Download Bovine rumen microbiome and its role in feed efficiency and

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
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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
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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)
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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
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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
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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!
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