Closed pulled straw vitrification of in vitro-produced and in vivo-produced bovine embryos

The objective of this study was to evaluate the efficiency of the closed pulled straw (CPS) method for cryopreserving in vitro-produced and in vivo-produced bovine (Bos taurus) embryos. Based on the open pulled straw (OPS) protocol, the top end of a CPS was closed by tweezers (heated in a flame) to prevent the cryoprotectant medium containing embryos from contacting the liquid nitrogen. Bovine in vitro or in vivo morulae and early blastocyst embryos were frozen by slow cryopreservation, OPS vitrification, or CPS vitrification. Morphology of postthawed embryos was evaluated, and normal embryos were used for successive culture for 72h. There were no significant differences between OPS and CPS freezing groups in postthawed in vitro-produced embryos with respect to rates of morphologically normal embryos (mean+/-SD, 87.9+/-5.2% vs. 85.4+/-4.9%), survival at 24h (58.0+/-6.8% vs. 56.3+/-4.4%), and survival at 72h (35.2+/-6.0% vs. 34.9+/-6.7%). However, both OPS and CPS vitrification resulted in higher postthaw rates of morphologically normal embryo and survival at 24 and 72h than those of the slow-freezing method (P<0.05). Similar results were obtained for in vivo-derived embryos. We concluded that CPS vitrification was a feasible method to cryopreserve both in vitro-derived and in vivo-derived bovine embryos. This method not only eliminated the risk of embryo contamination by preventing contact with liquid nitrogen but also retained the advantages of the OPS vitrification method.