Vitamin D3 Protects Ram Epididymal Sperm From Oxidative Stress and Improves 4°C Storage Quality

Abstract

Liquid storage of sperm increases oxidative stress (OS), reducing sperm quality. While vitamin D₃ (VD) has shown antioxidant and reproductive benefits in some species, limited studies have examined its effects on ram epididymal sperm, which are free from seminal plasma influences. This study explored how different doses of VD affect the epididymal sperm of Sanjabi rams under both normal and OS conditions and also during liquid storage at 4°C. Two experiments were conducted. The first tested three concentrations of VD (1.25, 6.25, and 31.25 µg/mL) under normal and OS conditions, analyzing antioxidant status by measuring total antioxidant capacity (TAC), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) levels. The second experiment involved adding the same VD concentrations to extenders used for storing sperm at 4°C, with evaluations of sperm motility, viability, membrane integrity, DNA integrity, and morphological defects at 24, 48, and 72 h. VD inclusion (particularly at 1.25 and 6.25 µg/mL) was associated with increased TAC and SOD activities (p < 0.05). These concentrations also reduced MDA levels (p < 0.05), reflecting decreased OS. During liquid storage, samples treated with VD demonstrated improved sperm motility, viability, and membrane integrity (p < 0.05). The concentration of 1.25 μg/mL consistently yielded the most favorable results, while 31.25 μg/mL caused increased morphological abnormalities and diminished DNA integrity over time. Adding low doses of VD to the extender enhanced sperm motility, viability, membrane stability, and DNA integrity during liquid storage. These findings suggest that VD, at the most effective concentrations within the tested range, may serve as a useful additive in ram sperm extenders for improving sperm preservation during liquid storage.