11. Vitrification of bovine oocytes and embryos

Improving pregnancy rates associated with the use of cryopreserved human oocytes would be an important advance in human assisted reproductive technology. Vitrification is frequently referred to as a novel technology of cryopreservation in embryology, although some young embryologists were born after its first successful application. Unfortunately, in spite of the accumulated evidence regarding its enormous potential value, most domestic animal and human laboratories use exclusively the traditional slow-rate freezing with its compromised efficiency and inconsistency. The purpose of this work is to challenge vitrification technology, in terms of survival, in vitro development and pregnancy rate in bovine oocytes and embryos to support or disprove the use of vitrification, and to outline its role among reproductive technologies. To provide evidence for the potential significance of vitrification, an advanced version of the “minimal volume approaches” is analyzed. This technology alone has resulted in more healthy human babies after cryopreservation of blastocysts than any other vitrification technique, and more successful oocyte vitrification resulting in normal births than any other cryopreservation method. This technique opens the way for widespread application for cryopreservation at all phases of oocyte and preimplantation embryo development in mammals. Vitrification allows glasslike solidification of a solution without ice crystal formation in the living cells. We have tested the survival rates of oocytes by a vitrification technique using a bovine model. In vitro matured oocytes were vitrified with ethylene glycol+dimethylsulfoxide+sucrose in MEM with Hepes on a thin plastic sticker. In experiment 1, the oocyte survival rates after vitrifying-warming, and the capability for fertilization and embryo development were examined in vitro. The survival rate was evaluated morphologically and by FDA fluorescence technique. Oocytes vitrified without cumulus cells had a 99% survival rate after thawing. The rate of embryo development to blastocyst arising from naked oocytes was not significantly different from fresh oocytes. In experiment 2, oocytes were matured and fertilized in vitro. They were cultured until blastocysts (n=109) and then vitrified. We thawed 77 blastocysts and evaluated them morphologically and by FDA fluorescence technique (100% survival). The 32 remaining blastocyst were thawed and transferred to receptor heifers at day 7 of standing oestrus. Transrectal ultrasonography for pregnancy diagnosis was done approximately 42 days after oestrus. Fifteen heifers were pregnant and 11 calves were born. These results of pregnancy and calves born of in vitro produced embryos showed no difference with the percentage that is normally obtained with fresh blastocysts. Our data indicates that in vitro matured bovine oocytes and embryos can be vitrified successfully. The absence of cumulus cells for vitrification does not affect oocyte survival rate after warming, and the behavior of vitrified and warmed oocytes is similar to fresh oocytes in terms of survival and embryo development.