Improving the quality and in vitro fertilization rate of frozen-thawed semen of buffalo (Bubalus bubalis) bulls with the inclusion of vitamin B12 in the cryopreservation medium

Abstract

This study was designed to evaluate the effects of vitamin B₁₂ in cryopreservation medium on frozen-thawed semen of buffalo (Bubalus Bubalis) bulls. Semen from five bulls (fertility-proven) were diluted in five aliquots not supplemented (control), or supplemented with 1, 2, 4, or 5 mg/mL of vitamin B₁₂ and evaluated using the Computer Assisted Sperm motion Analysis, antioxidant enzymes, lipid peroxidation (LPO), reactive oxygen species (ROS), ATP concentrations, and in vitro fertilization rate (%). Sperm progressive motility, rapid velocity (%), mitochondrial potential, and acrosome integrity were greater (P < 0.05) with supplementation of 4, and 5 mg/mL vitamin B₁₂ than the control sample. Similarly, compared with the control, samples with 5 mg/mL vitamin B₁₂ supplementation had markedly greater average-path, straight-line, and curved-line velocities (μm/sec). Semen samples supplemented with 2, 4 and 5 mg/mL vitamin B₁₂ had greater concentrations of GPx (U/mL) and SOD (U/mL), whereas LPO (μM/mL) was less (P < 0.05) compared with the control sample. Seminal plasma ROS concentrations (10⁴/25 × 10⁶) were less in the 5 mg/mL vitamin B₁₂ supplemented than control sample. Semen samples supplemented with 5 mg/mL of vitamin B₁₂ had greater concentrations of ATP than control and the 1 mg/mL vitamin B₁₂ supplemented sample. Semen samples supplemented with 5 mg/mL of vitamin B₁₂ had greater plasmalemma and DNA integrities (%) than the control sample. In summary, vitamin B₁₂ supplementation augments semen quality, as evidenced by values for CASA variables, antioxidant enzymes, and ATP concentrations, which may occur as a consequence of inhibition in LPO and ROS production by buffalo spermatozoa.

Keywords: Buffalo sperm; CASA; Lipid peroxidation; MMP; ROS; Semen quality; Vitamin B(12).