Background: Artificial insemination (AI) relies on liquid storage of boar semen for handling and timing to improve reproductive efficiency. However, storage promotes reactive oxygen species (ROS) accumulation in sperm, which damages the membranes, mitochondria, and DNA, leading to a loss of motility and integrity. The addition of antioxidants is a practical method for preventing ROS generation and functional loss. Accordingly, the effects of 1 nM Mitoquinone (MitoQ) on the viability, motility, and sperm protein function of boar sperm were evaluated. Methods: Semen extended with sperm was stored at 4℃ for up to 7 days in the presence of 1 nM MitoQ. Viability was assessed using SYBR-14/PI, and motility and kinematics (ALH, BCF, LIN, STR, VAP, VCL, VSL, and WOB) were measured using the computer-assisted sperm analysis (CASA) system. Western blotting was used to quantify phospho-AMPK (Thr172), total AMPK, and β-actin with α-tubulin as a loading reference. Results: Viability was higher in MitoQ from days 3 to 7 (p < 0.001). Total motility was also higher on day 1 (p < 0.01) and was better preserved on days 3,5 (p < 0.05) and 7 (p < 0.001). Kinematics showed reduced ALH and increased BCF with MitoQ, with higher LIN (day 3, p < 0.05) and STR (days 5 and 7, p < 0.05). VAP and VCL were transiently higher on day 1 and then lower than those of the control thereafter, whereas VSL and WOB were unchanged. The phospho-AMPK/total-AMPK ratio was higher with MitoQ on all measurement days (p < 0.001). β-actin did not differ on day 1, showed a numerical increase on day 3, and was higher on day 5 (p < 0.05). Conclusions: Viability and motility were preserved with 1 nM MitoQ, and sperm swimming was straighter and more efficient. The higher phospho-AMPK/total-AMPK ratio, together with preserved β-actin, points to AMPK-based energy control and a stable cytoskeleton. This supports follow-up studies on the dose, extender conditions, and fertility outcomes of boar artificial insemination.

ABSTRACT
181INTRODUCTION
MATERIALS AND METHODS
    Sperm preparation and MitoQ treatment
    Sperm viability analysis
    Sperm motility analysis
    Western blotting
    Statistical analysis
RESULTS
    Effect of MitoQ on boar sperm viability
    Effect of MitoQ on boar sperm motility
    Effect of MitoQ on boar sperm motility parameters
    Effect of MitoQ on antioxidant and stability proteins ofboar sperm
DISCUSSION
CONCLUSION
REFERENCES

• Min-Gi Han(Department of Stem Cells and Regenerative Technology, Konkuk University, Seoul 05029, Korea)
• Won-Young Lee(Department of Livestock, Korea National University of Agriculture and Fisheries, Jeonju 54874, Korea) Corresponding author
• Hyun-Jung Park(Department of Animal Biotechnology, Sangji University, Wonju 26339, Korea) Corresponding author