The dominance of the GSI-IX recipient oocyte��s cytoplasm is further highlighted by the loss of murine mtDNA as the non-supplemented embryos progressed to the blastocyst stage. In intra- and interspecific SCNT preimplantation embryos, where donor cell mtDNA has been reduced to residual levels through mtDNAspecific depletion, the preferential replication of donor cell mtDNA is attributed to the persistent expression of mtDNA-specific replication factors, such as POLGA, POLGB and TFAM. This contrasts with in vitro fertilised embryos, which first express these factors at the blastocyst stage. However, in more genetically diverse models, such as caprine-ovine iSCNT embryos, both POLGA and TFAM were also Navitoclax Bcl-2 inhibitor upregulated but, as with the non-supplemented murine-porcine crosses, donor cell mtDNA was almost totally eliminated by the later stages of preimplantation development. This suggests that the donor cell was replicating the genetically more diverse recipient oocyte mtDNA throughout preimplantation development, as also evident in our more divergent model at the blastocyst stage. Nevertheless, our strategy of depleting recipient oocytes to approximately 5% of their original mtDNA content and supplementing them with murine ESC extract containing mitochondria not only led to increased development to blastocyst, but vastly increased the amount of murine mtDNA present throughout preimplantation development. We chose to use mitochondria from undifferentiated ESCs, as they are similar to those of the oocyte and preimplantation embryo. They are spherical and oval in shape with poorly developed cristae indicating an inability to support OXPHOS whilst differentiated cells possess elongated mitochondria with well-developed cristae capable of supporting OXPHOS. Consequently, as shown in Figure 4C, murine ESC extract containing immature mitochondria enhanced embryonic development in porcine oocytes and their development to blastocyst whilst murine somatic cell extract containing more mature mitochondria had an adverse effect on preimplantation development. This outcome is supported by other reports, which, for example have demonstrated that development rates to blastocyst of murine parthenogenetically activated oocytes supplemented with murine somatic mitochondria were lower than in non-supplemented controls and those supplemented with oocyte cytoplasm. In the iSCNT-supplemented embryos, there were slight decreases in overall mtDNA copy number between the 2 and 4 cell stages of preimplantation development. However, murine mtDNA levels remained constant during this period with levels then increasing throughout the remaining stages of preimplantation development.