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This paper reviews current knowledge on two feedstuffs, that is, insect meal and fish by-products, as alternatives to conventional animal protein sources. After an introductory part that highlights the need for sustainable development of animal production, the alternative protein sources are discussed. In particular, after providing some indications on their production and supply focussing on EU, a SWOT (Strengths, Weaknesses, Opportunities, and Threats) analysis was performed to identify the key factors that could help or impair the development of both protein sources production sectors. Finally, future perspectives are presented. The use of processed animal proteins derived from insects in farmed fish feeding is recognised by the EU legislation that authorises the use of proteins from seven insect species and the allowed substrates to rear insects. Insects have several advantages in nutritional value and the amino acid composition of their proteins generally meet animal requirements for good growth and health. The SWOT analysis indicated that insect meals can be considered as feed functional ingredients with beneficial properties that depend on the insect species, rearing system adopted, and the substrate used for their growth. Insects are expected to be increasingly used as a replacement for conventional animal-derived proteins, especially in aquafeeds. In the section regarding fishery and aquaculture by-products, the potential use of raw materials obtained during seafood processing is discussed. Peptides and amino acids recovered from as hydrolysed proteins can be used in animal feeds to partially substitute conventional protein feedstuffs thus providing nutrients, bioactive compounds and feed additives for animals. The SWOT analysis identified opportunities and weaknesses. Both the alternative protein sources are promising alternative feed ingredients for livestock production. Highlights The sustainable development of animal production sector needs alternative protein sources for feeds formulation. Insects and fishery- and aquaculture by-products represent optimal alternative protein sources. A SWOT analysis has identified the key factors for the development of both protein sources production sectors.

Over the last years, the potential use of Black Soldier Fly meal (BSF) as a new and sustainable aquafeed ingredient has been largely explored in several fish species. However, only fragmentary information is available about the use of BSF meal-based diets in sturgeon nutrition. In consideration of a circular economy concept and a more sustainable aquaculture development, the present research represents the first comprehensive multidisciplinary study on the physiological effects of a BSF diet during sturgeon culture in an aquaponic system. Siberian sturgeon ( Acipenser baerii ) juveniles were fed over a 60-days feeding trial on a control diet (Hi0) and a diet containing 50% of full-fat BSF meal respect to fish meal (Hi50). Physiological responses of fish were investigated using several analytical approaches, such as gas chromatography-mass spectrometry, histology, Fourier Transformed Infrared Spectroscopy (FTIR), microbiome sequencing and Real-time PCR. While aquaponic systems performed optimally during the trial, Hi50 group fish showed lower diet acceptance that resulted in growth and survival reduction, a decrease in hepatic lipids and glycogen content (FTIR), a higher hepatic hsp70.1 gene expression and a worsening in gut histological morphometric parameters. The low feed acceptance showed by Hi50 group sturgeon highlighted the necessity to improve the palatability of BSF-based diet designed for sturgeon culture.

This study investigated the effects of dietary inclusion levels of full-fat Hermetia illucens prepupae meal (H) on growth and gastrointestinal integrity in rainbow trout (Oncorhynchus mykiss). A 98-day study was conducted using triplicate groups of trout (initial body weight, 137 ± 10.5 g) kept in 1-m3 tanks in a flow-through well water system. Three dietary treatments were prepared: one based on fishmeal and purified protein-rich vegetable ingredients (H0), and two experimental diets including graded levels of H meal (25% and 50%, referred to as H25 and H50, respectively). At the end of the feeding trial, no differences were observed in growth performance and plasma metabolite levels, with the biometric data confirmed by the liver expression of the genes involved in somatic growth regulation (igf1 and mstn1a). In the H50 group, a three-fold up regulation of liver hsp70 was observed. An activation of the stress/immune response (il-10, tnf-α, and tlr-5) was observed in medium intestine in the H25 and H50 groups (p < 0.05) together with a villi length reduction detected through histological analyses. Liver histology and Fourier Transform Infrared Imaging (FTIRI) spectroscopy highlighted an increase in lipid deposition. These findings suggest that caution should be taken into account when 50% replacement of conventional ingredients with H is selected.

Black Soldier Fly (BSF) meal is considered as an alternative, emerging and sustainable ingredient for aquafeed production. However, results on fish physiological responses are still fragmentary and often controversial, while no studies are available on fish behavior in response to these new diets. The present work represents the first comprehensive multidisciplinary study aimed to investigate zebrafish physiological and behavioural responses to BSF-based diets. Five experimental diets characterized by increasing inclusion levels (0, 25, 50, 75 and 100% respect to fish meal) of full fat BSF prepupae meal were tested during a 2-months feeding trial. Prepupae were cultured on coffee silverskin growth substrate enriched with a 10% Schizochytrium sp. to improve insects’ fatty acids profile. The responses of zebrafish were assayed through biometric, histological, gas chromatographic, microbiological, spectroscopic, molecular and behavioural analyses. Results evidenced that BSF-based diets affected fish fatty acid composition, while behavioural tests did not show differences among groups. Specifically, a 50% BSF inclusion level diet represented the best compromise between ingredient sustainability and proper fish growth and welfare. Fish fed with higher BSF inclusions (75 and 100%) showed hepatic steatosis, microbiota modification, higher lipid content, fatty acid modification and higher expression of immune response markers.

The production of insects on an industrial scale has attracted the attention of the research and agricultural industry as novel protein sources. To detect the presence of Gryllodes sigillatus (GS) in feed and food, a real-time PCR method based on the mitochondrial cytochrome b (CYB) gene is proposed by this study. Forty DNA samples of animal and plant origin were used to confirm the specificity of the qPCR system. The detection method’s performance was evaluated on different processed GS matrices including native GS (UnGS) and different commercial products: crunchy roasted samples (RoGS), insect meal mixtures (ACGS) and energetic snacks containing GS (GSS). Data on sequencing were aligned with the reference gene to confirm the PCR products. The regression curve (y = −3.394 x + 42.521; R2 = 0.994, d.f. 14) between Ct values and Log DNA concentrations of Gryllodes sigillatus resulted in an efficiency of 96.4%. The severity of the technological processing treatments and the matrix structure affected the intensity of the PCR signal with the same amount of insect DNA as observed by different y-intercepts of the three-regression lines for RoGS, ACGS, and GSS. The real-time PCR method resulted in robust and sensitive outcomes able to detect low amounts of GS DNA (5 g/100 g) in a complex matrix, making it suitable for detecting the presence or absence of labeled Gryllodes sigillatus material both in feed and food.

Microalgae have been used as live food in aquatic species. In recent years, the interest in microalgae has considerably increased, thanks to the evolution of production techniques that have identified them as an ecologically attractive aquafeed ingredient. The present study provides the first data about the effects of dietary inclusion of a microalgae consortium grown in a high-rate algal pond system on zootechnical performance, morphometric indices, and dietary nutrient digestibility as well as morphology and functionality of the digestive system of European sea bass, Dicentrarchus labrax . A dietary treatment including a commercial mono-cultured microalgae ( Nannochloropsis sp.) biomass was used for comparison. Six hundred and thirty-six European sea bass juveniles (18 ± 0.28 g) were randomly allotted into 12 experimental groups and fed 4 different diets for 10 weeks: a control diet based on fish meal, fish oil, and plant protein sources; a diet including 10% of Nannochloropsis spp. biomass (100 g/kg diet); and two diets including two levels (10% and 20%) of the microalgal consortium (100 and 200 g/kg diet) . Even at the highest dietary inclusion level, the microalgal consortium (200 g/kg diet) did not affect feed palatability and fish growth performance. A significant decrease in the apparent digestibility of dry matter, protein, and energy was observed in diets including 10 and 20% of the microalgal consortium, but all fish exhibited a well-preserved intestinal histomorphology. Moreover, dietary inclusion with the microalgal consortium significantly increased the enzymatic activity of maltase, sucrase-isomaltase, and ɤ-glutamil transpeptidase in the distal intestine of the treated European sea bass. Algal consortium grown using fish farm effluents represents an attempt to enhance the utilization of natural biomasses in aquafeeds when used at 10 % as substitute of vegetable ingredients in diet for European sea bass.

Gracilaria gracilis and Nannochloropsis oceanica, single or blended, were tested in European seabass (Dicentrarchus labrax) diets. A control (CTRL) diet was compared with experimental diets including either 8% G. gracilis (GRA8), 8% N. oceanica (NAN8), or a blend of 4% of each alga (NAN4GRA4). After 106 days of feeding, growth, nutrient utilization, antioxidant defense, immunological status, and end-product quality were evaluated. All fish exhibited similar feed intake (1.4–1.5%), body weight, growth, and feed conversion ratio (1.6). Dietary inclusion N. oceanica did not affect digestible N intake and gain. Fish fed GRA8 had the lowest digestible N and energy intake (P < 0.05), and simultaneously the highest nitrogen retention efficiency and energy retention efficiency, resulting in a N and energy gain similar to all other treatments. All fish had well-preserved intestinal morphology; feeding NAN8 resulted in a significant increase in neutral goblet cells compared with GRA8. Fish fed the algal diets had significantly lower (P < 0.05) hepatosomatic index (1.7–1.8 vs 2.1) and plasma triglyceride levels than CTRL, but whole body composition remained similar among treatments. The liver total antioxidant capacity of fish fed NAN8 was significantly higher than that of fish fed GRA8 but did not differ significantly from the CTRL group. NAN4GRA4 resulted in lower values of total glutathione, glutathione peroxidase, and alternative complement. N. oceanica decreased fillet springiness; however, with NAN4GRA4, the muscle fillet became less resilient. G. gracilis and N. oceanica biomass, either used single (8%) or blended (4% each), can be valuable natural ingredients for partial replacement of fish meal in European seabass diets.

The expected future demand for highly nutrient animal food products will push the animal production system to search for new sources of high-quality protein feedstuffs. In this scenario, economic and environmental issues will have to be considered while reducing the competition with the plant-based human food chains. Legume grains and some oilseed cakes, by-products from the oil industry, are the main protein sources for ruminants and terrestrial monogastrics such as pigs and poultry. Their relevant role will hold in the next decades, but it is necessary to increase the diversification of sources that can be grown profitably throughout the world, including European countries. Microalgae are a promising source of protein and other nutrients for animal feeding. However, an amazing richness of biologically active substances makes these organisms very interesting as feed ingredients, as their role go far beyond the supply of nutrients. Due to the limited usage of microalgae as human foodstuffs or food ingredients, low competition between microalgae-based feed and food chains is predictable. This review aims to synthesise current knowledge on minor pulses and other protein-rich plant products and microalgae, as alternative ingredients to the conventional animal protein sources, focussing on their production, availability, and nutritional values. Points of strength, weakness, opportunity and threat related to the use of these protein sources in animal feeding are separately analysed through a SWOT approach to underlie future needs in terms of research and/or technological development that could help valorise these nutrient sources as feed ingredients.

The inclusion of chitin-containing ingredients in rainbow trout (Oncorhynchus mykiss) diets has been claimed to be beneficial for fish health, but controversial results on growth performance and nutrient availability have been reported in literature. The present study aimed to evaluate in vitro the effect of increasing chitin level (1.5%, 3% and 4.5%) to simulate increasing insect meal inclusion in aquafeed (15%, 30% and 45%) on the protein degradation using a species-specific digestive enzymatic extract. The first in vitro assay investigated if and to what extent increasing chitin levels in a high digestible blend of purified proteins could affect protein hydrolysis. A second in vitro assay investigated the interference on protein hydrolysis by the same chitin levels included in a complete aquafeed formulation. The coefficients of protein degradation, soluble protein clearance and amino acids released over time were evaluated. The hydrolysis was maintained for 90 min and samples were collected at 0, 15, 30, 60 and 90 min. The overall results showed no substantial differences in terms of protein degradation and amino acids released after 90 min, indicating that an inclusion level of up to 4.5% chitin in the diet does not interfere with the in vitro protein bioaccessibility. All the evaluated parameters resulted in higher values in the protein blends than in the complete aquafeed; not due to the chitin presence but probably to the presence of other nutrients, like carbohydrates and lipids which interfered with the protein degradation.

To evaluate the effects of feeding a Hermetia illucens (HI) larvae meal on the different intestinal traits of hens, and to determine the toxic elements’ concentration in the insect meal and diets, 162 hens were randomly allotted to three groups. The control received a corn-soybean meal-based diet (SBM); the HI25 and HI50 groups received two diets in which the 25% and 50% of the dietary protein were replaced by the HI protein, respectively. The duodenal and jejunal villi height and villi/crypt were higher (p < 0.01) in the SBM than in the HI groups. The ileal villi height was higher (p < 0.05) in the SBM and HI25 groups than the HI50. The HI50 group exhibited a lower duodenal maltase activity. The intestinal alkaline phosphatase (IAP) activity linearly decreased in the duodenum and jejunum as the dietary insect meal inclusion increased. The HI50 group had a higher acetate and butyrate level than the SBM. The levels of cadmium (Cd), lead (Pb), mercury (Hg), and arsenic (As) in the diets and insect meal were lower than the maximum values established by the EU Commission. The 25% soybean protein replacement with Hermetia illucens larvae meal in the diet of laying hens was more suitable and closer to the optimal level than 50%.