Interaction between Dietary Vitamin D3 and Vitamin K3 in Gilthead Seabream Larvae (Sparus aurata) in Relation to Growth and Expression of Bone Development-Related Genes.

Vitamins D and K are essential fat-soluble nutrients that intervene in bone development processes among other biological functions. The present study is aimed at investigating the potential combined effect of dietary supplementation with vitamin D3 (cholecalciferol) and vitamin K3 (menadione) in gilthead seabream (Sparus aurata) larvae. For that purpose, seabream diets were supplemented with different combinations of vitamin D3/vitamin K3 (mg/kg diet) as follows: 0.00/0, 0.06/70, 0.06/170, 0.13/70, 0.13/170, 0.40/70, and 0.40/170. Feeding gilthead seabream larvae (22 days post hatch) for 21 days with the diets supplemented with 0.06-0.13 mg/kg vitamin D3 and 70 mg/kg vitamin K3 (diets 0.06/70 and 0.13/70) led to the highest larval growth and survival and the highest expression of important biomarkers of both bone development and health, such as bmp2, osx, and mgp, and calcium homeostasis, such as pthrp and casr. However, the increased supplementation with both vitamins at 0.40 mg/kg vitamin D3 and 170 mg/kg vitamin K3 (diet 0.40/170) reduced larval growth and survival, downregulated bmp2 and pthrp expressions, and upregulated osx and mgp, causing an unbalance in the relative expression of these genes. The results of the present study have shown the interaction between vitamin D3 supplementation and vitamin K3 supplementation in larval performance and gene expression related to bone development and calcium homeostasis, denoting the significance of a correct balance between both vitamins in larval diets.

High Levels of Vitamin A in Plant-Based Diets for Gilthead Seabream (Sparus aurata) Juveniles, Effects on Growth, Skeletal Anomalies, Bone Molecular Markers, and Histological Morphology.

Substitution of fish-based ingredients may alter the nutritional profile of the feeds, including the vitamin contents, ultimately leading to unbalanced vitamin supply. Vitamin A plays an essential role in epithelium preservation, cell differentiation, reproduction, and vision. It also intervenes in skeletogenesis through chondrocytes development. Therefore, low levels of vitamin A may cause poor growth and abnormal bone development among other symptoms. Besides, in gilthead seabream excess vitamin A altered bone structure and homeostasis, indicating that an upper level for vitamin A in feeds for this species must be defined. For this purpose, a practical plant-based diet (FM 10% and FO 6%) containing five increasing levels of vitamin A (24,000, 26,000, 27,000, 31,000, and 37,000 IU/kg) supplemented as retinyl acetate was formulated to identify the effects of high levels of vitamin A for gilthead seabream juveniles. The trial was conducted with 450 total fish distributed into 15 tanks, where each diet was tested in triplicates for 70 days. At the end of the trial, samples were taken for analyses of vitamin A-relevant markers. At the end of the trial the high levels of vitamin A supplementation did not cause a reduction in growth, whereas no significant effect was observed for the feed efficiency, specific growth rate, and feed convertion ratio. Although not significant, retinol content in liver showed a tendency to increase with the elevation of dietary vitamin A levels. Although minor, the highest level of vitamin A dietary content (37,000 IU/kg) caused a significant increase in caudal vertebrae partial fusion as well as caudal vertebrae malformations. Increasing dietary vitamin A was related to a reduction in the occurrence of microhemorrhages in the liver and a reduction in the presence of eosinophils associated to the pancreas. Overall, the results of the present study suggested that gilthead seabream juveniles fed a plant-based diet are able to tolerate very high levels of vitamin A supplementation when supplemented as retinyl acetate. Nevertheless, further supplementation should be avoided in order to reduce the prevalence of anomalies affecting the caudal vertebrae.

High-level biocidal products effectively eradicate pathogenic γ-proteobacteria biofilms from aquaculture facilities.

The use of effective biocides as disinfectants is essential in aquaculture facilities. However, while most biocides act effectively on free-living planktonic pathogens, they are seldom useful against biofilms. In this study, we evaluate the biocidal efficacy and antimicrobial specific contact time of three disinfectants, Virkon™Aquatic (VirA), peracetic acid (PerA) and hydrogen peroxide (HydP), on Vibrio anguillarum, V. harveyi, V. alginolyticus, and Photobacterium damselae subspecies piscicida against their both life phases. By using the minimum inhibitory, bactericidal, and eradication concentrations of disinfectants acting on the free-living planktonic state (MIC; MBC) and biofilms (MBIC; MBEC), we determined the in vitro susceptibility of each bacterial strain against three different individual concentrations of VirA, PerA, and HydP added at 1, 5, and 10 min intervals. PerA and VirA had the highest bactericidal efficacies against the free-living planktonic state and biofilm of all bacteria. Kinetically, PerA gave a positive result more quickly in both cases regardless of the strain in question, while the weakest HydP required longer than 10 min to act effectively. Moreover, we conducted a short in vivo safety trial by pouring the suggested MIC of each disinfectant into tanks containing juvenile Gilthead seabream (Sparus aurata). A significant mortality after 24 h was observed pointing to the potential risk a mishap of these chemicals might cause to fish. Nevertheless, collectively, our results support the inclusion of biocides within biosecurity protocols in aquaculture facilities and highlight PerA as the most effective disinfectant for fighting against biofilms produced by V. anguillarum, V. harveyi, V. alginolyticus or P. damselae subsp. piscicida.

Parental LC-PUFA biosynthesis capacity and nutritional intervention with alpha-linolenic acid affect performance of Sparus aurata progeny.

Environmental factors such as nutritional interventions during early developmental stages affect and establish long-term metabolic changes in all animals. Diet during the spawning period has a nutritional programming effect in offspring of gilthead seabream and affects long-term metabolism. Studies showed modulation of genes such as fads2, which is considered to be a rate-limiting step in the synthesis of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). However, it is still unknown whether this adaptation is related to the presence of precursors or to limitations in the pre-formed products, n-3 LC-PUFA, contained in the diets used during nutritional programming. This study investigated the combined effects of nutritional programming on Sparusaurata through broodstock diets during the spawning period and in broodfish showing higher or lower fads2 expression levels in the blood after 1 month of feeding with a diet containing high levels of plant protein sources and vegetable oils (VM/VO). Broodfish showing high fads2 expression had a noticeable improvement in spawning quality parameters as well as in the growth of 6 month old offspring when challenged with a high VM/VO diet. Further, nutritional conditioning with 18:3n-3-rich diets had an adverse effect in comparison to progeny obtained from fish fed high fish meal and fish oil (FM/FO) diets, with a reduction in growth of juveniles. Improved growth of progeny from the high fads2 broodstock combined with similar muscle fatty acid profiles is also an excellent option for tailoring and increasing the flesh n-3 LC-PUFA levels to meet the recommended dietary allowances for human consumption.

Time-critical influences of gestational diet in a seahorse model of male pregnancy.

Sex role reversal is not uncommon in the animal kingdom but is taken to the extreme by the Syngnathidae, in which male pregnancy is one of the most astonishing idiosyncrasies. However, critical and time-dependent environmental effects on developing embryos, such as those extensively studied in mammalian pregnancy, have not been investigated in the male pregnancy context. Here, we tested the hypothesis that seahorse pregnancy is subject to 'critical windows' of environmental sensitivity by feeding male long-snouted seahorses (Hippocampus reidi) a diet deficient in polyunsaturated fatty acids during specific periods before and during pregnancy. Despite embryos being nourished principally by maternally supplied yolk, we found that offspring morphology, fatty acid composition and gene expression profiles were influenced by paternal diet in a manner that depended critically on the timing of manipulation. Specifically, reception of a diet deficient in polyunsaturated fatty acids in the days preceding pregnancy resulted in smaller newborn offspring, while the same diet administered towards the end of pregnancy resulted in substantial alterations to newborn gene expression and elongation of the snout at 10 days old. Although paternal diet did not affect 10 day survival, the observed morphological alterations in some cases could have important fitness consequences in the face of natural selective pressures such as predation and food availability. Our results demonstrate that, under male pregnancy, fine-scale temporal variation in parental diet quality and subsequent critical window effects should not be overlooked as determinants of developing offspring fitness.

Long-chain PUFA profiles in parental diets induce long-term effects on growth, fatty acid profiles, expression of fatty acid desaturase 2 and selected immune system-related genes in the offspring of gilthead seabream.

The present study investigated the effects of nutritional programming through parental feeding on offspring performance and expression of selected genes related to stress resistance in a marine teleost. Gilthead seabream broodstock were fed diets containing various fish oil (FO)/vegetable oil ratios to determine their effects on offspring performance along embryogenesis, larval development and juvenile on-growing periods. Increased substitution of dietary FO by linseed oil (LO) up to 80 % LO significantly reduced the total number of eggs produced by kg per female per spawn. Moreover, at 30 d after hatching, parental feeding with increasing LO up to 80 % led to up-regulation of the fatty acyl desaturase 2 gene (fads2) that was correlated with the increase in conversion rates of related PUFA. Besides, cyclo-oxygenase 2 (cox2) and TNF-α (tnf-α) gene expression was also up-regulated by the increase in LO in broodstock diets up to 60 or 80 %, respectively. When 4-month-old offspring were challenged with diets having different levels of FO, the lowest growth was found in juveniles from broodstock fed 100 % FO. An increase in LO levels in the broodstock diet up to 60LO raised LC-PUFA levels in the juveniles, regardless of the juvenile's diet. The results showed that it is possible to nutritionally programme gilthead seabream offspring through the modification of the fatty acid profiles of parental diets to improve the growth performance of juveniles fed low FO diets, inducing long-term changes in PUFA metabolism with up-regulation of fads2 expression. The present study provided the first pieces of evidence of the up-regulation of immune system-related genes in the offspring of parents fed increased FO replacement by LO.

Increased parasite resistance of greater amberjack (Seriola dumerili Risso 1810) juveniles fed a cMOS supplemented diet is associated with upregulation of a discrete set of immune genes in mucosal tissues.

The main objective of this study was to determine the effect of two forms of mannan oligosaccharides (MOS: Bio-Mos® and cMOS: Actigen®, Alltech Inc, USA) and their combination on greater amberjack (Seriola dumerili) growth performance and feed efficiency, immune parameters and resistance against ectoparasite (Neobenedenia girellae) infection. Fish were fed for 90 days with 5 g kg-1 MOS, 2 g kg-1 cMOS or a combination of both prebiotics, in a Seriola commercial base diet (Skretting, Norway). At the end of the feeding period, no differences were found in growth performance or feed efficiency. Inclusion of MOS also had no effect on lysozyme activity in skin mucus and serum, but the supplementation of diets with cMOS induced a significant increase of serum bactericidal activity. Dietary cMOS also reduced significantly greater amberjack skin parasite levels, parasite total length and the number of parasites detected per unit of fish surface following a cohabitation challenge with N. girellae, whereas no effect of MOS was detected on these parameters. Of 17 immune genes studied cMOS dietary inclusion up-regulated hepcidin, defensin, Mx protein, interferon-γ (IFNγ), mucin-2 (MUC-2), interleukin-1ß (IL-1B), IL-10 and immunoglobulin-T (IgT) gene expression in gills and/or skin. MOS supplementation had a larger impact on spleen and head kidney gene expression, where piscidin, defensin, iNOS, Mx protein, interferons, IL-1ß, IL-10, IL-17 and IL-22 were all upregulated. In posterior gut dietary MOS and cMOS both induced IL-10, IgM and IgT, but with MOS also increasing piscidin, MUC-2, and IL-1ß whilst cMOS induced hepcidin, defensin and IFNγ. In general, the combination of MOS and cMOS resulted in fewer or lower increases in all tissues, possibly due to an overstimulation effect. The utilization of cMOS at the dose used here has clear benefits on parasite resistance in greater amberjack, linked to upregulation of a discrete set of immune genes in mucosal tissues.

Effects of Dietary Lipid Composition and Fatty Acid Desaturase 2 Expression in Broodstock Gilthead Sea Bream on Lipid Metabolism-Related Genes and Methylation of the fads2 Gene Promoter in Their Offspring.

Polyunsaturated fatty acids (PUFA) in parental diets play a key role in regulating n-3 LC-PUFA metabolism of the offspring. However, it is not clear whether this metabolic regulation is driven by the precursors presented in the diet or by the parental ability to synthesize them. To elucidate this, broodstocks of gilthead sea bream with different blood expression levels of fads2, which encodes for the rate-limiting enzyme in the n-3 LC-PUFA synthesis pathway, were fed either a diet supplemented with alpha-linolenic acid (ALA, 18:3n-3) or a control diet. The progenies obtained from these four experimental groups were then challenged with a low LC-PUFA diet at the juvenile stage. Results showed that the offspring from parents with high fads2 expression presented higher growth and improved utilization of low n-3 LC-PUFA diets compared to the offspring from parents with low fads2 expression. Besides, an ALA-rich diet during the gametogenesis caused negative effects on the growth of the offspring. The epigenetic analysis demonstrated that methylation in the promoter of fads2 of the offspring was correlated with the parental fads2 expression levels and type of the broodstock diet.

Lipid digestion capacity in gilthead seabream (Sparus aurata) from first feeding to commercial size.

To characterise the progression of lipid digestion capacity in gilthead seabream across life cycle, the activities of bile salt-activated lipase (BAL) and phospholipase A2 (PLA2) were determined in the digestive tracts of cultured gilthead seabream from first feeding to marketable size (49 µg to 300 g). Four trials were undertaken with gilthead seabream of different ages, fed on diets with fishmeal and fish oil as the main dietary protein and lipid sources and 21-25% lipid contents. Larvae of 4 days after hatching (dah) to 9 dah were fed rotifers with different fatty acid profiles: control (2.8% eicosapentaenoic acid, EPA; 1.6% docosahexaenoic acid, DHA; 5.4% n-3 long-chain polyunsaturated fatty acids, n-3 LC-PUFAs; and 0.2% arachidonic acid, ARA), low EPA (1.38% EPA, 1.6% DHA, 3.9% n-3 LC-PUFA and 0.4% ARA) or low LC-PUFA (0.7% EPA, 1.0% DHA, 1.8% n-3 LC-PUFA and 0.0% ARA) (% dry weight). Larvae fed the low-LC-PUFA diet showed a significantly lower growth at 10 dah. BAL activities were significantly higher in larvae fed the control diet than in those fed low-EPA and low-LC-PUFA diets at 9 dah. BAL activity increased with age across life cycle (49 µg to 300 g). PLA2 activity could not be detected in larvae but increased with age in juvenile and adult gilthead seabream (86 g to 295 g), similar to BAL. Results suggested a correspondence between the stimulation of lipid digestion capacity and growth performance in gilthead seabream by dietary essential fatty acids, particularly by EPA when DHA requirements are met in the diet especially in the very early stages of life cycle, when the progression of BAL and PLA2 activities could be used as indicators of the nutritional status of cultured gilthead seabream larvae. Finally, regarded that PLA2 activity was not detected in 4-dah to 44-dah gilthead seabream larvae, future works are suggested to assess the dietary effect on PLA2 activity and the PLA2 activity pattern along the larval stage of this species using a more sensitive detection method.

The importance of phospholipids combined with long-chain PUFA in formulated diets for pikeperch (Sander lucioperca) larvae.

Dietary phosphoglycerides and n-3 long-chain PUFA (LC-PUFA) play important functions in the development of pikeperch (Sander lucioperca) larvae. This study aimed to determine optimal dietary levels of soyabean lecithin (SBL)-derived phospholipids (PL) in starter feeds for pikeperch larvae 10-30 d post-hatch (DPH) and examine performance and ontogeny by additional supplementation of n-3 LC-PUFA in the form of Algatrium DHA 70 (glyceride product; 660-700 mg/g DHA; EPA 60-75 mg/g). In total, six isoproteic and isoenergetic extruded diets were formulated with increasing levels of PL (3·7, 8·3 or 14·5 % wet weight (w.w.), respectively); however, three of the diets were supplemented with three levels of Algatrium DHA 70 (0·6, 2·0 or 3·4 %, respectively). Liver proteomic analyses of larvae at 30 DPH were included for effects of PL and primarily DHA on performance, physiological expression and interactions in larval proteins. In addition, bone anomalies, digestive enzymatic activity, candidate gene expression and skeleton morphogenesis were examined. Results confirmed the importance of dietary PL levels of at least 8·2 % w.w., and an additional beneficiary effect of supplementation with DHA plus EPA. Thus, combined supplementation of SBL (up to 14·51 % w.w. PL) and n-3 LC-PUFA (1·004 % DM DHA and 0·169 % DM EPA) in the form of TAG resulted in highest growth and lowest incidence of anomalies, improved digestive enzyme activity and had differential effect on liver proteomics. The results denote that essential fatty acids can be supplemented as TAG to have beneficial effects in pikeperch larvae development.

Fatty Acid Composition and Fatty Acid Associated Gene-Expression in Gilthead Sea Bream (Sparus aurata) are Affected by Low-Fish Oil Diets, Dietary Resveratrol, and Holding Temperature.

To sustainably produce marine fish with a high lipid quality rich in omega-3 fatty acids, alternative sources of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are being identified. Moreover, the use of bioactive compounds that would stimulate the in vivo fatty acid synthesis, such as resveratrol (RV), would reduce the dependence on fish oil in aquafeeds. Gilthead sea bream (Sparus aurata) were fed four experimental diets combining two fish oil levels (6% dry matter (DM); 2% DM) with or without 0.15% DM resveratrol supplementation (F6, F2, F6 + RV, F2 + RV) for two months. Additionally, the fish were challenged either at 19 °C or 23 °C. A higher water temperature promoted their feed intake and growth, resulting in an increased crude lipid content irrespective of dietary treatment. The fatty acid composition of different tissues was significantly affected by the holding temperature and dietary fish oil level. The dietary RV significantly affected the hepatic EPA and DHA content of fish held at 19 °C. The observed effect of RV may be partly explained by alterations of the mRNA steady-state levels of ∆6-desaturase and ß-oxidation-related genes. Besides the relevant results concerning RV-mediated regulation of fatty acid synthesis in marine fish, further studies need to be conducted to clarify the potential value of RV to enhance fillet lipid quality.

Nano-delivery of trace minerals for marine fish larvae: influence on skeletal ossification, and the expression of genes involved in intestinal transport of minerals, osteoblast differentiation, and oxidative stress response.

Currently, the larviculture of many marine fish species with small-sized larvae depends for a short time after hatching, on the supply of high-quality live zooplankton to ensure high survival and growth rates. During the last few decades, the research community has made great efforts to develop artificial diets, which can completely substitute live prey. However, studies aimed at determining optimal levels of minerals in marine larvae compound feeds and the potential of novel delivery vectors for mineral acquisition has only very recently begun. Recently, the agro-food industry has developed several nano-delivery systems, which could be used for animal feed, too. Delivery through nano-encapsulation of minerals and feed additives would protect the bioactive molecules during feed manufacturing and fish feeding and allow an efficient acquisition of active substances into biological system. The idea is that dietary minerals in the form of nanoparticles may enter cells more easily than their larger counterparts enter and thus speed up their assimilation in fish. Accordingly, we evaluated the efficacy of early weaning diets fortified with organic, inorganic, or nanoparticle forms of trace minerals (Se, Zn, and Mn) in gilthead seabream (Sparus aurata) larvae. We tested four experimental diets: a trace mineral-deficient control diet, and three diets supplemented with different forms of trace minerals. At the end of the feeding trial, larvae growth performance and ossification, and the level of expression of six target genes (SLC11A2ß, dmt1, BMP2, OC, SOD, GPX), were evaluated. Our data demonstrated that weaning diets supplemented with Mn, Se, and Zn in amino acid-chelated (organic) or nanoparticle form were more effective than diets supplemented with inorganic form of minerals to promote bone mineralization, and prevent skeletal anomalies in seabream larvae. Furthermore, nanometals markedly improved larval stress resistance in comparison to inorganic minerals and upregulated mRNA copy number of OC gene. The expression of this gene was strongly correlated with mineralization degree, thus confirming its potency as a good marker of bone mineralization in gilthead seabream larvae.

Histochemical localization of zinc in the retina cells of gilthead sea bream (sparus aurata) fed different presentations of zinc.

To describe the distribution of zinc in the retina of a representative marine fish species and to determine whether the intracellular deposition amount correlates with the presentation of the zinc included in the practical diets (organic, inorganic, encapsulated, fish meal, and Control diet), we examined the precise localization of endogenous zinc in the gilthead sea bream (Sparus aurata) retina by autometallography. As observed by light microscopy, reaction products were widely distributed throughout the retina, including the outer segments of photoreceptors, except in the nuclear layers. Differing from other species previously studied, zinc depositions were not different between the outer and inner retina, and the retinal ganglion cell layer showed reaction products with a characteristic disposition surrounding the neuronal soma. An additional finding in this species was the rich disposition around photoreceptors, so abundant that it outlines the shape of the rods and cones. With regards to the diet, the zinc organic formulation was able to produce a higher amount of precipitates, followed by fish meal and encapsulated zinc diets. The inorganic and the Control diet produced a basal zinc deposition in the same layers above mentioned although less evident and similar to that determined in other species fed with non-supplemented diets.

Parental nutritional programming and a reminder during juvenile stage affect growth, lipid metabolism and utilisation in later developmental stages of a marine teleost, the gilthead sea bream (Sparus aurata).

Nutrition during periconception and early development can modulate metabolic routes to prepare the offspring for adverse conditions through a process known as nutritional programming. In gilthead sea bream, replacement of fish oil (FO) with linseed oil (LO) in broodstock diets improves growth in the 4-month-old offspring challenged with low-FO and low-fishmeal (FM) diets for 1 month. The present study further investigated the effects of broodstock feeding on the same offspring when they were 16 months old and were challenged for a second time with the low-FM and low-FO diet for 2 months. The results showed that replacement of parental moderate-FO feeding with LO, combined with juvenile feeding at 4 months old with low-FM and low-FO diets, significantly (P<0·05) improved offspring growth and feed utilisation of low-FM/FO diets even when they were 16 months old: that is, when they were on the verge of their first reproductive season. Liver fatty acid composition was significantly affected by broodstock or reminder diets as well as by their interaction. Moreover, the reduction of long-chain PUFA and increase in α-linolenic acid and linoleic acid in broodstock diets lead to a significant down-regulation of hepatic lipoprotein lipase (P<0·001) and elongation of very long-chain fatty acids protein 6 (P<0·01). Besides, fatty acid desaturase 2 values were positively correlated to hepatic levels of 18 : 4n-3, 18 : 3n-6, 20 : 5n-3, 22 : 6n-3 and 22 : 5n-6. Thus, this study demonstrated the long-term nutritional programming of gilthead sea bream through broodstock feeding, the effect of feeding a 'reminder' diet during juvenile stages to improve utilisation of low-FM/FO diets and fish growth as well as the regulation of gene expression along the fish's life-cycle.

Effects of thermal stress on the expression of glucocorticoid receptor complex linked genes in Senegalese sole (Solea senegalensis): Acute and adaptive stress responses.

The present study examined the short and mid-term effects of a rise in temperature from 18°C to 24°C on the expression of genes related to the stress response regulation in juveniles of Senegalese sole, Solea senegalensis. The animals were exposed to a temperature increase of 6°C, after 1month of acclimation at 18°C. After this process, samples of different tissues were collected from a total of 96 fish at four sampling points: 1h, 24h, 3days and 1week. The transcript levels of a set of genes involved in the stress response such as glucocorticoid receptors 1 and 2, corticotrophin-releasing factor, corticotrophin-releasing factor binding proteins, proopiomelanocortin A and B, and cellular stress defense (heat shock protein 70, 90AA and 90AB) were quantified at these sampling points. Additionally, blood samples were also taken to measure the circulating plasma cortisol concentration. Thermal stress induced by increasing temperature prompted an elevation of plasma cortisol levels in juvenile Senegalese sole after 1h as a short-term response, and a consecutive increase after one week, as a mid-term response. Senegalese sole seemed to respond positively in terms of adaptive mechanisms, with a rapid over-expression of grs and hsps in liver and brain, significantly higher after one hour post stress, denoting the fast and acute response of those tissues to a rapid change on temperature. The ratio hsp90/gr also increased 24h after thermal shock, ratio proposed to be an adaptive mechanism to prevent proteosomal degradation of GR. As a mid-term response, the elevation of brain crfbp gene expression one week after thermal shock could be an adaptive mechanism of negative feedback on HPI axis. Taken together, these data suggested an initial up-regulation of the glucocorticoid receptor complex linked genes in response to a temperature increase in Senegalese sole, with heat shock protein 90 potentially being a regulatory factor for the glucocorticoid receptor in the presence of cortisol.

Embryonic developmental plasticity in the long-snouted seahorse (Hippocampus reidi, Ginsburg 1933) in relation to parental preconception diet.

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 10000 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<0.05) than those produced by wild-fed males. When both males and females were fed with commercial diet, their offspring were significantly smaller than those from the other treatment groups. When commercial diet-fed females were mated with wild-fed males, the offspring showed distortion of the snout:head length ratio. These results support the view that the preconception diet received by males and females differentially affects embryonic development.

Effects of dietary concentrated mannan oligosaccharides supplementation on growth, gut mucosal immune system and liver lipid metabolism of European sea bass (Dicentrarchus labrax) juveniles.

The study assesses the effects of dietary concentrated mannan oligosaccharides (cMOS) on fish performance, biochemical composition, tissue fatty acid profiles, liver and posterior gut morphology and gen expression of selected parameters involved on the intestinal immune response and liver lipid metabolism of European sea bass (Dicentrarchus labrax). For that purpose, specimens of 20 g were fed during 8 weeks at 0 and 1.6 g kg(-1) dietary cMOS of inclusion in a commercial sea bass diet. Dietary cMOS enhanced fish length, specific and relative growth without affecting tissue proximate composition. However, cMOS supplementation altered especially liver and muscle fatty acid profiles by reducing levels of those fatty acids that are preferential substrates for ß-oxidation in spite of a preferential retention of long chain polyunsaturated fatty acids (LC-PUFA), such as 20:4n-6 or 22:5n-6, in relation to the down-regulation of delta 6/5 desaturase gene expression found in liver. Besides, dietary cMOS supplementation reduced posterior gut intestinal folds width and induced changes on the gene expression level of certain immune-related genes mainly by down regulating transforming growth factor ß (TGFß) and up-regulating immunoglobulin (Ig), major histocompatibility complex class II (MHCII), T cell receptor ß (TCRß) and Caspase 3 (Casp-3). Thus, dietary cMOS inclusion at 0.16% promoted European sea bass specific growth rate and length, stimulated selected cellular GALT-associated parameters and affected lipid metabolism in muscle and liver pointing to a higher LC-PUFA accumulation and promoted ß-oxidation.

Dietary vegetable oils: effects on the expression of immune-related genes in Senegalese sole (Solea senegalensis) intestine.

The decreased availability of fish oil, traditionally used as oil source in marine aquafeeds, has lead to the search for alternatives oils. Vegetable oils (VO) are being extensively used as lipid sources in marine fish diets, inducing an imbalance on certain dietary fatty acids. Alteration on the dietary ratio of w-6/w-3 has been described to have detrimental effects on fish immunity. Senegalese sole has high susceptibility to stress and diseases, and little is known on the effects of dietary VO on its immunity. In this study, Senegalese sole juveniles were fed diets (56% crude protein, 12% crude lipid) containing linseed (100LO), soybean (100SO) or fish (100FO) oils as unique oil source. Growth, cortisol and intestinal fatty acid composition were determined after 90 days. Moreover, at the final of the experiment a stress test (5 min of net chasing) was carried out. To evaluate the effect of diets and stress on intestine immunology, expression profiles of a set of 53 immune-related genes using RT-qPCR was also performed. The use of VO did not induced changes in fish growth, but affected fatty acid profile of intestine and expression of immune-related genes. The use of SO (rich in n-6 fatty acids) induced an over-expression of those genes related to complement pathway, recognizing pathogen associated to molecular patterns, defensive response against bacteria, defensive response against viruses, antigen differentiation, cytokines and their receptors. This general over-expression could indicate an activation of inflammatory processes in fish gut. When a stress was applied, a decrease of mRNA levels of different immune-related genes with respect to the unstressed control could be observed in fish fed 100FO. However, fish fed 100LO, with a higher ALA/LA ratio, seemed to ameliorate the effects of combined effects of FO substitution plus stressful situation whereas fish fed 100SO did not show this type of response.

Improved health and growth of fish fed mannan oligosaccharides: potential mode of action.

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.

Enhanced intestinal epithelial barrier health status on European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides.

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.