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.

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.

Distinguishing the Effects of Water Volumes versus Stocking Densities on the Skeletal Quality during the Pre-Ongrowing Phase of Gilthead Seabream (Sparus aurata).

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.

Exploring Omega-3's Impact on the Expression of Bone-Related Genes in Meagre (Argyrosomus regius).

Dietary supplementation with Omega-3 fatty acids seems to promote skeletal health. Therefore, their consumption at imbalanced or excessive levels has offered less beneficial or even prejudicial effects. Fish produced in aquaculture regimes are prone to develop abnormal skeletons. Although larval cultures are usually fed with diets supplemented with Omega-3 Long Chain Polyunsaturated fatty acids (LC-PUFAs), the lack of knowledge about the optimal requirements for fatty acids or about their impact on mechanisms that regulate skeletal development has impeded the design of diets that could improve bone formation during larval stages when the majority of skeletal anomalies appear. In this study, Argyrosomus regius larvae were fed different levels of Omega-3s (2.6% and 3.6% DW on diet) compared to a commercial diet. At 28 days after hatching (DAH), their transcriptomes were analyzed to study the modulation exerted in gene expression dynamics during larval development and identify impacted genes that can contribute to skeletal formation. Mainly, both levels of supplementation modulated bone-cell proliferation, the synthesis of bone components such as the extracellular matrix, and molecules involved in the interaction and signaling between bone components or in important cellular processes. The 2.6% level impacted several genes related to cartilage development, denoting a special impact on endochondral ossification, delaying this process. However, the 3.6% level seemed to accelerate this process by enhancing skeletal development. These results offered important insights into the impact of dietary Omega-3 LC-PUFAs on genes involved in the main molecular mechanism and cellular processes involved in skeletal development.

Selection for high growth improves reproductive performance of gilthead seabream Sparus aurata under mass spawning conditions, regardless of the dietary lipid source.

Genetic selection programmes in gilthead seabream mainly focus on traits related to growth, disease resistance, skeletal anomalies, or fillet quality. However, the effect of selection for growth on the reproductive performance of seabream broodstock has not received much attention. The present study aimed to determine the effect of selection for growth traits, high (HG) or low (LG) growth, and broodstock feeding with fish oil (FO diet) or rapeseed oil (RO diet) as main lipid sources, on reproductive performance of gilthead seabream. For the first part of the spawning season (Phase I) HG and LG broodstock were fed a commercial diet and the HG broodstock produced a higher number of larvae and higher viable eggs, hatching and larval survival rates than LG broodstock, affecting egg fatty acid profiles. For the second part of the study (Phase II) broodstock were fed one of the two diets containing FO or RO. Fecundity in terms of viable eggs, hatchlings, and larvae produced, as well as fertilization rates, were improved in HG broodstock. Some fatty acids such as 18:0, 20:2n-6, 20:3n-3 or EPA/ARA were also affected by the growth selection. According to the two-way ANOVA analysis, feeding the RO diet did not significantly affect fecundity parameters, but slightly reduced fertilization and hatching rates in HG broodstock. Nevertheless, HG broodstock showed better spawning quality parameters than LG broodstock, even when they were fed the RO diet. Egg fatty acid profiles reflected diet composition, although DHA contents were not affected. In conclusion, broodstock selected for high growth had a positive effect on broodstock performance, and FO replacement by RO did not markedly affect reproduction providing that fatty acid contents were sufficient to fulfill the essential fatty acid requirements of gilthead seabream broodstock.

Reversal of Doxorubicin-Induced Bone Loss and Mineralization by Supplementation of Resveratrol and MitoTEMPO in the Early Development of Sparus aurata.

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.

Organic Selenium (OH-MetSe) Effect on Whole Body Fatty Acids and Mx Gene Expression against Viral Infection in Gilthead Seabream (Sparus aurata) Juveniles.

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.

Erratum: Ferosekhan et al. Influence of Genetic Selection for Growth and Broodstock Diet n-3 LC-PUFA Levels on Reproductive Performance of Gilthead Seabream, Sparus Aurata. Animals 2021, 11, 519.

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

Dietary Phytogenics and Galactomannan Oligosaccharides in Low Fish Meal and Fish Oil-Based Diets for European Sea Bass (Dicentrarchus labrax) Juveniles: Effects on Gill Structure and Health and Implications on Oxidative Stress Status.

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 ß-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.

Influence of Genetic Selection for Growth and Broodstock Diet n-3 LC-PUFA Levels on Reproductive Performance of Gilthead Seabream, Sparus aurata.

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.

Influence of Parental Fatty Acid Desaturase 2 (fads2) Expression and Diet on Gilthead Seabream (Sparus aurata) Offspring fads2 Expression during Ontogenesis.

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.

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.

Reproductive performance of gilthead seabream (Sparus aurata) broodstock showing different expression of fatty acyl desaturase 2 and fed two dietary fatty acid profiles.

Previous studies have shown that it is possible to nutritionally program gilthead seabream offspring through fish oil (FO) replacement by vegetable oils (VO) in the broodstock diet, to improve their ability to grow fast when fed low fish meal (FM) and FO diets during grow-out phase. However, in those studies broodstock performance was reduced by the VO contained diet. Therefore, the present study aimed to determine if it is possible to replace FO by a mixture of FO and rapeseed oil (RO) with a specific fatty acid profile in broodstock diets, without altering gilthead seabream broodstock reproductive performance. Besides, the study also aimed to evaluate the reproductive performance of broodstock with different expression of fatty acid desaturase 2 gene (fads2) a key enzyme in synthesis of long chain polyunsaturated fatty acids. For that purpose, broodfish having either a high (HD) or low (LD) expression of fads2 were fed for three months during the spawning season with two diets containing different fatty acid profiles and their effects on reproductive hormones, fecundity, sperm and egg quality, egg biochemical composition and fads2 expression were studied. The results showed that blood fads2 expression in females, which tended to be higher than in males, was positively related to plasma 17ß-estradiol levels. Moreover, broodstock with high blood fads2 expression showed a better reproductive performance, in terms of fecundity and sperm and egg quality, which was correlated with female fads2 expression. Our data also showed that it is feasible to reduce ARA, EPA and DHA down to 0.43, 6.6 and 8.4% total fatty acids, respectively, in broodstock diets designed to induce nutritional programming effects in the offspring without adverse effects on spawning quality. Further studies are being conducted to test the offspring with low FM and FO diets along life span.

The Relationship between the Expression of Fatty Acyl Desaturase 2 (fads2) Gene in Peripheral Blood Cells (PBCs) and Liver in Gilthead Seabream, Sparus aurata Broodstock Fed a Low n-3 LC-PUFA Diet.

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.

Assessment of dietary supplementation with galactomannan oligosaccharides and phytogenics on gut microbiota of European sea bass (Dicentrarchus Labrax) fed low fishmeal and fish oil based diet.

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.

Stearoyl-CoA desaturase (scd1a) is epigenetically regulated by broodstock nutrition in gilthead sea bream (Sparus aurata).

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.

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.

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.

Dietary phytogenics and galactomannan oligosaccharides in low fish meal and fish oil-based diets for European sea bass (Dicentrarchus labrax) juveniles: Effects on gut health and implications on in vivo gut bacterial translocation.

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.

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.