Unconventional protein sources for aquafeeds: towards a circular economy approach

The growing aquaculture sector is linked to a continuous search for nutritionally balanced, and economically and environmentally sustainable ingredients in order to reduce the dependence on conventional marine and vegetal protein sources. In the last decades, several novel potential ingredients, namely insects, processed animal proteins and dried microalgae biomasses, have been tested in aquafeed formulations, achieving promising results. The aim of the present thesis was to investigate and analyse the effects of feeding rainbow trout (Oncorhynchus mykiss), gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) with diets containing either insect meal (Hermetia illucens, HI), poultry by-product meal (PBM), crustacean meal (Louisiana red claw crayfish meal, RCM), or dried microalgae biomasses (Tisochrysis lutea, Tetraselmis suecica sp. and Arthrospira platensis meals, DMB), singly or in combination, in replacement of conventional marine and vegetal proteins. Five trials were carried out. The first trial was conducted on gilthead sea bream to evaluate the effect of replacing fishmeal (FM) with HI larva meal on lipid composition, specifically on the fatty acid profiles of the total lipids, of the triglyceride (TG) fraction, and of TGs in the sn-2 position. In addition, the influence of the dietary treatment on the marketable indexes and on the physical characteristics of the fillets were evaluated. Results revealed that the diet including HI produced minimal and not commercially relevant variations in the fillet quality characteristics. In addition, the HI inclusion in the diet of gilthead sea bream did not substantially modify the presence of important fatty acids, such as EPA and DHA, in the sn-2 position of the fillet TGs. The second trial aimed to compare growth, gene expression involved in appetite regulation, physical characteristics and chemical composition of gilthead sea bream fed alternative protein sources. Specifically, ten isoproteic, isolipidic, and isoenergetic diets were formulated. The experimental diets were prepared by replacing graded levels of vegetal proteins from a vegetal-based control feed (CV) with proteins from: a commercial defatted HI pupae meal, PBM singly, at different percentages of inclusion (H10, H20, H40, P20, P40), or in combination (H10P30); red swamp crayfish meal (RCM10); a blend (2:1, w:w) of Tisochrysis lutea and Tetraselmis suecica dried microalgae biomasses (MA10). A marine ingredient-rich diet was also set as control (CF). The increase of feed intake, feed convertion ratio and ghre gene expression was observed in fish fed diet MA10. Overall, no detrimental effects of H, PBM, and RCM were observed in fish fatty acid profile, resulting to be comparable to CV. Thus, this trial demonstrated the possibility to introduce H, PBM and RCM as partial replacement of vegetal proteins in the diet for S. aurata. In the third trial, performed on rainbow trout, four experimental diets were formulated starting from a basal diet rich in vegetal protein sources (CV), and then replacing 10% of the dietary crude protein with either RCM or dried microbial biomass from Tetraselmis suecica (TS) or Artrhospira platensis (AP). A diet with 0.25% of conventional feed additives (nucleotides and sodium butyrate, CVplus) was also formulated in order to compare the effect of the new ingredients as functional supplements. By means of a multidisciplinary approach, fish responses to the different dietary formulations were evaluated in terms of growth performance, gut welfare and immune response. The results obtained showed that all the experimental diets globally improved fish responses compared to the CV diet. CVplus and AP fish growth was not impaired, but the gut health status was highly compromised. Differently, the TS and RCM diets led to a slight worsening of zootechnical parameters compared to the CV diet, but were able to improve the overall welfare and to preserve the structural integrity of the distal intestine. The fourth trial compared the nutrient-energy retention, digestive function, growth performance and welfare of rainbow trout fed isoproteic (42%), isolipidic (24%), fishmeal-free diets (CV). In the experimental diets, graded levels (10, 30, 60%) of protein from PBM and HI pupae meal, either singly or in combination, replaced the plant protein contained in the control diet. A fishmeal-based diet was also tested (CF) as reference diet. Nitrogen retention improved with moderate or high levels of dietary PBM and HI relative to CV. The enzymatic activity in the gut brush border was negligibly affected by the diets. Gastric chitinase was up-regulated after the ingestion of HI60 feed. The peptide and amino acid transport genes of the gut were differently regulated by protein source and level in diets. Serum cortisol was unaffected and the metabolic changes stayed within the physiological range. High PBM and high HI inclusion in diet formulations lowered the leukocyte respiratory burst activity and increased the lysozyme activity. In conclusion, moderate to high PBM and HI inclusions in fishmeal-free diets, either singly or in combination, improved gut function and nutrient retention, resulting in better growth performances and good welfare of rainbow trout. Finally, in the fifth trial, performed under commercial conditions (farm Ittica Caldoli, Foggia, Italy), European seabass were fed two isoproteic (45%) and grossly isolipidic (20%) diets for 63 days. Control group (CG) fish were fed a commercial diet and the fish of the other group were fed a diet (SSH) containing 10% HI, 30% PBM, and a low quantity of marine proteins. The response of the fish to the diets was evaluated by assessing the hepatic enzymatic activity, and the physical and nutritional traits. Results obtained showed that the SSH diet did not impair the marketable and physical characteristics of fish. Higher glucose 6-phosphate dehydrogenase, aspartate aminotransferase and 3-hydroxyacyl CoA dehydrogenase activities activities were also registered in the SSH group than in the CG. Fillet fatty acid profile was also unaffected by diet, except for C18:4n-3 and C12:1n-11 contents, that were lower in the SSH diet. Overall, regarding the nutritional quality of fillets, the sum of the eicosapentaenoic and docosahexaenoic acid contents in fillets from the SSH group matched the daily intake ranges recommended by the World Health Organization. In conclusion, the SSH diet, which in previous experimental trials showed promising results, demonstrated to be a suitable alternative to a common commercial feed for E. seabass feeding even when administrated in a commercial farm.