fmicb-09-00032 January 29, 2018 Time: 17:3 # 1 ORIGINAL RESEARCH published: 31 January 2018 doi: 10.3389/fmicb.2018.00032 The Dynamic Distribution of Small-Tail Han Sheep Microbiota across Different Intestinal Segments Hao Zhang1,2†, Mingxu Shao3†, He Huang4, Shujuan Wang1,2, Lili Ma1,2, Huining Wang1,2, Liping Hu5*, Kai Wei1,2* and Ruiliang Zhu1,2* 1 Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, Tai’an, China, 2 Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Shandong Agricultural University, Tai’an, China, 3 Lianyungang Entry-Exit Inspection and Quarantine Bureau, Lianyungang, China, 4 Shandong New Hope Liuhe Co., Ltd., New Hope Group, Qingdao, China, 5 Animal Disease Prevention and Control Center of Shandong Province, Animal Husbandry and Veterinary Bureau of Shandong Province, Jinan, China The sheep intestinal tract is characterized by a diverse microbial ecosystem that is vital Edited by: for the host to digest diet material. The importance of gut microbiota (GM) of animals George Tsiamis, has also been widely acknowledged because of its pivotal roles in the health and well- University of Patras, Greece being of animals. However, there are no relevant studies on GM of small-tail Han sheep, Reviewed by: Suzanne Lynn Ishaq, a superior mutton variety domestic in China. In this study, the structure and distribution University of Oregon, United States of gut microflora were studied by high-throughput sequencing technology. Results Dimitris Tsaltas, showed a significant difference between jejunum and cecum, jejunum, and rectum. Cyprus University of Technology, Cyprus Meanwhile, the cecum and rectum not only display higher species richness but also *Correspondence: exhibit higher similarity of the bacterial diversity than that of the jejunum based on the Ruiliang Zhu results of abundance-based coverage estimator (ACE), Chao1, and Shannon indexes. [email protected]; [email protected] Firmicutes and Bacteroidetes were the predominant phyla in cecum and rectum, while Kai Wei higher relative abundances of Firmicutes and Cyanobacteria were observed in jejunum. [email protected] At the genus level, Bacteroidetes, Ruminococcus, Lactobacillus, Flavonifractor, and Liping Hu [email protected] Clostridium were the dominant genera in the cecum and rectum. An obvious dynamic †These authors have contributed distribution of Lactobacillus is continuously decreasing from the jejunum to the cecum, equally to this work. then to the rectum, whereas the result of Bacteroides is completely inverse. In addition, this study also found many kinds of bacteria associated with the production of volatile Specialty section: This article was submitted to fatty acids (VFA) colonized in the large intestine. This study is the first to investigate the Systems Microbiology, distribution of intestinal flora in small-tail Han sheep. The findings provide an important a section of the journal Frontiers in Microbiology indication for diagnosis and treatment of intestinal diseases in small-tail Han sheep, as Received: 17 August 2017 well as offer a direction for the development of intestinal microecological preparations. Accepted: 09 January 2018 Keywords: gut microbiota, small-tail Han sheep, high-throughput sequencing, dynamic distribution, volatile fatty Published: 31 January 2018 acids Citation: Zhang H, Shao M, Huang H, Wang S, Ma L, Wang H, Hu L, INTRODUCTION Wei K and Zhu R (2018) The Dynamic Distribution of Small-Tail Han The gut microbiota (GM) is a diverse and complex community of microorganisms referred to as Sheep Microbiota across Different Intestinal Segments. the “forgotten organ.” Studies indicate that the gut microbiome is a signaling hub that integrates Front. Microbiol. 9:32. environmental inputs, such as diet, with genetic and immune signals affecting the host metabolism, doi: 10.3389/fmicb.2018.00032 immunity, and response to infection (Thaiss et al., 2016). For instance, the mammalian distal Frontiers in Microbiology| www.frontiersin.org 1 January 2018| Volume 9| Article 32 fmicb-09-00032 January 29, 2018 Time: 17:3 # 2 Zhang et al. Dynamic Distribution of Intestinal Microbiota intestine is a bioreactor containing anaerobic bacteria that are populations of microorganisms in these three intestinal segments, capable of degrading a variety of indigestible polysaccharides as well as the dynamic distribution of the related bacteria in the (Bäckhed et al., 2005). The GM is also known to provide other entire intestinal tract. beneficial functions for the host, including the recycling of bile salts, production of vitamin K, and production of exogenous alkaline phosphatase (Yolton and Savage, 1976; Gilliland MATERIALS AND METHODS and Speck, 1977; Ramotar et al., 1984). The gastrointestinal tract harbors a complex population of microbes that play a Ethics Statement fundamental role in the development of the immune system and The animal procedures were approved by the Animal Care and animal health (Gillor et al., 2008; Schuijt et al., 2013). Moreover, Use Committee of Shandong Agricultural University (Permit the intestinal microbial species restrain pathogens by producing number: 20010510) and performed in accordance with the antimicrobials (such as bacteriocins and some metabolites), “Guidelines for Experimental Animals” of the Ministry of Science competing for luminal nutrients and attachment sites, as well and Technology (Beijing, China). This study did not involve any as producing signaling molecules, which can modulate gene endangered or protected species. expression of other bacteria (Sturme et al., 2002). Recent studies have shown that the study of GM has become one of the most Animals and Sample Collection popular topics in the 21st century. Considering the multifaceted Three male 12-month-old healthy small-tail Han sheep were effects of GM, understanding animal GM for maintaining their obtained from a commercial feedlot (Shandong Province, China). health is important. The sheep’s diets were composed of 58.21% green hay, 16.77% The GM exhibits a large quantity and a complex composition corn, 12.53% alfalfa, 8.24% soybean meal, wheat bran 3.13%, known as the second genome of the body. In the past, 0.11% CaHPO4, 0.90% NaCl, and 0.11% premix. The animals traditional culture-dependent methods would be indispensable to were fed under the standard livestock management practices. understand the types of the GM. However, more than 50% of GM Then, the sheep were euthanized by intravenous injection of cannot be cultured out of the gut. Thus, scientists cannot make euthanasia solution, and the fresh samples (10 g) of different a description particularly about GM. To date, high-throughput intestinal segments, including jejunum (J1, J2, and J3), cecum DNA sequencing technology offers a convenient and fast tool (C1, C2, and C3), and rectum (R1, R2, and R3) were collected in describing the secret between the animal body and the GM simultaneously. The samples were transported to laboratory (Shendure and Lieberman, 2012; Marx, 2013). For instance, within 2 h in ice and stored at −80◦C. the study by Round and Mazmanian(2009) demonstrated that developmental aspects of the adaptive immune system are influenced by bacterial colonization of the gut. Some studies DNA Extraction, PCR Amplification and proved that the gut communities are influenced not only by Illumina MiSeq Sequencing geographically and culturally distinct settings but also by age Genomic DNA was extracted from each sample by using (Dominguez-Bello et al., 2011; Yatsunenko et al., 2012) with the TIANamp Genomic DNA Kit (TIANGEN Bio-Tek Co., Ltd., assistance of high-throughput sequencing technology. Moreover, Beijing, China) following the manufacturer’s instructions, and the in recent years, gut microbes and the relationship between extracts were purified by using DNA Purification Kit (Tiangen children’s growth and development is gradually becoming a DNA gel extraction kit, China). Subsequently, generation frontier research field. Blanton et al.(2016) have shown that sequencing library preparations and Illumina MiSeq sequencing Lactobacillus plantarum strains in the intestinal flora can were conducted at GENEWIZ, Inc. (Beijing, China). The quality maintain growth hormone activity through the signaling pathway of the DNA was checked by 1% (w/v) agarose gel electrophoresis of the liver, which can overcome the growth hormone resistance. and the concentration of DNA was measured with a UV–Vis Dominguez-Bello et al.(2016) demonstrated that “microbial spectrophotometer (Nano-Drop 2000, United States). 5–50 ng bath” can repair the intestinal flora of cesarean section newborn. DNA was used to generate amplicons using a MetaVxTM Undoubtedly, these studies, which are good for the development Library Preparation kit (GENEWIZ Inc., South Plainfield, NJ, of clinical medicine, are all inseparable from high-throughput United States). The different hypervariable regions of the 16S sequencing technology. rRNA (V3, V4, and V5) were amplified using the special Small-tail Han sheep, a local variety, is widely raised in China primers (V3 and V4 regions for forward primers containing because of its performance advantages, such as fattening ability, the sequence 50-CCTACGGRRBGCASCAGKVRVGAAT-30 and low fat rate, strong disease resistance, and genetic performance reverse primers 50-GGACTACNVGGGTWTCTAATCC-30, V4 stability. The preservation of these excellent properties is not and V5 regions for forward primers containing