Dietary L-Citrulline Supplementation Promotes Rumen Development and Modulates the Microbiota-Metabolome Axis in Suckling Hu Lambs.

Abstract

The suckling phase is the critical window for rumen functional maturation, yet amino-acid-based interventions tailored to this stage remain scarce. L-citrulline (L-cit) bypasses hepatic first-pass metabolism, is converted to L-arginine peripherally, and resists ruminal microbial degradation, making it a candidate functional additive for early-life ruminants. This study evaluated whether dietary L-cit at 2 g·lamb^-1·d^-1 would improve rumen development and metabolic function in suckling Hu lambs. Twenty male Hu lambs were randomly assigned to a control (CON) or L-cit group (n = 10/group) and reared for 45 d (3 d adaptation + 42 d treatment). Growth and starter intake were assessed in all lambs; six lambs per group (n = 6) were subsequently slaughtered for rumen morphometry, gas chromatography-flame ionization detection (GC-FID) volatile fatty acid (VFA) quantification, 16S rRNA gene sequencing, and liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics. L-cit increased average daily starter intake by 25.96% (p = 0.036) and produced a 20.00% numerical but non-significant increase in average daily gain (ADG) (p = 0.203; Cohen's d = 0.58). Rumen weight, volume, and papillary length, width, density, and epithelial thickness were all elevated (p < 0.05), whereas muscular thickness was unaffected (p = 0.162). Total VFA, acetate, propionate (+37.64%, p < 0.001), and butyrate were higher in the L-cit group; the molar proportion of propionate rose from 21.41% to 24.75%, and the acetate-to-propionate ratio declined from 2.90 to 2.44 (p = 0.005). Microbial richness (Chao1, Observed species) increased without altered evenness, and linear discriminant analysis effect size (LEfSe) identified L-cit-driven enrichment of propionate-generating and fiber-degrading genera, including Prevotellaceae_UCG-004, Ruminobacter, and the NK4A214_group. Of 539 differential metabolites (147 of which were annotated to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database), KEGG enrichment highlighted linoleic acid metabolism and purine metabolism as the biologically interpretable targets. Microbiota-metabolite correlations linked L-cit-enriched genera to up-regulated metabolites such as adenine. Dietary L-cit at 2 g·lamb^-1·d^-1 enhances starter intake, promotes rumen epithelial development, promotes a shift toward enhanced propiogenic fermentation within an acetate-dominant profile, and remodels the microbiota-metabolome axis, supporting its application as a functional additive during the suckling phase of ruminants. Because epithelial barrier integrity, oxidative stress, and inflammatory markers were not directly measured, these findings should be interpreted as morphological and association-based evidence, and further functional validation is required.