Manipulating rumen microbiome and fermentation through interventions during early life: a review:Rumen manipulation in early life

The nutritional manipulations of the rumen microbiome to enhance productivity and health are rather limited by the resilience of the ecosystem once established in the mature rumen. Based on recent studies, it has been suggested that the microbial colonisation that occurs soon after birth opens a possibility of manipulation with potential to produce lasting effects into adult life. This paper presents the state-of-the-art in relation to early-life nutritional interventions by addressing three areas: the development of the rumen as an organ in regards to the nutrition of the new-born, the main factors that determine the microbial population that first colonizes and establishes in the rumen, and the key immunity players that contribute to shaping the commensal microbiota in the early stage of life to understand host-microbiome specificity. The development of the rumen epithelium and muscularization are differently affected by the nature of the diet and special care should be taken with regards to transition from liquid (milk) to solid feed. The rumen is quickly colonized by all type of microorganisms straight after birth and the colonization pattern may be influenced by several factors such as presence/absence of adult animals, the first solid diet provided, and the inclusion of compounds that prevent/facilitate the establishment of some microorganisms or the direct inoculation of specific strains. The results presented show how early-life events may be related to the microbial community structure and/or the rumen activity in the animals post-weaning. This would create differences in adaptive capacity due to different early life experiences and leads to the idea of microbial programming. However, many elements need to be further studied such as: the most sensitive window of time for interventions, the best means to test long term effectiveness, the role of key microbial groups and host-immune regulations

Effect of progressive inoculation of fauna-free sheep with holotrich protozoa and total-fauna on rumen fermentation, microbial diversity and methane emissions

Rumen methanogenesis represents an energy waste for the ruminant and an important source of greenhouse gas; thus, integrated studies are needed to fully understand this process. Eight fauna-free sheep were used to investigate the effect of successive inoculation with holotrich protozoa then with total fauna on rumen methanogenesis. Holotrichs inoculation neither altered rumen fermentation rate nor diet digestibility, but increased concentrations of acetate (+15%), butyrate (+57%), anaerobic fungi (+0.82 log), methanogens (+0.41 log) and methanogenesis (+54%). Further inoculation with total fauna increased rumen concentrations of protozoa (+1.0 log), bacteria (+0.29 log), anaerobic fungi (+0.78 log), VFA (+8%), ammonia and fibre digestibility (+17%) without affecting levels of methanogens or methanogenesis. Rumen methanogens population was fairly stable in terms of structure and diversity, while the bacterial community was highly affected by the treatments. Inoculation with holotrich protozoa increased bacterial diversity. Further inoculation with total fauna lowered bacterial diversity but increased concentrations of certain propionate and lactate-producing bacteria, suggesting that alternative H(2) sinks could be relevant. This experiment suggests that holotrich protozoa have a greater impact on rumen methanogenesis than entodiniomorphids. Thus, further research is warranted to understand the effect of holotrich protozoa on methane formation and evaluate their elimination from the rumen as a potential methane mitigation strategy

Study of methanogen communities associated with different rumen protozoal populations

Protozoa-associated methanogens (PAM) are considered one of the most active communities in the rumen methanogenesis. This experiment investigated whether methanogens are sequestrated within rumen protozoa, and structural differences between rumen free-living methanogens and PAM. Rumen protozoa were harvested from totally faunated sheep, and six protozoal fractions (plus free-living microorganisms) were generated by sequential filtration. Holotrich-monofaunated sheep were also used to investigate the holotrich-associated methanogens. Protozoal size determined the number of PAM as big protozoa had 1.7–3.3 times more methanogen DNA than smaller protozoa, but also more endosymbiotic bacteria (2.2- to 3.5-fold times). Thus, similar abundance of methanogens with respect to total bacteria were observed across all protozoal fractions and free-living microorganisms, suggesting that methanogens are not accumulated within rumen protozoa in a greater proportion to that observed in the rumen as a whole. All rumen methanogen communities had similar diversity (22.2 ± 3.4 TRFs). Free-living methanogens composed a conserved community (67% similarity within treatment) in the rumen with similar diversity but different structures than PAM (P < 0.05). On the contrary, PAM constituted a more variable community (48% similarity), which differed between holotrich and total protozoa (P < 0.001). Thus, PAM constitutes a community, which requires further investigation as part of methane mitigation strategies

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

Functional Resilience and Response to a Dietary Additive (Kefir) in Models of Foregut and Hindgut Microbial Fermentation In Vitro

Stability in gut ecosystems is an important area of study that impacts on the use of additives and is related with several pathologies. Kefir is a fermented milk drink made with a consortium of yeast and bacteria as a fermentation starter, of which the use as additive in companion and livestock animals has increased in the last few years. To investigate the effect of kefir milk on foregut and hindgut digestive systems, an in vitro approach was followed. Either rumen fluid or horse fecal contents were used as a microbial inoculate and the inclusion of kefir (fresh, autoclaved, or pasteurized) was tested. Gas production over 72 h of incubation was recorded and pH, volatile fatty acids (VFAs), lactate and ammonia concentration as well as lactic acid (LAB) and acetic acid bacteria, and yeast total numbers were also measured. Both direct and indirect (by subtracting their respective blanks) effects were analyzed and a multivariate analysis was performed to compare foregut and hindgut fermentation models. Addition of kefir boosted the fermentation by increasing molar concentration of VFAs and ammonia and shifting the Acetate to Propionate ratio in both models but heat processing techniques like pasteurization or autoclaving influenced the way the kefir is fermented and reacts with the present microbiota. In terms of comparison between both models, the foregut model seems to be less affected by the inclusion of Kefir than the hindgut model. In terms of variability in the response, the hindgut model appeared to be more variable than the foregut model in the way that it reacted indirectly to the addition of different types of kefirpublishersversionPeer reviewe

The Role of Ciliate Protozoa in the Rumen

First described in 1843, Rumen protozoa with their striking appearance were assumed to be important for the welfare of their host. However, despite contributing up to 50% of the bio-mass in the rumen, the role of protozoa in rumen microbial ecosystem remains unclear.Phylogenetic analysis of 18S rDNA libraries generated from the rumen of cattle, sheep, and goats has revealed an unexpected diversity of ciliated protozoa although variation in gene copy number between species makes it difficult to obtain absolute quantification. Despite repeated attempts it has proven impossible to maintain rumen protozoa in axenic culture. Thus it has been difficult to establish conclusively a role of ciliate protozoa in rumen fibre degradation. The development of techniques to clone and express ciliate genes in phage, together with bioinformatic indices to confirm the ciliate origin of the genes has allowed the isolation and characterisation of fibrolytic genes from rumen protozoa. Elimination of the ciliate protozoa increases microbial protein supply by up to 30% and reduces methane production by up to 11%. Our recent findings suggest that holotrich protozoa play a disproportionate role in supporting methanogenesis whilst the small entodiniium are responsible for much of the bacterial protein turnover. As yet no method to control protozoa in the rumen that is safe and practically applicable has been developed, however a range of plant extract capable of controlling if not completely eliminating rumen protozoa have been described

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

Does Dietary Mitigation of Enteric Methane Production Affect Rumen Function and Animal Productivity in Dairy Cows?

It has been suggested that the rumen microbiome and rumen function might be disrupted if methane production in the rumen is decreased. Furthermore concerns have been voiced that geography and management might influence the underlying microbial population and hence the response of the rumen to mitigation strategies. Here we report the effect of the dietary additives: linseed oil and nitrate on methane emissions, rumen fermentation, and the rumen microbiome in two experiments from New Zealand (Dairy 1) and the UK (Dairy 2). Dairy 1 was a randomized block design with 18 multiparous lactating cows. Dairy 2 was a complete replicated 3 x 3 Latin Square using 6 rumen cannulated, lactating dairy cows. Treatments consisted of a control total mixed ration (TMR), supplementation with linseed oil (4% of feed DM) and supplementation with nitrate (2% of feed DM) in both experiments. Methane emissions were measured in open circuit respiration chambers and rumen samples were analyzed for rumen fermentation parameters and microbial population structure using qPCR and next generation sequencing (NGS). Supplementation with nitrate, but not linseed oil, decreased methane yield (g/kg DMI; P<0.02) and increased hydrogen (P<0.03) emissions in both experiments. Furthermore, the effect of nitrate on gaseous emissions was accompanied by an increased rumen acetate to propionate ratio and consistent changes in the rumen microbial populations including a decreased abundance of the main genus Prevotella and a decrease in archaeal mcrA (log10 copies/g rumen DM content). These results demonstrate that methane emissions can be significantly decreased with nitrate supplementation with only minor, but consistent, effects on the rumen microbial population and its function, with no evidence that the response to dietary additives differed due to geography and different underlying microbial populations

Multi-omics in vitro study of the salivary modulation of the goat rumen microbiome

Ruminants are able to produce large quantities of saliva which enter into the rumen and salivary components exert different physiological functions. Although previous research has indicated that salivary immunoglobulins can partially modulate the rumen microbial activity, the role of the salivary components other than ions on the rumen microbial ecosystem has not been thoroughly investigated in ruminants. To investigate this modulatory activity, a total of 16 semi-continuous in vitro cultures with oats hay and concentrate were used to incubate rumen fluid from four donor goats with autoclaved saliva (AUT) as negative control, saliva from the same rumen fluid donor (OWN) as positive control, and either goat (GOAT) or sheep (SHEEP) saliva as experimental interventions. Fermentation was monitored throughout 7 days of incubation and the microbiome and metabolome were analysed at the end of this incubation by Next-Generation sequencing and liquid chromatography coupled with mass spectrometry, respectively. Characterisation of the proteome and metabolome of the different salivas used for the incubation showed a high inter-animal variability in terms of metabolites and proteins, including immunoglobulins. Incubation with AUT saliva promoted lower fermentative activity in terms of gas production (−9.4%) and highly divergent prokaryotic community in comparison with other treatments (OWN, GOAT and SHEEP) suggesting a modulatory effect derived from the presence of bioactive salivary components. Microbial alpha-diversity at amplicon sequence variant (ASV) level was unaffected by treatment. However, some differences were found in the microbial communities across treatments, which were mostly caused by a greater abundance of Proteobacteria and Rikenellacea in the AUT treatment and lower of Prevotellaceae. These bacteria, which are key in the rumen metabolism, had greater abundances in GOAT and SHEEP treatments. Incubation with GOAT saliva led to a lower protozoal concentration and propionate molar proportion indicating a capacity to modulate the rumen microbial ecosystem. The metabolomics analysis showed that the AUT samples were clustered apart from the rest indicating different metabolic pathways were promoted in this treatment. These results suggest that specific salivary components contribute to host-associated role in selecting the rumen commensal microbiota and its activity. These findings could open the possibility of developing new strategies to modulate the saliva composition as a way to manipulate the rumen function and activity