Background: Sperm quality and the number of sperm introduced into the uterus during artificial insemination (AI) are pivotal factors influencing pregnancy outcomes. However, there have been no reports on the relationship between sperm concentration at AI and sperm quality in Hanwoo cattle. In this study, we examined sperm quality and pregnancy rates after AI using sperm inseminated at different concentrations. Methods: We evaluated the motility, viability, and acrosomal membrane integrity of sperm at different concentrations (10, 15, 18, and 20 million sperm/straw) in 0.5-mL straws. Subsequently, we compared the pregnancy rates after AI with different sperm concentrations. Results: After freeze-thawing, sperm at the assessed concentrations showed similar viability and acrosomal membrane integrity. After AI, cattle in the 10 million group had significantly lower pregnancy rates compared to those in the 18 and 20 million groups. Conversely, there were no statistically significant variances observed between cattle in the 10 and 15 million groups. Conclusions: Sperm at concentrations of 10, 15, 18 and 20 million per straw exhibited comparable motility, viability, and acrosomal membrane integrity. However, a concentration of at least 18 million sperm per straw is required to achieve a consistent rate of pregnancy rate in Hanwoo cattle after AI.

INTRODUCTION
MATERIALS AND METHODS
    Semen collection and semen freezing procedure
    Evaluation of frozen-thawed spermatozoa motility
    Evaluation of the viability and acrosomal membraneintegrity of frozen-thawed spermatozoa
    Synchronization of estrus in Hanwoo cows andartificial insemination
    Assessment of pregnancy after artificial insemination
    Statistical analysis
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

• Sung-Sik Kang(Hanwoo Research Institute, National Institute Animal Science, Rural Development Administration, Pyeongchang 25340, Korea) Corresponding author
• Sang-Rae Cho(Animal Genetic Resources Research Center, National Institute Animal Science, Rural Development Administration, Hamyang 50000, Korea)
• Ui-Hyung Kim(Poltury Research Institute, National Institute Animal Science, Rural Development Administration, Pyeongchang 25342, Korea)
• Yonghwan Kim(Hanwoo Research Institute, National Institute Animal Science, Rural Development Administration, Pyeongchang 25340, Korea, Department of Animal Resources Science, Kongju National Universuty, Yesan 32439, Korea)
• Seok-Dong Lee(Hanwoo Research Institute, National Institute Animal Science, Rural Development Administration, Pyeongchang 25340, Korea)
• Myung-Suk Lee(Hanwoo Research Institute, National Institute Animal Science, Rural Development Administration, Pyeongchang 25340, Korea)
• Eunju Kim(Hanwoo Research Institute, National Institute Animal Science, Rural Development Administration, Pyeongchang 25340, Korea)
• Jeong-Il Won(Hanwoo Research Institute, National Institute Animal Science, Rural Development Administration, Pyeongchang 25340, Korea)
• Shil Jin(Hanwoo Research Institute, National Institute Animal Science, Rural Development Administration, Pyeongchang 25340, Korea)
• Hyoun-Ju Kim(Hanwoo Research Institute, National Institute Animal Science, Rural Development Administration, Pyeongchang 25340, Korea)
• Sungwoo Kim(Hanwoo Research Institute, National Institute Animal Science, Rural Development Administration, Pyeongchang 25340, Korea)
• Sun-Sik Jang(Hanwoo Research Institute, National Institute Animal Science, Rural Development Administration, Pyeongchang 25340, Korea)
• Seunghoon Lee(Animal Biotechnology Division, National Institute Animal Science, Rural Development Administration, Wanju 55365, Korea)