32 NEXT-GENERATION SEQUENCING DISCLOSES DIFFERENCES IN microRNA EXPRESSION PROFILES OF BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED BY HAND-MADE CLONING AND IN VITRO FERTILIZATION

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.