved in Tris-based egg yolk extender at four caffeine concentrations. Post-thaw samples were incubated at 37 °C with 5% CO 2 and assessed at 0, 1, 2, 3, and 4 h for motility (CASA), morphology, viability, and acrosome integrity. The data obtained were subsequently analyzed using a general linear model ANOVA with Tukey’s post hoc test. Caffeine at 3 mM produced the highest total and progressive motility across all incubation times, while 5 and 8 mM improved acrosome integrity but increased tail abnormalities and accelerated motility decline. Viability was not significantly influenced by caffeine concentration. Prolonging the incubation time significantly reduced all semen quality parameters (P < 0.01). A concentration of 3 mM caffeine per 20 mL of Tris-based egg yolk extender at an incubation time of 0–1 h yielded the best results with respect to total motility, progressive motility, and straight-line velocity. Higher concentrations (5 and 8 mM) improved acrosome integrity but compromised motility and morphological integrity during extended incubation.