The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.
PLoS One. 2023 Aug 11;18(8):e0289342. doi: 10.1371/journal.pone.0289342. eCollection 2023.
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.
早期妊娠诊断是最大限度提高奶牛群生产力的关键。我们之前的研究表明，在小母牛妊娠 8 天内就可以检测到 miR-26 水平的升高。本研究的目的是确定：1）与失败妊娠相比，成功妊娠的泌乳牛在人工授精后早期，血浆 miR-26 水平是否明显升高；2）成功妊娠牛中 miRNA 水平的早期升高是否可以用白细胞（WBC）中 miRNA 表达的变化来解释，推测是胚胎衍生的干扰素 tau（IFNt）的作用所致；3）血浆 miRNA 水平是否可以作为一种可靠的早期妊娠预测指标，可在牛群水平上使用。在发情期人工授精前（D0）和 9 天和 18 天，共采集了 34 头奶牛（泌乳次数 1 至 4）的血液样本，然后在 D32 通过超声确认妊娠状态。此外，还从非妊娠奶牛（n=4）中收集白细胞，并在体外用重组绵羊 IFNt（0-100pg/ml）刺激。通过 qPCR 定量测定 miRNA 和 ISG15 的水平，ISG15 是一种已知的 IFNt 诱导基因。与 D0 相比，在随后被证实怀孕的奶牛（n=12）中，D9 时血浆 miR-26a 的增加幅度更大（P=0.04），而在未怀孕的奶牛（n=22）中则没有增加。WBC 中 miR-26a 的表达不受妊娠状态或体外 IFNt 刺激的影响（P>0.1），而 ISG15 的表达则明显增加（P<0.0001），这两种情况均出现在随后被证实怀孕的动物的 D18 时采集的 WBC 样本中，以及在体外用 IFNt 刺激后的 WBC 中。最后，ROC 分析显示，D9 或 D18 时的 miR-26a 可以预测妊娠结局，但准确性低于 D18 时的 WBC ISG15（似然比，2.3 比 15.4）。综上所述，在成熟奶牛的早期妊娠期间，血浆 miR-26a 水平会适度升高，这可能不是由 WBC 中 miRNA 水平的变化或 IFNt 的作用引起的。此外，与 ISG15 相比，miR-26a 水平的变化可能无法为奶牛的早期妊娠诊断提供准确的检测。
