33 BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED BY HAND-MADE CLONING AND IN VITRO FERTILIZATION DIFFER IN THEIR GLOBAL TRANSCRIPTOME PROFILE

Although the blastocyst rate obtained with nuclear transferred (NT) embryos is higher than that obtained following in vitro fertilization (IVF) in buffalo, the live birth rate of NT embryos is <2% across different farm animal species compared with a birth rate >40% obtained with IVF embryos. This is believed to be due primarily to incomplete or incorrect nuclear reprogramming of the donor somatic cell by the oocyte, which results in aberrant embryonic gene expression. We compared the global transcriptome profile of buffalo blastocysts produced by hand-made cloning (HMC) and IVF using next-generation sequencing (NGS) for discovering transcripts that are differentially expressed between the 2 types of embryos. NT blastocysts were produced using fibroblast donor cells obtained from ear skin of a buffalo bull. The semen of the same bull was used for producing genetically half-identical IVF blastocysts. Total RNA was isolated from 3 pools of Day 8 NT and IVF blastocysts, with each pool containing 40 blastocysts. Complementary DNA library was prepared and subjected to NGS on Illumina HiSEqn 2000 (Illumina Inc., San Diego, CA, USA). The reads generated were aligned to Bos taurus reference genome, UMD 3.1. Differential expression analysis between the 2 blastocysts types at a minimum of 2-fold change revealed that 5557 transcripts were differentially expressed, of which 584 were unique to NT blastocysts, 709 were unique to IVF blastocysts, and 4264 were expressed in both types of blastocysts. Among these transcripts, at a significance level of P < 0.05, 331 transcripts were differentially expressed between the 2 blastocyst types, of which 19 were unique, 188 were down-regulated, and 143 were up-regulated in NT blastocysts. One-way ANOVA with Benjamini and Hochberg false discovery rate (FDR) correction was applied to determine the statistically significant differentially expressed transcripts. Nine of the differentially expressed transcripts (at minimal 2-fold change, P < 0.05), from different functional classes (RELN, NDRG1, SULT1A1, MAP1LC3A, MTHFD1L, PCBD1, PPA2, MGST1 and PRPH) were subjected to quantitative real-time PCR analysis for validation of NGS data. Gene expression level of RELN, NDRG1, SULT1A1, MAP1LC3A, PPA2, MGST1, and PRPH was found to be up-regulated while that of MTHFD1L and PCBD1 was down-regulated (P < 0.05) in NT embryos compared with IVF embryos. This pattern and the magnitude of relative gene expression level were found to be similar to that observed in NGS. These results indicate that the gene expression profile of NT embryos is very different from that of their IVF counterparts. Further analysis of these differentially expressed transcripts can help in identification of gene functional classes and pathways that are affected by the inefficient reprogramming of donor nuclei in NT embryos. Normalizing the expression of some of the differentially expressed genes may help in improving the cloning efficiency.