The percent values of ADF were greater overall in the adult feces relative to the yearlings samples (Fig. 1A). Moreover, Days 0 (pre-supplement) and 7 (mid-supplement) demonstrated lower ADF than Day 28 for mature horses. ADIA content (silica) within the feces was greater in the yearling samples (Fig. 1B), even when comparing silica to ADF ratio (Fig. 1C), yet no significant day-to-day differences were seen in either age group.
QIIME2 was used to analyze diversity of the microbial populations in the samples. Data were rarefied to 4364 reads with all collected samples included. A rarefaction plot revealed abundance of amplification sequence variants (ASVs) found within each group with depths plateauing between 200 and 400 ASVs (Figure S2A). Faith’s Phylogenetic Diversity – a measure of richness of taxonomical diversity taking into account levels of phylogeny – was greatest on Day 7 for adults and yearlings and at its least on Day 21 for adults and yearlings (Figure S2B). Shannon H Scores – measurements of evenness of diversity amongst samples – were above 7.0 for all groups and indicated that many species were represented (richness) within similar proportions (evenness) (Figure S2C). An Unweighted Unifrac PcoA Beta Diversity Plot demonstrates that there was little distinct clustering by day of study or by age (Figure S2D).
The most abundant phyla included Firmicutes, Proteobacteria, Bacteroidetes, Verrucomicrobia, Fibrobacteres, Spirochaetes, and Actinobacteria, which corroborated findings from our previous study (16) (Fig. 2). For Phylum Fibrobacteres, there was a significant peak at Day 28 of the study, roughly 18 days after psyllium supplementation (Fig. 2C). Additionally, a dip appeared in bacteria of the phyla Firmicutes at Day 14 with a concurrent spike in bacteria of the phyla Proteobacteria (Fig. 2D-E).
Microbial abundance taxa tables at the level of taxonomical families were inputted into LEfSe (21). Several microbial families were identified in yearlings as having significantly changed in at least one time point, including bacteria in the families Methylophilaceae, Burkholderiaceae, Saprospiraceae, Neisseriaceae, Fibrobacteraceae, and Paraprevotellaceae; of these, only Fibrobacteraceae and Paraprevotellaceae families represented abundances greater than 1% (Table S2). In mature mares, changes were found within bacterial families Victivallaceae, Bacteroidaceae, Moraxellaceae, as well as archaea families Methanobacteriaceae and Methanocorpusculaceae, with Victivallaceae, Bacteroidaceae, and Moraxellaceae reaching abundances above 1% (Table S3). Thus, overall LEfSe results demonstrated changes in microbial families with relatively minor differences in levels of abundance.
Finally, the data were also applied to a tool called PICRUSt in order to infer functional profiles of microbial populations present in the samples (20). During psyllium supplementation, there was increased mycothiol biosynthesis, catechol degradation, and urea cycle activity (Fig. 3).