Blastocyst Cell Number and Allocation Affect the Developmental Potential and Transcriptome of Bovine Somatic Cell Nuclear Transfer Embryos

Cloning cattle using somatic cell nuclear transfer (SCNT) is inefficient. Although the rate of development of SCNT embryos in vitro is similar to that of fertilized embryos, most fail to develop into healthy calves. In this study, we aimed to identify developmentally competent embryos according to blastocyst cell composition and perform transcriptome analysis of single embryos. Transgenic SCNT embryos expressing nuclear-localized HcRed gene at day 7 of development were imaged by confocal microscopy for cell counting and individually transferred to recipient heifers. Pregnancy rates were determined by ultrasonography. Embryos capable of establishing pregnancy by day 35 had an average of 117 ± 6 total cells, whereas embryos with an average of 128 ± 5 cells did not establish pregnancy (P < 0.05). A lesser average number of 41 ± 3 cells in the inner cell mass (ICM) also resulted in pregnancies (<0.05) than a greater number of 48 ± 2 cells in the ICM. Single embryos were then subjected to RNA sequencing for transcriptome analysis. Using weighted gene coexpression network analysis, we identified clusters of genes in which gene expression correlated with the number of total cells or ICM cells. Gene ontology analysis of these clusters revealed enriched biological processes in coenzyme metabolic process, intracellular signaling cascade, and glucose catabolic process, among others. We concluded that SCNT embryos with fewer total and ICM cell numbers resulted in greater pregnancy establishment rates and that these differences are reflected in the transcriptome of such embryos.