Psyllium supplementation is associated with changes in the fecal microbiota of horses

Objective Prophylactic supplementation of psyllium husk is recommended to enhance passage of ingested sand from the gastrointestinal tracts of horses. We hypothesized that psyllium supplementation would increase fecal sand passage and favorably alter bacterial populations in the hindgut. Six yearlings and six mature mares were fed a psyllium supplement in the diet daily for seven days. Voluntarily-voided feces were collected over the course of 29 days, prior, during, and after treatment. Feces were analyzed for acid detergent fiber (ADF) and acid detergent insoluble ash analyses. Microbial DNA was also isolated, and the V4 region of the 16S ribosomal RNA gene was PCR-amplified and sequenced using MiSeq technology. Results Fecal ADF concentration was greater in adults while silica concentration was greater in yearlings. Mature mare fecal ADF decreased during and just after supplementation but thereafter increased. No changes in silica levels were noted in either group over time. Fecal microbial population phylogenetic diversity was greatest mid-supplementation and lowest at 11 days post-supplementation. Functional profiles of the microbial communities presented some benefits for psyllium supplementation. These findings provide compelling evidence for further detailed studies of prophylactic psyllium supplementation.


Introduction
Sand enteropathy is a prominent form of colic in which sand accumulates in the large colon of horses living in environments rich in sandy soil. Treatments for sand enteropathy include intravenous or oral uids and the administration of laxatives like magnesium sulfate, mineral oil, and psyllium to promote the evacuation of sand from a horse's GIT (1,2). Husk from the Plantago ovata plant is considered an effective prophylactic dietary supplement as approximately 50-60% of husk mass creates a hydrocolloid gel that increases fecal output which could push sand out of the GIT (3). Moreover, formulations of psyllium adhere to sand -a feature particularly valuable for landscaping and agricultural uses (4).
Psyllium also has intestinal anti-in ammatory and stimulatory gut motility properties in rodents and rabbits (5,6). In the horse, the combination of positive effects of psyllium all help to ameliorate the symptoms of sand enteropathy.
Since horses are hindgut fermenters, GI microbes play essential roles in the catabolism of complex food compounds, particularly brolytic bacteria fermenting structural carbohydrates of the plant cell wall of forages (7). Commensal microbes can promote a healthy GIT and reduce the ability of pathogenic organisms to colonize (7). It has yet to be determined how psyllium affects equine GIT microbes.
Changes in human gut microbial populations have been documented with psyllium supplementation, particularly for constipated individuals for which psyllium supplementation was associated with increased stool water and signi cant changes in fecal microbial populations (8).
We examined the effect of psyllium on silica output and GIT microbial populations before, during, and after supplementation. We hypothesized that psyllium supplementation would lead to increased fecal output of silica and to changes in fecal microbial populations. Acid detergent insoluble ash (ADIA) was used to determine silica levels in the feces. This strategy differs from previous techniques -large colon radiographic assessments and/or mesh lters separation of sand from feces (1,2,(9)(10)(11). Radiography is a popular diagnosis tool for sand colic because it provides information about location and severity within the GIT (10,12); however, the scope of this study was to understand sand clearance, even before the animals experienced colic, to explore psyllium's prophylactic potential. Furthermore, there are concerns about the accuracy of the quantitation of sand using otation and mesh lters. Fecal microbial DNA was isolated for PCR ampli cation and sequencing of the V3/V4 region of the 16S ribosomal RNA gene to determine represented microbial populations in each sample. All ndings were compared by age group and by study day.

Horses, Psyllium Supplementation, Fecal Sample Collection and Storage
Twelve horses -6 yearlings and 6 mature mares (adult age mean 21.5 +/-6.9 years) -were used in this study (Table S1). The animal care was approved by the university's Animal Care and Use Committee. Body weights were calculated by averaging weight tape and scale values (13). Horse diets included wheat hay for yearlings at morning and evening feedings while mares received alfalfa hay at morning feedings and wheat hay at evening feedings. A commercial psyllium product was supplemented with the daily morning ration balancer according to the manufacturer-recommended dosage for body weight (312.5 mg/kg) over the course of 7 days. Voluntarily-voided fecal samples were collected over 29 days (Days 0, 3,7,9,11,14,21,28). Days 0 and 3 were pre-supplementation; psyllium supplementation commenced on Day 4 and concluded on Day 10. Fecal samples were collected by taking feces from the upper middle portion of freshly voided manure piles in order to avoid any contamination from ground soil. Fecal samples were stored at -20 °C until analysis.

Dry Sample Analyses
Fecal samples were dried in a forced-air oven at 55 °C for 8 hours to achieve ≥ 85% dry matter. Samples were then ground in a Wiley mill through a 1-mm screen to ne particulates and analyzed using a Fibertec™ FT122/FT121 manual ber determination system (Foss Analytics, Denmark). ADIA methods were used to determine the effectiveness of psyllium in regards to sand removal by isolating insoluble matter (silica) within the feces (14,15).

Results
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 signi cant 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 rare ed to 4364 reads with all collected samples included. A rarefaction plot revealed abundance of ampli cation 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).
Combined barcoded libraries were submitted to the University of California Davis Genome Center DNA Technologies Core for 250 bp paired-end sequencing using the Illumina MiSeq platform. Raw sequence data are freely available at the Sequence Read Archive (SRA): Bio Project PRJNA649589. Ampli ed DNA sequences for each sample were analyzed using QIIME2 (Quantitative Insights Into Microbial Ecology) (19). Outputs from QIIME2 were then applied to the LEfSe (Linear Discriminant Analysis Effect Size) tool and PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) software (20, 21) ( Figure S1).

Statistical analyses
Fecal nutritional analysis data were analyzed with Graphpad Prism 6.0 (La Jolla, CA, USA) and with jamovi (22) with two-way ANOVA ( xed factors: age, day, age-day interaction; dependent variables: percent ber, percent insoluble ash, PICRUSt prediction) using Tukey-multiple test corrections for contrasts. Taxonomy data were analyzed with Friedman tests (across days within yearlings and within adults) and with Kruskal-Wallis tests (across groups each day), both with Dunn's multiple comparison tests; signi cance was assessed p < 0.05. (16) (Fig. 2). For Phylum Fibrobacteres, there was a signi cant 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 identi ed in yearlings as having signi cantly 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 pro les of microbial populations present in the samples (20). During psyllium supplementation, there was increased mycothiol biosynthesis, catechol degradation, and urea cycle activity (Fig. 3).

Discussion
Contrary to our hypothesis, fecal silica analyses (ADIA) from this study did not demonstrate any increase in silica excretion with manufacturer-recommended prophylactic doses of psyllium husk crumbles. Other studies providing therapeutic doses of psyllium as well as combinations of additional therapeutics (prebiotics and probiotics) demonstrated increased clearance of silica with supplementation (2,9,11,23).
We did see greater levels of fecal silica excreted from the yearlings. However, these contrasts could be associated with differences in forages fed to yearlings and adults. Higher relative fecal ADF levels in the mature horses seem to indicate either better forage fermentation in the yearlings, reduced fermentation in the mature mares, or possibly better GIT health in the yearling contributing to more overall sand clearance and greater fermentation.
Microbial populations were analyzed from the standpoint of diversity, populations present, as well as their possible functional roles within the GIT. During and after psyllium supplementation, fecal archaea and bacteria were present in abundances similar to most adult horses (7,16,24). In considering Faith's Phylogenetic Diversity, the samples with the richest diversity were those collected during midsupplementation on Day 7. Others have demonstrated that greater diversity was positively correlated with a healthy horse GIT, and our ndings suggest that prophylactic psyllium was bene cial for improving microbial diversity (25,26). However, population changes were not dramatic from the standpoint of percent abundance of comprising microbes. LEfSe results indicated that even statistically signi cant changes in microbial abundances were relatively small with slight reductions in Burkholderiaceae. Slight increases were seen in Fibrobacteraceae and Paraprevotellaceae in yearlings, which contain essential cellulose-degrading bacteria and bacteria found in the GIT of pasture forage-fed horses, respectively (27,28). In adults, there was a slight reduction in methanogenic archaean Methanocorpusculaceae but slight increases in methanogenic bacteria Methanobacteriaceae, which captures hydrogen and improves fermentation e ciency, and bacterial Moraxellaceae commonly found in younger horses and foals (16,26,27,29). PICRUSt analysis demonstrated that during psyllium supplementation there was increased mycothiol biosynthesis activity in the GIT. Mycothiol is a protective antioxidant produced by bacteria in the Phylum Actinobacteria (30). Moreover, an upturn in urea cycle activity is indicative of increased urea utilization by gut bacteria as a nitrogen source during psyllium supplementation (31). Finally, catechol degradation pathways were elevated in GIT bacteria at the time of supplementation because psyllium is catechol rich. Furthermore, four days after the last dose of psyllium at Day 14 there was a spike in bacteria from the phyla Proteobacteria and a drop in bacteria from the Phyla Firmicutes; these changes could indicate that after psyllium was no longer present in the GIT, bacterial populations readjusted to gut environment changes. These ndings demonstrate that microbial populations were adapting to the psyllium supplied within the GIT. Thus, there is utility to further characterization of the effects of psyllium supplementation on sand excretion and promoting GIT health in horses, particularly at higher levels of supplementation either for prophylaxis or treatment of sand colic (9,11).

Limitations
This study does have several limitations. A small number of horses were included (n = 12 horses, 6 in each group). Additionally, more psyllium dosages could have been considered beyond the one manufacturer-recommended prophylactic dose. Furthermore, complete GIT sand quantities excreted were not measured but instead concentrations of ADF and silica in the feces. Thus, total quantities of silica excreted with psyllium supplementation cannot be provided from our measurements. Moreover, the yearlings and mares had differing diets contributing to differences between the forage ber characteristics in the groups -likely impacting fermentation, the microbial community, and overall gut motility comparisons between yearling and adult groups. We also did not account for breed or sex differences in our analyses. Additionally, we only evaluated microbial populations using the V3/V4 region of the 16S rRNA gene and did not carry out a metagenomic study. Author's contributions: AB, MJM, and EJP were responsible for conceptualization and hypotheses. Feeding trial and sample collections were conducted by AB, TNB, NM, and MJM. Microbial DNA isolation was performed by JDH, AB, TNB, and NM. Fecal nutritional analyses was performed by AB and JDH. Data analysis was performed by MJM and AB in consultation with EJP and EAM. MJM and EAM were responsible for funding acquisition. Manuscript was written by MJM and AB. All authors contributed to edits and revisions. Figure 1 Nutritional Evaluation of Fecal Samples. Overall percent acid detergent ber (ADF) in feces was greater for mature horses (A); for mature horses ADF concentration was lower at Days 0, 7, and 11, relative to Day 28 of the study. Percent silica concentration was comparatively greater overall for yearlings (B), even when normalized by ADF in a silica:ADF ratio (C). Box color representations are: red, pre-supplementation; yellow, mid-supplementation; blue, post-supplementation. Two-way ANOVA (day, age) analysis was performed with Tukey's multiple comparison tests for contrasts: a, p < 0.05 compared with Day 28; ***, p < 0.001.