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Abstract Background Aquaculture, traditionally a form of biotechnology, has evolved to integrate innovative biotechnological applications, such as advanced feed formulations, aimed at improving the growth performance and health of farmed fish species. In the present study, the effects of feeding rainbow trout with novel feed formulations were investigated. Fish growth, gut and liver morphology, the concentration of fatty acids in the fillet, and volatile fatty acids in the gut were assessed. The study also validated scenarios from in vivo experiments using a nutrient-based model called FEEDNETICS™. This globally used model serves as a tool for data interpretation and decision support in the context of precision fish farming. Methods Alternative protein and oil sources, including poultry by-product meal (PBM) and natural algae oil, were explored as sustainable replacements for fishmeal (FM) and fish oil (FO). A 90-day feeding trial was conducted using rainbow trout, comparing two isoproteic, isolipidic and isoenergetic diets. The control diet contained 15% FM, 5% PBM, and 8% FO, while the test diet replaced FM with 15% PBM and 5% feather meal hydrolysate (FMH), and fully substituted FO with VeraMaris® natural algae oil and rapeseed oil. Results PBM successfully replaced FM protein without negatively affecting feed intake, growth performance or feed utilization in trout. The combination of PBM and natural algae oil was well tolerated by the trout and showed no negative effects on gut health. A detailed analysis of fatty acids in the fillet revealed that PUFAs of the n3 and n6 series were significantly higher in the PBM group than in the FM group. Values of fatty acid-related health indexes, including atherogenicity index, and thrombogenicity index, confirmed the high nutritional value of trout filet, thus representing a healthy product for human. In addition, the predictions using the FEEDNETICS™ indicated that the tested novel alternative formulations are economically viable. The validation of the model for fish growth resulted in a Mean Absolute Percentage Error (MAPE) of 8%. Conclusions The FEEDNETICS™ application enhances our ability to optimize feeding strategies and improve production efficiency in the aquaculture industry. VeraMaris® algae oil and PBM could serve as viable and sustainable raw materials for fish feed, promoting environmentally friendly aquaculture practices.

Before calcification begins, the early embryonic and fetal skeletal development of both mammalian Homo sapiens and the chondrichthyan fish Raja asterias consists exclusively of cartilage. This cartilage is formed and shaped through processes involving tissue segmentation and the frequency, distribution, and orientation of chondrocyte mitoses. In the subsequent developmental phase, mineral deposition in the cartilage matrix conditions the development further. The stiffness and structural layout of the mineralized cartilage have a significant impact on the shape of the anlagen (early formative structure of a tissue, a scaffold on which the new bone is formed) and the mechanical properties of the skeletal segments. The fundamental difference between the two studied species lies in how calcified cartilage serves as a scaffold for osteoblasts to deposit bone matrix, which is then remodeled. In contrast, chondrichthyans retain the calcified cartilage as the definitive skeletal structure. This study documents the distinct mineral deposition pattern in the cartilage of the chondrichthyan R. asterias, in which calcification progresses with the formation of focal calcification nuclei or “tesserae”. These are arranged on the flat surface of the endo-skeleton (crustal pattern) or aligned in columns (catenated pattern) in the radials of the appendicular skeleton. This anatomical structure is well adapted to meet the mechanical requirements of locomotion in the water column. Conversely, in terrestrial mammals, endochondral ossification (associated with the remodeling of the calcified matrix) provides limb bones with the necessary stiffness to withstand the strong bending and twisting stresses of terrestrial locomotion. In this study, radiographs of marine mammals (reproduced from previously published studies) document how the endochondral ossification in dolphin flippers adapts to the mechanical demands of aquatic locomotion. This adaptation includes the reduction in the length of the stylopodium and zeugopodium and an increase in the number of elements in the autopodium’s central rays.

Abstract Background Sustainable aquaculture relies on multiple factors, including water quality, fish diets, and farmed fish. Replacing fishmeal (FM) with alternative protein sources is key for improving sustainability in aquaculture and promoting fish health. Indeed, great research efforts have been made to evaluate novel feed formulations, focusing especially on the effects on the fish gut microbiome. Few studies have explored host-environment interactions. In the present study, we evaluated the influence of novel insect-based (Tenebrio molitor) fish diets on the microbiome at the water-fish interface in an engineered rainbow trout (Oncorhynchus mykiss) farming ecosystem. Using 16S rRNA gene metabarcoding, we comprehensively analyzed the microbiomes of water, tank biofilm, fish intestinal mucus, fish cutis, and feed samples. Results Core microbiome analysis revealed the presence of a highly reduced core shared by all sample sources, constituted by Aeromonas spp., in both the control and novel feed test groups. Network analysis showed that samples were clustered based on the sample source, with no significant differences related to the feed formulation tested. Thus, the different diets did not seem to affect the environment (water and tank biofilm) and fish (cutis and intestinal mucus) microbiomes. To disentangle the contribution of feed at a finer scale, we performed a differential abundance analysis and observed differential enrichment/impoverishment in specific taxa, comparing the samples belonging to the control diet group and the insect-based diet group. Conclusions Omic exploration of the water-fish interface exposes patterns that are otherwise undetected. These data demonstrate a link between the environment and fish and show that subtle but significant differences are caused by feed composition. Thus, the research presented here is a step towards positively influencing the aquaculture environment and its microbiome.

Fish protein hydrolysates used in larval diets have been prepared from a variety of fish species, with different enzymes used to hydrolyze the protein. This study’s objectives were to determine the effect of the dietary inclusion of fish muscle hydrolysates obtained from species-specific muscle/enzymes—versus hydrolysates produced from muscle/enzymes of a different species—on the growth performance, survival, skeletal development, intestinal peptide uptake, and muscle-free amino acid (FAA) composition of larval Walleye (Sander vitreus). Eight protein products were obtained for this study, comprising an unhydrolyzed and hydrolyzed product from each combination of muscle/enzymes from Walleye and Nile tilapia (Oreochromis niloticus). Four diets were produced, and the dietary protein was provided in a 50/50 ratio of unhydrolyzed and hydrolyzed protein from the respective muscle/enzyme combination. Four groups were fed one of the corresponding formulated diets, and two groups of larvae, fed a commercial starter diet and Artemia, respectively, served as reference groups. Larval Walleye fed the diet containing protein produced with the species-specific muscle and enzymes had a significantly higher weight after the study—30% higher than any other group. A significant interaction effect between muscle and enzyme sources on the growth of Walleye larvae was observed. The species-specific combination also led to a significant increase in postprandial FAA and indispensable amino acid concentrations in muscle. No significant differences were observed between the hydrolysate-fed groups in survival, deformity occurrence, or peptide uptake. Each hydrolysate-based diet significantly reduced skeletal deformities and survival compared to the commercial diet. The results of this study suggest that species-specific muscles and enzymes produce a more optimal dietary protein source for larval fish than non-species-specific products. Further research should focus on improving the physical properties of the formulated diets to reduce possible leaching of hydrolyzed protein and improve the survival of fish larvae.

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Many organisms exhibit social behaviors and are part of some scheme of social structure. Zebrafish are highly social, shoaling fish and therefore, social isolation may have notable impacts on their physiology and behavior. The objective of this study was to evaluate the effects of social isolation on feed intake, monoaminergic system related gene expression, and intestinal health of juvenile zebrafish fed a high-inclusion soybean meal based diet. At 20 days post-fertilization zebrafish were randomly assigned to chronic isolation (1 fish per 1.5 L tank) or social housing (6 fish per 9 L tank) with 18 tanks per treatment group (n = 18). Dividers were placed between all tanks to prevent visual cues between fish. Zebrafish were fed a commercial fishmeal based diet until 35 days post-fertilization and then fed the experimental high-inclusion soybean meal based diet until 50 days post-fertilization. At the end of the experiment (51 days post-fertilization), the mean total length, weight, and weight gain were not significantly different between treatment groups. Feed intake and feed conversion ratio were significantly higher in chronic isolation fish than in social housing fish. Expression of monoaminergic and appetite-related genes were not significantly different between groups. The chronic isolation group showed higher expression of the inflammatory gene il-1b, however, average intestinal villi width was significantly smaller and average length-to-width ratio was significantly higher in chronic isolation fish, suggesting morphological signs of inflammation were not present at the time of sampling. These results indicate that chronic isolation positively affects feed intake of juvenile zebrafish and suggest that isolation may be useful in promoting feed intake of less-palatable diets such as those based on soybean meal.

Recent research has revealed the significant impact of novel feed ingredients on fish gut microbiota, affecting both the immune status and digestive performance. As a result, analyzing the microbiota modulatory capabilities may be a useful method for assessing the potential functionality of novel ingredients. Therefore, this study aimed to evaluate the effects of dietary polychaete meal (PM) from Alitta virens on the autochthonous and allochthonous gut microbiota of European seabass (Dicentrarchus labrax). Two diets were compared: a control diet with 25% fishmeal (FM) and a diet replacing 40% of fishmeal with PM, in a 13-week feeding trial with juvenile fish (initial weight of 14.5 ± 1.0 g). The feed, digesta, and mucosa-associated microbial communities in fish intestines were analyzed using high-throughput sequencing of the 16S rRNA gene on the Illumina MiSeq platform. The results of feed microbiota analyses showed that the PM10 feed exhibited a higher microbial diversity than the FM diet. However, these feed-associated microbiota differences were not mirrored in the composition of digesta and mucosal communities. Regardless of the diet, the digesta samples consistently exhibited higher species richness and diversity than the mucosa samples. Overall, digesta samples were characterized by a higher abundance of Firmicutes in PM-fed fish. In contrast, at the gut mucosa level, the relative abundances of Mycobacterium, Taeseokella and Clostridium genera were lower in the group fed the PM10 diet. Significant differences in metabolic pathways were also observed between the FM and PM10 groups in both mucosa and digesta samples. In particular, the mucosal pathways of caffeine metabolism, phenylalanine metabolism, and sulfur relay system were significantly altered by PM inclusion. The same trend was observed in the digesta valine, leucine, and isoleucine degradation and secretion pathways. These findings highlight the potential of PM as an alternative functional ingredient in aquafeeds with microbiota modulatory properties that should be further explored in the future.

The objective of this study was to determine if selected fish genotypes could benefit from the use of functional additives in novel aqua feed formulations to improve growth performance, gut microbiota, immune response, and disease resistance in fish. Two batches of juvenile European sea bass selected for high growth (HG; selected sires x selected dams), and wild types (WT; wild sires x selected females) were fed a “future diet” coated with three different functional additives for 12 weeks as follows: (i) 2 weeks with a high dose, followed by (ii) 10 weeks with a low dose. The functional additives tested were a mixture of probiotics (PROB), organic acids (ORG), and phytogens (PHYTO). A pathogen challenge test (Vibrio anguillarum) and a stress condition (overcrowding) were performed after each dose. At the end of the feeding experiment, fish from the HG group performed better than fish from the WT group in terms of body weight, relative growth, SGR, and DGI. The results of the two challenge tests performed after two weeks of high dose and ten weeks of low dose showed a significant effect of diet on fish survival. GALT-associated gene expression analysis revealed an interaction between the genotype and diet for il-1β in the distal gut. Finally, regarding the gut microbiota, discriminant analysis showed no clear separation between fish fed the future diet and those fed the same diet with experimental additives. Nevertheless, the relative abundance of certain taxa varied between experimental groups. For example, fish fed the ORG diet had higher relative abundance of Streptococcus in both genotypes, whereas fish fed the PHYTO diet had higher abundance of Lactobacillales. In contrast, fish fed PROB had lower abundance of Pseudomonas and Acinetobacter.

This study addressed the urgent need for sustainable protein sources in aquaculture due to the depletion of marine resources and rising costs. Animal protein sources, particularly poultry by-product meal (PBM) and insect exuviae meal, were investigated as viable alternatives to fishmeal (FM). The research study confirmed the successful replacement of FM with a combination of PBM and insect exuviae meal (up to 50%) in the diet of European seabass without compromising growth, feed conversion, gut health, and liver fat content. In particular, growth was robust with both PBM formulations, with the 25% PBM diet showing better results. Histological examinations showed good gut and liver health, contradicting the concerns of previous studies. This paper emphasizes the importance of holistic analyzes that go beyond growth parameters and include histomorphological investigations. The results show that PBM in combination with insect/exuviae meal is well tolerated by seabass, which is consistent with reports in the literature of it mitigating negative effects on gut health. A detailed analysis of the microbiota revealed a decrease in the Firmicutes/Proteobacteria ratio due to an increase in potentially pathogenic bacteria. However, the formulation containing insect exuviae partially counteracted this effect by preserving the beneficial Lactobacillus and promoting the synthesis of short-chain fatty acids (SCFAs), particularly butyrate. Chitin-rich components from insect exuviae were associated with improved gut health, which was supported by the increased production of SCFAs, which are known for their anti-inflammatory properties. This paper concludes that a combination of PBM and insect/exuviae meal can replace up to 50% of FM in the diet of seabass, supporting sustainable aquaculture practices. Despite some changes in the microbiota, the negative effects are mitigated by the addition of insect exuviae, highlighting their potential as a prebiotic to increase fish productivity and contribute to a circular economy in aquaculture.

The aquaculture industry is looking for sustainable alternatives to conventional fish meals in fish feed, and insect-based meals are proving to be a promising solution. These meals are nutritionally optimal as they have a high protein content and an ideal amino acid profile. However, the presence of chitin, a component of the insect exoskeleton in these meals presents both an opportunity and a challenge. Chitosan, a derivative of chitin, is known to improve the physiological functions of fish, including growth, immunity, and disease resistance. While chitin and its derivative chitosan offer several physiological benefits, their presence can affect the digestibility of feed in some fish species, making the inclusion of insect-based meals in aquafeeds complex. While studies suggest positive effects, some problems, such as reduced growth rates in certain species, emphasize the need for further research on chitin digestion in fish. Chitinase, an enzyme that breaks down chitin, is being investigated as a potential solution to improve the nutritional value of insect meals in aquafeed. This review provides a comprehensive analysis of the applications, benefits, and challenges of using chitinase in aquaculture, highlighting the enzyme’s role in improving feed digestibility, disease control, and environmental sustainability. Extensive research is required to fully understand the potential of chitinase enzymes in aquaculture and to optimize their applications in this dynamic field. Overall, this review provides insight into the evolving landscape of insect-based meals and the applications of chitinase enzymes within sustainable aquaculture practices.

In aquafeeds in which plant proteins are used to replace fishmeal, exogenous methionine (Met) sources are demanded to balance the amino acid composition of diets and meet the metabolic fish requirements. Nonetheless, since different synthetic Met sources are commercially available, it is important to determine their bioavailability and efficacy. To address this issue, we conducted a two-month feeding trial with rainbow trout (Oncorhynchus mykiss), which were fed diets supplemented with five different forms of Met: Met-Met, L-Met, HMTBa, DL-Met, and Co DL-Met. No differences in growth performance were found in trout fed with different Met forms, but changes in the whole-body composition were found. In particular, Met-Met and L-Met promoted a significant body lipid reduction, whereas the protein retention was significantly increased in fish fed with HMTBa and Co DL-Met. The latter affected the hepatic Met metabolism promoting the trans-sulfuration pathway through the upregulation of CBS gene expression. Similarly, the L-Met enhanced the remethylation pathway through an increase in BHMT gene expression to maintain the cellular demand for Met. Altogether, our findings suggest an optimal dietary intake of all tested Met sources with similar promoting effects on fish growth and hepatic Met metabolism. Nevertheless, the mechanisms underlying these effects warrant further investigation.

Abstract Alternative nutrient sources to fishmeal for fish feed, such as insect meals, represent a promising sustainable supply. However, the consequences for fish digestive function have not been exhaustively investigated. In the present study we evaluated the effect of partial fishmeal substitution with 10% Hermetia illucens (Hi10) larvae meal on the neuromuscular function of proximal and distal intestine in gilthead sea bream. In animals fed with insect meal, weight and growth parameters were similar to controls fed with conventional fishmeal. In addition, no anomalies in intestinal gross morphology and no overt signs of inflammation were observed. The gastrointestinal transit was significantly reduced in Hi10 fed animals. In the proximal and distal intestine longitudinal muscle, Hi10 feeding downregulated the excitatory cholinergic and serotoninergic transmission. Sodium nitroprusside-induced inhibitory relaxations increased in the proximal intestine and decreased in the distal intestine after Hi10 meal. Changes in the excitatory and inhibitory components of peristalsis were associated with adaptive changes in the chemical coding of both proximal and distal intestine myenteric plexus. However, these neuromuscular function alterations were not associated with considerable variations in morphometric growth parameters, suggesting that 10% Hi meal may represent a tolerable alternative protein source for gilthead sea bream diets.

Functional ingredients have profiled as suitable candidates for reinforcing the fish antioxidant response and stress tolerance. In addition, selective breeding strategies have also demonstrated a correlation between fish growth performance and susceptibility to stressful culture conditions as a key component in species domestication processes. The aim of the present study is to evaluate the ability of a selected high-growth genotype of 300 days post-hatch European sea bass (Dicentrarchus labrax) juveniles to use different functional additives as endogenous antioxidant capacity and stress resistance boosters when supplemented in low fish meal (FM) and fish oil (FO) diets. Three isoenergetic and isonitrogenous diets (10% FM/6% FO) were supplemented with 200 ppm of a blend of garlic and Labiatae plant oils (PHYTO0.02), 1000 ppm of a mixture of citrus flavonoids and Asteraceae and Labiatae plant essential oils (PHYTO0.1) or 5000 ppm of galactomannan-oligosaccharides (GMOS0.5). A reference diet was void of supplementation. The fish were fed the experimental diets for 72 days and subjected to a H2O2 exposure oxidative stress challenge. The fish stress response was evaluated through measuring the circulating plasma cortisol levels and the fish gill antioxidant response by the relative gene expression analysis of nfΚβ2, il-1b, hif-1a, nd5, cyb, cox, sod, cat, gpx, tnf-1α and caspase 9. After the oxidative stress challenge, the genotype origin determined the capacity of the recovery of basal cortisol levels after an acute stress response, presenting GS fish with a better pattern of recovery. All functional diets induced a significant upregulation of cat gill gene expression levels compared to fish fed the control diet, regardless of the fish genotype. Altogether, suggesting an increased capacity of the growth selected European sea bass genotype to cope with the potential negative side-effects associated to an H2O2 bath exposure.

The sustainable development of modern aquaculture must rely on a significant reduction of the fish meal (FM) used in aquafeed formulations. However, FM substitution with alternative ingredients in diets for carnivorous fish species often showed reduced nutrient absorption, significantly perturbed metabolisms, and histological changes at both hepatic and intestinal levels. In the present study, rainbow trout (Oncorhynchus mykiss) were fed three different experimental aquafeeds. A control diet with higher FM content (27.3%) than two test formulations in which FM was substituted with two more sustainable and promising alternatives: insect meal (Hermetia illucens larvae = 10.1%, FM = 11.6%) and poultry by-products meal (PBM = 14.8%; FM = 11.7%). Combined metabolomics and proteomics analyses of fish liver, together with histological examination of liver and intestine demonstrated that a well-balanced formulation of nutrients in the three diets allowed high metabolic compatibility of either substitution, paving the way for a deeper understanding of the impact of novel raw materials for the fish feed industry. Results show that the main metabolic pathways of nutrient absorption and catabolism were essentially unaltered by alternative feed ingredients, and also histological alterations were negligible. It is demonstrated that the substitution of FM with sustainable alternatives does not have a negative impact on fish metabolism, as long as the nutritional requirements of rainbow trout are fulfilled.

Aquaculture is the fastest-growing agricultural industry in the world. Fishmeal is an essential component of commercial fish diets, but its long-term sustainability is a concern. Therefore, it is important to find alternatives to fishmeal that have a similar nutritional value and, at the same time, are affordable and readily available. The search for high-quality alternatives to fishmeal and fish oil has interested researchers worldwide. Over the past 20 years, different insect meals have been studied as a potential alternate source of fishmeal in aquafeeds. On the other hand, probiotics—live microbial strains—are being used as dietary supplements and showing beneficial effects on fish growth and health status. Fish gut microbiota plays a significant role in nutrition metabolism, which affects a number of other physiological functions, including fish growth and development, immune regulation, and pathogen resistance. One of the key reasons for studying fish gut microbiota is the possibility to modify microbial communities that inhabit the intestine to benefit host growth and health. The development of DNA sequencing technologies and advanced bioinformatics tools has made metagenomic analysis a feasible method for researching gut microbes. In this review, we analyze and summarize the current knowledge provided by studies of our research group on using insect meal and probiotic supplements in aquafeed formulations and their effects on different fish gut microbiota. We also highlight future research directions to make insect meals a key source of proteins for sustainable aquaculture and explore the challenges associated with the use of probiotics. Insect meals and probiotics will undoubtedly have a positive effect on the long-term sustainability and profitability of aquaculture.

Abstract Background Aquaculture must continue to reduce dependence on fishmeal (FM) and fishoil in feeds to ensure sustainable sector growth. Therefore, the use of novel aquaculture feed ingredients is growing. In this regard, insects can represent a new world of sustainable and protein-rich ingredients for farmed fish feeds. Accordingly, we investigated the effects of full replacement of FM with Tenebrio molitor (TM) larvae meal in the diet of rainbow trout (Oncorhynchus mykiss) on fish gut and skin microbiota. Methods A feeding trial was conducted with 126 trout of about 80 g mean initial weight that were fed for 22 weeks with two isonitrogenous, isolipidic, and isoenergetic extruded experimental diets. Partially defatted TM meal was included in one of the diets to replace 100% (TM 100) of FM, whereas the other diet (TM 0) was without TM. To analyse the microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and Qiime pipeline were used to identify bacteria in the gut and skin mucosa, and in the diets. Results The data showed no major effects of full FM substitution with TM meal on bacterial species richness and diversity in both, gut mucosa- and skin mucus-associated microbiome. Skin microbiome was dominated by phylum Proteobacteria and especially by Gammaproteobacteria class that constituted approximately half of the bacterial taxa found. The two dietary fish groups did not display distinctive features, except for a decrease in the relative abundance of Deefgea genus (family Neisseriaceae) in trout fed with insect meal. The metagenomic analysis of the gut mucosa indicated that Tenericutes was the most abundant phylum, regardless of the diet. Specifically, within this phylum, the Mollicutes, mainly represented by Mycoplasmataceae family, were the dominant class. However, we observed only a weak dietary modulation of intestinal bacterial communities. The only changes due to full FM replacement with TM meal were a decreased number of Proteobacteria and a reduced number of taxa assigned to Ruminococcaceae and Neisseriaceae families. Conclusions The data demonstrated that TM larvae meal is a valid alternative animal protein to replace FM in the aquafeeds. Only slight gut and skin microbiota changes occurred in rainbow trout after total FM replacement with insect meal. The mapping of the trout skin microbiota represents a novel contribution of the present study. Indeed, in contrast to the increasing knowledge on gut microbiota, the skin microbiota of major farmed fish species remains largely unmapped but it deserves thorough consideration.

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.

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 present study aimed to investigate the growth performance, whole-body proximate composition, and intestinal microbiome of rainbow trout strains when selected and non-selected for weight gain on all-plant protein diets. A 2x2 factorial design was applied, where a selected (United States) and a non-selected (ITA) rainbow trout strain were fed using either an all-plant protein (PP) or a commercial low-FM diet (C). Diets were fed to five replicates of 20 (PP) or 25 (C) fish for 105 days. At the end of the trial, growth parameters were assessed, and whole fish (15 pools of three fish/diet) and gut samples (six fish/diet) were collected for whole-body proximate composition and gut microbiome analyses, respectively. Independent of the administered diet, the United States strain showed higher survival, final body weight, weight gain, and specific growth rate when compared to the ITA fish (p < 0.001). Furthermore, decreased whole-body ether extract content was identified in the PP-fed United States rainbow trout when compared to the ITA strain fed the same diet (p < 0.001). Gut microbiome analysis revealed the Cetobacterium probiotic-like genus as clearly associated with the United States rainbow trout, along with the up-regulation of the pathway involved in starch and sucrose metabolism. In summary, the overall improvement in growth performance and, to a lesser extent, whole-body proximate composition observed in the selected rainbow trout strain was accompanied by specific, positive modulation of the intestinal microbiome.

This study’s objectives were to determine the effect of Largemouth Bass (LMB) muscle hydrolysates obtained using same-species digestive enzymes and the degree of LMB muscle hydrolysis when included in the first feeds of growth performance and survival, skeletal development, intestinal peptide uptake, and muscle-free amino acid composition of larval LMB. LMB muscle was mixed with digestive enzymes from adult LMB, and hydrolyzed for 1.5, 3, and 6 h, respectively. Five diets were produced, the intact diet containing non-hydrolyzed muscle and four diets with 37% muscle hydrolysate inclusion. Those diets were characterized by their level of each hydrolysate (presented as a ratio of 1.5, 3, and 6 Ts hydrolysates): 1:1:1, 1:3:6, 1:3:1, 6:3:1 for diets A, B, C, and D, respectively. To account for gut development, one group of larval LMB was fed a weekly series of diets B, C, and D to provide an increasing molecular weight profile throughout development. This group was compared against others that received either; (1) diets D, C, and B; (2) diet A; or (3) intact diet. The initial inclusion of the hydrolysates significantly improved the total length of the larval LMB; however, neither the hydrolysate inclusion nor the series of dietary molecular weight profiles improved the overall growth of larval LMB. The inclusion of hydrolysates significantly decreased the occurrence of skeletal deformities. The degree of hydrolysis did not have a significant effect on the parameters measured, except for intestinal peptide uptake, which was increased in the group that received the most hydrolyzed diet at the final time of sampling. The lack of overall growth improvement suggests that while the hydrolysates improve the initial growth performance, further research is necessary to determine the optimal molecular weight profile, hydrolysate inclusion level, and physical properties of feeds for larval LMB.

The aim of the present study is to evaluate the potential of two functional additives as gill endogenous antioxidant capacity boosters in European sea-bass juveniles fed low-FM/FO diets when challenged against physical and biological stressors. For that purpose, two isoenergetic and isonitrogenous diets with low FM (10%) and FO (6%) contents were supplemented with 5000 ppm plant-derived galactomannan–oligosaccharides (GMOS) or 200 ppm of a mixture of garlic and labiate plant essential oils (PHYTO). A control diet was void from supplementation. Fish were fed the experimental diet for nine weeks and subjected to a confinement stress challenge (C challenge) or a confinement stress challenge combined with an exposure to the pathogen Vibrio anguillarum (CI challenge). Both GMOS and PHYTO diets attenuated fish stress response, inducing lower circulating plasma cortisol and down-regulating nfκβ2 and gr relative gene-expression levels in the gill. This attenuated stress response was associated with a minor energetic metabolism response in relation to the down-regulation of nd5 and coxi gene expression.

Using a circular economy concept, the present study investigated the use of crude glycerol, a primary by-product of biodiesel production, as a low-priced nutrient source for heterotrophic cultivation of the fungus-like protist Schizochytrium limacinum SR21 strain. The whole biomass of this oleaginous microorganism, rich in docosahexaenoic acid (DHA) and high-quality proteins, was then paired with a vegetable oil (VO) source and used to replace fish oil (FO) in European sea bass (Dicentrarchus labrax) feeds. Four nutritionally balanced diets were formulated: diet FO (a FO-based diet), diet VO + 0 (a VO-based diet without S. limacinum), and diets VO + 5 and VO + 10 that were VO-based feeds supplemented with 5 and 10% of S. limacinum, respectively. After a 3-month feeding trial, fish of all dietary groups tripled their initial weight, but growth and feeding efficiencies of D. labrax were not significantly different among treatments. Although the formulated diets were balanced for polyunsaturated fatty acids (PUFAs), fish fed with feeds containing either VO or VO plus 5 and 10% of S. limacinum biomass had significantly higher levels of PUFAs in the flesh than fish fed the FO-based diet. Values of health-related lipid indexes, such as atherogenicity index, thrombogenicity index, and flesh lipid quality as well as n-6/n-3 and PUFAs/SFAs ratios confirmed the high nutritional value of sea bass filet, thus representing a healthy product for human consumption. Although the PUFAs/SFAs ratio showed a significantly higher value in fish fed with VO-based diets supplemented with S. limacinum than in those fed with FO diet, suggesting a better filet quality, the n-6/n-3 ratio clearly indicated that filet quality of dietary group FO was best (value of 0.55) and that of group VO + 10 second best (value of 0.98). We also evaluated the nutritional regulation of Δ6-desaturase (or fads2) gene expression in European sea bass liver. European sea bass fed the VO + 0 diet had the highest number of mRNA copies for Δ6-desaturase (or fads2), fish fed with diet VO + 10 the lowest. Our study adds to the growing body of literature concerning the use of thraustochytrid biomass as a valid alternative to marine-derived raw materials for European sea bass feeds.

An effective replacement for fish meal (FM) and fish oil (FO) based on plant-based raw materials in the feed of marine fish species is necessary for the sustainability of the aquaculture sector. However, the use of plant-based raw materials to replace FM and FO has been associated with several negative health effects, some of which are related to oxidative stress processes that can induce functional and morphological alterations in mucosal tissues. This study aimed to evaluate the effects of dietary oligosaccharides of plant origin (5,000 ppm; galactomannan oligosaccharides, GMOS) and a phytogenic feed additive (200 ppm; garlic oil and labiatae plant extract mixture, PHYTO) on the oxidative stress status and mucosal health of the gills of juvenile European sea bass (Dicentrarchus labrax). The experimental diets, low FM and FO diets (10%FM/6%FO) were supplemented with GMOS from plant origin and PHYTO for 63 days. GMOS and PHYTO did not significantly affect feed utilization, fish growth, and survival. GMOS and PHYTO downregulated the expression of β-act, sod, gpx, cat, and gr in the gills of the fish compared with that in fish fed the control diet. The expression of hsp70 and ocln was upregulated and downregulated, respectively, in the GMOS group compared with that in the control group, whereas the expression of zo-1 was downregulated in the PHYTO group compared with that in the GMOS group. The morphological, histopathological, immunohistochemical, and biochemical parameters of the fish gills were mostly unaffected by GMOS and PHYTO. However, the PHYTO group had lower incidence of lamellar fusion than did the control group after 63 days. Although the tissular distribution of goblet cells was unaffected by GMOS and PHYTO, goblet cell size showed a decreasing trend (−11%) in the GMOS group. GMOS and PHYTO significantly reduced the concentration of PCNA+ in the epithelium of the gills. The above findings indicated that GMOS and PHYTO in low FM/FO-based diets protected the gill epithelia of D. labrax from oxidative stress by modulating the expression of oxidative enzyme-related genes and reducing the density of PCNA+ cells in the gills of the fish.

The present research tested the effects of dietary nisin-producing Lactococcus lactis on growth performance, feed utilization, intestinal morphology, transcriptional response, and microbiota in gilthead sea bream (Sparus aurata). A feeding trial was conducted with fish weighting 70–90 g. Fish were tagged with passive, integrated transponders and distributed in nine 500 L tanks with 40 fish each. Fish were fed for 12 weeks with either a control (diet A) or experimental diets (diets B and C) in triplicate (3 tanks/diet). Extruded pellets of diets B and C were supplemented with a low (2 × 109 CFU/kg) and a high (5 × 109 CFU/kg) dose of probiotic, respectively. No significant differences were found between groups for the feed conversion ratio or specific growth rates. However, the final body weight of fish fed diet C was significantly higher than the control group with intermediate values for fish fed diet B. Histological analysis conducted using a semi-quantitative scoring system showed that probiotic did not alter the morphology of the intestine and did not trigger inflammation. With regard to the transcriptomic response, a customized PCR array layout was designed to simultaneously profile a panel of 44 selected genes. Significant differences in the expression of key genes involved in innate and acquired immunity were detected between fish fed probiotic and control diets. To analyze the microbiota associated to the feeds and the gut autochthonous microbial communities, we used the Illumina MiSeq platform for sequencing the 16S rRNA gene and a metagenomics pipeline based on VSEARCH and RDP databases. The analysis of gut microbiota revealed a lack of colonization of the probiotic in the host’s intestinal mucosa. However, probiotic did modulate the fish gut microbiota, confirming that colonization is not always necessary to induce host modification. In fact, diets B and C were enriched with Actinomycetales, as compared to diet A, which instead showed a higher percentage of Pseudomonas, Sphyngomonas, and Lactobacillus genera. These results were confirmed by the clear separation of gut bacterial community of fish fed with the probiotic from the bacterial community of control fish group in the beta-diversity and PLS-DA (supervised partial least-squares discriminant analysis) analyses.

Nutritional Programming (NP) has been shown to counteract the negative effects of dietary plant protein (PP) by introducing PP at an early age towards enhancement of PP utilization during later life stages. This study explored the effect of NP and its induction time on growth, expression of appetite-stimulating hormones, and any morphological changes in the gut possibly responsible for improved dietary PP utilization. At 3 days post-hatch (dph) zebrafish were distributed into 12 (3 L) tanks, 100 larvae per tank. This study included four groups: 1) The control (NP-FM) group received fishmeal (FM)-based diet from 13-36 dph and was challenged with PP-based diet during 36-66 dph; 2) The NP-PP group received NP with dietary PP in larval stage via live food enrichment during 3-13 dph followed by FM diet during 13-36 dph and PP diet during 36-66 dph; 3) The T-NP group received NP between 13-23 dph through PP diet followed by FM diet during 23-36 dph and PP diet during 36-66 dph; and 4) The PP group received PP diet from 13-66 dph. During the PP challenge the T-NP group achieved the highest weight gain compared to control and PP. Ghrelin expression in the brain was higher in T-NP compared to NP-FM and NP-PP, while in the gut it was reduced in both NP-PP and T-NP groups. Cholecystokinin expression showed an opposite trend to ghrelin. The brain neuropeptide Y expression was lower in NP-PP compared to PP but not different with NP-FM and T-NP groups. The highest villus length to width ratio in the middle intestine was found in T-NP compared to all other groups. The study suggests that NP induced during juvenile stages improves zebrafish growth and affects digestive hormone regulation and morphology of the intestinal lining-possible mechanisms behind the improved PP utilization in pre-adult zebrafish stages.

Nutritional Programming (NP) has been studied as a means of mitigating the negative effects of dietary plant protein (PP), but the optimal timing and mechanism behind NP are still unknown. The objectives of this study were: 1) To determine whether zebrafish (Danio rerio) can be programmed to soybean meal (SBM) through early feeding and broodstock exposure to improve SBM utilization; 2) To determine if NP in zebrafish affects expression of genes associated with intestinal nutrient uptake; 3) To determine if early stage NP and/or broodstock affects gene expression associated with intestinal inflammation or any morphological changes in the intestinal tract that might improve dietary SBM utilization. Two broodstocks were used to form the six experimental groups. One broodstock group received fishmeal (FM) diet (FMBS), while the other was fed ("programmed with") SBM diet (PPBS). The first ((+) Control) and the second group ((-) Control) received FM and SBM diet for the entire study, respectively, and were progeny of FMBS. The last four groups consisted of a non-programmed (FMBS-X-PP and PPBS-X-PP) and a programmed group (FMBS-NP-PP and PPBS-NP-PP) from each of the broodstocks. The programming occurred through feeding with SBM diet during 13-23 dph. The non-control groups underwent a PP-Challenge, receiving SBM diet during 36-60 dph. During the PP-Challenge, both PPBS groups experienced significantly lower weight gains than the (+) Control group. NP in early life stages significantly increased the expression of PepT1 in PPBS-NP-PP, compared to PPBS-X-PP. NP also tended to increase the expression of fabp2 in the programmed vs. non-programmed groups of both broodstocks. The highest distal villus length-to-width ratio was observed in the dual-programmed group, suggesting an increase in surface area for nutrient absorption within the intestine. The results of this study suggest that NP during early life stages may increase intestinal absorption of nutrients from PP-based feeds.

There is an increasing interest from the aquafeed industry in functional feeds containing selected additives that improve fish growth performance and health status. Functional feed additives include probiotics, prebiotics, organic acids, and phytogenics (substances derived from plants and their extracts). This study evaluated the effects of dietary inclusion of a mucilage extract rich in galactomannan oligosaccharides (GMOS), a mixture of garlic and labiatae-plants oils (PHYTO), and a combination of them (GMOSPHYTO), on gut microbiota composition of European sea bass (Dicentrarchus labrax) fed with a low fishmeal (FM) and fish oil (FO) diet. Three experimental diets and a control diet (plant-based formulation with 10% FM and 6% FO) were tested in a 63-days feeding trial. To analyze the microbiota associated to feeds and the intestinal autochthonous (mucosa-adhered) and allochthonous (transient) microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and QIIME2 pipeline were used. Metabarcoding analysis of feed-associated bacteria showed that the microbial communities of control (CTRL) feed deeply differed from those of experimental diets. The number of reads was significantly lower in CTRL feed than in other feeds. The OTU (operational taxonomic unit) number was instead similar between the feeds, ranging from 42 to 50 OTUs. The variation of resident gut microbiota induced by diet was lower than the variation of transient intestinal microbiota, because feedstuffs are a major source of allochthonous bacteria, which can temporarily integrate into the gut transient microbiome. However, the composition of transient bacterial communities was not simply a mirror of feed-borne bacteria. Indeed, the microbial profile of feeds was different from both faecal and mucosa profiles. Our findings suggest that the dietary inclusion of GMOS (0.5%) and PHYTO (0.02%) in a low FM and FO diet induces changes in gut microbiota composition of European sea bass. However, if on allochthonous microbiota the combined inclusion of GMOS and PHYTO showed an antagonistic effect on bactericidal activity against Vibrionales, at mucosa level, only GMOSPHYTO diet increased the relative abundance of Bacteroidales, Lactobacillales, and Clostridiales resident bacterial orders. The main beneficial effects of GMOS and PHYTO on gut microbiota are the reduction of coliforms and Vibrionales bacteria, which include several potentially pathogenic species for fish, and the enrichment of gut microbiota composition with butyrate producer taxa. Therefore, these functional ingredients have a great potential to be used as health-promoting agents in the farming of European sea bass and other marine fish.

Abstract Background Due to the paucity of oceanic resources utilized in the preparation of diets for cultured fish, commercial feed producers have been trying to replace fishmeal (FM) using alternative protein sources such as vegetable protein meals (VMs). One of the main drawbacks of using VMs in fish feed is related to the presence of a variety of anti-nutritional factors, which could trigger an inflammation process in the distal intestine. This reduces the capacity of the enterocytes to absorb nutrients leading to reduced fish growth performances. Methods We evaluated the mitigating effects of butyrate and taurine used as feed additives on the morphological abnormalities caused by a soybean meal (SBM)-based diet in the distal intestine of sea bass (Dicentrarchus labrax). We used three experimental diets, containing the same low percentage of FM and high percentage of SBM; two diets were supplemented with either 0.2% sodium butyrate or taurine. Histological changes in the intestine of fish were determined by light and transmission electron microscopy. Infiltration of CD45+ leucocytes in the lamina propria and in the submucosa was assessed by immunohistochemistry. We also quantified by One-Step Taqman® real-time RT-PCR the messenger RNA (mRNA) abundance of a panel of genes involved in the intestinal mucosa inflammatory response such as TNFα (tumor necrosis factor alpha) and interleukins: IL-8, IL-1β, IL-10, and IL-6. Results Fish that received for 2 months the diet with 30% soy protein (16.7% SBM and 12.8% full-fat soy) developed an inflammation in the distal intestine, as confirmed by histological and immunohistochemistry data. The expression of target genes in the intestine was deeply influenced by the type of fish diet. Fish fed with taurine-supplemented diet displayed the lowest number of mRNA copies of IL-1β, IL-8, and IL-10 genes in comparison to fish fed with control or butyrate-supplemented diets. Dietary butyrate caused an upregulation of the TNFα gene transcription. Among the quantified interleukins, IL-6 was the only one to be not influenced by the diet. Conclusions Histological and gene expression data suggest that butyrate and taurine could have a role in normalizing the intestinal abnormalities caused by the SBM, but the underling mechanisms of action seem different.

Antibiotic use during adolescence may result in dysbiosis-induced neuronal vulnerability both in the enteric nervous system (ENS) and central nervous system (CNS) contributing to the onset of chronic gastrointestinal disorders, such as irritable bowel syndrome (IBS), showing significant psychiatric comorbidity. Intestinal microbiota alterations during adolescence influence the expression of molecular factors involved in neuronal development in both the ENS and CNS. In this study, we have evaluated the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TrkB) in juvenile mice ENS and CNS, after a 2-week antibiotic (ABX) treatment. In both mucosa and mucosa-deprived whole-wall small intestine segments of ABX-treated animals, BDNF and TrKB mRNA and protein levels significantly increased. In longitudinal muscle-myenteric plexus preparations of ABX-treated mice the percentage of myenteric neurons staining for BDNF and TrkB was significantly higher than in controls. After ABX treatment, a consistent population of BDNF- and TrkB-immunoreactive neurons costained with SP and CGRP, suggesting up-regulation of BDNF signaling in both motor and sensory myenteric neurons. BDNF and TrkB protein levels were downregulated in the hippocampus and remained unchanged in the prefrontal cortex of ABX-treated animals. Immunostaining for BDNF and TrkB decreased in the hippocampus CA3 and dentate gyrus subregions, respectively, and remained unchanged in the prefrontal cortex. These data suggest that dysbiosis differentially influences the expression of BDNF-TrkB in the juvenile mice ENS and CNS. Such changes may potentially contribute later to the development of functional gut disorders, such as IBS, showing psychiatric comorbidity.

European sea bass were fed four low FM/FO (10%/6%) diets containing galactomannan oligosaccharides (GMOS), a mixture of garlic oil and labiatae plants oils (PHYTO), or a combination of both functional products (GMOSPHYTO) for 63 days before exposing the fish to an intestinal Vibrio anguillarum infection combined with crowding stress. In order to evaluate functional diets efficacy in terms of gut health maintenance, structural, cellular, and immune intestinal status were evaluated by optical and electron microscopy and gene expression analyses. A semi-automated software was adapted to determine variations in goblet cell area and mucosal mucus coverage during the challenge test. Feeding with functional diets did not affect growth performance; however, PHYTO and GMOS dietary inclusion reduced European sea bass susceptibility to V. anguillarum after 7 days of challenge testing. Rectum (post-ileorectal valve) showed longer (p = 0.001) folds than posterior gut (pre-ileorectal valve), whereas posterior gut had thicker submucosa (p = 0.001) and higher mucus coverage as a result of an increased cell density than rectum. Functional diets did not affect mucosal fold length or the grade of granulocytes and lymphocytes infiltration in either intestinal segment. However, the posterior gut fold area covered by goblet cells was smaller in fish fed GMOS (F = 14.53; p = 0.001) and PHYTO (F = 5.52; p = 0.019) than for the other diets. PHYTO (F = 3.95; p = 0.049) reduced posterior gut goblet cell size and increased rodlet cell density (F = 3.604; p = 0.068). Dietary GMOS reduced submucosal thickness (F = 51.31; p = 0.001) and increased rodlet cell density (F = 3.604; p = 0.068) in rectum. Structural TEM analyses revealed a normal intestinal morphological pattern, but the use of GMOS increased rectum microvilli length, whereas the use of PHYTO increased (p≤0.10) Ocln, N-Cad and Cad-17 posterior gut gene expression. After bacterial intestinal inoculation, posterior gut of fish fed PHYTO responded in a more controlled and belated way in terms of goblet cell size and mucus coverage in comparison to other treatments. For rectum, the pattern of response was similar for all dietary treatments, however fish fed GMOS maintained goblet cell size along the challenge test.

This study investigated, for the first time, the effects of replacement of fishmeal (FM) with insect meal from Hermetia illucens (HI) on the transcript levels of three genes involved in methionine (Met) metabolism in rainbow trout (Oncorhynchus mykiss) liver. Two target genes—betaine-homocysteine S-methyltransferase (BHMT) and S-adenosylhomocysteine hydrolase (SAHH)—are involved in Met resynthesis and the third one—cystathionine β synthase (CBS)—is involved in net Met loss (taurine synthesis). We also investigated the levels of two Met metabolites involved in the maintenance of methyl groups and homocysteine homeostasis in the hepatic tissue: S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). Three diets were formulated, an FM-based diet (HI0) and two diets in which 25% (HI25) and 50% (HI50) of FM was replaced with HI larvae meal. A 78-day feeding trial involved 360 rainbow trout with 178.9 ± 9.81 g initial average weight. Dietary replacement of up to 50% of FM with HI larvae meal, without any Met supplementation, did not negatively affect rainbow trout growth parameters and hepatic Met metabolism. In particular, Met availability from the insect-based diets directly modulated the transcript levels of two out of three target genes (CBS, SAHH) to maintain an optimal level of one-carbon metabolic substrates, i.e., the SAM:SAH ratio in the hepatic tissue.

In aquaculture research, one important aim of gut microbiota studies is to provide the scientific basis for developing effective strategies to manipulate gut microbial communities through the diet, promoting fish health and improving productivity. Currently, there is an increasing commercial and research interest towards the use of organic acids in aquafeeds, due to several beneficial effects they have on growth performance and intestinal tract’s health of farmed fish. Among organic acids, monoglycerides of short-chain fatty acids (SCFAs) and medium-chain fatty acids (MCFAs) have attracted particular research attention also for their bacteriostatic and bactericidal properties. Accordingly, the present study aimed to evaluate the potential beneficial effects of SCFA and MCFA monoglycerides, used as a feed additive, on fish growth performance, and intestinal microbiota composition. For this purpose, a specific combination of short- and medium-chain 1-monoglycerides (SILOhealth 108Z) was tested in 600 juvenile gilthead sea bream (Sparus aurata) of about 60 g mean initial weight that were fed for 90 days with plant-based diets. Two isoproteic and isolipidic diets were formulated. The control fish group received a plant-based diet, whereas the other group received the same control feed, but supplemented with 0.5% of SILOhealth 108Z. The Illumina MiSeq platform for high-throughput amplicon sequencing of 16S rRNA gene and QIIME pipeline were used to analyse and characterize the whole microbiome associated both to feeds and S. aurata intestine. The number of reads taxonomically classified according to the Greengenes database was 394,611. We identified 259 OTUs at 97% identity in sea bream fecal samples; 90 OTUs constituted the core gut microbiota. Firmicutes, Proteobacteria and Actinobacteria represented the dominant phyla in both experimental groups. Among them, relative abundance of Firmicutes and Proteobacteria were positively and negatively affected by dietary SCFA monoglycerides supplementation, respectively. In summary, our findings clearly indicated that SILOhealth 108Z positively modulated the fish intestinal microbiota by increasing the number of beneficial lactic acid bacteria, namely, Lactobacillus, and reducing Gammaproteobacteria, which include several potential pathogenic bacteria. The specific composition of 1-monoglycerides of short- and medium-chain fatty acids contained in SILOhealth 108Z could thus have a great potential as a feed additive in aquaculture.

We evaluated the chemical coding of the myenteric plexus in the proximal and distal intestine of gilthead sea bream (Sparus aurata), which represents one of the most farmed fish in the Mediterranean area. The presence of nitric oxide (NO), acetylcholine (ACh), serotonin (5-HT), calcitonin-gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal peptide (VIP) containing neurons, was investigated in intestinal whole mount preparations of the longitudinal muscle with attached the myenteric plexus (LMMP) by means of immunohistochemical fluorescence staining. The main excitatory and inhibitory neurochemicals identified in intestinal smooth muscle were ACh, SP, 5HT, and NO, VIP, CGRP. Some neurons displayed morphological features of ascending and descending interneurons and of putative sensory neurons. The expression of these pathways in the two intestinal regions is largely superimposable, although some differences emerged, which may be relevant to the morphological properties of each region. The most important variances are the higher neuronal density and soma size in the proximal intestine, which may depend on the volume of the target tissue. Since in the fish gut the submucosal plexus is less developed, myenteric neurons substantially innervate also the submucosal and epithelial layers, which display a major thickness and surface in the proximal intestine. In addition, myenteric neurons containing ACh and SP, which mainly represent excitatory motor neurons and interneurons innervating the smooth muscle were more numerous in the distal intestine, possibly to sustain motility in the thicker smooth muscle coat. Overall, this study expands our knowledge of the intrinsic innervation that regulates intestinal secretion, absorption and motility in gilthead sea bream and provides useful background information for rational design of functional feeds aimed at improving fish gut health.

Animal by-product meals from the rendering industry could provide a sustainable and commercially viable alternative to fishmeal (FM) in aquaculture, as they are rich in most essential amino acids and contain important amounts of water-soluble proteins that improve feed digestibility and palatability. Among them, poultry by-product meal (PBM) have given encouraging results in rainbow trout (Oncorhynchus mykiss). However, the introduction of new ingredients in the diet needs to be carefully evaluated since diet is one of the main factors affecting the gut microbiota, which is a complex community that contributes to host metabolism, nutrition, growth, and disease resistance. Accordingly, we investigated the effects of partial replacement of dietary FM with a mix of animal by-product meals and plant proteins on intestinal microbiota composition of rainbow trout in relation to growth and feeding efficiency parameters. We used 1540 trout with an initial mean body weight of 94.6 ± 14.2 g. Fish were fed for 12 weeks with 7 different feed formulations. The growth data showed that trout fed on diets rich in animal by-product meals grew as well as fish fed on control diet, which was rich in FM (37.3%) and PBM-free. High-throughput 16S rRNA gene amplicon sequencing (MiSeq platform, Illumina) was utilised to study the gut microbial community profile. After discarding Cyanobacteria (class Chloroplast) and mitochondria reads a total of 2,701,274 of reads taxonomically classified, corresponding to a mean of 96,474 ± 68,056 reads per sample, were obtained. Five thousand three hundred ninety-nine operational taxonomic units (OTUs) were identified, which predominantly mapped to the phyla of Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. The ratio between vegetable and animal proteins proved to play a central role in determining microbiome profiles and Firmicutes and Proteobacteria phyla were particularly discriminatory for diet type in trout. Plant ingredients favoured a higher Firmicutes:Proteobacteria ratio than animal proteins. Acceptable abundance of Firmicutes was guaranteed by including at least 25% of vegetable proteins in the diet regardless of animal protein source and percentage. In summary animal by-product meals, as replacements to FM, gave good results in terms of growth performances and did not induce significant changes in gut microbial richness, thus proving to be a suitable protein source for use in rainbow trout aqua feed.

Substituting fishmeal (FM) with vegetable meal (VM) can markedly affect the mineral composition of feeds, and may require additional mineral supplementation. Their bioavailability and optimal supplementation levels depend also on the form of delivery of minerals. The aim of the study was to determine the effect of different delivery forms of three major trace elements (Zn, Mn and Se) in a marine teleost. Gilthead sea bream juveniles of 22.5 g were fed a VM-based diet for 12 weeks that was either not supplemented with these minerals or supplemented with inorganic, organic, or encapsulated inorganic forms of minerals in triplicate and compared to a FM-based diet. Our results showed that mineral delivery form significantly affected the biochemical composition and morphology of posterior vertebrae. Supplementation of VM-based diets with inorganic forms of the target minerals significantly promoted growth, increased the vertebral weight and content of ash and Zn, enhanced bone mineralization and affected the vertebral shape. Conversely, encapsulation of inorganic minerals reduced fish growth and vertebral mineral content, whereas supplementation of organic minerals, enhanced bone osteogenesis by upregulating bone morphogenetic protein 2 (bmp2) gene and produced vertebrae with a larger length in relation to height. Furthermore, organic mineral forms of delivery downregulated the expression of oxidative stress related genes, such as Cu/Zn superoxide dismutase (Cu/Zn sod) and glutathione peroxidase 1 (gpx-1), suggesting thus that dietary minerals supplemented in the organic form could be reasonably considered more effective than the inorganic and encapsulated forms of supply.

Taurine (Tau) is an amino sulfonic acid, which is widely distributed in animal tissues, whereas it is almost lacking in plants with the exception of certain algae, seaweeds, and few others. In the aquafeed industry, Tau is mainly used as a feed additive to promote growth in marine fish species with limited cysteine sulfinate decarboxylase activity. In particular, Tau supplementation is required in feeds in which fishmeal (FM) is substituted with high percentages of plant-derived protein sources such as soybean meals (SBM) that have much lower levels of Tau than FM. In addition to being a growth promoter, Tau exert powerful antioxidant properties being a scavenger of the reactive oxygen species (ROS). Under sustained swimming conditions, an intracellular increase in ROS production can occur in fish red muscle where the abundance of mitochondria (the main site of ROS formation) is high. Accordingly, this study aimed at investigating the effects of dietary Tau on European seabass (Dicentrarchus labrax) growth and oxidative stress response induced by swimming exercise. Individually tagged fish of 92.57 ± 20.33 g mean initial weight were fed two experimental diets containing the same low percentage of FM and high percentage of SBM. One diet was supplemented with 1.5% of Tau. Tau supplemented in the diet had a positive effect on fish growth, and enhanced swimming performance and antioxidant status. Two swim endurance tests were performed during the feeding trial. Metabolic oxygen consumption (MO2) was measured during exercise at incremental swimming speeds (0.7, 1.4, 2.1, 2.8, 3.5, and then 4.2 BL (body length) s−1, until fatigue). Fish maximal sustainable swimming speed (Ucrit) was determined too. To investigate the antioxidant effect of dietary Tau, we also measured ROS production in fish blood by RBA (respiratory burst activity) assay and quantified the expression of genes coding for antioxidant enzymes by qPCR (quantitative polymerase chain reaction) , such as SOD (superoxide dismutase), GPX (glutathione peroxidase), and CAT (catalase) in red muscle and liver. There was a significant effect of Tau upon Ucrit during exercise. Additionally, ROS production was significantly lower in fish fed with Tau supplemented diet, supporting the role of Tau as ROS scavenger. The protective effect of Tau against oxidative stress induced by forced swimming was denoted also by a significant decrease in antioxidant enzymes gene expression in fish liver and muscle. Taken together these results demonstrate that Tau is beneficial in low FM-based diets for seabass.

The worldwide growing consumption of proteins to feed humans and animals has drawn a considerable amount of attention to insect rearing. Insects reared on organic wastes and used as feed for monogastric animals can reduce the environmental impact and increase the sustainability of meat/fish production. In this study, we designed an environmentally closed loop for food supply in which fruit and vegetable waste from markets became rearing substrate for Hermetia illucens (BSF— black soldier fly). A vegetable and fruit-based substrate was compared to a standard diet for Diptera in terms of larval growth, waste reduction index, and overall substrate degradation. Morphological analysis of insect organs was carried out to obtain indications about insect health. Processing steps such as drying and oil extraction from BSF were investigated. Nutritional and microbiological analyses confirmed the good quality of insects and meal. The meal was then used to produce fish feed and its suitability to this purpose was assessed using trout. Earthworms were grown on leftovers of BSF rearing in comparison to a standard substrate. Chemical analyses of vermicompost were performed. The present research demonstrates that insects can be used to reduce organic waste, increasing at the same time the sustainability of aquaculture and creating interesting by-products through the linked bio-system establishment.

This study evaluated the effects of dietary insect meal from Hermetia illucens larvae on autochthonous gut microbiota of rainbow trout (Oncorhynchus mykiss). Three diets, with increasing levels of insect meal inclusion (10%, 20%, and 30%) and a control diet without insect meal were tested in a 12-week feeding trial. To analyze the resident intestinal microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and QIIME pipeline were used. The number of reads taxonomically classified according to the Greengenes database was 1,514,155. Seventy-four Operational Taxonomic Units (OTUs) at 97% identity were identified. The core of adhered intestinal microbiota, i.e., OTUs present in at least 80% of mucosal samples and shared regardless of the diet, was constituted by three OTUs assigned to Propiobacterinae, Shewanella, and Mycoplasma genera, respectively. Fish fed the insect-based diets showed higher bacterial diversity with a reduction in Proteobacteria in comparison to fish fed the fishmeal diet. Insect-meal inclusion in the diet increased the gut abundance of Mycoplasma, which was attributed the ability to produce lactic and acetic acid as final products of its fermentation. We believe that the observed variations on the autochthonous intestinal microbiota composition of trout are principally due to the prebiotic properties of fermentable chitin.

Bacteria that inhabit the epithelium of the animals' digestive tract provide the essential biochemical pathways for fermenting otherwise indigestible dietary fibers, leading to the production of short-chain fatty acids (SCFAs). Of the major SCFAs, butyrate has received particular attention due to its numerous positive effects on the health of the intestinal tract and peripheral tissues. The mechanisms of action of this four-carbon chain organic acid are different; many of these are related to its potent regulatory effect on gene expression since butyrate is a histone deacetylase inhibitor that play a predominant role in the epigenetic regulation of gene expression and cell function. In the present work, we investigated in the European sea bass (Dicentrarchus labrax) the effects of butyrate used as a feed additive on fish epigenetics as well as its regulatory role in mucosal protection and immune homeostasis through impact on gene expression. Seven target genes related to inflammatory response and reinforcement of the epithelial defense barrier [tnfα (tumor necrosis factor alpha) il1β, (interleukin 1beta), il-6, il-8, il-10, and muc2 (mucin 2)] and five target genes related to epigenetic modifications [dicer1(double-stranded RNA-specific endoribonuclease), ehmt2 (euchromatic histone-lysine-N-methyltransferase 2), pcgf2 (polycomb group ring finger 2), hdac11 (histone deacetylase-11), and jarid2a (jumonji)] were analyzed in fish intestine and liver. We also investigated the effect of dietary butyrate supplementation on histone acetylation, by performing an immunoblotting analysis on liver core histone extracts. Results of the eight-week-long feeding trial showed no significant differences in weight gain or SGR (specific growth rate) of sea bass that received 0.2% sodium butyrate supplementation in the diet in comparison to control fish that received a diet without Na-butyrate. Dietary butyrate led to a twofold increase in the acetylation level of histone H4 at lysine 8, but showed no effect on the histone H3 at Lys9. Moreover, two different isoforms of histone H3 that might correspond to the H3.1 and H3.2 isoforms previously found in terrestrial animals were separated on the immunoblots. The expression of four (il1 β, il8, irf1, and tnfα) out of seven analyzed genes related to mucosal protection and inflammatory response was significantly different between the two analyzed tissues but only il10 showed differences in expression due to the interaction between tissue and butyrate treatment. In addition, butyrate caused significant changes in vivo in the expression of genes related to epigenetic regulatory mechanisms such as hdac11, ehmt2, and dicer1. Statistical analysis by two-way ANOVA for these genes showed not only significant differences due to the butyrate treatment, but also due to the interaction between tissue and treatment.

Dietary fatty acids have been shown to exert a clear effect on the stress response, modulating the release of cortisol. The role of fatty acids on the expression of steroidogenic genes has been described in mammals, but little is known in fish. The effect of different fatty acids on the release of cortisol and expression of stress-related genes of European sea bass (Dicentrarchus labrax) head kidney, induced by a pulse of adenocorticotrophin hormone (ACTH), was studied. Tissue was maintained in superfusion with 60 min of incubation with EPA, DHA, arachidonic acid (ARA), linoleic acid or α-linolenic acid (ALA) during 490 min. Cortisol was measured by RIA. The quantification of stress-related genes transcripts was conducted by One-Step TaqMan real-time RT-PCR. There was an effect of the type of fatty acid on the ACTH-induced release of cortisol, values from ALA treatment being elevated within all of the experimental period. The expression of some steroidogenic genes, such as the steroidogenic acute regulatory protein (StAR) and c-fos, were affected by fatty acids, ALA increasing the expression of StAR after 1 h of ACTH stimulation whereas DHA, ARA and ALA increased the expression of c-fos after 20 min. ARA increased expression of the 11β-hydroxylase gene. Expression of heat shock protein 70 (HSP70) was increased in all the experimental treatments except for ARA. Results corroborate previous studies of the effect of different fatty acids on the release of cortisol in marine fish and demonstrate that those effects are mediated by alteration of the expression of steroidogenic genes.

The objective of the present study was to examine the effect of diets with descending fish meal (FM) inclusion levels and the addition of salt to the diet containing the lowest FM level on growth performances, feed conversion ratio, and intestinal solute carrier family 6 member 19 (SLC6A19) and oligopeptide transporter 1 (PEPT1) transcript levels, in freshwater-adapted European sea bass (Dicentrarchus labrax). We first isolated by molecular cloning and sequenced a full-length cDNA representing the neutral amino acid transporter SLC6A19 in sea bass. The cDNA sequence was deposited in GenBank database (accession no. KC812315). The twelve transmembrane domains and the ‘de novo’ prediction of the three-dimensional structure of SLC6A19 protein (634 amino acids) are presented. We then analysed diet-induced changes in the mRNA copies of SLC6A19 and PEPT1 genes in different portions of sea bass intestine using real-time RT-PCR. Sea bass were fed for 6 weeks on different diets, with ascending levels of fat or descending levels of FM, which was replaced with vegetable meal. The salt-enriched diet was prepared by adding 3 % NaCl to the diet containing 10 % FM. SLC6A19 mRNA in the anterior and posterior intestine of sea bass were not modulated by dietary protein sources and salt supplementation. Conversely, including salt in a diet containing a low FM percentage up-regulated the mRNA copies of PEPT1 in the hindgut. Fish growth correlated positively with the content of FM in the diets. Interestingly, the addition of salt to the diet containing 10 % FM improved feed intake, as well as specific growth rate and feed conversion ratio.

Aquatic hypoxia is a frequent event and in fish a complex set of physiological and bi- ochemical alterations are employed to cope with this environmental stress. Many of these adjustments depend to a large extent on changes in the expression of genes that encode for physiologically relevant proteins. Genes that are induced by hypoxia appear to share a common mode of transcriptional regula- tion. This induction depends upon activation of a transcription factor, the hypoxia inducible factor-1 (HIF-1), which is a heterodimer composed of two subunits: α ‧ and β. In this study we report first on the molecular cloning and characterization of HIF-1α ‧ in sea bass (Dicentrarchus labrax). The full-length sea bass cDNA for HIF-1α was isolated and deposited in the GenBank with accession no. DQ171936. It consists of 3317 base pairs (bp) carrying a single open-read- ing frame that encompasses 2265 bp of the coding region and 1052 bp of the 3’ UTR. We then utilized the real-time PCR technology to monitor dynamic changes in levels of HIF-1α ‧ tran- scripts, in response to acute and chronic hypoxic stress. The number of HIF-1α ‧ mRNA copies were signifi- cantly increased in response to both acute (1.9 mg/L, dissolved oxygen for 4 hours) and chronic (4.3 mg/L, DO for 15 days) hypoxia in sea bass, whereas it remained unchanged in fish exposed to hyperoxic (DO 13.5±1.2 mg/L, 155 % saturation) conditions. This is the first study carried out to investigate the behaviour of HIF-1α gene transcripts during hypoxia in representative of marine hypoxia-sensitive fish species.

Astaxanthin is one of the major carotenoids in aquatic animals including salmonid fishes and is the preferred pigments added to salmon feed. It’s also a powerful antioxidant compared to other carotenoids and that may confer numerous health benefits. The aim of the present experi- ment was to investigate the effect of Astaxanthin deposition on the lipids peroxidation by studying the Malondialdeide (MDA) level in muscle of rainbow trout (Oncorhynchus mykiss). The Astaxanthin concentrations in fish fed with a commercial sources as Lucantin®Pink (BASF Ludwigshafen, Ger- many) reached values to 5.76±0.18x10-3 mg/g after 50 days feeding, while the MDA concentration de- creased from 1.56x103 to 0.45x103 ng/g. The correlation between MDA and Astaxanthin concentrations decreased linearly and confirmed the antioxidant properties of the pigment by reducing the lipids peroxidation.

BACKGROUND: Calpains, a superfamily of intracellular calcium-dependent cysteine proteases, are involved in the cytoskeletal remodeling and wasting of skeletal muscle. Calpains are generated as inactive proenzymes which are activated by N-terminal autolysis induced by calcium-ions. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we characterized the full-length cDNA sequences of three calpain genes, clpn1, clpn2, and clpn3 in channel catfish, and assessed the effect of nutrient restriction and subsequent re-feeding on the expression of these genes in skeletal muscle. The clpn1 cDNA sequence encodes a protein of 704 amino acids, Clpn2 of 696 amino acids, and Clpn3 of 741 amino acids. Phylogenetic analysis of deduced amino acid sequences indicate that catfish Clpn1 and Clpn2 share a sequence similarity of 61%; catfish Clpn1 and Clpn3 of 48%, and Clpn2 and Clpn3 of only 45%. The domain structure architectures of all three calpain genes in channel catfish are similar to those of other vertebrates, further supported by strong bootstrap values during phylogenetic analyses. Starvation of channel catfish (average weight, 15-20 g) for 35 days influenced the expression of clpn1 (2.3-fold decrease, P

Shellfish aquaculture is a widespread activity in the Italian peninsula. However, only two bivalve species are mainly cultured along the coastline of that country: the Mediterranean mussel Mytilus galloprovincialis and the Manila clam Venerupis philippinarum (Ruditapes philippinarum). By contrast, just a few other mollusc species of commercial interest are scarcely reared at a small-scale level. After analysing the current status of Italian shellfish production, this paper reports and discusses the potential for culturing several different invertebrate species [i.e., the European flat oyster Ostrea edulis, the grooved carpet shell Venerupis decussata (Ruditapes decussatus), the razor clams Ensis minor and Solen marginatus, the cephalopod Octopus vulgaris, and the purple sea urchin Paracentrotus lividus] in this country. In addition, a detailed overview of the progress made in aquacultural techniques for these species in the Mediterranean basin is presented, highlighting the most relevant bottlenecks and the way forward to shift from the experimental to the aquaculture phase. Finally, an outlook of the main economic and environmental benefits arising from these shellfish culture practices is also given.

Fundamentally, in land based mediterranean aquaculture, two techniques are applied to supply water with oxygen: paddling water aeration and application of pure oxygen. The two oxygenation techniques result in quite different PO2 regimens and, consequently, different fish growth performance and gill morphology. Data exist showing a reduction in total respiratory surface (RSA) and increasing gas diffusion distance (GDD) in gills of sea bass (Dicentrarchus labrax, L.) farmed under elevated PO2 regimens. That such a modification might have an effect on the ion regulation has been defined elsewhere as osmorespiratory compromise. In this study, European sea bass previously acclimatized to two PO2 regimens, mild hypoxia and mild hyperoxia (70-80% and 130-140% of the saturation value, respectively), were challenged for 1 hour with hypo-osmotic plus manipulation stress in two separate trials. During the first trial, when only Na+ loss was determined, the ion efflux during the first 5 min resulted in a rate of 163.72±31 and 112.23±87 nmol g-1min-1 from hypoxia and hyperoxia sea bass groups, respectively, and, if sustained, would approach 15.3 and 11.2% per hour of the total body Na+, respectively. During the second trial, in which both Na+ and Cl- loss were determined, after 60 min the Na+ loss was shown to be 76.86±12 and 179.28±32 nmol g-1 min-1 for the fish previously acclimatized to hyperoxia and hypoxia regimens, respectively, whereas for Cl- this loss was 62.02±11 and 157.28±28 nmol g-1min-1, respectively. Our data are compatible with the hypothesis of an osmotic advantage of sea bass exposed to an elevated PO2 regimen, achievable with application of pure oxygen, instead of simple water aeration.

Eurasian perch (P. fluviatilis) is a very important fish species in Varese lake (N-W Italy). Since the second half of 20th century, perch catches in the lake have steadily decreased and by the end of the ‘80s the species resulted clearly endangered. The purpose of this study was to investigate growth, mortality and feeding conditions of perch postlarvae, reared in illuminated floating cage in Varese lake, to obtain fingerlings for a restocking program. In June 2006 and 2007, groups of 280 and 300 pre-weaned post-larvae (average body weight 0.64±0.09 g and 0.25±0.08 g respectively P