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LC Supplementation Increases the Population of Prevotella in
Release date:2018-01-28 17:26    Author:admin    Click: 
Low-Molecular-Weight Chitosan Supplementation Increases the Population of Prevotella in the Cecal Contents of Weanling Pigs
 
Ting Yu 1, 2†, Yu Wang 1, 3†, Shicheng Chen 4†, Min Hu 5, Zhiling Wang 1, Guozhong Wu 6, Xianyong Ma 1, Zhuang Chen 2* and Chuntian Zheng 1*
 
1 Key Laboratory of Animal Nutrition and Feed Science (South China) of Ministry of Agriculture, State Key Laboratory of Livestock and Poultry Breeding, Guangdong Public Laboratory of Animal Breeding and Nutrition, Guangdong Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, China, 2 Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou, China, 3 Hebei Depond Animal Health Care Science and Technology Co., Ltd, Shijiazhuang, China, 4 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, United States, 5 Guangdong Key Laboratory of Integrated Agro-Environmental Pollution Control and Management, Guangdong Institute of Eco-Environmental Science & Technology, Guangzhou, China, 6 Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China
 

Low-molecular-weight chitosan (LC) promoted growth in weaned piglets as an alternative to feed-grade antibiotics. To investigate the influence of LC supplementation on piglets’ gut microbiome and compare the differences in community composition between LC and antibiotics with ZnO addition, we assessed the cecal microbial community by 16S rRNA gene sequencing with three treatments consisting of basal diet (CTR group), basal diet with low-molecular-weight chitosan (LC group), and basal diet with antibiotic and ZnO (AZ group). LC decreased pH more than AZ did in the cecum (both compared to CTR). Beta diversity analysis showed that community structure was distinctly different among the CTR, LC, and AZ treatments, indicating that either LC or AZ treatment modulated the piglet microbiota. Bacteroidetes, Firmicutes, and Proteobacteria dominated the community [>98% of operational taxonomic units (OTUs)] in piglet cecal contents. Compared to CTR, both LC, and AZ increased the relative abundance of Bacteroidetes while they decreased the count of Firmicutes and AZ decreased the population of Proteobacteria. In CTR the top four abundant genera were Prevotella (∼10.4%), Succinivibrio (∼6.2%), Lactobacillus (∼5.6%), and Anaerovibrio (5.4%). Both LC and AZ increased the relative abundance of Prevotella but decreased the ratio of Lactobacillus when they compared with CTR. Moreover, LC increased the relative abundance of Succinivibrio and Anaerovibrio while AZ decreased them. The microbial function prediction showed LC enriched more pathways in the metabolism of cofactors and vitamins than CTR or AZ did. LC may potentially function as an alternative to feed-grade antibiotics in weaned piglets due to its beneficial regulation of the intestinal microbiome.

Keywords: low-molecular-weight chitosan, weaned piglets, cecal microflora, microbial community, 16S rDNA sequencing

INTRODUCTION
The piglets at an early weaning stage face a dramatic life change in the food source, the immune system as well as the environmental and social status (Pluske et al., 1997; Lallès et al., 2007).These stressful events often cause digestive disorders, nutrient malabsorption and a high incidence of diarrhea in piglets (Madec et al., 1998; Boudry et al., 2004; Fairbrother et al., 2005). Antibiotics and zinc oxide (ZnO) have been widely supplemented into the piglet diets, which improve the growth rates and decrease the diarrhea rates (Barton, 2000; Zhu et al., 2017). However, the continuous addition of antibiotics and ZnO leads to negative consequences including the drug accumulation in livestock products, environmental contamination and bacterial antibiotic resistance (Barton, 2000; Vahjen et al., 2015).
  Therefore, alternative diet supplements have been investigated to replace antibiotics and ZnO (Turner et al., 2001). Among the alternative supplements, chitosan (∼1,000 kDa of molecular weight) and its derivatives low-molecular-weight chitosan (LC or LMWC, <150 kDa) or chito-oligosaccharide (COS, <5 kDa), can be obtained from chitin after the physical, chemical, and enzymatic conversions (Yin et al., 2009). They have been widely applied in chemical, medicinal, food, and agricultural industries such as food and feed additive (Yin et al., 2009; Vinsová and Vavríková, 2011). Due to the properties of nontoxic, biocompatibility and biodegradability as the few alkaline polysaccharides with positive charge (Yin et al., 2009; Vinsová and Vavríková, 2011), they have been reported to possess many beneficial biological properties (e.g., anti-microbial, anti-tumor, anti-oxidant, anti-diabetic, anti-obesity, cholesterol lowering, immunity regulation, and metal ion adsorption in animals; Yin et al., 2009; Vinsová and Vavríková, 2011).
  As the lowest molecular weight and the highest water-soluble, COS as a feed additive was more widely studied than chitosan and LC in animals (Jung et al., 2006; Han et al., 2007; Liu et al., 2008, 2010; Yang et al., 2012; Zhou et al., 2012; Kong et al., 2014; Xiao et al., 2014; Xiong et al., 2015). Several studies reported that COS (100∼1,000mg/kg) promoted animal growth, increased feed digestibility, reduced the incidence of diarrhea, anti-oxidative, enhanced immunity, and improved intestinal surface barrier function in weaned piglets (Jung et al., 2006; Han et al., 2007; Liu et al., 2008, 2010; Yang et al., 2012; Zhou et al., 2012; Kong et al., 2014; Xiao et al., 2014; Xiong et al., 2015). More importantly, COS protected against pathogenic infections (Escherichia coli and Staphylococcus aureus) and enhanced commensal bacteria members (lactobacilli and bifidobacteria) to maintaining the healthy gastrointestinal microflora in animals (Jung et al., 2006; Han et al., 2007; Liu et al., 2008; Yang et al., 2012; Kong et al., 2014). However, a newly reported that low dosage of COS supplementation at 30 mg/kg had no effects on promoting growth performance and even have compromised the intestinal barrier integrity (Xiong et al., 2015). The effects of LC as a feed additive on animals remain largely unknown. However, LC (∼12 kDa) had much potent antimicrobial activity than did COS, including against pathogens E. coli, S. aureus, Pseudomonas aeruginosa, Salmonella enterica serovar Typhi, and Bacillus cereus (Tsai et al., 2004). Moreover, compared to that in COS treatment, diet supplementation of LC increased more lipid metabolism and intestinal disaccharidase activities in obese rats induced by high-fat-diet (Chiu et al., 2017). However, the effects of LC on microbiome profiles in piglets remain unknown. Previous information on gut microbiota affected by the probiotic LC was fragmentary and the investigations were mostly limited in the culture-based method (Tsai et al., 2004; Jung et al., 2006; Han et al., 2007; Liu et al., 2008; Yang et al., 2012; Kong et al., 2014).
  Our preliminary data showed that LC (20–30 kDa) at a dosage of 50mg/kg improved the animal growth performance, intestinal tract health and anti-oxidant in weaned piglets (unpublished data). In this study, it was hypothesized that the diets containing LC influenced the piglet gut microbiome and might partly exhibit similar effects as in-feed antibiotics and ZnO. High-throughput sequencing of 16S rRNA gene was performed to investigate the microbial community structure variation of cecal bacteria in the weaned piglets with LC supplement and compared with that in antibiotics and ZnO supplement. The study helps to understand the effects of feed supplement on intestinal bacterial communities and facilitate studies of the alternative strategy for treating diarrhea in piglets. Given the similar gut bacterial communities between human beings and sows, our study here also contributes to understanding the effects of LC supplement on modulating the complexity of animal microbial communities and their functional properties in influencing health and disease.

MATERIALS AND METHODS
Animals and Sample Collection
All experimental procedures were carried out with the approval (IACUC-150701) of the Animal Care and Use Committee in Guangdong Academy of Agricultural Sciences, China. A total of 60 male weaned piglets (Duroc × Landrace × Large White) with an average weight of 6 kg and 21-day old were used in this study. The control (CTR) group was the piglets fed the basal diet (Supplementary Table S1); the antibiotics and ZnO (AZ) treatment group was fed the basal diet supplemented with aureomycin (30 mg/kg), polymyxin E (12 mg/kg) and ZnO (3,000 mg/kg); the LC group was those given the basal diet added with low-molecular-weight (20,000∼30,000 Da) chitosan (50 mg/kg), which was provided by Jiaxing Korui Biotech Co. Ltd, Zhejiang, China (http://www.korui-china.com/). The product of LC (KR901, Korui) is fabricated by physical methods and in the form of fine powder, water insoluble but soluble in dilute acidic solution, and recommended dosage 50 g/T for piglet’s feed. Five replicates were used for each treatment (4 piglets per replicate, totaling 20 animals). The feeding trials lasted for 28 days. The food and water were daily processed ad lib. At day 28, 1 randomly selected piglet in individual replicate for each treatment (total 5 animals/treatment) were slaughtered under anesthesia. The contents of cecum were collected and proceeded. The cecal pH was immediately determined.

DNA Extraction, Library Construction and 16S rDNA Sequencing
Microbial genomic DNA was extracted from 200 mg of the sample using the QIAamp DNA stool minikit (Qiagen, Germany) according to the manufacturer’s recommendation. DNA quality was evaluated by the agarose gel electrophoresis. The V4 hypervariable regions (the forward primer was 520F 5-AYTGGGYDTAAAGNG-3 and the reverse primer was 802R 5-TACNVGGGTATCTAATCC-3) of 16S rDNA were PCR amplified from microbial genomic DNA (Caporaso et al., 2011). Briefly, 2 µL of diluted DNA sample (∼20 ng/µL) was used for PCR amplification (25 µL mixtures). The PCR conditions were as follows: one pre-denaturation cycle at 98◦C for 2 min, 25 cycles of denaturation at 98◦C for 15 s, annealing at 50◦C for 30 s, and elongation at 72◦C for 30 s, and one post-elongation cycle at 72◦C for 5 min. The PCR amplicon products were purified using 2% agarose gels and used to construct the sequencing library. The libraries of amplicons were attached to Illumina sequencing adapters using the NEBNext UltraTM II DNA Library Prep Kit for Illumina (E7645L), purified using AMPure XP beads (Biomek, USA) and quality controlled on an Agilent 2100 Bioanalyzer (Agilent, USA). The pooled libraries were pair-end sequenced on the Illumina MiSeq platform with using 2 × 250 bp MiSeq reagent kit v2 (Illumina, USA).
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