Growth, Survival, and Intestinal Health Alterations in Mediterranean Yellowtail (Seriola dumerili) Due to Alternatives to Fishmeal and Fish Oil.

Fishmeal and fish oil substitution in aquafeeds might have adverse effects on fish growth and health, mainly in carnivorous species, such as Mediterranean yellowtail (Seriola dumerili). Mediterranean yellowtail shows great potential as an alternative aquaculture species due to its fast growth and high price on the market, but the need for high-quality protein and fatty acid content in its diets is limiting its production. In order to improve the sustainability of its production, this study was conducted with 360 fish of 35 g to evaluate the effects on fish growth and health. Six diets were used: one control diet without replacement, three with FM replacement (FM66, FM33, and FM0) (33%, 66%, and 100% FM replacement), and two with FO replacement (FO50 and FO0) (50% and 100% FO replacement). The substitution of FM was with vegetable (VM) (corn gluten) and animal (AM) (krill and meat meal) meals. The reductions in FM and FO of up to 33 and 0%, respectively, did not affect the growth and survival of S. dumerili at the intestinal morphology level, except for the anterior intestine regarding the lower villi length and width and the posterior intestine regarding the lower width of the lamina propria. On the other hand, the substitution of fish ingredients in the diet affects liver morphology, indicating alterations in the major diameter of hepatocytes or their nuclei. Finally, diet did not affect the gut microbiota with respect to the control, but significant differences were found in alpha and beta diversity when FO and FM microbiota were compared. A 66% FM replacement and total FO replacement would be possible without causing major alterations in the fish.

Organic Ingredients as Alternative Protein Sources in the Diet of Juvenile Organic Seabass (Dicentrarchus labrax).

The use of organic ingredients as a source of protein in aquaculture diets has gained significant attention due to the growing demand for organic seafood products. This study aimed to evaluate the potential for the use of organic ingredients as protein sources in the diet of juvenile organic seabass (Dicentrarchus labrax). A total of 486 juvenile seabass with an average weight of 90 g were fed six diets containing varied organic proteins. The control group (CON) was fed a diet with conventional fishmeal from sustainable fisheries as the primary protein source. The other five groups were fed diets with different compositions: organic Iberian pig meal byproduct (IB diet), a combination of organic Iberian pig meal byproduct and insect meal (IB-IN diet), a mix of organic Iberian pig meal byproduct and organic rainbow trout meal byproduct (IB-TR diet), a blend of organic rainbow trout meal byproduct and insect meal (TR-IN), and a mixed diet containing all of these protein sources (MIX diet). Over a 125-day feeding trial, growth performance, feed utilisation, feed digestibility, and histological parameters were assessed. The results showed that the fish fed the control diet had the highest final weight and specific growth rate, followed by the fish fed the TR-IN and IB-TR diets. The IB-TR diet had the highest apparent digestibility coefficients (ADCs) for protein, while the TR-IN diet had the lowest. Histological analysis revealed that fish fed the control diet had the largest nucleus diameter and hepatocyte diameter. Use of IN seems to penalise performance in several ways. Fish fed diets containing insect meal grew less, and those diets had lower digestibility. Fish fed the TR and IB diets grew at rates near that of the control, and the feed had acceptable digestibility.

Hepatic, Muscle and Intestinal Oxidative Status and Plasmatic Parameters of Greater Amberjack (Seriola dumerili, Risso, 1810) Fed Diets with Fish Oil Replacement and Probiotic Addition.

The present study was conducted to investigate the effects of dietary fish oil replacement with a mixture of vegetable oils and probiotic supplementation on plasma biochemical parameters, oxidative stress, and antioxidant ability of Seriola dumerili. Specimens with an initial weight of 175 g were used. Four feeds were formulated with 0% (FO-100), 75% (FO-25), and 100% (FO-0 and FO-0+ with the addition of Lactobacillus probiotics) substitution of fish oil with a mixture of linseed, sunflower, and palm oils. After 109 days, no significant differences were observed in the activity of antioxidant enzymes in the liver, foregut, and hindgut, only glucose-6-phosphate dehydrogenase activity in the liver was higher in the fish fed the FO-100 diet than in those fed the FO-0 diet. No significant differences were observed in the total, reduced, and oxidized glutathione and the oxidative stress index in the liver. In addition, lipid peroxidation in the liver and red muscle values were higher in the fish fed the FO-100 diet than in the fish fed the FO-0+ diet, however, the foregut of the fish fed the FO-100 diet presented lower values than that of the fish fed the FO replacement diet, with and without probiotics. There were significant differences in cholesterol levels in the FO-100 group; they were significantly higher than those observed with the fish diets without fish oil. To sum up, fish oil can be replaced by up to 25% with vegetable oils in diets for Seriola dumerili juveniles, but total fish oil substitution is not feasible because it causes poor survival. The inclusion of probiotics in the FO-0+ diet had no effects on the parameters measured.

Effect of Additives Inclusion in Gilthead Seabream (Sparus aurata L.) Diets on Growth, Enzyme Activity, Digestibility and Gut Histology Fed with Vegetable Meals.

The fishmeal replacement by vegetable meals or other alternative sources, without affecting fish performance and productivity, is one of the principal challenges in aquaculture. The use of hydrolyzed porcine mucosa (HPM) and nucleotide (NT) concentrates, as feed additives in gilthead seabream (Sparus aurata L.) non-fishmeal diets was assessed in order to determine the possible effects on growth, feed efficiency, protein digestion, and gut histology when these were included in a plant-based diet (HPM 1% and 2%, P1 and P2; NT 250 and 500 ppm, N250 and N500), in comparison with two control diets, AA0 (100% plant-protein-based diet) and FM100 (100% fishmeal-protein-based diet). Diets were assayed in triplicate and the growth assay lasted 134 days. Results showed a significant improvement in all groups in terms of final weight and specific growth rate in comparison with the AA0 group. An improvement in the feed conversion ratio and the protein efficiency ratio was also observed when the additives were included in lower percentages (P1 and N250) compared to the FM100 group. Significant differences were found in hepatosomatic index, villi thickness, and goblet cells. Thus, the inclusion of NT and HPM was tested as beneficial for the improvement of efficiency of plant feed in seabream.

Successful Inclusion of High Vegetable Protein Sources in Feed for Rainbow Trout without Decrement in Intestinal Health.

The aquaculture of carnivorous fish is in continuous expansion, which leads to the need to reduce the dependence on fishmeal (FM). Plant proteins (PP) represent a suitable protein alternative to FM and are increasingly used in fish feed. However, PP may lead to stunted growth and enteritis. In the current study, the effect of high FM substitution by PP sources on the growth, mortality and intestinal health of rainbow trout (Oncorhynchus mykiss) was evaluated in terms of the histological intestine parameters and expression of genes related to inflammation (IL-1ß, IL-8 and TGF-ß) and immune responses (Transferrin, IgT and IFN-γ). The results show that a total substitution registered lower growth and survival rates, probably due to a disruption to the animal's health. Confirming this hypothesis, fish fed FM0 showed histological changes in the intestine and gene changes related to inflammatory responses, which in the long-term could have triggered an immunosuppression. The FM10 diet presented not only a similar expression to FM20 (control diet), but also similar growth and survival. Therefore, 90% of FM substitution was demonstrated as being feasible in this species using a PP blend of wheat gluten (WG) and soybean meal (SBM) as a protein source.

An Approach to the Spanish Consumer's Perception of the Sensory Quality of Environmentally Friendly Seabass.

Seabass is one of the leading aquaculture species in Europe. Sensory analysis is essential for new product development. This research focused on establishing and differentiating the opinion of consumers about seabass quality obtained with organic feeding. Fish were fed for 196 days with four treatments (a control diet with 30% fishmeal and three diets with different levels of fishmeal supplemented with organic vegetable ingredients: 25%, 30% and 35%). Experimental diets were compared with commercial samples from the retail industry that were considered as "adequate quality for fish". Two sensory analyses were carried out, check-all-that-apply (CATA) to obtain feedback on consumers' characterization towards a different type of fish evaluated and projective mapping (PM) to measure the similarity among a set of products and establish a comparison between results provided by both methods. According to the CATA results, white color, softness, meaty taste and juicy texture were considered relevant attributes, also showing a good relationship with an adequate cooked fish description. A penalty analysis confirmed that the previous characteristics were considered essential while fibrous was an undesirable attribute. The projective mapping showed a similar sensory configuration to the CATA, corroborating these findings that showed that commercial fish were placed in a position away from the rest of the treatments, and the organic diet with a higher level of fishmeal (35%) was the most distant from the control diet.

Intestinal Explant Cultures from Gilthead Seabream (Sparus aurata, L.) Allowed the Determination of Mucosal Sensitivity to Bacterial Pathogens and the Impact of a Plant Protein Diet.

The interaction between diet and intestinal health has been widely discussed, although in vivo approaches have reported limitations. The intestine explant culture system developed provides an advantage since it reduces the number of experimental fish and increases the time of incubation compared to similar methods, becoming a valuable tool in the study of the interactions between pathogenic bacteria, rearing conditions, or dietary components and fish gut immune response. The objective of this study was to determine the influence of the total substitution of fish meal by plants on the immune intestinal status of seabream using an ex vivo bacterial challenge. For this aim, two growth stages of fish were assayed (12 g): phase I (90 days), up to 68 g, and phase II (305 days), up to 250 g. Additionally, in phase II, the effects of long term and short term exposure (15 days) to a plant protein (PP) diet were determined. PP diet altered the mucosal immune homeostasis, the younger fish being more sensitive, and the intestine from fish fed short-term plant diets showed a higher immune response than with long-term feeding. Vibrio alginolyticus (V. alginolyticus) triggered the highest immune and inflammatory response, while COX-2 expression was significantly induced by Photobacterium damselae subsp. Piscicida (P. damselae subsp. Piscicida), showing a positive high correlation between the pro-inflammatory genes encoding interleukin 1ß (IL1-ß), interleukin 6 (IL-6) and cyclooxygenase 2(COX-2).

Recovery of Fatty Acid Composition in Mediterranean Yellowtail (Seriola dumerili, Risso 1810) fed a Fish-Oil Finishing Diet.

The present study evaluated the effects of wash-out on the fatty acid (FA) composition in the muscles of Mediterranean yellowtail. After 109 days during which fish were fed either a fish oil (FO)-based diet (FO 100) or a diet (FO 0) in which FO was completely substituted by vegetable oils, all fish were subjected to a wash-out with FO 100 diet for 90 days. The FA profile of muscles in fish fed FO 0 diet at the beginning of the experiment reflected that of dietary vegetable oils, rich in linoleic acid (LA), and α-linolenic acid (ALA), and was deficient in AA (arachidonic acid), EPA (eicosapentaenoic acid), and DHA (docosahexaenoic acid). No essential FA were fully restored in fish previously fed FO 0 diet on 45th or 90th day of wash-out. At the end of wash-out, the FA composition showed that AA, EPA, and DHA in the white muscles increased by +33%, +16%, and +43% (p < 0.001), respectively. Similarly, AA and DHA in the red muscles increased by +33% and +41% respectively, while EPA remained similar to fish fed FO 0 diet exclusively. Therefore, a 90-d wash-out can partially improve the FA profile in muscles of Mediterranean yellowtail previously fed vegetable oil-based diets.

Impact of high dietary plant protein with or without marine ingredients in gut mucosa proteome of gilthead seabream (Sparus aurata, L.).

The digestive tract, particularly the intestine, represents one of the main sites of interactions with the environment, playing the gut mucosa a crucial role in the digestion and absorption of nutrients, and in the immune defence. Previous researches have proven that the fishmeal replacement by plant sources could have an impact on the intestinal status at both digestive and immune level, compromising relevant productive parameters, such as feed efficiency, growth or survival. In order to evaluate the long-term impact of total fishmeal replacement on intestinal mucosa, the gut mucosa proteome was analysed in fish fed with a fishmeal-based diet, against plant protein-based diets with or without alternative marine sources inclusion. Total fishmeal replacement without marine ingredients inclusion, reported a negative impact in growth and biometric parameters, further an altered gut mucosa proteome. However, the inclusion of a low percentage of marine ingredients in plant protein-based diets was able to maintain the growth, biometrics parameters and gut mucosa proteome with similar values to FM group. A total fishmeal replacement induced a big set of underrepresented proteins in relation to several biological processes such as intracellular transport, assembly of cellular macrocomplex, protein localization and protein catabolism, as well as several molecular functions, mainly related with binding to different molecules and the maintenance of the cytoskeleton structure. The set of downregulated proteins also included molecules which have a crucial role in the maintenance of the normal function of the enterocytes, and therefore, of the epithelium, including permeability, immune and inflammatory response regulation and nutritional absorption. Possibly, the amino acid imbalance presented in VM diet, in a long-term feeding, may be the main reason of these alterations, which can be prevented by the inclusion of 15% of alternative marine sources. SIGNIFICANCE: Long-term feeding with plant protein based diets may be considered as a stress factor and lead to a negative impact on digestive and immune system mechanisms at the gut, that can become apparent in a reduced fish performance. The need for fishmeal replacement by alternative ingredients such as plant sources to ensure the sustainability of the aquaculture sector has led the research assessing the intestinal status of fish to be of increasing importance. This scientific work provides further knowledge about the proteins and biologic processes altered in the gut in response to plant protein based diets, suggesting the loss of part of gut mucosa functionality. Nevertheless, the inclusion of alternative marine ingredients was able to reverse these negative effects, showing as a feasible option to develop sustainable aquafeeds.

Fatty Acid Signatures in Different Tissues of Mediterranean Yellowtail, Seriola dumerili (Risso, 1810), Fed Diets Containing Different Levels of Vegetable and Fish Oils.

The study aimed to evaluate how replacing different proportions of fish oil (FO) with vegetable oils (VO) in the diet of Mediterranean yellowtail, Seriola dumerili (Risso, 1810), affects the fatty acids (FA) signature, i.e.; overall FA profile, in different tissues. A total of 225 Mediterranean yellowtail juveniles (initial live weight: 176 ± 3.62 g) were fed for 109 days with one of three diets: A control diet (FO 100), with FO as the only lipid source, or diets with 75% and 100% of FO replaced with a VO mixture. At the end of the feeding trial, the brains, muscles, livers, and visceral fat were sampled in four fish per tank (12 per treatment), and their fat were extracted and used for FA analysis. The FA signatures of red and white muscle, liver, and visceral fat tissues changed when the dietary FA source changed, whereas FA signatures in the brain were rather robust to such dietary changes. These new insights might help evaluate whether key physiological functions are preserved when fish are fed diets with low FO levels, as well as define the dietary FA requirements of Mediterranean yellowtail to improve the sustainability of the production and welfare of the fish.

Long-term feeding with high plant protein based diets in gilthead seabream (Sparus aurata, L.) leads to changes in the inflammatory and immune related gene expression at intestinal level.

BACKGROUND: In order to ensure sustainability of aquaculture production of carnivourous fish species such as the gilthead seabream (Sparus aurata, L.), the impact of the inclusion of alternative protein sources to fishmeal, including plants, has been assessed. With the aim of evaluating long-term effects of vegetable diets on growth and intestinal status of the on-growing gilthead seabream (initial weight = 129 g), three experimental diets were tested: a strict plant protein-based diet (VM), a fishmeal based diet (FM) and a plant protein-based diet with 15% of marine ingredients (squid and krill meal) alternative to fishmeal (VM+). Intestines were sampled after 154 days. Besides studying growth parameters and survival, the gene expression related to inflammatory response, immune system, epithelia integrity and digestive process was analysed in the foregut and hindgut sections, as well as different histological parameters in the foregut. RESULTS: There were no differences in growth performance (p = 0.2703) and feed utilization (p = 0.1536), although a greater fish mortality was recorded in the VM group (p = 0.0141). In addition, this group reported a lower expression in genes related to pro-inflammatory response, as Interleukine-1ß (il1ß, p = 0.0415), Interleukine-6 (il6, p = 0.0347) and cyclooxigenase-2 (cox2, p = 0.0014), immune-related genes as immunoglobulin M (igm, p = 0.0002) or bacterial defence genes as alkaline phosphatase (alp, p = 0.0069). In contrast, the VM+ group yielded similar survival rate to FM (p = 0.0141) and the gene expression patterns indicated a greater induction of the inflammatory and immune markers (il1ß, cox2 and igm). However, major histological changes in gut were not detected. CONCLUSIONS: Using plants as the unique source of protein on a long term basis, replacing fishmeal in aqua feeds for gilthead seabream, may have been the reason of a decrease in the level of different pro-inflammatory mediators (il1 ß, il6 and cox2) and immune-related molecules (igm and alp), which reflects a possible lack of local immune response at the intestinal mucosa, explaining the higher mortality observed. Krill and squid meal inclusion in vegetable diets, even at low concentrations, provided an improvement in nutrition and survival parameters compared to strictly plant protein based diets as VM, maybe explained by the maintenance of an effective immune response throughout the assay.

Inclusion of alternative marine by-products in aquafeeds with different levels of plant-based sources for on-growing gilthead sea bream (Sparus aurata, L.): effects on digestibility, amino acid retention, ammonia excretion and enzyme activity.

The search for new sustainable aquafeeds for the species with greater economic importance, such as the gilthead sea bream in Europe, is one of the main challenges in the aquaculture sector. The present work tested fishmeal replacement by a mixture of plant meals at different levels, as well as the use of marine by-products with attractant properties and high-quality protein in high plant protein diets. In order to do that, effects on growth and biometric parameters, digestibility, amino acid retention, excreted ammonia and proteases and amylase activity were assessed, using six different diets: FM100 (100% of protein provided by fishmeal), FM50 (50% of replacement), FM25 (75% of replacement) and FM0 (100% of replacement), but also FM25+ (75% of replacement and 15% of squid and krill meal inclusion), and FM0+ (100% of replacement and 15% of squid and krill meal inclusion). In group FM0, a clear impact of dietary changes was observed on growth, survival and ammonia excretion. Amino acid retention in group FM0+ was also significantly affected, which can be explained by the limited content of certain amino acids in this diet. On the other hand, no significant differences were observed in most biometric parameters or in enzyme activity. In conclusion, complete fishmeal replacement can be achieved by using a mixture of plant-based sources, but supplementation with complementary marine ingredients can prevent detrimental effects on growth, survival, nutritional parameters and protein metabolism.

Effects of dietary inclusions of red beet and betaine on the acute stress response and muscle lipid peroxidation in rainbow trout.

This study evaluates the effects of red beet (RB) and betaine on rainbow trout submitted to an acute stress challenge. A control diet was compared with four experimental diets in which red beet (14 and 28%) and betaine (0.9 and 1.63%) were incorporated in different concentrations according to a factorial design. Cortisol in plasma and fin, glucose and lactate plasma levels, and malondialdehide (MDA) in muscle were all measured before the stress challenge and 30 min and 6 and 12 h after the stress challenge as parameters to determine the diet effects. RB and betaine had no effect on cortisol, glucose, and MDA basal levels. However, lactate basal levels were significantly lower on fish fed with RB and betaine. Thirty minutes after the stress challenge, there was a significant increase in plasma and fin cortisol, glucose and lactate concentrations, although fish fed with diets containing RB and betaine showed significantly higher plasma cortisol values. MDA values of fish fed with 14% RB and 0.9% betaine were significantly higher than MDA values from fish fed with 28% RB and 1.63% betaine. After 6 and 12 h, plasma and fin cortisol and lactate levels recovered in a similar trend. Glucose plasma levels recovered in almost all groups 12 h after the stress. Also, MDA values recovered basal levels after 6 and 12 h. RB and betaine did not enhance the tolerance to the stress challenge compared to the control group, although the presence of these ingredients had no negative effect on any of the stress indicators.

Red beet and betaine as ingredients in diets of rainbow trout (Oncorhynchus mykiss): effects on growth performance, nutrient retention and flesh quality.

The objective of the study was to evaluate the impact of different concentrations of dietary red beet and betaine on the growth performance and fish flesh quality of rainbow trout. Therefore, a control diet was compared with four diets in which two levels of red beet (14% and 28%) and betaine (0.9% and 1.63%) were incorporated in combination. The study was set up with an average body weight of 69 ± 2.2 g and finished when fish reached commercial weight (175-250 g) after 105 d. The impact of the diets was studied based on the growth performance, biometric indexes, proximal composition, protein and fat retention efficiencies and apparent nutrient digestibility by fish reared on a recirculation system. Further estimates were the effect of red beet and betaine on the flesh proximate composition and quality of the final product (water activity, colour, texture, thiobarbituric acid reactive substances and sensory characteristics). Results showed that inclusion of 14% red beet and 0.9% betaine did not affect growth, nutritive or biometric parameters and nutrient retention when compared with the control diet. However, higher levels of red beet and betaine had negative effects on growth and nutritive parameters. The tested ingredients enhanced quality parameters regardless of the concentration used. After feeding the red beet and betaine, fish flesh showed lower water activity and better textural and colour properties than the control and also a dose-dependent effect on lipid oxidation was observed.

Pea protein concentrate in diets for sharpsnout sea bream (Diplodus puntazzo): effects on growth and health status.

Four diets for sharpsnout sea bream juveniles (14 g body weight) with four levels of air-processed pea protein concentrate (PPC) (0, 160, 320 and 487 g/kg diet) were tested in triplicate. The experimental diets were isonitrogenous (43% crude protein) and isolipidic (19% ether extract) and the fish were fed to satiation twice a day. After 125 d, fish growth was diminished by the inclusion of PPC. Feed conversion did not show significant differences in any treatment. Neither the body analyses nor the protein and individual essential amino acid retention efficiencies were affected by the inclusion of PPC in the diet. However, histological gut examinations revealed noticeable differences. Fish fed the diet with the highest inclusion level of PPC presented the longest villous length and the most goblet cells, and the width of the lamina propria increased in the anterior intestine. Although no negative changes in nutritive parameters were detected, these alterations might affect nutrient transport, with negative consequences for fish growth. It was concluded that the PPC in the amounts tested here is an inappropriate substitute for fishmeal in diets for sharpsnout sea bream juveniles.

Potential use of high levels of vegetal proteins in diets for market-sized gilthead sea bream (Sparus aurata).

The effect of partial or total dietary substitution of fishmeal (FM) by vegetal protein sources on growth and feed efficiency was carried out in on-growing gilthead sea bream (mean initial weight 131 g). The Control diet (FM 100) contained FM as the primary protein source, while in Diets FM 25 and FM 0 the FM protein was replaced at 75% and 100%, respectively, by a vegetable protein mixture consisting of wheat gluten, soybean meal, rapeseed meal and crystalline amino acids. Diets FM 25 and FM 0 also contained krill meal at 47 g/kg in order to improve palatability. At the end of the trial (after 158 d), fish survival was above 90%. Final weight and the specific growth rate were statistically lower in fish fed the Control diet (361 g and 0.64%/d), compared with 390-396 g and 0.69-0.70%/d after feeding vegetal diets. No significant differences were found regarding feed intake and feed conversion ratio. The digestibility of protein and amino acids (determined with chromium oxide as indicator) was similar in all diets. The blood parameters were not significantly affected by treatments. The activity of trypsin and pepsin was significantly reduced after feeding Diet FM 0. In the distal intestine, the villi length in fish fed Diet FM 25 was significantly longer and the intestine of the fish fed the FM 100 diet showed a smaller number of goblet cells. In conclusion, a total FM substitution by a vegetal mix supplemented with synthetic amino acids in on-growing sea bream is feasible.