Background: Porcine embryonic development is widely utilized in the medical industry. However, the blastocyst development rate in vitro is lower compared to in vivo . To address this issue, various supplements are employed. Extracellular vesicles (EVs) play the role of communicators that carry many bioactive cargoes. Additionally, the contents of EVs can vary on the estrous cycle. Methods: We compared the effects of adding EVs derived from porcine uterine fluid (UF), categorized as non-EV (G1), EVs in estrus (G2) and EVs in diestrus (G3). After in vitro culture (IVC) was performed in three different groups, cleavage rate and blastocyst development rate were examined. In addition, glutathione (GSH) and reactive oxygen species (ROS) levels were measured 2 days after activation to assess oxidative stress. Results: Using NTA and cryo-TEM, we confirmed the presence of EVs with sizes ranging from 30 nm to 200 nm, that the particles were suitable for analysis for analysis. In IVC data, the highest cleavage rate was observed in G2, which was significantly different from G1 but not significantly different from the next highest, G3. Similarly, the highest blastocyst development rate was observed in G2, which was significantly different from G1 but not significantly different from the next highest, G3. Conclusions: These results indicate that estrus derived EVs contain biofactors beneficial for early blastocyst development, including GSH which protects the blastocyst from oxidative stress. Additionally, although diestrus-derived EVs are expected to have some effect on blastocyst development, it appeared to be less effective than estrus-derived EVs.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Chemicals
    EV isolation and characterization
    In vitro maturation (IVM) oocytes
    Parthenogenetic activation & in vitro culture (IVC) withsupplement of EVs
    Measurement of embryos glutathione and reactiveoxygen species levels
    Statistical analysis
RESULTS
    EVs isolation and characterization
    Comparison of embryonic development based on thepresence of EVs
    Measure of oxidative stress in embryos
DISCUSSION
CONCLUSION
REFERENCES

• Shuntaro Miura(Department of Materials Science and Bioengineering, Nagaoka University of Technology, Niigata 940-2188, Japan, College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea)
• Heejae Kang(College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea)
• Seonggyu Bang(College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea)
• Ayeong Han(College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea)
• Islam M. Saadeldin(College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea, Department of Comparative Medicine, King Faisal Specialist Hospital & Research Center, Riyadh 11211, Saudi Arabia)
• Sanghoon Lee(College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea)
• Koichi Takimoto(Department of Materials Science and Bioengineering, Nagaoka University of Technology, Niigata 940-2188, Japan)
• Jongki Cho(College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea) Corresponding author