Prevotella and succinate treatments altered gut microbiota, increased laying performance, and suppressed hepatic lipid accumulation in laying hens

Abstract

Background: This work aimed to investigate the potential benefits of administering Prevotella and its primary metabolite succinate on performance, hepatic lipid accumulation and gut microbiota in laying hens.

Results: One hundred and fifty 58-week-old Hyline Brown laying hens, with laying rate below 80% and plasma triglyceride (TG) exceeding 5 mmol/L, were used in this study. The hens were randomly allocated into 5 groups and subjected to one of the following treatments: fed with a basal diet (negative control, NC), oral gavage of 3 mL/hen saline every other day (positive control, PC), gavage of 3 mL/hen Prevotella melaninogenica (10⁷ CFU/mL, PM) or 3 mL/hen Prevotella copri (10⁷ CFU/mL, P. copri) every other day, and basal diet supplemented with 0.25% sodium succinate (Succinate). The results showed that PM and P. copri treatments significantly improved laying rate compared to the PC (P < 0.05). The amount of lipid droplet was notably decreased by PM, P. copri, and Succinate treatments at week 4 and decreased by P. copri at week 8 (P < 0.05). Correspondingly, the plasma TG level in Succinate group was lower than that of PC (P < 0.05). Hepatic TG content, however, was not significantly influenced at week 4 and 8 (P > 0.05). PM treatment increased (P < 0.05) the mRNA levels of genes PGC-1β and APB-5B at week 4, and ACC and CPT-1 at week 8. The results indicated enhanced antioxidant activities at week 8, as evidenced by reduced hepatic malondialdehyde (MDA) level and improved antioxidant enzymes activities in PM and Succinate groups (P < 0.05). Supplementing with Prevotella or succinate can alter the cecal microbiota. Specifically, the abundance of Prevotella in the Succinate group was significantly higher than that in the other 4 groups at the family and genus levels (P < 0.05).

Conclusions: Oral intake of Prevotella and dietary supplementation of succinate can ameliorate lipid metabolism of laying hens. The beneficial effect of Prevotella is consistent across different species. The finding highlights that succinate, the primary metabolite of Prevotella, represents a more feasible feed additive for alleviating fatty liver in laying hens.

Keywords: Fatty liver; Gut microbiota; Laying hen; Prevotella; Succinate.

Fig. 1

Effect of Prevotella (3 × 10⁷ CFU) and sodium succinate (0.25%) on plasma parameters of hens. A and F Triglyceride (TG). B and G Total cholesterol (TCH). C and H Glucose (GLU). D and I Low-density lipoprotein-cholesterol (LDL-C). E and J High-density lipoprotein-cholesterol (HDL-C). The assessment measures were conducted at two different time points: at week 4 (A–E) and week 8 (F–J). ^a−cMeans with different letter differ significantly (P < 0.05)

Fig. 2

Prevotella (3 × 10⁷ CFU) and sodium succinate (0.25%) suppressed the hepatic lipid accumulation at week 4. A Liver index. B Hepatic triglyceride (TG) content. C Hepatic total cholesterol (TCH) content. D Abdominal fat pad index. E Liver morphology (upper), and H&E staining (lower). F Hepatic ORO staining. G The ratio of lipid droplets in ORO staining. ^a−cMeans with different letters differ significantly (P < 0.05)

Fig. 3

Prevotella (3 × 10⁷ CFU) and sodium succinate (0.25%) suppressed the hepatic lipid accumulation at week 8. A Liver index. B Hepatic triglyceride (TG) content. C Hepatic total cholesterol (TCH) content. D Abdominal fat pad index. E Liver morphology (upper) and H&E staining (lower). F Hepatic ORO staining. G The ratio of lipid droplets in ORO staining. ^a,bMeans with different letters differ significantly (P < 0.05)

Fig. 4

Effect of Prevotella and sodium succinate on enzymes activities related to hepatic lipid metabolism. A and F Fatty acid synthase (FAS) activity. B and G Malic enzyme (ME) activity. C and H Lipoprotein lipase (LPL) activity. D and I Hepatic lipase (HL) activity. E and J Free fat acid (FFA) content. The assessment measures were conducted at two different time points: at week 4 (A–E) and at week 8 (F–J). ^a−cMeans with different letters differ significantly (P < 0.05)

Fig. 5

Prevotella and sodium succinate regulated hepatic lipid metabolism and mitochondrial function related mRNA expression. The assessment measures were conducted at two different time points: at week 4 (A and B) and at week 8 (C and D). ^a−bMeans with different letters differ significantly (P < 0.05)

Fig. 6

Prevotella and sodium succinate improved the hepatic antioxidant activities. A and G Malondialdehyde (MDA) content. B and H Glutathione (GSH) concentration. C and I Total antioxidant capacity (T-AOC) concentration. D and J Glutathione peroxidase (GSH-Px) activity. E and K Superoxide dismutase (SOD) activity. F and L Catalase (CAT) activity. The assessment measures were conducted at two different time points: at week 4 (A–F) and at week 8 (G–L). ^a,bMeans with different letters differ significantly (P < 0.05)

Fig. 7

Effect of Prevotella and sodium succinate on the gut microbiota. A Venn diagram illustrated the overlap of operational taxonomic unit (OTU) in all tested chicken. B The composition of gut microbiota of community bar plot analysis on phylum level. C Heatmap hierarchical cluster analysis based on the 20 most abundant phylum among groups. D Linear discriminant analysis effect size (LEfSe) analyses (LDA score ≥ 4.0) at the phylum level to the genus level. E Cladogram generated from LEfSe analysis (LDA score ≥ 4.0). Different color nodes represent the significant enriched microbial groups in the corresponding treatment groups which have a significant impact on the differences among groups, and the diameters of the circles are proportional to the axon’s abundance

Fig. 8

Effect of Prevotella and sodium succinate on the gut microbiota. A–D Alpha diversity among groups (Faith PD, Chao1, Shannon, and Simpson index). E Principal coordinate analysis (PCoA) based on Bray–Curtis dissimilarity. F Partial least squares discriminant analysis (PLS-DA). G The relative abundance of Prevotellaceae. H The relative abundance of Prevotella. ^a,bMeans with different letters differ significantly (P < 0.05)

Fig. 9

Prevotella and sodium succinate influenced the correlations between intestinal flora and physicochemical parameters. A Redundancy analysis (RDA), parameters are represented by arrows. The longer the arrows are, the greater the correlation coefficients are. The Angle between the arrow line and the ordering axis represents the correlation between an environmental factor and the ordering axis. The smaller the Angle is, the higher the correlation will be. The larger the dot, the higher the corresponding species abundance. B Spearman's correlation analysis between serum biochemical indexes and cecal microbiota at the phylum level, R-values are shown in different colors in the heatmap, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. C The genus-level microbiome Spearman’s correlation network, the circles represent a species, the size represents its relative abundance, and different colors represent different phylum species classification. The lines between the circles represent significant correlation between the two species (P < 0.05), and the lines color red represents positive correlation and blue represents negative correlation. The thicker the lines, the greater the absolute value of the correlation coefficient. D Phylogenetic tree and heatmap, on the left, different colored branches of the evolutionary tree represent different phyla, and each end branch represents an OTU, annotated with the genus classification to which the corresponding OTU belongs, the heat map on the right represents the normalized abundance, with larger values representing higher relative abundance