High Pregnancy and Calving Rates with a Limited Number of Transferred Handmade Cloned Bovine Embryos.

A major obstacle of widespread commercial application of bovine somatic cell nuclear transfer is the low overall efficiency, that is, healthy calf-late pregnancy per transferred embryo rate. In this study, we report a series of experiments with a limited number of embryos created with handmade cloning (HMC) and transferred without or after open pulled straw vitrification. Embryo reconstruction was performed by using in vitro matured oocytes and adult ear skin fibroblasts. In two experiments, a total of 53 D7 blastocysts were developed from 188 reconstructed embryos. Fresh transfer of seven blastocysts into six recipients has resulted in three early pregnancies, two of them developed over 90 days and eventually resulted in healthy calves (33% offspring/transfer rate). In another two experiments, a total of 11 D7 blastocysts were obtained from 36 reconstructed embryos. Out of these, eight have reexpanded 18 hours after vitrification and warming. Transfer of these blastocysts into eight recipients has resulted in four early pregnancies and two live births; 25% offspring/transfer rate. These results indicate that low overall efficiency may not be an intrinsic feature of cattle cloning, and selection of the right procedures may help to overcome the actual limitations.

Vitrification in ART ­ getting closer?

Background: Twenty five years after the first successful application in mammalian embryology vitrification seems to emerge now from the "new", "experimental", "immature" category and has become a method of choice for routine use both in domestic animals and humans. Review: The aim of this review is to summarize major steps of this regretfully slow development, outline the actual possibilities and limitations, highlight some promising perspectives, and deal also with factors that have hampered and still hamper the widespread application. The unique feature of vitrification in cryobiology is the total elimination of ice formation both in the sample and the entire solution surrounding it. This radical approach requires some drastic measures including high cryoprotectant concentration and high cooling/warming rates, although theoretically none of these factors are absolute requirements for vitrification. Initial efforts to improve vitrification results were focused on decreasing the toxic and osmotic effect of cryoprotectants. In the second period, the increase of cooling and warming rates has become the major goal. Subsequent changes in the equilibration parameters have improved the protection of the whole sample and allowed efficient cryopreservation of challenging structures including mature and immature oocytes, early stage domestic animal embryos and human blastocysts. In spite of proven superiority over other cryopreservation approaches, vitrification is still surrounded by a suspicious atmosphere, as technical problems, financial limitations and legal restrictions compromise its well-deserved acknowledgment. Ongoing debate about liquid nitrogen-mediated disease transfer issues illustrates this controversial situation well: although the danger is negligible, never proven, and there are established solutions to eliminate even the theoretical possibility, the incipient legislative ban of all open systems may compromise efficiency and reduce benefits of vitrification in the most important fields including bovine blastocyst and human oocyte cryopreservation. On the other hand, some supplementary techniques including low pressure treatment of liquid nitrogen or high pressure treatment of samples may further improve the efficiency of vitrification, and eliminate the ­ already slight ­ differences between the developmental competence of fresh and cryopreserved oocytes and embryos. Conclusion: During the past decade vitrification has become the method of choice for cryopreservation of oocytes and embryos in all mammalian species. It may offer special benefits for samples that are difficult to cryopreserve with other methods including porcine embryos, domestic animal and human oocytes, and human blastocysts. Full exploitation of these possibilities may considerably increase the overall efficiency and broaden the application fields of in vitro procedures in ART; however, its acceptance requires an open-minded approach from both specialists and authorities.