Cleavage of One- And Two-Celled Rabbit Eggs in Vitro After Removal of the Zona Pellucida

The following experiment was performed to study cleavage in one- and two-celled rabbit eggs after removal of the zona pellucida. Adult rabbits were killed between 18 and 30 hr after natural mating. Under sterile conditions, the one- and two-celled eggs were flushed from the Fallopian tubes with Hanks' salt solution buffered with tris-citrate buffer. They were washed twice, then transferred into a 1% solution of Pancreatin (L. Light & Co) or the protease Pronase (Calbiochem) (Mintz, 1962) for between 5 and 30 min to remove the albumen coat and the zona pellucida. Pronase was more efficient than Pancreatin especially in digesting the albumen layer, although it was seldom that either enzyme removed the zona completely. The remaining part of the zona pellucida was removed by drawing the eggs in and out of a fine Pasteur pipette until the zona split and the egg could be drawn gently out. Examples