Cryoprotective Effects of α-Tocopherol, Quercetin and Their Combination on Sperm Functional Integrity during Cryopreservation of Murrah Buffalo (Bubalus bubalis) Semen

Abstract

The study was conducted during August, 2022 to April, 2024 at frozen semen bank, Bassi, Jaipur and department of Veterinary Physiology and Biochemistry, Post Graduate Institute of Veterinary Education and Research (PGIVER) Jaipur, Rajasthan, India to study the cryoprotective impacts on the combined supplementation for superior protective effects over the individual treatments in cryopreserved Murrah Buffalo (Bubalus Bubalis) bull semen. Total ejaculates were collected and evaluated. Ejaculate was processed separately and divided into four equal aliquots designated as Control (no antioxidant), T₁ (α-tocopherol @ 0.65 mg ml^-1), T₂ (quercetin @ 75 µM), and T₃ (α-tocopherol @ 0.65 mg ml^-1+quercetin @ 75 µM). Samples were cooled to 5°C in a cold cabinet, packed in 0.25 ml French straws, frozen, and stored in liquid nitrogen (-196°C). Frozen straws were thawed individually at 37°C for 30 s before evaluation. Significant differences (p<0.05) were observed among the treatment groups for all evaluated semen quality parameters, including progressive motility (%), Live spermatozoa (%), Intact acrosome (%) and Abnormal spermatozoa (%) at all stages of cryopreservation, including post-dilution (PD), post-equilibration (PE), and post-thaw (PT) stages. Among the treatments, the combination group (T₃) exhibited significantly superior results compared to the individual antioxidant treatments (T₁ and T₂), whereas T₂ showed significantly better performance than T₁ for most of the evaluated parameters. The present study concluded that the combination of α-tocopherol and quercetin exerts the most pronounced protective effect against oxidative stress and cryoinjury, compared to their individual supplementation, thereby improving semen quality during cryopreservation in Murrah buffalo bulls.