71 CIRCULATING microRNAs AS POTENTIAL BIOMARKERS OF EARLY PREGNANCY IN HIGH-PRODUCING DAIRY COWS

Abstract

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.