Temporal Metagenomic and Metabolomic Characterization of Fresh Perennial Ryegrass Degradation by Rumen Bacteria

Abstract

Understanding the relationship between ingested plant material and the attached microbiome is essential for developing methodologies to improve ruminant nutrient use efficiency. We have previously shown that perennial ryegrass (PRG) rumen bacterial colonization events follow a primary (up to 4 h) and secondary (after 4 h) pattern based on the differences in diversity of the attached bacteria. In this study, we investigated temporal niche specialization of primary and secondary populations of attached rumen microbiota using metagenomic shotgun sequencing as well as monitoring changes in the plant chemistry using mid-infrared spectroscopy (FT-IR). Metagenomic Rapid Annotation using Subsystem Technology (MG-RAST) taxonomical analysis of shotgun metagenomic sequences showed that the genera Butyrivibrio, Clostridium, Eubacterium, Prevotella, and Selenomonas dominated the attached microbiome irrespective of time. MG-RAST also showed that Acidaminococcus, Bacillus, Butyrivibrio, and Prevotella rDNA increased in read abundance during secondary colonization, whilst Blautia decreased in read abundance. MG-RAST Clusters of Orthologous Groups (COG) functional analysis also showed that the primary function of the attached microbiome was categorized broadly within “metabolism;” predominantly amino acid, carbohydrate, and lipid metabolism and transport. Most sequence read abundances (51.6, 43.8, and 50.0% of COG families pertaining to amino acid, carbohydrate and lipid metabolism, respectively) within these categories were higher in abundance during secondary colonization. Kyoto encyclopedia of genes and genomes (KEGG) pathways analysis confirmed that the PRG-attached microbiota present at 1 and 4 h of rumen incubation possess a similar functional capacity, with only a few pathways being uniquely found in only one incubation time point only. FT-IR data for the plant residues also showed that the main changes in plant chemistry between primary and secondary colonization was due to increased carbohydrate, amino acid, and lipid metabolism. This study confirmed primary and secondary colonization events and supported the hypothesis that functional changes occurred as a consequence of taxonomical changes. Sequences within the carbohydrate metabolism COG families contained only 3.2% of cellulose activities, on average across both incubation times (1 and 4 h), suggesting that degradation of the plant cell walls may be a key rate-limiting factor in ensuring the bioavailability of intra-plant nutrients in a timely manner to the microbes and ultimately the animal. This suggests that a future focus for improving ruminant nutrient use efficiency should be altering the recalcitrant plant cell wall components and/or improving the cellulolytic capacity of the rumen microbiota.

Invited review: Application of meta-omics to understand the dynamic nature of the rumen microbiome and how it responds to diet in ruminants

“…In accordance with this model, it has been observed that little biomass is degraded during primary colonization, with the majority of degradation occurring after this (Piao et al, 2014;Huws et al, 2016). It has also been hypothesized that decreasing microbial richness during the shift from primary to secondary colonization is due to niche specialization of Clostridiales and Bacteroidales, which play a specific role in lignocellulose degradation during secondary colonization Mayorga et al, 2016;Rubino et al, 2017).…”

“…Bacteria preferentially attach to damaged sites on the plant surface, whereas fungi are able to physically disrupt the ingested material via rhizoidal growth. Microbial attachment is absolutely essential for the development of the complex microbial populations required for feed digestion in the rumen and occurs via a multistep process: (1) displacement of the epiphytic microbiome by rumen microbes (time <1 h), (2) establishment of a primary colonizing community of generalist microbes that primarily metabolize accessible carbohydrates (time 1 h to 4 h), (3) loss of some primary colonizers and selection of secondary colonizers specialized in digesting hemicellulose and cellulose (time > 4 h; Figure 2) (Piao et al, 2014;Huws et al, 2016;Mayorga et al, 2016). Within this community are taxa including Butyrivibrio, Fibrobacter, Olsenella and Prevotella that do not change significantly in abundance during primary and secondary colonization Liu et al, 2016).…”

Changes in the Solid-, Liquid-, and Epithelium-Associated Bacterial Communities in the Rumen of Hu Lambs in Response to Dietary Urea Supplementation

“…This result is consistent with findings regarding the rumen solid and liquid fractions of dairy cattle fed ryegrass or white clover (Bowen et al, 2018) and a TMR (forage:concentrate = 70:30, forage = prewilted grass and maize silage) (De Mulder et al, 2017) and the rumen contents and epithelium of dairy cattle fed a TMR (forage:concentrate = 55:45, forage = corn silage and corn stover) (Liu et al, 2016). Prevotella represents one of the most abundant genera in the rumen; this genus exhibits genetic and metabolic diversity (Bekele et al, 2010) and plays roles in carbohydrate utilization (Dehority, 1966;Cotta, 1992;Gardner et al, 1995;Kabel et al, 2011), nitrogen metabolism (Kim et al, 2017), and fiber degradation (Mayorga et al, 2016). The results of these study suggest the importance of Prevotella spp.…”

A Systems Biology Approach Reveals Differences in the Dynamics of Colonization and Degradation of Grass vs. Hay by Rumen Microbes with Minor Effects of Vitamin E Supplementation

“…Several studies have described strategies to maximize forage quality ( Minson, 1990 ) and described the main differences between fresh grass and hay in terms of rumen digestion of nutrients using cannulated animals ( Petit and Tremblay, 1992 ; Holden et al, 1994 ). More recent studies based on molecular techniques have defined the bacterial colonization pattern for fresh grass ( Edwards et al, 2007 ; Huws et al, 2013 , 2016 ; Mayorga et al, 2016 ) and grass hay ( Piao et al, 2014 ) but no direct comparison between grass and hay has been reported. These studies focused only on the feed colonization by solid-associated microbes which under ordinary feeding conditions account for 75% of the microbial matter in the rumen ( Forsberg and Lam, 1977 ; Craig et al, 1987 ) and are responsible for most of the endoglucanase (90%), xylanase (88%), amylase (70%) and protease activity (75%) in the rumen ( McAllister et al, 1994a ) as well as cellulose and hemicellulose ( Williams and Strachan, 1984 ).…”