186 Impact of Sericea lespedeza extract on the quality of cooled and cryopreserved bull spermatozoa.

Abstract Artificial insemination (AI) has been extensively utilized in cattle to enhance reproductive efficiency and improve the genetic merit of offspring. Cryopreservation of semen for AI is associated with excessive production of reactive oxygen species (ROS) resulting in a reduction in sperm motility, viability, and subsequent impairment in sperm fertility. Tannins, a well-known antioxidant compounds, have been used effectively to protect spermatozoa against freezing damage. Sericea lespedeza (SL); a tannin-rich legume, can offer a cost-effective and a potential source of antioxidants. Our aim was to evaluate the impact of supplementing semen extender with SL extract on the quality of cooled and frozen-thawed bull spermatozoa. Semen was collected from five mature Angus bulls by electroejaculation once/week for 5 weeks. Semen samples were then pooled and diluted in AndroMed® extender supplemented with 0, 0.25, 2, 5, and 10% of SL aqueous extract. The final sperm cell concentration was set at 70 x 106 spermatozoa. Then, diluted semen was either cooled at 4°C for 72 h or cryopreserved. For cryopreservation, semen was equilibrated for 3 h at 4°C and then loaded into 0.25-mm French straws. Straws were then placed 4 cm above LN2 vapor for 10 min before being stored in LN2. Following one week, straws were thawed at 37°C for 30 sec. Sperm motility and kinematic parameters were evaluated by CASA at 24, 48, and 72 h post-cooling and in frozen/thawed semen. Acrosome integrity (with FITC/PNA), mitochondrial activity (FITC/Rhodamine 123), and lipid peroxidation (with BODIPY C11) were assessed in frozen/thawed semen. Data were analyzed by general linear model and one-way ANOVA. The results showed that sperm motility and kinematics decreased (P< 0.05) in a time dependent manner, and the lowest values were seen at 72 h. However, supplementing semen extender with 0.25%, 2%, or 5% SL extract improved (P< 0.05) sperm motility at 72 h compared to the control (0%) and those supplemented with 10% supplemented SL extract. In frozen/thawed semen, sperm motility was higher (P< 0.05) in 5% SL extract group than in control one (45.6±4.5% vs. 23.4±4.3%, respectively). Sperm kinematics include VCL, VAP, DCL, DSL, DAP, ALH were also higher in 5% SL extract group than other groups. Percentage of spermatozoa with intact acrosome was higher (P< 0.05) in 5% SL extract supplemented group than in the control (83.4±0.9 vs. 74.3±0.3). No significant differences were observed in mitochondrial activity and lipid peroxidation among the different groups. In conclusion, inclusion of 5% SL extract during cooling and freezing of bull semen enhanced sperm motility, kinematics parameters, and acrosome integrity. These results assure the protective role played by SL in protecting sperm against cooling and cryopreservation inflicted damage in livestock.