The global problem of climate change and the greenhouse effect has become increasingly severe, and the abatement of greenhouse gases has received great attention from the international community. Methane produced by ruminants during digestion not only aggravates the greenhouse effect but also causes a waste of feed energy.

The compound 3-nitrooxypropanol (3-NOP) is a promising methane inhibitor, which performs well in inhibiting methane emission and does not affect animal feed intake and digestibility. However, it causes a significant increase in hydrogen production while suppressing methane emission, resulting in a waste of feed energy. Vitamin B₁₂ is a key factor in the propionate production pathway and thus plays an important role in regulating the hydrogen utilization pathway. In this study, the effects of 3-NOP combined with vitamin B₁₂ supplementation on rumen fermentation and microbial compositional structure in dairy cattle were investigated by simulating rumen fermentation in vitro. Experiments were performed using a 2 × 2-factorial design: two 3-NOP levels (0 or 2 mg/g dry matter) and 2 vitamin B₁₂ levels (0 or 2 mg/g dry matter). Three experiments were performed, each consisting of 4 treatments, 4 replicates, and 4 blanks containing only inoculum. The combined supplementation of 3-NOP and vitamin B₁₂ reduced methane emission by 12% without affecting dry matter digestibility. The combined addition of 3-NOP and vitamin B₁₂ significantly increased the concentration of propionate and reduced the concentration of acetate and the acetate to propionate ratio. At the bacterial level, 3-NOP increased the relative abundances of Christensenellaceae_R-7_group and Lachnospiraceae_NK3A20_group. Vitamin B₁₂ increased the relative abundances of unclassified_f__Prevotellaceae and Prevotellaceae_UCG-003 and decreased the relative abundance of Lachnospiraceae_NK3A20_group. At the archaeal level, the combination of 3-NOP and vitamin B₁₂ increased the relative abundances of Methanobrevibacter_ sp._ Abm4, OTU1125, and OTU95 and decreased the relative abundances of uncultured_methanogenic_archaeon_g__Methanobrevibacter, OTU1147, OTU1056, and OTU55. The results indicated that 3-NOP combined with vitamin B₁₂ could alleviate rumen hydrogen emission and enhance the inhibition of methane emission compared with 3-NOP alone.

This study explored the combined effects of vitamin B₁₂ and fumarate supplementation on methane (CH₄) emission and propionate synthesis in dairy cows through simulated rumen fermentation in vitro. The experimental animals were 3 cows with an average milk yield of 23 ± 2.8 kg/d, a body weight of 618 ± 100 kg, and a parity of 3 ± 1 that were selected as rumen fluid donors. The TMR diet fed to cows is a fermentation substrate. Experiments adopted 2 × 2 factorial design, including control group, vitamin group (1 mg/g DM vitamin B₁₂), fumarate group (100 mg/g DM), and combined addition group (1 mg/g DM vitamin B₁₂ and 100 mg/g DM). All treatments had no effect on the dry matter degradation (DMD). Both vitamin B₁₂ and fumarate reduced CH₄ emission, increased the propionate concentration, and reduced the acetate/propionate ratio without any observed interaction. Vitamin B₁₂ made Prevotella and Prevotellaceae_ UCG‐003 increase in quantity, and fumarate increased the abundance of Succinivibrionaceae_UCG‐002 and Selenomonas, both of which are propionate‐producing bacteria. At the species level, the supplementation of vitamin B₁₂ and fumarate slightly changed the abundance of some strains, but it was not statistically significant. Shifts in the abundance of propionate‐producing bacteria and methanogenic archaea species suggest an increase in propionate production and a decrease in CH₄ emission. In conclusion, the addition of vitamin B₁₂ and fumarate changed the fermentation mode of the rumen and reduced the emission of CH₄ by affecting the structure of the rumen microbial community, but no obvious interaction was found between the two.

Vitamin B₁₂, an important cofactor involved in propionate formation, is synthesized exclusively by bacteria and archaebacteria. Humans need to intake vitamin B₁₂ through food, and dairy products are generally the best source of vitamin B₁₂. In the present study, the effects of vitamin B₁₂ supplementation in diets on in vitro ruminal fermentation characteristics and microbial communities were investigated to provide a reference for increasing the vitamin B₁₂ content in milk by dietary supplementation. A completely randomized design was carried out using the in vitro rumen culture technique, and 5 vitamin B₁₂ dose levels (0, 0.5, 1.0, 2.0, and 4 mg/g of dry matter) were used. The results showed that vitamin B₁₂ supplementation in diets decreased acetate: propionate ratio and butyrate concentration. The change in the acetate: propionate ratio can be attributed to the increased relative abundances of the Proteobacteria phylum and the Negativicutes class, both of which are involved in propionate metabolism. The decrease in butyrate concentration can likely be attributed to a reduction in relative abundance of species belonging to the Clostridia class, which are known as the predominant butyrate producers in the mammalian intestine. In addition, vitamin B₁₂ supplementation in diets reduced the CH₄ production by altering the species composition of the archaeal community. In conclusion, dietary supplementation of vitamin B₁₂ resulted in rumen perturbation. In vivo studies should be conducted cautiously when evaluating the effects of vitamin B₁₂ supplementation on the synthesis and absorption of it, as well as its content in milk.

This study aimed to investigate the inhibitory effects of rottlerin, a plant-derived polyphenol compound, on methane (CH₄) production in dairy cow rumen fermentation by targeting methyl-coenzyme M reductase (MCR). Molecular docking and virtual screening revealed that rottlerin exhibited a high binding affinity (-8.300 kcal/mol) with key residues in the catalytic site of MCR (GLY-142 and GLN-230). In vitro rumen fermentation showed that rottlerin significantly reduced CH₄ production in a dose-dependent manner (up to 32.4%), while maintaining stable pH, dry matter digestibility, and VFA concentration. Microbial community analysis revealed a reduction in the relative abundance of Methanobrevibacter, a dominant hydrogenotrophic methanogen, and a simultaneous increase in Candidatus_Methanomethylophilus (P < 0.05), a methylotrophic methanogen, indicating a metabolic shift favoring methylotrophic rather than hydrogenotrophic methanogenesis. The in vitro MCR inhibition assay showed a concentration dependent inhibition of MCR activity (half-maximal inhibitory concentration [IC₅₀] = 23.72 μM), suggesting a direct interaction between rottlerin and MCR, consistent with the docking results. These findings highlight the potential of rottlerin as a naturally occurring, MCR-targeted feed additive for sustainable methane mitigation in dairy cows.