The present study was aimed to determine the effects of green tea extract (GTE) and beta-mercaptoethanol (β-ME) supplementation in boar sperm freezing extender on in vitro fertilization (IVF) and reactive oxygen species (ROS) and glutathione (GSH) levels of presumptive zygotes (PZs). Experimental groups were allocated into lactose egg yolk (LEY) without antioxidant (control), GTE (1,000 mg/l in LEY) and β-ME (50 μM in LEY). In freezing, spermatozoa extended with LEY were cooled to 5°C for 3 h and then kept at 5°C for 30 min following dilution with LEY containing 9% glycerol and 1.5% Equex STM. The final sperm concentration was 1 × 108/ml. Spermatozoa were loaded into straws and frozen in nitrogen vapor for 20 min. For IVF, oocytes were matured in NCSU-23 medium and co-cultured with spermatozoa following thawing at 37°C for 25 sec. At 12 h following IVF, IVF parameters (sperm penetration and monospermy) were evaluated. In addition, GSH and ROS levels of PZs were determined by Cell Tracker Blue CMF2HC and DCHFDA, respectively. IVF parameters did not show any significant difference among the experimental groups. GSH and ROS levels of PZs were not significantly different between groups. In conclusion, antioxidant supplementation in boar sperm freezing could not influence IVF parameters, ROS and GSH levels of PZs.

INTRODUCTION
 MATERIALS and METHODS
  1. Chemicals
  2. Preparation of antioxidants
  3. Collection of semen
  4. Sperm freezing-thawing protocol
  5. Oocyte collection and In vitro maturation (IVM)
  6. In vitro fertilization (IVF) and In vitro culture (IVC)
  7. Assessment of sperm penetration and presence of two pronuclei (PN)
  8. Measurement of intracellular ROS and GSH levels of PZs
  9. Statistical analyses
 RESULTS and DISCUSSION
 REFERENCES

• Sang-Hyoun Park(Department of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Chonbuk National University)
• Yubyoel Jeon(Department of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Chonbuk National University)
• Il-Jeoung Yu(Department of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Chonbuk National University) Correspondence