Use of a unique freezing technique in the freezing of bovine embryos

Abstract

The cryopreservation of embryos has been tried with variable success. Embryos are often damaged during the cryopreservation process and/or thawing, either by the formation of large ice crystals or by increased intracellular concentration of solutes and accompanying changes that result from dehydration of cells during cryopreservation. Freezing damage decreases the pregnancy rate when embryos are transferred into recipients. Fast freezing minimizes damage from solution effects; however, it leads to the formation of large ice crystals that cause severe mechanical damage. In contrast, slow freezing prevents large ice crystal formation, but it leads to increased damage from solution effects. Therefore, further experiments are needed to improve the pregnancy rates following embryo cryopreservation and transfer.

Recently, a new unique freezing technology (UFT) has been developed for the freezing of food stuffs and other products. Two studieswere conducted in order to determine if this UFT can be used in the cryopreservation of cattle embryos for the improvement of embryo survival and pregnancy rates. Preliminary studies in embryo survival using the UFT were conducted with in vitro matured/fertilized bovine embryos. These studies were followed by experiments with embryos flushed from superovulated cattle, frozen using the UFT, and then compared to development of both fresh (control) embryos and embryos traditionally frozen in Liquid Nitrogen (LN).

Data analysis for the comparison of the Control group (never frozen). Liquid Nitrogen group (direct plunge into LN) and UFT at all percentage levels (2%, 4%, 6%, 8% and 10%) of Dimethyl Sulphoxide (DMSO) revealed a difference in expansion rates (P > 0.018) and for hatching rates (P > 0.009). The comparison of LN with all the levels of DMSO using the UFT failed to reveal a difference for expansion rates (P > 0.44); however, a difference was observed for the hatching rates (P > 0.06). Comparison of the 8% DMSO group, which gave the highest overall expansion and hatching rates, using the UFT versus LN did not reveal a difference for expansion (TP > 0.21) or for hatching rates (P > 0.29); however, a fold increase was observed. All DMSO groups using the UFT did not differ for expansion rates (P > 0.56), but differences were noted for hatching rates (P > 0.03), with the 8% DMSO group yielding the highest hatching percentage rate. Data analysis was not conducted for the superovulation studies due to less than expected numbers of embryos recovered from the donor cows.

The data suggest the UFT can be used in the cryopreservation of bovine embryos, and of the techniques used, the methods similar to standard vitrification seemed to work best. These data suggest the UFT is a viable alternative to current cryopreservation techniques; however, future studies are needed to define the optimum techniques, cryoprotectants, and thawing procedures for use with the UFT.