Application of hollow fiber vitrification for cryopreservation of bovine early cleavage stage embryos and porcine morula-blastomeres

Abstract

A novel hollow fiber vitrification (HFV) method was applied to materials that have previously been difficult to cryopreserve, thereby expanding the potential application of this method. The results showed that zona-free porcine morulae and their isolated blastomeres remained viable even after vitrification. The rate of development to blastocysts after vitrification was similar for zona-free and zona-intact morulae (21/23, 91.3% for both). Vitrified blastomeres had a developmental potential equal to that of non-vitrified blastomeres (blastocyst formation rate after reaggregation: 16/17, 94.1% for both). The HFV method was also effective for the cryopreservation of in vitro matured/fertilized bovine embryos at the 2- to 4-cell, 8- to 16-cell and morula stages. The blastocyst formation rates of vitrified embryos (66.1-82.5%) were similar to those of non-vitrified embryos (74.5-82.5%). These results indicate that this novel HFV method is an effective tool for embryo cryopreservation that can enhance current practices in reproductive biology.

Fig. 1.

Development of porcine zona-free morulae after cryopreservation by the HFV method. A, B: Zona-free (A) and zona-intact morulae (B) loaded in the hollow fiber. C–F: Zona-free (C, E) and zona-intact morulae (D, F) were cultured in microwells (arrowhead) after being vitrified or as the non-vitrified controls. Scale bar = 100 µm.

Fig. 2.

Cryopreservation of isolated morula-blastomeres by the HFV method and post-rewarming development. A: Isolated morula-blastomeres loaded in a hollow fiber. B−D: Non-vitrified group. The isolated morula-blastomeres (B), blastomere aggregate in a microwell (C) and blastocyst developed from the control aggregate (D). E−G: Vitrified group. The isolated morula-blastomeres before vitrification (E) and after aggregation following vitrification/rewarming (F) and the blastocyst developed from the aggregate (G). Scale bar = 1 mm for A and 100 µm for B–G.

Fig. 3.

In vitro development of IVM/IVF bovine embryos vitrified using the HFV method at early developmental stages. Bovine IVM/IVF embryos at the 2- to 4-cell, 8- to 16-cell and morula stages were vitrified/rewarmed, and their subsequent development was compared with that of non-vitrified controls. Scale bar = 100 µm.