Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Bovine rumen microbiome and its role in feed efficiency and methanogenesis Dr. Sinéad M. Waters Principal Research Scientist Animal and Bioscience Research Department Teagasc Grange Ireland 8th June, 2017 2nd IS-FOOD International Workshop, Public University of Navarra, Pamplona, Spain Teagasc • Teagasc – the Irish Agriculture and Food Development Authority • National body providing integrated research, advisory and training services to the agriculture and food industry and rural communities • Research and Innovation • : Teagasc: Priority Programmes 1. Food 2. Animal and Grassland – Animal and Bioscience Research Department 3. Crops, Environment and Land Use 4. Advisory 5. Education Irish Agriculture, Food and Drink Sector • 7.6% of Ireland’s economy • 12.3% of Exports • 8.6% of total employment • In 2015, Irish agri-food and drink exports increased by an estimated 3% to €10.8 bn Future challenges? Grass: Unexploited Potential Overview • What is the rumen and its microbiome? • How do we study the rumen microbiome? • Influence of host feed efficiency on the rumen microbiome • Role of the rumen microbiome in enteric methanogenesis • Factors affecting the rumen microbiome • Development of the rumen microbiome • Summary and future research strategies Introduction • Major challenge in Agriculture Feeding a rapidly increasing global population projected to rise to ca. 9 bn by 2050 International pressure to reduce the environmental footprint The Rumen Microbiome Why is it important? Role of Cattle Forage High Quality Milk and Meat Rich Sources Amino Acids, Vitamins & Minerals Ruminants - unique in their ability to convert plant material into high quality meat and milk protein for humans Types of feed Grass is cheap and plentiful ‘healthy’ Ireland’s advantage The Bovine Rumen • Part of digestive tract in ruminants • Natural fermentation tank and feed reservoir • A ~200 litre fermentation chamber which is highly adapted to the breakdown of lignocellulose of plants • 150L liquid • Microorganisms adhere to feed particles and mucosal of rumen • Microbial community influenced by host age, health, diet and geographical location The bovine gastrointestinal tract 1. 2. 3. 4. Rumen Reticulum Omasum Abomasum The rumen forms the larger part of the bovine GIT First chamber in the alimentary canal of ruminant animals Primary site for microbial fermentation of ingested feed Rumen microbiome Bacteria Anaerobic Fungi Ciliate Protozoa Methanogenic Archaea 1010 to 1011cells/ml <105 cells/ml <105 cells/ml 106 to 108 cells/ml The rumen is highly adapted to the breakdown of lignocellulose of plants Ruminants most efficient digesters of lignocellulose • Plant biomass – most abundant renewable resource available • Lignocellulosic biomass - major source of ruminant production • Crucial step – bioconversion of lignocellulosic feedstock (cellulose and hemicellulose) to fermentable sugar • Cellulolytic microorganisms – bacteria, fungi and protozoa A symbiotic relationship exists between host animal and rumen microbial community • Rumen microbes - regulate rumen pH and fermentation of cellulose, hemicellulose and fiber to end products utilizable by the host, ie volatile fatty acids (VFA) eg acetate, propionate, butyrate • Host animal - provides substrate and a suitable anaerobic environment Ruminants • Foregut fermenters • Absorption of volatile fatty acids enhanced by a good blood supply to the walls of the rumen • Papillae – increase surface area Rumen Fermentation • Breakdown sugars, starches, cellulose, hemicelluloses • Pyruvate reduced anaerobically SCFA/VFAs • Main VFAs – acetate, propionate and butyrate • CH4 byproduct of rumen fermentation CO2 + 4 H2 → CH4 + 2H2O Degradation of lignocellulosic material • An array of enzymes are required to degrade lignocellulosic biomass – heterogeneity in composition and structure – 3 classes of enzymes involved in cellulose breakdown • Endo-β-1,4-glucanase • Cellobiohydrolase • β-glucosidase CAZymes • Carbohydrate-Active Enzymes – build and breakdown complex carbohydrates • GH5 and GH9 – most diverse and predominant family of cellulases in rumen • Can be expressed by eukaryotic microorganisms in the rumen – Fungi & protozoa Studying the rumen microbiome Rumen sample collection Rumen fistulation Stomach tubing Slaughter Technologies to study the rumen microflora • Majority of rumen microbes unculturable and require anaerobic environment Traditionally • Anaerobic chambers and culturing • Culture-independent molecular techniques PCR-DGGE Sub-cloning sequencing Quantitative PCR Next generation sequencing 454 pyrosequencing Technologies to study the rumen microflora Illumina next generation sequencing technology Host and microbial genomes sequencing using Next-Gen technologies – Microbial composition and potential function Genomics “Omics” Technologies Metabolomics Understanding and manipulating rumen function Identify key microbial metabolites produced in the rumen Proteomics Levels of active gene expression at a point in time – host and microbial metabolic activity Transcriptomics Translational activity – protein production at a point in time by the rumen microbiota Influence of host feed efficiency on the rumen microbiome Beef Profitability: A Function of Inputs and Outputs Feed - up to 80% of the variable costs in beef production major factor determining cost competitiveness Improved nutrient utilisation from feed = enhanced productivity and economic efficiency Measure of feed efficiency - Residual Feed Intake (RFI) High RFI – low feed efficiency; Low RFI – high feed efficiency Rumen microbial fermentation plays a pivotal role in host nutrition Diet digestibility and fermentation account for 19% of the variation in RFI (Herd and Arthur, 2009) Does the Rumen microbiome play a role in host feed efficiency? Rumen microflora is influenced by RFI phenotype • Rumen microbiome has been shown to be associated with host RFI • Effect of RFI on bacterial profiles was influenced by diet • A core bacterial community exists in the rumen of feed efficient and inefficient animals with minor populations altered due to phenotypic RFI • Prevotella abundance was higher in inefficient animals • Microbiome genes and species accurately predicted the animals' feed efficiency phenotype (dairy cows) – reflective of better energy and carbon channeling to the animal and lowering methane emissions to the atmosphere Guan et al., 2008; Hernandez-Sanabria et al., 2010; Carberry et al., 2012; Rius et al., 2012; McCann et al., 2014; Myer et al., 2015; Ben-Shabat et al., 2016 Application of microbiota data in feed efficiency breeding strategies? ‘the host animal controls its own microbiota to a significant extent and open up the implementation of effective breeding strategies using rumen microbial gene abundance as a predictor for difficult-to-measure traits, such as feed efficiency, on a large number of hosts’ Roehe et al., 2016 Microbial transcriptome and feed efficiency • Differences in the active rumen microbiota between H- and L-RFI steers • Difference in abundance of CAZymes between the 2 RFI groups • Essential roles in occupying rumen ecological niches Li et al., 2017 Role of the rumen microbiome in enteric methanogenesis Enteric methane production from rumen fermentation • Methane is produced by methanogenic archaea which convert the hydrogen produced by ruminal fermentation into methane – Hydrogen produced ciliated protozoa • Maintains rumen pH by preventing accumulation of hydrogen ions in the rumen • Methane production in the rumen is an energetically wasteful process, accounting for loss in up to 15% of dietary gross energy Methane and Agriculture Methane (CH4) • Important greenhouse gas (GHG), GWP100 (Global warming potential over 100 years) = 25, • Methane is 25 times more potent than carbon dioxide • Atmospheric lifetime 12 years • Accounts for almost 2/3 of agricultural GHGs • Production /transport of fossil fuels, livestock, rice cultivation, decay of organic waste in solid waste landfills Global Legislation Kyoto Protocol (2008-2012) • Developed nations to reduce their GHG emission by 5.2% collectively with respect to 1990 levels • Second commitment period 2013-2020; Doha Agreement Paris Agreement 2015 Limit global warming to well below 2°C relative to pre-industrial levels and pursue efforts to limit the temperature increase to 1.5°C European Legislation • 2030 Climate change and Energy Package • 40% reduction in GHG emission from the 1990 levels • EU Low carbon economy in 2050 • Keeping the global temperature increase well below 2°C • EU cut GHG emission by 80% relative to 1990 levels • Milestones - 40% by 2030 and 60% by 2040. Overall Aim: creating a European economy that is climate friendly and less energy consuming What are we doing in Ireland to reduce GHG from agriculture? Agriculture accounts for approximately 30% of Irish greenhouse gas production Three main greenhouse gasses from agriculture are: Methane, nitrous oxide and carbon dioxide Agricultural emissions are in steady decline and are 9% lower than 1990 levels Irish grass based beef production systems are relatively carbon efficient 37 Teagasc Presentation Footer What are we doing in Ireland to reduce GHG from agriculture? Farmers are adopting a number of practices and technologies can significantly improve efficiency, improve profitability and lower GHG emissions • Carbon Navigator – all farmers involved » - Longer grazing season, » - Younger age at first calving » - Higher calving rate » - Improved growth rates » - Nitrogen efficiency » - Slurry management • Beef Data and Genomics Programme (BDGP) 2015-2022 » Genomic selection breeding programme launched in Ireland in 2010 » Farmers are recording more information on their animals and improving efficiency of production, use of AI Teagasc Presentation Footer Measuring methane …and in sheep! Significant differences in enteric methane reported in cattle divergent in RFI more feed efficient (low RFI) cattle produce less CH4 • Feed efficient cattle produced: 25% less CH4 on high conc. diets (Hegarty et al., 2007) 28% less CH4 on high conc. diets (Nkrumah et al., 2006) • 27% less CH4 on high quality pasture (Jones et al., 2011) Methane (g/d) vs. DMI (kg/d) (Fitzsimons et al., 2013) 300 8.2 8 g/d 280 CH4 7.8 DMI 7.6 7.4 270 kg/d 290 7.2 260 7 6.8 250 6.6 240 6.4 High Low RFI group • Reduction in CH4 (g/d) of low RFI cattle is function of reduced DMI Hyslop et al. (2013) Are rumen methanogens influenced by host feed efficiency? • Total methanogen concentrations in the rumen contents did not differ due to host feed efficiency (Carberry et al., 2014) • Differences existed in the composition of the methanogenic community (Carberry et al., 2014) – Various genotypes of Methanobrevibacter smithii - more abundant in cattle of high compared to low feed efficiency across diets • An uncultured Succinivibrionaceae species associated with the lower methane emissions and higher concentrations of propionate (McCabe et al., 2015) • Relative abundance and methanogenic functionality of Archaea and Methanobrevibater gottschalkii clade increased in less feed efficient animals (McCabe et al., 2015; McGovern et al., 2017 In Press) Rumen microbiome influences host feed efficiency! Factors affecting the rumen microbiome Factors that affect microbial community - Animal type (species, breed, age, rumen development) - Host specificity (animal individuality) - Diet (ingredients and chemical composition) - Rumen phase (liquid, solid, epimural) - Plane of nutrition, sampling time Factors resulting from study METHODOLOGY - Sample collection - Site of sampling - DNA isolation and analysis Rumen microorganisms – many unknowns and unculturables • ~77% rumen microorganisms unknown and non-cultured • Global Rumen Census – (Henderson et al., 2015) – 742 samples from 32 countries – Microbial community similar worldwide – Differ due to host species – Diet primary factor responsible for compositional variation Factors: Animal species Henderson et al., 2015 Scientific Reports Factors: Animal species Henderson et al., 2015 Scientific Reports Factors: Animal species Cow-reindeer rumen content exchange Same diet 6 weeks RUMEN CONTENT EXCHANGE • Proportion of microbial community in the rumen specific to cows or reindeer • Core microbiome common to both • After digesta exchange microbial communities in the rumen of reindeer more similar to that of cows than original population • Evidence of changes in species specific to reindeer over time consistent with a host animal effect. FP7- RuminOmics Project www.ruminomics.eu 9 weeks Factors: Breed 23 OTUs 180 clones • Same herd • Same diet 55 OTUs 185 clones King et al., 2011 Appl. Envir. Microbiol. 20 OTUs 12 OTUS OTU: Operational taxonomic unit Used to categorise bacteria based on sequence similarity Possible mechanisms of host control of the gut microbiome • pH: no studies on saliva production/composition and microbiome • Rumen size / retention time • ‘Low-methane yield sheep have smaller rumens and shorter rumen retention time’ Goopy et al., 2014 • Host immunity • Early-life Rumen Development • First weeks of life calf functions largely as a monogastric, as the rumen develops • Solid feed stimulates rumen development • Calf relies on milk/milk replacer for energetic requirements – until 4-6 weeks old • Hindgut microbiota responsible for degradation of milk solids (up to 20%) in this period(Drackley, 2016) 53 Rumen Development Goal at weaning: Smooth transition from monogastric to ruminant Functioning Rumen Physical Development Machinery for VFA absorption Microbial Community Machinery for VFA production Rumen Development Undeveloped rumen (Day 0) Developing rumen (Week 18) Developed rumen (24mths) O’Hara et al., 2017 In press 55 Microbial Colonisation Mature Complexity & Homogeneity among individuals increases with age Initial Innoculation: Week 1: Week 2: Weeks 3-6 0-24h:, facultative anaerobes fibrolytic bacteria and methanogens (d2-8) Anaerobic fungi detected (d8-12) More homogeneous population community continues to evolve Over 8000 microbial protein families identified – functional capacity Ciliate protozoa Differences in 6mth vs. 2 year old animals 48h: Anaerobic bacteria R. albus , methanogens (d1) VFAs and enzyme activity detected (d2) Weaning to Adulthood Stabilises. Still highly responsive to dietary change Metabolically active microbiota established before exposure to solid diet Age and management appear to be major determinants of rumen microbial establishment Rumen Development MATURE . 3-4 months 8 weeks 0-2 weeks ? Nutritional Intervention • The microbial ecosystem once established in the mature rumen is very resilient • Microbial colonization occurs soon after birth – sterile at birth? • Early life events may be related to the microbial community structure and/or the rumen activity in the animals post-weaning RumenStability - Yáñez-Ruiz et al., (2015) Frontiers. Microbiol. Current and Future Research Strategies Significant international research efforts focus on metagenome and metatranscriptomic analysis – to understand the composition and diversity of the rumen microbiome Culturing and metabolically and biochemically characterising rumen microbes – use of rumen simulated media Microbial genome sequencing – enhance databases Identify the carbohydrate active enzymes (CAZymes) responsible for plant biomass digestion in the rumen – biotechological exploitation Integrate microbiome information into BREEDING strategies Conclusion • Beef and dairy output need to be increased however expansion is restrained by international and European environmental legislation • A requirement to improve feed efficiency to reduce feed costs while decreasing methane emissions • Ruminants - unique in their ability to convert low quality forage into high quality meat and milk protein for humans – Fermentation process carried out by rumen microbes • Rumen microflora influenced by host species, diet and breed but can potentially be manipulated in early life • Need improved knowledge and exploitation of the rumen microbiome – Feeding strategies – breeding for an efficient rumen microbiome Funding Acknowledgements – The Research Team and international collaborators Prof David Kenny Dr Chris Creevey University of Aberystwyth 61 Teagasc Presentation Footer Dr Ciara Carberry Eoin O’Hara Emily McGovern Dr Milka Popova, INRA, France Prof Leluo Guan Dr Alan Kelly University of Alberta University College Dublin, Ireland Thank you for your attention! 62 Teagasc Presentation Footer