[III-15-08] Effect of two-step combined zona pellucida removal on developmental competence of mouse embryos
Aims: Zona removal (ZR) is necessary for blastomere separation, chimeric embryo production, hand-made cloning, and lipofection. To obtain zona free (ZF) embryos, acid Tyrode (AT) or Pronase (P) are commonly used. However, digesting time and further toxicity are concerned. Therefore, a quick and stable ZR is important for further study. In this study, we aimed the comparative study of ZR by using AT, P, and AT-P as well as further development and embryo quality. M&M: After IVF, derived 2-cell embryos were used for this study. ZF embryos treated by AT, P and AT-P were cultured in the handmade WOW. ZR time, blastocyst rate, total cell number (TCN), apoptosis-related gene expression, and TUNEL assay were investigated. Results: Although the treatment time of AT was shorter than P, a wrinkled blastomere surface was observed. Blastocyst rate was significantly decreased by AT and P, whereas AT-P treatment reduced treatment time with less harmful effect to blastomere morphology, increased blastocyst rate, TCN than AT and P. Apoptosis cell numbers were lower in AT-P than AT. Conclusion: Combined AT-P can reduce ZR time with less toxic effect and improve development and quality.