Effects of L-citrulline supplementation in the basal diet on reproductive performance, serum metabolites, and microbial community structure in Simmental cows

The experiment was designed to evaluate the influence of dietary supplementation with L-Citrulline (L-Cit) on reproductive outcomes, serum reproductive hormone profiles, ruminal and intestinal microbial communities, serum metabolites, and their metabolic associations in Simmental cows, thereby providing a theoretical basis for its application in ruminant production. All cows were screened by vaginal mucus microscopy (no pathogenic bacteria detected) and B-ultrasound examination (no ovarian cysts or uterine inflammation observed) to confirm the absence of reproductive disorders. 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 min) 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 h 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% (<i>p <</i> 0.05). In experimental Group II, serum concentrations of gonadotropin-releasing hormone (GnRH) and follicle-stimulating hormone (FSH) measured 10 h after cows accepted mounting rose by 15.79% (<i>p <</i> 0.05) and 35.71% (<i>p <</i> 0.01), respectively, relative to the control group. Analysis of 16S rRNA sequences revealed significant differences in ruminal microbial taxa, including <i>Verrucomicrobiota, Lentisphaeria, Oligosphaeraceae, vadinBE97_g_norank</i>, <i>vadinBE97</i>, <i>Eubacterium_ruminantium_group</i>, and <i>Prevotellaceae_g_norank</i>. In the intestine, significant differences were observed in <i>Lachnospiraceae</i>, <i>Lachnospirales</i>, <i>Marvinbryantia</i>, <i>Desulfovibrionia</i>, <i>Desulfovibrionaceae</i>, and <i>Desulfobacterota</i>. KEGG enrichment analysis based on Liquid Chromatography-Mass Spectrometry (LC-MS) <i>data indicated upregulation</i> 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. Within the experimental framework, dietary supplementation with L-Cit combined with the two-shot PG synchronization protocol reshaped the ruminal microbial community structure of Simmental cows, improved glucose metabolic efficiency and xenobiotic degradation capacity, and promoted the synthesis and release of reproductive hormones. Consequently, estrus and conception rates were enhanced.