Across farm animal species, the live birth rate obtained with somatic cell nuclear transfer (SCNT) embryos is only <2% compared with >40% obtained with in vitro fertilization (IVF) embryos, primarily due to incomplete nuclear reprogramming which results in aberrant embryonic gene expression. We used RNA sequencing to compare the global transcriptome profile of SCNT and IVF buffalo blastocysts. SCNT blastocysts expressed 17,061 transcripts, of which 941 were unique whereas, IVF blastocysts expressed 17,303 transcripts, of which 1,183 were unique. At ≥2‐folds change (p < .05), 331 transcripts were differentially expressed in the two groups among which, 19 were unique, 188 were downregulated and 143 were upregulated in SCNT compared with IVF blastocysts. Many genes affecting pluripotency, trophectoderm development, developmental regulation, and epigenetic modifications were upregulated in SCNT compared with IVF blastocysts. Among the four functional categories analyzed, epigenetic regulators were the most affected. Most of the WNT signaling pathway genes were upregulated whereas, the inhibitors of this pathway, such as DKK1, were downregulated in SCNT blastocysts, suggesting that this pathway is overexpressed in SCNT embryos. Gene Ontology analysis revealed that 25 biological processes, 20 molecular functions, and 24 cellular compartment categories were enriched in SCNT blastocysts. This data can help identify reprogramming errors for improving cloning efficiency.

We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes. Based on gene stability measure (M value) and pairwise variation (V value), calculated by geNorm analysis, the most stable ICGs were RPS15, HPRT1, and ACTB for cloned blastocysts, HMBS, UBC, and HPRT1 for IVF blastocysts and RPS15, GAPDH, and HPRT1 for both the embryo types analyzed together. RPS18 was the least stable gene for both cloned and IVF blastocysts. Following NormFinder analysis, the order of stability was RPS15 = HPRT1>GAPDH for cloned blastocysts, HMBS = UBC>RPS23 for IVF blastocysts, and HPRT1>GAPDH>RPS15 for cloned and IVF blastocysts together. These results suggest that despite overlapping of the three most stable ICGs between cloned and IVF blastocysts, the panel of ICGs selected for normalization of qPCR data of cloned and IVF blastocyst-stage embryos should be different.

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Mammalian embryo development is a complex process with a series of critical events taking place at every stage of development. It is an established fact that the birth rate of animals produced by nuclear transfer (NT) is far less (<2%) than that of IVF embryos (40%) after successful embryo transfers in different farm animal species. Micro(mi)RNAs are small non-coding RNAs of 17 to 25 nucleotides that alter the function of their target genes by either degrading them or inhibiting their expression. MiRNAs play a vital role during mammalian embryo development and may be adding to the extremely low birth rate and abnormalities in cloned animals. The present study was done with an objective of comparing the miRNA expression profiles of pre-implantation buffalo blastocysts produced by handmade cloning (HMC) and IVF. We hypothesised that there may be differences in the profiles of miRNAs expressed between the 2 groups that contribute to higher success rate in IVF group compared with HMC. Next-generation sequencing (NGS) was done to generate and compare the miRNA profiles and further discern the differentially expressed miRNAs between the 2 groups of blastocysts. For this study, NT blastocysts were produced using fibroblast donor cells isolated from ear skin of a buffalo bull. To produce genetically half-identical IVF blastocysts, the semen of the same bull was used. The oocytes used for generation of both HMC and IVF blastocysts were aspirated from buffalo ovaries obtained from abattoir. Total RNA was isolated from HMC and IVF blastocysts in 4 pools of each group. Each pool consisted of 40 blastocysts. A MiRNA cDNA library was prepared which was then subjected to NGS on Illumina HiSEqn 2000 (Illumina Inc., San Diego, CA, USA). Bos taurus genome was taken as reference to align the reads generated. The data from NGS was validated by RT-qPCR, taking 10 miRNAs (mir-15a, mir-23a, mir-128, mir-130a, mir-133a, mir-194, mir-196b, mir-200b, mir-431 and mir-451). The results positively validated the NGS data. Differential expression analysis of miRNAs between the 2 types of blastocysts revealed that the number of differentially expressed miRNAs with fold change of ≥ 2.0 were 74, with 52 miRNAs up-regulated in HMC and 22 miRNAs up-regulated in IVF. At significance level of P < 0.2, there were 2 miRNAs (mir-202 and mir-133a) that were uniquely expressed in IVF blastocysts and 8 miRNAs (mir-219, mir-451, mir-497, mir-33a, mir-2448, mir-592, mir-187, and mir-502a) that were uniquely expressed in HMC blastocysts. According to the gene ontology analysis, mir-202 is involved in negative regulation of apoptosis and positive regulator of cell growth, whereas mir-133a is involved in generating immunity. Absence of mir-202 and mir-133a expression from HMC blastocysts may be contributing to high apoptosis and other abnormalities in them compared with IVF counterparts. The NGS results indicate that the miRNA profiles of HMC and IVF blastocysts show huge differences. Further analysis of these differentially expressed miRNAs may open the door to miRNA therapies for treating the HMC blastocysts by regulating the expression of critical miRNAs in HMC blastocysts, thereby improving the success rate of cloning.

Inhibition of ERK/MAPK pathway has been shown to decrease DNA methylation via down-regulation of DNA methyltransferases (DNMTs) in several studies suggesting that this pathway plays an important role in regulation of DNA methylation. We examined the relative expression level of seven important genes related to ERK/MAPK pathway and DNMTs (DNMT1, DNMT3a and DNMT3b) by quantitative real-time PCR in buffalo blastocysts produced by Hand-made cloning and compared it with that in blastocyst-stage embryos produced by in vitro fertilization (IVF). The expression level of six of seven genes related to ERK/MAPK pathway examined i.e., p21RAS, RAF1, AKT1, ERK2, PIK3R2 and c-Myc was significantly higher (p < 0.05) in cloned than in IVF embryos. However, the expression level of FOS was lower (p < 0.005) in cloned than in IVF embryos. The relative expression level of DNMT3a and DNMT3b but not that of DNMT1 was significantly higher (p < 0.05) in cloned than in IVF embryos. These results indicate that the cloned embryos exhibit an abnormal expression of several important genes related to ERK/MAPK pathway and DNMTs. Although a direct link between ERK/MAPK pathway and DNMTs was not examined in the present study, it can be speculated that ERK/MAPK pathway may have a role in regulating the expression of DNMTs in embryos, as also observed in other tissues.