Rumen microbiota inoculation indicates collaborative mechanisms enhancing propionate supply to alleviate weaning stress in lambs

BACKGROUND: The transition from milk to solid feed during weaning often imposes metabolic stress on young ruminants due to energy deficits. Previous studies suggest that ruminal microbiota transplantation from adults to juveniles can alleviate weaning stress, but the underlying mechanisms remain poorly defined. RESULTS: In this study, 48 Hu lambs were randomly assigned to two groups (n = 24 each): an inoculated group (Inoc) that received lyophilized ruminal microbiota and a control group (Ctrl) that received no inoculation. We evaluated rumen fermentation characteristics, blood metabolites, hepatic glycogen levels, expression of hepatic gluconeogenic genes, and shifts in the rumen microbiome at three key time points-the end of weaning, 1 and 2 weeks post-weaning. Oral inoculation significantly elevated rumen propionate concentration, upregulated the gene expression of hepatic pyruvate carboxylase (EC 6.4.1.1) and glucose-6-phosphatase (EC 3.1.3.9), and increased hepatic glucose production. Microbiome analysis revealed increased colonization by lactic acid-producing bacteria (e.g., Olsenella and Sharpea) and propionate producers, such as Megasphaera elsdenii, alongside enriched families associated with propionate production, including Prevotellaceae, Succinivibrionaceae, and Erysipelotrichaceae. Genome-resolved metagenomics further demonstrated an increased abundance of metagenome-assembled genomes (MAGs) carrying polysaccharide utilization loci (PULs) and genes involved in lactate-to-propionate conversion. Notably, the inoculation promoted co-occurrence of functionally complementary MAGs-such as s_Megasphaera elsdenii (MAG98), s_Bilifractor sp902797025 (MAG125), s_Prevotella sp002391185 (MAG342), and s_Prevotella sp900540375 (MAG298)-that carry a wide repertoire of genes involved in polysaccharide degradation and lactate-to-propionate fermentation. In vitro co-culture experiments with Megasphaera elsdenii and Bilifractor porci confirmed their synergistic role in promoting propionate production. CONCLUSIONS: This study demonstrates that oral inoculation of pre-weaned lambs with starter feed-adapted adult rumen microbiota facilitates the establishment of a microbial consortium capable of enhanced lactate and propionate production, thereby enhancing hepatic gluconeogenesis and energy homeostasis, which ultimately mitigates weaning stress. This approach may offer a promising strategy to facilitate dietary transition and enhance metabolic resilience in young ruminants during weaning by modulating rumen microbial composition toward a propionate-producing community. Video Abstract.