University of Plymouth PEARL https://pearl.plymouth.ac.uk 04 University of Plymouth Research Theses 01 Research Theses Main Collection 2017 The impacts of wheat gluten products and short-chain fructooligosaccharides on the health and production of juvenile rainbow trout (Oncorhynchus mykiss) Voller, Samuel W. http://hdl.handle.net/10026.1/9826 University of Plymouth All content in PEARL is protected by copyright law. Author manuscripts are made available in accordance with publisher policies. Please cite only the published version using the details provided on the item record or document. In the absence of an open licence (e.g. Creative Commons), permissions for further reuse of content should be sought from the publisher or author. The impacts of wheat gluten products and short-chain fructooligosaccharides on the health and production of juvenile rainbow trout (Oncorhynchus mykiss) by Samuel W. Voller A thesis submitted to Plymouth University in partial fulfilment for the degree of Doctor of Philosophy (September 2016) This work was supported by Tereos Syral, Marckolsheim, France and Plymouth University. I Copyright statement This thesis copy has been supplied on the condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the author’s prior consent. II | P a g e Abstract: The impacts of wheat gluten products and short-chain fructooligosaccharides on the health and production of juvenile rainbow trout (Oncorhynchus mykiss) Samuel W. Voller Through the implementation of in vivo feeding trials, the efficacy of three wheat gluten (WG) products, vital (Amytex®), hydrolysed (Merripro®) and soluble hydrolysed (Solpro®) wheat gluten as replacement of soy protein concentrate, and scFOS prebiotic (Profeed®) supplementation were analysed to assess their impacts on intestinal health and production of juvenile rainbow trout. Microbial community analysis in experiment one revealed a degree of diet based modulation with 7.5% and 15% inclusions of wheat gluten (WG) products. Bacterial species diversity was significantly reduced with 15% hydrolysed wheat gluten (HWG) inclusion compared to the plant protein control and 15% vital wheat gluten (VWG) treatments, with sequenced OTUs dominated by the phylum Firmicutes and possible promotion of probiotic species. No detrimental effects were observed on intestinal morphology. These findings led onto a longer duration feed trial with a more holistic, higher resolution approach. Experiment two revealed modulation of the allochthonous intestinal microbiota, with increased proportions of Enterococcus and Weissella in the 10% and 20% VWG treatments. Bacillus and Leuconostoc relative abundances were significantly increased with 10% HWG and soluble hydrolysed (Sol) wheat gluten inclusions. HSP 70 transcripts were significantly down-regulated in all WG treatments compared to the basal soy protein concentrate treatment (SPC) and increased intraepithelial leukocyte counts were observed with 10% VWG inclusion. Growth performance was unaffected by 10% dietary inclusions of WG, however, FCR’s were significantly improved in the 20% VWG treatment compared to the 10% HWG and Soluble treatments. This led to the investigation of increased inclusion levels of WG products in experiment three. All WG treatments in experiment three yielded significantly improved growth performance. Somatic indices were significantly increased with 30% blended WG inclusion compared to the SPC treatment. Modulation of allochthonous intestinal microbiota was observed to a lower degree than the previous experiments, with a dose response observed with increasing blended WG inclusion. In the final experiment two basal diets (SPC and 20% Blended) and two scFOS supplemented diets (SPC + FOS and 20% Blended + FOS) were investigated for the effect on growth performance, gut health and allochthonous microbial population. Growth performance was unaffected, however, modulation of the allochthonous microbial population was observed with an apparent synergistic effect of scFOS supplementation in WG diets. This synergistic trend was also observed in the transcription level expression of immune relevant genes. 20% WG inclusion with additional scFOS supplementation observed significant down regulation of the pro-inflammatory cytokine TNF-α, as well as HSP 70, CASP 3 and Glute ST compared to the 20% Blend treatment. The present research demonstrates dietary inclusions of WG products, solely or blended, at the expense of soy protein concentrate to modulate the allochthonous microbial population, potentially promoting probiotic species, whilst reducing the levels of intestinal stress in juvenile rainbow trout. Supplementation of the prebiotic scFOS modulated the microbial populations, enhancing the proportion of potential probiotic species, and combined with WG inclusions, reduce intestinal and oxidative stress and inflammation biomarkers, with no observed deleterious effects. III | P a g e Home Office statement All experimental work involving animals complied with the 1986 Animals Scientific Procedures Act, operating under Home Office project license PPL 30/2644 and personal license PIL 30/10401. All experimental work involving animals further complied with the Plymouth University Animal Welfare and Ethical Review Committee. IV | P a g e Contents Copyright statement ................................................................... Error! Bookmark not defined. Abstract ....................................................................................... Error! Bookmark not defined. Home Office statement ............................................................. IError! Bookmark not defined. Contents .....................................................................................................................................V List of Tables ............................................................................... Error! Bookmark not defined. List of figures ............................................................................... Error! Bookmark not defined. Acknowledgements ..................................................................... Error! Bookmark not defined. Author’s declaration ................................................................... Error! Bookmark not defined. Presented Work .......................................................................... Error! Bookmark not defined. Abbreviations………………………………………………………………………………………………………………....XVIII CHAPTER 1. Introduction ........................................................... Error! Bookmark not defined. 1.1. Aquaculture; an overview ................................................ Error! Bookmark not defined. 1.1.1. Salmonids ................................................................... Error! Bookmark not defined. 1.1.2. Sustainability and aquafeed ...................................... Error! Bookmark not defined. 1.2. Alternative plant protein sources ................................... Error! Bookmark not defined. 1.2.1. Plant protein sources ................................................. Error! Bookmark not defined. 1.3. Wheat gluten ................................................................... Error! Bookmark not defined. 1.3.1.Amino acid profile ....................................................... Error! Bookmark not defined. 1.3.2. Growth performance ................................................. Error! Bookmark not defined. 1.3.3.Health ......................................................................... Error! Bookmark not defined. 1.3.4. Lipid metabolism........................................................ Error! Bookmark not defined. 1.4. Intestinal microbiota of fish ............................................ Error! Bookmark not defined. 1.4.1. Indigenous intestinal microbiota of rainbow trout. .. Error! Bookmark not defined. 1.4.2. Intestinal microbiota and nutrition ........................... Error! Bookmark not defined. 1.4.3. Intestinal microbiota and immunity .......................... Error! Bookmark not defined. 1.5.Prebiotics .......................................................................... Error! Bookmark not defined. 1.6. scFOS ................................................................................ Error! Bookmark not defined. 1.6.1. Growth performance ................................................. Error! Bookmark not defined. V | P a g e 1.6.3. Health and immunology ............................................ Error! Bookmark not defined. 1.7.Conclusions ....................................................................... Error! Bookmark not defined. 1.8 Thesis objectives and aims ............................................... Error! Bookmark not defined. CHAPTER 2. General methodologies ....................................... Error! Bookmark not defined.2 2.1. Overview ........................................................................ Error! Bookmark not defined.2 2.2. Experimental animals and housing ............................... Error! Bookmark not defined.2 2.3. Experimental diets and formulation ............................. Error! Bookmark not defined.4 2.4. Experimental feeding .................................................... Error! Bookmark not defined.5 2.5. Growth performance and feed utilisation ...................... Error! Bookmark not defined. 2.6. Sampling protocol and fish dissection ...........................