Effects of Pasture Grass, Silage, and Hay Diet on Equine Fecal Microbiota.

The research study delves into the impact of three distinct forage feeds—pasture grass, silage, and hay—on the fecal microbiota of horses. By examining the microbial community in the horses’ gastrointestinal tract, the study elucidates the role different diets play in shaping these communities, highlighting significant changes in bacterial diversity.

Objective:

The primary aim of this research is to investigate the effects of three different forage feeds—grass at pasture, silage, and hay—on the horse fecal microbiota.

Introduction:

The introduction emphasizes the evolutionary significance of horses and their adaptability in terms of dietary needs. The fecal microbiota serves as a common tool to study the influence of diet on the equine intestinal microbiota. The research underscores the traditional reliance on pastures and hay as primary feed sources for horses. However, in specific regions like Xinjiang in northwest China, silage has gained prominence as a feed choice. This preference for silage stems from its economic advantages and storage convenience compared to other feed types.

Materials and Methods:

2.1. Ethnic Announcement:

• Location: Guanzhong Stud farm, Shanxi province, China.
• Licensing: The study had a welfare license (No.AW11101202-2-1) from the Animal Care and Use Committee of China Agricultural University.

2.2. Animal Selection and Experimental Design:

• Participants: 24 mares and 12 stallions of the Guanzhong breed.
• Selection: Randomly selected from a Horse Ranch in Shanxi Province.
• Average Weight: 391 kg (SD ± 21.4 kg) as per the estimation formula by Carroll and Huntington.
• Health: No illness or antibiotic administration history for 6 months prior.
• Age Range: 3 to 15 years with a mean age of 7 years.
• Body Condition: Mean score of 4.8 (SD ± 0.6) based on the Henneke scoring system.
• Pre-experiment Diet: Same commercial hay with corn-based concentrates.
• Experimental Diets: Local pasture ryegrass, ryegrass silage, and second-cutting ryegrass hay.
• Feed Analysis: Nutritive content of each diet was assessed at the Animal Nutrient Analysis Center of China Agricultural University.

2.3. Fecal Sampling:

• Sampling: Fecal samples collected manually from each horse’s mid-rectum at the feeding trial’s end over three days.
• Handling: Samples temporarily stored in ice, then processed in the lab where the central part of each fecal sample was stored at −80 °C.

2.4. DNA Preparation:

• Method: Using the E.Z.N.A.® soil DNA Kit, bacterial genomic DNA was extracted from fecal samples.
• Assessment: DNA concentration and purity were evaluated using the NanoDrop 2000 UV-vis spectrophotometer.

2.5. High-Throughput Sequencing:

• Target: V3–V4 region of the 16S rRNA gene.
• Sequencing: Paired-end reads sequenced on the Illumina MiSeq PE300 platform/NovaSeq PE250 platform.

2.6. Bioinformatics Analysis:

• Data Processing: The 16S rRNA gene sequencing reads were filtered, merged, and clustered.
• Analysis: Various tools and databases like QIIME, RDP Classifier, and Silva rRNA were used for sequence identification and comparison.
• Diversity Analysis: Employed MOTHUR for alpha diversity analysis.
• Comparative Analysis: PCoA used to assess bacterial community variations between groups, along with ANOSIM and LEfSe for significant differences.

Results:

General Health of Horses:

The body condition and weight of all the horses remained consistent throughout the feeding trial. Additionally, no clinical abnormalities were observed in any of the horses during the trial.

3.1 Sequencing Quality Data:

• A total of 2,463,742 sequence reads were detected across all samples.
• After quality control, 1,181,628 sequences remained, ranging from 32,823 to 71,449 per sample.
• At the 97% threshold level, 3,644 OTUs (Operational Taxonomic Units) were identified, which were classified across various bacterial taxonomic levels.
• The rarefaction curve suggested that the sequencing depth was sufficient to capture the bacterial community from the samples.

3.2 Microbial Composition Analysis:

• The dominant bacterial populations were visualized using stacked histograms at different taxonomic levels.
• At the phylum level, the most abundant phyla were Firmicutes (59.8%), Bacteroidetes (25.2%), and Verrucomicrobia (6.4%), among others.
• Notably, the relative abundance of some phyla like Verrucomicrobia and Spirochaetes varied significantly between the groups.
• At the family level, Lachnospiraceae and Oscilliospiraceae were the most prevalent in all groups but showed significant differences between the groups.
• At the genus level, some genera showed significant variations between the groups, with Streptococcus being notably lower in group P.

3.3 Alpha Diversity Analysis:

• The Chao and Shannon indices were used to analyze microbial species richness and diversity, respectively.
• Group P had the richest microbial community based on the Chao index, while group H had the lowest.
• Both group S and H had significantly lower microbial diversity than group P based on the Shannon index.

3.4 Beta Diversity and LEfSe Analysis:

• The beta diversity analysis, using the Bray–Curtis distance, showed distinct bacterial composition clusters for each group based on their dietary regimens.
• The LEfSe (Linear Discriminant Analysis Effect Size) identified distinct microbial taxa associated with each feeding pattern.
• 414 taxa showed significant differences between the groups, with 26 of them having a notably high LDA score.
• Specific bacterial taxa were identified as highly associated with each dietary group.

Discussion:

The study delves into the factors influencing the microbial community in the gastrointestinal tract of horses. There was a noticeable shift in the fecal bacterial diversity and structure when horses were subjected to different diets, more so when supplements were introduced into their regimen. The study highlighted the distinct microbial compositions associated with each diet group, as represented by a PCoA plot with specific clusters. The research identifies two predominant bacterial phyla, Firmicutes and Bacteroidetes, that play a pivotal role in the equine intestinal ecosystem.

Experimental Setup:

• The study aimed to standardize various factors to ensure a consistent effect of diet on the horses’ gut microbiota. The feeding trial was synchronized and maintained over an 8-week period to allow adaptation to the new diets.
• To minimize differences in microbial communities between male and female horses, a 2:1 mare to stallion ratio was maintained in each group.
• The study selected horses with similar body conditions and ages to reduce variance due to obesity and aging effects.
• The amount of feed given to the horses was based on recommended standards. However, estimating pasture grass intake was challenging.

Microbial Diversity and Diet:

• Diet plays a crucial role in shaping the fecal bacterial diversity of horses.
• Significant differences were observed in the richness and diversity of fecal microbiota across the three diet groups. Horses on pasture showed the highest microbial richness and diversity, while those on hay had the least.
• A diverse microbial community is vital for maintaining healthy gut flora and preventing the overgrowth of harmful bacteria. The high diversity in the pasture group could be due to the variability of feed available in natural grazing conditions.
• Lower microbial diversity in humans has been linked to gastrointestinal disorders. Similarly, horses with reduced microbial diversity have been linked to conditions like colic.

Microbial Composition Variations:

• Microbial compositions varied across the groups. Silage, being a fermented product, might impact the hindgut microbiota differently compared to hay and fresh grass.
• The study noted differences in the relative abundance of certain bacteria across groups based on the type of diet. For instance, horses on silage had the highest relative abundance of Euryarchaeota, a phylum associated with methane production.
• Firmicutes and Bacteroidetes were the dominant phyla in all groups, indicative of a healthy intestinal status. However, the abundance of Fibrobacteres, a fibrolytic phylum, varied across groups.
• The Lachnospiraceae family, which plays a significant role in fiber digestion, varied in abundance between groups. It was most abundant in horses fed hay, possibly due to the high dry matter content in hay.
• The Oscillospiraceae family, which might be influenced by dietary ingredients like crude protein, also varied in abundance across groups.
• The Streptococcaceae family, associated with lactic acid production and sometimes linked to laminitis in horses, had notably lower levels in the pasture group. Its role in horse health remains to be fully understood.
• Distinct bacterial taxa associated with silage-fed horses were identified, but further research is needed to understand their roles in gut health.

Knowledge Gaps:

• The presence of several unranked or unclassified bacterial genera across all groups highlights the existing gaps in our understanding of the equine intestinal ecosystem. Advanced bacterial identification methods could provide deeper insights into equine gut microbiota.

Conclusion:

The research concludes that the diet significantly influences the microbial community in the gastrointestinal tract of horses. Different diets, especially when supplemented, induce variations in fecal bacterial diversity and structure. The study underscores the need to consider diet when analyzing the equine intestinal ecosystem, especially given the distinct roles of bacterial phyla like Firmicutes and Bacteroidetes.