This present study was conducted to investigate protective effect of discontinuous Percoll gradient containing alpha-linolenic acid (ALA) before freezing process on viability, acrosome damage, mitochondrial activity, and oxidative stress of frozen-thawed boar spermatozoa. The separation of spermatozoa by discontinuous Percoll gradient was performed by different concentration of Percoll solution (45/90%) containing ALA combined with bovine serum albumin (BSA), and collected sperm in each Percoll layer was cryopreserved. To evaluate viability, acrosome damage, mitochondrial activity, and reactive oxygen species (ROS) level of frozen-thawed sperm, flow cytometry was used. Morphological abnormalities were observed under light microscope. In results, viability of sperm from 90% Percoll layer was higher than control and 45% Percoll group (p < 0.05). Separated sperm in 90% Percoll layer had lower acrosome damage and morphological abnormalities than control as well as viability, whereas 45% Percoll group was higher (p < 0.05). Similar with acrosome damage and abnormalities, mitochondrial activity was slightly enhanced and the population of live sperm with high ROS level was decreased by 90% Percoll separation, however, there was no significant difference. Supplementation of 3 ng/mL ALA into Percoll solution increased sperm viability and decreased population of live sperm with high ROS compared to control (p < 0.05). In conclusion, discontinuous Percoll gradient before freezing process could improve efficiency of cryopreservation of boar sperm through selection of sperm with high freezing resistance, and supplement of ALA during Percoll gradient might contribute suppression of ROS generation via stabilizing of plasma membrane during cryopreservation.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONFLICTS OF INTEREST
ACKNOWLEDGEMENTS
AUTHOR CONTRIBUTIONS
AUTHOR’S POSITION AND ORCID NO
REFERENCES

• Doo-San Kim(College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea)
• Yong Hwangbo(College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea)
• Hee-Tae Cheong(College of Veterinary Medicine, Kangwon National University, Chuncheon 24341, Korea)
• Choon-Keun Park(College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea) Corresponding author