IntroductionExcessive intake of lactate caused by improper use of silage in animal husbandry has adverse effects on rumen fermentation, such as rumen acidosis. The speed of absorption and metabolism of D-lactate in rumen epithelial cells was slower than that of L-lactate, making D-lactate more prone to accumulate and induce rumen acidosis. Therefore, this study was conducted to explore the effects of dietary D-lactate levels on rumen fermentation of beef cattle and its mechanism in an in vitro system.MethodsThis experiment was adopted in single-factor random trial design, with 5 days for adaptation and 3 days for sample collection. Three treatments (n = 8/treatment) were used: (1) D-LA (0.3%), basal fermentation substrate with 0.3% (dry matter, DM basis) D-lactate; (2) D-LA (0.75%), basal fermentation substrate with 0.75% (DM basis) D-lactate; and (3) D-LA (1.2%), basal fermentation substrate with 1.2% (DM basis) D-lactate.ResultsWith the dietary D-lactate levels increased, the daily production of total gas, hydrogen and methane, as well as the ruminal concentrations of acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, total volatile fatty acid and D-lactate increased (p < 0.05), but the ruminal pH and acetate/propionate ratios decreased (p < 0.05). Principle coordinate analysis based on Bray-Curtis distance showed that increasing dietary D-lactate levels could significantly affect the structure of rumen bacterial community (p < 0.05), but had no significant effect on the structure of rumen eukaryotic community (p > 0.05). NK4A214_group, Ruminococcus_gauvreauii_group, Eubacterium_oxidoreducens_group, Escherichia-Shigella, Marvinbryantia and Entodinium were enriched in D-LA (1.2%) group (p < 0.05), as well as WCHB1-41, vadinBE97, Clostridium_sensu_stricto_1, Anaeroplasma and Ruminococcus were enriched in D-LA (0.3%) group (p < 0.05). Changes in the composition of ruminal microorganisms affected rumen metabolism, mainly focus on the biosynthesis of glycosaminoglycans (p < 0.05).DiscussionOverall, feeding whole-plant corn silage with high D-lactate content could not induce rumen acidosis, and the metabolization of dietary D-lactate into volatile fatty acids increased the energy supply of beef cattle. However, it also increased the ruminal CH4 emissions and the relative abundance of opportunistic pathogen Escherichia-Shigella in beef cattle. The relative abundance of Verrucomicrobiota and Escherichia-Shigella may be influenced by glycosaminoglycans, reflecting the interaction between rumen microorganisms and metabolites.