Comparative Metabolite Fingerprinting of the Rumen System during Colonisation of Three Forage Grass (Lolium perenne L.) Varieties

The rumen microbiota enable ruminants to degrade complex ligno-cellulosic compounds to produce high quality protein for human consumption. However, enteric fermentation by domestic ruminants generates negative by-products: greenhouse gases (methane) and environmental nitrogen pollution. The current lack of cultured isolates representative of the totality of rumen microbial species creates an information gap about the in vivo function of the rumen microbiota and limits our ability to apply predictive biology for improvement of feed for ruminants. In this work we took a whole ecosystem approach to understanding how the metabolism of the microbial population responds to introduction of its substrate. Fourier Transform Infra Red (FTIR) spectroscopy-based metabolite fingerprinting was used to discriminate differences in the plant-microbial interactome of the rumen when using three forage grass varieties (Lolium perenne L. cv AberDart, AberMagic and Premium) as substrates for microbial colonisation and fermentation. Specific examination of spectral regions associated with fatty acids, amides, sugars and alkanes indicated that although the three forages were apparently similar by traditional nutritional analysis, patterns of metabolite flux within the plant-microbial interactome were distinct and plant genotype dependent. Thus, the utilisation pattern of forage nutrients by the rumen microbiota can be influenced by subtleties determined by forage genotypes. These data suggest that our interactomic approach represents an important means to improve forages and ultimately the livestock environment

Using the forces of hydrodynamic countercurrent chromatography for the study of bacteriophages

Bacteriophages (phages) are viruses that target bacteria, with the ability to lyse and kill host bacterial cells. Due to this, they have been of some interest as a therapeutic since their discovery in the early 1900s, but with the recent increase in antibiotic resistance, phages have seen a resurgence in attention. Current methods of isolation and purification of phages can be long and tedious, with caesium chloride concentration gradients the gold standard for purifying a phage fraction. Isolation of novel phages requires centrifugation and ultrafiltration of mixed samples, such as water sources, effluent or faecal samples etc, to prepare phage filtrates for further testing. We propose countercurrent chromatography as a novel and alternative approach to use when studying phages, as a scalable and high-yield method for obtaining phage fractions. However, the full extent of the usefulness and resolution of separation with this technique has not been researched; it requires optimization and ample testing before this can be revealed. Here we present an initial study to determine survivability of two phages, T4 and ϕX174, using only water as a mobile phase in a Spectrum Series 20 HPCCC. Both phages were found to remain active once eluted from the column. Phages do not fully elute from the column and sodium hydroxide is necessary to flush the column between runs to deactivate remaining phages

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

Increasing the efficiency of utilization of fresh and preserved forage is a key target for ruminant science. Vitamin E is often used as additive to improve product quality but its impact of the rumen function is unknown. This study investigated the successional microbial colonization of ryegrass (GRA) vs. ryegrass hay (HAY) in presence of zero or 50 IU/d supplementary vitamin E, using a rumen simulation technique. A holistic approach was used to link the dynamics of feed degradation with the structure of the liquid-associated (LAB) and solid-associated bacteria (SAB). Results showed that forage colonization by SAB was a tri-phasic process highly affected by the forage conservation method: Early colonization (0?2 h after feeding) by rumen microbes was 2? faster for GRA than HAY diets and dominated by Lactobacillus and Prevotella which promoted increased levels of lactate (+56%) and ammonia (+18%). HAY diets had lower DM degradation (-72%) during this interval being Streptococcus particularly abundant. During secondary colonization (4?8 h) the SAB community increased in size and decreased in diversity as the secondary colonizers took over (Pseudobutyrivibrio) promoting the biggest differences in the metabolomics profile between diets. Secondary colonization was 3? slower for HAY vs. GRA diets, but this delay was compensated by a greater bacterial diversity (+197 OTUs) and network complexity resulting in similar feed degradations. Tertiary colonization (>8 h) consisted of a slowdown in the colonization process and simplification of the bacterial network. This slowdown was less evident for HAY diets which had higher levels of tertiary colonizers (Butyrivibrio and Ruminococcus) and may explain the higher DM degradation (+52%) during this interval. The LAB community was particularly active during the early fermentation of GRA and during the late fermentation for HAY diets indicating that the availability of nutrients in the liquid phase reflects the dynamics of feed degradation. Vitamin E supplementation had minor effects but promoted a simplification of the LAB community and a slight acceleration in the SAB colonization sequence which could explain the higher DM degradation during the secondary colonization. Our findings suggest that when possible, grass should be fed instead of hay, in order to accelerate feed utilization by rumen microbespublishersversionPeer reviewe

Cut-lengths of perennial ryegrass leaf-blades influences in vitro fermentation by the anaerobic fungus neocallimastix frontalis

Anaerobic fungi in the gut of domesticated and wild mammalian herbivores play a key role in the host’s ability to utilize plant biomass. Due to their highly effective ability to enzymatically degrade lignocellulose, anaerobic fungi are biotechnologically interesting. Numerous factors have been shown to affect the ability of anaerobic fungi to break down plant biomass. However, methods to reduce the non-productive lag time in batch cultures and the effect of leaf-blade cut-length and condition on the fungal fermentation are not known. Therefore, experimentation using a novel gas production approach with pre-grown, axenic cultures of Neocallimastix frontalis was performed using both fresh and air-dried perennial ryegrass leaf-blades of different cut-lengths. The methodology adopted removed the lag-phase and demonstrated the digestion of un-autoclaved leaf-blades. Fermentation of leaf-blades of 4.0 cm cut-length produced 18.4% more gas yet retained 11.2% more apparent DM relative to 0.5 cm cut-length leaf-blades. Drying did not affect fermentation by N. frontalis, although an interaction between drying and leaf-blade cut-length was noted. Removal of the lag phase and the use of un-autoclaved substrates are important when considering the biotechnological potential of anaerobic fungi. A hypothesis based upon sporulation at cut surfaces is proposed to describe the experimental results

A Multi-Kingdom Study Reveals the Plasticity of the Rumen Microbiota in Response to a Shift From Non-grazing to Grazing Diets in Sheep

Increasing feed efficiency is a key target in ruminant science which requires a better understanding of rumen microbiota. This study investigated the effect of a shift from a non-grazing to a grazing diet on the rumen bacterial, methanogenic archaea, fungal, and protozoal communities. A systems biology approach based on a description of the community structure, core microbiota, network analysis, and taxon abundance linked to the rumen fermentation was used to explore the benefits of increasing depth of the community analysis. A total of 24 sheep were fed ryegrass hay supplemented with concentrate (CON) and subsequently ryegrass pasture (PAS) following a straight through experimental design. Results showed that concentrate supplementation in CON-fed animals (mainly starch) promoted a simplified rumen microbiota in terms of network density and bacterial, methanogen and fungal species richness which favored the proliferation of amylolytic microbes and VFA production (+48%), but led to a lower (ca. 4-fold) ammonia concentration making the N availability a limiting factor certain microbes. The adaptation process from the CON to the PAS diet consisted on an increase in the microbial concentration (biomass of bacteria, methanogens, and protozoa), diversity (+221, +3, and +21 OTUs for bacteria, methanogens, and fungi, respectively), microbial network complexity (+18 nodes and +86 edges) and in the abundance of key microbes involved in cellulolysis (Ruminococcus, Butyrivibrio, and Orpinomyces), proteolysis (Prevotella and Entodiniinae), lactate production (Streptococcus and Selenomonas), as well as methylotrophic archaea (Methanomassiliicoccaceae). This microbial adaptation indicated that pasture degradation is a complex process which requires a diverse consortium of microbes working together. The correlations between the abundance of microbial taxa and rumen fermentation parameters were not consistent across diets suggesting a metabolic plasticity which allowed microbes to adapt to different substrates and to shift their fermentation products. The core microbiota was composed of 34, 9, and 13 genera for bacteria, methanogens, and fungi, respectively, which were shared by all sheep, independent of diet. This systems biology approach adds a new dimension to our understanding of the rumen microbial interactions and may provide new clues to describe the mode of action of future nutritional interventions

Forage grass growth under future climate change scenarios affects fermentation and ruminant efficiency

With an increasing human population access to ruminant products is an important factor in global food supply. While ruminants contribute to climate change, climate change could also affect ruminant production. Here we investigated how the plant response to climate change affects forage quality and subsequent rumen fermentation. Models of near future climate change (2050) predict increases in temperature, CO2, precipitation and altered weather systems which will produce stress responses in field crops. We hypothesised that pre-exposure to altered climate conditions causes compositional changes and also primes plant cells such that their post-ingestion metabolic response to the rumen is altered. This “stress memory” effect was investigated by screening ten forage grass varieties in five differing climate scenarios, including current climate (2020), future climate (2050), or future climate plus flooding, drought or heat shock. While varietal differences in fermentation were detected in terms of gas production, there was little effect of elevated temperature or CO2 compared with controls (2020). All varieties consistently showed decreased digestibility linked to decreased methane production as a result of drought or an acute flood treatment. These results indicate that efforts to breed future forage varieties should target tolerance of acute stress rather than long term climate