Background: Modern dairy diets have shifted from being forage-based to grain and energy dense. However, feeding high-starch diets can lead to a metabolic disturbance that is linked to dysregulation of the gastrointestinal microbiome and systemic inflammatory response. Plant flavonoids have recently attracted extensive interest due to their anti-inflammatory effects in humans and ruminants. Here, multi-omics analysis was conducted to characterize the biological function and mechanisms of citrus flavonoids in modulating the hindgut microbiome of dairy cows fed a high-starch diet. Results: Citrus flavonoid extract (CFE) significantly lowered serum concentrations of lipopolysaccharide (LPS) proinflammatory cytokines (TNF-α and IL-6), acute phase proteins (LPS-binding protein and haptoglobin) in dairy cows fed a high-starch diet. Dietary CFE supplementation increased fecal butyrate production and decreased fecal LPS. In addition, dietary CFE influenced the overall hindgut microbiota’s structure and composition. Notably, potentially beneficial bacteria, including Bacteroides, Bifidobacterium, Alistipes, and Akkermansia, were enriched in CFE and were found to be positively correlated with fecal metabolites and host metabolites. Fecal and serum untargeted metabolomics indicated that CFE supplementation mainly emphasized the metabolic feature “sphingolipid metabolism.” Metabolites associated with the sphingolipid metabolism pathway were positively associated with increased microorganisms in dairy cows fed CFE, particularly Bacteroides. Serum lipidomics analysis showed that the total contents of ceramide and sphingomyelin were decreased by CFE addition. Some differentially abundant sphingolipid species were markedly associated with serum IL-6, TNF-α, LPS, and fecal Bacteroides. Metaproteomics revealed that dietary supplementation with CFE strongly impacted the overall fecal bacterial protein profile and function. In CFE cows, enzymes involved in carbon metabolism, sphingolipid metabolism, and valine, leucine, and isoleucine biosynthesis were upregulated. Conclusions: Our research indicates the importance of bacterial sphingolipids in maintaining hindgut symbiosis and homeostasis. Dietary supplementation with CFE can decrease systemic inflammation by maintaining hindgut microbiota homeostasis and regulating sphingolipid metabolism in dairy cows fed a high-starch diet.

Supplementing citrus flavonoid extract to the diet of dairy cows significantly improved rumen fermentation and lactational performance via regulating the rumen microbiota and metabolite profiles.

Methane (CH₄) production from ruminants accounts for 16% of the global greenhouse gas emissions and represents 2% to 12% of feed energy. Mitigating CH₄ production from ruminants is of great importance for sustainable development of the ruminant industry. H₂ is the primary substrate for CH₄ production in the processes of ruminal methanogenesis. Sulfate reducing bacteria are able to compete with methanogens for H₂ in the rumen, and consequently inhibit the methanogenesis. Enhancing the ruminal sulfate reducing pathway is an important approach to mitigate CH₄ emissions in ruminants. The review summarized the effects of sulfate and elemental S on ruminal methanogenesis, and clarified the related mechanisms through the impacts of sulfate and elemental S on major ruminal sulfate reducing bacteria. Enhancing the activities of the major ruminal sulfate reducing bacteria including Desulfovibrio, Desulfohalobium and Sulfolobus through dietary sulfate addition, elemental S and dried distillers grains with solubles can effectively decrease the ruminal CH₄ emissions. Suitable levels of dietary addition with different S sources for reducing the ruminal CH₄ production, as well as maintaining the performance and health of ruminants, need to be investigated in the future.

The objectives of this study were to determine the effects of dietary supplementation with citrus flavonoid extracts (CFE) on milk performance, serum biochemistry parameters, fecal volatile fatty acids, fecal microbial community, and fecal metabolites in dairy cows. Eight multiparous lactating Holstein cows were used in a replicated 4 × 4 Latin square design (21-day period). Cows were fed a basal diet without addition (CON) or basal diet with added CFE at 50 (CFE50), 100 (CFE10), and 150 g/d (CFE150). Feeding CFE up to 150 g/d increased milk yield and milk lactose percentage. Supplementary CFE linearly decreased milk somatic cell count. Serum cytokines interleukin-1β (IL-1β), IL-2, IL-6, and tumor necrosis factor-α (TNF-α) concentrations decreased linearly as the levels of CFE increased. Cows in CFE150 had lower serum lipopolysaccharide and lipopolysaccharide binding protein compared with CON. These results indicate feeding CFE decreased systemic inflammation and endotoxin levels in dairy cows. Furthermore, feeding CFE linearly increased the concentrations of total volatile fatty acids, acetate, and butyrate in feces. The relative abundances of beneficial bacteria Bifidobacterium spp., Clostridium coccoides-Eubacterium rectale group, and Faecalibacterium prausnitzii in feces increased linearly with increasing CFE supplementation. The diversity and community structure of fecal microbiota were unaffected by CFE supplementation. However, supplementing CFE reduced the relative abundances of genera Ruminococcus_torques_group, Roseburia, and Lachnospira, but increased genera Bacteroides and Phascolarctobacterium. Metabolomics analysis showed that supplementary CFE resulted in a significant modification in the fecal metabolites profile. Compared with CON, fecal naringenin, hesperetin, hippuric acid, and sphingosine concentrations were greater in CFE150 cows, while fecal GlcCer(d18:1/20:0), Cer(d18:0/24:0), Cer(d18:0/22:0), sphinganine, and deoxycholic acid concentrations were less in CFE150 cows. Predicted pathway analysis suggested that "sphingolipid metabolism" was significantly enriched. Overall, these results indicate that citrus flavonoids could exert health-promoting effects by modulating hindgut microbiome and metabolism in lactating cows.

Six steers were used to study the effects of dietary supplementation with sodium sulfate (Na₂SO₄) on rumen fermentation, nutrient digestion, rumen microbiota and plasma metabolites. The animals were fed a basal ration added with Na₂SO₄ at 0 [sulfur (S) content 0.115% DM], 20 (S 0.185% DM) and 40 g/d (S 0.255%DM), respectively, in a replicate 3 × 3 Latin square design. Results indicated that supplementing Na₂SO₄ increased the ruminal concentration of total volatile fatty acids, the molar proportions of acetate and butyrate, the ruminal concentrations of microbial protein, SO₄^2--S and S^2--S and the digestibility of fiber while it decreased the molar proportion of propionate and the ruminal concentration of ammonia nitrogen. Supplementing Na₂SO₄ increased the diversity and the richness of rumen microbiota and the relative abundances of phylum Firmicutes, and genera Ruminococcus 2, Rikenellaceae RC9 gut group and Desulfovibrio whereas it decreased that of phylum Bacteroidetes and genera Prevotella 1, Prevotellaceae UCG-001 and Treponema 2. Supplementing Na₂SO₄ also increased the plasma concentrations of amino acids (L-arginine, L-methionine, L-cysteine, L-lysine), purine derivatives (xanthine, hypoxanthine), vitamins (thiamin, biotin) and lipids (acetylcarnitine and L-carnitine). It was concluded supplementation with Na₂SO₄ to steer ration was beneficial to improve the rumen fermentation, fiber digestibility and nutrient metabolism through modulating rumen microbial community. IMPORTANCE Essential elements such as nitrogen and sulfur greatly affect rumen fermentation and metabolism in ruminants. However, little knowledge is available on the effects of sulfur on rumen microbiota and plasma metabolome. Results of the present trial demonstrated that supplementation with sodium sulfate to steer ration markedly improved rumen fermentation, fiber digestibility and metabolism of amino acids, purine derivatives and vitamins through the impacts on the ruminal microbiome. The facts obtained from the present trial clarified the possible mechanisms of the positive effects of sulfur on rumen fermentation and nutrient utilization.

As a natural fermentation system, the rumen ecosystem of ruminants can efficiently degrade plant cellulose, indicating that the rumen microbiome offers an opportunity for anaerobic digestion to utilize biomass wastes containing cellulose. Knowledge of the response of the in situ microbial community to citrus pomace during anaerobic fermentation will help improve the current understanding of citrus biomass waste utilization.