38 FULL TERM DEVELOPMENT IN A COW CARRYING A NUCLEAR TRANSFER EMBRYO DERIVED FROM FIBROBLASTS AND OOCYTES OF ITS OWN CLONE

Abstract

Cloned animals are a unique model for studying the role of maternal immune response in failure of pregnancies often reported in bovine somatic cloning. Major histocompatibility complex class 1 (MHC1) has been shown to be down-regulated in placentomes during pregnancy, and abnormal MHC1 expression by the fetal membranes has been suggested to be the cause of pregnancy failure in bovine clones. We designed this study to investigate whether pregnancy losses would be decreased in cloned cows transferred with their genetically identical conceptus. Bovine fibroblasts were established from a skin biopsy on an adult Holstein cow from which a clone of 12 females had previously been derived. Five heifers of this set of clones were used for providing oocytes through (OPU) after stimulation, and seven were used as recipient heifers. Fibroblasts from this genotype were thawed, grown to confluency, and then used as the source of donor cells for NT. Recipient oocytes were matured in vitro before enucleation at 20–22 h post maturation (hpm). Embryos were reconstructed by fusion with donor cells at 23–24 hpm and activated in 10 µg/mL cycloheximide and 5 µg/mL cytochalasin B for 5 h after fusion, and then co-cultured on Vero cells for 7 days in microdrops of B2 medium. A control group of NT embryos was produced simultaneously with the same cell line and unrelated oocytes from slaughterhouse ovaries. Embryos were transferred into synchronous recipients (one blastocyst/recipient). The pregnancies were followed by ultrasonography. The reconstructed embryos fused at the same rate in both groups of oocytes, but the cleavage rate was significantly lower in the clone group (Table 1). The reason for this low rate is unclear. However, pregnancy rates after transfer of NT blastocysts were similar up to term between the related and non-related groups of transplanted embryos. These results suggest that the maintenance of pregnancy in bovine is not linked to genetic disparity between fetus and dam. More investigations are required to elucidate why the in vitro development was so low, and whether pregnancies established in cows with their genetically identical conceptus are less prone to abortion or physiopathological developments.

Table 1. Development of NT embryos with clone or unrelated cell/oocyte/ recipient genotype