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Our previous research revealed the advantages of separate feeding (SF) systems compared to total mixed ration (TMR) in terms of ruminal methane (CH 4 ) production. The purpose of this experiment was to confirm the advantage of SF as a nutritional strategy for CH 4  mitigation, and to determine the effects of different feeding systems (TMR and SF) on the rumen microbiome and associated metagenome of two different breeds and on CH 4  emissions. We randomly allocated four Holstein (305 ± 29 kg) and four Hanwoo steers (292 ± 24 kg) to two groups; the steers were fed a commercial concentrate with tall fescue (75:25) as TMR or SF, in a crossover design (two successive 22-day periods). Neither feeding systems nor cattle breeds had an effect on the total tract digestibility of nutrients. The TMR feeding system and Hanwoo steers generated significantly more CH 4  ( P  < 0.05) and had a higher yield [g/d and g/kg dry matter intake (DMI)] compared to the SF system and Holstein steers. A larger rumen acetate:propionate ratio was observed for the TMR than the SF diet ( P  < 0.05), and for Hanwoo than Holstein steers ( P  < 0.001), clearly reflecting a shift in the ruminal H 2  sink toward CH 4  production. The linear discriminant analysis (LDA) effect size (LEfSe) revealed a greater abundance (α < 0.05 and LDA > 2.0) of operational taxonomic units (OTUs) related to methanogenesis for Hanwoo steers compared to Holstein steers. Kendall’s correlation analysis revealed wide variation of microbial co-occurrence patterns between feeding systems, indicating differential H 2  thermodynamics in the rumen. A metagenome analysis of rumen microbes revealed the presence of 430 differentially expressed genes, among which 17 and 27 genes exhibited positive and negative associations with CH 4  production, respectively ( P  < 0.001). A strong interaction between feeding system and breed was observed for microbial and metagenomic abundance. Overall, these results suggest that the TMR feeding system produces more CH 4 , and that Hanwoo cattle are higher CH 4  emitters than SF diet and Holstein cattle, respectively. Interestingly, host-associated microbial interactions differed within each breed depending on the feeding system, which indicated that breed-specific feeding systems should be taken into account for farm management.

Feeding Systems and Host Breeds Influence Ruminal Fermentation, Methane Production, Microbial Diversity and Metagenomic Gene Abundance