Effect of caffeine on functions of cooling-stored ram sperm in vitro

Caffeine is a well-known sperm motility stimulator, however, its effects on cooling-stored ram semen are unknown. The aim of the study was to examine the effect of caffeine on selected motility and viability indices of cooling-stored ram spermatozoa. Sperm ejaculates from 4 rams were diluted (1:3) in a Triladyl extender. Samples were stored for 96 h at 4-5 °C in two sets. In the first set used for motility analysis, caffeine at concentrations of 1, 2 or 4 mmol·l-1 was added to sperm aliquots on the day of analysis. In the second set used for viability assay, caffeine at the same concentrations (1, 2 or 4 mmol·l-1) was added at the beginning of storage. Control was left without caffeine addition. Sperm motility was analyzed at 0, 24, 48 and 72 h of cooling-storage. Viability assays were done after 72–96 h of cooling-storage. Caffeine significantly (P < 0.05) increased sperm motility and progressive movement and maintained this value for 72 h. Caffeine at the dose of 2 mmol·l-1 and 4 mmol·l-1 significantly (P < 0.05) reduced the proportion of dead/necrotic sperm detected by propidium iodide and proportion of apoptotic sperm detected by Yo-Pro-1, respectively. No effect of caffeine on plasma membrane integrity was noted. Proportion of sperm with membrane destabilization (annexin V-Fluos) was reduced by caffeine given at 1 and 4 mmol·l-1 compared to control. Our study for the first time demonstrates that caffeine maintains motility and viability of cooling-stored ram sperm for longer time compared to control. Methylxanthines, membrane integrity, membrane stability, apoptosis, motility Progress in the use of artificial insemination is related to search for substances with the potential ability to improve the fertilizing capacity of spermatozoa. Caffeine is a cyclic nucleotide phosphodiesterase inhibitor which stimulates sperm motility, fructolysis, and respiration and also increases cyclic adenosine monophosphate (cAMP) titres (Fraser and Monks 1990). Positive correlation between the intracellular concentration of cAMP and the rates of progressive motility, fructose utilization, and protein synthesis by ram spermatozoa was observed (Salem et al. 1992). The effects of caffeine are concentrationdependent and species-specific. Studies on bulls revealed that 4 mmol·l-1 of caffeine is an ideal concentration for semen fortification to improve the preservability and postthaw sperm motility (Singh and Raina 2000). At higher concentrations (10 mmol·l-1) caffeine induces an increase in intracellular calcium and the immediate hyperactivation of incubated ram sperm (Colas et al. 2010). Compared to fresh semen, cooled semen is more predisposed to decrease in sperm motility and higher incidence of morphological alterations, which may reduce sperm fertility and increase embryonic loses (Aisen et al. 2002; Gil et al. 2003). The data on caffeine effect on frozen-thawed semen are available; however, there are no reports on the effects of caffeine on cooling-stored ram semen evaluated in relation to indices of sperm viability and motility. The aim of the study was to examine effect of caffeine on selected variables of coolingstored ram semen. Sperm motility (CASA) and selected characteristic of sperm viability (sperm membrane integrity and membrane stability, sperm apoptosis) following liquid storage of sperm under hypothermic conditions were evaluated. ACTA VET. BRNO 2014, 83: 019–025; doi:10.2754/avb201483010019 Address for correspondence: Eliška Špaleková Animal Production Research Centre Nitra Hlohovecká 2, 951 41 Lužianky, Slovak Republic Phone: +421 376 546 196 E-mail: [email protected] http://actavet.vfu.cz/ Materials and Methods