24Developmental competence determines the oocyte capacity to support initial embryo 25 growth, but the molecular mechanisms underlying this phenomenon are still ill-26 defined. Changes in microRNA (miRNA) expression pattern have been described 27 during follicular growth in several species. Therefore, aim of this study was to 28 investigate whether miRNA expression pattern in cow oocyte and follicular fluid (FF) 29 is associated with the acquisition of developmental competence. Samples were 30 collected from ovaries with more than, or fewer than, 10 mid-antral follicles (H-and 31 L-ovaries) because previous studies demonstrated that this parameter is a reliable 32 predictor of oocyte competence. After miRNA deep sequencing and bioinformatic 33 data analysis, we identified 58 miRNAs in FF and 6 in the oocyte that were 34 differentially expressed between H-and L-ovaries. Overall, our results indicate that 35 miRNA levels both in FF and in the ooplasm must remain within specific thresholds 36 and that changes in either direction compromise oocyte competence. Some of the 37 miRNAs found in FF (miR-769, miR-1343, miR-450a, miR-204, miR-1271 and miR-38 451) where already known to regulate follicle growth and their expression pattern 39 indicate that they are also involved in the acquisition of developmental competence. 40Some miRNAs were differentially expressed in both compartments but with opposite 41 patterns, suggesting that miRNAs do not flow freely between FF and oocyte. Gene 42Ontology analysis showed that the predicted gene targets of most differentially 43 expressed miRNAs are part of a few signalling pathways. Regulation of maternal 44 mRNA storage and mitochondrial activity seem to be the processes more 45 functionally relevant in determining oocyte quality. In conclusion, our data identified a 46 few miRNAs in the follicular fluid and in the ooplasm that modulate the oocyte 47Recently, microRNAs (miRNAs), which regulate gene expression at the mRNA level, 65 have been associated with folliculogenesis and oogenesis [10, 11]. MiRNAs, which 66 range in size from 18 to 25 nucleotides (nt), have been found in the different 67 compartments of ovarian follicles, including granulosa cells [12, 13], theca cells [14], 68 follicular fluid and the oocyte itself [15]. Studies on the role of miRNAs during follicle 69 development in humans [16][17][18], mice [19, 20], cattle [10, 21, 22], pigs [23] and 70horses [24] suggest that they regulate the cellular differentiation processes which 71 occur during follicular development. 72 Follicular fluid and germinal vesicle oocyte collection 111Ovaries were collected at a commercial abattoir and were transported to the 112 laboratory in warmed (27-30°C) Dulbecco Phosphate Buffered Saline (PBS). Ovaries 113 were classified into low and high antral follicle count categories according to the 114 methods used in previous works [28, 29]. Briefly, the ovaries were assigned to high 115 antral follicle count ovaries (H ovaries) when more than 10 mid-antral follicles (2-5 116 mm in dia...

Endurance exercise induces metabolic adaptations and has recently been reported associated with the modulation of a particular class of small noncoding RNAs, microRNAs, that act as post-transcriptional regulators of gene expression. Released into body fluids, they termed circulating miRNAs, and they have been recognized as more effective and accurate biomarkers than classical serum markers. This study examined serum profile of miRNAs through massive parallel sequencing in response to prolonged endurance exercise in samples obtained from four competitive Arabian horses before and 2 h after the end of competition. MicroRNA identification, differential gene expression (DGE) analysis and a protein-protein interaction (PPI) network showing significantly enriched pathways of target gene clusters, were assessed and explored. Our results show modulation of more than 100 miRNAs probably arising from tissues involved in exercise responses and indicating the modulation of correlated processes as muscle remodeling, immune and inflammatory responses. Circulating miRNA high-throughput sequencing is a promising approach for sports medicine for the discovery of putative biomarkers for predicting risks related to prolonged activity and monitoring metabolic adaptations.

Lactation induces changes in the metabolic status of postpartum dairy cows that negatively affects the likelihood of pregnancy establishment. At present, pregnancy diagnosis with confidence is only possible after the third week of gestation. Earlier diagnosis could facilitate earlier re-breeding, reduce calving intervals, and improve profits for the industry. MicroRNAs (miRNAs) released in body fluids have been identified as minimally invasive biomarkers of several diseases. In addition, distinct miRNA profiles have been directly related to specific stages of human pregnancy. The aim of this study was to profile circulating miRNAs in the blood of high-producing dairy cows in order to identify biomarkers of early pregnancy. In-calf primiparous Holstein-Friesian cows (n = 22) with a similar economic breeding index were used. At calving, cows were randomly assigned to one of two groups: (1) lactating (n = 11; milked twice per day) or (2) non-lactating (n = 11; dried off immediately). Around 65 to 75 days postpartum (dpp), oestrous cycles were synchronized and a single embryo from a superovulated Holstein-Friesian donor was transferred at Day 7 post-oestrus. Plasma samples were analysed at Day 13 (initiation of conceptus elongation) and at Day 19 (initiation of implantation). Pregnancy rate, established by the presence of conceptus at Day 19, was 5/11 (45%) for lactating and 8/11 (73%) for non-lactating cows, respectively. Circulating miRNA levels were profiled in 4 animals per group in non-lactating pregnant, and lactating pregnant and nonpregnant cows at selected timepoints using Illumina HiSEqn 2000 (Illumina Inc., San Diego, CA, USA) for smallRNA sequencing. Annotation and discovery of miRNAs were done using MirDeep2, and read counts were analysed using edgeR to identify differentially expressed miRNAs. Target genes analysis was run with miRWalk and pathways interactions were built using Cytoscape (P ≤ 0.05). Differentially abundant miRNAs between lactating and non-lactating cows were found at both time points (FDR ≤ 0.05). At Day 13, non-lactating cows had a distinct miRNA profile compared with lactating cows showing 8 differentially expressed miRNA (6 v. pregnant and 2 v. nonpregnant cows). At Day 19, no significant differences were found within pregnant cows, but 5 differentially expressed miRNAs were identified between pregnant and nonpregnant cows, regardless of metabolic status. Interestingly, one miRNA, bta-mir140, was up-regulated in non-lactating pregnant cows from Day 13 onwards compared with nonpregnant cows. Furthermore, the same miRNA was up-regulated in lactating pregnant v. nonpregnant at Day 19. Among bta-mir140 target genes, CD274, SLC44A4, CXCL12, and SIRPA were strictly associated with immune tolerance. In conclusion, the maternal plasma miRNome may represent an early indicator of pregnancy status. In particular, the up-regulation of bta-mir140 in pregnant cows suggests that this miRNA may be a good candidate as an early biomarker of fertility. Furthermore, the positive correlation between this miRNA and pathways involved in T-cell response may indicate a role of immune tolerance in preventing rejection at the initiation of implantation.

Oocyte developmental competence is correlated with antral follicle count through ill-defined mechanisms. Oocytes from ovaries with fewer than 10 mid-antral follicles of 2 to 6 mm in diameter (low group) show reduced competence compared with those from ovaries with more than 10 follicles (high group). To unravel mechanisms underlying this phenomenon, this work explored the role of follicular fluid microRNAs (miRNAs, short non-coding RNAs regulating gene expression at the post-transcriptional level). A total of 3 pools of 300 µL of follicular fluid (FF) were collected from mid-antral follicles of low (L) and high (H) groups, respectively. Following miRNA extraction and library preparation, deep sequencing was carried out on Illumina HisEqn 2000 (Illumina Inc., San Diego, CA, USA). Differentially expressed miRNAs were identified with R package edgeR (http://bioconductor.org/packages/release/bioc/html/edgeR.html). Target genes of differentially expressed miRNAs were predicted with DIANA miRPath using homologous human miRNA and gene union options (P < 0.01). Gene ontology (GO) analysis was carried out by Cytoscape (http://www.cytoscape.org/) using a bovine database. In total, 1279 miRNAs were identified in FF: 805 ± 139 in L and 862 ± 36 in H (P > 0.05). We found that 27 miRNAs were differentially expressed (false discovery rate ≤0.001): 17 were up-regulated in L and 10 in H. Up-regulated miRNAs in L group were predicted to target 121 genes, 39 of which are specific for ovarian function (e.g. BCL2, FOXO3, KIT, TP53, and PTK2). The GO analysis indicated that these genes were kinases, anti-apoptotic and oncogenic factors, and enriched stress-activated MAPK cascade mediated by oxygen reactive species, G1/S transition checkpoint, cellular response to interleukin-1, and negative regulation of cellular adhesion. Overexpressed miRNAs in H group were predicted to target 92 genes, 22 of which (e.g. MAPK, APC, JNK, PKA) are involved in folliculogenesis. These genes were represented by kinases, apoptotic and cytoskeleton remodelling factors, and enriched very important ovarian processes such as cell cycle, ephrin receptor, smoothened and phosphatidylinositol 3-kinase activity GO processes. Only 7 target genes were common between the 2 groups, 2 of which were important in ovarian functionality (CDKN1A and ITGA5). Interestingly, overexpressed miRNAs in both groups regulate several genes involved in processes apparently not related to folliculogenesis. Finally, regulation can be exerted also by low levels of specific miRNAs such as miR-320, which was reduced in L group and is known to be associated with premature ovarian senescence in women and decreased developmental potential of mouse oocytes. Our results indicate that the different oocyte quality is associated with a different miRNA blueprint, which may alter the expression of several genes relevant for intra- and extra-ovarian processes. Further studies will be necessary to determine if FF miRNAs can act outside the ovary and if their levels can be detected in the bloodstream thereby becoming possible noninvasive, real-time markers to determine oocyte quality in living animals. This study was supported by FP7-KBBE-2012-FECUND-312097.