ORIGINAL RESEARCH article
Front. Microbiol.
Sec. Microorganisms in Vertebrate Digestive Systems
This article is part of the Research TopicNew Horizons in Gut Microbiome Research for Enhancing Livestock ProductivityView all 40 articles
Effects of L-Citrulline Supplementation in the Basal Diet on Reproductive Performance, Serum Metabolites, and Microbial Community Structure in Simmental Cows
Provisionally accepted
Changgeng LiHui ChenJiaqi LiuWeijie Zao
琛 樊Waike LvGuozhu XuZhantao LuXihu WangXiaojun Liu
Guodong Zhao*- Xinjiang Agricultural University, Ürümqi, China
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A total of 240 multiparous Simmental cows, 3–4 years of age with an average body weight of 470 ± 15 kg, were randomly allocated into three groups of 80 animals each: a control group, Group Ⅰ, and Group Ⅱ. The control group received only the basal diet, whereas Group Ⅰ and Group Ⅱ were provided with the basal diet supplemented with 7 g/d and 14 g/d of L-Cit per cow, respectively. Day 0 of the trial was defined by the first intramuscular injection of prostaglandin (PG, 0.4 mg per cow), after which L-Cit supplementation commenced at 72-h intervals. On day 10, a second PG injection (0.4 mg per cow) was administered, and supplementation was discontinued on day 11. Estrus expression was monitored, and pregnancy was assessed on day 35 post-insemination using B-mode ultrasonography. During the experimental period, six cows were randomly selected from each group. Blood samples were collected from the coccygeal vein prior to the morning feeding on days 1 and 11 using plain tubes (without anticoagulant). The samples were centrifuged at 720 × g (approximately 3,000 rpm for 15 minutes) to separate the serum. The obtained serum was stored at -20°C for subsequent analysis of reproductive hormones.In addition, blood, rumen fluid, and fecal samples were obtained 10 hours after mounting to support untargeted metabolomic profiling and microbial community analysis. Compared with the control group, the estrus rate in experimental Group Ⅰ increased by 12.5% (P < 0.05). In experimental Group Ⅱ, serum concentrations of gonadotropin-releasing hormone (GnRH) and follicle-stimulating hormone (FSH) measured 10 hours after cows accepted mounting rose by 15.79% (P < 0.05) and 35.71% (P < 0.01), respectively, relative to the control group. Analysis of 16S rRNA sequences revealed significant differences in ruminal microbial taxa, including Verrucomicrobiota, Lentisphaeria, Oligosphaeraceae, vadinBE97_g_norank, vadinBE97, Eubacterium_ruminantium_group, and Prevotellaceae_g_norank. In the intestine, significant differences were observed in Lachnospiraceae, Lachnospirales, Marvinbryantia, Desulfovibrionia, Desulfovibrionaceae, and Desulfobacterota. KEGG enrichment analysis based on Liquid Chromatography-Mass Spectrometry(LC-MS) data indicated upregulation of the arginine biosynthesis pathway in the experimental groups, whereas metabolites associated with the tricarboxylic acid (TCA) cycle were notably elevated in experimental Group Ⅰ.
Keywords: L-Citrulline (L-Cit), reproductive hormones, Ruminal microbiota, Simmental cows, Untargeted metabolism
Received: 08 Nov 2025; Accepted: 15 Jan 2026.
Copyright: © 2026 Li, Chen, Liu, Zao, 樊, Lv, Xu, Lu, Wang, Liu and Zhao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Guodong Zhao
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