{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T20:16:02Z","timestamp":1789503362017,"version":"build-2803163510"},"reference-count":39,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2024,5,28]],"date-time":"2024-05-28T00:00:00Z","timestamp":1716854400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Japan Society for the Promotion of Science","award":["15K14838"],"award-info":[{"award-number":["15K14838"]}]},{"name":"Japan Society for the Promotion of Science","award":["18K05989"],"award-info":[{"award-number":["18K05989"]}]},{"name":"Japan Society for the Promotion of Science","award":["21K05933"],"award-info":[{"award-number":["21K05933"]}]},{"name":"Japan Society for the Promotion of Science","award":["R2-60"],"award-info":[{"award-number":["R2-60"]}]},{"name":"Ito Foundation","award":["15K14838"],"award-info":[{"award-number":["15K14838"]}]},{"name":"Ito Foundation","award":["18K05989"],"award-info":[{"award-number":["18K05989"]}]},{"name":"Ito Foundation","award":["21K05933"],"award-info":[{"award-number":["21K05933"]}]},{"name":"Ito Foundation","award":["R2-60"],"award-info":[{"award-number":["R2-60"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Circulating microRNAs (miRNAs) were investigated as biomarkers for the diagnosis of early pregnancy in cattle. The levels of prospective miRNA biomarkers and the features of extracellular vesicles (EVs) in the blood were evaluated. In Study 1, plasma samples from cows 21 days after artificial insemination (AI) were examined using RT-qPCR to determine the levels of seven circulating miRNAs. Only the levels of miR-126-3p were significantly lower in the pregnant group than in the non-pregnant group. In Study 2, among individuals not pregnant at the first AI, the miRNA levels were compared between the individuals pregnant at the second AI and those who remained non-pregnant. The miR-25 levels were significantly higher in the pregnant group at the second AI than in the pregnant group at the first AI; miR-19b, miR-27b, and miR-29a levels were also high. In the non-pregnant group, changes were absent in the miRNA levels in the same individual between the first and second AIs. In Study 3, Western blotting and RT-qPCR showed the presence of miRNAs in EVs and their levels were lower than in plasma. Thus, circulating miR-126-3p may serve as a biomarker for the diagnosis of early pregnancy in cattle. In addition, the expression of some miRNAs tended to be higher during pregnancy than during non-pregnancy in the same individual, suggesting their potential as an index to determine pregnancy and non-pregnancy rates using a comparative method.<\/jats:p>","DOI":"10.3390\/ani14111592","type":"journal-article","created":{"date-parts":[[2024,5,28]],"date-time":"2024-05-28T13:32:55Z","timestamp":1716903175000},"page":"1592","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Potential of Circulating miRNA Biomarkers and Exosomes for Early Pregnancy Diagnoses in Cattle"],"prefix":"10.3390","volume":"14","author":[{"given":"Chiaki","family":"Ninomiya","sequence":"first","affiliation":[{"name":"Cooperative Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Iwate, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hitomi","family":"Yoshino","sequence":"additional","affiliation":[{"name":"Graduate School of Veterinary Sciences, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Iwate, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toshina","family":"Ishiguro-Oonuma","sequence":"additional","affiliation":[{"name":"Cooperative Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Iwate, Japan"},{"name":"Graduate School of Veterinary Sciences, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Iwate, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kosuke","family":"Iga","sequence":"additional","affiliation":[{"name":"Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ikenodai 2, Tsukuba 305-8602, Ibaraki, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7643-0689","authenticated-orcid":false,"given":"Tomomi","family":"Kanazawa","sequence":"additional","affiliation":[{"name":"Cooperative Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Iwate, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1457-4967","authenticated-orcid":false,"given":"Toru","family":"Takahashi","sequence":"additional","affiliation":[{"name":"Cooperative Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Iwate, Japan"},{"name":"Graduate School of Veterinary Sciences, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Iwate, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2012-8533","authenticated-orcid":false,"given":"Keiichiro","family":"Kizaki","sequence":"additional","affiliation":[{"name":"Cooperative Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Iwate, Japan"},{"name":"Graduate School of Veterinary Sciences, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Iwate, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,5,28]]},"reference":[{"key":"ref_1","first-page":"367","article-title":"Pregnancy determination in cattle: A review of available alternatives","volume":"21","author":"Lucy","year":"2011","journal-title":"Proc. Appl. Reprod. Strateg. Beef Cattle"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/j.cvfa.2015.09.006","article-title":"Methods for and implementation of pregnancy diagnosis in dairy cows","volume":"32","author":"Fricke","year":"2016","journal-title":"Vet. Clin. N. Am. Food Anim. Pract."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"10292","DOI":"10.3168\/jds.2017-12959","article-title":"A 100-year review: Practical female reproductive management","volume":"100","author":"Stevenson","year":"2017","journal-title":"J. Dairy Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3766","DOI":"10.3168\/jds.2018-15668","article-title":"Symposium review: Immunological detection of the bovine conceptus during early pregnancy","volume":"102","author":"Ott","year":"2019","journal-title":"J. Dairy Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"958540","DOI":"10.1155\/2013\/958540","article-title":"Early pregnancy diagnosis in bovines: Current status and future directions","volume":"2013","author":"Balhara","year":"2013","journal-title":"Sci. World J."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"274","DOI":"10.3168\/jds.S0022-0302(07)72628-0","article-title":"Regulation of interferon-stimulated genes in peripheral blood leukocytes in pregnant and bred, nonpregnant dairy cows","volume":"90","author":"Gifford","year":"2007","journal-title":"J. Dairy Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1016\/j.anireprosci.2010.05.010","article-title":"Measurement of interferon-Tau (IFN-\u03c4) Stimulated Gene Expression in Blood leukocytes for Pregnancy Diagnosis within 18\u201320d after Insemination in Dairy Cattle","volume":"121","author":"Green","year":"2010","journal-title":"Anim. Reprod. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Kizaki, K., Shichijo-Kizaki, A., Furusawa, T., Takahashi, T., Hosoe, M., and Hashizume, K. (2013). Differential neutrophil gene expression in early bovine pregnancy. Reprod. Biol. Endocrinol., 11.","DOI":"10.1186\/1477-7827-11-6"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Matsuyama, S., Kojima, T., Kato, S., and Kimura, K. (2012). Relationship between quantity of IFNT estimated by IFN-stimulated gene expression in peripheral blood mononuclear cells and bovine embryonic mortality after AI or ET. Reprod. Biol. Endocrinol., 10.","DOI":"10.1186\/1477-7827-10-21"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.tvjl.2017.10.007","article-title":"Evaluation of interferon-stimulated genes in peripheral blood granulocytes as sensitive responders to bovine early conceptus signals","volume":"229","author":"Toji","year":"2017","journal-title":"Vet. J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.theriogenology.2017.11.014","article-title":"A predictive threshold value for the diagnosis of early pregnancy in cows using interferon-stimulated genes in granulocytes","volume":"107","author":"Yoshino","year":"2018","journal-title":"Theriogenology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"106283","DOI":"10.1016\/j.anireprosci.2020.106283","article-title":"Use of a prediction method for early pregnancy status utilizing receiver operating characteristic curve analysis of peripheral blood leukocyte interferon-stimulated genes in Japanese-Black cattle","volume":"214","author":"Yoshino","year":"2020","journal-title":"Anim. Reprod. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Yoshino, H., Kizaki, K., Hirata, T.I., Iga, K., Matsuda, H., Yamanouchi, T., Hashiyada, Y., Imai, K., Ishiguro-Oonuma, T., and Kanazawa, T. (2023). Interferon-stimulated gene expression in peripheral blood leucocytes as a convenient prediction marker for embryo status in embryo-transferred Japanese Black cows during the peri-implantation period. Vet. Sci., 10.","DOI":"10.3390\/vetsci10070408"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3031","DOI":"10.3168\/jds.2015-10595","article-title":"Associations between pregnancy-associated glycoproteins and pregnancy outcomes, milk yield, parity, and clinical diseases in high-producing dairy cows","volume":"99","author":"Mercadante","year":"2016","journal-title":"J. Dairy Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1016\/j.prevetmed.2017.07.004","article-title":"A bayesian latent class model to estimate the accuracy of pregnancy diagnosis by transrectal ultrasonography and laboratory detection of pregnancy-associated glycoproteins in dairy cows","volume":"145","author":"Fosgate","year":"2017","journal-title":"Prev. Vet. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1016\/S0092-8674(04)00045-5","article-title":"MicroRNAs: Genomics, biogenesis, mechanism, and function","volume":"116","author":"Bartel","year":"2004","journal-title":"Cell"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3509","DOI":"10.1158\/0008-5472.CAN-05-0298","article-title":"MicroRNA biogenesis and cancer","volume":"65","author":"Gregory","year":"2005","journal-title":"Cancer Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/j.addr.2015.05.001","article-title":"An overview of microRNAs","volume":"87","author":"Hammond","year":"2015","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1437","DOI":"10.1126\/science.1102513","article-title":"Argonaute2 is the catalytic engine of mammalian RNAi","volume":"305","author":"Liu","year":"2004","journal-title":"Science"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.gpb.2015.02.001","article-title":"Exosome and exosomal microRNA: Trafficking, sorting, and function","volume":"13","author":"Zhang","year":"2015","journal-title":"Genom. Proteom. Bioinform."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1038\/ncb2210","article-title":"MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins","volume":"13","author":"Vickers","year":"2011","journal-title":"Nat. Cell Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"5003","DOI":"10.1073\/pnas.1019055108","article-title":"Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma","volume":"108","author":"Arroyo","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"657086","DOI":"10.1155\/2015\/657086","article-title":"Exosomes: Novel biomarkers for clinical diagnosis","volume":"2015","author":"Lin","year":"2015","journal-title":"Sci. World J."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1186\/s12943-018-0897-7","article-title":"Effect of exosomal MiRNA on cancer biology and clinical applications","volume":"17","author":"Sun","year":"2018","journal-title":"Mol. Cancer"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"106706","DOI":"10.1016\/j.domaniend.2021.106706","article-title":"Analysis of circulating microRNA during early gestation in Japanese Black cattle","volume":"79","author":"Ono","year":"2022","journal-title":"Domest. Anim. Endocrinol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Ioannidis, J., and Donadeu, F.X. (2017). Changes in circulating microRNA levels can be identified as early as Day 8 of pregnancy in cattle. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0174892"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"731","DOI":"10.1002\/mrd.22856","article-title":"Circulating microRNA as candidates for early embryonic viability in cattle","volume":"84","author":"Pohler","year":"2017","journal-title":"Mol. Reprod. Dev."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1016\/j.theriogenology.2018.08.020","article-title":"Exploring maternal Serum microRNAs during early pregnancy in cattle","volume":"121","author":"Gebremedhn","year":"2018","journal-title":"Theriogenology"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Ioannidis, J., and Donadeu, F.X. (2016). Circulating MiRNA signatures of early pregnancy in cattle. BMC Genom., 17.","DOI":"10.1186\/s12864-016-2529-1"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1111","DOI":"10.1007\/s13258-018-0668-2","article-title":"Profiling and identification of pregnancy-associated circulating microRNAs in dairy cattle","volume":"40","author":"Markkandan","year":"2018","journal-title":"Genes Genom."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.domaniend.2011.09.004","article-title":"Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its expected roles in the bovine endometrium during gestation","volume":"42","author":"Mishra","year":"2012","journal-title":"Domest. Anim. Endocrinol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2007","DOI":"10.1002\/jcp.25854","article-title":"Interplay between MiRNAs and human diseases","volume":"233","author":"Paul","year":"2018","journal-title":"J. Cell. Physiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"12955","DOI":"10.1038\/s41598-018-31099-w","article-title":"Association of plasma microRNA expression with age, genetic background and functional traits in dairy cattle","volume":"8","author":"Ioannidis","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"922","DOI":"10.1016\/j.prp.2017.05.010","article-title":"Deregulation of MiR-126-3p in basal-like breast cancers stroma and its clinical significance","volume":"213","author":"Li","year":"2017","journal-title":"Pathol. Res. Pract."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1038\/nature06487","article-title":"Endogenous human microRNAs that suppress breast cancer metastasis","volume":"451","author":"Tavazoie","year":"2008","journal-title":"Nature"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"704","DOI":"10.1038\/s41419-018-0713-8","article-title":"Placental exosome-mediated Bta-MiR-499-Lin28B\/Let-7 axis regulates inflammatory bias during early pregnancy","volume":"9","author":"Zhao","year":"2018","journal-title":"Cell Death Dis."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2287","DOI":"10.2337\/db18-1360","article-title":"Diabetic nephropathy alters the distribution of circulating angiogenic microRNAs among extracellular vesicles, HDL, and Ago-2","volume":"68","author":"Florijn","year":"2019","journal-title":"Diabetes"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1007\/978-1-4939-2550-6_15","article-title":"A protocol for exosome isolation and characterization: Evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods","volume":"1295","author":"Greening","year":"2015","journal-title":"Methods Mol. Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"789","DOI":"10.7150\/thno.18133","article-title":"Progress in exosome isolation techniques","volume":"7","author":"Li","year":"2017","journal-title":"Theranostics"}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/14\/11\/1592\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:49:39Z","timestamp":1760107779000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/14\/11\/1592"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,5,28]]},"references-count":39,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2024,6]]}},"alternative-id":["ani14111592"],"URL":"https:\/\/doi.org\/10.3390\/ani14111592","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,5,28]]}}}