Effects of concentrate supplementation on fecal microbiota, serum metabolomics, and proteomics in grazing Yili horses

Technical Summary: Effects of Concentrate Supplementation on Fecal Microbiota, Serum Metabolomics, and Proteomics in Grazing Yili Horses

Problem Statement
The Yili horse, a dual-purpose breed in China, relies on natural grazing, which is subject to seasonal fluctuations and rainfall variations that can compromise pasture quality and nutrient availability. While natural pastures provide roughage, they may fail to meet the specific energy and protein requirements of early-lactation mares, potentially restricting health management and the development of mare milk resources. Although concentrate supplementation is a known strategy to address nutritional gaps, there is a lack of integrated research examining how such supplementation affects the complex interplay between hindgut fermentation, microbial community structure, and host systemic metabolism (metabolomics and proteomics) in grazing equids.

Methodology
The study employed a controlled 100-day experiment (10-day adaptation, 90-day trial) with 16 early-lactation Yili mares (3–4 years old, ~391.5 kg) randomly assigned to two groups:

• Grazing Group (G): Grazed naturally on pasture.
• Grazing + Supplementation Group (GS): Received 2 kg/day of a formulated concentrate supplement (52% corn, 20% soybean meal, 12% bran, 10% barley, plus minerals/vitamins) in addition to natural grazing.

Data Collection and Analysis:

• Samples: Fecal samples (for fermentation parameters, pH, and 16S rRNA sequencing) and blood samples (for biochemical indices, metabolomics, and proteomics) were collected on day 90.
• Fecal Analysis: Measured volatile fatty acids (VFAs) via gas chromatography and assessed microbial diversity using PacBio full-length 16S rRNA sequencing.
• Serum Analysis:
  • Biochemistry: Automated analyzer for glucose, lipids, liver enzymes, and bilirubin.
  • Metabolomics: Untargeted UPLC-MS/MS (ESI+ and ESI-) to identify serum metabolites.
  • Proteomics: DIA-based LC-MS/MS (Orbitrap Astral) to quantify serum proteins.
• Statistics: Independent t-tests for biochemical data; OPLS-DA for metabolomics; LEfSe for microbiota; and correlation analyses (Spearman) to link microbiota, fermentation, metabolites, and proteins.

Key Results

• Fecal Fermentation and Microbiota:

  • The GS group exhibited significantly higher fecal propionate and butyrate concentrations, lower fecal pH, and a lower acetate-to-propionate ratio compared to the G group.
  • Microbial diversity (Simpson index) was higher in the GS group.
  • Genus-level shifts: The GS group showed significantly higher relative abundances of Treponema and Oliverpabstia, while the G group had higher abundances of Lachnoclostridium, Roseburia, Anaerosacchariphilus, and Coprococcus.
  • Correlation analysis indicated that Treponema was positively correlated with butyrate and negatively with pH, whereas fiber-degrading genera (Lachnoclostridium, Roseburia) were negatively correlated with fermentation products in the supplemented group.

• Serum Biochemistry:

  • Concentrate supplementation significantly increased serum glucose (GLU).
  • It significantly decreased total bilirubin (TBIL), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C).
  • No significant differences were found in other indices like urea, ALT, AST, or triglycerides.

• Serum Metabolomics:

  • 487 differential metabolites were identified (78 upregulated, 409 downregulated in GS).
  • KEGG enrichment highlighted pathways related to the tricarboxylic acid (TCA) cycle, nucleotide metabolism, bile secretion, vitamin B6 metabolism, and pyruvate metabolism.
  • Specific increases in the GS group included estrone-3-sulfate, fumaric acid, uric acid, and citric acid. Decreases included cis-aconitate, inosine, and guanine.

• Serum Proteomics:

  • 31 differentially expressed proteins (DEPs) were identified (20 upregulated, 11 downregulated in GS).
  • Upregulated: Peroxiredoxin-5 (PRDX5), lactoylglutathione lyase (GLO1), transketolase, ATP1A1, and several immune-related proteins (e.g., immunoglobulin light chain fragments, alpha-1-antitrypsin).
  • Downregulated: Phospholipid transfer protein, annexin, kallikrein-related peptidase 11, and specific immunoglobulin fragments.
  • Functional enrichment pointed to metabolic processes, cellular responses to stimulus, and immune system processes, specifically involving pyruvate metabolism and the pentose phosphate pathway.

Key Contributions
This study provides a multi-omics perspective on how concentrate supplementation alters the physiological state of grazing Yili horses. By integrating fecal microbiota, fermentation parameters, serum metabolomics, and proteomics, the research elucidates the mechanistic link between dietary starch intake and host metabolic adaptation. It demonstrates that supplementation shifts hindgut fermentation toward propionate and butyrate production and modulates serum profiles to enhance glucose availability and antioxidant capacity while altering lipid and bile acid metabolism.

Significance and Claims
The authors conclude that moderate concentrate supplementation (2 kg/day) effectively alters fecal fermentation patterns and microbial composition in grazing Yili horses. The study claims that these dietary changes regulate pathways related to serum glucose and lipid metabolism, bile acid metabolism, antioxidant responses, and energy metabolism. Specifically, the upregulation of antioxidant proteins (PRDX5, GLO1) and enzymes involved in the pentose phosphate pathway suggests an enhanced capacity for metabolic buffering and detoxification.

The paper posits that these findings suggest moderate concentrate supplementation may improve the nutritional and metabolic status of grazing Yili horses, particularly during lactation. The study aims to provide a theoretical basis for precision nutritional management and feeding strategies that optimize horse health while maintaining grassland ecological stability. The authors explicitly note that while Treponema abundance increased (a genus sometimes associated with inflammation), the study did not measure histological changes, so this potential risk is noted with caution.