Annals of Microbiology (2018) 68:185–194 https://doi.org/10.1007/s13213-018-1329-z ORIGINAL ARTICLE The significance of the diversity and composition of the cecal microbiota of the Tibetan swine Weiping Yang1,2 & Haiyun Xin1,3 & Fangjun Cao1 & Jinxing Hou4 & Li Ma1 & Lijuan Bao1 & Fangyuan Wang1 & Zhantao Yu1 & Binyun Cao1 Received: 22 August 2017 /Accepted: 22 February 2018 /Published online: 9 March 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018 Abstract The Tibetan swine (TIS) is a non-ruminant herbivore with high disease resistance. Also, it has the ability to digest plants with high fiber content. However, it is not known whether any relationship exist between these characteristics of the TIS and its cecal microbiota. Thus, this study aims to investigate the cecal microbiota of the adult TIS using high-throughput sequencing techniques in order to explore possible relationships between these unique characteristics of the TIS (high disease resistance and ability to digest high fiber plants) and its cecal microbiota. PIC pigs (lean type) were chosen as controls. The results show that 75,069 valid sequences of the 16S rRNA gene at V4-V5 region were obtained in the cecal content of TIS. They were composed of 15 phyla, 70 genera and divided into 660 Operational Taxonomic Units (OTUs). Bacteroidetes and Firmicutes were the predominant phyla in both breeds, but TIS had more Bacteroidetes than Firmicutes. Also, 42.4% of the cecal bacteria were found to be unclassified and uncultured. Many cellulolytic bacteria were also found in the two breeds. TIS (88.10%) had much higher abundance in the core bacterial communities than PIC pigs (81.29%), and the proportion of Bacteroides and Spirochaetes that can effectively degrade cellulose were 6.01 and 6.40% higher than PIC pigs, respectively, while Proteobacteria that are closely related to gastrointestinal diseases were 1.61% lower than PIC pigs. Thus, the disease resistance of the TIS and its ability to digest plants with high fiber content may be related to high abundance of core bacterial communities as well as the large number of unknown and unclassified bacteria. Keywords Cecal microbiota . 16S rRNA gene . High-throughput sequencing . Tibetan swine . Disease resistance . Herbivorous characteristics Highlights • Comparing the cecal microbiota of a Tibetan and PIC (lean- type) pig. • The core bacterial communities were different in these two breeds. • Tibetan swine had a higher proportion of digestion related bacteria phyla. Weiping Yang and Haiyun Xin are joint first authors. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-018-1329-z) contains supplementary material, which is available to authorized users. * Binyun Cao 3 Institute of Animal Science, Guangdong Academy of Agricultural [email protected] Sciences, No.1 Da feng Road, Tianhe District, Guangzhou, Guangdong 510640, People’s Republic of China 1 College of Animal Science and Technology, Northwest A&F University, No.22 Xinong Road, Yangling 712100, Shaanxi, 4 Animal Engineering Branch, Yangling Vocational & Technical ’ People s Republic of China College, No. 10 Xinong Road, Yangling, Shaanxi 712100, People’s 2 Life sciences college, Luoyang Normal University, No.6 Jiqing Republic of China Road, Yibin District, Luoyang 471934, Henan, People’sRepublicof China 186 Ann Microbiol (2018) 68:185–194 Introduction farms. All the five pigs of each breed were reared under the same standard feeding and management conditions. The diet The microbial communities in the gastroenteric tracts of of the TIS consisted of 90% of green fodder and 10% of humans and animals greatly influence their digestion, metabo- soybeans and wheat bran, while the diet of PIC pigs consisted lism, and disease resistance (Turnbaugh et al. 2009). Firmicutes of compound feed. The pigs had no history of gut infectious and Bacteroidetes are the two dominant phyla in the gut bacte- disease, and no antimicrobial administration occurred during rial communities of many ruminant and non-ruminant animals. the feeding process. They were fed an antibiotic-free diet. The Changes in their composition and ratio greatly affect digestion, cecal content samples from the TIS and PIC pigs were collect- metabolism, and substance absorption of animals (Turnbaugh ed from the Tibetan swine Slaughterhouse of Shaanxi Huayi et al. 2009;Lietal.2012;Looftetal.2012). Moreover, changes Industrial Co., Ltd. and the Shaanxi Benxiang Pig in the proportion of Lactobacilli and Enterobacteria could be Slaughterhouse, respectively. After the pigs were killed with used as a gut health indicator for animals (Castillo et al. 2007). sodium pentobarbital (50 mg/1 kg BW), the cecum was ligat- The diversity and composition of the gut microbial communi- ed at both ends (~ 100 g/sample) and was immediately re- ties of animals are related to several factors, including animal moved from the peritoneal cavity, placed into aseptic ziplock species (Pajarillo et al. 2014), age (Castillo et al. 2007), envi- baggies, and stored in foamed plastic containers filled with dry ronment (Wu et al. 2012), diet type (Yan et al. 2013;Chenetal. ice. All samples were transported to the laboratory within 2014), dietary fiber content (Castillo et al. 2007;Liuetal.2012; 30 min for microbial genomic DNA extraction. The DNA Zhang et al. 2016), as well as antibiotics content in the diet was kept frozen at − 80 °C until it was needed. (Looftetal.2012, 2014). The experimental design and procedures were approved by The Tibetan swine (TIS) is a Qinghai (China) native, the Animal Care and Use Committee of Northwest A&F plateau-dwelling herbivore. Ninety percent of its nutrients University. The cecal samples were collected with the permis- can be obtained from forage grasses. The TIS has a large sion of Hongzhou Wang and Junfang Yan, the director of intestine like other non-ruminant monogastric animals, and Tibetan Pig Slaughterhouse of Shaanxi Huayi Industrial Co., its structure and function are at an intermediate stage between Ltd. and Shaanxi Benxiang Pig Slaughterhouse, respectively. those of carnivorous and herbivorous animals. Most undigest- The study did not involve endangered or protected species. ed feed components and endogenous secretions are fermented by microorganisms in the large intestine to provide the neces- DNA extraction and PCR amplification sary nutrients for the animal (Wenk 2001). About 1011–1012 microbial cells live in each gram of cecal After mixing the cecal contents of each pig, the genomic DNA content of pigs, comprising of 400 to 500 different types was extracted from 200 mg of samples using the E.Z.N.A.® (Castillo et al. 2007). However, more than 80% of the bacterial Stool DNA Kit (OMEGA, USA) according to the manufac- species are not yet identified (Leser et al. 2002). At present, turer’s protocols and the concentration was measured using a studies on TIS microbiota mainly focus on the isolation and NanoDrop Spectrophotometer ND1000 (Thermo Scientific, identification of bacteria that can degrade cellulose (Meng USA). The V4-V5 region of the bacterial 16S rRNA gene et al. 2014;Yangetal.2014;Maetal.2015) or secrete anti- was amplified by PCR using primers 515F 5′-barcode- bacterial peptide (Xin et al. 2017). Xiao et al. (2017)found GTGCCAGCMGCCGCGG)-3′ and 907R 5′-CCGT that the immunologic characteristics can be transferred by gut CAATTCMTTTRAGTTT-3′ (Sun et al. 2013; Pitta et al. microbiota, suggesting the vital role of microbiota in immune 2014). The PCR procedure was as follows: initial denaturation phenotype programming, and making us suppose the relation- at 95 °C for 2 min; 30 cycles of 95 °C for 30 s, 55 °C for 30 s, ship of host microbiota with disease resistance and other char- and 72 °C for 30 s, and a final extension at 72 °C for 5 min acteristics. Therefore, we investigated the diversity and com- where the barcode is an eight-base sequence unique to each position of the bacterial community in the cecum of the TIS sample. PCR reactions were performed in triplicate involving using Miseq high-throughput sequencing analysis in order to 20 μL mixtures containing 4 μL of 5× FastPfu buffer, 2 μLof explore possible relationships between the bacterial commu- 2.5 mM dNTPs, 0.8 μLofeachprimer(5μM), 0.4 μLof nity of the TIS and its unique characteristics. FastPfupolymerase,and10ngoftemplateDNA. Illumina MiSeq sequencing Materials and methods Amplicons were extracted from 2% agarose gels and purified Collection of cecal content samples using the AxyPrep DNA Gel Extraction Kit (TaKaRa) accord- ing to the manufacturer’s instructions. They were quantified Five healthy male adult TIS (8 months) and five healthy male using QuantiFluor™ -ST (Promega, USA). The purified adult PIC (5 months) pigs were obtained from two different amplicons were pooled in equimolar concentrations and Ann Microbiol (2018) 68:185–194 187 paired-end sequenced (2 × 250) on an Illumina MiSeq plat- chimeric sequences were removed using UCHIME; 75,069 form according to the standard protocol. and 74,759 sequences were left for further analysis of the TIS and PIC pigs, respectively. Further analysis identified Processing and analysis of sequencing data 660 and 668 OTUs from the TIS and PIC pigs, respectively. The total number of optimized reads, OTUs, statistical species Raw fastq files were demultiplexed and quality-filtered using richness, and diversity estimations for each sample are pre- QIIME (Caporaso et al. 2010) (version 1.17) in accordance sented in Table 1. with the following criteria: (i) 250 bp reads were truncated at OTUs and microbial diversity indices were used to con- any site receiving an average quality score of < 20 over a 10- struct rarefaction and Shannon curves to compare the micro- bp sliding window, and the truncated reads below 50 bp were bial community richness and diversity of the different samples discarded. (ii) The exact barcode matching, two nucleotide at different sequencing depths by means of the Mothur soft- mismatch in primer matching, and reads containing ambigu- ware.