Background: Typical difficulties encountered during in vitro fertilization (IVF) to produce embryos in pigs include poor pronucleus formation and poor-quality fertilized embryos because of high polysperm invasion. In this study, we evaluated the effects of supplementation with apple seed extract (ASE) and coculture systems on porcine in vitro-fertilized embryo culture. Methods: Slaughterhouse-derived ovaries were used to obtain cumulus-oocyte complexes (COCs). COCs were conventionally used to perform IVF. We examined the differences in apoptosis and metabolism during development following addition of ASE to normal culture and coculture systems. Matrix metalloproteinases (MMPs), cell development-related factors, and apoptotic proteins were compared in porcine embryos produced under different conditions. Results: The expression of genes related to insulin-like growth factor (IGF) signaling was increased in the coculture system. In the ASE group, early apoptosis and necrosis were reduced in fertilized embryos and the late survival rate increased. Supplementation of the coculture system with ASE led to increased expression of BCL-2 and decreased expression of Casp-3 in the cytoplasm, thereby lowering the apoptosis rate and inducing MMP expression. In addition, compared with the extract-supplemented group in normal culture, the activity of MMP-2 decreased in the coculture system supplemented with ASE, activity of MMP-9 increased, and the expression of dynactin p62 and BrdU in the cytoplasm was higher than that in the other groups. Conclusions: The coculture system increased the activity of the embryonic cytoplasm compared with the non-coculture system. Supplementation with ASE may induce cell activity and inhibit the expression of apoptotic factors.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Oocyte maturation
    Sperm processing and IVF
    In vitro culture
    Gelatin zymography
    ELISA
    Immunofluorescence
    Statistical analysis
RESULTS
    Analysis of embryonic cell death factor activityaccording to ASE treatment
    Action of cytoplasmic and cell cycle activators
    Analysis of activity and expression control of MMPs inembryonic cells
DISCUSSION
CONCLUSION
REFERENCES

• Min-Jee Oh(General Graduate School of Animal Life Convergence Science, Hankyong National University, Anseong 17579, Korea, Institute of Applied Humanimal Science, Hankyong National University, Anseong 17579, Korea)
• Baasanjav Batmunkh(General Graduate School of Animal Life Convergence Science, Hankyong National University, Anseong 17579, Korea)
• Ji-Yeon Mo(School of Animal Life Convergence Science, Hankyong National University, Anseong 17579, Korea)
• Sang-Hwan Kim(General Graduate School of Animal Life Convergence Science, Hankyong National University, Anseong 17579, Korea, School of Animal Life Convergence Science, Hankyong National University, Anseong 17579, Korea, Institute of Applied Humanimal Science, Hankyong National University, Anseong 17579, Korea) Corresponding author