In the quest to make aquaculture a more sustainable and cost-effective animal production industry, fishmeal was gradually replaced by alternative, often vegetable meals. Aquafeeds have nowadays very low percentages of marine raw materials inclusion and instead, soybean meal, insect and poultry meals make up for the majority of protein sources. These feed formulations, thoroughly studied and balanced so that growth performance and feed efficiency are not jeopardized, have nevertheless inherent properties that compromise fish physiology at certain extents. A well-known bottleneck of vegetable ingredient is their content in antinutrients (saponins, phytosterols, etc) to which fish intestinal barrier mounts an inflammatory response. Rainbow trout, Oncorhynchus mykiss, is particularly susceptible to these compounds, and when faced with these alternative diets for the first time, develops a plant-protein-induced enteritis. In the meantime before fish eventually adapt to the presence of these antinutrients, inflammation means an extra energy expense (deviated from growth and other meaningful physiological processes), compromised intestinal epithelial structure and nutrients uptake. This project, TRYPinGUT, means to develop a nutritional strategy that does not affect the backbone of these feed formulations and is able to inhibit the side effects of an exacerbated inflammatory response. Based on the hypothesis that TRP dietary supplementation will directly (via metabolism in gut immune cells) and/or indirectly (via its breakdown pathway in gut serotonin-producing cells) modulate the gut immune response to these antigens, TRYPinGUT aims at i) characterizing rainbow trout plant-based diet-induce enteritis; ii) determining whether enteritis itself, or enteritis-induced changes in gut serotonergic activity triggers a central response; iii) assessing the modulatory effect of TRP supplementation in enteritis and in the brain-gut axis and iv) unveiling metabolic pathways through which TRP mediates its effects. To achieve these major goals, a plan of methods subdivided in four tasks is presented in this proposal. Workpackage 2 (WP2) is related to preliminary work needed to carry out analytical processes in WP3. It aims at producing polyclonal antibodies against two TRP enzymes and against a T-cell co-receptor, to be used in gut samples immunohistochemistry. A feeding trial with juvenile rainbow trout will be conducted in WP3, where the effects of TRP-supplemented plant-based diets (different supplementation doses) will be evaluated in terms of systemic and gut immune response, and the brain-gut axis in what the serotonergic activity and the neuroendocrine response are concerned. The most promising results will then be tested in another in vivo trial (WP4) where the same treatment (dose and feeding time) shall be compared to a pulse feeding regime, in an attempt to reduce feed expenses.
Analytical work will comprise molecular biology techniques (qPCR, cloning, recombinant protein and antibody production), humoral and gut immune parameters and the haematological profile, gut immunohistochemistry to detect immune and enterochromaffin cells and to study TRP metabolic fate, HPLC to evaluate brain serotonergic activity, and shotgun proteomics.
TRYPinGUT is meant to be an integrative innovative approach, as it assesses the modulatory effect of a dietary ingredient not only on its target tissue (gut) but also in associated organs/physiological processes (brain and the HPI-axis; systemic immune response), evaluating fish response as a whole.
The innovative character and strength of this project sits on an appropriate fish model that has been extensively used in the study of the HPI-axis function and that, especially during its juvenile phase, is very susceptible to vegetable ingredients; a new inflammation setting that enables the evaluation of local and direct effects (enteritis, where the inflamed tissue is the first organ being exposed to TRP supplement and has its own immune equipment); a proposal that not only tests different administration doses but also optimizes feeding regimes; and an holistic high throughput approach, where both brain and gut proteomic responses to enteritis and TRP supplementation are studied and compared.
With this proposal, it is expected to optimize a TRP dietary supplementation as an immune-nutritional strategy that is cost-effective and efficient in overcoming diet-induced enteritis in rainbow trout. By taking into account other welfare-related physiological responses, this study is also expected to better characterize gut inflammation and to gather new data on other possible enteritis side-effects.