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Dietary Energy Level Affects the Composition of Cecal Microbiota of Starter Pekin Ducklings

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Dietary Energy Level Affects the Composition of Cecal Microbiota of Starter Pekin Ducklings Original Paper Czech J. Anim. Sci., 63, 2018 (1): 24–31 doi: 10.17221/53/2017-CJAS Dietary Energy Level Affects the Composition of Cecal Microbiota of Starter Pekin Ducklings Jun-Qiang Liu1, Yan-Hong Wang1, Xing-Tang Fang1, Ming Xie3, Yun-Sheng Zhang3, Shui-Sheng Hou3, Hong Chen1, Guo-Hong Chen2, Chun-Lei Zhang1,2* 1Institute of Cellular and Molecular Biology, School of Life Sciences, Jiangsu Normal University, Xuzhou, P.R. China 2College of Animal Science and Technology, Yangzhou University, Yangzhou, P.R. China 3Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, P.R. China *Corresponding author: [email protected] ABSTRACT Liu J.-Q., Wang Y.-H., Fang X.-T., Xie M., Zhang Y.-S., Hou S.-S., Chen H., Chen G.-H., Zhang C.-L. (2018): Dietary energy level affects the composition of cecal microbiota of starter Pekin ducklings. Czech J. Anim. Sci., 63, 24–31. In this study, we evaluated the phylogenetic diversity of the cecal microbiota of 3-week-old ducklings fed three diets differing in metabolizable energy. The contents of the ceca were collected from ducklings of different groups. The ceca bacterial DNA was isolated and the V3 to V4 regions of 16S rRNA genes were amplified. The amplicons were subjected to high-throughput sequencing to analyze the bacterial diversity of different groups. The predominant bacterial phyla were Bacteroidetes (~65.67%), Firmicutes (~17.46%), and Proteobacteria (~10.73%). The abundance of Bacteroidetes increased and that of Firmicutes decreased with increasing dietary energy level. The diversity decreased (Simpson diversity index and Shannon diversity index) with the increase in dietary energy level, but the richness remained constant. Notably, Brachyspira bacteria were detected with a very high relative abundance (4.91%) in ceca of ducks fed a diet with 11.30 MJ/kg metabolizable energy, sug- gesting that low energy content may affect their colonization in cecum. Keywords: duck; bacterial diversity; dietary energy Ducks have a global population exceeding 1 bil- 2012). In addition to digestion, microflora in the lion individuals ranking second only to chickens gastrointestinal tract of poultry influences health according to annual poultry production in 2014 and wellbeing (Stanley et al. 2012). according to FAO’s corporate database. Like other In the past few years, the gastrointestinal mi- birds, ducks have proportionally smaller intestines crobiomes of economically important birds such and shorter transit times than mammals, but do as chicken (Callaway et al. 2009; Danzeisen et al. not appear to be less efficient at digestion than 2011; Singh et al. 2013), duck (Swayne and McLaren their mammalian counterparts (McWhorter et 1997), turkey (Scupham et al. 2008), goose (Lu et al. 2009), which rely on microbes in their gas- al. 2009), ostrich (Matsui et al. 2010) have been trointestinal tract to accomplish this (Flint et al. revealed by high-throughput next-generation se- Supported by the China Postdoctoral Science Foundation (2013M541739), and the MaTS-Waterfowl System (CARS-43-1). 24 Czech J. Anim. Sci., 63, 2018 (1): 24–31 Original Paper doi: 10.17221/53/2017-CJAS quencing technologies. These studies indicate that Different groups were given feed with a different avian intestinal microbiomes are more complex ME content (Table 1). Nine ducks were randomly and diverse than previously thought, and greatly selected from each group and sacrificed on day 21. expand the perspective on the avian intestinal Ducks were fasted for 16 h before the sacrifice. microbiome in terms of species composition, di- Ceca were immediately collected and kept on ice. versity, and community structure. The contents of the ceca were collected by gently A recent report revealed that diet plays a more squeezing the organ and were snap frozen in liquid important role than host genotype in shaping microbial ecology (Carmody et al. 2015). Diet Table 1. Ingredients and chemical composition of the 1 energy level variation induces rapid changes in the experimental diets bacterial composition of the human and mouse gut Treatment Diet microbiota, and changes the energy gain from the group A group B group C gastrointestinal tract (Daniel et al. 2014). Dietary Ingredients2 changes can increase performance of chickens Corn 54.21 59.41 56.41 by optimizing the composition of bacterial spe- cies within the microbiota of the distal intestine Corn DDGS 15.00 15.90 14.80 (Bedford and Cowieson 2012). However, the ef- Soybean meal 3.00 3.00 3.00 fect of dietary energy level on poultry intestinal Peanut meal 18.50 16.9 18 microbiota remains unclear. Rice hulls 4.50 0 0 Compared with chicken, the duck gastrointestinal Soybean oil 0 0 3.00 microbiome is largely undiscovered. Two reports Dicalcium phosphate 1.40 1.40 1.40 from a French laboratory revealed that Firmicutes Limestone 1.10 1.10 1.10 and Bacteroidetes are the dominant phyla in the Salt 0.30 0.30 0.30 bacterial community of ducks at 12–14 weeks, and Premix3 1.00 1.00 1.00 the richness and diversity is significantly affected dl-Methionine 0.16 0.16 0.16 by overfeeding and dietary probiotic (Vasai et al. l-Threonine 0.18 0.18 0.18 2014). Early life events are critical for the dynamics of microbiota development (Dore et al. 2015). As l-Tryptophan 0.046 0.048 0.047 4 meat is the major product of ducks slaughtered Nutrients at 4–6 weeks, discovering the diversity of intes- ME (MJ/kg) 11.30 11.93 12.58 tinal microbiota in young ducks is important for Crude protein (%) 19.13 19.15 19.15 implementing a strategy for improving gut health dl-Methionine (%) 0.45 0.45 0.45 and performance. l-Lysine (%) 0.60 0.60 0.60 In this study we characterized the phylogenetic l-Tryptophan (%) 0.22 0.22 0.22 diversity of the cecal microbiota of ducks at 3 weeks, l-Threonine (%) 0.75 0.75 0.75 and investigated the effects of dietary metaboliz- Calcium (%) 0.84 0.84 0.84 able energy (ME) on cecal microbiota using high- Non-phytic acid 0.38 0.38 0.38 throughput sequencing after amplification of the phosphorus (%) V3–V4 region of the bacterial 16S rRNA gene. DDGS = distillers dried grains with solubles, ME = metabo- lizable energy MATERIAL AND METHODS 1inclusions of feed ingredients are in kg 2supplied per 100 kg diet as-fed 3 Experimental animals and diets. Thirty healthy provided per kg of diet: vitamin B2 4.5 mg, niacinamide newly-hatched male Pekin ducklings with similar 40 mg, d-pantothenic acid 9 mg, choline chloride 500 mg, weight were divided into three groups. In each vitamin B12 20 μg, vitamin E (dl-α-tocopheryl acetate) 30 mg, group, 10 birds were randomly assigned to one vitamin K 2.3 mg, vitamin A 12 500 IU, vitamin D3 5000 IU, cage (3 × 3 m), and raised on wire floors that biotin 0.1 mg, folic acid 0.5 mg, FeSO4·H2O 147 mg, MnO2 contained a self-feeder and water source in an 100 mg, CuSO4·5H2O 40 mg, ZnSO4·H2O 143 mg, Na2SeO3 air-controlled house. One 100-W light bulb per 0.5 mg, KI 2 mg, ethoxyquin 125 mg cage was provided for ducklings up to 10 days. 4ajusted to 87.3% dry matter 25 Original Paper Czech J. Anim. Sci., 63, 2018 (1): 24–31 doi: 10.17221/53/2017-CJAS nitrogen. All samples were stored at –80°C. All Illumina reads were analyzed using the mothur experimental procedures were approved by the software package, Version 1.34.4 (Schloss et al. Ethics Committee for Experimental Procedures 2009). Chimeric sequences were removed using on Animals of the Jiangsu Normal University. the USEARCH software based on the UCHIME DNA extraction. Genomic DNA was extracted algorithm (Dore and Blottiere 2015). Sequences using the QIAamp DNA Stool Mini Kit (Qiagen, were further analyzed using the open source soft- Germany) by following the manufacturer’s instruc- ware package QIIME, Version 1.9.1 (Caporaso tions, with some modifications. Mucosal con- et al. 2010) and the Ribosomal Database Project tents were first treated with 25 mg/ml of lysozyme (East Lansing, USA) (Cole et al. 2003). Operational (Vivantis Technologies, Malaysia) in lysis buffer Taxonomic Units (OTUs) were picked using de (20 mM Tris-Cl, pH 8.0; 2 mM EDTA, pH 8.0; 1% novo OTU picking protocol with a 97% similar- Triton X-100) for 30 min at 37°C. Samples were ity threshold. The alpha and beta diversities were then treated with DNase-free RNase (Epicentre, analyzed in QIIME Version 1.9.1 software as per USA). DNA concentration and quality were deter- QIIME defaults for that version (Caporaso et al. mined using a NanoDrop 2000 spectrophotometer 2010). The detailed protocol was referred to by (Thermo Fisher Scientific, USA) and gel electro- Sergeant et al. (2014). phoresis, respectively. Samples were then stored Statistical analysis. Data were analyzed using at –80°C until required. the GLM procedure of SPSS software for MS Win- High-throughput sequencing and analysis dows by one-way ANOVA. Differences between of 16S rRNA gene amplicons. PCR amplifica- groups were considered significant when P-values tions were carried out using 50 μl of reaction were < 0.05. ® mixtures which contained 25 μl NEBNext High- Fidelity 2X PCR Master Mix (New England Bio- labs, USA) (containing 2.0 mM MgCl2), 25 μM RESULTS primer, and 50 ng DNA template. The PCR re- action included an initial denaturation step at Characterization of the duck cecal microbiota. 98°C for 30 s followed by 25 cycles of 98°C for After stringent quality trimming on raw data, 10 s, 65°C for 30 s, 72°C for 30 s, and final exten- 549 992 sequences (20 370 ± 5363 sequences/ sion step at 72°C for 5 min in S1000 Thermal sample), with an average read length of 450 bp, Cycler (Bio-Rad Laboratories, USA).
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