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Fish meal is increasingly substituted in diets for gilthead sea bream with ingredients of terrestrial origin which may affect the mineral content and availability. Among these minerals, copper (Cu) is an essential trace element whose excess may have a potential toxic effect. Since ingredients of terrestrial origin have higher Cu levels than marine ones it is important to define the optimal dietary supply of Cu. Therefore, the aim of this study was to evaluate optimal dietary inclusion level of Cu in low FM-FO diets for gilthead sea bream fingerlings. Five practical diets with low FM (10%) and FO (6%) contents were respectively supplemented with 5 levels of CuSO4 to provide 5.5, 7.4, 9.3, 11.0 and 32.0 mg Cu kg−1 diet. Sea bream fingerlings (12.6 ± 1.4 g, mean ± SD) were distributed in 15 tanks with 30 fish per tank in triplicates and randomly assigned one of the dietary treatments. The fish were fed three times a day until apparent visual satiation for 42 days. Growth was recorded at the end of the trial and samples were taken for biochemical, mineral, histology, X-ray and hepatic gene expression analyses. The results obtained suggest that gilthead sea bream fed practical diets based on plant protein sources that provide at least 5.5 mg Cu kg−1 need no additional Cu supplementation, whereas dietary contents of 11–32 mg Cu kg−1 negatively affected gilthead sea bream performance by reducing growth, increasing oxidative risk and inducing hepatic damage and cholestasis. Dietary Cu levels did not affect body weight, SGR, TGC or FCR, denoting that the level in the non-supplemented diet (5.5 mg Cu kg−1) was enough to cover the requirements for growth. However, increasing dietary Cu levels from 5.5 to 9.3 mg/kg-1 up-regulated cat gene expression. On the contrary elevation of dietary Cu levels up to 11.0 and 32.0 mg Cu kg−1 tended to reduce growth and increased liver steatosis, broken cell margin, peripheral nuclei and sinusoid dilatation which are the markers of hepatic damage and cholestasis denoting potential toxic effects of Cu.

Systemic granulomatosis has already been reported in meagre larvae with an adequate feeding protocol and enrichment media preventing its appearance in the first weeks of life. Afterwards, the control of this disease could be prevented through nutritional components of the inert food, being the antioxidants the key to success. For this reason, in the present study, meagre larvae were reared from 30 days post hatching (dph) with five isonitrogenous and isolipidic experimental microdiets with different levels of vitamin E and C: C– (40 mg kg−1 E, 100 mg kg−1C), C+ (400 mg kg−1 E, 1000 mg kg−1C), Krill (400 mg kg−1 E, 1000 mg kg−1C and substitution of fish oil by krill oil), EC (200 mg kg−1 E, 500 mg kg−1C) and EECC (800 mg kg−1 E, 2000 mg kg−1C). Prior to this, larvae were co-fed with rotifers and Artemia following a protocol which prevented the appearance of granulomas, as previously demonstrated. The substitution of fish oil by krill oil significantly increased levels of eicosapentaenoic acid (EPA, 16.6%) and docosahexaenoic acid (DHA, 17.6%) in meagre, consequently increasing the peroxidation index, which in turn translated into a higher incidence of granulomas. Although even low levels of vitamin E and C (40 mg kg−1 E, 100 mg kg−1C; C-) allowed the adequate growth of larvae, these levels were not enough to prevent the appearance of granulomas, requiring superior levels of both antioxidant vitamins (800 mg kg−1 E and 2000 mg kg−1C) to mitigate systemic granulomatosis. This mitigation was simultaneous with the reduction of thiobarbituric acid reactive substances TBARs content in larvae, which were highly correlated with the appearance of granulomas (R2 = 0.892, y = 0.0446× + 0.0756). A strong negative correlation was observed between the dietary levels of vitamin E (y = −0.0098× + 11.174, R2 = 0.8766, p value = .019, r = −0.93) and vitamin C (y = −0.0022× + 6.4777, R2 = 0.9278, p value = .003, r = −0.96) and the percentage of larvae with granulomas. The results showed that the occurrence of systemic granulomatosis seems to be associated to the larvae peroxidation status, so that high dietary levels of vitamin E and C (800 and 2000 mg kg−1, respectively; Diet EECC), reduced lipid peroxidation and completely prevented the appearance of granulomas in meagre larvae at 44 dph.

The present study aimed to determine the effects of dietary inclusion of selenium (Se) in the form of either inorganic Se (sodium selenite, NaSe) or organic Se (hydroxy-selenomethionine, OH-SeMet) on gilthead seabream (Sparus aurata). Triplicate groups of 150 fish each (6.2 ± 0.04 g initial body weight) were distributed in 15 tanks and fed one of five experimental diets for 63 days. A control diet (C) without Se supplementation (0.8 mg Se/kg diet), was formulated and served as a basal diet for the other 4 experimental feeds, supplemented with either 0.2 or 0.5 mg Se/kg supplied in the form of NaSe or OH-SeMet. Fish growth performance, biochemical composition of liver and muscle and Se content, malondialdehyde (MDA) concentration in muscle and hepatopancreas, hepatocyte morphology and lysozyme activity in the serum were studied. Furthermore, expression of related hepatic genes, such as manganese superoxide dismutase (Mn-sod), catalase (cat), heat shock protein 70 (hsp70), and glutathione peroxidase (gpx) was also analyzed. After the trial, fish were exposed to an acute and chronic confinement stress. Blood samples for cortisol analysis were taken at 0 and 2 h after the acute stress and after 7 days of chronic stress. The highest growth rate was observed in fish fed OH-SeMet at a level of 0.2 mg/kg, but with no significant difference with fish fed the control diet with no-added Se. The lowest growth was observed in fish fed NaSe up to 0.5 mg/kg. Increase in dietary Se, particularly in the form of OH-SeMet, led to an increase in Se contents in liver and muscle. Furthermore, dietary inclusion of OH-SeMet, led to a significant (p In summary, supplementation of Se up to 0.2 mg/kg (1–1.1 mg/kg analyzed dietary Se), particularly in the form of OH-SeMet, led to a beneficial effect on growth, maintenance of hepatic morphology and improved protection of juvenile gilthead seabream from acute or chronic stress. Besides, OH-SeMet was found to be more effective than NaSe in protection against oxidative stress in fish muscle.

Meagre (Argyrosomus regius) is a promising new aquaculture species, however with some bottlenecks to high-scale commercial production. One of those constraints are the scarce information about its nutritional requirements, which difficult the production of optimized diets and can lead to pathological alterations in liver, such as hepatic steatosis. Furthermore, meagre production is widely affected by granulomatosis, an infectious or non-infectious disease that may compromise fish performance and appearance. The cause of non-infectious granulomatosis is still unclear, but nutritional deficiencies are potential explanations. Since essential fatty acids are nutrients required to sustain vital functions, growth, but also immune status and disease resistance, the present work evaluated, in a 30-day feeding trial, the effect of 5 dietary n-3 long-chain polyunsaturated fatty acid levels (0.8, 1.4, 2.0, 2.6 and 3.5% in dry weight of the diet) on liver health status of meagre fingerlings. Fish fed the lowest n-3 LC-PUFA diets (0.8 and 1.4%) showed a higher hepatosomatic index, as well as a higher hepatic lipid infiltration, which resulted in severe hepatic steatosis. Oleic acid and docosahexaenoic acid were the most important fatty acids driving the variability of meagre hepatic fatty acid composition and, consequently, hepatic histological alterations. Although fatty acid composition of phospholipids was less affected by the dietary n-3 LC-PUFA, meagre fed 0.8% n-3 LC-PUFA tended to present lower levels of essential fatty acids and higher levels of oleic acid in polar lipids. Moreover, the diet with the lowest n-3 LC-PUFA content (0.8%) led to a higher incidence of hepatic granulomas, suggesting a possible relation between essential fatty acid deficiency and hepatic granulomatosis in meagre. These results suggest that the inclusion of vegetable oils in diets for meagre fingerlings should be taken with careful, guaranteeing that, at least, 2.0% DW of n-3 LC-PUFA is available in feeds, in order to maintain the normal hepatic function in meagre.

Elevated mortality during early stages and high occurrence of skeletal anomalies are major concerns in Greater amberjack (Seriola dumerili) juvenile's production. Identification of nutritional requirements during live prey feeding stages, particularly those related with n-3 HUFA would help to formulate specific diets for this species improving larval survival and reducing skeleton anomalies. In this study S. dumerili larvae were reared from 17 to 35 days post hatching (dph). Larvae were fed Artemia sp. containing five different n-3 HUFA levels from 2.7 to 20.5% TFA. Growth, survival, skeletal anomalies occurrence and larval biochemical composition were determined. Best growth, final survival and survival after air stress test were achieved in the range of dietary n-3 HUFA concentrations, between 5 and 20%TFA. The lowest occurrence of skeletal anomalies was achieved in fish fed 12% n-3 HUFA content in Artemia, and the occurrence of cranial anomalies was correlated to increased dietary n-3 HUFA levels. Besides, whole body larval fatty acid profiles showed a positive correlation with dietary n-3 HUFA. Based on the overall results the recommended n-3 HUFA dietary level during Artemia feeding for larval S. dumerili was suggested between 12 and 17%TFA.

The present work investigated how the combination of poultry oil with microalgae oils, rich in eicosapentaenoic acid and docosahexaenoic acid (ED diets) or n-6 docosapentaenoic acid and n-3 docosahexaenoic acid (DD diets) modulates hepatic lipid metabolism in gilthead sea bream juveniles. Diets were tested using two different fishmeal contents (15% and 7.5%) and compared against a fish oil-based diet (CTRL) and two negative control diets based on poultry oil as lipid source (PO diets). After 74 days of feeding, sea bream fed 15% FM ED or DD diets showed similar daily growth index to those fed CTRL, while those fed PO diets caused reduced growth. Fish livers reflected the highest contents in n-3 long-chain polyunsaturated fatty acids when fed CTRL, ED or DD diets, which down-regulated fas, scd-1a, fads2, lpl and cpt1, reducing hepatic lipid accumulation and hepatocytes size. In contrast, fish fed PO diets showed the lowest deposition of n-3 long-chain polyunsaturated fatty acids and the highest oleic acid in liver, leading with the highest hepatosomatic index due to increased liver lipids. Therefore, these fish revealed a severe hepatic steatosis associated with an increased expression of lipogenesis-related genes, particularly fas, lpl and sbrep1. Furthermore, PO diets seemed to activate desaturation pathways in fish livers, reflected by the highest accumulation of fatty acids that are products from desaturases and the highest fads2 and scd-1a expressions. The reduction of the dietary fishmeal content to 7.5% lowered fish growth, although hepatic lipid metabolism seemed to be more affected by FO replacement than FM replacement. Combining microalgae with poultry oil could be an alternative lipid and essential fatty acid source to fish oil in marine fish diets.

The negative effects of oxidative damage on larval development, growth and survival are among the factors markedly affecting marine fish larvae production success. Whereas α-tocopherol (vit E) and l -ascorbic acid (vit C) are strong antioxidants, the potential antioxidant role of taurine (Tau) in fish diets and its relation with other antioxidant nutrients needs further clarification. The present study aimed to determine the potential interactions between the dietary levels of vit E plus vit C and Tau on survival, growth, bone development and oxidative metabolism in gilthead seabream larvae. Six micro-bound diets containing two levels of Tau combined with three levels of vit E plus vit C were manually fed 24 times/day to 16 dph larvae for 15 days. Elevation of dietary vit E and vit C over 3000 mg/kg, led to unbalanced body vit C/vit E ratio and up-regulation of genes coding for antioxidant enzymes, the highest incidence of bone anomalies and the lowest larval survival. Increase in dietary vit C + vit E up to 1800 mg/kg, down-regulated GPX expression, up-regulated OC expression and reduced maxillary bone anomalies. Increase in dietary Tau up to 7.1 g/kg significantly improved larval growth, down-regulated antioxidant enzyme genes and reduced the incidence of bone anomalies. Moreover, the pro-oxidative effects of the high supplementation of vit C and E where mitigated by increase in dietary Tau levels. Finally, increase in dietary vit E, vit C and Tau up to 1783 mg/kg, 1921 mg/kg and 7 g/kg lead to the highest larval survival and growth, the highest OC gene expression and the lowest incidence of bone anomalies, including lordosis, kyphosis, branchiostegal and maxillary bones anomalies.

A well-balanced diet adapted to fulfil the specific nutritional requirements of greater amberjack (Seriola dumerili) would contribute to optimize reproduction and spawning quality. The main objective of the present study was to examine the effects of supplemental histidine, taurine and protein in broodstock diets, on egg quality of greater amberjack. Twelve broodstocks were distributed in three 40 m3 circular tanks and fed three different diets, one high in histidine, another in taurine and the third one in protein contents. Overall the best spawning quality was obtained histidine levels were elevated from 1 to 1.5%, increasing the relative fecundity, fertilization rates, egg viability, hatching rates, larval survival, egg protein contents, and egg and larval size, denoting the importance of this amino acid for embryo and larval development. The increase of taurine from 0.3 to 1.1% in diets for greater amberjack increased the relative fecundity in comparison to fish fed higher protein levels. Fertilization rates tend to increase with the elevation of dietary taurine and were not significantly different from those of broodstock fed higher histidine levels. Besides, dietary taurine increased egg diameter and taurine contents. Increase in dietary protein contents from 51 to 56% lead to an increase protein content in egg, as well as a larger yolk sac volume, but did not improve any of the spawning quality parameters. In conclusion, the results of this study have pointed out the importance of raising histidine contents in broodstock diets from 1 to 1.5% to optimize the reproductive performance of greater amberjack. Besides, the study showed that taurine levels in broodstock diets increased fecundity, maintaining good fertilization rates, but further studies must be conducted to determine the optimum taurine dietary levels.

Systemic granulomatosis is a growing disease with a high morbidity, which affects to the majority of farmed meagre (Argyrosomus regius). The impossibility of isolating any infectious agents has hypothesized a nutritional origin of the disease. In order to try to elucidate the nutritional origin of granulomas, juvenile meagre were fed for 15 weeks with six diets containing different levels of vitamin E and C and with or without addition of vitamin K: Diet 0 (basal premix, no K, 150 mg kg‐−1 E, 20 mg kg‐−1 C), K (added 23 mg kg‐−1 K), EC (300 mg kg‐−1 E, 70 mg kg‐−1 C), KEC (23 mg kg‐−1 K, 300 mg kg‐−1 E, 70 mg kg‐−1 C), EECC (450 mg kg‐−1 E, 230 mg kg‐−1 C) and KEECC (23 mg kg‐−1 K, 450 mg kg‐−1 E, 230 mg kg‐−1 C). The diet EC significantly increased meagre growth in terms of final weight and length. Fish fed the highest levels of vitamin E and C presented lower percentage of granulomas in liver and heart than fish fed diet 0. The scored severity of granulomatosis in liver and kidney (main affected organs) tended to be lower with dietary increase of vitamin E, C and addition of vitamin K (from 1.83 diet 0 to 1.3 diet KEECC and from 0.91 diet 0 to 0.39 diet KEECC). In liver, the diet KEECC significantly increased catalase expression compared with diet 0. In kidney tnfα expression was significantly up-regulated in fish fed diet EECC and KEECC. In heart, low vitamin E and C levels (300 and 70 mg kg‐−1, respectively) significantly increased superoxide dismutase and glutathione peroxidase expression and high addition increased the expression of tnfα and cox-2 (0 or 23 mg kg‐−1 K, 450 mg kg‐−1 E and 230 mg kg‐−1 C, diet EECC and KEECC). The results show that combination of high dietary content of vitamin K and antioxidant vitamins E and C (23, 450 and 230 mg kg‐−1, respectively) influenced in the incidence of the granulomatosis, which suggests that this pathology could be mediated by nutritional factors.

The establishment of well-balanced diets that meet nutrient requirements is important to optimize a large-scale production of new aquaculture species. This is the case of meagre (Argyrosomus regius), a promising new aquaculture species, with great potential owing to its high growth rate, feed efficiency and easy adaptation to captivity. Knowledge on the nutritional requirements of this species is still scarce, namely regarding essential fatty acids, which are required to sustain growth, development, immune status and survival. A feeding trial was performed with meagre fingerlings (2.8 g ± 0.23) testing 5 increasing dietary n-3 LC-PUFA levels (0.8, 1.4, 2.0, 2.6 and 3.6% DM) with the purpose of evaluating the n-3 LC-PUFA requirements for fast growth of meagre fingerlings. Meagre reflected very high specific growth rates (4.1 to 4.6%) and low feed conversion ratios (0.7 to 0.8), thus highlighting its great potential for aquaculture production. Fish fed 0.8% n-3 LC-PUFA showed the lowest growth, which was significantly improved by increasing the dietary n-3 LC-PUFA levels up to 2.0–2.6%. DHA and ARA were preferentially retained over EPA in whole fish body. Fish fed 0.8% n-3 LC-PUFA showed an up-regulation of fads2 and elovl5 relative gene expressions. Thus, meagre seems to have active Δ6 desaturases and Elovl5, but their activities being insufficient to produce DHA and EPA from PUFA precursors to sustain fast growth, at least under the experimental conditions tested. Young meagre shows a typical marine requirement for n-3 LC-PUFA, estimated to be, at least, 2.0% DM of the diet.

A sustainable growth of the aquaculture sector implies the use of sustainable novel raw materials as replacers of the traditional fishmeal (FM) and fish oil (FO) ingredients. This fact has led to the development of sustainable and functional diets as part of a management strategy to reduce the effects on fish growth performance and health derived from low FM/FO dietary contents. In this sense, Antarctic krill (Euphausia superba) is considered a potential candidate in dietary inclusions to potentiate fish growth and health status. In this study, European sea bass (Dicentrarchus labrax) were fed a practical diet with either a 15% fishmeal content (KM0; control diet) or the same diet substituted by 30% (KM5; 50 g KM/kg diet) or 50% (KM7.5; 75 g KM/kg diet) Antarctic krill meal (KM) for 12 weeks in triplicates. At the end of the feeding trial, growth performance, liver morphology, liver proximate composition, lipid classes and fatty acid profiles, as well as the expression of hepatic genes related with lipid metabolism were evaluated. Fish fed KM-based diets presented higher (p 0.05) biochemical composition and fatty acid profile. However, smaller hepatocellular area and lower grade of cytoplasm vacuolization as well as a better alignment around sinusoidal spaces were found. The analyses of liver lipid classes revealed a positive correlation between the level of dietary KM and the pigmented material such as astaxanthin and free fatty acid content, as well as a negative correlation with the cholesterol levels. The expression of hepatic genes studied demonstrated a downregulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr) and delta-6-desaturase (fads2) expression levels in fish fed KM-based diets. Besides, gene expression levels of fatty acid binding protein 7 (fabp7) and lipoprotein lipase (lpl) were significantly correlated with KM dietary levels. Altogether, these results profile KM as a potential promoter of growth and liver health in European sea bass fed low fish meal and oil diets.

The aim of this study was to evaluate the combination of maximum replacement of fishmeal (FM) and fish oil (FO) by alternative meals (AM) and oils (AO) to develop nutritious, but more eco-friendly diets for European sea bass ( Dicentrarchus labrax ) juveniles. European sea bass of 9.8 ± 1.5 g and 9.1 ± 0.5 cm were fed nine isonitrogenous (45% crude protein) and isolipidic (21%) diets containing gradually reduced levels of FM and FO as follows (%FM/%FO): 58/15, 20/6, 20/3, 10/6, 10/3, 5/6, 5/3 and 0/0. Another diet, 0/0 + , was similar to the 0/0 but supplemented with LC-PUFA from microbial and marine sources. After 90 days of feeding, European sea bass fed the 0/0 diet showed reduced (P + diet). Reduction of FM/FO down to 5/3 did not significantly affect fish survival or condition factor, whereas reduction to 5/6 did neither affect sea bass length or feed conversion and reduction down to 10/3 did not affected body weight, DGI and perivisceral fat or visceral weight. FM reductions down to 5% significantly reduced growth rate. FO substitution by vegetable oils (VO) led to reduced growth and increased liver to body weight ratios, liver lipid deposition and hepatocellular size. In liver and muscle, FO substitution increased n-6 fatty acids (FA) and n-6/n-3 ratio and reduced saturated FA and n-3 LC-PUFA, whereas monounsaturated FA were not increased in proportion to dietary contents. Moreover, FO reduction increased the products from Δ6 desaturase and elongases, Elovl6 and Elovl5. In conclusion, the results of this study show that it is possible to reduce dietary FM and FO levels down to 10% and 3% in mostly plant-based diets without affecting growth performance of European sea bass juveniles.

The main objective of this study was to assess the effects of graded levels of replacement of fishmeal (FM) and fish oil (FO) by terrestrial meals (TM) and vegetable oils (VO) on gut health of European sea bass (Dicentrarchus labrax) based on gut morphology, gut-associated immune system (GALT) and gut microbiota composition. Reducing the dietary FM and FO levels down to 10 and 3%, respectively did not affect the growth of European sea bass. Increase in TM and VO markedly affected anterior and posterior gut morphology and fatty acid composition, which mirrored the dietary fatty acid profile. FO replacement by VO increased lipid deposition in anterior gut lamina propria, denoting an effect in lipid metabolism and lipoprotein synthesis. In posterior gut, FM replacements by TM induced an engrossment of the submucosa, an up-regulation of the proinflammatory cytokines IL-1β and TNFα genes, a relatively high mucus production and changes in mucosal microbiota profiles. Finally, supplementation of 0%FM-0%FO diets with LC-PUFA contributed to maintain gut homeostasis in terms of GALT response and microbiota profiles alterations. Statement of relevance FM and FO appropriate dietary levels for European seabass

Seven experimental isonitrogenous (50%) and isolipidic (16%) diets with different levels of α-tocopherol acetate (16, 100, 190, 285, 430, 880 and 1300 mg kg − 1 ) were tested during 72 days to evaluate growth performance, tissue composition, fillet oxidation and liver histology in meagre juveniles, Argyrosomus regius . Growth performance, feed conversion ratio (FCR) and tissue composition were similar among treatments ( P > 0.05). In the liver, no major differences were recorded in lipid and fatty acid composition but higher lipid vacuolization were observed in diets E100, E190 and E880. Muscle fatty acid profiles showed an increment of the highly unsaturated fatty acid (HUFA) and a decrease of the saturated fatty acid with the increase of dietary vitamin E, which was accompanied with a reduction of the muscle TBARS responses. Therefore, is suggested that diets for this species should be supplemented with 451 mg kg − 1 of DL-α-tocopherol acetate (496 UI of vitamin E), as determine by broken-line regression analysis of muscle TBARS, to provide good overall growth performance and improved fish quality and storage stability. Moreover, results suggest that vitamin E deficiency or excess may deteriorate fish health. Statement of relevance The optimization of the dietary vitamin E level will contribute to formulate a suitable diet for meagre, a candidate for European aquaculture diversification, that until now is being fed with diets specific for other species. This study will narrow the knowledge gap that exists regarding meagre nutritional needs.

Aquaculture, the fastest growing food production sector cannot continue to rely on finite stocks of marine fish as the primary source of the omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA; 20:5n3) and docosahexaenoic acid (DHA; 22:6n-3), for feeds. A four-month feeding trial was conducted to investigate the impact of a de novo oil, with high levels of EPA and DHA, obtained from transgenic Camelina sativa on growth performance, tissue fatty acid profiles, and expression of lipid metabolism genes when used as a replacement for fish oil in feed for European seabass (Dicentrachus labrax). Triplicate groups of 50 juvenile fish (initial weight 16.7 ± 0.92 g) per tank were fed for 4 months with one of three isolipidic and isoproteic experimental diets consisting of a standard diet containing a commercial blend of fish oil and rapeseed oil (CFO), a diet containing transgenic Camelina oil (TCO), or a blend of fish oil and rapeseed oil with enhanced levels of EPA and DHA (EFO) formulated to match the n-3 LC-PUFA profile of the TCO feed. Final weight of fish fed the GM-derived oil was not different to fish fed either CFO or EFO. Slight lower growth performance of fish fed TCO at the beginning of the trial was related to transient reduced feed intake, possibly caused by glucosinolates in the raw Camelina sativa oil. The GM-derived oil improved the nutritional quality of the fish fillet by enhancing total n-3 PUFA levels compared to the fish fed the other two feeds, and maintained flesh EPA and DHA at the same levels as in fish fed the diets containing fish oil. The metabolic response in liver and intestine was generally relatively mild although diets TCO and EFO seemed to trigger a metabolic response consisting of an up-regulation of both β-oxidation (cpt1a) and fatty acid transport (fabp1), possibly reflecting higher levels of LC-PUFA. Overall, the present study indicated that an oil of terrestrial origin, Camelina sativa, when engineered to contain high levels of EPA and DHA can replace fish oil in feeds for European seabass with no detrimental impact on growth or feed efficiency, while also maintaining or increasing tissue n-3 LC-PUFA contents.

Manganese (Mn) is an essential metal for fish and requirements have been established for several finfish but not for gilthead sea bream. Thus, the present study aims to establish the optimal dietary supplementation level of Mn in gilthead sea bream fingerlings fed vegetable based diets. Gilthead sea bream fingerlings (weight 12.6 ± 1.5 g, mean ± S.D.) were fed five practical diets high in vegetable ingredients (fish meal: 10%, fish oil: 6%). The diets were supplemented to contain 19, 27, 30, 41 and 66 mg Mn kg−1 as MnSO4. Four hundred and fifty sea bream fingerlings were randomly distributed in 15 tanks and fed one of the five diets until apparent satiation three times per day for 42 days. Growth parameters including feed intake, thermal growth coefficient and feed conversion ratio were calculated. At the end of the trial, samples were taken for biochemical, mineral, histological and gene expression analyses. After the feeding trial, fish almost tripled their weight, but dietary Mn levels did not affect growth parameters or survival. The high fish meal substitution levels led to high Mn contents in the basal diet (19 mg Mn kg−1 diet), that seemed to be sufficient to promote sea bream growth. Body lipid composition, protein and ash were not affected by the dietary Mn. Similarly, whole body, liver and vertebrae mineral contents were not affected by Mn supplementation. Morphological characteristics of liver had no significant differences among dietary Mn levels. However, increase of Mn contents beyond 30 mg Mn kg−1 down-regulated mnsod expression. Expression of cat gene was not affected. Overall, results suggest that the Mn content present in the basal diet (19 mg Mn kg−1) was sufficient to cover the requirements in juvenile gilthead sea bream fed practical plant-based diets, although results from oxidative status markers might point out the need to increase supplementation levels beyond this point when fish are under conditions that may affect their oxidative status.

Dietary lipids and fatty acids composition play an important role in the reproductive processes, embryo development and larval survival in marine fish. Long-chain polyunsaturated fatty acids (LC-PUFA) are especially important for egg and larval quality. The main objective of the present study was to evaluate the effects of increasing dietary n-3 LC-PUFA levels on spawning performance and egg quality of greater amberjack (Seriola dumerili). Sixteen mature broodfish were distributed in four 40 m3 circular tanks and fed four diets containing different levels of n-3 LC-PUFA: diet D1 with 1% of the dry weight basis (dw), diet D2 with 1.7% dw, diet D3 with 2.3% dw and diet D4 with 3% dw. The fecundity was significantly higher (P

The main objective of this study was to evaluate the stress response of the greater amberjack, Seriola dumerili, by determining plasma and skin mucus cortisol levels, as physiological indicator of fish stress, and skin mucin-2 gene expression as indicator of skin mucin production, under three different potential stressors related to aquaculture practices: temperature, handling and fasting. For trial 1, 45 greater amberjack juveniles (19.5 ± 4.1 g) were divided in three graded rearing temperatures (17, 22 and 26 °C) and maintained for 120 days, in order to define the basal cortisol level for this species after a long-term thermal acclimation within the range described for this species. Trial 2 was conducted under two different rearing densities and two different handling protocols with 222 fish (425.02 ± 36.9 g) divided in 4 treatments by triplicate for 90 days, in order to study the effect of routine handling protocols on the selected stress markers for this species. Trial 3 used 72 fish (302.27 ± 21.4 g) divided in two experimental groups, one fed to apparent satiety and another one subjected to fasting for 10 days (triplicates for each group). After trials 2 and 3, a shallow water challenge was conducted to further study the adaptive response of greater amberjack juveniles to combined stress situations. Plasma and skin mucus were obtained for cortisol determination at 1, 24 and 72 h (trial 2) and 1, 3 and 24 h (trial 3) post stress challenge. Additionally, skin was dissected out for determining muc-2 gene expression. Long-term acclimation to temperature did not affect plasma or mucus cortisol levels and muc-2 gene expression, being basal levels of circulating plasma cortisol around 7.5 ng/mL. A high stocking density (8 kg m−3) induced an elevation (p

The complete removal of fishmeal (FM) and fish oil (FO) is required to promote the sustainable development of aquaculture and for that, fast growing high quality fish that are fed without FM and FO are necessary. Early nutritional programming may allow the production of fish better adapted to utilize diets with vegetable meals (VM) and oils (VO). The main objective of this study was to research in the potential value of fatty acids as modulators of early nutritional programming in marine fish for a better utilization of VO/VM. For that purpose gilthead sea bream (Sparus aurata) broodstock were fed four different replacement levels of FO by linseed oil (LO) and their effect on fecundity and spawn quality, egg composition, Δ-6-desaturase (Δ6D) gene expression, progeny growth performance and their growth response to a challenge with diets low in FO and FM, but high in VO and VM. The results showed that feeding gilthead sea bream broodstock with high LO diets had very long-term effects on the progeny. Thus, FO replacement by LO up to 80–100% in broodstock diets for gilthead sea bream not only reduced fecundity and spawn quality, but also growth of 45 dah and 4-month-old juveniles, as well as Δ6D gene expression. However, when the 4 month-old juveniles were fed with a low FM and FO diet, even those from broodstock fed only 60% replacement of FO by LO showed a higher growth and feed utilization than juveniles from parents fed FO. These results demonstrate the interesting potential of early nutritional programming of marine fish by broodstock feeding to improve long-term performance of the progeny. Further studies are being conducted to determine optimum nutrient levels in the broodstock diets and the molecular mechanisms implied to develop effective nutritional intervention strategies for this species. Statement of relevance This study demonstrates for the first time in fish the potential of broodstock nutrition to conduct early nutritional programming of culture fish for a better utilization of low fish meal and fish oil diets by the progeny, showing its effect not only during reproduction and larval development but also during on-growing.

To date, European abalone aquaculture relies mostly on locally harvested fresh seaweeds which nutritional quality and abundance vary greatly, hence affecting abalone growth. Abalone artificial diets generally include fishmeal, limiting their utilization in ecologically sustainable aquaculture and affecting abalone quality and acceptance by the consumers. A six month feeding trial was conducted to assess the nutritional value of four different dried seaweed meals: Ulva lactuca (Chlorophyta), Gracilaria cornea (Rhodophyta), Laminaria digitata (Phaeophyta) and Palmaria palmata (Rhodophyta), as ingredients to all-vegetable-based formulated feeds for abalone Haliotis tuberculata coccinea (33.1 ± 0.8 mm and 4.7 ± 0.6 g). A mixed fresh algae diet of G. cornea and U. Ulva rigida, reared in an Integrated Multi-Trophic Aquaculture (IMTA) system, served as control. Survival rates were very high (95–98%), regardless of the diet fed. Enriched fresh algae produced a significantly higher growth for H. tuberculata coccinea (169% weight gain) than all the artificial diets (49–84% WG). Comparison among abalone fed the different formulated diets showed that the inclusion of P. palmata improved growth, condition index and dietary protein utilization. On the contrary, the use of L. digitata markedly reduced the efficiency of dietary protein since the protein-related nutritional index (PER), the percentage of protein deposited in the foot muscle as well as the meat to shell ratio recorded for animals fed this diet were the lowest, despite a higher feed intake. Large differences were found in the FA profile of fresh algae as compared with the three formulated diets. The n-3/n-6 ratio was much higher in the fresh algae and, consequently, in the foot tissues of abalone fed this diet in comparison to the one of those fed the formulated ones. The elevated contents of 20:4n-6 in the abalone fed the experimental diets and 20:5n-3 in abalone fed the fresh algae, as well as their respective metabolites, suggest that abalone have the ability to desaturate and elongate LA to ARA and ALA to EPA. Further studies are required to improve the growth obtained with these vegetable based diets, especially concerning the use of different seaweed combinations and inclusion levels, as well as the diet processing methods to improve diet water stability. Statement of relevance Development of vegetable based diets for abalone.

The longfin yellowtail ( Seriola rivoliana ) is a preferred species for aquaculture diversification in the Canary Islands. The aim of the present study was obtain a large number of spawns whit hormonal induction, which would provide reliable numbers of juveniles for the sustainable development of longfin yellowtail culture. Two females with oocytes bigger than 500 μ, and four males, previously captured from the wild were used for this study. Females and males of 10.7 ± 1.4 and 8.5 ± 0.9 kg body weight and 86.2 ± 5.3 and 78.9 ± 1.9 cm total length, respectively, were acclimated in 10 m 3 tanks. Fish were injected three times per month between June and November with 20 μg kg − 1 of GnRHa. During the experimental period, temperature ranged from 21.9 ± 0.2 °C in June to 23.5 ± 0.4 °C in November. Broodstock feeding consisted in Atlantic mackerel ( Scomber scombrus ) at 2% BW, three times per week. A total of 20,189,700 eggs (944,326 eggs female kg − 1 ) were obtained with fifteen injections and 33 spawns (2.2 ± 0.7 spawns per injection). A significant positive was found, between the numbers of eggs obtained each month and the average monthly temperature. Latency period was approximately of 28 h and 30 min. A highly significant negative statistical relation ( P − 1 of GnRHa every 10 days in fluent males and 500 μm oocytes bearing females of longfin yellowtail broodstock were successful to obtain a high induction efficiency and excellent spawn quality.

Two trials were conducted to evaluate the nutrient digestibility of river crab (RC) ( Procambarus clarkii ) and marine crab (MC) ( Chaceon affinis ) meals in diets for red porgy ( Pagrus pagrus ) and the effect on ammonia-N excretion and P and Ca retentions. In trial I, the apparent digestibility coefficient (ADC) of nutrients and energy contents in RC and MC meals were determined. Results showed that, the ADC values for protein, lipids, ash, dry matter and gross energy were significantly higher for MC than for RC meal. Both crab meals were efficiently digested despite their high chitin and ash contents. In trial II, red porgy were fed over 6 months with five diets: a control diet (CD) based on high quality fish meal (FM), and four diets where FM protein was replaced at a rate of 10% or 20% by protein derived from either RC or MC meal (RC10, RC20, MC10 and MC20). Replacement of the FM protein by RC or MC proteins had no effect both on protein utilisation and ammonia-N excretion rates, which ranged from 105 to 119 mg N–NH 4 + kg − 1 day − 1 for all diets. A linear increase was observed in the dietary Ca/P ratio by the increase of the dietary RC and MC meal inclusions, resulting in a reduction in the whole fish body content of P, Ca and ash with interesting similar whole body Ca/P ratio (1.59–1.63) for all treatments. Accordingly, Ca, P and ash retention rates were significantly reduced by increasing the dietary proportion of both crab meals. Moreover, a negative relationship between fish growth and final whole body Ca, P and ash contents was obtained. However, no significant differences were observed in total P excretion value (kg P t − 1 ).

The inclusion of complementary antioxidative factors, such as selenium (Se), could counteract the high oxidation risk in early weaning diets high in polyunsaturated fatty acids (PUFA). The present study investigated the effects of graded levels of Se derived yeast with krill phospholipids (KPL) on skeletal development, survival, stress resistance, oxidative status and biochemical composition of seabream larvae. Seabream larvae were completely weaned at 16 dph and fed five microdiets for 30 days with different levels of Se: 2SE, 4SE, 6SE, 8SE and 12SE (1.73, 3.91, 6.41, 8.47, 11.65 mg kg− 1 dietary dry weight, respectively). Increases in Se up to 11.65 mg kg− 1 dietary dry weight significantly improved survival rate (54%) and stress resistance, but did not affect larval growth. Seabream larvae fed diets supplemented with 12SE (11.65 mg kg− 1) showed a gradual increase in this mineral according to dietary Se levels, denoting the progressive absorption of this nutrient. The degree of larval lipid oxidation, as indicated by malondialdehyde (MDA) content and antioxidant enzyme (AOE) gene expression, was significantly lower in larvae fed 8SE and 12SE diets compared to those fed 2SE and 4SE diets. Furthermore, a reactive response as a result of Se inclusion was observed by the increase in osteocalcin, osteonectin, osteopontin, alkaline phosphatase and matrix gla protein gene expression in larval tissues, suggesting a well skeletal development. These results denoted the high efficiency of Se as an antioxidant factor and the importance of the inclusion of adequate levels (11.65 mg Se kg− 1 diet) in early weaning diets.

The present study was conducted to evaluate the effect of feeding marine crab (SMC) meal and sea urchin (SU) meal on growth performance, ammonia-N excretion, skin colouration and final flesh quality and lipid oxidation of red porgy ( Pagrus pagrus ). Fish were fed with five diets: a control-diet (CD); two diets replacing 10% and 20% fish meal protein by protein from SMC meal (SMC10, SMC20), and two diets with 8% and 16% inclusion of SU meal (SU8, SU16). The inclusion of both SMC and SU meals increased fish appetite response reflected in a significantly higher feed intake and final weight. Results were even better for fish fed on SU diets, with improved feed conversion (FCR) and protein utilization (PER). Ammonia-N excretion rates decreased in fish fed on SMC20, SU8 and SU16 diets. Fish skin colouration improved by SMC meal inclusion, while SU meal promoted yellowness colouration in skin. Higher muscle content of arachidonic fatty acid (ARA), was concomitant with the increased level of SMC in diets. The same trend was also found in fillet of fish fed on SU meal diets, with the highest values in ARA content. Results of TBARS in raw fillets showed that the dietary inclusion of SMC meal delayed lipid oxidation compared to FM and SU diets during refrigerated storage.

The present study was conducted to evaluate the effect of using river crab ( Procamburus clarkii ) meal (RC) and marine crab ( Chaceon affinis ) meal (MC) on the red porgy ( Pagrus pagrus ) fillet fatty acid profile and quality. Red porgy were fed during 193 days with five diets: a control diet based on high-quality fish meal (CD); and four diets replacing 10% and 20% fish meal protein by protein from both RC and MC meals (RC10, RC20 and MC10, MC20). Fillet fat content resulted significantly higher in MC20 fish than the rest of diets. Feeding with both RC and MC meals slightly increased muscle contents of polyunsaturated (PUFA), arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) fatty acids and n − 3/n − 6 ratio as well as decreased n − 9 fatty acids, although difference is only significant for the ARA content. After inclusion of both crab meals, values of atherogenicity index (AI) resulted similar among treatments while thrombogenecity index (TI) was reduced, but not significantly, reflecting the higher n − 3 fatty acid content in fillets from crab diets. Sensory analysis showed that red porgy fillets of all treatments were very well appreciated with some differences detected in only MC20 fillets, which had significantly higher scores in the attributes of oily odour and flavour, cohesiveness aspect and firmness texture compared to the rest of diets. RC meal dietary inclusion did not promote any changes in sensory evaluation panel. Instrumental muscle texture did not revealed differences between treatments. Results from TBARS of raw fillets showed that the inclusion of both crab meals clearly delays lipid oxidation compared to a FM based diet, during refrigerated storage at 4 °C. Thus, for 4 and 7 days of storage, fillets of RC or MC diets, attained significantly lower TBARS values than those fed on CD.

Escapes of farmed fish into the ecosystem have recently received a marked interest from scientists for their competition with wild populations or the risk of genetic contamination. Fatty acid profiles have been proposed as bio-indicators due to the inclusion of terrestrial oils in aquafeed ingredients. This study evaluated 1) the effect of wasted food on the fatty acid content of a farm-associated fish and 2) the suitability of fatty acid profiles as a bioindicator of aquaculture-ecosystem interactions, using the bogue ( Boops boops ) as a model. This species is an opportunistic fish usually associated with–or even found in–sea farms, and with a high natural occurrence along the Mediterranean and Atlantic coasts. The results showed that fish farms had a direct effect on the condition index, muscle and whole lipid content of bogue inside or around the sea cage, and this effect disappeared completely at 3 km from the cages. Despite the fact that aquafeeds also affected the bogue fatty acid profiles by increasing linoleic and oleic acids and reducing DHA, these profiles were very similar to those of bogue sampled close to a sewage outfall, denoting that these fatty acids are not exclusively influenced by aquaculture and therefore should not be considered as good biomarkers for escapees. However, bogues influenced by aquaculture were higher in linolenic acid than those found further than 3 km from the farm or close to the sewage, suggesting this fatty acid as a better indicator of aquaculture influence.

In the present study, the effects of long term feeding dietary conjugated linoleic acid (CLA) on growth, liver and perivisceral fat morphology, lipid metabolism enzymes and prostaglandin production potential were assessed. Two hundred and forty-three European sea bass (152.4 ± 1.96 g) were randomly allocated in 9 indoor 1 m 3 fiberglass tanks (4.1 kg/m 3 ) and assigned three diets containing graded amounts of CLA (0, 0.5 and 1%) for 20 weeks. Dietary CLA did not affect growth performance or tissue proximate composition although increased the saturated fatty acid fraction in fish fed with 1% CLA in muscle. Muscle and liver fatty acid profiles were slightly altered by CLA inclusion, mainly by the increase of the CLA isomers and by the increase of 18:0 and the decrease of 16:1n-7. Liver lipogenic and lipolytic enzymes were not affected by dietary CLA. Hepatocyte area was reduced in fish fed with 1% of CLA, as well as adipocyte area in fish fed with 0.5% of CLA where adipocyte number by unit of area also increased. Head kidney prostaglandins' production potential was reduced in fish fed with 1% of CLA, having also a negative correlation with dietary CLA level of inclusion. The present results suggest that CLA can be incorporated up to 1% in 150 g European sea bass for 20 weeks without affecting fish performance or enzyme activities and at the time improves liver and adipose tissue morphology.

Four diatoms species Amphora sp., Navicula incerta , Nitzschia sp. and Proschkinia sp., were tested as food for Haliotis tuberculata coccinea post-larvae. Larvae were settled on Ulvella lens and Ulva rigida germlings at a density of 0.5 larvae cm 2 of substrate. Growth and survival, in each dietary treatment, were monitored during ten weeks. Experimental nursery tanks were weekly inoculated with 2 L of the different diatom inoculums (10 5 –10 6 cells mL − 1 ). Proximate biochemical compositions were significantly different for the four diatom species. Amphora sp. presented a significantly higher lipid and protein contents with 7.11 ± 0.29% DW and 18.50 ± 0.97% DW respectively, while Proschkinia sp. presented the lowest protein content and the highest ash content. The fatty acid profiles of the diatoms were variable but remained typical of the diatom family. Larvae contained significantly more lipids (25.30 ± 0.93% DW) than juveniles. The different fatty acid compositions found for the various diatom species were dissimilar to the ones of larvae, juveniles and substrates; (Courtois de Vicose et al., in press-a) the fatty acid compositions of these last two not being different among treatments. 16:4n − 3, 20:4n − 6 (arachidonic acid, ARA) and 22:5n − 3 (docosapentaenoic acid, DPA) were the most abundant fatty acids in juveniles while 20:5n − 3 (ecosapentaenoic acid, EPA) was the dominant polyunsaturated fatty acid (PUFA), present at a comparable level in all diatoms. This suggests that the post-larvae and juveniles are capable to absorb a selection of fatty acids from the food source or that they are able to synthesise them from precursor dietary fatty acids. Growth rates obtained in the present study are presenting different phases and diatom diets made little difference to growth rate of post-larvae until reaching around 0.8–1 mm SL, after which divergence in growth rate among diets became significant. Post-larvae fed the diatom with the highest protein and lipid contents ( Amphora sp.) showed the best daily growth rate (DGR) of 50 ± 3 μm over the feeding trial, highlighting the joint influence of diatoms’ protein and lipid content on growth. Around 2 mm SL, juveniles exhibited increased growth rates, in all treatments, reaching 94 μm day − 1 , indicating a possible shift in nutrition towards green macroalgae germlings and suggesting that U. lens and U. rigida are used as a complement to the diatom diet at this stage. 18:2n − 6 and 18:3n − 3 found in high amount in all juveniles as well as in all substrates could be an indication of that nutritional shift and could be involved in growth enhancement.

The octopus, Octopus vulgaris , is one of the main targets for aquaculture diversification in Mediterranean countries. However, the development of octopus farming is limited by the lack of information regarding nutritional requirements of this species during its life cycle. In this study, five diets were tested on the biological performance (growth, protein retention and biochemical composition) of individually reared octopuses (n = 8 per diet), including three single diets constituted by: an endemic crab (the white crab, Plagusia depressa ), a commercial crab imported frozen (the blue crab, Portunus pelagicus ), and bogue ( Boops boops ) discarded from fish farms (aquaculture by-product), as well as two mixed diets, containing a 60–40% of blue crab-bogue and white crab-bogue, respectively. The rearing period lasted 8 weeks. Octopuses that fed on a mixed diet constituted by blue crab-bogue showed a higher growth than those feeding on bogue as a single food item. No significant differences in growth were observed among individuals feeding on single food items. Highest protein retention was observed in octopuses fed on diets containing discarded bogue, associated with a high lipid and monoenes content in this food item, underlying the use of lipid as energy source in O. vulgaris . However, discarded bogue was deficient in ARA in comparison with octopus tissues, which did not seem to affect growth during the experimental period. These findings underline the potential of aquaculture by-products, particularly bogue, as an adequate diet for culturing O. vulgaris .

Abalone Haliotis tuberculata coccinea Reeve (1846), is a target species for diversification of European aquaculture production. Taking into account that sustainable, eco-friendly production methods are to be a part of future expansion of the abalone industry, growth performance of juvenile abalone reared in an integrated culture system was evaluated and compared with that of abalone fed non-enriched macroalgae. Four macroalgae treatments, three monospecific: Ulva rigida (UN), Hypnea spinella (HN) and Gracilaria cornea (GN) and a composite one (MN), were produced out of fishpond wastewater effluents, while other four control treatments consisted of the same species reared in fresh seawater (U; H; G; M). Seaweeds reared in fishpond wastewater effluents increased their protein content from 11–17% to 29–34%. Lipids consisted mainly of saturated fatty acids (SFA) (43–60%), palmitic acid being the most abundant fatty acid (40–47%). Highest EPA percentage was found in red algae H. spinella (6.9%), being ten times higher than that of U. rigida (0.7%). All the algae tested contained very low levels of arachinodic acid (0.1–1.6%) and docosahexaenoic acid (0.5–3%). Protein levels in foot muscle (74–76%) did not differ significantly (P G. cornea led to the lowest growth performance probably due to the lowest feed intake. The results clearly indicate that H. tuberculata coccinea growout can efficiently take place in an integrated-culture system suggesting that on-farm seaweed-abalone production could be part of future development of the abalone industry in the Canary Islands.

The physiological response to stressors in fish, including hormonal profiles and associated tissue responsiveness, is less documented. The aim of this study was to evaluate feeding gilthead sea bream ( Sparus aurata ) with diets containing linseed oil (LO) and soybean oil (SO) as substitutes to fish oil (FO) and their effect on fatty acid profile of head kidney and the consequent effect on stress response to a crowding challenge. Fish were fed 8 experimental diets with different levels of substitution 0% (FO), 70% (70LO, 70SO, 20LO50SO and 50LO20SO) and 100% (100LO, 100SO and 50LO50SO) over a period of 8 months. At the end of the feeding trial, samples of head kidney were collected for biochemical analysis and the fish were challenged by a crowding test. During the challenge, samples of plasma for cortisol analysis were collected at 0 h, 2 h, 5 h, 24 h, 48 h and 1 week in order to study acute and chronic stress responses. Results showed that fish fed vegetable oils (VO) had significantly decreased ARA, EPA, DHA and n-3 HUFA, while LA, LNA and total C18 PUFAs were significantly increased. The basal cortisol levels were significantly increased in fish fed 70LO, 100LO, 50LO20SO and 50LO50SO. The physiological response to crowding was significantly affected by the diet. After 2 h of crowding, all the treatments showed higher cortisol, with fish fed 100LO had significantly the highest response registering 131.38 pg/ml. After 5 h and 24 h, plasma cortisol was reduced in all treatments except in 50LO20SO. After 48 h of crowding, the plasma cortisol was increased in all treatments with the maximum value seen in fish fed 100LO (72.12 pg/ml). These levels were decreased in fish fed FO, 70LO, 100LO and 50L050SO after 1 week of crowding, but remained higher in fish fed 70SO, 100SO, 20LO50SO and 50LO20SO. In conclusion, fish fed LO diets showed the same response pattern as the control but with higher intensity regaining the basal levels after 1 week as the control, while fish fed SO had a slow response but changed the pattern characterized by a lower response at the beginning and longer recuperation without regaining the control value even after 1 week.

The post mortem degradation processes that take places in fish muscle gradually modify the initial state of freshness. Tissue degradation is accompanied by drastic myofibrillar proteolysis produced as a consequence of the activation of proteolytic enzymes. In this study, structural changes were correlated with the textural properties of sea bream (Sparus aurata) muscle during 14 days of post mortem cold storage through the immunohistochemical detection of muscle filament proteins (desmin, actin and dystrophin), as well as endoproteases (μ-calpain, m-calpain) and their endogenous inhibitor (calpastatin). Actin and desmin were detected in fish muscle as late as 10 days post mortem (dpm) while dystrophin could not be detected after 4 dpm. In contrast, labelling for both calpains and calpastatin persisted during the entire storage period. Fracturability was clearly reduced after 7 dpm, while greatest changes in hardness, gumminess and chewiness were observed during the first week of storage. Our immunohistochemical observations indicate the disappearance of cytoskeleton proteins at different times post mortem. Thus, actin and desmin persist even when the muscle tissue shows a deteriorated appearance and texture, while dystrophin vanishes soon after the death of the fish. Detachment between myofibres and the myocommata was concomitant with the loss of dystrophin and also corresponded to the time when the reduction in flesh hardness observed was most significant. On the contrary, after 4 days of storage, calpain activity remained practically unaltered in refrigerated muscle specimens.

Despite being proposed as a potential candidate for diversification of marine aquaculture, limited larval survival and the occurrence of elevated levels of skeletal deformities restrict the commercial production of red porgy. The present study was conducted to determine the effect of rotifer enrichment, particularly on DHA, on growth, survival and occurrence of skeleton deformities in this species. The study included two trials, in one the viability of commercial emulsions for rotifer enrichment was evaluated whereas in another the effect of the elevation of DHA in rotifer emulsions on the larvae was studied. No significant differences were found in growth between larvae fed different rotifers in both trials suggesting that 1.9% dw DHA is enough to fulfil the DHA requirements of red porgy larvae for maximum growth. However, a marked positive effect of rotifer DHA content supplementation on larval survival was found. A low larval survival was found when rotifers had a high DPA content, in agreement with the results obtained for other Sparids. This fatty acid was accumulated in red porgy larval tissues in high levels (0.79% dw DPA) when rotifers rich in DPA were the main source of food. Although the external appearance of the juveniles seemed to be normal, X-ray observations showed elevated levels of bone abnormalities associated, in both trials, to low DHA content in the live prey. Among different anomalies, the presence of fused vertebrae was the most frequent deformity for both rearing trials. Although DHA content in the rotifers used for feeding was the only difference a 50% reduction in the number of deformed fish for each type of deformity studied was obtained when the larvae fed higher DHA levels, denoting the important role of this FA in the prevention of deformities at the rotifer feeding stage. Further studies are needed to elucidate the importance of essential fatty acids on the development of bone deformities in fish, since the functions of HUFA are different, and their absolute levels and ratios among them can lead to very different effects in fish metabolism, including bone formation.

Two single cell heterotrophs were produced as alternative sources of docosahexaenoic acid (DHA; 22:6 n-3) to fisheries-derived oils. Schizochytrium G13/2S or Crypthecodinium cohnii biomasses, either homogenised (S H , C H , respectively) or non-homogenised (S, C), were tested in gilthead seabream ( Sparus aurata ) microdiets. In Trial 1 the biomases of both species algae were used in replacement of fish oil, whereas in Trial 2 all dietary lipids, including those derived from squid meal, were replaced by the algal biomass in all diets except for control diet. Larval dietary acceptance, overall survival, air-exposure stress survival, growth and disease resistance were not significantly affected by the source of DHA employed when only fish oil was substituted. However, complete substitution of all dietary lipids by the algal biomass, resulted in reduced growth and survival which was apparently related to other dietary imbalances besides simply DHA content, such as changes in the DHA:EPA (eicosapentaenoic acid; 20:5 n-3) ratio. After 15 and 21 d feeding the experimental diets to S. aurata larvae in Trial 1 (0.1 mg initial larval dry weight) and Trial 2 (0.15 mg initial larval dry weight), respectively, the algal biomass inclusion or homogenisation did not significantly affect the DHA deposition in fish body (14.4 ± 0.3% total fatty acids ± SEM and 28.4 ± 0.4, average values for Trials 1 and 2, respectively). Higher arachidonic acid levels were found in larvae fed Schizochytrium sp. diets in Trial 2 and were related to the retro-conversion from docosapentaenoic acid (DPA; 22:5 n-6) which was high in these diets. These results show the potential of single cell heterotrophs as alternative DHA sources for fish oil in microdiets for gilthead seabream but also point out the necessity of EPA sources to completely replace fisheries-derived oils.

Red porgy, Pagrus pagrus, is a candidate for aquaculture diversification in the Mediterranean area. The main restriction for the development of this species, under cultured conditions, is the loss of their natural red–pink coloured skin. Several studies, using different dietary astaxanthin sources have given positive results on red porgy skin coloration. However, there is scarce information on the time required for cultured red porgy to obtain adequate skin colour when astaxanthin is included in the diet. This study aims to evaluate the effect of supplementing adult red porgy diet with shrimp shell meal, as a source of esterified astaxanthin, during different periods prior to harvest, on skin colour and skin carotenoid concentration. The experiment was carried out for 180 days, testing a control diet with no carotenoids and a shrimp shell meal diet (SM) with an inclusion of 16% shrimp shell meal in substitution of fish meal. Four treatment groups were established. Control treatment fish fed throughout the experiment on the control diet and SM60, SM120 and SM180 treatment fish fed first on the control diet and thereafter for 60, 120 and 180 days before harvesting on the SM diet. Growth was higher in fish from SM180 treatment group in comparison to control fish. Skin redness (a⁎) increased with increasing feeding period, and skin yellowness (b⁎) only until 120 days of feeding with SM diet. Whereas skin lightness (L⁎) does not seem to be influenced by dietary inclusion of shrimp shell meal for different periods of time. Skin carotenoid concentration also augmented with increasing feeding with SM diet. As a result, cultured red porgy fed for a period of 120 to 180 days, prior to harvest, on the SM diet may achieve similar skin coloration to wild red porgy, only differing in skin carotenoid concentration.

Developmental changes in swimming speed were analysed in the seabream (Sparus aurata) larvae. Four feeding regimes using live preys (rotifer Brachionus plicatilis) enriched with fish oil, soybean oil, linseed oil and rapeseed oil, differing in fatty acid profile, were tested during the first weeks of larval life. There was an increase in burst swimming speed and cruise swimming speed during the visual stimulus experiment at day 16th of life in the present study in agreement with the better eye development in larvae of this age. Swimming activity before stimulus was significantly reduced when larvae were fed rotifers enriched with vegetable oils. Larvae fed with rotifers enriched with fish oil reacted with a higher burst swimming speed after a visual stimulus than after the sound stimulus (159.5 SL/s vs. 18.30 SL/s) denoting the importance of the vision during this period of development not only for predation but also for the burst. The reduction in dietary essential fatty acid contents, by the enrichment with vegetable oils, delays the appearance of response to visual stimulus, in agreement with the minor DHA content in eyes and brains of these larvae and suggesting a delay in the functional development of brain and vision.

This study examines the utility of Passive Integrated Transponder (PIT) tagging system in fingerlings of gilthead seabream, Sparus auratus L. Two different body positions (abdominal, dorsal) and four weight classes (2–3 g, 3–4 g, 4–5 g, 5–8 g) were tested in three experiments. Tag loss rate, as well as its effect on growth and mortality rates, was determined. Times of handling and healing were also assessed. Fish tagged in the abdominal cavity showed a significantly lower tag loss rate (14%) than those given dorsal muscle implants (40%). No differences were found in growth within experiment between tagged and untagged fish, their final mean weight ranging between 6–39 g and 6–38 g, respectively. There was no difference in mortality between tagged and untagged fish at any position (0–3.4% and 0–2.1%, respectively). Fish smaller than 3 g suffered significantly higher mortality (14.3%), indicating that the PIT tags are not suitable for very small individuals. PIT retention rate was 100% above 4 g, irrespective of the personal expertise in tagging. Mean tag application time was 19 s per fish. Mean wound healing time was close to 20 days. Hence, these results showed that PIT tagging of gilthead seabream above 3 g is feasible because it does not affect growth or mortality.

To determine the effect of long term feeding of diets with high levels of vegetable oils gilthead seabream juveniles were fed for 7 months with several diets containing vegetable oils at two inclusion levels. Anchovy oil was the only lipid source of FO (fish oil) diet, whereas in diets 60RO, 60LO and 60SO, 60% of fish oil was substituted by rapeseed (RO), linseed (LO) or soybean (SO) oils, respectively, and 80% was substituted by linseed or soybean oils in diets 80LO and 80SO. Afterwards, all fish were fed only a fish oil diet to determine the progressive evolution of fillet fatty acid profiles, since fish muscle fatty acid composition changes by feeding vegetable oils affecting its nutritional value for human health. Results showed that substitution by vegetable oils of up to 60% fish oil in diets for gilthead seabream does not affect growth and feed utilization even after a long feeding period. However, 80% substitution of fish oil significantly reduced growth. Feeding vegetable oils reduced muscle contents of docosahexaenoic acid (DHA) and arachidonic acid (ARA) to a lower degree than their reduction in the diet, denoting their importance, reduction of eicosapentaenoic acid (EPA) in muscle being more pronounced. Moreover, re-feeding with a FO diet for 60 days effectively recovered muscle DHA and ARA contents, whereas those of EPA were not recovered even after 90 days. Linoleic acid was strongly retained even after “wash out”. Fish fillets were very well accepted by the panel of judges. Dietary soybean oil inclusion produced significantly less shiny and less hard fillets, but more juicy and adhesive than FO fillets, with a slight earthy flavour when fish were fed 80SO diet. However linseed oil inclusion only increased shininess when fish were fed 80LO diet. A significantly higher chroma value was obtained for 80LO fish fillets, although it was not noticeable to the human eye.

In the present study 75 g European sea bass were fed for 8 months with different diets (22% dietary lipid content) containing vegetable oils at two inclusion levels. The control diet (Diet FO) contained anchovy oil as the only lipid source; in diets 60RO, 60LO, and 60SO of fish oil was substituted by rapeseed, linseed or soybean oils, respectively; finally, in diet 80LO, 80% of the fish oil was substituted by linseed oil. Fish were fed to apparent satiation three times a day. All fish were individually weighed once per month. Lipid and fatty acid composition of diets and fish fillets were determined at the beginning, middle and end of the experimental period. Once the commercial size was reached, all fish were fed a 100% FO containing diet during 150 days. No significant differences were found in feed intake. Fish fed the diets containing 80% linseed oil or 60% rapeseed oil had significant ( P After 150 days of re-feeding period with a 100% fish oil diet, DHA levels were restored in those fish previously fed diets containing vegetable oils, but EPA levels remained lower when compared with fish fed 100% FO diet. Flesh content of linoleic and linolenic acids remained higher in those fish previously fed soybean and linseed oil containing diets, respectively.

A 60-day feeding trial was conducted to assess the suitability of three red algae, Hynea spinella, Hynea musciformis and Gracilaria cornea, as potential feed for the culture of juvenile abalone, Haliotis tuberculata coccinea R. Seaweeds were reared in a biofiltration unit with fishpond waste water effluents. The three algal species were found to contain high protein contents which would be related to its production under the high nitrogen culture conditions of the biofilter system. Protein and carbohydrate contents were highest in H. musciformis and lowest in G. cornea. Survival rates of juvenile abalone were very good, regardless of the algae fed. Feed intake of H. spinella was highest, followed by H. musciformis. Growth rates of abalone were within the range obtained under commercial conditions, final shell length and weight being significantly highest in animals fed H. spinella and lowest in those fed G. cornea. Feeding G. cornea lead to the lowest growth performance due to the lowest feed intake, whereas feed conversion ratios were significantly highest for H. musciformis and protein efficiency ratios were higher for both H. spinella and G. cornea. This study suggested the good potential of any of the three red seaweeds tested—successfully produced by the biofilter system, their nutritional composition being similar to other macroalgae used as feed for abalone and matching the abalone protein and lipid requirements—hence promoting growth and survival. Nevertheless, the biofilter produced macroalgae H. spinella showed the highest dietary value for juvenile of H. tuberculata coccinea.

Whereas wild specimens exhibit a red pink silver colour, under captivity red porgy (Pagrus pagrus) skin turns dark grey, being directly associated with lower acceptance by the consumers and lower market value. This study was conducted to evaluate the effect of diet supplementation with two carotenoid sources, at two different concentrations, on growth and skin coloration. Fish of 44 g mean initial weight were fed the following diets: control (with no added carotenoids), CTX 40 and CTX100 (diets with 40 and 100 mg of synthetic canthaxanthin/kg); SM20 and SM40 (diets with 20 and 40 mg of astaxanthin from shrimp shell meal/kg). Three samplings were carried out in this study, at the beginning and after 75 and 105 days of feeding. At each sampling, individual weight and colour measurements were performed. Colour was evaluated in three different body zones: front lateral (zone I), front dorsal (zone II) and caudal fin (zone III). Growth was not influenced by the inclusion of carotenoids in red porgy diet. Only astaxanthin from shrimp shell meal, particularly at 40 mg/kg diet, was able to give red porgy skin an overall reddish coloration, suggesting a better utilization of astaxanthin from this source. Shrimp shell meal diets enhanced reddish hue and chroma values, nevertheless hue values at days 75 and 105 did not show a marked difference and chroma attained after 75 days of feeding time tended to be reduced at day 105, suggesting skin colour saturation after a certain feeding time. Carotenoid supplementation did not influence lightness of red porgy skin in the present experiment. From the three evaluated zones, zone I of fish fed SM40 diet presented lower hue, chroma and lightness variation; therefore, it could be proposed as a control zone for future studies. The results of this study suggests that the inclusion of astaxanthin, from shrimp shell meal, in red porgy commercial diets significantly improves skin coloration and markedly enhances the commercial value of this cultured species.

In many cultured fish species, particularly in those new for aquaculture, unpredictable and variable reproductive performance is an important limiting factor for the successful mass production of juveniles. An improvement in broodstock nutrition and feeding has been shown to greatly improve not only egg and sperm quality but also seed production. Gonadal development and fecundity are affected by certain essential dietary nutrients, especially in continuous spawners with short vitellogenic periods. Thus, during the last two decades, more attention has been paid to the level of different nutrients in broodstock diets. However, studies on broodstock nutrition are limited and relatively expensive to conduct. Lipid and fatty acid composition of broodstock diet have been identified as major dietary factors that determine successful reproduction and survival of offspring. Some fish species readily incorporate dietary unsaturated fatty acids into eggs, even during the course of the spawning season. Highly unsaturated fatty acids (HUFA) with 20 or more carbon atoms affect, directly or through their metabolites, fish maturation and steroidogenesis. In some species, HUFA in broodstock diets increases fecundity, fertilization and egg quality. As in higher vertebrates, vitamin E deficiency affects reproductive performance, causing immature gonads and lower hatching rate and survival of offspring. For example, elevation of dietary α-tocopherol levels has been found to reduce the percentage of abnormal eggs and increase fecundity in the gilthead seabream ( Sparus aurata ). Ascorbic acid has also been shown to play an important role in salmonid reproduction, where the dietary requirement of broodstock was higher than that of juveniles. Among different feed ingredients, cuttlefish, squid and krill meals are recognized as valuable components of broodstock diets. The protein component of cuttlefish and squid together with their optimal concentration of HUFA appear to be responsible for their positive effect on reproductive performance. Both polar and nonpolar lipid fractions of raw krill were found to effectively improve egg quality.

In a previous study feeding larval gilthead seabream on microdiets, an improved growth rate was observed when dietary n−3 HUFA (n−3 highly unsaturated fatty acids) were contained in the polar lipid fraction. A beneficial effect of dietary lecithin on n−3 HUFA incorporation to the larval polar lipids was also observed. To confirm those results and in order to obtain additional information on larval gut and liver histological structure, 11-day-old larvae were fed 4 microdiets differing in lipid composition. In diets N′ and NL′, the main sources of n−3 HUFA were neutral lipids, and polar lipids in diets P′ and PL′. Three lecithin levels were tested, being the highest in diet NL′ and the lowest in diet P′. The total n−3 HUFA level in diets N′ and NL′ was enough to meet the essential fatty acid requirement of larval gilthead seabream obtained in a previous study with a triacylglyceride based diet, while diets P′ and PL′ had a total n−3 HUFA level lower than the minimum determined. A reduction in the larval essential fatty acid requirement to 1.5% d.w. was obtained by feeding dietary n−3 HUFA present in the polar lipid fraction. Feeding larvae with microdiets with low polar lipid levels produced an accumulation of lipid droplets in the enterocytes and a high number of lipid vacuoles in the hepatocytes producing nuclear migration and suggesting a need of dietary polar lipid for lipid transport.

Red porgy larvae, like other sparids such as red seabream are visual feeders. The normal development of the visual system is essential for successful prey capture and predator avoidance, leading to increased larval growth and survival. The aim of this work is to characterise the development of visual organs in relation to changes in the digestive system and feeding habits. Twenty-five larvae from hatching to day 29 were daily collected from the rearing tank, fixed in formalin, embedded in paraffin, 5 μm sectioned and stained with haematoxylin and eosin (H&E) and Periodic Acid Shift Reactive-Haematoxiline (PAS-Hx). Light microscopy was used to study changes in ocular morphology with respect to digestive system development. At hatching, eye and digestive systems of Pagrus pagrus larvae have no function. However, at day 3 post-hatch, when the mouth opens, the larvae must be ready for prey capture and digestion. Despite this, few day 3 larvae had food in the digestive tract. At day 4 photoreceptors were well developed in the eye, pigmentation pattern was complete and thus the visual system was completely ready for prey capture. This development coincided with detection of digestive activity in the midgut and most of larvae starting to take food. The results of this study suggest that adequate development of the visual system is important to establish the start of exogenous feeding. Besides, the appearance of rod cells increases larval photosensitivity and suggests that changes in lighting regimes could be necessary throughout the larval phase.

Effect of eight diets comparing three different lipid levels (15, 22 and 27%) and two fish meal qualities were studied on growth and liver histology. Fish meal quality was judged by the content of biogenic amines and temperature processing techniques. The experiment included a comparison of pelleted feed with extruded feed for the 22% lipid diet. A total of 1140 gilthead seabream of 70 g average initial body weight were randomly stocked in 500-l fiberglass tanks in duplicate groups of 60 fish. After 2 months of experiment, the fish were transferred to 1-m3 tanks. Fish were fed twice a day to apparent satiation for 6 months until they reached about 400 g (commercial size). Fish fed diets containing high quality fish meal showed, in general, a higher growth than those fish fed with low quality fish meal. For diets containing high quality fish meal, the fish fed 22 and 27% dietary lipid had significantly higher growth than those fish fed 15% dietary lipid. On the contrary, in diets containing low quality fish meal, only fish fed 27% dietary lipid showed significantly the higher growth rate. Fish fed the pelletized diets showed a lower growth than those fish fed extruded diets. Livers from fish fed diets containing high quality fish meal and 27% lipid showed foci of swelling hepatocytes that were not found for low quality fish meal at the same dietary lipid content. Ultrastructurally, these foci were characterized to present irregular nuclei displaced to periphery of hepatocytes and large lipid droplets in the cytoplasm. Livers from fish fed high and low fish meal qualities with 22% lipid showed similar morphological characters of hepatocytes to those that fed 15% lipid, but the difference was observed in the nuclei displacement.

Red porgy can be considered as a potential candidate to diversify aquaculture production of sparids, due both to the importance of red porgy fisheries and market within Europe, and to its phylogenetic closeness to gilthead seabream. Spontaneously spawned eggs were obtained from a broodstock of wild individuals captured in Canarian waters. Different rearing trials were conducted to study the success of: (a) different egg stocking densities (100, 150 and 200 eggs/l); (b) first feeding with rotifers enriched with different products (Powersh-fish oil, freeze-dried shark eggs and Selco supplemented with Nannochloropsis sp); and (c) supply of rotifers to larval cultures at different concentrations (5, 7 and 10 rotifers/ml). In all cases, larval feeding lasted for 14 days. Larval length was measured at the time of mouth opening, at day 10 of life, and at the end of the experiments. Larval survival was estimated at the end of each experiment by counting all the remaining live larvae. Total lipid and fatty acid composition of rotifers and larvae were analyzed and discussed in relation to larval growth and survival. In this study, egg densities between 100 and 150 eggs/l and the use of rotifers enriched with Powersh-W, containing a high level of 22:6n−3, at concentrations of 5–7 rotifers/ml produced the best larval growth and survival.