105 Improving embryo parthenogenetic development in pigs by blastomere exchange

Chromosomal errors and epigenetic imprinting problems have been observed during the in vitro production of parthenogenetic embryos. Despite their inability to lead to pregnancies with full-term development, these embryos serve as valuable models for other techniques, such as SCNT. Parthenogenetic embryos were used to obtain preliminary results on the effect of blastomere exchange (BE). In recent years, our group has developed the technology of embryonic aggregation following SCNT using the microwell system with intimate contact between zona-free blastomeres, which seems to improve developmental outcomes. However, the BE between embryos was not evaluated, which could optimize the number of required oocytes. BE between different embryos may compensate for epigenetic deviation. Therefore, an experiment was performed in which 2-cell parthenogenetic embryos were desegregated, and their blastomeres were individually combined in a microwell with 2 or 4 blastomeres from different embryonic origins. To do that, cumulus–oocyte complexes were obtained from pig ovaries and subsequently matured in vitro for 44 h, followed by cumulus denudation and activation through electric pulses. After 24 h of culture, embryos were incubated in 1.5 mg/mL pronase (P-8811) in TALP-H for 1–2 min to remove the zona pellucida (ZP). Two-cell embryos were then desegregated and combined in the microwell system with blastomeres from different parthenogenetic embryos. The embryos were distributed into four treatment groups: two blastomeres per well (2B group), four blastomeres per well (4B group), zona-free control group (parthenogenetic embryos, ZP control), and a control group (zona-included parthenogenetic embryos, control group). All experimental groups were cultured in vitro in porcine zygote medium (PZM-3; Yoshioka et al., 2002 Biol. Reprod. 66, 112–119) 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. Results are shown in Table 1. Significant differences were observed in blastocyst rates per well among the groups (Table 1). The 2B group showed poor outcomes, possibly due to the manipulation and lack of contact between blastomeres in the microwell. In contrast, the 4B group exhibited significant greater blastocyst development in comparison with the 2B group, suggesting compensation for chromosomal and epigenetic errors. In conclusion, the exchange of parthenogenetic blastomeres holds the potential to enhance the efficiency of in vitro embryo production, potentially reducing the number of required oocytes and embryos. Further experiments are needed to validate this improvement in SCNT embryo production.