Effects of slitting the zona pellucida and its subsequent sealing on freeze-thaw survival of Day 7 bovine embryos

The effects of slitting the zona pellucida and its subsequent sealing by either embedding in agar or surrounding with an additional zona pellucida on the development of frozen/thawed Day 7 bovine embryos were investigated in vitro and in vivo. A total of 225 embryos was frozen and thawed rapidly as controls (Group 1), after slitting the zona pellucida (Group 2), after slitting and subsequent sealing of the zona pellucida with agar (Group 3), or after slitting the zona pellucida (Grothen transferring the embryo into an additional zona pellucida (Group 4). The survival rate (embryos classified morphologically as excellent, good, or poor) was 95.1, 95.4, 92.2, and 94.3% for Groupsl, 2, 3, and 4, respectively. Culture of 145 embryos in vitro for 60 h revealed that 57.1, 59.5, 47.4, and 57.1% developed to hatching and hatched blastocysts in Groups 1, 2, 3, and 4, respectively. Within Group 3, however, a significantly (P < 0.05) lower percentage of the embryos continued to develop when the agar was not removed after thawing (31.8%) compared with embryos from which the agar had been removed (68.8%). After nonsurgical transfer of 78 embryos, the pregnancy rate was significantly (P < 0.05) lower (8.3%) with embryos of Group 3 compared with controls (61.5%) or embryos of Group 2 (42.9%). No significant difference existed between controls and embryos of Group 2. We conclude that an intact zona pellucida prior to rapid freezing is not essential for the survival of Day 7 bovine embryos.