An Investigation into Rumen Fungal and Protozoal Diversity in Three Rumen Fractions, during High-Fiber or Grain-Induced Sub-Acute Ruminal Acidosis Conditions, with or without Active Dry Yeast Supplementation

Abstract

Sub-acute ruminal acidosis (SARA) is a gastrointestinal functional disorder in livestock characterized by low rumen pH, which reduces rumen function, microbial diversity, host performance, and host immune function. Dietary management is used to prevent SARA, often with yeast supplementation as a pH buffer. Almost nothing is known about the effect of SARA or yeast supplementation on ruminal protozoal and fungal diversity, despite their roles in fiber degradation. Dairy cows were switched from a high-fiber to high-grain diet abruptly to induce SARA, with and without active dry yeast (ADY, Saccharomyces cerevisiae) supplementation, and sampled from the rumen fluid, solids, and epimural fractions to determine microbial diversity using the protozoal 18S rRNA and the fungal ITS1 genes via Illumina MiSeq sequencing. Diet-induced SARA dramatically increased the number and abundance of rare fungal taxa, even in fluid fractions where total reads were very low, and reduced protozoal diversity. SARA selected for more lactic-acid utilizing taxa, and fewer fiber-degrading taxa. ADY treatment increased fungal richness (OTUs) but not diversity (Inverse Simpson, Shannon), but increased protozoal richness and diversity in some fractions. ADY treatment itself significantly (P < 0.05) affected the abundance of numerous fungal genera as seen in the high-fiber diet: Lewia, Neocallimastix, and Phoma were increased, while Alternaria, Candida Orpinomyces, and Piromyces spp. were decreased. Likewise, for protozoa, ADY itself increased Isotricha intestinalis but decreased Entodinium furca spp. Multivariate analyses showed diet type was most significant in driving diversity, followed by yeast treatment, for AMOVA, ANOSIM, and weighted UniFrac. Diet, ADY, and location were all significant factors for fungi (PERMANOVA, P = 0.0001, P = 0.0452, P = 0.0068, Monte Carlo correction, respectively, and location was a significant factor (P = 0.001, Monte Carlo correction) for protozoa. Diet-induced SARA shifts diversity of rumen fungi and protozoa and selects against fiber-degrading species. Supplementation with ADY mitigated this reduction in protozoa, presumptively by triggering microbial diversity shifts (as seen even in the high-fiber diet) that resulted in pH stabilization. ADY did not recover the initial community structure that was seen in pre-SARA conditions.

Keywords: SARA; dairy cattle; fungal ITS; mothur; protozoal 18S; rumen pH.

Figure 1

Relative abundance of rumen fungi genera for cows receiving a high fiber (HF) or high grain (HG) diet, with (Y) or without (C) yeast supplementation. Treatments include high-fiber control (HFC), high-fiber yeast (HFY), high-grain control (HGC), and high-grain yeast (HGY).

Figure 2

Relative abundance of rumen protozoal species for cows receiving a high fiber (HF) or high grain (HG) diet, with (Y) or without (C) yeast supplementation. Treatments include high-fiber control (HFC), high-fiber yeast (HFY), high-grain control (HGC), and high-grain yeast (HGY).

Figure 3

Linear Discriminant Analysis of significant fungal OTUs in the epimural (E), fluid (F), and solid (S) fractions for cows receiving two dietary treatments with or without yeast supplementation under SARA conditions. Error bars represent standard deviation for OTUs with multiple LDA values. Treatments include high-fiber control (HFC), high-fiber yeast (HFY), high-grain control (HGC), and high-grain yeast (HGY).

Figure 4

Linear Discriminant Analysis of significant protozoal OTUs in the epimural (E), fluid (F), and solid (S) fractions for cows receiving two dietary treatments with or without yeast supplementation under SARA conditions. Error bars represent standard deviation for OTUs with multiple LDA values. Treatments include high-fiber control (HFC), high-fiber yeast (HFY), high-grain control (HGC), and high-grain yeast (HGY).

Figure 5

Non-metric Multidimensional Scaling (nMDS) plot for rumen fungi from cows receiving two dietary treatments with or without yeast supplementation under SARA conditions. Lowest stress = 0.13, R² = 0.93.

Figure 6

Non-metric Multidimensional Scaling (nMDS) plot for rumen protozoa from cows receiving two dietary treatments with or without yeast supplementation under SARA conditions. Lowest stress = 0.19, R² = 0.80.

Figure 7

Significant Pearson's correlations between diet, active dry yeast supplementation, and rumen location, with the top 20 fungal and protozoa OTUs.