Lean Breed Landrace Pigs Harbor Fecal Methanogens at Higher Diversity and Density than Obese Breed Erhualian Pigs

The diversity of fecal methanogens of Erhualian (obese type) and Landrace (lean type) pigs was examined using separate 16S rRNA gene libraries for each breed. A total of 763 clones were analyzed; 381 from the Erhualian library and 382 from the Landrace library were identified belonging to the genus Methanobrevibacter. Others were identified belonging to the genus Methanosphaera. The two libraries showed significant differences in diversity (P < 0.05) and composition (P < 0.0001). Only two operational taxonomic units (OTUs) were found in both libraries, whereas six OTUs were found only in the Erhualian library and 23 OTUs were found only in the Landrace library. Real-time PCR showed that the abundance of fecal methanogens in Landrace pigs was significantly higher than that in Erhualian pigs (P < 0.05). Results showed that the Landrace pig (lean) harbored a greater diversity and higher numbers of methanogen mcrA gene copies than the Erhualian pig (obese). These differences may be related to the fatness or leanness in these two pig breeds. The results provide new leads for further investigations on the fat storage of pigs or even humans.


Introduction
Methanogenic archaea exist widely in the gastrointestinal (GI) tract of many vertebrates and invertebrates including humans [1][2][3]. Methanogens can use hydrogen and other products such as formate, methanol, acetate to reduce carbon dioxide to methane. Methane formation not only contributes to global warming as a greenhouse gas, but also represents an energy loss for the animal.
Pigs were estimated to typically lose 1.2% of ingested energy due to methane formation [4]. Furthermore, it was shown in a germ-free mouse model that methanogens play an important role in energy metabolism and adipose deposition through the re-colonization or co-colonization of the human fecal isolate Methanobrevibacter smithii and Bacteroides thetaiotaomicron into the GI tract of mice [5,6]. Further research in humans showed that the diversity of Mbb. smithii concentration was higher in anorexic patients than in a lean population [7]. It was also reported that the GI tract microbiota from obese individuals was depleted in Mbb. smithii [8].
Archaea in the human GI tract comprise mainly members of the order Methanobacteriales, which are H 2 -oxidizing methanogens. Interestingly, the number of H 2 -utilizing methanogens was significantly higher in obese individuals than in lean or postgastric-bypass individuals [9]. These reports may suggest some relationship between the composition and abundance of GI tract methanogenic communities, and the host's energy metabolism, which subsequently relates to the fatness or leanness of the host.
Pigs share a high similarity with humans with respect to the anatomy, physiology and metabolism of the digestive system [10]. Thus, the composition of GI tract microbiota of obese and lean pigs could reflect that of corresponding human phenotypes. Erhualian and Landrace breeds are typically obese and lean pigs [11][12][13][14], respectively, thus their energy metabolism might be distinctive. Erhualian pig, a sister breed to Meishan (both belonging to Taihu pig), Archaea is a local porcine breed mainly located around the Taihu Lake area of China and is characterized by increased fat storage, tasty meat quality and high fertility [15]. In contrast, Landrace is an "alien" breed, which is usually used as a sire in the breeding of commercial pork production for the breed's high growth rate and lean meat percentage. According to previous research described above, the distinction between the two breeds may be partially contributed by their different gut microbiota. Recently, it has been found that obese Meishan pigs showed an increased relative abundance of Firmicutes and lower numbers of Bacteroidetes [16], in line with several reports from humans as described above. Although the diversity and abundance of bacteria between obese and lean pigs have been shown to vary [12,16], there is no evidence has been shown regarding the variation of intestinal methanogens between the two phenotypes. Considering the key role of gut methanogens during the microbial fermentation, we hypothesized that the two breeds will have different composition and density of GI tract methanogens.
Therefore, this study investigated the phylogenetic diversity and community structure of methanogens in the feces from Erhualian (obese) and Landrace (lean) pigs by analyzing 16S rRNA gene sequences from two clone libraries, one for each breed. In addition, the density of methanogens was quantified by real-time PCR targeting the mcrA gene. The results shown here could provide new leads towards understanding and control of the role of GI tract microbiota in fat storage of pigs and potentially humans.

Sample Sources and
Processing. All Erhualian and Landrace pigs were raised at a commercial farm in Jiangsu Province using the same feed and under the same environmental conditions. Piglets were weaned 45 days after birth. Fecal samples from 3 suckling (40 d) piglets, 4 weaned (50 d) pigs, 4 growing (70 d) pigs, and 4 sows (11 to 12 months) of Erhualian breed, and 4 suckling piglets, 3 weaned pigs, 3 growing pigs, and 4 sows of Landrace breed were collected. Animals were randomly selected from different litters. Approximately 10 g of feces from each pig was collected into a sterilized 15 mL centrifuge tube and stored at −20 • C until further processing.

DNA Extraction, PCR Amplification, and Clone Library
Construction. Nucleic acids were extracted from 0.5 g of fecal material, based on the bead-beating method described by Zoetendal et al. [17]. The extracted DNA was purified with a PCR Clean-Up system (Promega, USA) and stored at −20 • C.
Primers Met86F and Met1340R were used to amplify archaeal 16S rRNA genes [18]. The amplification was initiated with a denaturation at 94 • C for 3 min, then followed by 40  polymerase. The product was purified using a PCR Clean-Up system (Promega, USA).
To construct 16S rRNA gene clone libraries, equal quantities of purified PCR products from animals of same breed (i.e., Erhualian, Landrace) were pooled. Cloning of pooled amplicons into Escherichia coli TOP10 using the pGEM-T Easy vector (Promega, USA), and screening of transformants using RFLP analysis of the cloned 16S rRNA genes by restriction digestion with endonucleases Hae III, Alu I and Hpa II, was done as described previously [19]. Clones with identical RFLP patterns were defined as one phylotype. One representative clone from each RFLP pattern was sequenced in both directions commercially (Invitrogen, China).

Estimation of Archaeal Diversity and Phylogenetic
Analysis. Based on a species-level sequence identity criterion of 98% [20], MOTHUR [21] was used to assign sequences across the two libraries to operational taxonomic units (OTUs). As part of the MOTHUR suite of programs, Shannon Index was used to analyze diversity and Libshuff analysis was used to compare population structure between the two libraries. The sampling effort in each library for species-level OTUs was evaluated by calculating the coverage (C) according to the equation C = 1 − (n/N), where n is the number of OTUs represented by a single clone and N is the total number of clones analyzed in the library [22]. GenBank's BLAST program [23] was used to presumptively identify the nearest validly described neighbor of each methanogen sequence. Lastly, a neighbor-joining tree was constructed using the phylogenetic software PHYLIP (ver 3.69) with 1,000 bootstrap resamplings [24].

Quantification of Total Fecal Methanogens of Each Breed by Real-Time PCR.
The abundance of fecal methanogens was determined with real-time PCR using an Applied Biosystems 7300HT Real-Time PCR System (Applied Biosystems, CA, USA). Primers targeting the mcrA gene [25] were used for the specific detection of methanogenic archaea. DNA samples extracted from each fecal sample of each breed (total 15 Erhualian pigs and 14 Landrace pigs) as described above were used for the real-time PCR amplification of mcrA gene. DNA from cells of a pure culture of Methanobrevibacter smithii supplied by CSIRO Livestock Industry (Brisbane, Australia) was also extracted with a Genomic DNA Purification Kit (Promega, USA). The concentrations of the above DNA samples extracted from fecal materials, or from cells of the pure culture, were determined in triplicate with a NanoDrop ND-1000 UV Spectrophotometer (NanoDrop Technologies, USA) and the mean values were calculated. Serial dilutions of DNA extracted from Methanobrevibacter smithii cells were used to generate a standard curve. A reaction mixture (10 µL) consisted of 5 µL of IQ SYBR Green Supermix (Roche, Basel, Switzerland), 0.5 µL of each primer (10 µM), and 1 µL of template DNA (100 ng/µL). PCR was performed with an initial denaturation step of 94 • C for 2 min, followed by 30 cycles of 94 • C for 30 s, 60 • C for 15 s and 68 • C for 1 min. Archaea 3 Differences in the abundance of total fecal methanogens between Erhualian and Landrace pigs were tested for significance with an One-Sample t-test method using the statistical software SPSS 16.0. Differences were considered significant when P < 0.05.

Nucleotide Sequences and Accession Numbers.
Phylotypes were designated by using the prefix LGM (Laboratory of Gastrointestinal Microbiology) followed by either "Er" or "La" to represent the two pig breeds, Erhualian and Landrace, respectively; a number to indicate the unique phylotype (e.g., phylotype 7 from the Erhualian pig breed is LGM-Er7).
The nucleotide sequences reported in this paper have been deposited in the GenBank database under accession numbers. HM573393 to HM573406 (Erhualian) and HM573407 to HM573449 (Landrace).

The Density of Total mcrA Gene Copies in the Feces of the Two Breed Pigs.
Quantitative real-time PCR showed that the number of mcrA gene copies in the feces of Landrace pigs 8.80 ± 0.91 (Log10 (mcrA gene copies per gram of wet weight)) was significantly higher than that in the Erhualian pigs (8.23 ± 0.63, P < 0.05).

Sequence Analysis of the Two Archaeal 16S rRNA
Gene Clone Libraries. A total of 381 cloned archaeal 16S rRNA gene amplicons, obtained from fecal samples taken from Erhualian pigs at different life stages, were analyzed. Sequence examination of these clones revealed eight different OTUs ( Table 1). The majority of sequences (368/381) were most closely related to members belonging to the genus Methanobrevibacter with sequence identities ranging from 96.9% to 99.9%. One hundred and forty-one sequences (37%) were assigned to OTU11 (Table 2) and related to Methanobrevibacter gottschalkii and Methanobrevibacter millerae, while 111 sequences (29%) were assigned to OTU13 and related to Methanobrevibacter smithii (Table 2). Clone LGM-Er7 (OTU2) was distantly related to Methanobrevibacter millerae with 96.9% identity, but had 97.7% identity to uncharacterized Methanobrevibacter clones from the foregut of the Tammar wallaby [26].
LGM-La42 (OTU30) and LGM-La43 (OTU31) were distantly related to Methanosphaera cuniculi and Methanobrevibacter smithii with 95.3% identity, respectively, but had only 96.3% and 96.2% to their nearest neighbor, clones from the Holstein and Jersey dairy cows [28]. It is important to note that OTUs except OTU11 and OTU13 were only found in this library (Table 1, Figure 1).

Methanobrevibacter thauri
LGM-Erhualian14 ON-CAN.10 (DQ123880) Methanobrevibacter smithii (CP000678) LGM-Landrace19 LGM-Landrace3 LGM-Landrace16 LGM-Landrace15 Methanobrevibacter millerae ZA-10 (AY196673) LGM-Landrace20 LGM-Landrace40 LGM-Landrace34 LGM-Landrace39 LGM-Erhualian13 LGM-Erhualian12 LGM-Erhualian11 LGM-Erhualian1 Methanobrevibacter gottschalkii PG (U55239) LGM-Erhualian6 LGM-Erhualian5 LGM-Erhualian2 LGM-Erhualian3 LGM-Erhualian9 Methanobrevibacter wolinii SH (U55240) Methanobrevibacter olleyae (AY615201) LGM-Erhualian8 LGM-Landrace13    Methanogens can produce methane from substrates such as H 2 and CO 2 and formate, which could also be used for the formation of propionate and acetate. For ruminants, it is widely established that the formation of methane results in a loss of energy available for the host [33,34]. Thus, a highly dense and diverse methanogen community as observed in fecal samples may also suggest an energy loss, which may consequently affect energy metabolism and body fat mass formation. It is also possible that although methane formation may represent a small portion of energy for a pig growth, it may affect metabolic pathway network and consequently affect the energy metabolism. In total, 763 clones were examined from the two 16S rRNA gene clone libraries, revealing 53 phylotypes assigned Archaea 7 to 31 OTUs (Table 1). OTUs 11 and 13 were the only OTUs found in both pig breeds (Table 2) and accounted for 66% and 24% of the clones from Erhualian and Landrace pigs, respectively. Interestingly, clones belonging to OTU11 were nearly 35 times higher in Erhualian library than Landarce library. OTUs 11 and 13 combined for 70% of all OTUs in Erhualian library, but only 36% of all OTUs in the Landrace library ( Table 2).
The comparison of OTUs between the two libraries ( Table 2) showed that Methanobrevibacter-like sequences (96.6%) were dominant in the feces of Erhualian pigs, whereas the proportion was 56.5% for Landrace pigs. Furthermore, Methanosphaera-like sequences accounted for 3.4% in Erhualian and 43.5% in Landrace. This is consistent with previous findings that Methanobrevibacter species are the predominant methanogen in the hindgut of monogastric animals [27,[35][36][37][38][39][40][41][42] or in the rumen of sheep [31,[43][44][45], cattle [45][46][47][48] and also in some cultivation studies [49]. Nevertheless, in the rumen of sheep and bovine, the most dominant methanogens belonged to genus Methanobrevibacter, and the density of Methanosphaera-like species was much less than that of Methanobrevibacterlike species [45,46]. While in the rumen of sheep from Western Australia, Methanosphaera stadtmanae was only found in a minority of sheep [31]. In our previous study, the proportion of Methanosphaera stadtmanae 16S rRNA sequences was very small in Duroc × Landrace × Yorkshire pig feces [27]. However, in the present study, Landrace pigs had 10 times more Methanosphaera-like methanogens than Erhualian pigs, while Methanosphaera-like species were the second dominant methanogens behind Methanobrevibacter in the feces of both pig breeds.
Our libraries also contained several yet unidentified euryarchaeotic sequences. Fourteen OTUs (LGM-La1 and 11, 3, 4 and 22, 12, 14, 21, 27, 33, 35, 36, 37, 42, 43 and LGM-Er4) were most likely represent yet unknown methanogenic species and strains (Table 1). Interestingly, in a previous study we found that populations related to Aciduliprofundum boonei and Thermoplasma acidophilum were present, in addition to the Methanobrevibacter and Methanosphaera populations, in the feces of Duroc × Landrace × Yorkshire pigs [27]. Methanogens related to the Thermoplasmatales clade were also found in some ruminants [19,32,46,50], whereas in the current study, most sequences were associated with the two genera Methanobrevibacter and Methanosphaera. This suggests that there might be a difference in the diversity of gut methanogens between pure breeds and hybrids, and hybridization of different breeds might introduce a certain alteration of the methanogenic diversity into the intestine.

Conclusion
The current study provided the first account on the abundance and phylogenetic diversity of methanogens found in Erhualian (obese) and Landrace (lean) pig feces based on 16S rRNA gene clone library analysis. Landrace pigs have a markedly higher density of methanogens than the Erhualian pigs (P < 0.05). The diversity of methanogens of Landrace pigs was also significantly higher than that of Erhualian animals (P < 0.05) with Methanobrevibacter as the most dominant genus in both breeds, and Methanosphaera being the second most dominant methanogen in Landrace pigs. The functional roles of these methanogens in the pig gut, and whether observed differences in methanogen diversity and density are related to the pig fat or energy metabolism, need further investigation.