Effects of Duodenal Infusion of L-Citrulline on Plasma Metabolism, Fecal Microbiota Structure, and Reproductive Hormones in Ewes

This experiment aimed to investigate the metabolism of L-Citrulline (L-Cit) in the intestinal tract of ewes and its effects on fecal microbiota composition, plasma metabolism, and reproductive hormone levels. Twelve 18-month-old non-pregnant multiparous Turpan black ewes weighing 51.65 kg ± 2.49 kg were selected and randomly assigned to a control group (Con) and an experimental group (L-Cit), with six ewes in each group. Both groups were fed identical nutrient-dense rations. In the Con group, 100 mL of saline was administered through the duodenal fistula, while the L-Cit group received an additional 0.25 g/kg BW−1 of L-Cit solution. On day 7, the crude protein and amino acid concentrations in feces and urine were assessed using total feces and urine collection methods. Fecal and blood samples were collected to evaluate microbiological and reproductive hormone indices, with blood samples also collected for plasma non-targeted metabolomics analysis two hours post-infusion. Compared to the Con group, the L-Cit group exhibited a significant reduction in crude protein content in feces (p < 0.05) and a highly significant decrease in urine (p < 0.01). Nitrogen metabolism indices did not differ significantly between groups (p > 0.05), but the L-lysine content in feces was significantly higher in the L-Cit group (p < 0.05). 16S rRNA sequencing revealed no significant PCA separation between the two groups. However, the relative abundance of Lachnospiraceae_NK3A20_group, Oscillibacter, and Mogibacterium was significantly higher in the Con group (p < 0.01), while SP3-e08, Parvibacter, Anaerosporobacter, Butyricimonas, and Peptococcus were more abundant in the L-Cit group (p < 0.05). LC-MS analysis showed significant up-regulation of purine and nucleotide metabolism pathways in the L-Cit group (p < 0.05). Plasma levels of estradiol (E2), progesterone (P4), gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were significantly elevated in the L-Cit group at both 1 and 2 h post-infusion (p < 0.01). These results suggest that duodenal infusion of L-Cit enhances intestinal nitrogen utilization, alters specific bacterial populations, promotes purine and nucleotide metabolism, and stimulates reproductive hormone secretion in ewes.