Effect of additional cytoplasm injection on the cloned bovine embryo organelle distribution and stress mitigation

Although somatic cell nuclear transfer (SCNT) is a critical component of animal cloning, this approach has several issues. We previously introduced the cytoplasm injection cloning technology (CICT), which significantly improves the quality and quantity of cloned embryos. This study examined the residual status of fused cytoplasmic organelles, such as the endoplasmic reticulum (ER) and lysosomes, in the CICT group during early embryo development. We found that extra-cytoplasmic organelles stained using the ER-Tracker™ Green dye and LysoTracker™ Deep Red probe were fused and dispersed throughout the recipient oocyte and were still visible in day 8 blastocysts. We screened for ER stress, autophagy, and apoptosis-related genes to elucidate the association between the added organelles and improved embryo quality in CICT-cloned embryos. We found that CHOP, ATF4, ATG5, ATG7, and LC3 genes showed non-significantly up- or downregulated expression between CICT- and in vitro fertilization (IVF)-derived embryos but showed significantly (p < 0.05) upregulated expression in SCNT-cloned embryos. Surprisingly, a non-significant difference in the expression of some genes, such as ATF6 and caspase-3, was observed between the CICT- and SCNT-cloned embryos. Our findings imply that compared to conventional SCNT cloning, CICT-derived cloned embryos with additional cytoplasm have much higher organelle activity, lower autophagy, lower rates of apoptosis, and higher embryo development rates.