Effects of succinate supplementation on rumen fermentation, microbiota, and propionate-associated metabolism in vitro

Succinate is an intermediate in the ruminal succinate-propionate pathway, but its direct effects as an exogenous additive on rumen fermentation, bacterial community structure, and energy-related metabolites remain insufficiently characterized. This study evaluated whether graded succinate supplementation changes in vitro rumen fermentation and propionate-associated microbial and metabolite profiles.

Rumen fluid collected from three fistulated Chinese Holstein cows was pooled within each weekly fermentation run and incubated with alfalfa substrate and sodium succinate at 0, 4, 8, or 12 mmol/L. Gas-production kinetics, fermentation characteristics, bacterial community composition, targeted energy metabolites, and correlation networks were analyzed.

Succinate supplementation altered gas-production kinetics, with 24-h and potential gas production peaking at 8 mmol/L. Acetate, propionate, butyrate, and total VFA concentrations increased, whereas pH and NH₃-N concentration were not significantly affected. Succinate supplementation was associated with shifts in bacterial relative abundance, including changes in Succiniclasticum, Selenomonas, Butyrivibrio, Anaeroplasma, Pseudobutyrivibrio, Desulfovibrio, Fibrobacter, and Corynebacterium. Targeted metabolomics comparing SA0 and SA3 showed FDR-significant changes in metabolites mapped to glycolysis/gluconeogenesis, pyruvate metabolism, the tricarboxylic acid cycle, and acetate metabolism.

These findings indicate that succinate supplementation was associated with increased propionate concentration and coordinated changes in bacterial taxa and targeted metabolites related to propionate-associated metabolism in vitro. Because methane production, metabolic flux, enzyme activity, gene expression, and absolute microbial abundance were not directly measured, the results should be interpreted as association-level evidence. Further in vivo studies and direct functional measurements are needed to determine whether succinate can be used reliably to modulate rumen fermentation.