Association between blood miR-26a levels following artificial insemination, and pregnancy outcome in dairy cattle

Early pregnancy diagnosis is key to maximise productivity of dairy herds. We previously showed that an increase in the levels of miR-26 could be detected as soon as day 8 of pregnancy in heifers. The aims of this study were to determine whether 1) plasma miR-26 levels would be distinctly elevated, retrospectively, early after artificial insemination in lactating cows with successful compared to failed pregnancies, 2) the early increase in miRNA levels in cows with successful pregnancy could be accounted for by changes in miRNA expression in white blood cells (WBCs), presumably induced by the effects of embryo-derived interferon tau (IFNt), and 3) plasma miRNA levels may provide a reliable early predictor of pregnancy that could be used at a herd level. Blood samples were taken from a total of 34 dairy cows (lactation number 1 to 4) before (D0) and 9 and 18 days after artificial insemination at oestrus, followed by confirmation of pregnancy status by ultrasound on D32. In addition, WBCs collected from non-pregnant cows (n = 4) were stimulated in vitro with recombinant ovine IFNt (0-100 pg/ml). Levels of miRNAs and ISG15, a known IFNt-induced gene, were quantified by qPCR. Relative to D0, a larger increase in plasma miR-26a (P = 0.04) occurred on D9 in cows later confirmed to be pregnant (n = 12) than in cows with a failed pregnancy (n = 22). Expression of miR-26a in WBCs was not affected (P>0.1) by pregnancy status or IFNt stimulation in vitro, in contrast to ISG15 expression which increased markedly (P<0.0001) both in WBC samples collected on D18 from animals later confirmed to be pregnant, and in WBCs after stimulation with IFNt in vitro. Finally, ROC analyses revealed that miR-26a on D9 or D18 could predict pregnancy outcome with much lower accuracy than WBC ISG15 on D18 (Likelihood ratio, 2.3 vs 15.4). In summary, a modest increase in plasma miR-26a levels occurs during early pregnancy in mature dairy cows which may not accounted for by changes in miRNA levels in WBCs or the effects of IFNt. Moreover, compared to ISG15, changes in miR-26a levels may not provide an accurate test for early diagnosis of pregnancy in cows.