23 Improving SCNT development in pigs by blastomere exchange

Although somatic cell nuclear transfer (SCNT) technology was developed more than 20 years ago, cloning efficiency remains low. Failures in the reprogramming of the donor cell result in embryos with aberrant epigenetic patterns and low developmental rates. Chromosomal errors and epigenetic imprinting problems have been observed during SCNT production of embryos. In recent years, our group has developed the technology of embryonic aggregation following SCNT using the microwell system, which seems to improve developmental outcomes. However, blastomere exchange (BE) between embryos was not evaluated, which could optimize the number of required oocytes with different epigenetic origins. BE between different embryos may compensate for epigenetic deviation. Therefore, an experiment was performed in which 2-cell SCNT embryos were disaggregated, and their blastomeres were individually combined in a microwell with 4 blastomeres from different embryonic origins. To do this, clone embryos were produced using porcine oocytes, and oocyte collection, IVM, and SCNT procedures were performed as reported. Twenty-four hours after activation, 2-cell embryos were then disaggregated and combined in the microwell system with blastomeres from different SCNT embryos. The embryos were distributed into four treatment groups: one embryo per microwell control group (1E group), two embryos per microwell (2E group), four blastomeres per microwell (4B group), and a zona-free control group (parthenogenetic embryos without BE, ZP control). All experimental groups were cultured in vitro in porcine zygote medium (PZM-3) for 7 days at 38.5°C and 5% CO₂ in a humidified atmosphere, and cleavage and blastocyst rates were recorded. Statistical analysis was conducted using a chi-squared test, with significance set at P < 0.05. The cleavage rates at 24 h were as follows: 1E (2/22, 9%), 2E (8/47, 17%), 4B (4/22, 18%), and control (35/59, 59%). The blastocyst rates per microwell at Day 7 were 1E (11/22, 50%), 2E (58/94, 61%), 4B(14/22, 63%), and ZP control (12/59, 20%). No significant differences were observed between groups, suggesting that with a smaller number of reconstituted embryos, we can achieve the same percentage of blastocysts but with four different epigenetic origins, assuming they will better compensate for epigenetic errors arising from reprogramming. In conclusion, the exchange of SCNT blastomeres holds the potential to enhance the efficiency of in vitro embryo production possibly due to epigenetic compensation derived from different embryonic origins. This process may potentially reduce the number of oocytes and embryos required. In the future, an evaluation of the different epigenetic profiles should be carried out. Further, embryo transfers still need to be performed to confirm this improvement in SCNT embryo production.