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

Timing

Sampling

type

• Morning feeding (-1h to +6h)

• Evening feeding

• Morning/evening combined

• Stomach tubing

• Rumen fistula

• At slaughter

• Sampling site within the rumen

Feces

Regurgi-tated

sample

Sampling and storage options

Sample fraction

Storage

• Liquids

• Solids

• Mixed rumen content

• Frozen (-20C, -80C, N2)

• Freeze-dried

• Mixed with cryo-protectants(ethanol, RNA later, formalin, PBS/glycerol)

Non invasive methods =(?) Rumen sampling

Buccal

swabs

DNA

extraction

Before v after feeding

**

*

*

0.009

0.002

0.043

Perc

enta

ge o

f th

e c

om

munity

Timing

Henderson, Sun et al., unpublished pasture-fed sheep, n = 10, paired t-test

*

*

*

<0.001

0.038

Perc

enta

ge o

f th

e c

om

munity

Fistula v stomach tube

*

Perc

enta

ge o

f th

e c

om

munity

Sampling

type

Henderson et al., PLOS ONE (accepted) ryegrass-fed cows, n = 14, paired t-test

*

*

Perc

enta

ge o

f th

e c

om

munity

Whole v liquid v solid

*

*

Perc

enta

ge o

f th

e c

om

munity

Sample fraction

*

*

Henderson et al., PLOS ONE (accepted) ryegrass-fed cows, n = 14, paired t-test

*

Perc

enta

ge o

f th

e c

om

munity

Freeze-dried v frozen only

**

*

*

0.003 0.005

0.002 0.011

*

*0.041

0.035

0.001

Perc

enta

ge o

f th

e c

om

munity

Perc

enta

ge o

f th

e c

om

munity

Storage

0.041

0.017 0.008

0.009 0.002

0.007 <0.001

*

*

*

*

Muetzel, Henderson et al., unpublished lucerne chaff-fed sheep, n = 6, paired t-test

Perc

enta

ge o

f th

e c

om

munity

Without cryo-protectant fresh

Regurgitated fresh

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

Cryo-protectantsStorage

Animal

1

Animal

2

Regurgitated PBS-glycerol ETOH Just frozen

-80C -80C RT -80C McKain et al. short communication sheep, n=2

Non-invasive sample collectionFeces

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 extractionP

erc

enta

ge o

f th

e c

om

munity

DNA

extraction

Henderson et al., PLOS ONE (accepted)

Perc

enta

ge o

f th

e c

om

munity

Take home message

Every sampling technique will represent the rumen microbial communityIt 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• ...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

RuminOmics consortium

Katerina Fliegerova

Jakub Mrazek

Marialuisa Callegari

Paolo Bani

Sara Federici

Aurelie Bonin

Frederic Boyer

Pierre TaberletVahideh Heidarian Miri

Michael Zethof

Sarah Lewis

Sam Noel

Peter Janssen

Funding

PGgRc

Ministry for Primary Industries

SLMACC

Dairy NZ

AgResearch

Pierre Taberlet

Kevin Shingfield

John Wallace

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 8:125Dowd 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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