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The replacement of fishmeal (FM) with economical and nutritious FM alternatives including plant- and animal-based protein ingredients has become a global research priority. However, the presence of several anti-nutritional factors (ANFs) in these alternatives may impair nutrient utilization in fish. The supplementation of exogenous protease as feed additives could be an effective approach to improve the nutrient digestibility of these alternative proteins. Proteases are protein-digesting enzymes that catalyze the hydrolysis of complex protein macromolecules into simpler amino acids. Exogenous protease supplementation stimulates the activities of endogenous proteolytic enzymes for better nutrient digestion and absorption in fish. This review article summarizes the optimum protease supplementation levels in various fish species and its beneficial effects on growth performance, nutrient digestibility, intestinal morphology, digestive enzyme activities, whole-body composition, physiochemical parameters, immunity, and gene expression of fish. Moreover, the interactive effects of exogenous protease with organic acids and probiotics are also discussed. Overall, the supplementation of exogenous protease in fish feed is a viable solution for poor nutrient utilization of FM alternatives. Important gaps, however, exist in the current knowledge, particularly with regard to the effect of protease supplementation in animal-based proteins as most of the studies have focused on the effect of protease supplementation on plant-based proteins as FM alternatives. Moreover, additional studies focused on the effect of protease supplementation on the digestive enzyme activities, immunity, gut microbes and antioxidant parameters of fish will provide the basis for a better understanding of the mode of action of protease. The scarce knowledge in these areas limits the scope of protease supplementation as a feed additive in the aquaculture sector. [ABSTRACT FROM AUTHOR]

The study evaluated the effects of dietary phosphorus supplementation on the fishmeal replacement with Clostridium autoethanogenum protein (CAP) in the diet of L. vannamei. Four isonitrogenous and isolipid diets were formulated: the PC diet contains 25% fishmeal, the NC, P1 and P2 diets were replaced 40% fishmeal with CAP and supplemented with 0, 0.8 and 1.6% NaH 2 PO 4 respectively (equivalent to dietary phosphorus level of 0.96%, 1.12% and 1.27%). Sampling and V. parahaemolyticus challenge test were conducted after 50-day-feeding (initial shrimp weight 1.79 ± 0.02 g). The results showed that there were no significant differences in the growth performance of shrimp among the 4 groups. The expressions of dorsal in the gut were significantly lower in shrimp fed the P1 and P2 diets than shrimp fed the NC diet and the expression of peroxinectin in the gut was lower in shrimp fed the NC diet than others. The cumulative mortality of shrimp after V. parahaemolyticus challenge was significantly lower in shrimp fed the P2 diet than those fed the NC diet. After the challenge, genes expressions related to the prophenoloxidase activating system (proPO , lgbp , ppaf) were inhibited in the hepatopancreas of shrimp fed NC diet but activated in shrimp fed the P1 diet compared to those fed the PC diet. The AKP and T-AOC activities were higher in shrimp fed the P2 diet than those fed the other diets. The thickness of muscle layer of shrimp fed the P1 diet was thicker than that in the other groups, and significant stress damage happened in the midgut of the shrimp fed the NC diet. The abundance of Pseudoalteromonas , Haloferula and Ruegeria in shrimp fed the P1 diet was higher than those fed the other diets, while Vibrio in shrimp fed the P2 diet was higher than those fed the other diets. This indicated that a low fishmeal diet with dietary phosphorus level of 1.12% could improve the histology, enhance immune response, and increase the abundance of beneficial bacteria in the gut of shrimp. The low fishmeal diet with dietary phosphorus level of 1.27% could improve disease resistance and antioxidant capacity, but there was a possibility of damage to the gut histology as well as increasing abundance of Vibrio in the gut microbiota of shrimp. • The feasibility of supplementing Dietary phosphorus based on the 40% fishmeal replacement with C. autoethanogenum protein in the diet of Pacific white shrimp. • Dietary phosphorus level of 1.12% could improve the health of the gut of shrimp, while the level of 1.27% could enhance the disease resistance. [ABSTRACT FROM AUTHOR]

We evaluated the growth, gut histomorphometry, selected immune responses and resistance of Nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae infection when fed VILIGEN™, an additive blend containing sodium butyrate, dehydrated hydrolysed yeast and zinc proteinate. Increasing doses (0.00, 0.60, 1.20, 2.40 and 4.80 g kg−1) were added to a basal diet and randomly assigned to six groups, each with 28 fish. The fish were fed to satiation twice daily for 60 days, infected via gavage with S. agalactiae at 1.0 × 109 CFU ml−1, and mortality was recorded for 14 days. Dietary inclusion of VILIGEN™ promoted higher weight gain, gut‐fold perimeter and the number of goblet cells. Through polynomial regression, the optimal inclusion level for these variables was estimated at 2.80, 3.30 and 3.50 g kg−1, respectively. However, there was no clear effect of the additive on the immune responses tested before bacterial infection or in the survival after challenge. This is the first study on VILIGEN™ supplementation in fish diets. Higher growth and altered gut morphology were seen with the inclusion of VILIGEN™ in the range of 2.80 to 3.50 g kg−1 but the response to the bacterial challenge was not clearly affected. [ABSTRACT FROM AUTHOR]

To develop specific recommendations on the feeding strategies for farmed lumpfish, four duplicate groups (n = 70; N = 280) of lumpfish with a mean ± SD start weight of 30.4 ± 5.3 g were distributed among eight sea cages (5 × 5 × 5 m) each stocked with 220 one year old Atlantic salmon with a mean (± SD) weight of 1485.9 ± 213.2 g. The fish were fed either pelleted feed at 3% BW−1 or with feed blocks at 1.5%, 2% and 3% BW−1 respectively for 66 days. There were clear differences in growth performance between the four treatment groups with lumpfish fed with pelleted feed attaining the highest weight gain. There were only minor histopathological changes observed between the dietary treatments with mainly mild focal and multifocal inflammation observed in sampled tissues. There were no significant differences in liver vacuolisation between the dietary groups and baseline samples with most of the livers evaluated appeared to be vacuolized within what is regarded as normal range for all four treatment groups. Only minor differences in welfare score between the four treatment groups were found. The incidence of cataracts varied between the treatment groups and was correlated to differences observed in growth between the four groups. Lumpfish fed once daily from automatic feeders with pelleted feed had the highest incidence and severity of cataracts. This study suggests that feeding lumpfish with moderate amounts of feed blocks (≤ 2% BW−1) may be advantageous for maintaining slow growth and good welfare in salmon cages, and this should be further tested in large scale studies in commercial salmon farms. • We assessed growth, cataract development, welfare score and gut histopathology in lumpfish fed either pelleted feed or with feed blocks. • Lumpfish fed with pelleted feed attaining the highest weight gain. • Lumpfish fed with pelleted feed had the highest incidence and severity of cataracts. • Only minor histopathological changes observed between the dietary treatments. • Feeding lumpfish with moderate amounts of feed blocks (≤ 2% B−1) in salmon cages is recommended. [ABSTRACT FROM AUTHOR]

Abstract Three duplicate groups of individually tagged lumpfish (mean initial weight: 22.3 ± 2.5 g) were fed either daily (7DW); four days per week (4DW) or three days per week (3DW) at a feeding rate of 2% body weight day−1 for a period of 126 days. There were significant differences in growth rates between the groups with the 7DW fish having the highest growth rates. Cataract prevalence was 53% lower in the 3DW group compared to the 7DW group. Histological examination showed that in some individuals in all three groups there was moderate expansion of the lamina propria in the mid and hind gut regions of the intestine with tissue most likely to represent fibrous tissue with scattered leucocytes. The severity of inflammation appeared to increase the more frequent the fish were fed with the 7DW fish having the highest inflammation score in these tissues. Feeding fish daily also resulted in higher levels of liver vacuolisation and chronic inflammation of the lamina propria in the mid and hind gut region of the intestines. Results from the present study show that restricted feeding regimes can be used to control growth and improve gut and eye health in lumpfish. Highlights • We assessed growth, cataract development and gut histopathology in lumpfish feed 7, 4 and 3 days per week (DW). • Growth and cataract prevalence was lowest in the 3DW group. • Liver vacuolisation and chronic inflammation in the gut was higher in the 7DW group. [ABSTRACT FROM AUTHOR]

Changing trends in fish feed formulation, with progressively higher inclusion levels of plant ingredients, are invariably introducing more fibre despite the fact that this component cannot be utilized by most fish. The effects of increasing insoluble dietary fibre level on growth, nutrient utilisation, blood parameters and gut health in European sea bass ( Dicentrarchus labrax L.) were studied over a period of 117 days. Moreover, investigation on digesta transit time through gastrointestinal evacuation pattern and digesta characteristics (moisture of digesta) were studied. Five isoproteic diets were formulated to contain increasing insoluble fibre levels, neutral detergent fibre, NDF (7.2, 8.9, 11.5, 13.1 and 15.5%) derived by the inclusion of sunflower hulls and soybean hulls. No significant differences due to fibre inclusion levels were observed in final body weight, specific growth rate, feed intake, feed conversion rate, protein and lipid efficiency. No significant differences in serum total protein, glucose, triglycerides, alkaline phosphatase and inorganic phosphorous were found. All the histological sections showed normal intestinal architecture, and inflammatory and/or degenerative changes were not present in any histological section from all subjects examined. The investigation into gastrointestinal evacuation pattern revealed no significant differences between treatments, however higher dietary fibre levels seem to increase the time required to empty the stomach while the time required to empty 90% of the hindgut content was similar in all the treatments: around 46–47 h. No differences were found between diets in the moisture content of digesta along the digestive tracts. We can conclude that the different insoluble fibre levels tested in this trial have no effects on overall performances and feed efficiency in European sea bass. Results from blood biochemistry profile and histology confirm good nutritional and health status of fish under all feeding treatments. The inclusion of fibre had no influence on digesta transit time. In formulation of feed for the on-growing of European sea bass insoluble fibre derived from sunflower hulls and soybean hulls can be included at a level of up to 15.5%. [ABSTRACT FROM AUTHOR]

Abstract: Three duplicate groups of individually tagged lumpfish (mean initial weight: 20.0 ± 4.0 g) were fed three commercially available diets (A, B and C) for a period of 147 days. There were significant differences in growth rates between the groups with fish fed diet A having the highest growth rates whilst fish fed diet C achieved the lowest growth. Lumpfish fed diets A and B had lower prevalence and severity of cataracts compared with fish fed diet C, suggesting a possible dietary effect on cataract, alone or in combination with other environmental factors. There was a significant difference in the degree of liver vacuolization between the three dietary treatments at the end of the study period with fish fed diets A and B showing moderate to severe vacuolization compared with liver tissue from fish fed diet C, which had milder vacuolizations. There was little or no inflammation in pyloric caeca, midgut and hindgut and similarly for epithelial necrosis or vacuolization for all three dietary treatments at the end of the study. Fish fed diet A had the longest intestinal folds whilst fish fed diet C had the shortest. [ABSTRACT FROM AUTHOR]

The present study was carried out to evaluate growth, gut histology and gut bacterial community of gilthead sea bream ( Sparus aurata ) fed with increasing dietary soybean meal (SBM) levels in a low fishmeal (FM) based diet, in comparison with a control diet. Five isoproteic and isolipidic experimental diets were formulated to contain increasing levels of SBM (0, 100, 200, and 300 g kg −1 named S0, S10, S20 and S30, respectively) with 150 g kg −1 of FM, and one control diet (C) without SBM and containing 350 g kg −1 of FM. Sixty sea bream (initial body weight 75.9 ± 1.9 g, n = 900) per tank were reared in a recirculation system at 23.0 ± 1.0 °C and fed to satiation. The trial was run in triplicate and lasted 100 days. At the end of the trial fish fed the S30 diet showed a higher ( P ≤ 0.05) specific growth rate (SGR) compared to S0 (SGR, 1.17 ± 0.03, 1.20 ± 0.01, 1.22 ± 0.01, 1.25 ± 0.01 and 1.21 ± 0.04 for S0, S10, S20, S30 and C, respectively), and a higher feed intake (FI) compared to S0, S10 and S20. Sea bream fed the C diet had a higher ( P ≤ 0.05) FI compared to S0 (FI, 1.40 ± 0.01, 1.45 ± 0.01, 1.44 ± 0.03, 1.51 ± 0.03 and 1.46 ± 0.02 for S0, S10, S20, S30 and C, respectively). No significant differences in feed conversion rate, protein efficiency ratio, gross protein efficiency and gross lipid efficiency among the treatments were detected. No specific histopathological changes indicative of soy-induced enteritis were observed in the intestine of any fish examined. Gut bacterial community of the distal intestine content was analyzed by Next-Generation Sequencing. At the phylum level, the gut bacterial community was dominated by Firmicutes (relative abundance 71%), while the most represented family was Lactobacillaceae (26%). Even if no significant differences ( P ≤ 0.05) in the gut bacterial community α and β-diversity according to the different diets were detected, Cyanobacteria and Lactobacillaceae progressively increased from diet C to diet S30. In conclusion results of growth, nutrient utilization, gut histology and gut bacterial community indicate that SBM can be successfully incorporated up to a level of 300 g kg −1 with the inclusion of 150 g kg −1 of FM, without any deleterious effects on growth, protein utilization and gut health during the on-growing of sea bream. [ABSTRACT FROM AUTHOR]

Abstract: A trial on turbot (Psetta maxima) juveniles was undertaken to evaluate the effect of replacing dietary fishmeal (FM) with a complementary mixture of plant proteins (PP) consisting of soybean meal (SBM), wheat gluten meal (WGM) and corn gluten meal (CGM). Four practical diets were formulated to progressively replace 25% (PP25), 39% (PP39), 52% (PP52) and 66% (PP66) of FM protein. Forty animals per tank (initial weight 24.2±4.4g) were randomly distributed into twelve 450-liter square tanks connected to a closed recirculation system (temperature 18±1°C). The diets were tested in triplicate for 77days. Final weight ranged from 126.6g (PP25) to 99.5g (PP66). Voluntary feed intake of group PP66 (55.5gfish−1) was significantly lower than in PP25. Specific growth rate in fish fed diet PP25 (2.14% day−1) was significantly higher than those found both in PP52 (2.01% day−1) and PP66 (1.82% day−1) groups. Feed conversion ratio (FCR) in groups PP25 and PP39 (0.66–0.67) was lower in comparison with those of the other groups (FCR 0.70–0.74). None of the diets affected whole-body composition and hepatosomatic index. Condition factor significantly decreased with increasing dietary PP (2.06, 2.02, 1.97 and 1.91) whereas a significant increase in viscerosomatic index was observed in fish fed PP52 and PP66 (6.70–6.95) in comparison with the other two groups (6.31–6.21). Reduced protein retention was found in groups fed diet PP52 and PP66, with protein efficiency ratio (PER) ranging from 2.71 to 2.63 and gross protein efficiency (GPE) from 40.3 to 40.0, in comparison with the other two groups (PER 2.83 and GPE 45.5–45.8), even though apparent digestibility coefficients were not reduced. Ammonia excretion, (g total ammonia nitrogen 100gproteinintake−1), was significantly higher for group PP25 (3.41) than group PP52 (2.61). Gut histology examined in four different sites of intestine (pyloric caeca, proximal, intermediate and distal intestine) revealed no noticeable differences among fish of the various diet groups. In conclusion, substitution of a mixture of SBM, WGM and CGM for up to 52% of FM protein did not reduce feed intake, and at 39% substitution, turbot maintained optimal growth rate and nutrient utilization. Worsened FCR of fish fed diets containing higher plant protein levels was not associated with a reduced digestibility of ingredients or alterations of gut histology. [Copyright &y& Elsevier]

The present study was performed to determine the effect of soybean meal (SBM) on the performance and gut histology of gilthead sea bream and European sea bass. Three isonitrogenous and isolipidic extruded diets (crude protein, 470 g kg−1 diet; crude fat, 200 g kg−1 diet) were formulated containing 0 (0 SBM), 180 (180 SBM) and 300 (300 SBM) g kg−1 diet SBM and tested on both species in two separate experiments. Fish at an initial average weight of around 18 g were randomly allocated to 800 L square tanks connected to a closed recirculating system. The trials lasted 80 days for sea bream and 89 days for sea bass. Fish were hand-fed to apparent satiation. Increasing the level of SBM had no significant effects on the specific growth rate, feed intake and feed conversion rate in both the species. In the sea bream distal intestine, lamina propria was moderately and diffusely expanded in some fish due to an increase in cell infiltration represented by mononuclear cells, this finding being more frequent in animals fed diet 300 SBM. No other morphological alterations in intestinal folds, enterocytes or other inflammation signs were noticed in the sea bream distal intestine of any group. No histological differences were found in sea bass in any experimental group. [ABSTRACT FROM AUTHOR]