In the present study, we examined the effect of straw size on spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing. Hanwoo semen was collected from three bulls and diluted with an animal protein-free extender, divided into two groups, namely, 10 million spermatozoa in 0.25 mL and 20 million spermatozoa in 0.5 mL straw, and cryopreserved. In Experiment 1, the motility and motility parameters of the frozenthawed spermatozoa were evaluated. After freezing-thawing, the spermatozoa motility parameters fast progressive, straight line velocity, and average path velocity were compared between the 0.25 mL straw and 0.5 mL straw groups. They were 35.2 ± 1.0 and 32.3 ± 0.7%, 34.6 ± 0.7 and 31.8 ± 0.5 μm/s, 51.4 ± 1.3 and 47.1 ± 1.1 μm/s, 0.25 mL straw and 0.5 mL straw groups, respectively. In Experiment 2, the viability, acrosome membrane integrity, and mitochondrial membrane potential of the frozen-thawed spermatozoa were assessed. After freezing-thawing, the percentages of spermatozoa with live, intact acrosomes and high mitochondrial membrane potential were compared between the in 0.25 mL straw and 0.5 mL straw groups. They were 48.0 ± 2.6% and 35.6 ± 2.8% between the 0.25 mL straw and 0.5 mL straw groups. In Experiment 3, the plasma membrane integrity of frozen-thawed spermatozoa was compared. After freezingthawing, the plasma membrane integrity was higher for the in 0.25 mL straw group than the 0.5 mL straw group. They were 62.0 ± 2.2 and 54.1 ± 1.3% between the 0.25 mL straw and 0.5 mL straw groups. In conclusion, our results suggest that freezing semen in 0.25 mL straw improves the relative motility, viability, and acrosomal, mitochondrial membrane potential, and plasma membrane integrity of Hanwoo bull spermatozoa.

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

• Sung-Sik Kang(Hanwoo Research Institute, National Institute Animal Science (NIAS), Rural Development Administration (RDA), Pyeongchang 25340, Korea)
• Ui-Hyung Kim(Hanwoo Research Institute, National Institute Animal Science (NIAS), Rural Development Administration (RDA), Pyeongchang 25340, Korea)
• Myung-Suk Lee(Hanwoo Research Institute, National Institute Animal Science (NIAS), Rural Development Administration (RDA), Pyeongchang 25340, Korea)
• Seok-Dong Lee(Hanwoo Research Institute, National Institute Animal Science (NIAS), Rural Development Administration (RDA), Pyeongchang 25340, Korea)
• Sang-Rae Cho(Hanwoo Research Institute, National Institute Animal Science (NIAS), Rural Development Administration (RDA), Pyeongchang 25340, Korea) Corresponding author