Dietary modulation of the rumen microbiome drives the expression of metabolic and methanogenic pathways in Bos indicus

Diet influences ruminal methane emissions by modulating the composition and activity of the rumen microbiome. However, how diet shapes the functional capacity of the rumen microbiome in Nelore cattle ( Bos indicus ), a key tropical beef breed, remains unclear. This study used metatranscriptomics to investigate how dietary supplementation with agro-industrial by-products affects the active rumen microbiome and its association with residual methane emissions. Rumen samples from 50 Nelore cattle fed either a conventional or by-product-based diet revealed that the active microbiome was dominated by bacteria (88.4% ± 3.16%) and archaea (11.6% ± 3.16%), with no significant taxonomic differences between diets. Despite this, functional profiling identified genes from 193 pathways and 3,512 gene families, with distinct metabolic signatures between diets. Specifically, six pathways and 87 gene families were unique to the conventional diet, while seven pathways and 210 gene families were unique to the by-product diet. The associations between gene families enriched under each diet with residual methane emission revealed that the expression of two gene families exhibited negative correlations, while five were positively correlated with methane emission under conventional diet. In the by-product diet, we identified five gene families positively associated with methane emissions and 14 negatively associated. These results demonstrate that diet alters rumen microbial functions with methane mitigation potential, without affecting taxonomic composition. IMPORTANCE Understanding how diet modulates the functional activity of the rumen microbiome is essential for developing strategies to mitigate methane emissions in cattle. This study provides novel insights into how feeding agro-industrial by-products to Nelore cattle ( Bos indicus ), a key tropical beef breed, reshapes the functional profile of the rumen microbiome. Although no taxonomic shifts were detected, animals fed the by-product diet exhibited a greater number of microbial functions associated with lower methane production potential. These findings suggest that diet-driven modulation of microbial metabolism could contribute to strategies aimed at reducing methane emissions. Moreover, the use of by-products supports circular economy principles, enhancing the sustainability and economic resilience of tropical livestock systems. This work emphasizes the importance of examining the active microbiome through RNA rather than solely profiling taxonomic composition without considering microbial activity. It also contributes to unveiling microbial functions to support future methane mitigation and sustainable feeding strategies.