Effect of Extender Supplementation With Caffeine on Arabian Stallion’s Semen Quality After Freezing

Abstract

In the current study, we aimed to investigate the effect of adding different levels of caffeine to the diluent on the Arabian stallion’s sperm quality post cryopreservation. Semen cryopreservation is an essential part of artificial insemination in Arabian horses. In most of cases, the stallion selection is based on desirable phenotypic and physical traits rather than on fertility. This approach may have resulted in the propagation of low-fertility genotypes that are unable to withstand the various stresses associated with cryopreservation such as freezing-thawing stress, oxidative stress, and osmotic stress. Caffeine has been shown to act as a phosphodiesterase inhibitor which may increase the level of cAMP in sperm and subsequently, increase the formation of adenosine triphosphate, which is important for sperm motility. Because it contains xanthine and theobromine, caffeine has antioxidant properties that increase sperm motility and their fertilizability. Five healthy stallions aged 4 to 10 years have been used for semen collection. Semen collections has been done twice weekly by artificial vagina. The ejaculates were evaluated for general progressive motility and sperm concentration. Sperm concentration and motility were determined using the CASA system (ISAS program, Prosser R+D, Paterna, Valencia, Spain). Samples with a minimum concentration of 200 × 106 sperm/ml and motility > 60% used for this study. The filtered semen of each ejaculate was diluted (1:1) and divided into 4 aliquots. The aliquots were centrifuged at 800 g for 10 minutes, the seminal plasma was removed, and each sample was re-suspended with FH-20 (0) without any supplementation as a control or with caffeine at a concentration of 0.5 mM, 1 mM, 3 mM or 5 mM. The final semen concentration after dilution was 200 × 106 sperm/ml. All samples were assessed before freezing in liquid nitrogen. The frozen thawed semen was assessed for general and progressive motility and then evaluated using the ISAS program and assessed for plasma membrane integrity, morphology defects, acrosome integrity, and viability. Descriptive analyses were determined for the evaluated variables. Statistical comparisons between groups were performed with one-way analysis of variance (ANOVA) (p < 0.05). The addition of 1 mM caffeine to the extender gave better results in total motility (TM), progressive motility (PM) and vitality. However, all studied caffeine concentrations had good effects on straight line velocity (VSL), straightness (STR), linearity (LIN), wobble (WOB) and HOST with no effects on acrosome integrity. It could be concluded that caffeine has a beneficial effect on Arabian stallion semen cryopreservation when added at a concentration of 1 mM to the extender.