Feeding citrus flavonoid extracts decreases bacterial endotoxin and systemic inflammation and improves immunometabolic status by modulating hindgut microbiome and metabolome in lactating dairy cows

Abstract

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.

Keywords: Citrus flavonoid; Dairy cow; Fecal microbiota; Systemic inflammation; Volatile fatty acid.

Graphical abstract

Fig. 1

Similarity of the fecal bacterial structure between dairy cows fed citrus flavonoid extracts at 0 (CON) and 150 g/d (CFE150). (A) PCoA plot based on unweighted UniFrac. (B) PCoA plot based on weighted UniFrac distance. The effect of CFE supplementation on the clustering pattern of fecal microbiota was evaluated via ANOSIM. Significance was declared with P < 0.05.

Fig. 2

The fecal bacteria taxa that had significant differences between dairy cows fed citrus flavonoid extracts at 0 (CON) and 150 g/d (CFE150). (A) Differential taxa at phylum level. (B) Differential taxa at genus level. Positive differences denote greater relative abundances of fecal bacteria at the phylum level and at the genus level in CFE150, while negative differences denote fewer relative abundances in CFE150.

Fig. 3

The Spearman correlation matrix between fecal differentially abundant bacteria and fecal volatile fatty acids and serum indices. Asterisks denote significant difference between fed the control diet (CON) and citrus flavonoid extracts at 150 g/d (CFE150) (∗0.01 < P ≤ 0.05; ∗∗0.001< P ≤ 0.01; ∗∗∗P ≤ 0.001). TVFA = total volatile fatty acids; LPS = lipopolysaccharide; ADPN = adiponectin.

Fig. 4

Analysis of the fecal microbiota in dairy cows fed citrus flavonoid extracts at 0 (CON) and 150 g/d (CFE150). The PCA (A) and OPLS-DA (C), and OPLS-DA permutation test (E) in the positive model, and plot of PCA (B) and OPLS-DA (D) and OPLS-DA permutation test (F) in the negative model, for the fecal metabolites. R²Y, goodness-of-fit parameter; Q², predictive ability parameter.

Fig. 5

The pathway analysis of fecal differentially expressed metabolites based on the comparison of dairy cows fed citrus flavonoid extracts at 0 (CON) versus 150 g/d (CFE150). The x axis denotes the impact values in of pathways the topology analysis, and larger bubbles indicate higher impact values. The y axis denotes the P-value (-lnP) of the metabolic pathway in the enrichment analysis, and darker bubbles indicate higher levels of pathway enrichment.

Fig. 6

The Spearman correlation matrix between fecal differentially abundant bacteria and fecal differentially expressed metabolites. Asterisks denote significant difference between dairy cows fed the control diet (CON) and citrus flavonoid extracts at 150 g/d (CFE150), ∗0.01 < P ≤ 0.05; ∗∗0.001 < P ≤ 0.01; ∗∗∗P ≤ 0.001.

Fig. 7

A working mechanism to illustrate the hindgut bacteria and metabolites that might be associated with the improvement of immunometabolic status in lactating dairy cows. AdipoR = adiponectin receptor; ER = endoplasmic reticulum; IL = interleukin; LBP = lipopolysaccharide binding protein; LPS = lipopolysaccharide; TLR4 = toll-like receptor 4; TNF-α = tumor necrosis factor α; SM = sphingomyelin; S1P = sphingosine 1-phosphate; VFA = volatile fatty acids.