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

PLOS ONE RESEARCH ARTICLE Association between blood miR-26a levels following artificial insemination, and pregnancy outcome in dairy cattle Thomas Tzelos¤, Seungmee Lee, Alex Pegg, F. Xavier Donadeu ID* The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Tzelos T, Lee S, Pegg A, Donadeu FX (2023) Association between blood miR-26a levels following artificial insemination, and pregnancy outcome in dairy cattle. PLoS ONE 18(8): e0289342. https://doi.org/10.1371/journal. pone.0289342 Editor: Muhammad Mazhar Ayaz, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, PAKISTAN Received: March 14, 2023 Accepted: July 8, 2023 Published: August 11, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0289342 Copyright: © 2023 Tzelos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ¤ Current address: Moredun Research Institute, Pentlands Science Park, Penicuik, United Kingdom * Abstract 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. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. PLOS ONE | https://doi.org/10.1371/journal.pone.0289342 August 11, 2023 1 / 11 PLOS ONE Funding: This study was funded by a Biotechnology and Biological Sciences Research Council award to FXD (BB/T004037/1) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. miR-26 and early pregnancy Introduction Early pregnancy diagnosis is key to maximize reproductive efficiency in precision animal farming. This is particularly important in dairy cattle since pregnancy rates in modern commercial herds continue to be unacceptably low (typically �40%), with an average of three services required per pregnancy [1]. Most pregnancy losses in high-producing dairy cows occur during the first three weeks following insemination [2]. Failure to promptly re-inseminate before the next oestrus, i.e. within 21 days post-insemination, leads to extended calving intervals and a significant reduction in overall herd milk production [3]. Critically, even with more recent advances such as electronic monitoring system, detection of oestrus in dairy farms remains overall inefficient [4], and confirmation of pregnancy using available tools such as transrectal ultrasonography or PAG detection in blood or milk is only possible at 24 days or later. As a result, opportunities to re-inseminate cows that fail to become pregnant after service are often missed unnecessarily, with important consequences for reproductive efficiency and milk productivity. Detection of Early Pregnancy Factor, Interferon Stimulated Genes (ISGs) and pregnancy-associated miRNAs have been proposed for pregnancy diagnosis before 21 days in cattle, although the accuracy of such assays for discriminating successful from failed pregnancies has been shown to be limited or, in the case of miRNAs, has not been evaluated properly (reviewed by [5]). Body fluid miRNAs provide distinct advantages as tissue function biomarkers. Clinical applications using biofluid miRNA levels as diagnostic biomarkers exist or are being developed for a wide array of human diseases, and applications have also actively been explored in animals [6,7], for example, for pregnancy detection in cattle [8–12]. Our group was the first to provide a detailed characterisation of the bovine blood miRNome and to report changes in circulating miRNA profiles associated with early pregnancy in cattle [8]. Using RNA-sequencing in dairy heifers we identified 77 miRNAs which levels changed significantly in plasma during the first 60 days of pregnancy [9]. Of note, a consistent finding in our previous studies was an increase in miR-26a levels during early pregnancy, which could be detected as soon as day 8 after insemination but not after sham-insemination in heifers [8,9]. Whether similar changes in miR-26a also occur during early pregnancy in lactating cows has not been established. Moreover, a distinct increase in levels of a related miRNA, miR-26b, during early pregnancy was separately reported in bovine blood [13] and milk [14] samples, whereas miR-26a levels increased in blood serum of day (...truncated)