The vitrification of embryos is essential for animal reproduction and significantly contributes to assisted reproductive technologies, enabling fast cryopreservation without ice crystal formation. Mitochondria, vital organelles in cellular metabolism, are responsible for critical functions like ATP synthesis, calcium regulation, and apoptotic signaling. Preserving mitochondrial integrity is essential for ensuring embryonic strength. Studies demonstrate that vitrification, a widely used cryopreservation method, can markedly impair mitochondrial function in mammalian embryos. This study examines the efficacy of novel/modified antifreeze peptide as a biocompatible agent when used in an appropriate concentration with base vitrification media. Blastocysts vitrified in base media as well as supplemented with the peptide exhibited significantly enhanced post-thaw survival rates, attaining re-expansion and hatching rates of 96.89 ± 4.2% and 88.31 ± 1.3%, respectively, in contrast to 79.38 ± 3.7% and 52.57.9 ± 0.8% observed in the control group. Furthermore, peptide-treated BLs demonstrated elevated expression of PGC1α, BCL2, and Sirt-1, which are the key genes related to mitochondrial membrane potential and anti-apoptotic factors. The mitochondrial function was maintained, and the levels of reactive oxygen species (ROS) and the expression of genes such as Cyto-c, caspases 3, and caspase 9 were markedly diminished in the embryos vitrified with peptide. These findings highlight the ability of this modified peptide to preserve mitochondrial integrity and reduce oxidative stress, hence enhancing the survival of blastocysts post-vitrification.

Abstract
Introduction
Materials and Methods
    1. Vitrification and thawing procedure
    2. Evaluation of embryo viability
    3. Oocyte aspiration and IVM
    4. In vitro fertilization
    5. In vitro culture
    6. Immunofluorescence staining
    7. RNA extraction and quantitative real-time PCR(qPCR)
    8. Implantation assay
    9. Statistical analysis
Results and Results and Discussion
    1. Effect of peptide addition on developmentalcompetence of blastocyst
    2. Comparative effect of peptide on slow-freezingand vitrification
    3. Effect of peptide on freeze-thaw (vitrification)cycle of the blastocyst
    4. Protective effect of peptide on mitochondrialhealth during the freeze-thaw cycle
    5. Effect of peptide on apoptosis and embryo survivalduring vitrification
Acknowledgment
References

• Muhammad Tayyab Khan(Graduate Student, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Zaheer Haide(Graduate Student, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Chalani Dilshani Perera(Graduate Student, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Seo Hyeon Lee(Graduate Student, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Seung Eun Lee(Graduate Student, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Seo Young Hyeon(Graduate Student, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Il Keun Kong(Graduate Student, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea, Professor, Institute of Agriculture and Life Science, Gyeongsang National University, Gyeongnam Province, Korea, Researcher, Thekingkong Corp. Ltd., Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea) Corresponding author
• Muhammad Idrees(Researcher, Thekingkong Corp. Ltd., Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea)
• Sung Woo Kim(Researcher, Dairy Science Division, National Institute of Animal Science, Rural Development Administration, Cheonan, Chungcheongnam-do 31000, Korea)
• Jun Kyu Son(Researcher, Hanwoo Research Center, National Institute of Animal Science, Rural Development Administration, Pyeongchang-gun, Gangwon-do, 25340, Korea)