The use of a two-step removal protocol and optimized culture conditions improve development and quality of zona free mouse embryos

The zona pellucida (ZP) plays an important role in both the fertilization and embryonic development. For successful handling of early stage blastomeres for differentiation analysis, production identical twins or quadruplets, nuclear transfer gene introduction requires removal ZP (ZPR). Although single use either acidic Tyrode's solution pronase are commonly used ZPR, long-term exposure to these agents can result inhibition development with collapse three-dimensional blastomere structure. Here, we demonstrate benefits using a two-step combined ZPR method, which relies upon customized well-of-well (cWOW) system smaller well size, on developmental competence quality free (ZF) mouse embryos. We first isolated 2-cell embryos acid then cultured commercially available cWOW, had microwell size. rate blastocyst was significantly increased by cWOW when compared other culture systems. Then evaluated protocol, relying proteinase K, subsequent system. treatment alone reduced time, morphology became wrinkled, significant decrease associated number apoptotic cells expression apoptosis-related genes were observed. Using K time decreased rate, but did not induce increase cell expression. In contrast, method improved increasing total wells reducing experiments. These results suggest that protocol is beneficial toxic effects ZF embryo suitable