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

Abstract

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.