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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.

Macrophage aggregates (MAs) are structures in the spleen, kidney and sometimes liver of fishes which have various functions such as recycling/storing/detoxification of cellular wastes and exogenous substances. They have been also reported to be important in the specific immune response and are used as health indicators. Changes in MA density, size and pigment content have been used in national and local monitoring programs in the U.S. as indicators of contaminant exposure. However, MA number and structure can also be affected by other factors, including general stress or nutritional status of fish. An experiment was conducted to study the effects of vitamin E and n −3 highly unsaturated fatty acid (HUFA) deficiencies and stocking density on spleen and kidney MAs of gilthead seabream, one of the most important species for Mediterranean aquaculture. Fish were held at two stocking densities, high and low, and fed experimental diets. Diet NE had no supplemental vitamin E, Diet NFA was deficient in n −3 HUFA and Diet C was a control diet. Number, size and shape factor of MAs were measured using image analysis. The percentage of tissue occupied by MAs was calculated from these measurements. The results showed that high stocking density alone increased the number of splenic but not kidney MAs of fish fed the control diet. A deficiency of n −3 HUFA alone also increased the number of splenic but not kidney MAs at both stocking densities. Vitamin E deficiency alone had no significant effect on MAs in either organ. However, the combined effect of vitamin E deficiency and high stocking density increased the number and size of kidney but not splenic MAs. This study indicates that specific dietary deficiencies can influence MA accumulation and that splenic MAs may be more responsive to general stress than kidney MAs.

Three day-old Sparus aurata larvae were fed rotifers ( Brachionus plicatilis ) previously enriched with four lipid emulsions containing different percentages (10–30%) of n −3 highly unsaturated fatty acids (HUFA) but a fixed docosahexaenoic (DHA; 22:6 n −3) to eicosapentaenoic acid (EPA; 20:5 n −3) ratio of about 2/1. The n −3 HUFA content of the enriched rotifers varied from 0.9 to 3.0% on a dry weight basis (DWB). In addition, the four groups of rotifers displayed a constant DHA/EPA ratio of around 1.4/1. After 7 days of feeding, no significant differences in larval growth were observed among the treatments. One week later, larval total length was significantly improved by the elevation of the n −3 HUFA level in the diet from 0.9 to 1.5% DWB, with further increment not exerting any apparent effect on larval development. There was some loss of total lipids in the larvae during the first week of feeding which particularly affected the levels of n −3 HUFA in the polar lipid fraction. These losses were clearly restored from day 7 to 14 of feeding. At the end of the experimental period, larvae fed rotifers contributing less n −3 HUFA, contained lower levels of total and polar lipids, as well as lower levels of n −3 HUFA in the polar lipids. In conclusion, under our experimental conditions, the minimum requirement for n −3 HUFA of 17 day-old S. aurata larvae appears to be met by 1.5% in rotifers DWB, almost four times less than the value previously reported by our group when using rotifers with DHA/EPA ratios 2–3 times less favourable to DHA than to EPA.

In a previous study, several histological alterations were found in the liver of gilthead seabream fed with a diet containing 30% soybean meal (SBM). In the current study, SBM containing diets were supplemented with either potassium phosphate, zinc sulfate or phytase ( Aspergillus ficuum ), or increasing the dietary n −3/ n −6 fatty acids ratio to meet that of a fish meal based diet. Diet composition did not affect fish growth, feed efficiency (FE) or protein productive value (PER). Phosphorous supplementation significantly reduced hepatosomatic indexes (HSI), although it did not alter liver lipid content. Both phosphorous supplementation and correcting the dietary n −3/ n −6 fatty acid ratio significantly altered the lipid and protein content in fish muscle. Only these two treatments and principally the corrected dietary n −3/ n −6 fatty acid ratio, improved the liver histological alterations observed in fish fed with SBM based diets.

To study the potential of fluorescence based assays in the study of lipid digestion in fish, acyl esters of 4-methylumbelliferone and 1-acyl-2-[6 (7 nitro-1,3 benzoxadiazol-4-yl)amino]caproyl labeled phosphatidylcholine compounds (NBD-PC) were used as substrates for the assay of neutral lipase and phospholipase, respectively, in the gut contents of turbot. 4-Methylumbelliferyl hepatanoate (4-MUH) was hydrolysed at a higher rate than the butyrate or oleate esters whilst the hexanoic (C6) ester of NBD-PC was a more convenient substrate for the phospholipase assay than the dodecanoic (C12) ester. Neutral lipase activity was almost 10% higher when 50 mm potassium phosphate buffer pH 7.8 was used instead of 0.01 m citrate/sodium phosphate buffer pH 7.2. Both assays were very sensitive: neutral lipase and phospholipase activities were detectable at a minimum protein concentration in the digesta of 0.04 and 1.25 mg/ml, respectively. When the variations in lipolytic activities with gut segment and with size of fish were examined neutral lipase activity was always found to be higher in the hindgut and rectum segments than in the foregut. Although phospholipase activity was also found to be highest in the hindgut of the largest fish examined (av. wt. 182.3g), in fish of average weight 8g fish the activity was similar in all three segments. In the digesta from the whole gut of smaller fish (av. wt. 0.2, 0.6 and 1.43g) neutral lipase and phospholipase activities increased with increasing body mass when expressed as per ml of digesta. It is concluded that fluorescence-based assays are applicable to the study of lipid digestion in fish of different size.

Studies on the alternative complement pathway (ACP) activity in fish have suggested the particular relevance of this pathway as a non-specific immune mechanism being its activation directly related with the availability of certain nutrients. In the present study ACP activity in gilthead seabream fed different dietary levels of a-tocopherol and n-3 HUFA deficiencies was studied under different stress conditions (overcrowding and repetitive chasing). A reduction in ACP activity was found when diets with deficiencies in α-tocopherol and n-3 HUFA were assayed at any of the experimental conditions assayed. Levels of plasma cortisol were similar to those of fish after crowding stress subjected to dietary deficiencies at low stocking density, suggesting that dietary deficiencies produced a chronic elevation of cortisol. When fish were subjected to repetitive chasing, plasma cortisol from fish fed control diet showed a progressive increase whereas plasma cortisol levels from both dietary deficient groups showed a transitory peak 4 days after the beginning of the repetitive chasing. However, ACP activity levels of fish fed deficient diets remained lower than the control group and constant during chasing period. These results suggest that both a-tocopherol and n-3 HUFA nutritional deficiencies rather than stress are an important determinant of fish complement activity.

Bone and fins development in Seriola rivoliana were studied from cleared and stained specimens from 3 to 33 days after hatching. The vertebral column began to mineralize in the neural arches at 4.40 ± 0.14 mm Standard Length (SL), continued with the haemal arches and centrums following a cranial-caudal direction. Mineralization of the caudal fin structures started with the caudal rays by 5.12 ± 0.11 mm SL, at the same time that the notochord flexion occurs. The first dorsal and anal fin structures were the hard spines (S), and lepidotrichium (R) by 8.01 ± 0.26 mm SL. The metamorphosis was completed by 11.82 ± 0.4 mm SL. Finally, the fin supports (pterygiophores) and the caudal fins were completely mineralized by 16.1 ± 0.89 mm SL. In addition, the meristic data of 23 structures were provided. Results from the present study might be used as a practical guide for future studies on this field with S. rivoliana or in related species.

Two feeding experiments were conducted to evaluate the suitability of two commercial microdiets as a complementary food for the rearing of larval gilthead seabream Sparus aurata. The effect of these diets on growth, survival and total lipid composition, as well as the histological structure of the liver were examined. The results of the first trial highlighted water quality as an important factor for the survival of microdiet-fed larvae. An increase in water flow rate improved larval survival despite a reduction in live food supply. An effect of tank design on larval growth and survival was observed also and seems to be related with the light level inside the tanks; wider light grey tanks gave better results than narrower black ones. Microdiet feeding significantly improved larval growth and increased hepatocyte diameter and presence of PAS positive vacuoles suggesting an increased glycogen storage in comparison with that of larvae fed live prey only. Artificial diets provided the larvae with a higher amount of lipids and polyunsaturated fatty acids of the n-3 series, resulting in a higher DHA content in the total lipid of the larvae fed on microdiets.

The effect of fish meal quality on growth, feed efficiency, protein digestibility and fillet composition was studied in a growth experiment with gilthead seabream. The experiment included a comparison of pelleted feed with extruded feed. The fish were fed three extruded diets which varied in the quality of the fish meal used in the respective feeds: low quality fish meal, good quality fish meal, and a mixture (50%) of the two fish meal qualities. Fish meal quality was judged by protein digestibility as measured with mink as the test animal and by the content of biogenic amines. A fourth experimental group was fed a pelleted feed with the same mixed fish meal blend. The experiment started with 70 g seabream and lasted for 3 months until the fish reached about 160 g. All groups showed good growth and feed efficiency during the experimental period, with daily specific growth rates (SGR) of 0.90–1.00% and feed efficiencies (FE; fish weight gain per feed offered) from 0.58 to 0.66. The fish fed the feed with low quality fish meal showed significantly poorer feed efficiency than the other groups, and there was a significant correlation between feed efficiency obtained in seabream and the protein digestibility as measured with mink. No difference was observed among groups fed the extruded diets, for growth, fillet composition or amount of liver or viscera, although a significant correlation was obtained between SGR and protein digestibility in mink. Fillet lipid content was higher in fish fed the extruded feed, compared with those fed the corresponding pelleted diet. Protein digestibilty in seabream showed no difference among the extruded feeds, while the pelleted feed was significantly lower.

The effects of live food ( Artemia nauplii ) and dietary supplementation of digestive enzymes (pancreatin) on ingestion and assimilation of dry microdiet (MD) and nutrient deposition and larval growth were studied in 20–39 day old seabass ( Dicentrarchus labrax ) larvae. Four treatments were tested at each age: (i) MD with Artemia nauplii; (ii) MD supplemented with digestive enzymes and Artemia nauplii; (iii) MD supplemented with digestive enzymes; (iv) MD without enzyme supplementation. Ingestion and assimilation of the MD were tested by trials using a radioisotope method. Another trial was conducted to investigate the effect of MD in larval growth and body composition. Addition of Artemia to MD-fed seabass larvae positively affected rates of assimilation and growth, regardless of the age of the fish. No effect of dietary enzymes on MD assimilation or larval growth was observed. However, addition of Artemia and dietary enzyme supplementation affected lipid and protein deposition in the larvae. In conclusion, live Artemia given together with MD enhanced the efficiency of MD in seabass, by promoting the assimilation and deposition of dietary nutrients in the larval body. The factors responsible in Artemia and the possible mechanisms involved are discussed.

To investigate the specific component of squid meal which enhances spawning quality, gilthead seabream broodstock were fed four isonitrogenous, isolipidic and isocaloric diets containing fat-soluble or fat-insoluble extracts from fish and squid meals for 2 months during the spawning season. A positive effect of the fat-insoluble fraction of squid meal was observed on the total number of eggs, egg viability and the number of larvae produced daily per kilogram of female. Increased dietary levels of eicosapentaenoic acid correlated with the number of fertilized eggs produced, whereas elevation of n − 3 highly unsaturated fatty acid levels was correlated with a reduction in the percentage of eggs with more than one lipid droplet. The results of this study highlight the value of squid meal as a good protein and lipid source in diets for gilthead seabream broodstock.

Nowadays, aquaculture industry still confronts several disease-related problems mainly caused by viruses, bacteria and parasites. In the last decade, the use of mannan oligosaccharides (MOS) in fish production has received increased attention due to its beneficial effects on fish performance and disease resistance. This review shows the MOS use in aquaculture with a specific emphasis on the effectiveness of the several MOS forms available in the market related to disease resistance, fish nutrition and the possible mechanisms involved. Among the main beneficial effects attributed to MOS dietary supplementation, enhanced fish performance, feed efficiency and pathogen protection by potentiation of the systemic and local immune system and the reinforcement of the epithelial barrier structure and functionality are some of the most commonly demonstrated benefits. These combined effects suggest that the reinforcement of the intestinal integrity and functionality, together with the stimulation of the innate immune system, are the primary mode of action of MOS in fish. However, the supplementation strategy related to the structure of the MOS added, the correct dose and duration, as well as fish species, size and culture conditions are determinant factors to achieve improvements in health status and growth performance.

The expression and regulation of intestinal oligopeptide transporter (PepT)-1 when vegetable sources are used as a substitute for fish meal in the diet of marine fish has not yet been explored. In the present study, as part of our ongoing work on elucidating PepT1 gene expression in relation to different dietary treatments, we have now isolated and deposited in Genbank database (accession no. GU733710) a cDNA sequence representing the PepT1 in the sea bream (Sparus aurata). The “de novo” prediction of the three-dimensional structure of PepT1 protein is presented. We also analyzed diet-induced changes in the expression of PepT1 mRNA via real-time RT-PCR using the standard curve method. Sea bream were fed for 140 days with one of the following four diet formulations (43% protein/21% lipid): a control fast growth-promoting diet (C), and three diets with the same formulation but in which 15% of the fish meal was substituted by protein concentrates either from lupine (LPC), chick pea (CPC), or green pea (PPC). Fish fed PPC had significantly (p < 0.05) lower levels of PepT1 transcripts in the proximal intestine than the controls, whereas PepT1 transcript levels in fish fed LPC or CPC were not significantly different from the controls. Although growth was similar between fish fed with different diets during the first 72 days of feeding, growth of the fish fed with PPC was reduced during the second part of the trial and was significantly (p < 0.05) lower than fish fed LPC and CPC diets by the end of the experiment. Correlation between these results and fish growth performances highlights that the intestinal PepT1 mRNA level may serve as a useful marker of the dietary protein quality and absorption efficiency.

The aim of this study was to determine if algal products rich in DHA or ARA are able to completely replace fish oil in microdiets for marine fish larvae, gilthead seabream and if extra supplementation with EPA may further enhance larval performance. For that purpose, 20day-old gilthead seabream larvae of 5.97 +/- 0.4mm mean total length and 0.12 +/- 0.001mg mean dry body weight were fed with five microdiets tested by triplicate: a control diet based on sardine oil; a diet containing AquaGrow (R) DHA (diet DHA) to completely substitute the sardine oil; a diet containing AquaGrow (R) ARA (diet ARA); a diet containing both products, AquaGrow (R) DHA and AquaGrow (R) ARA to completely substitute the fish oil; and, a diet containing both products, AquaGrow (R) DHA and AquaGrow (R) ARA, together with an EPA source. Temperature, air and salinity activity tests were also performed to detect larval resistance to stress. At the end of the experiment, final survivals did not differ among groups. The microorganism produced DHA was able to completely replace fish oil in weaning diets for gilthead seabream without affecting survival, growth or stress resistance, whereas the inclusion of microorganism produced ARA did not improve larval performance. Moreover, addition of EPA to diets with total replacement of fish oil by microorganism produced DHA and ARA, significantly improved growth in terms of body weight and total length. The results of this study denoted the good nutritional value of microorganisms produced DHA as a replacement of fish oil in weaning diets for gilthead seabream, without a complementary addition of ARA. However, dietary supplementation of EPA seems to be necessary to further promote larval performance.

The greater amberjack Seriola dumerili (Risso 1810) is a marine pelagic species with a high interest for aquaculture due to its rapid growth and high commercial value. However, during grow out, monogenean parasites cause occasional mass mortalities in farmed fish and there is not so much published data on the optimum temperature for growth and immunological parameters of this fish species fingerlings. Two hundred and twenty five juveniles were kept in triplicated in three experimental recirculated aquaculture system (RAS) at 17, 22 and 26 oC, each RAS controlling 3 tanks. Fish were fed a commercial diet to apparent satiety during four months. Growth was monitored each month. At the end of the experimental period samples of intestine, skin, blood and skin mucus were obtained. Growth, feed efficiency and immunological parameters, such as lysozyme and bactericidal activity have been studied to observe the relations between the different growth rates produced by the temperature and the immune system in serum and skin mucus. Besides, samples form skin and intestine were also obtained for histological studies, including the mucus production potential by analysing the goblet cells and the distribution of GALT in posterior intestine. Fish held at 26oC presented the best growth and feed utilization, being the results obtained for immune potential discussed also in terms of temperature influence.

Low dietary levels of raw marine ingredients in marine fish species diets induce variable side-effects on fish growth performance, welfare and health, depending mainly on the terrestrial protein and oil source used, dietary levels and fish species studied. Particularly, high dietary levels of vegetable meals (VM) and oils (VO) blends, may induce intestinal dysfunction by altering tissue morphology, intestinal functionality, microbiota populations or gut associated lymphoid tissue (GALT) homeostasis. On the other hand, alternative feed ingredients such as prebiotics and probiotics have demonstrated to promote fish performance and disease resistance through potentiating GALT efficiency and functionality. Thus, the main objective of this study is to evaluate the effects of dietary mannan oligosaccharides (Biomos® and ActigenTM), Pediococcus acidilactici (BAC; Bactocell ®) and their combination in low FM and FO diets for european sea bass juveniles. Fish of 19.7±0.09g were distributed in 18 tanks and fed for 90 days six experimental diets with a low content of FM and FO, containing graded and combined levels of MOS and BAC as follows: HP; LP; B; HPB; LPB and C (Control). After 60 and 90 days of feeding, fish were sampled for growth, gut morphometry and histopathology, gut mucus production and expression of GALT related genes. Besides, samples were taken for gut iNOS and TNFα immunohistochemistry (IHQ) and for in situ hybridation (ISH) studies of MHCβ, TCRβ, CD4 and CD8. Fish fed diets HP, LP and LPB presented higher final weight. Two-way ANOVA attributed this effect to MOS regardless of dietary BAC inclusion. The use of prebiotic induced an increase on number of goblet cells in posterior gut (Fig.1). IHQ analyses performed against iNOS and TNFα showed the use of prebiotics induce an inflammatory-like pattern in the apical side of gut villi. Results for ISH showed that combined use of pre and probiotic induced a wider distribution of TCR-β marked cells, with special emphasis in the lamina propria. Expression of GALT-related genes, will be also discussed. Fig.1. Posterior gut (Alcian Blue-PAS). (a) LPB and (b) C diets t=90days. Note the higher number of goblet cell for fish fed LPB diet Acknowledgements: This study has been funded by the Spanish Ministry of Economy and Competitiveness, Project AGL2012-39919 (PROINMUNOIL)

Docosahexaenoic acid (DHA) is an essential fatty acid necessary for many biochemical, cellular and physiological functions in fish. However, high dietary levels of DHA increase free radical injury in sea bass (Dicentrarchus labrax) larvae muscle, even when vitamin E (α-tocopherol, α-TOH) is increased. Therefore, the inclusion of other nutrients with complementary antioxidant functions, such as vitamin C (ascorbic acid, vitC), could further contribute to prevent these lesions. The objective of the present study was to determine the effect of vitC inclusion (3,600 mg/kg) in high DHA (5 % DW) and α-TOH (3,000 mg/kg) microdiets (diets 5/3,000 and 5/3,000 + vitC) in comparison to a control diet (1 % DHA DW and 1,500 mg/kg of α-TOH; diet 1/1,500) on sea bass larvae growth, survival, whole body biochemical composition and thiobarbituric acid reactive substances (TBARS) content, muscle morphology, skeletal deformities and antioxidant enzymes, insulin-like growth factors (IGFs) and myosin expression (MyHC). Larvae fed diet 1/1,500 showed the best performance in terms of total length, incidence of muscular lesions and ossification degree. IGFs gene expression was elevated in 5/3,000 diet larvae, suggesting an increased muscle mitogenesis that was confirmed by the increase in the mRNA copies of MyHC. vitC effectively controlled oxidative damages in muscle, increased α-TOH larval contents and reduced TBARS content and the occurrence of skull deformities. The results of the present study showed the antioxidant synergism between vitamins E and C when high contents of DHA are included in sea bass larvae diets.

The potential muscle regeneration after nutritional dystrophy caused by high dietary DHA contents in fish and the physiological pathways involved are still unknown. To better understand this process, an experiment was conducted for 3 weeks in 14 day-old European sea bass larvae using different DHA ratios (1 or 5%). After this period, part of the larvae fed 5% DHA diet was switched to 1% DHA diet ("wash-out") for another 2 weeks. Larvae fed 5% DHA diet showed altered oxidative status as indicated by the highest TBARS values, antioxidant enzymes (AOE) expression and incidence of muscular lesions. Accordingly, "washed-out" larvae showed lower dry weight and α-TOH content. IGF-I gene expression was elevated in 5% DHA larvae at 35 dph, suggesting increased muscle mitogenesis that was corroborated by the increase in myosin heavy chain expression. It can be concluded that high dietary DHA contents alter the oxidative status and cause muscular lesions in European sea bass larvae, with morphological and molecular aspects of mammalians muscular degenerative disease.

The study assesses the effects of dietary mannan oligosaccharides (MOS) in European sea bass (Dicentrarchus labrax) posterior intestinal lipid class composition and its possible relation to the potential prostaglandins production and Gut Associated Lymphoid Tissue (GALT) stimulation. Fish were fed 4 g kg(-1) MOS (Bio-Mos(®) Aquagrade, Alltech, Inc., USA) for eight weeks. Fish fed MOS presented higher (P ≤ 0.05) weight gain, total length, and specific and relative growth rates than fish fed the control diet. Stimulated posterior gut of fish fed MOS showed higher (P ≤ 0.05) prostaglandins production than fish fed the control diet. Lipid class analyses of posterior gut revealed a reduction (P ≤ 0.05) in the neutral lipid fraction in fish fed MOS compared to fish fed the control diet, particularly due to a reduction (P ≤ 0.05) in triacylglycerols content. The polar lipid fraction increased (P ≤ 0.05) in fish fed MOS compared to fish fed the control diet, mainly due to an increase (P ≤ 0.05) in phosphatidylethanolamine and phosphatidylcoline contents. Light microscopy of posterior gut revealed increased number or goblet cells as well as higher level of infiltrated eosinophilic granulocytes for fish fed MOS. Transmission electron microscopy qualitative observations revealed a better preserved cytoarchitecture of the intestinal epithelial barrier in the posterior gut of fish fed MOS. Posterior gut of fish fed MOS presented more densely packed non-damaged enterocytes, better preserved tight junctions structure, healthier and more organized microvilli, and a higher presence of infiltrated lymphocytes and granulocytes compared fish fed the control diet. The present study indicates that dietary MOS enhances European sea bass posterior gut epithelial defense by increasing membrane polar lipids content in relation to a stimulation of the eicosanoid cascade and GALT, promoting posterior gut health status.

The objective of the present study was to determine the effect of Se inclusion in high-DHA and vitamin E microdiets (5 g DHA/100 g dry weight and 300 mg vitamin E/100 g dry weight; 5 g DHA/100 g dry weight and 300 mg vitamin E/100 g dry weight supplemented with Se) in comparison with a control diet (1 g DHA/100 g dry weight and 150 mg vitamin E/100 g dry weight) on sea bass larval growth, survival, biochemical composition, malonaldehyde (MDA) content, muscle morphology and antioxidant enzymes (AOE), insulin-like growth factors (IGF) and myosin expression. For a given DHA and vitamin E dietary content, Se inclusion favoured larval total length and specific growth rate, and reduced the incidence of muscular lesions, MDA contents and AOE gene expression. In contrast, IGF gene expression was elevated in the 5/300 larvae, suggesting an increased muscle mitogenesis that was corroborated by the increase in mRNA copies of myosin heavy chain. The results of the present study denoted the beneficial effect of Se not only in preventing oxidative stress, as a glutathione peroxidase cofactor, but probably due to other as yet unknown physiological functions.

Rotifers were enriched for 24 h with a lipid emulsion and the algae Nannochloropsis sp. Some of the rotifers were treated with antibiotics (chloramphenicol and oxolinic acid). Larval sizes were recorded initially, at day 7 and at the end of the experiment. Larval survival rates were estimated at the end of the experiment. Rotifers and larvae were analysed for lipid and moisture as well as the fatty acid profile of total lipids. The number of bacterial colonies from rotifer and larvae culture was also recorded. The best growth rate was found in larvae reared in a recirculating system with addition of phytoplankton and antibiotics, although no significant differences (P < 0.05) were apparent with the same treatment without antibiotics. Larvae reared in a flow-through system with phytoplankton showed the best survival rates, but larvae receiving rotifers treated with antibiotics showed the lowest survival rates. The n−3 HUFA levels in rotifers and larvae treated with antibiotics were significantly lower than those in treatments without these chemicals. The results of this study suggest that antibiotics should not be used indiscriminately in hatcheries as they may exert an adverse effect on larval cultures.

The results of studies on the essential fatty acid requirement of larval gilthead seabream differ in the recommended n −3 HUFA levels in rotifers ( Brachionus plicatilis ) necessary to obtain optimal larval growth and survival. Some authors have suggested that the total lipid content of the rotifers may influence the fatty acid requirement of the larvae. In order to study this hypothesis, an experiment was conducted by feeding gilthead seabream larvae microdiets containing combinations of two lipid and two essential fatty acid levels. Fifteen-day-old gilthead seabream larvae were fed with 4 microparticulated diets containing the following lipid and n −3 HUFA levels: (A) 12.44% and 0.82%; (B) 12.72% and 2.16%; (C) 19.45% and 0.74%; and (D) 20.03% and 2.05%. Larvae were fed the experimental diets together with a small amount of rotifers cultured on baker's yeast for 12 days. Larvae fed Diet A had the lowest growth rate ( P n −3 HUFA (B and D) supported the best survival rates regardless of their lipid content. These results showed that for the lipid levels tested, the larval n −3 HUFA requirement did not decrease as the lipid level in the diet decreased.

Pink dentex ( D. gibbosus ) can be considered as a potential candidate to diversify the culture of Sparidae, due both to the importance of dentex fisheries and market within Europe, and to its phylogenetic closeness to the gilthead sea bream ( Sparus aurata , Linnaeus, 1758). First trials to obtain D. gibbosus spawnings under captive conditions were carried out at the Planta Experimental de Cultivos Marinos (Instituto Canario de Ciencias Marinas, Gran Canaria, Canary Islands, Spain) in May, 1991. Broodstock management conditions determining egg quality, embryonic and larval development of D. gibbosus up to day 31 are described for the first time. Larval allometric characteristics were studied. The equation y =2.321×e 0.03975 x represents larval growth under experimental conditions. A comparison of egg size, hatching time and size of newly hatched larvae in several Sparidae under culture conditions is also presented. Results on broodstock acclimation, natural spawnings and larval growth rates suggest that pink dentex has a high potential for commercial culture.

Two experiments were carried out to study the effect of n-3 HUFA levels in rotifers on survival, growth, activity and fatty acid composition of gilthead bream, Sparus aurata (L.), larvae. From the third to the 15th day after hatching, gilthead bream larvae received one of the three kinds of rotifers containing different percentages of n–3 HUFA. Moisture, crude lipid, saponifiable matter and fatty acid composition of total lipids of rotifers and larvae were determined. A good correlation was found between larval growth and n–3 HUFA levels in rotifers. Larval survival was also significantly improved by the elevation of the n–3 HUFA levels in rotifers. A high occurrence of hydrops was registered in larvae fed with EFA-deficient rotifers. The n–3 HUFA levels in the larvae were increased by the elevation of n–3 HUFA contents in rotifers. However, n–9 fatty acids in the larvae remained almost constant, regardless of the different 18:1n–9 contents in rotifers. Therefore, the ratio of oleic acid to n–3 HUFA, known to be an indicator of the EFA deficiency in fish, was reduced by the elevation of the n–3 HUFA levels in rotifers.

Dietary omega − 3 long chain polyunsaturated fatty acids (n − 3 LCPUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have a marked effect on fish behavior. There is limited information on the mechanisms involved in this effect and its relation to neuron development and functioning. Deficiency of n − 3 LCPUFA reduces fish escape swimming. Mauthner cells (M-cell) are neurons responsible for initiating an escape response. The aim was to compare the effect of dietary DHA and EPA on escape behavior and neuronal activity of sea bream larvae. We studied burst swimming speed as a measure of behavior. M-cell activity was studied by ChAT immuno-fluorescence. Feeding the lowest n − 3 LCPUFA levels a lower burst swimming speed. Increase in dietary EPA did not significantly improve escape response. Elevation of dietary DHA was correlated with a higher burst speed denoting the importance of this nutrient for escape swimming. Incorporation of DHA into larval tissues was proportional to DHA dietary levels and significantly correlated with burst speed. In addition, a higher immunoreactivity to ChAT, associated to a higher neural activity, was found in M-cell of larvae fed higher dietary DHA contents. These results show first evidence of n − 3 LCPUFA on fish neuronal activity and their implications in behavior, denoting that DHA boosts escape swimming and this effect is at least partly mediated by the increase in neural activity of M-cell.

In previous studies, we observed dystrophic alterations in muscle of 48-day-old sea bass fed imbalanced docosahexaenoic acid (DHA) and vitamin E diets. To understand the whole pathological process associated with oxidative stress, a histological study was performed by feeding 14-day-old sea bass larvae with microdiets containing different ratios of DHA/vitamin E (1/150, 5/150 and 5/300) for a period of 21 days. Larvae fed diet 1/150 showed no lesions in contrast to larvae fed diets 5/150 and 5/300 where the highest incidence of muscle lesions and thiobarbituric acid reactive substances (TBARS) content was observed. Semithin sections revealed focal lesions consisting of degenerated fibres with hypercontracted myofilaments and extensive sarcoplasm vacuolization affecting both red and white muscle. Ultrathin sections of degenerating muscle fibres showed diffuse dilatation of sarcoplasmic reticulum, disorganized myofilaments and autophagic vacuoles containing myelin figures and dense bodies. Additionally, some macrophages were observed among injured fibres as numerous satellite cells. Results from the study agree with those obtained from previous work, proving the pathological potential of free radicals in sea bass larvae musculature. Moreover, high vitamin E inclusion could not completely protect cell membranes from free radicals action.

The mode of action of highly unsaturated fatty acids (HUFA) in regulating gilthead sea bream (Sparus aurata) head kidney (HK) cortisol production was studied throughin vitrotrials using a dynamic superfusion system. Fish were previously fed with different diets containing several inclusion levels of linseed oil (LO) or soyabean oil (SO) for 26 weeks. Five diets were tested; anchovy oil was the only lipid source for the control diet (fish oil, FO) and two different substitution levels (70 and 100 %) were tested using either LO or SO (70LO, 70SO, 100LO and 100SO). Fatty acid compositions of the HK reflected the dietary input, thus EPA, DHA, arachidonic acid andn-3 HUFA were significantly (P P P in vitrocortisol release, and this effect is partly mediated by COX and/or LOX metabolites.

In gilthead sea bream Sparus aurata held in groups of two, five or 10 fish, social hierarchies were observed. Subordinate S. aurata were characterized by elevation of basal levels of plasma cortisol, together with a reduced immunological potential. Subordinate fish also showed lower feed intake, feed utilization and lower growth. Fatty acid composition was also affected by social status, with a lower content of saturated acids, oleic and eicosapentaenoic, in muscle and liver of fish considered as subordinate. Results show that social hierarchy acts as a stressor in S.aurata.

Commercial gilthead sea bream feeds are highly energetic, fish oil traditionally being the main lipid source. But the decreased fish oil production together with the increased prices of this oil encourages its substitution by vegetable oils, imposing new nutritional habits to aquaculture species. Partial replacement of fish oil by vegetable oils in diets for marine species allows good feed utilization and growth but may affect fish health, since imbalances in dietary fatty acids may alter fish immunological status. The effect of dietary oils on different aspects of fish immune system has been reported for some species, but very little is known about the effect of dietary oils on immune-related genes expression in fish. Thus, the objective of this study was to elucidate the role of dietary oils on the expression of two pro-inflammatory cytokines, Tumor Necrosis Factor-α (TNF-α) and Interleukine 1β (IL-1β) on intestine and head kidney after exposure to the bacterial pathogen Photobacterium damselae sp. piscicida. For that purpose, 5 iso-nitrogenous and iso-lipidic diets (45% crude protein, 22% crude lipid content) were formulated. Anchovy oil was the only lipid source used in the control diet (FO), but in the other diets, fish oil was totally (100%) or partially (70%) substituted by linseed (rich in n-3 fatty acids) or soybean (rich in n-6 fatty acids) (100L, 100S, 70L, 70S). Fish were fed experimental diets during 80 days and after this period were exposed to an experimental intestinal infection with the pathogen. Serum and tissue samples were obtained at pre-infection and after 1, 3 and 7 days of infection. RNA was extracted and cDNA was synthesized by reverse transcription from intestine and head kidney and the level expression of TNF-α and IL-1β were assayed by using quantitative real time PCR. The expression level of genes analysed was represented as relative value, using the comparative Ct method (2(-ΔΔCt)). Serum anti-bacterial activity was measured as serum bactericidal capacity and lysozyme activity. Reduction of FO tends to reduce basal (pre-infection) genetic expression of both cytokines. However, complete FO replacement caused an over expression of both pro-inflammatory cytokines, particularly after 3 days of induced infection in fish fed soybean oil based diets. On the other hand, fish fed diets with low content of n-6 fatty acids showed better serum bactericidal capacity after infection, suggesting that the substitution of fish oil by vegetable oils containing high levels of n-6 fatty acids may induce imbalances on fish immune response, leading to a lower potential response against infections.

Marine fish larvae fed microdiets have not, at this stage, matched the growth and survival performances demonstrated by larvae fed live feeds such as rotifers and Artemia. This chapter discusses the issues related to the use of microdiets as a sole or partial feed for marine fish larvae. The techniques and methods of manufacturing microdiet particles, chemical and physical properties and the relationship to the ingestion and digestion are described. The chapter also looks at the physiological development and the nutritional requirements of larvae. Advances in feeding regimes such as co-feeding and feeding systems are also reviewed.

The aim of the present study was to investigate the hypothesis that parental periconception nutrition in adult seahorses affects the development and growth of their offspring. We tested the hypothesis that because seahorse embryos develop inside the male’s brood pouch, manipulation of the male’s diet would affect offspring growth and development independently of the female’s diet. Adult males and females were fed separately with either wild-caught crustaceans or commercial aquarium diet for 1 month before conception to influence the periconception environment. Approximately 10 000 offspring were obtained from four different treatment groups (Male/Wild or Male/Commercial × Female/Wild or Female/Commercial). Weights, physical dimensions and fatty acid profiles of the newborns were determined. Offspring produced when the males receiving commercial diet were mated with wild-fed females were larger (P

Lipid and docosahexaenoic acid (DHA) accumulation into Schizochytrium G13/2S was studied under batch and continuous culture. Different glucose and glutamate concentrations were supplemented in a defined medium. During batch cultivation, lipid accumulation, 35% total fatty acids (TFA) occurred at the arithmetic growth phase but ceased when cell growth stopped. When continuous culture was performed under different glutamate concentrations, nitrogen-growth-limiting conditions induced the accumulation of 30-28% TFA in Schizochytrium. As the dilution rate decreased from 0.08 to 0.02 h(-1), both cell dry weight and TFA content of the cell increased. Under a constant dilution rate of 0.04 h(-1), carbon-limiting conditions decreased the TFA to 22%. Fatty acid profile was not affected by the different nutrient concentrations provided during continuous culture. Consequently, lipid accumulation can be induced through the carbon and nitrogen source concentration in the medium to maximise the TFA and subsequently DHA productivity by this microorganism.

The aim of the present study was to determine the influence of the dietary fatty acid profile on the lipoprotein composition in sea bream fed different vegetable oils. Six experimental diets were formulated combining fish oil with three vegetable oils (soybean, rapeseed, linseed) in order to obtain 60– 80 % (w/w) fish-oil replacement. VLDL, LDL and HDL in plasma samples were obtained by sequential centrifugal flotation. The lipid class, protein content and fatty acid composition of each lipoprotein fraction were analysed. HDL was the predominant lipoprotein in sea bream plasma containing the highest proportion of protein (34 %) and phosphatidylcholine. LDL presented a high content of cholesterol, whereas triacylglycerol comprised a larger proportion of VLDL. The lipid class of the lipoprotein fractions was affected by the dietary vegetable oils. Thus, a high dietary inclusion of soyabean and linseed oil (80 %) increased the cholesterol in HDL and LDL in comparison to fish oil. Similarly, the triacylglycerol concentration of VLDL was increased in fish fed 80 % soyabean and linseed oils owing to the low n-3 highly unsaturated fatty acid content of these diets. Lipoprotein fatty acid composition easily responded to dietary fatty acid composition. VLDL was the fraction more affected by dietary fatty acid, followed by LDL and HDL. The n-3 highly unsaturated fatty acid content increased in the order VLDL less than LDL and less than HDL, regardless of dietary vegetable oils. Sea bream: Lipoproteins: VLDL: LDL: HDL: Vegetable oils: Cholesterol: Triacylglycerols: Phosphatidylcholine: Sterol esters: n-3 highly unsaturated fatty acids

Despite the good growth performance of several fish species when dietary fish oil is partly replaced by vegetable oils, recent studies have reported several types of intestinal morphological alterations in cultured fish fed high contents of vegetable lipid sources. However, the physiological process implied in these morphological changes have not been clarified yet, since alterations in the physiological mechanisms involved in the different processes of lipid absorption could be responsible for such gut morphological features. The objective of the present study was to investigate the activities of reacylation pathways in fish, the glycerol-3-phosphate and the monoacylglycerol pathways, in order to clarify the intestinal triacylglycerol (TAG) and phospholipid biosynthesis to better understand the morphological alterations observed in the intestine of fish fed vegetable oils. Intestinal microsomes of sea bream fed different lipid sources (fish, soyabean and rapeseed oils) at three different inclusion levels were isolated and incubated with L-[ 14 C(U)]glycerol-3-phosphate and [1- 14 C]palmitoyl CoA. The results showed that in this fish species the glycerol-3-phosphate pathway is mainly involved in phospholipid synthesis, whereas TAG synthesis is mainly mediated by the monoacylglycerol pathway. Feeding with rapeseed oil reduced the reacylation activity in both pathways, explaining the high accumulation of lipid droplets in the supranuclear portion of the intestinal epithelium, whereas soyabean oil enhanced phosphatidylcholine synthesis, being associated with the increase in VLDL found in previous studies. Intestinal microsomes: Vegetable oils: Sea bream: Monoacylglycerol pathway: Glycerol-3-phosphate pathway: Cytidine diphosphate nucleotides: Triacylglycerols: Phospholipids

The aim of this study was to investigate the effects of different levels of substitution of fish oil by vegetable oils rich in oleic, linoleic and linolenic acids on gilthead seabream plasma and leukocyte fatty acid compositions and prostaglandin (PG) and leptin production. Juvenile seabream of 24 g initial body mass were fed four iso-energetic and iso-proteic experimental diets for 281 days. Fatty acid composition of plasma lipids was markedly affected by the inclusion of vegetable oils (VO). ARA (arachidonate), EPA (eicosapentaenoate) and DHA (docosahexaenoate) were preferentially incorporated into polar lipids of plasma, and DHGLA (di-homogammalinoleate) accumulated with increased vegetable oil inclusion. Dietary treatments resulted in alterations of DHGLA/ARA ratios, but not ARA/EPA. ARA-derived PGE(2) production in plasma was not affected by vegetable oils, in agreement with similar eicosanoid precursor ratio (ARA/EPA) in leukocytes total lipids and plasma phospholipids among fish fed with the different dietary treatments. Feeding vegetable oils leads to a decrease in plasma EPA which in turn reduced plasma PGE(3) concentration. Moreover, PGE(3) was the major prostaglandin produced in plasma of fish fed fish oil based diet. Such findings point out the importance of EPA as a precursor of prostaglandins in marine fish, at least for the correct function of the blood cells, and correlates well with the predominant role of this fatty acid in immune regulation in this species. A negative correlation was found between plasma PGE(2) and leptin plasma concentration, suggesting that circulating levels of leptin may act as a metabolic signal modulating PGE(2) release. The present study has shown that increased inclusion of vegetable oils in diet for gilthead seabream may profoundly affect the fatty acid composition of plasma and leukocytes, specially HUFA (highly unsaturated fatty acids), and consequently the production of PGE(3), which can be a major PG in plasma. Alteration in the amount and type of PG produced can be at least partially responsible for the changes in the immune system and health parameters of fish fed diets with high inclusion of VO.

As in mammals, seahorse embryos develop internally but, unlike in mammals, this process occurs within a paternal structure (the brood pouch). Functionally, the brood pouch supports developing embryos through placenta-like interactions, but as egg quality is determined by the female's diet, the seahorse system offers opportunities to study the effect of the male's diet on embryo development while varying the female's diet independently. In this study, we investigated the hypothesis that development of seahorse embryos is affected differentially by independent manipulation of the male and female parental diets. Adult males and females were fed separately with either wild-caught crustaceans (Male-W or Female-W, respectively) or commercial aquarium diet (Male-C or Female-C, respectively) for 1 month before conception and during the subsequent pregnancy (approximately 15 days). Dietary unsaturated fatty acid content (18:3n-3) and (20:4n-6) in the W diet was approximately double that in the C diet. In total, 5231 first-brood offspring were obtained from 4 treatment groups formed from (1) Male-W × Female-W; (2) Male-C × Female-W; (3) Male-W × Female-C; and (4) Male-C × Female-C. Each treatment was replicated with 4 couples. Newborns (10 from each brood) were weighed and dimensions measured. Fifteen-day postnatal survival rates were determined from 40 offspring/couple (N at Day 0 = 160/treatment) and fatty acid profiles were evaluated. Data were analysed by nested analyses of covariance (ANCOVA); replicates were nested within treatments and individual offspring measurements were nested within replicates (male and female parental sizes were used as covariates). Offspring produced by the Male-C × Female-W were ~10% taller (both as standard length and tail length; P

Several enrichment conditions (type and concentration of oil in culture medium and time of enrichment) were tested in order to study their influence on rotifer (Brachionus plicatilis) lipid and fatty acid composition as well as on its population profile. Rotifers pre-fed on baker's yeast (Saccharomyces cerevisiae) were enriched with lipid emulsions containing triacylglycerols (TAG) or methyl ester (ME) mixtures at three different concentrations. Rotifers were sampled after 0 (yeast-fed rotifer), 3, 6, 12 and 24h enrichment. At every concentration and time assayed, the highest lipid levels were obtained when using TAG. In spite of the differences existing in saturated and monounsaturated fatty acid composition between the enrichment emulsions, rotifers tended to maintain the level of these fatty acids independently of the enrichment period and oil concentration. Increasing the enrichment period rather than the amount of oil present in the medium was found to be more efficient in increasing the n − 3 highly unsaturated fatty acids (n − 3 HUFA) level in rotifers. Rotifers reflected a better incorporation of eicosapentaenoic acid (EPA, 20:5n − 3) than of docosahexaenoic acid (DHA, 22:6n − 3), regardless of the ratio between both fatty acids in the emulsions. The use of ME as an enrichment source produced greater mortality in egg-carrying females than when using TAG., Sí

Two feeding trials were conducted to determine the minimum dietary protein level producing maximum growth, and the optimum protein to energy ratio in diets for red porgy (Pagrus pagrus) fingerlings, respectively. In the first trial, six isoenergetic diets were formulated with protein levels ranging from 400 to 650 g kg )1 in increments of 50 g kg )1 , and fed for 11 weeks to 2.8 g average initial weight fish. Weight gain, specific growth rate and feed efficiency were significantly higher with diets containing higher protein levels, when compared with dietary levels below 500 g kg )1 . The highest protein efficiency ratios were obtained in fish fed 500 g kg )1 dietary protein. The minimum dietary protein level producing maximum fish growth was found to be 500 g kg )1 .I n the second trial, 15 g average initial weight fish were fed for 12 weeks, six diets containing three different lipid levels (100, 150 and 200 g kg )1 ) combined with two protein levels (450 and 500 g kg )1 ). Weight gain values increased when dietary lipids increased from 100 to 150 g kg )1 , with a further decrease for 200 g kg )1 lipids in diets; the lowest fish growth being supported by 200 g kg )1 dietary lipids. Fish growth was significantly higher when dietary protein increased from 450 to 500 g kg )1 . There was no evidence of a protein-sparing effect of dietary lipids. Liver protein and lipid contents were low when compared with other fish species. All diet assayed produced high liver glycogen accumulation. The recommended protein and lipid levels in diets for red porgy fingerlings were 500 and 150 g kg )1 , respectively.

Linseed (LO) and soyabean (SO) oils were evaluated as fish-oil (FO) substitutes in the diets of marketable-sized gilthead sea bream (Sparus aurata). Practical diets were designed factorially with the lipid added as follows (%): FO 100, LO 60+FO 40, LO 80+FO 20, SO 60+FO 40, SO 80+FO 20. The effects of experimental diets on growth, fatty acids patterns in liver and muscle, flesh quality variables and activities of selected enzymes involved in lipid synthesis and catabolism were determined at the end of a 7-month trial. Fatty acid composition of liver and muscle generally reflected the fatty acid composition of the diets. Then−3 PUFA levels were significantly reduced by the inclusion of vegetable oils. This tendency was more pronounced for EPA than for docosahexaenoic acid. Then−3:n−6 fatty acid ratio reached the lowest values in fish fed the SO diets; this was associated with a higher liver lipid deposition. No differences were found in fillet texture and pH. However, under conditions of forced peroxidation, muscles from fish fed the SO diets had lower peroxidation levels. Vegetable oil substitution decreased lipogenesis in liver and this effect was greatest at the highest substitution level. In contrast, muscle β-oxidation enzymes had increased activities with vegetable oil substitution. Thus, the lower hepatic lipogenesis was correlated with an increased lipid utilisation in muscle. It is concluded that growth and lipid metabolism were affected by experimental diets.