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      Next generation sequencing for gut microbiome characterization in rainbow trout ( Oncorhynchus mykiss) fed animal by-product meals as an alternative to fishmeal protein sources

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          Abstract

          Animal by-product meals from the rendering industry could provide a sustainable and commercially viable alternative to fishmeal (FM) in aquaculture, as they are rich in most essential amino acids and contain important amounts of water-soluble proteins that improve feed digestibility and palatability. Among them, poultry by-product meal (PBM) have given encouraging results in rainbow trout ( Oncorhynchus mykiss). However, the introduction of new ingredients in the diet needs to be carefully evaluated since diet is one of the main factors affecting the gut microbiota, which is a complex community that contributes to host metabolism, nutrition, growth, and disease resistance. Accordingly, we investigated the effects of partial replacement of dietary FM with a mix of animal by-product meals and plant proteins on intestinal microbiota composition of rainbow trout in relation to growth and feeding efficiency parameters. We used 1540 trout with an initial mean body weight of 94.6 ± 14.2 g. Fish were fed for 12 weeks with 7 different feed formulations. The growth data showed that trout fed on diets rich in animal by-product meals grew as well as fish fed on control diet, which was rich in FM (37.3%) and PBM-free. High-throughput 16S rRNA gene amplicon sequencing (MiSeq platform, Illumina) was utilised to study the gut microbial community profile. After discarding Cyanobacteria (class Chloroplast) and mitochondria reads a total of 2,701,274 of reads taxonomically classified, corresponding to a mean of 96,474 ± 68,056 reads per sample, were obtained. Five thousand three hundred ninety-nine operational taxonomic units (OTUs) were identified, which predominantly mapped to the phyla of Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. The ratio between vegetable and animal proteins proved to play a central role in determining microbiome profiles and Firmicutes and Proteobacteria phyla were particularly discriminatory for diet type in trout. Plant ingredients favoured a higher Firmicutes: Proteobacteria ratio than animal proteins. Acceptable abundance of Firmicutes was guaranteed by including at least 25% of vegetable proteins in the diet regardless of animal protein source and percentage. In summary animal by-product meals, as replacements to FM, gave good results in terms of growth performances and did not induce significant changes in gut microbial richness, thus proving to be a suitable protein source for use in rainbow trout aqua feed.

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          Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing

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            Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies

            16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of ‘best available’ primer pairs for Bacteria and Archaea for three amplicon size classes (100–400, 400–1000, ≥1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.
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              Potential beneficial effects of butyrate in intestinal and extraintestinal diseases

              The multiple beneficial effects on human health of the short-chain fatty acid butyrate, synthesized from non-absorbed carbohydrate by colonic microbiota, are well documented. At the intestinal level, butyrate plays a regulatory role on the transepithelial fluid transport, ameliorates mucosal inflammation and oxidative status, reinforces the epithelial defense barrier, and modulates visceral sensitivity and intestinal motility. In addition, a growing number of studies have stressed the role of butyrate in the prevention and inhibition of colorectal cancer. At the extraintestinal level, butyrate exerts potentially useful effects on many conditions, including hemoglobinopathies, genetic metabolic diseases, hypercholesterolemia, insulin resistance, and ischemic stroke. The mechanisms of action of butyrate are different; many of these are related to its potent regulatory effects on gene expression. These data suggest a wide spectrum of positive effects exerted by butyrate, with a high potential for a therapeutic use in human medicine.
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                Author and article information

                Contributors
                Role: Data curationRole: Writing – original draft
                Role: ConceptualizationRole: Funding acquisitionRole: SupervisionRole: Writing – review & editing
                Role: Data curationRole: Formal analysis
                Role: Data curationRole: Formal analysisRole: SoftwareRole: Validation
                Role: MethodologyRole: ResourcesRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                6 March 2018
                2018
                : 13
                : 3
                : e0193652
                Affiliations
                [1 ] Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy
                [2 ] Inter-University Centre for Research in Protein Biotechnologies "The Protein Factory"- Polytechnic University of Milan and University of Insubria, Varese, Italy
                [3 ] Fondazione Parco Tecnologico Padano, Lodi, Italy
                [4 ] VRM S.r.l. Naturalleva, Cologna Veneta, Verona, Italy
                Universidade de Vigo, SPAIN
                Author notes

                Competing Interests: Naturalleva provided support by manufacturing the fish diets and conducting fish feeding trial. This does not alter our adherence to all PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0002-7532-7951
                Article
                PONE-D-17-35153
                10.1371/journal.pone.0193652
                5839548
                29509788
                f6cdb605-5504-457a-8de4-39bca9cb77a8
                © 2018 Rimoldi et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 28 September 2017
                : 15 February 2018
                Page count
                Figures: 4, Tables: 8, Pages: 29
                Funding
                We declare that the commercial company "Naturalleva" employs one of the authors, Fabio Brambilla, who receives a salary. We declare that Naturalleva provided support by manufacturing the fish diets and conducting fish feeding trial, but did not have any additional role in the study design, microbiota data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of Fabio Brambilla is articulated in the 'author contributions' section.
                Categories
                Research Article
                Biology and Life Sciences
                Nutrition
                Diet
                Medicine and Health Sciences
                Nutrition
                Diet
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Fish
                Osteichthyes
                Trout
                Biology and Life Sciences
                Anatomy
                Digestive System
                Gastrointestinal Tract
                Medicine and Health Sciences
                Anatomy
                Digestive System
                Gastrointestinal Tract
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbiome
                Biology and Life Sciences
                Genetics
                Genomics
                Microbial Genomics
                Microbiome
                Biology and Life Sciences
                Microbiology
                Microbial Genomics
                Microbiome
                Biology and Life Sciences
                Organisms
                Bacteria
                Gut Bacteria
                Biology and Life Sciences
                Zoology
                Fish Biology
                Fish Physiology
                Biology and Life Sciences
                Zoology
                Animal Physiology
                Vertebrate Physiology
                Fish Physiology
                Biology and Life Sciences
                Organisms
                Bacteria
                Biology and Life Sciences
                Biochemistry
                Lipids
                Fatty Acids
                Custom metadata
                All relevant data are within the paper.

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                Uncategorized

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