Introduction
Aquaculture represents the fastest-growing sector of animal production. However, its continued growth and long-term viability depends on developing innovative and sustainable aquafeed formulations. Chitin has recently emerged as a promising additive in aquafeeds, due to its potential to improve growth, intestinal health, and immunity of aquatic organisms. In fish, chitin can be degraded by endogenous enzymes (chitinases). However its metabolic response is dose- and particle size-dependent. At appropriate levels, chitin exhibits immunostimulatory properties and improve gut health. Reported effects include increased goblet cell density, enhanced mucus production, and strengthened intestinal barrier. At the molecular level, it modulates the expression of immune-related genes, including pro- and anti-inflammatory cytokines such as il1β, tnfα, and il10.
Traditionally derived from shrimp and yeast, chitin can also be obtained from insects, which are considered a promising protein source for animal nutrition. Notably, insect farming generates by-products such as pupal exuviae and the exoskeletons of deceased adults, which are rich in chitin. This presents an opportunity to develop value-added feed ingredients. Rainbow trout (Oncorhynchus mykiss) is the most extensively farmed freshwater species in Europe, and efforts to improve its sustainable production remain a central research focus.
Within this framework, the ACInA project integrates aquaculture, insect farming, and agriculture into a circular-economy model. The strategy aims to improve sustainability by valorizing waste and enhancing overall resource efficiency. Insects,reared on substrates derived from agri-food by-products, enable their conversion into high-value biomass. Insects' derivatives are used to formulate functional aquafeeds, while aquaculture waste are converted into fertilizers for agricultural use.
In this context, the present study aimed to extract chitin from insects and incorporate it as functional additive into diets for Rainbow trout, to evaluate its effects on fish growth performance and overall physiological responses.
Materials and Methods
Exuviae collection. Agricultural organic by-products were used as rearing substrate for Hermetia illucens in a vertical farming system (Baolfly S.r.l., Trento, Italy). Insect larvae were reared at 27–28 °C, 70% humidity, and in darkness. At the end of the cycle, exuviae and dead adults were collected, dried, and milled. The recovered products were used to formulate experimental trout diets.
Experimental diets. Starting from a commercial diet specific for rainbow trout (VRM S.r.l., Trento, Italy), which was used as Control diet (C ), five experimental diets were obtained: (i) C+ diet, (control diet + 3% w/w chitin); (ii) IM5 diet, (control diet + 3% w/w chitin + 5% insect meal); (iii) IM7 diet, (control diet + 3% w/w chitin + 7% insect meal); (iv) IM10 diet, (control diet + 3% w/w chitin + 10% insect meal).
Experimental design. Three hundred and seventy-five rainbow trout (initial body weight: 200-220g), were initially acclimatized for two weeks at the experimental fish facility Fondazione Edmund Mach; Centro di Trasferimento Tecnologico, San Michele a./A., Italy) and then divided into 12 open circulating tanks (800L; three tanks per experimental group), obtaining five experimental groups. Fish were hand-fed the experimental diets 5 days per week from 2% to 1.5% of the biomass adjusting it by measuring the mean body weight. At the end of the feeding trial, (upon reaching commercial size, 500g, 16 weeks) 5 fish per tank (15 per experimental group) were weighted and zootechnical parameters were measured. Samples from liver, intestine and stomach were obtained and properly stored for subsequent analyses.
Analyses. Biometric and zootechnical indexes: (i) survival rate, (ii) growth performances calculating the specific growth rate (SGR%), and weight gain (WG); (iii) feed conversion ratio (FCR), to evaluate feed efficiency; histological analyses of the liver to assess parenchymal architecture and lipid deposition, and of the distal intestine to evaluate histopathological indices and tissue integrity; molecular expression of genes involved in immune response (il1b, il10, tnfα) and on chitin digestion (chia) assessed in distal intestine and stomach, respectively.
Results
Survival was 100% across all experimental groups while no significant differences were observed in zootechnical performance, including WG, SGR and FCR. Liver histology showed a generally normal parenchymal structure in all groups. However, the degree of lipid accumulation varied among treatments. In the distal intestine, no significant differences were detected in the evaluated histopathological indices while supplemented diets affected the density of goblet cells and of supranuclear vacuoles. Considering molecular analysis, dietary treatments modulated immune-related gene expression, influencing the expression of inflammatory markers. Genes related to digestive or metabolic functions were also influenced in response to specific dietary components.
Discussion
Overall, fish maintained optimal health and growth across all dietary treatments, with no adverse effects on general welfare. Histological analyses confirmed normal tissue structure in key metabolic organs, indicating good physiological tolerance to the tested diets.
Dietary supplementation with insect meal proved to be an effective strategy, supporting positive physiological responses in fish. The moderate increase in hepatic lipid accumulation observed was not associated with pathological conditions and is likely related to the fatty acid profile of the ingredients. Differences in goblet cell abundance appears to reflect an adaptive response to chitin, a polysaccharide that can influence gut function by stimulating mucus production.
Chitin may also act as a natural immunostimulant, contributing to enhanced immune efficiency. Therefore, optimizing its inclusion level and physicochemical properties is crucial to improving the nutritional value and functional benefits of insect-based diets, while ensuring fish health and promoting more sustainable aquaculture practices.
ACInA project (Bando Ricerca e Sviluppo 2023/24 Fondazione Caritro project N° 2023.0598) is supported by Fondazione Cassa di Risparmio di Trento e Rovereto.