Effects of the zona pellucida, agar embedding, and culture on the survival of micromanipulated bovine embryos after freezing and thawing

Abstract

Three experiments were undertaken to compare how the integrity of the zona pellucida, embedding in agar, and incubation in the sheep oviduct, respectively, affected the survival of bovine embryos after micromanipulation, freezing, and thawing. In Experiment 1, the proportions of whole day 7 embryos that continued their development in vitro after being frozen without a zona pellucida (N=18), or with the zona pellucida opened over one-half (N=14), or one-fourth (N=14) of its circumference, or left intact (N=17) did not differ significantly (44, 43, 36, and 59%, respectively). Experiment 2 compared the survival of bisected day 7 embryos frozen with or without embedding in agar cylinders. In vitro, one of eight half-embryos (17%) survived in each group. In vivo, the survival rate was 0% (N=18) after freezing half-embryos in an agar cylinder and (11%) (N=18) for halves frozen without agar. In Experiment 3, embryos were bisected on day 5 or 6, embedded in agar, then cultured for 2 days or 1 day, respectively, in the sheep oviduct before freezing them on day 7. Of the 54 half-embryos transferred to the sheep oviducts, 27 (50%) were recovered, frozen, thawed, and transferred to recipients, resulting in the birth of 17 calves (63%), of which six pairs were identical twins. This survival rate was significantly higher than for control embryos collected at day 7, bisected, and frozen without agar embedding (16%;N=12). It was concluded that neither the zona pellucida nor agar embedding plays a critical role in cryoprotection and that efficient freezing of half-embryos depends more on bisection with minimal trauma and a recovery period in a supportive medium (such as the sheep oviduct) before freezing.