Effects of citric acid and heat-treated soybean meal on rumen fermentation characteristics, methane emissions, and microbiota: an in vitro study

This study aimed to assess the impact of citric acid (CA) and heat-treated soybean meal (SBM) on rumen fermentation characteristics, methane production, and microbiota through an <italic>in vitro</italic> experiment. Untreated SBM, heat-treated SBM (HSBM), CA-treated SBM (CSBM), and SBM treated with a combination of heat and CA (HCSBM). Parameters assessed in <italic>in vitro</italic> were gas production, methane emissions, dry matter degradability (IVDMD), crude protein degradability (IVCPD), ammonia nitrogen (NH₃-N), microbial crude protein (MCP), volatile fatty acids (VFA), pH, and microbiota composition. The HCSBM exhibited the lowest gas production and theoretical maximum gas production (<italic>p </italic>&lt; 0.01). Methane production (%) was significantly reduced in both CSBM and HCSBM (<italic>p </italic>&lt; 0.01), with the lowest methane emissions (mL/g  dry matter, DM) observed in HCSBM (<italic>p </italic>&lt; 0.01). The IVCPD was significantly reduced in both the HSBM and HCSBM groups (<italic>p </italic>&lt; 0.01). HCSBM had the lowest NH₃-N and MCP concentrations (<italic>p </italic>&lt; 0.01). Total VFA production was the lowest in HCSBM (<italic>p </italic>&lt; 0.01), with a higher proportion of acetate and lower proportions of propionate (<italic>p </italic>&lt; 0.01). HCSBM reduced the enrichment of Thermoplasmatota compared to HSBM (<italic>p </italic>&lt; 0.05) and decreased the enrichment of the coenzyme M biosynthesis pathway in the microbial functional profiles compared to SBM and CSBM (<italic>p</italic> &lt; 0.05). Additionally, an increase in fiber-degrading bacteria, particularly Fibrobacterota, was observed in HCSBM (<italic>p</italic> &lt; 0.05). These findings suggest that the combination of heat and CA treatment of SBM may effectively reduce ruminal protein degradation and methane emissions. Further <italic>in vivo</italic> studies are necessary to validate these results and assess their practical application in ruminant nutrition.