Bacillus subtilis and Macleaya cordata extract regulate the rumen microbiota associated with enteric methane emission in dairy cows

Background Ruminant livestock production is a considerable source of enteric methane (CH 4 ) emissions. In a previous study, we found that dietary inclusions of Bacillus subtilis (BS) and Macleaya cordata extract (MCE) increased dry matter intake and milk production, while reduced enteric CH 4 emission in dairy cows. The objective of this study was to further elucidate the impact of feeding BS and MCE on rumen methanogenesis in dairy cows using rumen metagenomics techniques. Results Sixty dairy cows were blocked in 20 groups of 3 cows accordingly to their live weight, milk yield, and days in milk, and within each group, the 3 cows were randomly allocated to 1 of 3 treatments: control diet (CON), control diet plus BS (BS), and control diet plus MCE (MCE). After 75 days of feeding experimental diets, 12 cows were selected from each treatment for collection of rumen samples for the metagenomic sequencing. Results showed that BS decreased ruminal acetate and butyrate, while increased propionate concentrations, resulting in decreased acetate:propionate ratio. The metagenomics analysis revealed that MCE reduced relative abundances of Methanobrevibacter wolinii, Methanobrevibacter sp. AbM4, Candidatus Methanomassiliicoccus intestinalis, Methanobrevibacter cuticularis, Methanomicrobium mobile, Methanobacterium formicicum, and Methanobacterium congolense . Both BS and MCE reduced relative abundances of Methanosphaera sp . WGK6 and Methanosphaera stadtmanae . The co-occurrence network analysis of rumen bacteria and archaea revealed that dietary treatments influenced microbial interaction patterns, with BS and MCE cows having more and stronger associations than CON cows. The random forest and heatmaps analysis demonstrated that the Halopenitus persicus was positively correlated with fat- and protein-corrected milk yield; Clostridium sp . CAG 269 , Clostridium sp. 27 14 , Haloarcula rubripromontorii , and Methanobrevibacter curvatus were negatively correlated with rumen acetate and butyrate concentrations, and acetate:propionate ratio, whereas Selenomonas rumiantium was positively correlated with those variables. Conclusions The present results provided new information for mitigation of enteric methane emissions of dairy cows by feeding BS and MCE to influence rumen microbial activities. This fundamental knowledge is essential for developing enteric CH4 reduction strategies to mitigate climate change and reduce dietary energy waste. 7hDgYvMWQEHgf6eUhi7c3N Video Abstract