The aim of this study was performed to evaluate the effects of ice-binding protein from the arctic yeast Leucosporidium (LeIBP) supplementation on cryopreservation of boar sperm. The collected semen was diluted (1.5×108/ml) in lactose egg yolk (LEY) and cooled at 5°C for 3 h. The cooled semen was then diluted (1×108/ml) in LeIBP containing LEY with 9% glycerol and maintained at 5°C for 30 min. The semen was divided into six experimental groups (control, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/ml of LeIBP). The straws were kept on above the liquid nitrogen (LN2) vapors for 20 minutes and then plunged into LN2. After thawing, computer-assisted sperm analysis was used for sperm motility and flow cytometry was performed to assess the viability, acrosome integrity (FITC-PSA/PI), ROS (DCF/PI), lipid peroxidation (BODIPY C11/PI) and apoptosis (Annexin V/PI), respectively. No significant responses were observed for sperm motility. However, sperm viability was significantly increased on 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). In addition, acrosome integrity was significantly increases LeIBP groups (P < 0.05) and both ROS and lipid peroxidation level were lower in all LeIBP groups than those of control (P < 0.05). On the other hand, a significant higher apoptosis rate was observed in 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). It can be assumed that a supplementation of LeIBP in boar sperm freezing extender is an effective method to increase the sperm qualities after cryopreservation.

Abstract
 INTRODUCTION
 MATERIALS AND METHODS
  1. Chemicals
  2. Preparation of Leucosporidium ice-binding protein (LeIBP)
  3. Collection of boar semen
  4. Sperm freezing-thawing protocol
  5. Assessment of sperm motility
  6. FITC-PSA/PI staining for Sperm viability and acrosome integrity
  7. Sperm intracellular ROS level
  8. BODIPY-C11 581/591 staining for sperm lipid peroxidation (LPO)
  9. Apoptosis rate [Phosphatidylserine (PS) translocation]
  10. Flow cytometry
  11. Statistical analyses
 RESULTS
  1. Effect of LeIBP supplementation in LEY extender on sperm motility of boar spermatozoa after cooling and post-thaw
  2. Effects of LeIBP supplementation on viability and acrosome integrity
  3. Effect of LeIBP supplementation on ROS level
  4. Effect of LeIBP supplementation on lipid peroxidation
  5. Effect of LeIBP supplementation on phosphatidylserine (PS) translocation
  DISCUSSION
   1. Effect of LeIBP on motility
   2. Effect of LeIBP on viability and acrosome integrity
   3. Effect of LeIBP on ROS level
   4. Effect of LeIBP on lipid peroxidation (LPO)
   5. Effect of LeIBP on PS translocation
   6. Conclusion
 REFERENCES

• Sang Hyoun Park(Animal Biotechnology Division, National Institute of Animal Science)
• Keon Bong Oh(Animal Biotechnology Division, National Institute of Animal Science)
• Sun-A Ock(Animal Biotechnology Division, National Institute of Animal Science, RDA)
• Sung June Byun(Animal Biotechnology Division, National Institute of Animal Science, RDA)
• Hwi-Cheul Lee(Animal Biotechnology Division, National Institute of Animal Science, RDA)
• Suresh Kumar(Animal Biotechnology Division, National Institute of Animal Science, RDA)
• Sung Gu Lee(Unit of Polar Genomics, Korea Polar Research Institute, KIOST)
• Jae-Seok Woo(Animal Biotechnology Division, National Institute of Animal Science, RDA) Corresponding Author