Effects of vitamin D supplementation in extender on sperm kinematics and apoptosis following the freeze-thaw process in normozoospermic and asthenozoospermic Holstein bulls

Background: Asthenozoospermia is a usual male infertility factor, characterized by decreased semen quality. It has been revealed that antioxidants improve sperm function, enhance endogenous antioxidant activities, and protect spermatozoa against oxidative damage during cryopreservation. This aimed to evaluate the effects of vitamin D on sperm kinematics and apoptosis in the semen of bulls with normozoospermia and asthenozoospermia after the freeze-thaw process. For this purpose, 32 semen samples of four Holstein bulls (normozoospermic, progressive motility > 70 %) and 32 semen samples of four bull (asthenozoospermic progressive motility < 40 %) were collected and pooled separately (normozoospermic and asthenozoospermic). Samples were then diluted into four equal aliquots of extender containing different vitamin D concentrations (0, 5, 10, and 50 ng/mL) and aspirated into a 0.5 mL straw. Results: The percentages of sperm progressive motility and viability were significantly higher (P < 0.05) in 50 ng/mL of vitamin D in normozoospermic group. Sperm kinematics parameters including curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP) were significantly higher in the high dose (50 ng/mL) vitamin D-treated group compared to the low dose vitamin D-treated group (5ng/mL) in normozoospermic bull semen samples. The supplementation of the semen extender with different concentrations of vitamin D could not increase the rate of acrosome integrity in normozoospermic bulls compared to the control group (P < 0.05). In the asthenozoospermic group, 10 ng/mL vitamin D-treated group could increase the rate of plasma membrane integrity compared to 5 ng/mL vitamin D-treated group (P < 0.05). The percentages of early-apoptosis (P = 0.049) and late-apoptosis (P = 0.005) were significantly higher in the asthenozoospermic than the normozoospermic group. Conclusions: The present study revealed that a high dose (50 ng/mL) of vitamin D protected normozoospermic bulls' sperms from the freezing procedure and lead to higher quality of frozen-thawed bull sperm.