Research Article Early Methanogenic Colonisation in the ...downloads.hindawi.com/Journals/Archaea/2014/547908.PdfResearch Article Early Methanogenic Colonisation in the Faeces of Meishan

Research ArticleEarly Methanogenic Colonisation in the Faeces of Meishan andYorkshire Piglets as Determined by Pyrosequencing Analysis

Yong Su1 Gaorui Bian1 Zhigang Zhu1 Hauke Smidt2 and Weiyun Zhu1

1 Laboratory of Gastrointestinal Microbiology College of Animal Science and Technology Nanjing Agricultural UniversityNanjing 210095 China

2 Laboratory of Microbiology Wageningen University Dreijenplein 10 Wageningen 6703 HB The Netherlands

Correspondence should be addressed to Weiyun Zhu zhuweiyunnjaueducn

Received 21 September 2013 Accepted 18 December 2013 Published 6 January 2014

Academic Editor Masaaki Morikawa

Copyright copy 2014 Yong Su et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Gutmethanogenic archaea ofmonogastric animals are considered to be related to energymetabolism and adipose deposition of thehost however information on their development in young piglets is limited Thus to investigate early methanogenic colonisationin the faeces ofMeishan and Yorkshire piglets faecal samples were collected from piglets at 1 3 7 and 14 days after birth and used toanalyse the methanogenic community with 16S rRNA gene pyrosequencing Results showed that the diversity of the methanogeniccommunity in the faeces of neonatal piglets decreased from one to 14 days of age as the total methanogen populations increasedThe age of piglets but not the breed significantly affected the diversity of the methanogenic community which was dominated bythe genus Methanobrevibacter From the ages of one to 14 days the abundance of M smithii-related operational taxonomic units(OTUs) increased significantly while the abundances of M thaueri- and M millerae-related OTUs decreased significantly Thesubstitution of M smithii for M thaueriM millerae was faster in Yorkshire piglets than in Meishan piglets These results suggestthat the early establishment ofmicrobiota in neonatal piglets is accompanied by dramatic changes in themethanogenic communityand that the changes vary among pigs of different genotypes

1 Introduction

Methanogenic archaea exist widely in the gastrointestinal(GI) tract of many vertebrates and invertebrates [1ndash3]Methanogens can use hydrogen and other compounds suchas formate methanol and acetate as electron donors forthe production of methane Methane formation not onlycontributes to global warming as a greenhouse gas but italso represents an energy loss for the animal [4] Recentlymethanogenic archaea in the gut of monogastric animalsincluding humans have been studied intensively becausegut methanogens are considered to be related to energymetabolism and adipose deposition of the host [5] Inaddition methane produced by methanogens might play animportant role in the pathogenesis of several intestinal dis-orders including colon cancer inflammatory bowel diseaseirritable bowel syndrome and diverticulosis [6] Thus faronly limited reports on gut methanogens in pigs have beenavailable Based on archaeal 16S ribosomal RNA (rRNA) gene

clone library analysis methanogens belonging to the genusMethanobrevibacter were found to be predominant in pigfaeces [7 8]

The GI microbiota of newborn animals play a funda-mentally important role in the development of intestinalfunction and the innate immune system [9ndash12]The infant gutecosystem undergoes a dramatic transition from an essen-tially sterile state to extremely dense colonisation endingwith the establishment of an adult-like microbial community[13 14] In contrast to the gut microbiota of adult animals themicrobiota of neonates are more variable and less stable overtime The fragile ecological system is not only a disease riskto the newborn gut but it can also have a long-term effecton itrsquos later life health [15ndash17] Comparing the gut bacterialcommunities of neonatal animals and humans which havebeen intensively studied information on gut methanogeniccommunities of neonatal monogastric animals such as pigs isstill limited

Hindawi Publishing CorporationArchaeaVolume 2014 Article ID 547908 10 pageshttpdxdoiorg1011552014547908

2 Archaea

The Meishan and Yorkshire breeds are typical obeseand lean pigs respectively thus their energy metabolismmight be distinctive It has been found that obese Meishanpigs harbour relatively higher numbers of Firmicutes andlower numbers of Bacteroidetes compared to lean breeds[18] Moreover a recent study showed that lean Landracepigs harboured a greater diversity of methanogens and ahigher number of methanogen mcrA gene copies than theobese Erhualian pigs [8] However it is not clear whetherthe different composition of gut methanogens in the variouspig breeds is related to the early methanogenic colonisationof newborn piglets Therefore the aim of this study was toinvestigate the development of methanogenic archaea in thefaeces of newborn Meishan and Yorkshire piglets by usinghigh throughput pyrosequencing analysis of PCR-amplified16S rRNA genes

2 Materials and Methods

21 Collection of Faecal Samples This study was approvedby the Nanjing Agricultural University Animal Care and UseCommittee All of theMeishan andYorkshire pigswere raisedon a commercial farm in Jiangsu province China Candidatesows with a similar expected delivery date were chosen fromboth breeds and injected intramuscularly with cloprostenol(02mg per sow) at 1000 am on day 113 of pregnancy toensure homochronous deliveries Four vaginally deliveredlitters of piglets (each litter with 10ndash12 piglets) for each pigbreed which delivered homochronously within two hourswere finally used in this study The same diets were formu-lated for Meishan and Yorkshire sows according to nutrientrequirements of the National Research Council Fresh faeceswere collected from the piglets at 1 3 7 and 14 days of age andimmediately stored at minus28∘C for further molecular analysis

22 DNA Extraction and PCR Amplification Total genomicDNA was isolated from the faecal samples using a com-mercially available stool DNA extraction kit according tothe instructions of the manufacturer (QIAamp DNA StoolMini Kit Qiagen Hilden Germany) The concentration ofthe extracted DNA was determined using a NanoDrop 1000spectrophotometer (Thermo Scientific IncWilmington DEUSA)

To analyse the taxonomic composition of the methano-genic community Archaea-specific primers (Arch344F 51015840-ACG GGG YGC AGC AGG CGC GA-31015840 and Arch915R 51015840-GTGCTCCCCCGCCAATTCCT-31015840) targeting the V3ndashV6region of the 16S rRNAgenewere chosen for the amplificationand subsequent pyrosequencing of the PCR products [19 20]The PCRs were carried out in triplicate in 50 120583L reactionswith 10 120583L 5-fold reaction buffer 50 ng of DNA 04mM ofeach primer 05U Pfu polymerase (TransStart-FastPfu DNAPolymerase TransGen Biotech) and 25mM dNTPs Theamplification program consisted of an initial denaturationstep at 95∘C for 2min This was followed by 30 cycles whereone cycle consisted of 95∘C for 30 s (denaturation) 58∘Cfor 90 s (annealing) 72∘C for 30 s (extension) and a finalextension of 72∘C for 5min PCR products were visualised

on agarose gels (2 in TBE buffer) containing ethidiumbromide and purifiedwith aDNAgel extraction kit (AxygenChina)

23 Pyrosequencing and Bioinformatics Prior to sequencingtheDNA concentration of each PCR product was determinedusing a Quant-iT PicoGreen double-stranded DNA assay(Invitrogen Germany) and was quality-controlled on anAgilent 2100 Bioanalyzer (Agilent USA) Amplicon pyrose-quencing was performed from the A end using a 454RocheA sequencing primer kit on a Roche Genome Sequencer GS-FLX Titanium platform at Majorbio Bio-Pharm TechnologyCo Ltd Shanghai China

PCR-amplified fragments were blunted and tagged onboth ends with ligation adaptors that contained a unique10 bp sequence (sample specific barcode sequence) and ashort 4-nucleotide sequence (TCAG) called sequencing keywhich were recognised by the system software and thepriming sequences All pyrosequencing reads were binnedaccording to barcode and primer sequences The resultingsequences were further screened and filtered for qualitySequences that were shorter than 200 bp in length containedambiguous characters contained over two mismatches tothe primers or contained mononucleotide repeats of oversix nt were removed To assess bacterial diversity amongsamples in a comparable manner a randomly selected 2564-sequence (the lowest number of sequences in the 32 samples)subset from each sample was aligned using the ldquoalignseqsrdquocommand and compared with the SILVA archaeal database(SILVA version 108) The aligned sequences were furthertrimmed and the redundant reads were eliminated usingsuccessively the ldquoscreenseqsrdquo ldquofilterseqsrdquo and ldquouniqueseqsrdquocommands The ldquochimeraslayerrdquo command was used todetermine chimeric sequences The ldquodistseqsrdquo commandwas performed and unique sequences were clustered intooperational taxonomic units (OTUs) defined by 97 sim-ilarity [21] using CD-HIT-OUT program [22] We alsocalculated the coverage percentage usingGoodrsquosmethod [23]abundance-based coverage estimator (ACE) bias-correctedChao richness estimator and the Shannon and Simpsondiversity indices A heatmapwas generated using customPerlscripts All the analyses were performed using theMOTHURprogram (httpwwwmothurorg) [24] Principal coordi-nate analysis (PCoA) was conducted based on the weightedUniFrac distance [25]

24 Phylogenetic Analysis Sequences of OTUs with an abun-dance higher than 01with total readswere derived andusedfor construction of a phylogenetic tree Homology searchesof the GenBank DNA database were further performed witha BLAST search Sequences of OTUs-related species wereretrieved from the GenBank database Multiple sequencealignments were performed using ClustalX181 [26] Phylo-genetic analysis was performed with the MEGA 31 softwarepackage An unrooted phylogenetic tree was constructedusing the neighbour-joining method [27]

25 Real-Time PCR Quantification of Methanogenic ArchaeaQuantitative PCR was performed on an Applied Biosystems

Archaea 3

7300 Real-Time PCR System (ABI) using SYBR Green as thefluorescent dye The reaction mixture (25 120583L) consisted of125 120583L of IQ SYBR Green Supermix (Bio-Rad) 02 120583M ofeach primer set and 5 120583L of the template DNA The amountof DNA in each sample was determined in triplicate andthe mean values were calculated Archaea-specific primersArch 344 f [19]) and Arch806 [28] were used to quantify the16S rRNA gene of archaeal methanogens under the followingconditions an initial DNA denaturation step at 95∘C for10min followed by 40 cycles of denaturation at 95∘C for 15 sand primer annealing and extension at 60∘C for 1min DNAfrom cells of a pure culture of M smithii was used as thestandard Results are expressed as the numbers of 16 S rRNAgene copies per gram of faeces

26 Statistical Analysis The effects of pig breed and age onthe composition of the methanogenic archaeal communitywere tested for significance using a two-way analysis ofvariance (ANOVA) program in the Statistical Package forthe Social Sciences (SPSS170) Significant differences weredeclared when 119875 lt 005

3 Results

31Metrics of Pyrosequencing Analysis Across all 32 samples132 138 quality-trimmed sequences from a total of 165 338reads were classified as Archaea The average length ofthe quality-trimmed sequences was 483 bp The rarefactioncurves generated by MOTHUR plotting the number of readsagainst the number of OTUs indicated that using 2564 readsper sample (the minimum number of sequences passing allquality control measures across all samples) for the finalanalysis was adequate as the curves tended to approach thesaturation plateau (Figure 1)

32 Diversity Coverage number of OTUs and statisticalestimates of species richness for each group at a geneticdistance of 3 are presented in Table 1 The age of thepiglets significantly affected the diversity indices (Shannonand Simpson) and richness estimators (ACE and Chao) offaecal methanogenic archaeal community (119875 lt 005) therewas no significant difference between the pig breeds In bothbreeds the piglets harboured a higher diversity of faecalmethanogens at 1 and 3 days of age than at 7 and 14 days(119875 lt 005)

33 Taxonomic Composition Across all reads 9991 wereidentified as classMethanobacteria whileThermoplasmatalescomposed the remaining 009 Within class Methanobac-teria family Methanobacteriaceae was predominant repre-sented by generaMethanobrevibacter andMethanosphaera Avery high abundance of genus Methanobrevibacter (9501ndash100) was found in all samples thus further analysis wasperformed at the species (OTU) level

Clustered heat map analysis based on the archaeal com-munity profiles at the OTU level showed that most samplestaken from the piglets at 1 and 3 days of age were groupedtogether and separated from the samples taken at 7 and 14

0

50

100

150

200

250

300

350

400

450

0 500 1000 1500 2000 2500 3000

Num

ber o

f OTU

s

Number of sequences

Y1Y3Y7Y14

M1M3M7M14

Figure 1 Rarefaction curves comparing the number of reads withthe number of phylotypes (OTUs) found in the 16S rRNA genelibraries from faecal methanogens ofMeishan and Yorkshire pigletsM Meishan piglets Y Yorkshire piglets 1 3 7 and 14 represent theages of 1 3 7 and 14 days

days (Figure 2) In addition PCoA analysis also showed thatthe first principal coordinate (P1) which explains 440 ofthe variation separated the archaeal communities of mostof the piglets at 7 and 14 days from the samples of youngeranimals (Figure 3)

Sequences of predominant OTUs with a relative abun-dance higher than 05 of total reads and reference sequencesof Methanobrevibacter spp were used for construction ofthe phylogenetic tree All of the OTUs were closely relatedto genus Methanobrevibacter (Figure 4) Most of the OTUswere divided into two clustersmdashM smithiiM woesei clusterand M thaueriM millerae cluster OTUs 1 2 3 4 13 20and 28 were most closely related to M smithii whereasOTUs 5 6 7 8 9 14 19 29 and 30 were most closelyrelated toM thaueri OTUs with a relative abundance higherthan 01 of total reads were further used for a significancetest of relative abundance among different groups (Table 2)OTUs significantly affected by pig breed or age were mainlyrelated to M smithii M thaueri and M millerae (119875 lt005) Pig breed significantly affected the relative abundancesof M smithii- and M thaueri-related OTUs and pig agesignificantly affected the relative abundances of predominantM smithii- M thaueri- and M millerae-related OTUs(119875 lt 005) (Table 3) Interestingly it was observed that thesubstitution ofM smithii forM thaueriMmillerae occurredfaster in Yorkshire piglets than in Meishan piglets Sequencesmost closely related to M smithii became predominant atthe age of 7 days in Yorkshire piglets whereas this speciesdominated in the Meishan piglets only from day 14 In bothbreeds from day 1 to day 14 the relative abundance of Msmithii-related OTUs increased significantly in the faeces

4 Archaea

OTU13

Distance004

003

002

001

000

OTU16OTU15OTU18OTU34OTU31OTU47OTU33OTU42OTU35OTU32OTU28OTU43OTU40OTU37OTU45OTU44OTU41OTU30OTU27OTU36OTU39OTU38OTU26OTU25OTU46OTU29OTU24OTU22OTU21OTU20OTU17OTU23OTU12OTU19OTU14OTU10OTU11OTU9OTU7OTU6OTU8OTU5OTU4OTU3OTU2OTU1

Dist

ance

010

008

005

003

000

EM1

HM1

FM7

DY1

FM1

GM3

EM3

BY1

AY1

CY1

CY3

DY3 AY3

BY3

GM7

HM3

HM7

FM14

AY14

EM7

GM14

FM3

BY14

CY14

CY7

BY7

EM14

AY7

HM14

DY1

4

DY7

Amount

068

001

000

000

000

Methanobrevibacter smithiiMethanobrevibacter olleyaeMethanobrevibacter olleyaeMethanobrevibacter smithiiMethanobrevibacter smithiiMethanobrevibacter smithiiMethanobrevibacter spMethanobrevibacter spMethanobrevibacter spMethanobrevibacter spMethanobrevibacter spMethanobrevibacter smithiiMethanobrevibacter thaueriMethanobrevibacter thaueriMethanobrevibacter thaueriMethanobrevibacter spMethanobrevibacter milleraeMethanobrevibacter spMethanobrevibacter thaueriMethanobrevibacter alcaliphilumMethanobrevibacter smithiiMethanobrevibacter smithiiMethanobrevibacter smithiiMethanobrevibacter smithiiMethanobrevibacter smithiiMethanobrevibacter thaueriMethanobrevibacter thaueriMethanobrevibacter thaueriMethanobrevibacter olleyae

Methanobrevibacter smithiiMethanobrevibacter milleraeMethanobrevibacter woliniiMethanobrevibacter milleraeMethanobrevibacter thaueriMethanobrevibacter thaueriMethanobrevibacter woeseiMethanobrevibacter thaueriMethanobrevibacter thaueriMethanobrevibacter thaueriMethanobrevibacter thaueriMethanobrevibacter thaueriMethanobrevibacter thaueriMethanobrevibacter smithiiMethanobrevibacter smithiiMethanobrevibacter smithiiMethanobrevibacter smithii

Methanobrevibacter gottschalkii

Figure 2 Double dendrogram showing the distribution of methanogens in the faeces of Meishan and Yorkshire piglets (OTU level) Therelationship among samples was determined using Bray distance and the complete clustering method A total of 47OTUs with an abundancehigher than 03 within total methanogens were selected for the analysis The heatmap plot depicts the relative abundance of each OTU(variables clustering on the 119910-axis) within each sample (119909-axis clustering) The relative values for the OTUs are depicted by colour intensityin the legend at the top of the figure Clusters based on the distance of all samples along the 119909-axis and the different OTUs along the 119910-axisare indicated at the top and left of the figure respectively AndashD litter numbers of Yorkshire piglets EndashH litter numbers of Meishan piglets

while the relative abundances ofM thaueri- andMmillerae-related OTUs decreased significantly (119875 lt 005)

34 Quantification of 16S rRNA Gene Copies of MethanogenicArchaea The absolute numbers of the 16S rRNA gene copiesof methanogenic archaea in the faeces of the piglets weredetermined with real-time PCR assays (Figure 5) Pig breeddid not affect faecal methanogen populations however inboth breeds significant differences were found among sam-ples taken from the piglets at different ages The numbers of

faecal methanogen 16S rRNA gene copies found in the pigletsat 7 and 14 days were significantly higher than those found at1 and 3 days (119875 lt 005)

4 Discussion

Since Ley et al found that gut microbiota were associatedwith the energy metabolism of the host the role of gutmicrobiota in the metabolism of the host has received moreattention [29] While numerous studies have focused on two

Archaea 5

Table 1 Phylotype coverage and diversity estimation of the 16S rRNA gene libraries from faecal methanogens of Meishan and Yorkshirepiglets1

Breed Age (d) OTUs ACE Chao Shannon Simpson Coverage

Meishan

1 3403 6971 5642 4263 0045 09383 2358 4840 3983 3551 0085 09587 1930 4709 3431 2797 0207 096314 1798 5024 3539 2505 0279 0963

Yorkshire

1 4123 7834 6584 4732 0029 09283 4270 7911 6894 4789 0028 09247 1603 4168 2778 2311 0326 096814 1260 4349 2720 1764 0419 0972

SEM2 1377 25095 2140 1297 0168 0023

Effect (P value)Breed 0184 0409 0307 0664 0185 0236Age 0000 0048 0001 0000 0000 0001

Breed times age 0049 0346 0102 0063 0138 00921The operational taxonomic units (OTUs) were defined with 3 dissimilarity The coverage percentages richness estimators (ACE and Chao) and diversityindices (Shannon and Simpson) were calculated2SEM standard error of means 119899 = 4

HM3

DY14DY7

AY7AY14 CY7

EM14

HM14

HM7 GM1

GM7

GM3

BY1 DY1AY3

CY14BY14 EM7FM14

GM14

FM3BY7

DY3

EM3EM1

HM1

FM1

FM7

BY3

CY1

CY3

AY1

10

05

00

00 05 10

Axi

s2175

Axis1 440

minus05

minus08

minus05minus10

Figure 3 Principal coordinates analysis of weighted UniFrac valuesin the fecal methanogens of Meishan and Yorkshire piglets Sampleidentifiers are the same as in Figures 1 and 2

major bacterial groups (Firmicutes and Bacteroidetes) in thegut of animals it was shown in a germ-free mouse modelthat methanogens also play an important role in energymetabolism and adipose deposition [30] Unlike the potentialroles of methanogens in a hostrsquos energy metabolism and adi-pose deposition the diversity and structure of methanogeniccommunities in pig gut have not been well understood

Studies using 16S rRNA gene-based techniques indicatethat the predominant species in ruminants belong to thegenusMethanobrevibacter [31 32] Recently a methanogenicarchaeal 16S rRNA gene library of pig faeces was con-structed wherein the clones were mainly separated into threeclustersmdashMethanobrevibacter Methanosphaera and a groupof uncultivated archaea [7] In our previous study the genus

Methanobrevibacter was also found to dominate in the faecesof Erhualian and Landrace pigs [8] which is consistent withthe result of the present study on Yorkshire and Meishanpiglets However methanogenic genera besidesMethanobre-vibacter were seldomly detected in the present study whichsuggests that the colonisation of other methanogens mightoccur later than the colonization ofMethanobrevibacter spp

To date the diversity of methanogens in the human guthas been thought to be limited to several species amongwhich M smithii has been regarded as the main methaneproducer [33] Furthermore it has been shown thatM smithiiconcentration was higher in anorexic patients than in a leanpopulation [34] It was also reported that the gut microbiotafrom obese individuals were depleted in M smithii [35]These results indicate thatM smithiimight play an importantrole in host energy metabolism Similarly it was observedthat the abundance of M smithii-related OTUs within themethanogenic 16S rRNA gene library was significantly higherin lean Landrace pigs than in obese Erhualian pigs [8] In thepresent study we found that M smithii in the faeces of bothbreeds gradually became predominant during the first twoweeks after birth Furthermore the dominance ofM smithiiin the lean Yorkshire piglets occurred earlier than in obeseMeishan piglets which confirms the previous finding thatlean animals and humans may harbour more M smithii intheir gut than obese ones

The composition and diversity of neonatal microbiotaare variable and vulnerable during early life until a stableecosystem is established Facultative anaerobes such as Enter-obacteriaceae enterococci streptococci and staphylococcihave been reported to be the predominant intestinal micro-biota in infants during the first week [36 37] as they cansurvive in the oxygen-containing gut As oxygen is com-sumed anaerobic microorganisms such as RuminococcusBacteroides and Bifidobacterium gradually thrive [14 3839] An increasing diversity in the bacterial community wasalso observed during the development of gut microbiota innewborn piglets [40] In contrast to the bacterial community

6 Archaea

OTU7

OTU8

OTU9

OTU14

OTU19

OTU30

OTU5

OTU6

OTU29

OTU17

OTU12

OTU11

OTU10

OTU25

OTU18

OTU26

OTU28

OTU4

OTU13

OTU20

OTU2

OTU1

OTU3

OTU21

OTU23

OTU22

OTU24

OUT15OTU16

OTU27

Methanobrevibacter thaueri

Methanobrevibacter millerae

Methanobrevibacter woesei

Methanobrevibacter smithii

Methanobrevibacter wolinii

Methanobrevibacter ruminantiumMethanobrevibacter olleyae

Methanobrevibacter filiformisMethanobrevibacter arboriphilu

Methanobacterium aarhusenseMethanobacterium alcaliphilum

Methanosphaera stadtmanae

M smithiiM woeseicluster

M thaueriM millerae cluster

1790

7070

4474

97

32

29

799

89 70

70

78

97

71

8376

62

80

11

93

32

70

85

76

54

78

68

001

Methanobrevibacter gottschalkii

Figure 4 Unrooted phylogenetic tree of Methanobrevibacter spp reference strains and predominant OTUs in the libraries from faecalmethanogens of Meishan and Yorkshire piglets The reference bar indicates 1 sequence dissimilarity

we found that the diversity indices of the faecalmethanogeniccommunity of Meishan and Yorkshire piglets decreased from1 day to 14 days which suggests that the colonisation ofgut methanogens in neonatal piglets is the result of mutualselection between the methanogens and the host This resultis also consistent with the previous finding that the diversityof methanogens in the human and monogastric animal guthas been thought to be limited to several species

In the present study although the diversity of themethanogen populations decreased with the age of thenewborn piglets the numbers of methanogens increasedsignificantly and reached 109 copies of 16S rRNA gene pergram of faeces which is similar to values observed forfaeces of grown pigs and suckling piglets before weaning[8] Combined with the results of diversity and taxonomiccomposition this result suggests that a stable methanogeniccommunity is established in the gut of piglets at the age of 14days

Interestingly we found that from day 1 to day 14the abundance of M smithii-related OTUs in the faeces

increased significantly while the abundances of M thaueri-and Mmillerae-related OTUs decreased significantly Itwas found that M thaueri- and M millerae-related OTUsdominated in the methanogenic 16 S rRNA gene libraryfrom the faeces of the Meishan and Yorkshire piglets threedays after birth Although these two species could be foundin the hindgut of pigs [8 41] M smithii was the mostabundant species in previous studies [7 8] In the gutM smithii converts H

2 CO2 and formate into CH

4using

carbon as the terminal electron acceptor In contrast Mthaueri only grows and produces methane from H

2and

CO2and does not grow or produce methane from for-

mate acetate methanol trimethylamine or methanol withH2[41] Considering the potential role of M smithii in

energy metabolism this result indicates that the substitutionof M smithii for other Methanobrevibacter spp might beimportant to the gut microbial ecosystemWe found that thissubstitution occurred more quickly in the Yorkshire pigletsthan in theMeishan piglets Further studies are still needed tounderstand whether this difference is related to the differentphenotype of these two breeds

Archaea 7

Table 2 Relative abundances of predominant OTUs (percentage) in the 16S rRNA gene libraries from faecal methanogens of Meishan andYorkshire piglets1

OTUs Yorkshire Meishan SEM2 Effect (P value) Closestreference strainD1 D3 D7 D14 D1 D3 D7 D14 Breed Age Breed times age

OTU2 053 069 2063 579 020 083 139 320 170 0741 0046 0034 M smithiiOTU6 689 120 009 000 1016 068 341 008 507 0293 0001 0679 M thaueriOTU7 319 131 003 000 987 392 297 026 450 0024 0012 0392 M thaueriOTU10 364 157 002 000 614 322 160 010 323 0157 0011 0860 M woeseiOTU14 226 060 002 000 280 238 133 014 178 0108 0028 0731 M thaueriOTU17 256 222 001 001 074 239 017 016 176 0547 0017 0508 M milleraeOTU19 212 045 000 000 252 151 119 011 137 0097 0004 0738 M thaueriOTU20 020 064 326 257 020 003 012 022 188 0015 0204 0217 M smithiiOTU21 289 035 002 000 323 003 000 001 198 0997 0002 0984 M gottschalkiiOTU26 010 025 121 061 005 022 013 200 088 0812 0005 0010 M smithiiOTU27 149 149 008 001 098 019 024 002 103 0223 0049 0403 M alcaliphilumOTU30 067 109 003 000 115 121 014 003 093 0560 0032 0956 M thaueriOTU38 008 034 053 066 012 017 040 086 036 0864 0001 0542 M smithiiOTU39 003 019 055 100 003 012 041 052 053 0324 0026 0756 M smithiiOTU40 121 054 002 000 075 004 022 002 069 0409 0019 0594 M thaueriOTU44 066 049 002 000 106 020 011 002 055 0750 0005 0544 M milleraeOTU48 015 034 053 040 006 009 016 061 028 0134 0010 0083 M milleraeOTU52 056 005 001 000 100 040 019 002 053 0139 0012 0796 M thaueriOTU63 042 020 001 000 080 017 009 001 030 0077 0000 0088 M thaueriOTU69 059 020 002 000 051 006 020 001 027 0875 0000 0299 M thaueriOTU71 026 031 000 000 068 014 014 001 031 0285 0005 0146 M milleraeOTU73 008 013 060 035 001 008 003 025 027 0016 0038 0067 M smithiiOTU74 041 063 000 000 008 035 000 000 040 0243 0035 0697 M olleyaeOTU77 100 003 002 000 036 001 000 000 048 0219 0003 0278 M milleraeOTU78 038 015 002 000 051 009 020 002 023 0263 0000 0487 M thaueriOTU82 028 014 001 000 033 007 040 005 021 0075 0010 0047 M thaueriOTU85 025 013 000 000 039 035 010 002 026 0175 0047 0862 M thaueriOTU86 017 016 002 000 044 016 029 000 020 0024 0010 0153 M thaueriOTU88 033 004 000 000 049 020 012 000 025 0143 0002 0845 M thaueriOTU91 067 007 000 000 033 000 004 001 035 0399 0006 0572 M milleraeOTU93 051 024 000 000 018 017 000 000 029 0291 0045 0570 M milleraeOTU103 004 004 017 032 002 004 010 021 015 0277 0002 0821 M smithiiOTU104 026 010 000 000 024 022 010 000 017 0338 0010 0706 M thaueriOTU107 016 006 000 000 047 005 014 000 018 0007 0000 0017 M thaueriOTU109 016 038 003 000 020 004 003 002 019 0217 0038 0073 M milleraeOTU110 022 018 004 000 027 008 004 002 017 0892 0018 0786 M millerae1OTUs the abundances that were significantly affected by pig breed age or the interaction between breed and age are shown in this table2SEM standard error of means 119899 = 4

The microbiota of newborns mainly travel from themotherrsquos vagina skin and faeces [42 43] In addition dietcomposition is regarded as the main factor affecting gutmicrobiota It was reported that Erhualian (similar breed toMeishan) sows had higher concentrations of milk lactose andfat but lower concentration of milk protein as compared totraditional Western breeds [44] which may be a possiblereason for the existence of M thaueri and M millerae in

the faeces of newborn piglets in relatively high abundanceFurther studies are needed to reveal their potential role in thegut of newborn piglets

5 Conclusions

In conclusion the present study showed that themethanogenic community in the faeces of Meishan and

8 Archaea

Table 3 Relative abundances (percentage) of M smithii- M thaueri- M millerae- and M olleyae-related OTUs in the 16S rRNA genelibraries from faecal methanogens of Meishan and Yorkshire piglets

Breed Age (d) Relative abundance within total methanogensM smithii-related OTUs M thaueri-related OTUs M millerae-related OTUs M olleyae-related OTU

Meishan

1 181 6111 1843 1673 813 2679 2368 26257 2628 5754 729 000014 7851 752 699 0000

Yorkshire

1 368 3301 2772 10253 1689 1895 2487 5357 8841 079 374 01314 9427 002 085 0000

SEM1 4167 3178 1606 1453

Effect (P value)Breed 0003 0010 0969 0521Age 0000 0020 0009 0081

Breed times age 0017 0198 0715 01731SEM standard error of means 119899 = 4

0

1

2

3

4

5

6

7

8

9

10

Meishan Yorkshire

Lg (1

6S rR

NA

gen

e cop

ies

g)

D1D3

D7D14

lowast lowast

Figure 5 Real-time PCR quantification of 16S rRNA gene copiesof methanogenic archaea in the faeces of Meishan and Yorkshirepiglets lowast

119875

lt 005 119899 = 4

Yorkshire neonatal piglets was dominated by members ofthe genusMethanobrevibacter represented byM smithiiMthaueri and M millerae The structure of the methanogeniccommunity was significantly affected by the age of pigletshowever pig breed could also affect the substitution ofdifferentMethanobrevibacter spp

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research has received funding from the National BasicResearch Program of China (2012CB124705) the NationalNatural Science Foundation of China (30810103909)the Fundamental Research Funds for the CentralUniversities (KYZ201153) the European CommunityrsquosSeventh Framework Programme (FP72007-2013) under theGrant Agreement no 227549 and the China-EU Science andTechnology Cooperation Foundation (1008)

References

[1] J H P Hackstein and T A van Alen ldquoFecal methanogens andvertebrate evolutionrdquoEvolution vol 50 no 2 pp 559ndash572 1996

[2] B Morvan F Bonnemoy G Fonty and P Gouet ldquoQuantitativedetermination of H

2

-utilizing acetogenic and sulfate-reducingbacteria and methanogenic Archaea from digestive tract ofdifferent mammalsrdquo Current Microbiology vol 32 no 3 pp129ndash133 1996

[3] C Lin and T L Miller ldquoPhylogenetic analysis of Methanobre-vibacter isolated from feces of humans and other animalsrdquoArchives of Microbiology vol 169 no 5 pp 397ndash403 1998

[4] K A Johnson and D E Johnson ldquoMethane emissions fromcattlerdquo Journal of Animal Science vol 73 no 8 pp 2483ndash24921995

[5] B S Samuel E E Hansen J K Manchester et al ldquoGenomicand metabolic adaptations ofMethanobrevibacter smithii to thehuman gutrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 25 pp 10643ndash106482007

[6] D Roccarina E C Lauritano M Gabrielli F Franceschi VOjetti and A Gasbarrini ldquoThe role of methane in intestinaldiseasesrdquo American Journal of Gastroenterology vol 105 no 6pp 1250ndash1256 2010

[7] S-Y Mao C-F Yang and W-Y Zhu ldquoPhylogenetic analysis ofmethanogens in the pig fecesrdquoCurrentMicrobiology vol 62 no5 pp 1386ndash1389 2011

Archaea 9

[8] Y H Luo Y Su A D G Wright L L Zhang H Smidt andWY Zhu ldquoLean breed Landrace pigs harbor fecal methanogens athigher diversity and density than obese breed Erhualian pigsrdquoArchaea vol 2012 Article ID 605289 9 pages 2012

[9] M C Collado M Cernada C Bauerl M Vento and G Perez-Martinez ldquoMicrobial ecology and host-microbiota interactionsduring early life stagesrdquo Gut Microbes vol 3 pp 352ndash365 2012

[10] CH F Hansen D S NielsenM Kverka et al ldquoPatterns of earlygut colonization shape future immune responses of the hostrdquoPLoS ONE vol 7 no 3 Article ID e34043 2012

[11] J M Saavedra and A M Dattilo ldquoEarly development of intesti-nalmicrobiota implications for future healthrdquoGastroenterologyClinic of North America vol 41 pp 717ndash731 2012

[12] S Matamoros C Gras-Leguen F le Vacon G Potel and MF de la Cochetiere ldquoDevelopment of intestinal microbiota ininfants and its impact on healthrdquo Trends in Microbiology vol21 pp 167ndash173 2013

[13] S Fanaro R Chierici P Guerrini and V Vigi ldquoIntestinalmicroflora in early infancy composition and developmentrdquoActa Paediatrica vol 91 supplement 441 pp 48ndash55 2003

[14] C Palmer E M Bik D B DiGiulio D A Relman andP O Brown ldquoDevelopment of the human infant intestinalmicrobiotardquo PLoS Biology vol 5 no 7 article e177 2007

[15] P Panigrahi S Parida L Pradhan et al ldquoLong-term colo-nization of a Lactobacillus plantarum synbiotic preparation inthe neonatal gutrdquo Journal of Pediatric Gastroenterology andNutrition vol 47 no 1 pp 45ndash53 2008

[16] C L Thompson B Wang and A J Holmes ldquoThe immedi-ate environment during postnatal development has long-termimpact on gut community structure in pigsrdquo ISME Journal vol2 no 7 pp 739ndash748 2008

[17] M E Conroy H N Shi and W A Walker ldquoThe long-termhealth effects of neonatal microbial florardquo Current Opinion inAllergy and Clinical Immunology vol 9 no 3 pp 197ndash201 2009

[18] X Guo X Xia R Tang and K Wang ldquoReal-time PCRquantification of the predominant bacterial divisions in thedistal gut of Meishan and Landrace pigsrdquo Anaerobe vol 14 no4 pp 224ndash228 2008

[19] E O Casamayor R Massana S Benlloch et al ldquoChanges inarchaeal bacterial and eukaryal assemblages along a salinitygradient by comparison of genetic fingerprinting methods in amultipond solar salternrdquoEnvironmentalMicrobiology vol 4 no6 pp 338ndash348 2002

[20] M J L Coolen E C Hopmans W I C Rijpstra et alldquoEvolution of themethane cycle inAce Lake (Antarctica) duringthe Holocene response of methanogens and methanotrophs toenvironmental changerdquo Organic Geochemistry vol 35 no 10pp 1151ndash1167 2004

[21] E Stackebrandt and B M Goebel ldquoTaxonomic note a placefor DNA-DNA reassociation and 16S rRNA sequence analysisin the present species definition in bacteriologyrdquo InternationalJournal of Systematic Bacteriology vol 44 no 4 pp 846ndash8491994

[22] S Wu Z Zhu L Fu B Niu andW Li ldquoWebMGA a customiz-able web server for fast metagenomic sequence analysisrdquo BMCGenomics vol 12 article 444 2011

[23] J Good ldquoThe population frequencies of species and the estima-tion of population parametersrdquoBiometrika vol 40 pp 237ndash2641953

[24] P D Schloss S L Westcott T Ryabin et al ldquoIntroduc-ing mothur open-source platform-independent community-supported software for describing and comparing microbial

communitiesrdquoApplied and EnvironmentalMicrobiology vol 75no 23 pp 7537ndash7541 2009

[25] C Lozupone and R Knight ldquoUniFrac a new phylogeneticmethod for comparing microbial communitiesrdquo Applied andEnvironmentalMicrobiology vol 71 no 12 pp 8228ndash8235 2005

[26] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[27] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[28] K Takai and K Horikoshi ldquoRapid detection and quantificationof members of the archaeal community by quantitative PCRusing fluorogenic probesrdquo Applied and Environmental Microbi-ology vol 66 no 11 pp 5066ndash5072 2000

[29] R E Ley F Backhed P Turnbaugh C A Lozupone R DKnight and J I Gordon ldquoObesity alters gut microbial ecologyrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 31 pp 11070ndash11075 2005

[30] B S Samuel and J I Gordon ldquoA humanized gnotobiotic mousemodel of host-archaeal-bacterial mutualismrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 26 pp 10011ndash10016 2006

[31] S E Denman N W Tomkins and C S McSweeney ldquoQuan-titation and diversity analysis of ruminal methanogenic pop-ulations in response to the antimethanogenic compound bro-mochloromethanerdquo FEMS Microbiology Ecology vol 62 no 3pp 313ndash322 2007

[32] A-D G Wright C H Auckland and D H Lynn ldquoMoleculardiversity of methanogens in feedlot cattle from Ontario andPrince Edward Island Canadardquo Applied and EnvironmentalMicrobiology vol 73 no 13 pp 4206ndash4210 2007

[33] B Dridi M Henry A El Khechine D Raoult and MDrancourt ldquoHigh prevalence ofMethanobrevibacter smithii andMethanosphaera stadtmanae detected in the human gut usingan improved DNA detection protocolrdquo PLoS ONE vol 4 no 9Article ID e7063 2009

[34] F ArmougomMHenry B Vialettes D Raccah andD RaoultldquoMonitoring bacterial community of human gut microbiotareveals an increase in Lactobacillus in obese patients andMethanogens in anorexic patientsrdquo PLoS ONE vol 4 no 9Article ID e7125 2009

[35] MMillionMMaraninchiMHenry et al ldquoObesity-associatedgut microbiota is enriched in Lactobacillus reuteri and depletedin Bifidobacterium animalis and Methanobrevibacter smithiirdquoInternational Journal of Obesity vol 36 pp 817ndash825 2012

[36] H H Wenzl G Schimpl G Feierl and G SteinwenderldquoTime course of spontaneous bacterial translocation fromgastrointestinal tract and its relationship to intestinalmicroflorain conventionally reared infant ratsrdquo Digestive Diseases andSciences vol 46 no 5 pp 1120ndash1126 2001

[37] A K Benson S A Kelly R Legge et al ldquoIndividuality in gutmicrobiota composition is a complex polygenic trait shaped bymultiple environmental and host genetic factorsrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 107 no 44 pp 18933ndash18938 2010

[38] C F Favier W M de Vos and A D L Akkermans ldquoDevelop-ment of bacterial and bifidobacterial communities in feces ofnewborn babiesrdquo Anaerobe vol 9 no 5 pp 219ndash229 2003

10 Archaea

[39] P A Vaishampayan J V Kuehl J L Froula J L MorganH Ochman and M P Francino ldquoComparative metagenomicsand population dynamics of the gut microbiota in mother andinfantrdquo Genome Biology and Evolution vol 2 no 1 pp 53ndash662010

[40] S R Konstantinov A A Awati B A Williams et al ldquoPost-natal development of the porcine microbiota composition andactivitiesrdquo Environmental Microbiology vol 8 no 7 pp 1191ndash1199 2006

[41] T L Miller and C Lin ldquoDescription of Methanobrevibac-ter gottschalkii sp nov Methanobrevibacter thaueri sp novMethanobrevibacter woesei sp nov and Methanobrevibacterwolinii sp novrdquo International Journal of Systematic and Evolu-tionary Microbiology vol 52 no 3 pp 819ndash822 2002

[42] M G Dominguez-Bello E K Costello M Contreras et alldquoDelivery mode shapes the acquisition and structure of theinitial microbiota across multiple body habitats in newbornsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 26 pp 11971ndash11975 2010

[43] M-M Gronlund Ł Grzeskowiak E Isolauri and S SalminenldquoInfluence ofmotherrsquos intestinal microbiota on gut colonizationin the infantrdquo Gut Microbes vol 2 no 4 pp 227ndash233 2011

[44] Y Qin S Zou Y Xu P Wang and X Xu ldquoDynamic analysesof lactose and milk protein in Erhualian Sows and theircomparisonswithYorkshire sowsrdquo Journal of AnhuiAgriculturalUniversity vol 27 pp 167ndash170 2000 (Chinese)

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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