Introduction
The readily available and cost-effective plant protein-rich ingredients have become the main protein sources used in aquafeeds to substitute unsustainable marine-derived ones. However, the transition to plant-based diets is more difficult to achieve for carnivorous fish, like salmonids, in which certain plant protein-rich ingredients can cause adverse effects on growth, liver integrity and gut health. In this regard, functional feeds represent an interesting solution to mitigate these drawbacks, even at low dietary inclusion. Conventional feed additives, like nucleotides and sodium butyrate, have been shown to positively modulate immune response by preserving intestinal integrity in salmonids. Furthermore, a new generation of more sustainable ingredients can be intended as feed supplements due to their promising features. The dried microbial biomass of certain microalgae and cyanobacteria represents a source of vitamins, minerals, carotenoids and antioxidant compounds. Differently, the meal obtained by the Louisiana red swamp crayfish (Procambarus clarkii), a highly invasive alien species in Europe, can be exploited as an innovative feed supplement converting an ecological problem to a natural source of protein and bioactive compounds such as astaxanthin and chitin. The present study was aimed at exploring the nutraceutical effects of low dietary inclusions of conventional feed additives (nucleotides and sodium butyrate) or innovative potential feed supplements such as Louisiana red swamp crayfish meal (RCM) and dried microbial biomass from intact cells of Tetraselmis suecica (TS) and Artrhospira platensis (spirulina; AP) in fish meal-free diets during a 104 day feeding trial performed on rainbow trout (Oncorhynchus mykiss).
Materials and Methods
Four grossly isoproteic (42%), isolipidic (24%) and isoenergetic (23 MJ/kg) test diets were formulated starting from a basal one, named CV, characterized by a 10:90 ratio between marine (hydrolysed fish proteins used as feed stimulant) and vegetable protein sources. For conventional feed additives, a diet coined CVplus was prepared by adding sodium butyrate and nucleotides (0.2 and 0.05%, respectively) to CV. The test ingredients (RCM, TS and AP) were used to replace 10% of dietary crude protein supplied by the major plant protein-rich ingredients of the basal diet CV. A total of 750 rainbow trout (79.7 ± 13.5 g) were randomly distributed into 15 squared fiberglass tanks (1.6 m3; 3 tanks per dietary treatment). At the end of the trial, zootechnical parameters were measured and samples of blood, liver and distal intestine were carefully collected and properly stored for the following analyses. (i) Histology: a series of histo-pathological indexes were evaluated to assess the gut health status, with emphasis on mucosal fold height and inflammatory influx, and the degree of fat accumulation in hepatic parenchyma. (ii) Fourier Transform Infrared Imaging (FTIRI): to evaluate the biochemical composition of liver tissue, false colour images were created resulting from integration process of IR maps in the spectral regions of lipid, protein, and glycogen components. Spectra were extracted and band area ratios were calculated and statistically analysed. (iii) Real-time PCR: relative quantification of the expression of genes involved in immune response (tlr1, nfkb, il1b, il10, and tnfa) was performed on distal intestine samples. (iv) Blood chemistry: serum aliquots were stored in dry ice, then metabolites, transaminases and minerals were analysed by spectrophotometric assays. (v) Metataxonomic analyses on intestinal microbiota: faecal content was collected, frozen in liquid nitrogen and then analysed by Illumina Miseq sequencing of V3-V4 region of 16S rRNA gene, using Qiime2 for data analysis.
Results and Discussion
The provision of a diet rich in vegetal protein sources (CV) resulted in a severe impairment of distal intestine morphological and histopathological indexes, without affecting growth performances in relation to the best diets here compared. The CVplus diet did not result in a significant improvement of zootechnical parameters and showed only a marginal ameliorative effect on distal intestine health status that remained highly inflamed. Both AP and TS diets reduced microbiota diversity compared to the other groups but showed opposite results in terms of growth and gut. Despite no significant differences with CV and CVplus diets, fish fed AP diet showed improved zootechnical performances respect to those fed both RCM and TS dietary treatments, possibly due to an enhanced nutrient uptake and a higher digestible-nutrient intake as respectively evidenced by: (i) a significantly higher supranuclear vacuolization of distal intestine enterocyte; (ii) a higher nutrient digestibility of AP compared to that of TS, characterized by thick and cellulosic cell wall. Accordingly, fish fed TS diet were characterized by depressed growth performances compared to fish given CV, CVplus and AP diets and by a stronger selective pressure on microbiota, probably favouring those bacteria with specific cellulolytic activities. However, considering the distal intestine health status, providing TS diet resulted in a well-structured and more developed intestinal absorptive epithelium and led to a significant reduction of inflammatory influx respect to both CV and AP diets. In fact, the AP dietary inclusion was not able to fully contrast the negative side-effects of the vegetable ingredients on gut health, leading to a high degree of basal inflammatory influx. Finally, providing 10% dietary crude protein in the form of RCM did not affect zootechnical parameters compared to those observed for fish given diet CV, despite the high biogenic amines content could have led to a reduction in feed consumption and consequently in growth compared to CVplus and AP groups. On the other hand, RCM showed promising results due to its ameliorative effects on gut histological conditions, since it is a source of carotenoids (mainly astaxanthin) with a potential beneficial role on distal intestine. In addition, gut of fish fed RCM diet harboured high percentages of the Proteobacteria genus Deefgea (12%), a specific bacterial genus that provides a strong chitinolytic activity. Considering liver, both histological and spectroscopic analyses detected a low hepatic lipid accumulation in fish from all the dietary treatments. Based on blood chemistry analyses, the metabolic status, in terms of total proteins and lipids, were similar in all dietary groups, whereas a significant increasing of minerals content was observed in fish fed RCM, TS and AP diets.
As a conclusion, results from the present study demonstrated that both conventional feed additives and all the novel feed supplements investigated were able to ameliorate responses of rainbow trout when added to a vegetable-based diet.
This work was supported by SUSHIN (SUstainable fiSH feeds INnovative ingredients) project funded by Ager (AGER2-SUSHIN Cod 2016–0112)