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 the successful handling of early stage blastomeres for differentiation analysis, the production of identical twins or quadruplets, nuclear transfer or gene introduction requires the removal of the ZP (ZPR). Although single use of either acidic Tyrode's solution or pronase are commonly used for ZPR, long-term exposure to these agents can result in the inhibition of development with the collapse of the three-dimensional blastomere structure.