Background: Sperm cooling is a critical step in cryopreservation, as exposure to temperatures below 15℃ can induce cold shock-associated damage, including phospholipid scrambling and lipid peroxidation. Goat sperm are particularly vulnerable to cooling-induced injury because of their membrane characteristics. This study evaluated the effects of cooling rate and interval agitation during cooling in frozenthawed goat sperm. Methods: The samples were cooled at 0.5℃/min (0.5 Cool) or 0.1℃/min (0.1 Cool) and agitated every 15 min during cooling until 5℃, then, the samples were cryopreserved in liquid nitrogen. After thawing, sperm motility was assessed using computer-assisted sperm analysis (CASA) and plasma membrane integrity, acrosomal integrity, intracellular H2O2 levels, mitochondrial activity, mitochondrial membrane integrity, and mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry. Results: In results, there were no significant differences in sperm motility, plasma membrane integrity, acrosomal integrity, and mitochondrial function among the treatment groups. However, intracellular H2O2 levels were significantly lower in agitation-treated groups than non-agitated groups both 0.5 Cool and 0.1 Cool treatment groups (p < 0.05), whereas there were no significant differences between cooling rate groups. Conclusions: In conclusion, these results indicate that interval agitation during the cooling process could reduce intracellular reactive oxygen species in frozen-thawed goat sperm.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Animals and collection
    Sperm preparation
    Sperm cooling methods
    Motility
    Structural integrity
    Intracellular H2O2 levels
    Mitochondrial function
    Statistical analysis
RESULTS
    Effects of cooling rate and interval agitation on spermmotility
    Effects of cooling rate and interval agitation on spermstructural integrity
    Effects of cooling rate and interval agitation onintracellular H2O2 levels
    Effects of cooling rate and interval agitation on spermmitochondrial function
DISCUSSION
CONCLUSION
REFERENCES

• Yeoreum Yoon(College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea)
• Eunju Seok(College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea)
• Minwoo Sin(College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea)
• Chanjin Woo(College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea)
• Nahyeon Kang(College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea)
• Sang-Hee Lee(College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea, School of ICT, University of Tasmania, Hobart 7005, Australia) Corresponding author
• Eun-Do Lee(Animal Genetic Resources Research Center, National Institute of Animal Science, Rural Development Administration, Hamyang 50000, Korea)
• Kwan-Woo Kim(Animal Genetic Resources Research Center, National Institute of Animal Science, Rural Development Administration, Hamyang 50000, Korea) Corresponding author
• Seungjun Lee(College of Veterinary Medicine, Seoul National University, Seoul 08826, Korea)
• Jongki Cho(College of Veterinary Medicine, Seoul National University, Seoul 08826, Korea, Farm Animal Clinical Training and Research Center, Institutes of Green-Bio Science and Technology, Seoul National University, Pyeongchang 25354, Korea)