Reproductive efficiency using somatic cell nuclear transfer (SCNT) technology remains suboptimal. Of the various efforts to improve the efficiency, chromatin transfer (CT) and clone-clone aggregation (NTagg) have been reported to produce live cloned animals. To better understand the molecular mechanisms of somatic cell reprogramming during SCNT and assess the various SCNT methods on the molecular level, we performed gene expression analysis on bovine blastocysts produced via standard nuclear transfer (NT), CT, NTagg, in vitro fertilization (IVF), and artificial insemination (AI), as well as on somatic donor cells, using bovine genome arrays. The expression profiles of SCNT (NT, CT, NTagg) embryos were compared with IVF and AI embryos as well as donor cells. NT and CT embryos have indistinguishable gene expression patterns. In comparison to IVF or AI embryos, the number of differentially expressed genes in NTagg embryos is significantly higher than in NT and CT embryos. Genes that were differentially expressed between all the SCNT embryos and IVF or AI embryos are identified. Compared to AI embryos, more than half of the genes found deregulated between SCNT and AI embryos appear to be the result of in vitro culture alone. The results indicate that although SCNT methods have altered differentiated somatic nuclei gene expression to more closely resemble that of embryonic nuclei, combination of insufficient reprogramming and in vitro culture condition compromise the developmental potential of SCNT embryos. This is the first set of comprehensive data for analyzing the molecular impact of various nuclear transfer methods on bovine pre-implantation embryos.

Pre-implantation embryos produced by somatic cell nuclear transfer (SCNT) have varied developmental potentials. The majority of SCNT blastocysts do not develop to term, and the mechanisms inhibiting development are still largely unknown. Aggregation of cloned embryos has been attempted to compensate for the developmental deficiency of individual cloned embryos. In this report, we investigated the impact of aggregation of bovine cloned embryos at the four-cell stage on in vitro development and gene expression of the embryos. Cell numbers and development rate of aggregated (NTagg) and non-aggregated (NT) blastocysts were characterized and compared. The blastocyst formation after aggregation was modeled using the binominal distribution. The results indicate that aggregation enhances the blastocyst formation but does not increase the overall blastocyst rate. Additionally, utilizing microarray gene chip analysis 8.8% of 8,059 genes analyzed were differentially expressed between NTagg and NT blastocysts, with more than 80% of the differentially expressed genes up-regulated in the NTagg blastocysts. Up-regulated genes include those involved in transcription, biosynthesis and signaling such as TDGF1, HNFA, CAV1, GLU5, and CD81. Our results indicate that aggregation of bovine cloned embryos at an early stage promotes the in vitro development of the resulting pre-implantation embryos.

Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

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Somatic cell nuclear transfer (SCNT) technology is a powerful tool for preservation and propagation of superior genetics of livestock animals. Bovine oocytes derived from abattoirs are usually used in SCNT embryo production. The puncture of the zona pellucida during the nuclear transfer process has raised additional concerns about the risk of disease transmission through application of this new technology. The objective of this study was to use bovine viral diarrhea virus (BVDV) as a model to perform a comprehensive risk assessment on infectious disease transmission in the SCNT system. Thirteen batches of cumulus–oocyte complexes (COC; n = 550) were collected from several abattoirs over 6 months. Two hundred were tested for BVDV before cumulus cell removal. Cumulus cells were removed from the other 350 COC by gentle vortexing in 0.1% hyaluronidase in HEPES-M199 with Hanks’ salts. The cumulus-cell-free oocytes (CFO) were then washed 3 times with FBS-free HEPES-M199 with Hanks’ salts containing penicillin-streptomycin solution (5 μg mL–1). Both COC and CFO were pooled in groups (5/group) and tested for presence of BVDV using sensitive real-time PCR method. Only 2.5% of the COC were BVDV positive and all of the CFO were BVDV negative. Additionally, 293 embryos were produced from 14 different cell lines using the previously described SCNT procedure (Zhou et al. 2008 Mol Reprod Dev. 75, 1281–1289). These embryos were generated using in vitro-matured oocytes collected as 23 different batches over 7 months. The embryos were cultured in vitro to blastocyst stage and then tested for BVDV. All of the 293 SCNT embryos (100%) were BVDV negative. In conclusion, under these SCNT embryo production conditions, a small portion of COC were BVDV positive. However, all CFO and SCNT embryos were BVDV negative. Therefore, the risk of disease transmission using abattoir oocytes and SCNT procedure is relatively low and can be eliminated by using a combination of cumulus cell removal and adequate oocyte washing procedures. F. Arenivas, B. Findeisen, V. Farrar, E. Hwang helped with the SCNT embryo production for this study.