Determination of the effects of caffeine C1, 3. 7- trimethylxanthine in Tris-based egg yolk extender and different incubation intervals on the quality of cryopreserved bull semen

Abstract

Cryopreservation is a technique used to maintain the structure and function of cells by halting their metabolism and enzymatic activity at cryogenic temperatures. Cryopreservation induces structural and functional damage to bovine spermatozoa, resulting in reduced post-thaw fertility potential. Caffeine's ability to modify intracellular signaling pathways and mitigate the adverse effects of cryopreservation stress on spermatozoa makes it a promising supplement for improving the success rate of artificial insemination. This study evaluated the effects of caffeine supplementation (0, 3, 5, and 8 mM), post-thaw incubation time (0-4 hours), and their interaction on quality parameters of cryopreserved bull semen. Semen samples were assessed for total motility, progressive motility, non-progressive motility, non-motile sperm cells, viability, acrosome integrity, and sperm cell morphological abnormalities using standard microscopic techniques. Semen for this study was collected using an electro-ejaculator and analysed using a computer-aided sperm cell analyser. Data were analysed using a general linear model ANOVA in Minitab statistical software (Version 21.1.0). Incubation time significantly affected all evaluated parameters (P < 0.01). Caffeine significantly improved progressive motility (P < 0.05) and acrosome integrity (P < 0.01). The 3 mM caffeine treatment consistently produced the highest total and progressive motility throughout the incubation period. The results showed a decline in sperm velocity parameters (VCL, VSL, and VAP) over incubation time. In comparison, higher concentrations (5 and 8 mM) resulted in improved acrosome integrity but a faster decline in motility and an increase in tail abnormalities. Viability declined progressively in all treatments after 2 hours of incubation. These findings demonstrate that a concentration of 3 mM caffeine effectively enhances the functional performance of sperm cells, while excessive caffeine exerts cytotoxic effects. Future research is recommended to investigate whether the observed improvements result in enhanced in vivo fertility outcomes, including conception and calving rates.