{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T02:14:55Z","timestamp":1779156895153,"version":"3.51.4"},"reference-count":45,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2025,9,9]],"date-time":"2025-09-09T00:00:00Z","timestamp":1757376000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Livestock Promotional Funds of the Japan Racing Association (KS)"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Repeat breeder (RB) cows are subfertile animals and represent a major issue in the livestock industry. The influence of the gut microbiota on reproductive function and the gut microbiota\u2013gonadal axis has garnered scientific attention. This study aimed to evaluate the relationship between reproductive performance and gut microbiota in Holstein cows, depending on reproductive attempts (number of artificial inseminations or embryo transfers) and subsequent pregnancy outcomes. Fecal samples were collected from Holstein cows at the time of ovulation and classified into four groups: Normal pregnancy (n = 27, \u2264three reproductive attempts, subsequently fertile), Normal open (n = 25, \u2264three reproductive attempts, subsequently infertile), RB pregnancy (n = 38, over four reproductive attempts, subsequently fertile), and RB open (n = 60, over four reproductive attempts, subsequently infertile). The PLS-DA analysis revealed differences in classification among groups, especially between Normal pregnancy and RB open. Regarding \u03b1- and \u03b2-diversity analyses, these indices were significantly different among the four groups (p &lt; 0.05). Using LEfSe analysis, three genus-level bacteria (Bacillus, Ruminococcus, and Sphingobium) were identified as specific bacteria in the Normal pregnancy group, and eight genus-level bacteria (Huintestinicola, Intestinimonas, Neglecta, Oscillibacter, Zongyangia, Phocaeicola, Alistipes, and Acholeplasma) were identified as specific bacteria in both open groups (p &lt; 0.05). KEGG analysis predicted some pathways (Apoptosis, Lipopolysaccharide biosynthesis, etc.) to be enriched in both the open groups. MetaCyc analysis predicted amine and polyamine degradation\/biosynthesis as the specific changing pathways in the RB open group. This study demonstrated that the composition of the gut microbiota differs between normal fertility and repeated infertility conditions. Furthermore, even with fewer reproductive attempts, differences in gut microbiota composition were observed between cows that conceived and those that failed to conceive.<\/jats:p>","DOI":"10.3390\/ani15182637","type":"journal-article","created":{"date-parts":[[2025,9,9]],"date-time":"2025-09-09T08:40:15Z","timestamp":1757407215000},"page":"2637","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Differences in the Gut Microbiota in Long-Term Infertile Holstein Repeat Breeder Cows and Healthy Fertile Holstein Cows"],"prefix":"10.3390","volume":"15","author":[{"given":"Yui","family":"Kitagawa","sequence":"first","affiliation":[{"name":"Department of Animal Science, Tokyo University of Agriculture, Atsugi 243-0034, Kanagawa, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sayoko","family":"Oishi","sequence":"additional","affiliation":[{"name":"Department of Animal Science, Tokyo University of Agriculture, Atsugi 243-0034, Kanagawa, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karen","family":"Koshimizu","sequence":"additional","affiliation":[{"name":"Department of Animal Science, Tokyo University of Agriculture, Atsugi 243-0034, Kanagawa, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4945-0649","authenticated-orcid":false,"given":"Ryotaro","family":"Miura","sequence":"additional","affiliation":[{"name":"Department of Large Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069-0836, Hokkaido, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-1655-3937","authenticated-orcid":false,"given":"Seizo","family":"Hamano","sequence":"additional","affiliation":[{"name":"Artificial Insemination Association of Japan, Koto 135-0041, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7238-8997","authenticated-orcid":false,"given":"Hisataka","family":"Iwata","sequence":"additional","affiliation":[{"name":"Department of Animal Science, Tokyo University of Agriculture, Atsugi 243-0034, Kanagawa, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Koumei","family":"Shirasuna","sequence":"additional","affiliation":[{"name":"Department of Animal Science, Tokyo University of Agriculture, Atsugi 243-0034, Kanagawa, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.theriogenology.2007.09.001","article-title":"The use of embryo transfer to produce pregnancies in repeat-breeding dairy cattle","volume":"69","author":"Dochi","year":"2008","journal-title":"Theriogenology"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Perez-Marin, C.C., and Quintela, L.A. 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