The Effect of Ryegrass Silage Feeding on Equine Fecal Microbiota and Blood Metabolite Profile

Abstract

Silage is fed to horses in China and other areas in the world, however, knowledge about the impact of feeding silage on horse health is still limited. In the current study, 12 horses were assigned into two groups and fed ryegrass silage and ryegrass hay, respectively, for 8 weeks. High-throughput sequencing was applied to analyze fecal microbiota, while liquid chromatography-tandem mass spectrometry (LC-MS/MS) based metabolomics technique was used for blood metabolite profile to investigate the influence of feeding ryegrass silage (group S) compared to feeding ryegrass hay (group H) on equine intestinal and systemic health. Horses in group S had significantly different fecal microbiota and blood metabolomes from horses in group H. The results showed that Verrucomicrobia was significantly less abundant which plays important role in maintaining the mucus layer of the hindgut. Rikenellaceae and Christensenellaceae were markedly more abundant in group S and Rikenellaceae may be associated with some gut diseases and obesity. The metabolomics analysis demonstrated that ryegrass silage feeding significantly affected lipid metabolism and insulin resistance in horses, which might be associated with metabolic dysfunction. Furthermore, Pearson's correlation analysis revealed some correlations between bacterial taxa and blood metabolites, which added more evidence to diet-fecal microbiota-health relationship. Overall, ryegrass silage feeding impacted systemic metabolic pathways in horses, especially lipid metabolism. This study provides evidence of effects of feeding ryegrass silage on horses, which may affect fat metabolism and potentially increase risk of insulin resistance. Further investigation will be promoted to provide insight into the relationship of a silage-based diet and equine health.

Keywords: fecal microbiota; hay; horses; metabolome; silage.

FIGURE 1

Relative abundance of the dominant bacterial taxa (> 1%) at the phylum (A) and family (B) level of two groups. Student’s t-test was used to assess the difference of the relative abundance between the two groups. *P < 0.05, **P < 0.01. Red, group H; Blue, group S.

FIGURE 2

Alpha diversity indices of the fecal microbiota in horses fed silage (S, n = 6) and hay (H, n = 6), **P < 0.01. (A) Bacterial taxa richness or Chao index of each group (B) Bacterial taxa diversity of Shannon index of each group. Red, group H; Blue, group S.

FIGURE 3

Microbial community analysis of fecal microbiota in group S and group H. (A) Principal coordinates analysis (PCoA) was conducted based on Bray-Curtis distances with P-values of Anosim to emphasize the difference of fecal microbiota community in the two groups. Red dot: PCoA value of individual horse in group H, blue triangle: PCoA value of individual horse in group S. (B) LEfSe illustrates the difference of fecal microbiota between group S and group H at different levels. only taxa with LDA score of 3 or above are shown and ranked by the effect size in LEfSe. Red, group H; blue, group S.

FIGURE 4

Effect of ryegrass silage on horse metabolite profiles. (A) Principal component analysis (PCA) of blood metabolites of group S and group H. Green dot: PCA value of individual horse in group S. Orange rectangle: PCA value of individual horse in group H. (B) Metabolites discriminating between group S and group H with VIP > 1 were listed. Red dots indicate up-regulated metabolites. Green dots indicated down-regulated metabolites. (C) Heatmap of significantly altered metabolites in group S and group H. Individual samples (horizontal axis) and metabolites (vertical axis) are presented using hierarchical cluster analysis. The color scale is noted on the right side of the figure. Red and green color represent up-regulated and down-regulated metabolites, respectively, relative to the median metabolite level. (D) KEGG enrichment of each pathway related to this study was presented using rich factor. P-values and impact values are indicated on the horizontal axis and vertical axis, respectively. The sized and colors of the shapes represent the influence of silage diet on sample pathways relative to hay diet. The larger red shapes indicate greater impact of the diet on the pathway.

FIGURE 5

Heatmap of Pearson’s correlation between the fecal microbiota and blood metabolites significantly different between groups is illustrated. Red represents significantly positive correlation (P < 0.05), green represents significantly negative correlation (P < 0.05) and yellow represents that the correlation is not significant (P > 0.05).