Sampling and sample storage
Gemma Henderson (AgResearch, New Zealand)
With contributions from: Sandra Kittelmann, Faith Cox, Garry Waghorn, Stefan Muetzel, Xuezhao Sun, Michelle Kirk,
Michael Tavendale, Vahideh Heidarian Miri, Michael Zethof, Sarah Lewis, Sam Noel & Peter Janssen
&
Ilma Tapio (MTT, Finland)
With contributions from: Katerina Fliegerova, Aurelie Bonin, Marialuisa Callegari, Jakub Mrazek, Frederic Boyer, Pierre
Taberlet, Sara Federici, Paolo Bani, Kevin Shingfield & John Wallace
Joint RuminOmics and RMG Network Workshop
22/06/2013, Dublin, Ireland
Sampling and storage options
• Morning feeding (-1h to +6h)
• Evening feeding
• Morning/evening combined
Timing
Feces
Sampling
type
Regurgitated
sample
Sample
fraction
Buccal
swabs
DNA
extraction
Storage
Non invasive methods =(?) Rumen sampling
• Stomach tubing
• Rumen fistula
• At slaughter
• Sampling site within the
rumen
• Liquids
• Solids
• Mixed rumen content
• Frozen (-20C, -80C, N2)
• Freeze-dried
• Mixed with cryo-protectants
(ethanol, RNA later, formalin,
PBS/glycerol)
Percentage of the community
Timing
Before v after feeding
0.002
0.009
*
*
0.043
*
*
0.038
<0.001
Henderson, Sun et al., unpublished
*
*
pasture-fed sheep, n = 10, paired t-test
Fistula v stomach tube
Percentage of the community
Sampling
type
Henderson et al., PLOS ONE (accepted)
*
*
*
ryegrass-fed cows, n = 14, paired t-test
Whole v liquid v solid
Percentage of the community
Sample
fraction
Henderson et al., PLOS ONE (accepted)
*
*
*
*
*
ryegrass-fed cows, n = 14, paired t-test
0.001
0.035
0.041
Percentage of the community
Percentage of the community
Percentage of the community
Freeze-dried v frozen only
Storage
0.002
0.011
**
*
*
*
0.017
0.008
0.009
0.002
*
0.003
0.005
*
*
0.007
Muetzel, Henderson et al., unpublished
<0.001
0.041
*
*
lucerne chaff-fed sheep, n = 6, paired t-test
Storage
Cryo-protectants
In ethanol at RT
Without cryo-protectant in -80C
In PBS-glycerol in -80C
Regurgutated in -80C
Without cryo-protectant in -80C
Regurgitated in -80C
Without cryo-protectant fresh
Regurgitated fresh
Without cryo-protectant fresh
Regurgitated fresh
Regurgitated PBS-glycerol ETOH Just frozen
-80C
-80C
RT
-80C
McKain et al. short communication
Animal
1
Animal
2
sheep, n=2
Feces
Non-invasive sample collection
Families within the Firmicutes
Families within the Bacteroidetes
de Oliveira et al. 2013 Characterizing the microbiota across the gastrointestinal tract of a Brazilian
Nelore steer. Veterinary Microbiology 164: 307-314.
DNA extraction
Percentage of the community
DNA
extraction
Henderson et al., PLOS ONE (accepted)
Take home message
Every sampling technique will represent the rumen microbial community
It is not yet possible to tell which techniques truly represent the rumen microbial community
• There is no “right” or “wrong”
Design your study carefully and be aware of differences introduced by selection of even subtly
different protocols
• ...especially if looking for subtle changes
Large cohort studies should strive to use the same or comparable protocols
• Global Rumen Census
• RuminOmics
• ring test
Many more downstream variables to be considered
• Primers, data analysis, taxonomic frameworks, etc.
Acknowledgements
New Zealand
Sandra Kittelmann
Faith Cox
Garry Waghorn
Stefan Muetzel
Xuezhao Sun
Michelle Kirk
Michael Tavendale
Vahideh Heidarian Miri
Michael Zethof
Sarah Lewis
Sam Noel
Peter Janssen
RuminOmics consortium
Katerina Fliegerova
Jakub Mrazek
Marialuisa Callegari
Paolo Bani
Sara Federici
Aurelie Bonin
Frederic Boyer
Pierre Taberlet
Kevin Shingfield
John Wallace
Funding
PGgRc
Ministry for Primary Industries
SLMACC
Dairy NZ
AgResearch
Funding
RuminOmics
Thanks!
Other valuable references:
Callaway et al. 2010, J Anim Sci 88:3977
De Oliveira et al. 2013, Vet Microbiology 164:307
Dowd et al. 2008, BMC Microbiology 8:125
Fouts et al. 2012, PLOS ONE 7: e48289
Leedle et al. 1982, AEM 44:402
Li et al. 2009, J Appl Microbiol 107:1924
Li et al. 2012, Vet Microbiology 155:72
Lodge-Ivey et al. 2009, J Anim Sci 87: 2333
Prates et al. 2010, Anim Feed Sci Technol 155:186
Rosewarne et al. 2011, Microb Ecol 61:448
Shanks et al. 2011, Appl Environ Microbiol 77:2992
Wu et al. 2010, BMC Microbiology 10:206
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