104 Developmental competence of single blastomeres from bovine 8-cell stage embryos in monozygotic multiple blastocyst production

Based on inheritance of totipotency from zygote to early blastomeres, the maximum number of calves obtained through blastomere separation is quadruplets. However, the developmental competence of single blastomeres from 8-cell stage embryos and their potential to generate genetically identical calves remains unclear. In this study, we analysed the developmental competence of individual 2-, 4-, and 8-cell stage blastomeres in bovine embryos. In vitro-produced embryos were selected at 28, 36, and 48 h postinsemination for the 2-, 4-, and 8-cell stage, respectively (n = 42, 41, and 28). In the blastomere separation group (BS), the zona pellucida was removed with 0.05% pronase, the blastomeres were separated through pipetting, and cultured in a single blastomere. In the handling control group (HC), all blastomeres from the same embryo were reaggregated and cultured after separation (n = 86). Intact embryos were cultured in the control group (C; n = 88). Embryos of each group were cultured in time-lapse incubator in well-of-the-well culture dishes with CR1aa supplemented with 10% calf serum. Blastocyst cavity formation on Day 7 and the number of blastomeres at the onset of compaction was assessed retroactively using time-lapse cinematography (TLC). The DNA staining and immunofluorescence staining with anti-CDX2 or anti-SOX2 antibodies were performed on embryos with cavities on Day 8, and some of them were additionally subjected to RNA sequencing on Day 7. Both CDX2 and SOX2 were used as markers for trophectoderm (TE) and inner cell mass (ICM), respectively. The cavity formation rates were compared by chi-squared test. Cell counts at compaction were compared by t-test. The percentage of SOX2-positive cells to total cells (SOX2%) and gene expression were examined by one-way ANOVA followed by Tukey’s test, or Kruskal–Wallis test followed by Steel-Dwass test. P < 0.05 was considered statistically significant. The cavity formation rates of C, HC, 2, 4, and 8 BS were 78, 86, 77, 63, and 35%, respectively, and were significantly lower in 4 and 8 BS. The TLC revealed that cavitated embryos in the 4 and 8 BS had significantly more cells at compaction than noncavitated embryos (4 BS: 5.5 vs 3.8, 8 BS: 2.9 vs 2.5). The SOX2% was also significantly lower in 4 and 8 BS. Gene expression analysis indicated that changes in gene expression increased progressively from HC to 2, 4, and 8 BS, with particularly pronounced changes at 8 BS. Cell cycle-related pathways were frequently enriched in differential gene expression analysis between in HC and 8 BS. While TE-related gene expression levels did not differ significantly, ICM-related genes such as NANOG and OCT4 exhibited changes at 8 BS. In conclusion, when cultured in a single blastomere, blastomere competence for blastocyst formation is reduced from the 4-cell stage, and lineage differentiation in blastocysts is significantly impaired from the 8-cell stage. These findings emphasise the importance of cell-fate regulation in producing monozygotic multiplets using single blastomeres derived from 8-cell stage embryos.