The Equine Faecal Microbiota of Healthy Horses and Ponies in The Netherlands: Impact of Host and Environmental Factors

Abstract

Several studies have described the faecal microbiota of horses and the factors that influence its composition, but the variation in results is substantial. This study aimed to investigate the microbiota composition in healthy equids in The Netherlands under standard housing and management conditions and to evaluate the effect of age, gender, horse type, diet, pasture access, the season of sampling and location on it. Spontaneously produced faecal samples were collected from the stall floor of 79 healthy horses and ponies at two farms. The validity of this sampling technique was evaluated in a small pilot study including five ponies showing that the microbiota composition of faecal samples collected up to 6 h after spontaneous defaecation was similar to that of the samples collected rectally. After DNA extraction, Illumina Miseq 16S rRNA sequencing was performed to determine microbiota composition. The effect of host and environmental factors on microbiota composition were determined using several techniques (NMDS, PERMANOVA, DESeq2). Bacteroidetes was the largest phylum found in the faecal microbiota (50.1%), followed by Firmicutes (28.4%). Alpha-diversity and richness decreased significantly with increasing age. Location, age, season, horse type and pasture access had a significant effect on beta-diversity. The current study provides important baseline information on variation in faecal microbiota in healthy horses and ponies under standard housing and management conditions. These results indicate that faecal microbiota composition is affected by several horse-related and environment-related factors, and these factors should be considered in future studies of the equine faecal microbiota.

Keywords: age; diet; equine; faecal; gender; location; microbiota; pasture; pony; season.

Figure 1

Observed richness and alpha-diversity (Shannon diversity index) of the faecal microbiota over time of faecal samples collected from five ponies after exposure to room air. A decrease in observed richness alpha-diversity is visible after 12 h of air exposure (p = 0.062).

Figure 2

The effect of age on the observed richness and alpha-diversity (Shannon diversity index) on the faecal microbiota of 61 horses and ponies. A significant decrease, determined with linear modelling, in observed richness and alpha-diversity is visible in older horses.

Figure 3

Differentially abundant ASVs grouped by family for increasing age (by year) of the faecal microbiota in 61 horses and ponies. The log2 fold change (per year) in ASV abundance is shown on the x-axis. ASVs assigned to bacterial families on the left side of the plot are less abundant in horses with increasing age. ASVs assigned to families depicted on the right side of the plot are more abundant in horses with increasing age. NA = ASV belonging to an unknown family (colours indicate the phylum).

Figure 4

Differentially abundant ASVs grouped by family for horse type (ponies vs. horses) of the faecal microbiota in 61 horses and ponies. The log2 fold change in species abundance is shown on the x-axis. ASVs assigned to bacterial families on the left side of the plot are less abundant in ponies than horses, ASVs assigned to families depicted on the right side of the plot are more abundant in ponies than horses. NA = ASV belonging to an unknown family (colours indicate the phylum).

Figure 5

Relative abundance of phyla in the faecal microbiota of 61 horses and ponies in The Netherlands. (A) Summer (B) Winter.

Figure 6

Differentially abundant ASVs grouped by family for season of sampling (winter vs. summer) of the faecal microbiota in 61 horses and ponies. The log2 fold change in species abundance is shown on the x-axis. ASVs assigned to bacterial families on the left side of the plot are less abundant in samples collected in winter compared to samples collected in summer, ASVs assigned to families depicted on the right side of the plot are more abundant samples collected in the winter compared to sampled collected in the summer. NA = ASV belonging to an unknown family (colours indicate the phylum).

Figure 7

NMDS plot of beta-diversity showing the effect of location (farm) on the faecal microbiota composition of 35 horses. Red = Farm II, Blue = Farm I. NMDS stress level = 0.17.