Search

The interaction between vitamin D and vitamin K is crucial for regulating bone metabolism and maintaining calcium homeostasis across diverse animal species due to their complementary roles in calcium metabolism and bone health. However, research on this interaction of vitamin D and K in fish, particularly Mediterranean species like gilthead seabream, is limited or not studied. This study aimed to understand the effects of different dietary combinations of vitamin D3 and K3 on juvenile gilthead seabream. Accordingly, seabream juveniles were fed with varying combinations of vitamin D3/vitamin K3 (mg/kg diet) for 3 months: (0.07/0.01), (0.20/0.58), (0.19/1.65), (0.51/0.74), (0.56/1.00). At the end of the trial, survival, growth, body morphology, serum calcitriol, and vertebral mineral composition remained unaffected by varying vitamin levels, while gene expression patterns related to bone formation, resorption, and calcium regulation in various tissues were significantly influenced by both vitamins and their interaction. Gilthead seabream juveniles fed the 0.07/0.01 mg/kg diet upregulated calcium-regulating genes in the gills, indicating an effort to enhance calcium absorption to compensate for dietary deficiencies. Conversely, an increase in vitamin D3 and K3 up to 0.19 and 1.65 mg/kg, respectively, upregulated bone formation, bone remodeling, and calcium homeostasis-related gene expression in vertebra and other tissues. On the contrary, a dietary increase in these vitamins up to 0.56 mg/kg vitamin D3 and 1.00 mg/kg vitamin K3 downregulated calcium metabolism-related genes in tissues, suggesting an adverse interaction resulting from elevated levels of these vitamins in the diet. Hence, sustaining an equilibrium in the dietary intake of vitamin D3 and vitamin K3, in an appropriately combined form, may potentially induce interactions between the vitamins, contributing to favorable effects on bone development and calcium regulation in gilthead seabream juveniles.

Abstract This study describes the effects on the skeletal phenotype of two pivotal factors, density and water volume, don the hatchery (larval) phase of gilthead seabream (Sparus aurata) larvae previoulsy described for two model species (Danio rerio and Oryzias latipes) and sub‐adult (pre‐ongrowing) gilthead seabream (Sparus aurata). The experimental trial was conducted using single conditions in a pilot study, starting with a total of 615,385 eggs from the same batch. Three densities (LD low density: 25 larvae/L; MD medium density: 125 larvae/L; and HD high density: 250 larvae/L) and two water volumes (500 and 1000 L) were tested from spawning up to 60 days post‐hatching (dph) (average standard length: 1.79 cm; average dry weight: 27.11 mg). On the final samples, morphometric, anatomical, and histological data were collected for data pertinent to meristic counts and skeletal anomalies. The results (analyzed by means of multivariate analyses) indicated that the LD‐reared larvae were significantly longer and heavier than HD‐reared fish. Furthermore, LD‐reared gilthead seabream exhibited a significant reduction in the frequency of individuals with anomalies of jaws, vertebral body shape, and vertebral arches than the MD and HD conditions, which is in agreement with previous experiments carried out on model species and gilthead seabream sub‐adults.

Abstract Over the past 20 years, substantial progress has been made in improving feeds and feeding technologies for most aquaculture species. Notable improvements in feed conversion efficiency (through a better understanding of requirements and improved feed management) and ingredient sustainability (through increased capability to use a wider range of ingredients) have been achieved. While advances have been made in understanding the requirements of many of the main aquaculture species, there is still much to be done in defining requirements, especially for many of the species being farmed in the developing world. Gains in the efficiency of feeds are slowing for developed species, but potential gains are still appreciable for less developed species. There is a growing need to more precisely prescribe the required levels of essential nutrients and various additives in the diet based on age, genotype, environment, and immune status to deliver a “precision nutrition” approach to farming aquaculture species. There is still further need to diversify our ingredient options to provide greater resilience, as the sustainability of different feed ingredient sources, including possible climate change impacts, is becoming a growing issue. There is a growing demand for biocircularity in our feed ingredient supply chains. Ultimately, what is needed to sustain future feed ingredient needs are sustainable sources of cost‐effective protein, some essential amino acid additives, some omega‐3 fatty acid resources, and various minerals and vitamin additives. The increasing use of new and varied resources will ensure that food safety remains an important issue throughout the world. Feed manufacturing has evolved from a simplistic exercise to a highly complex science with state‐of‐the‐art engineering, but its application is not consistent across all sectors, as there is still widespread use of pelleting, mash, and trash fish feeding in the developing world. Similarly, feed management has also dichotomized between the developed and developing world, with a high reliance on manual skilled labor in the developing world, whereas more advanced aquaculture systems are becoming increasingly reliant on automated computer‐controlled feeding systems.

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.

Most commercial products available for Artemia sp. enrichment include high levels of docosahexaenoic acid (DHA) to boost its nutritional value, though with limited success. In this regard, the present study evaluated the alternative utilization of eicosapentaenoic acid (20 : 5n-3; EPA) oils to improve the n-3 long-chain highly unsaturated fatty acid content (n-3 LC-HUFA) in enriched Artemia sp. to feed greater amberjack (Seriola dumerili) larvae. Five experimental emulsions containing increasing levels of EPA from 0.8% to 60% of total fatty acids (TFA) and n-3 LC-HUFA (1.3%–70.6% TFA) were formulated. Each diet was fed to greater amberjack larvae (17–35 days posthatch (dph)) in three replicate 200-L tanks. The dietary EPA supplementation significantly improved larval growth during the feeding trial and survival at 35 dph (P

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.

Genetic improvement of feed efficiency is key to improve the economic and environmental sustainability of fish farming. However, it requires individual phenotypes of feed efficiency, which are difficult if not impossible to obtain when fish are reared in tanks or cages. Here, we applied and validated on gilthead sea bream a method to evaluate individual feed efficiency based on individual rearing of fish in aquariums under restricted feeding. We collected individual phenotypes of feed efficiency in aquariums on 538 sea bream (average weight = 54.50 g). Based on these individual phenotypes, fish (average weight = 174.6 g) were reared in groups of divergent phenotypes (high or low feed efficiency), validating that individual feed efficiency had an impact on group feed efficiency at a later stage. All 538 fish, their parents as well as 794 sibs reared in cages in a production environment, were genotyped on a 57k SNP array to estimate genomic heritability and correlations between traits. We showed that feed efficiency was heritable but did not find significant associated QTLs. We also showed that feed efficiency was negatively genetically correlated to viscera yield, indicating that the most efficient fish had less viscera than the least efficient ones. Altogether, these results support that measuring individual feed efficiency in aquariums under restricted feeding may be used as a reliable phenotyping method to genetically improve feed efficiency, despite the bias intrinsically linked to individual rearing.

Gilthead seabream (Sparus aurata) production is a highly valued aquaculture industry in Europe. The presence of skeletal deformities in farmed gilthead seabream represents a major bottleneck for the industry leading to economic losses, negative impacts on the consumers’ perception of aquaculture, and animal welfare issues for the fish. Although past work has primarily focused on the hatchery phase to reduce the incidence of skeletal anomalies, this work targets the successive pre-ongrowing phase in which more severe anomalies affecting the external shape often arise. This work aimed to test the effects of: (i) larger and smaller tank volumes, stocked at the same density; and (ii) higher and lower stocking densities maintained in the same water volume, on the skeleton of gilthead seabream fingerlings reared for ~63 days at a pilot scale. Experimental rearing was conducted with gilthead seabream juveniles (~6.7 ± 2.5 g), which were selected as ‘non-deformed’ based on external inspection, stocked at three different densities (Low Density (LD): 5 kg/m3; Medium Density (MD): 10 kg/m3; High Density (HD): 20 kg/m3) in both 500 L and 1000 L tanks. Gilthead seabream were sampled for growth performance and radiographed to assess the skeletal elements at the beginning and end of the experimental trial. Results revealed that (i) LD fish were significantly longer than HD fish, although there were no differences in final weights, regardless of the water volume; (ii) an increase in the prevalence of seabream exhibiting cranial and vertebral axis anomalies was found to be associated with increased density. These results suggest that farmers can significantly reduce the presence of some cranial and axis anomalies affecting pre-ongrown gilthead seabream by reducing the stocking density.

The aim of this study was to generate new knowledge on fish epigenetics, assessing the effects of linolenic acid (ALA) conditioning of broodstock in the offspring of the marine fish Sparus aurata. Attention was focused on gene organization, methylation signatures and gene expression patterns of fatty acid desaturase 2 (fads2) and stearoyl-CoA desaturase 1a (scd1a). Blat searches in the genomic IATS-CSIC database (www.nutrigroup-iats.org/seabreamdb) highlighted a conserved exon-intron organization, a conserved PUFA response region, and CG islands at the promoter regions of each gene. The analysed CpG positions in the fads2 promoter were mostly hypomethylated and refractory to broodstock nutrition. The same response was achieved after conditioning of juvenile fish to low water oxygen concentrations, thus methylation susceptibility at individual CpG sites seems to be stringently regulated in fish of different origin and growth trajectories. Conversely, the scd1a promoter was responsive to broodstock nutrition and the offspring of parents fed the ALA-rich diet shared an increased DNA-methylation, mainly in CpG sites neighbouring SP1 and HNF4α binding sites. Cytosine methylation at these sites correlated inversely with the hepatic scd1a expression of the offspring. Co-expression analyses supported that the HNF4α-dependent regulation of scd1a is affected by DNA methylation. The phenotypic output is a regulated liver fat deposition through changes in scd1 expression, which would also allow the preservation of fatty acid unsaturation levels in fish fed reduced levels of n-3 LC-PUFA. Collectively, these findings reveal a reliable mechanism by which parent’s nutrition can shape scd1a gene expression in the fish offspring.

This 10-week feeding experiment examined the effects of supplementing vitamin C (VC) and E (VE) in oxidized oil diets on growth, fatty acid composition, blood physiological indicators, innate immunity, antioxidant capacity, and liver and intestine histology of rainbow trout (Oncorhynchus mykiss). Four diets were produced including a fresh fish oil containing diet (FFO diet), an oxidized fish oil containing diet (OFO diet), and OFO diet supplemented with 500 mg kg–1 VC and 400 mg kg–1 VE (OFO+C500+E400 diet) or 1,000 mg kg–1 VC and 800 mg kg–1 VE (OFO+C1000+E800 diet). Four hundred and twenty fish averaging 46.9 ± 0.32 g were stocked into 12 tanks and fed the diets twice a day to visual satiety. The results showed no significant effect of OFO or vitamins supplementation on growth, feed intake and feed utilization (P > 0.05). The groups that received OFO and OFO+C1000+E800 diets had significantly (P < 0.05) lower hepatosomatic indices than the other groups. Muscle fatty acid composition showed decreased proportion of 15:0, 16:0, 16:1n-7, 16:2n-4, 18:1n-5, 18:2n-6 (linoleic acid) and total saturated fatty acids in OFO group. Whereas higher percentages of 20:3n-6 and 20:3n-3 were found in OFO group. In addition, the highest ratio of docosahexaenoic acid (DHA, 22:6n-3) to eicosapentaenoic acid (EPA, 20:5n-3) was detected in OFO+C500+E400 group. The highest serum triglyceride concentration was recorded in the OFO group. Serum aspartate aminotransferase activity increased in OFO and OFO+C1000+E800 groups compared to OFO+C500+E400 group. Furthermore, significantly higher alkaline phosphatase activity in blood was found in OFO and OFO+C500+E400 groups. Significantly lower serum lysozyme, antiprotease, superoxide dismutase, and catalase activities were recorded in OFO group compared to FFO and OFO+C500+E400 groups, and an opposite trend was observed for malondialdehyde concentration. Muscle VC and VE concentrations, and liver and intestine histology remained unaffected. To conclude, feeding diet containing oxidized oil with peroxide value of 182 meq kg–1 with/without VC and VE supplementation did not influence growth and tissue VC and VE concentrations of rainbow trout. However, supplementing 500 mg kg–1 VC and 400 mg kg–1 VE reversed the adverse effects of OFO on non-specific immune response and antioxidant capacity.

This study evaluated growth, body composition, antioxidant capacity, innate immunity and ammonia excretion of European grayling (Thymallus thymallus) fed diets containing different protein and lipid contents. Six diets were produced to contain 30, 40, or 50% protein and 10 or 20% lipid. Juvenile fish averaging 25.2 ± 0.28 g were stocked into eighteen 450-L circular tanks in a recirculating aquaculture system (RAS) and fed the test diets to satiation twice daily for 12 weeks. Fish weight gain (WG) was enhanced (P < 0.05) as dietary protein increased from 30% (229% WG) to 40% (262% WG) and plateaued thereafter. Enhancing protein and lipid content of diet led to reduced feed intake. Also, feed efficiency was improved by increasing dietary protein (by 40.8%) and lipid (by 16.5%) levels. An interaction of protein and lipid was found on whole-body lipid, and muscle lipid content increased as dietary lipid level increased. Muscle arachidonic acid (ARA), eicosapentaenoic acid (EPA, 20:5n-3) and total n-6 long-chain polyunsaturated fatty acids (LC-PUFA) contents enhanced by increasing dietary protein level. Moreover, increasing fat content of diet led to enhanced muscle linoleic acid, linolenic acid, total monounsaturated fatty acids (MUFA), total n-6, ratio of docosahexaenoic acid (DHA, 22:6n-3) to EPA and n-6/n-3. However, EPA, DHA, total n-6 LC-PUFA, total n-3, total n-3 LC-PUFA, and EPA/ARA ratio decreased at higher dietary lipid level. Serum triglyceride (TG) level and lactate dehydrogenase (LDH) activity decreased as dietary protein level increased. Increasing fat content of diet led to enhanced serum TG, cholesterol and glucose concentrations and reduced alanine aminotransferase, aspartate amino transferase and LDH activities. Serum malondialdehyde concentration was enhanced by increasing both dietary protein and lipid. Furthermore, serum myeloperoxidase activity was enhanced at higher dietary lipid level. Water ammonium nitrogen (NH4+-N) concentration was measured after 5 and 24 h of last feeding, and the results indicated the reduction of ammonia excretion as dietary lipid content increased. These findings suggest that 40% dietary protein can support optimal growth of juvenile European grayling reared in RAS and increasing lipid content from 10 to 20% can improve feed utilization and reduce ammonia excretion to the rearing water.

An effective replacement for fish meal (FM) and fish oil (FO) based on plant-based raw materials in the feed of marine fish species is necessary for the sustainability of the aquaculture sector. However, the use of plant-based raw materials to replace FM and FO has been associated with several negative health effects, some of which are related to oxidative stress processes that can induce functional and morphological alterations in mucosal tissues. This study aimed to evaluate the effects of dietary oligosaccharides of plant origin (5,000 ppm; galactomannan oligosaccharides, GMOS) and a phytogenic feed additive (200 ppm; garlic oil and labiatae plant extract mixture, PHYTO) on the oxidative stress status and mucosal health of the gills of juvenile European sea bass (Dicentrarchus labrax). The experimental diets, low FM and FO diets (10%FM/6%FO) were supplemented with GMOS from plant origin and PHYTO for 63 days. GMOS and PHYTO did not significantly affect feed utilization, fish growth, and survival. GMOS and PHYTO downregulated the expression of β-act, sod, gpx, cat, and gr in the gills of the fish compared with that in fish fed the control diet. The expression of hsp70 and ocln was upregulated and downregulated, respectively, in the GMOS group compared with that in the control group, whereas the expression of zo-1 was downregulated in the PHYTO group compared with that in the GMOS group. The morphological, histopathological, immunohistochemical, and biochemical parameters of the fish gills were mostly unaffected by GMOS and PHYTO. However, the PHYTO group had lower incidence of lamellar fusion than did the control group after 63 days. Although the tissular distribution of goblet cells was unaffected by GMOS and PHYTO, goblet cell size showed a decreasing trend (−11%) in the GMOS group. GMOS and PHYTO significantly reduced the concentration of PCNA+ in the epithelium of the gills. The above findings indicated that GMOS and PHYTO in low FM/FO-based diets protected the gill epithelia of D. labrax from oxidative stress by modulating the expression of oxidative enzyme-related genes and reducing the density of PCNA+ cells in the gills of the fish.

Doxorubicin is a widely used chemotherapeutic drug known to induce bone loss. The mechanism behind doxorubicin-mediated bone loss is unclear, but oxidative stress has been suggested as a potential cause. Antioxidants that can counteract the toxic effect of doxorubicin on the bone would be helpful for the prevention of secondary osteoporosis. We used resveratrol, a natural antioxidant, and MitoTEMPO, a mitochondria-targeted antioxidant, to counteract doxorubicin-induced bone loss and mineralization on Sparus aurata larvae. Doxorubicin supplemented Microdiets increased bone deformities, decreased mineralization, and lipid peroxidation, whereas Resveratrol and MitoTEMPO supplemented microdiets improved mineralization, decreased bone deformities, and reversed the effects of doxorubicin in vivo and in vitro, using osteoblastic VSa13 cells. Partial Least-Squares Discriminant Analysis highlighted differences between groups on the distribution of skeletal anomalies and mineralization of skeleton elements. Calcium and Phosphorus content was negatively affected in the doxorubicin supplemented group. Doxorubicin reduced the mRNA expression of antioxidant genes, including catalase, glutathione peroxidase 1, superoxide dismutase 1, and hsp90 suggesting that ROS are central for Doxorubicin-induced bone loss. The mRNA expression of antioxidant genes was significantly increased on resveratrol alone or combined treatment. The length of intestinal villi was increased in response to antioxidants and reduced on doxorubicin. Antioxidant supplements effectively prevent bone deformities and mineralization defects, increase antioxidant response and reverse doxorubicin-induced effects on bone anomalies, mineralization, and oxidative stress. A combined treatment of doxorubicin and antioxidants was beneficial in fish larvae and showed the potential for use in preventing Doxorubicin-induced bone impairment.

There is an increasing interest from the aquafeed industry in functional feeds containing selected additives that improve fish growth performance and health status. Functional feed additives include probiotics, prebiotics, organic acids, and phytogenics (substances derived from plants and their extracts). This study evaluated the effects of dietary inclusion of a mucilage extract rich in galactomannan oligosaccharides (GMOS), a mixture of garlic and labiatae-plants oils (PHYTO), and a combination of them (GMOSPHYTO), on gut microbiota composition of European sea bass (Dicentrarchus labrax) fed with a low fishmeal (FM) and fish oil (FO) diet. Three experimental diets and a control diet (plant-based formulation with 10% FM and 6% FO) were tested in a 63-days feeding trial. To analyze the microbiota associated to feeds and the intestinal autochthonous (mucosa-adhered) and allochthonous (transient) microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and QIIME2 pipeline were used. Metabarcoding analysis of feed-associated bacteria showed that the microbial communities of control (CTRL) feed deeply differed from those of experimental diets. The number of reads was significantly lower in CTRL feed than in other feeds. The OTU (operational taxonomic unit) number was instead similar between the feeds, ranging from 42 to 50 OTUs. The variation of resident gut microbiota induced by diet was lower than the variation of transient intestinal microbiota, because feedstuffs are a major source of allochthonous bacteria, which can temporarily integrate into the gut transient microbiome. However, the composition of transient bacterial communities was not simply a mirror of feed-borne bacteria. Indeed, the microbial profile of feeds was different from both faecal and mucosa profiles. Our findings suggest that the dietary inclusion of GMOS (0.5%) and PHYTO (0.02%) in a low FM and FO diet induces changes in gut microbiota composition of European sea bass. However, if on allochthonous microbiota the combined inclusion of GMOS and PHYTO showed an antagonistic effect on bactericidal activity against Vibrionales, at mucosa level, only GMOSPHYTO diet increased the relative abundance of Bacteroidales, Lactobacillales, and Clostridiales resident bacterial orders. The main beneficial effects of GMOS and PHYTO on gut microbiota are the reduction of coliforms and Vibrionales bacteria, which include several potentially pathogenic species for fish, and the enrichment of gut microbiota composition with butyrate producer taxa. Therefore, these functional ingredients have a great potential to be used as health-promoting agents in the farming of European sea bass and other marine fish.

The authors wish to make the following corrections to this paper [...]

The supplementation of fish diets with OH-SeMet reduces oxidative stress and modulates immune response against bacterial infection. However, despite the importance of essential polyunsaturated fatty acids in fish nutrition and their high risk of oxidation, the potential protective effect of OH-SeMet on these essential fatty acids has not been studied in detail. Moreover, while viral infection is very relevant in seabream production, no studies have focused the Se effects against viral infection. The aim of the present study was to assess the impact of dietary supplementation with OH-SeMet on gilthead seabream fatty acid profiles, growth performance and response against viral infection. Gilthead seabream juveniles (21.73 ± 0.27 g) were fed for 91 days with three experimental diets, a control diet without supplementation of Se (0.29 mg Se kg diet−1) and two diets supplemented with OH-SeMet (0.52 and 0.79 mg Se kg diet−1). A crowding stress test was performed at week 7 and an anti-viral response challenge were conducted at the end of the feeding trial. Selenium, proximate and fatty acid composition of diets and body tissues were analyzed. Although fish growth was not affected, elevation in dietary Se proportionally raised Se content in body tissues, increased lipid content in the whole body and promoted retention and synthesis of n-3 polyunsaturated fatty acids. Specifically, a net production of DHA was observed in those fish fed diets with a higher Se content. Additionally, both monounsaturated and saturated fatty acids were significantly reduced by the increase in dietary Se. Despite the elevation of dietary Se to 0.79 mg kg−1 not affecting basal cortisol levels, 2 h post-stress plasma cortisol levels were markedly increased. Finally, at 24 h post-stimulation, dietary OH-SeMet supplementation significantly increased the expression of the antiviral response myxovirus protein gene, showing, for the first time in gilthead seabream, the importance of dietary Se levels on antiviral defense.

European sea bass were fed four low FM/FO (10%/6%) diets containing galactomannan oligosaccharides (GMOS), a mixture of garlic oil and labiatae plants oils (PHYTO), or a combination of both functional products (GMOSPHYTO) for 63 days before exposing the fish to an intestinal Vibrio anguillarum infection combined with crowding stress. In order to evaluate functional diets efficacy in terms of gut health maintenance, structural, cellular, and immune intestinal status were evaluated by optical and electron microscopy and gene expression analyses. A semi-automated software was adapted to determine variations in goblet cell area and mucosal mucus coverage during the challenge test. Feeding with functional diets did not affect growth performance; however, PHYTO and GMOS dietary inclusion reduced European sea bass susceptibility to V. anguillarum after 7 days of challenge testing. Rectum (post-ileorectal valve) showed longer (p = 0.001) folds than posterior gut (pre-ileorectal valve), whereas posterior gut had thicker submucosa (p = 0.001) and higher mucus coverage as a result of an increased cell density than rectum. Functional diets did not affect mucosal fold length or the grade of granulocytes and lymphocytes infiltration in either intestinal segment. However, the posterior gut fold area covered by goblet cells was smaller in fish fed GMOS (F = 14.53; p = 0.001) and PHYTO (F = 5.52; p = 0.019) than for the other diets. PHYTO (F = 3.95; p = 0.049) reduced posterior gut goblet cell size and increased rodlet cell density (F = 3.604; p = 0.068). Dietary GMOS reduced submucosal thickness (F = 51.31; p = 0.001) and increased rodlet cell density (F = 3.604; p = 0.068) in rectum. Structural TEM analyses revealed a normal intestinal morphological pattern, but the use of GMOS increased rectum microvilli length, whereas the use of PHYTO increased (p≤0.10) Ocln, N-Cad and Cad-17 posterior gut gene expression. After bacterial intestinal inoculation, posterior gut of fish fed PHYTO responded in a more controlled and belated way in terms of goblet cell size and mucus coverage in comparison to other treatments. For rectum, the pattern of response was similar for all dietary treatments, however fish fed GMOS maintained goblet cell size along the challenge test.

Genetic selection in gilthead seabream (GSB), Sparus aurata, has been undertaken to improve the growth, feed efficiency, fillet quality, skeletal deformities and disease resistance, but no study is available to delineate the effect of genetic selection for growth trait on GSB reproductive performance under mass spawning condition. In this study, high growth (HG) or low growth (LG) GSB broodstock were selected to evaluate the sex steroid hormones, sperm, egg quality and reproductive performance under different feeding regime of commercial diet or experimental broodstock diet containing either fish oil (FO) or vegetable oil (VO) based diet. Under commercial diet feeding phase, broodstock selected for either high growth or low growth did not show any significant changes in the egg production per kg female whereas egg viability percentage was positively (p = 0.014) improved by the high growth trait broodstock group. The experimental diet feeding results revealed that both growth trait and dietary fatty acid composition influenced the reproductive performance of GSB broodstock. In the experimental diet feeding phase, we observed high growth trait GSB males produced a higher number of sperm cells (p < 0.001) and also showed a higher sperm motility (p = 0.048) percentage. The viable egg and larval production per spawn per kg female were significantly improved by the broodstock selected for high growth trait and fed with fish oil-based diet. This present study results signifies that gilthead seabream broodstock selected on growth trait could have positive role in improvement of sperm and egg quality to produce viable progeny.

Previous studies have shown that it is possible to increase the ability of marine fish to produce long-chain polyunsaturated fatty acid from their 18C precursors by nutritional programming or using broodstock with a higher fatty acyl desaturase 2 (fads2) expression. However, those studies failed to show the effect of these interventions on the expression of the fads2 gene in the developing egg. Moreover, there were no studies on the temporal expression of the fads2 during ontogeny in the gilthead sea bream (Sparus aurata). In order to determine the changes in expression of fads2 during ontogeny, gilthead sea bream broodstock with a high (HRO) or low (LRO) fads2 expression fed a diet previously used for nutritional programming, or a fish oil-based diet (LFO) were allowed to spawn. The samples were taken at the stages of spawning, morula, high blastula, gastrula, neurula, heart beating, hatch and 3 day-old first exogenous feeding larvae to determine fads2 expression throughout embryonic development. The results showed the presence of fads2 mRNA in the just spawned egg, denoting the maternal mRNA transfer to the developing oocyte. Later, fads2 expression increased after the neurula, from heart beating until 3-day-old larvae, denoting the transition from maternal to embryonic gene expression. In addition, the eggs obtained from broodstock with high fads2 expression showed a high docosahexaenoic acid content, which correlated with the downregulation of the fads2 expression found in the developing embryo and larvae. Finally, feeding with the nutritional programming diet with the partial replacement of fish oil by rapeseed oil did not affect the long chain polyunsaturated fatty acid (LC-PUFA) contents nor fads2 expression in the gilthead sea bream developing eggs.

The principle aim of this study is to elucidate the relationship between the fatty acid desaturase 2 gene (fads2) expression pattern in peripheral blood cells (PBCs) and liver of gilthead seabream (GSB), Sparus aurata broodstock in order to determine the possible use of fads2 expression as a potential biomarker for the selection of broodstock. This selection could be utilized for breeding programs aiming to improve reproduction, health, and nutritional status. Passive Integrated Transponder (PIT)-tagged GSB broodstock (Male-1.22 ± 0.20 kg; 44.8 ± 2 cm and female-2.36 ± 0.64 kg; 55.1 cm) were fed a diet containing low levels of fish meal and fish oil (EPA 2.5; DHA 1.7 and n-3 LC-PUFA 4.6% TFA) for one month. After the feeding period, fads2 expression in PBCs and liver of both male and female broodstock were highly significantly correlated (r = 0.89; p < 0.001). Additionally, in male broodstock, liver fads2 expression was significantly correlated (p < 0.05) to liver contents in 16:0 (r = 0.95; p = 0.04) and total saturates (r = 0.97; p = 0.03) as well as to 20:3n–6/20:2n–6 (r = 0.98; p = 0.02) a Fads2 product/precursor ratio. Overall, we found a positive and significant correlation between fads2 expression levels in the PBCs and liver of GSB broodstock. PBCs fads2 expression levels indicate a strong potential for utilization as a non-invasive method to select animals having increased fatty acid bioconversion capability, better able to deal with diets free of fish meal and fish oil.

Substituting fishmeal (FM) with vegetable meal (VM) can markedly affect the mineral composition of feeds, and may require additional mineral supplementation. Their bioavailability and optimal supplementation levels depend also on the form of delivery of minerals. The aim of the study was to determine the effect of different delivery forms of three major trace elements (Zn, Mn and Se) in a marine teleost. Gilthead sea bream juveniles of 22.5 g were fed a VM-based diet for 12 weeks that was either not supplemented with these minerals or supplemented with inorganic, organic, or encapsulated inorganic forms of minerals in triplicate and compared to a FM-based diet. Our results showed that mineral delivery form significantly affected the biochemical composition and morphology of posterior vertebrae. Supplementation of VM-based diets with inorganic forms of the target minerals significantly promoted growth, increased the vertebral weight and content of ash and Zn, enhanced bone mineralization and affected the vertebral shape. Conversely, encapsulation of inorganic minerals reduced fish growth and vertebral mineral content, whereas supplementation of organic minerals, enhanced bone osteogenesis by upregulating bone morphogenetic protein 2 (bmp2) gene and produced vertebrae with a larger length in relation to height. Furthermore, organic mineral forms of delivery downregulated the expression of oxidative stress related genes, such as Cu/Zn superoxide dismutase (Cu/Zn sod) and glutathione peroxidase 1 (gpx-1), suggesting thus that dietary minerals supplemented in the organic form could be reasonably considered more effective than the inorganic and encapsulated forms of supply.

The present study aimed to determine the spawning efficacy, egg quality and quantity of captive breed meagre induced with a single gonadotrophin‐releasing hormone agonist (GnRHa) injection of 0, 1, 5, 10, 15, 20, 25, 30, 40 or 50 µg kg-1 to determine a recommended optimum dose to induce spawning. The doses 10, 15 and 20 μg kg-1 gave eggs with the highest quality (measured as: percentage of viability, floating, fertilisation and hatch) and quantity (measured as: total number of eggs, number of viable eggs, number of floating eggs, number of hatched larvae and number of larvae that reabsorbed the yolk sac). All egg quantity parameters were described by Gaussian regression analysis with R2=0.89 or R2=0.88. The Gaussian regression analysis identified that the optimal dose used was 15 μg kg-1. The regression analysis highlighted that this comprehensive study examined doses that ranged from low doses insufficient to stimulate a high spawning response (significantly lower egg quantities, p-1 through to high doses that stimulated the spawning of significantly lower egg quantities and eggs with significantly lower quality (egg viability). In addition, the latency period (time from hormone application to spawning) decreased with increasing doses to give a regression (R2=0.93) which suggests that higher doses accelerated oocyte development that in turn reduced egg quality and quantity. The identification of an optimal dose for the spawning of meagre, which has high aquaculture potential, represents an important advance for the Mediterranean aquaculture industry.

Terrestrial protein and lipid sources are commonly used as substitutes for marine fishery-derived raw ingredients in fish diets. However, their use is related with several side-effects on marine fish performance, health, or disease resistance. Physical barriers of the skin, gills, and gut constitute the primary defense mechanism of fish. Skin mucus mucosal mucins, water, proteins, ions, and lipids determine the physical, chemical, and protective characteristics of skin mucus. Very little is known about the influence of diet composition on fish skin mucus fatty acid profile. Gilthead sea bream skin mucus contained 10% of total lipids (TL), which consisted of 50⁻60% neutral (NL) and 40⁻50% polar lipids (PL) fractions. Σn−3 long chain polyunsaturated fatty acids (LC-PUFA) deposition was preferential in the NL fraction, whereas Σn−6LC-PUFA accumulation was similar in both lipid classes. Docosahexaenoic acid (DHA; 22:6n−3) was the main LC-PUFA stored in skin mucus (14% TL) in relation to eicosapentaenoic acid (EPA; 20:5n−3) (2⁻3% TL) and arachidonic acid (ARA; 20:4n−6) (2% TL). This study denotes the importance of DHA as component of skin mucus lipids compared to other essential fatty acids, such as EPA and ARA, as well as importance of maintaining an adequate Σn−3/ Σn−6 ratio, regardless of dietary intake.

Background: The aim of this analysis was to ascertain the type of relationship between fish and seafood consumption, omega-3 polyunsaturated fatty acids (ω-3 PUFA) intake, and depression prevalence. Methods: Cross-sectional analyses of the PREDIMED-Plus trial. Fish and seafood consumption and ω-3 PUFA intake were assessed through a validated food-frequency questionnaire. Self-reported life-time medical diagnosis of depression or use of antidepressants was considered as outcome. Depressive symptoms were collected by the Beck Depression Inventory-II. Logistic regression models were used to estimate the association between seafood products and ω-3 PUFA consumption and depression. Multiple linear regression models were fitted to assess the association between fish and long-chain (LC) ω-3 PUFA intake and depressive symptoms. Results: Out of 6587 participants, there were 1367 cases of depression. Total seafood consumption was not associated with depression. The odds ratios (ORs) (95% confidence intervals (CIs)) for the 2nd, 3rd, and 4th quintiles of consumption of fatty fish were 0.77 (0.63⁻0.94), 0.71 (0.58⁻0.87), and 0.78 (0.64⁻0.96), respectively, and p for trend = 0.759. Moderate intake of total LC ω-3 PUFA (approximately 0.5⁻1 g/day) was significantly associated with a lower prevalence of depression. Conclusion: In our study, moderate fish and LC ω-3 PUFA intake, but not high intake, was associated with lower odds of depression suggesting a U-shaped relationship.

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 β-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.

Dietary fatty acids have been shown to exert a clear effect on the stress response, modulating the release of cortisol. The role of fatty acids on the expression of steroidogenic genes has been described in mammals, but little is known in fish. The effect of different fatty acids on the release of cortisol and expression of stress-related genes of European sea bass (Dicentrarchus labrax) head kidney, induced by a pulse of adenocorticotrophin hormone (ACTH), was studied. Tissue was maintained in superfusion with 60 min of incubation with EPA, DHA, arachidonic acid (ARA), linoleic acid or α-linolenic acid (ALA) during 490 min. Cortisol was measured by RIA. The quantification of stress-related genes transcripts was conducted by One-Step TaqMan real-time RT-PCR. There was an effect of the type of fatty acid on the ACTH-induced release of cortisol, values from ALA treatment being elevated within all of the experimental period. The expression of some steroidogenic genes, such as the steroidogenic acute regulatory protein (StAR) and c-fos, were affected by fatty acids, ALA increasing the expression of StAR after 1 h of ACTH stimulation whereas DHA, ARA and ALA increased the expression of c-fos after 20 min. ARA increased expression of the 11β-hydroxylase gene. Expression of heat shock protein 70 (HSP70) was increased in all the experimental treatments except for ARA. Results corroborate previous studies of the effect of different fatty acids on the release of cortisol in marine fish and demonstrate that those effects are mediated by alteration of the expression of steroidogenic genes.

The improvement of micro diets, for fish larvae, has been a major challenge in past years; however some problems remain without a resolution. Besides its tremendous growth rates, at early developmental stages, fish larvae present an immature digestive system and a progressive metamorphic process that suggest special nutritional requirements. It is therefore essential to provide fish larvae with easy and/or quickly metabolized protein sources, like protein hydrolisates, high bioavailable forms of vitamins and trace minerals. The low molecular weight of such compounds, in association to the high surface area of the micro-particles, makes them highly soluble and prone to water leaching losses. These nutrient losses, not only makes these nutrients unavailable to the larvae, but originate an important increase of organic matter in the rearing tanks, leading to detrimental water quality criteria. The development of high-performing micro-particulate diets for fish larvae requires a delicate balance between an adequate quantitative and qualitative nutritional supply and good water stability of the feeds, without compromising the palatability and the digestive breakdown. A series of binders, protein-based (e.g. fish gelatin, wheat gluten, spray-dried plasma) and carbohydrate-based (gums, alginates, pectins, starches, dextrin, polyols) were incorporated at graded levels (1, 2.5 and 5%) in a standard formulation for seabream larvae. Diets were manufactured by a proprietary low-shear extrusion process and afterwards processed to obtain a 200-400 micron fraction. All diets were analyzed for the following criteria: water leaching of nitrogenous compounds, turbidimetry and particle water stability. An additionally approach relied on the use of various encapsulation techniques (spray-drying, fluid-bed drying and vacuum coating) to incorporate trace minerals (Zn and Mn) and amino acids (taurine, lysine and methionine). Technological results show that the beneficial effects towards nutrient leaching and physical stability of the micro-pellets after immersion in water are highly dependent on binder type and less markedly on binder dose. Through the use of selected binders (e.g. guar gum and gelatin) in association to a spray-drying encapsulation process, we were able to significantly alter the release profile of Zn, Mn and taurine. Some of these prototypes were also tested in vivo with gilthead seabream larvae and clearly suggest that microencapsulation technologies that allow a controlled release profile of nutrients result in beneficial effects to larval growth and quality criteria. The results of several specific scenarios will be presented.