Fetal Bovine Serum (FBS) plays a crucial role in animal cell culture; however, the increasing number of bovine fetuses used and sacrificed solely for FBS collection has raised ethical concerns globally. The welfare of fetuses during FBS blood collection has become a key focus of debate among animal welfare and ethics organizations worldwide. Previous studies indicate that heat-inactivated coelomic fluid (HI-CF) from the earthworm Perionyx excavatus may serve as a viable FBS alternative in adherent cell cultures. This study evaluates the potential of HI-CF as an FBS substitute during the in vitro maturation (IVM) stage of bovine embryo culture, with a focus on improving developmental rate through antioxidation effects. In this study, 2% HI-CF was incorporated into IVM media, assessing its impact on cell growth, differentiation, and the expression of genes related to antioxidation. The group of 2% of HI-CF exhibited a trend toward increased cleavage and blastocyst development rates compared to the control group. Although antioxidant genes such as NRF2 and GSR showed no statistically significant differences between the control and treatment groups, a trend toward increased expression was observed. Conversely, GPX1 displayed a trend of decreased expression. Notably, IGF1 and NQO1 were significant upregulated (p < 0.05) in the 2% HI-CF group. Additionally, oocytes stained with H2DCFDA showed a significantly reduced ROS levels (p < 0.05) in the 2% HI-CF group compared with controls. These findings suggest that HI-CF's antioxidative effects support enhanced cell growth and blastocyst development rate, surpassing those observed with FBS. Consequently, HI-CF shows promise as an effective alternative to FBS in vitro maturation of bovine oocytes.

Abstract
Introduction
Material and Methods
    1. Reagents
    2. Animal ethics
    3. Oocyte collection and in vitro maturation
    4. In vitro fertilization and culture
    5. H2DCFDA staining for Reactive Oxygen Species(ROS)
    6. Extraction of mRNA and cDNA synthesis
    7. Real Time polymerase chain reaction (RT-PCR)
    8. Statistics analysis
Results and Discussion
    1. Optimal concentration of HI-CF for in vitromaturation of bovine oocytes
    2. Cleavage and Blastocyst development rate ishigher at 2% HI-CF
    3. Analysis of gene expression
    4. Effect of HI-CF on ROS is significantly lower inbovine embryos
Acknowledgements
References

• Ji Hyo Kim(Undergraduate Student, Division of Animal Science, Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Jun Seok Heo(Undergraduate Student, Division of Animal Science, Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Dong Baek Kim(Undergraduate Student, Division of Animal Science, Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Seo Hyeon Lee(Graduate Student, Division of Applied Life Science & Animal Science (BK21 Four), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Seung Eun Lee(Graduate Student, Division of Applied Life Science & Animal Science (BK21 Four), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Seo Young Hyeon(Undergraduate Student, Division of Animal Science, Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Song Park(Undergraduate Student, Division of Animal Science, Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea, Graduate Student, Division of Applied Life Science & Animal Science (BK21 Four), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea, Professor, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Il-Keun Kong(Undergraduate Student, Division of Animal Science, Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea, Graduate Student, Division of Applied Life Science & Animal Science (BK21 Four), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea, Professor, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea) Corresponding author