Implications of miRNA expression pattern in bovine oocytes and follicular fluids for developmental competence

Developmental competence determines the oocyte capacity to support initial embryo growth, but the molecular mechanisms underlying this phenomenon are still ill-defined. Changes in microRNA (miRNA) expression pattern have been described during follicular growth in several species. Therefore, aim of this study was to investigate whether miRNA expression pattern in cow oocyte and follicular fluid (FF) is associated with the acquisition of developmental competence. Samples were collected from ovaries with more than, or fewer than, 10 mid-antral follicles (H- and L-ovaries) because previous studies demonstrated that this parameter is a reliable predictor of oocyte competence. After miRNA deep sequencing and bioinformatic data analysis, we identified 58 miRNAs in FF and 6 in the oocyte that were differentially expressed between H- and L-ovaries. Overall, our results indicate that miRNA levels both in FF and in the ooplasm must remain within specific thresholds and that changes in either direction compromising oocyte competence. Some of the miRNAs found in FF (miR-769, miR-1343, miR-450a, miR-204, miR-1271 and miR-451) where already known to regulate follicle growth and their expression pattern indicate that they are also involved in the acquisition of developmental competence. Some miRNAs were differentially expressed in both compartments but with opposite patterns, suggesting that miRNAs do not flow freely between FF and oocyte. Gene Ontology analysis showed that the predicted gene targets of most differentially expressed miRNAs are part of a few signalling pathways. Regulation of maternal mRNA storage and mitochondrial activity seem to be the processes more functionally relevant in determining oocyte quality. In conclusion, our data identified a few miRNAs in the follicular fluid and in the ooplasm that modulate the oocyte developmental competence. This provides new insights that could help with the management of cattle reproductive efficiency

Profiling bovine blastocyst microRNAs using deep sequencing

MicroRNAs (miRNAs) are known to control several reproductive functions, including oocyte maturation, implantation and early embryonic development. Recent advances in deep sequencing have allowed the analysis of all miRNAs of a sample. However, when working with embryos, due to the low RNA content, miRNA profiling is challenging because of the relatively large amount of total RNA required for library preparation protocols. In the present study we compared three different procedures for RNA extraction and prepared libraries using pools of 30 bovine blastocysts. In total, 14 of the 15 most abundantly expressed miRNAs were common to all three procedures. Furthermore, using miRDeep discovery and annotation software (Max Delbr\ufcck Center), we identified 1363 miRNA sequences, of which bta-miR-10b and bta-miR-378 were the most abundant. Most of the 179 genes identified as experimentally validated (86.6%) or predicted targets (13.4%) were associated with cancer canonical pathways. We conclude that reliable analysis of bovine blastocyst miRNAs can be achieved using the procedures described herein. The repeatability of the results across different procedures and independent replicates, as well as their consistency with results obtained in other species, support the biological relevance of these miRNAs and of the gene pathways they modulate in early embryogenesis

The buffalo genome and the application of genomics in animal management and improvement

The publication of the human genome sequence in 2001 was a major step forward in knowledge necessary to understand the variations between individuals. For farmed species, genomic sequence information will facilitate the selection of animals optimised to live, and be productive, in particular environments. The availability of cattle genome sequence has allowed the breeding industry to take the first steps towards predicting phenotypes from genotypes by estimating a "genomic breeding value" (gEBV) for bulls using genome-wide DNA markers. The sequencing of the buffalo genome and creation of a panel of DNA markers has created the opportunity to apply molecular selection approaches for this species. The genomes of several buffalo of different breeds were sequenced and aligned with the bovine genome, which facilitated the identification of millions of sequence variants in the buffalo genomes. Based on frequencies of variants within and among buffalo breeds, and their distribution across the genome compared with the bovine genome, 90,000 putative single nucleotide polymorphisms (SNP) were selected to create an Axiom\uae Buffalo Genotyping Array 90K. This "SNP Chip" was tested in buffalo populations from Italy and Brazil and found to have at least 75% high quality and polymorphic markers in these populations. The 90K SNP chip was then used to investigate the structure of buffalo populations, and to localise the variations having a major effect on milk production

Sequencing of the Water Buffalo Genome: results and perspectives

The water buffalo (Bubalus bubalis) is important for small-holder producers in developing countries and an important source of products for specialized markets. Knowledge of the buffalo genome will help to unravel the evolutionary relationships among different breeds and will contribute to understanding the genetic control of phenotypic variation. Resulting genomic tools will help the genetic improvement of animals for milk and meat production. The International Buffalo Genome Project has three main phases: 1) the complete sequencing and assembly of the buffalo genome and its annotation; 2) SNP and variation detection in river and swamp buffalo genomes; 3) analysis of genetic variation among buffalo populations. The project has produced sequence for a female Mediterranean buffalo genome using Illumina GAII and Roche 454 high throughput sequencing. These sequences were assembled using the MaSuRCA assembler, obtaining a contig N50 of 21Kb and a scaffold N50 of 1.4Mb organized in 367K scaffolds, giving an estimated genome size of 2.7 Gbp. Eighty-six buffaloes from 8 breeds were sequenced to a depth between 5 and 12X by Illumina PE reads, yielding a total of 470X genome coverage. Four of these breeds (Mediterranean, Murrah, Jaffarabady and Nili Ravi) were analysed to yield a total of 22.2M SNPs. The within and across breeds allele frequencies for these SNPs were calculated and the minimum allele frequencies (MAF) were determined. These data were used to design an Axiom Buffalo Genotyping 90K array (Affymetrix). Preliminary comparative analyses of the buffalo genome assembly with the bovine sequence, highlights extensive conservation between genomes of cattle and buffalo, including a high degree of between species conservation of genomic and transcribed sequence. However, detailed analysis of the largest buffalo genome scaffolds suggests a complex history of genomic rearrangements since divergence. Finally, current estimates suggest that 13-18% of the buffalo genomic sequence is unique