Fish genetically selected for growth (GS) and reference (REF) fish were fed with CTRL (15% FM, 5–7% FO) or FUTURE (7.5% FM, 10% poultry meal, 2.2% poultry oil + 2.5% DHA-algae oil) diets during a 12-months production cycle. Samples from initial (t₀; November 2019), intermediate (t₁; July 2020) and final (t₂; November 2020) sampling points were used for Illumina 16S rRNA gene amplicon sequencing of the adherent microbiota of anterior intestine (AI). Samples from the same individuals (t₁) were also used for the gene expression profiling of AI by RNA-seq, and subsequent correlation analyses with microbiota abundances. Discriminant analyses indicated the gut bacterial succession along the production cycle with the proliferation of some valuable taxa for facing seasonality and different developmental stages. An effect of genetic background was evidenced along time, decreasing through the progression of the trial, namely the gut microbiota of GS fish was less influenced by changes in diet composition. At the same time, these fish showed wider transcriptomic landmarks in the AI to cope with these changes. Our results highlighted an enhanced intestinal sphingolipid and phospholipid metabolism, epithelial turnover and intestinal motility in GS fish, which would favour their improved performance despite the lack of association with changes in gut microbiota composition. Furthermore, in GS fish, correlation analyses supported the involvement of different taxa with the down-regulated expression of pro-inflammatory markers and the boosting of markers of extracellular remodelling and response to bacterium. Altogether, these findings support the combined action of the gut microbiome and host transcriptionally mediated effects to preserve and improve gut health and function in a scenario of different growth performance and potentiality.

The effect of different main dietary compositions on growth, anticipatory digestive enzyme activities, and oxidative status was studied in the proximal intestine of juvenile European sea bass. A control diet (C, 44% protein, 17.6% lipid, and 20% starch), three diets with increasing starch levels to test protein sparing (P36S36, P40S29, and P43S24), and two diets with high lipid content (L20S13 and L22S7) were tested. After 20 weeks, growth, digestive enzyme activities, lipid peroxidation, antioxidant enzyme activities, and G6PDH activity were measured after a 24-h fast. Sea bass fed P43S24 and L20S13 maintained an oxidative status like C fish, up-regulated CAT activity, and adjusted anticipatory protease activity. Instead, the lipid peroxidation increased in the L22S7 group, although CAT activity increased, whereas anticipatory total protease activity was downregulated. P40S29 also triggered LPO and CAT activity, but G6PDH levels diminished significantly. Moreover, an up-regulation in digestive enzyme activities was found. Finally, P36S36 fish showed less antioxidant enzyme activity and G6PDH, although their LPO tended to increase and their lipase and α-amylase activities were upregulated. In conclusion, the inclusion of carbohydrates up to 24% or lipids up to 20% is possible for this species if protein requirements are met without negative effects on growth.

High-fat diets (HFDs) enhance fish growth by optimizing nutrient utilization (i.e., protein-sparing effect); however, their potential negative effects have also encouraged the search for feed additives. This work has investigated the effects of an extract rich in a polyphenolic antioxidant, hydroxytyrosol (HT), supplemented (0.52 g HT/kg feed) in a HFD (24% lipid) in gilthead sea bream (Sparus aurata). Fish received the diet at two ration levels, standard (3% of total fish weight) or restricted (40% reduction) for 8 weeks. Animals fed the supplemented diet at a standard ration had the lowest levels of plasma free fatty acids (4.28 ± 0.23 mg/dL versus 6.42 ± 0.47 in the non-supplemented group) and downregulated hepatic mRNA levels of lipid metabolism markers (ppara, pparb, lpl, fatp1, fabp1, acox1, lipe and lipa), supporting potential fat-lowering properties of this compound in the liver. Moreover, the same animals showed increased muscle lipid content and peroxidation (1.58- and 1.22-fold, respectively, compared to the fish without HT), suggesting the modulation of body adiposity distribution and an enhanced lipid oxidation rate in that tissue. Our findings emphasize the importance of considering this phytocompound as an optimal additive in HFDs for gilthead sea bream to improve overall fish health and condition.

Background: Broodstock nutritional programming improves the offspring utilization of plant-based diets in gilthead sea bream through changes in hepatic metabolism. Attention was initially focused on fatty acid desaturases, but it can involve a wide range of processes that remain largely unexplored. How all this can be driven by a different genetic background is hardly underlined, and the present study aimed to assess how broodstock nutrition affects differentially the transcriptome and genome-wide DNA methylome of reference and genetically selected fish within the PROGENSA® selection program. Results: After the stimulus phase with a low fish oil diet, two offspring subsets of each genetic background received a control or a FUTURE-based diet. This highlighted a different hepatic transcriptome (RNA-seq) and genome-wide DNA methylation (MBD-seq) pattern depending on the genetic background. The number of differentially expressed transcripts following the challenge phase varied from 323 in reference fish to 2,009 in genetically selected fish. The number of discriminant transcripts, and associated enriched functions, were also markedly higher in selected fish. Moreover, correlation analysis depicted a hyper-methylated and down-regulated gene expression state in selected fish with the FUTURE diet, whereas the opposite pattern appeared in reference fish. After filtering for highly represented functions in selected fish, 115 epigenetic markers were retrieved in this group. Among them, lipid metabolism genes (23) were the most reactive following ordering by fold-change in expression, rendering a final list of 10 top markers with a key role on hepatic lipogenesis and fatty acid metabolism (cd36, pitpna, cidea, fasn, g6pd, lipt1, scd1a, acsbg2, acsl14, acsbg2). Conclusions: Gene expression profiles and methylation signatures were dependent on genetic background in our experimental model. Such assumption affected the magnitude, but also the type and direction of change. Thus, the resulting epigenetic clock of reference fish might depict an older phenotype with a lower methylation for the epigenetically responsive genes with a negative methylation-expression pattern. Therefore, epigenetic markers will be specific of each genetic lineage, serving the broodstock programming in our selected fish to prevent and mitigate later in life the risk of hepatic steatosis through changes in hepatic lipogenesis and fatty acid metabolism.

Fish oil is commonly replaced by vegetable oils in sea bream diets, but little is known about their effects on intestinal health regarding oxidative stress biomarkers. The negative effects of lipid peroxidation on digestive mucosa could have consequences in animal nutrition and welfare. In this study, five isonitrogenous (46%) and isolipidic (22%) diets with 75% of vegetable oils inclusion were evaluated: soybean oil (S) alone or different mixtures containing soybean oil with linseed (SL), linseed and rapeseed (SLR), linseed and palm (SLP), and linseed, rapeseed, and palm (SLRP). Gilthead sea bream juveniles were fed twice a day for 18 weeks. Pyloric caeca and proximal intestine samples were collected 24 h post feeding for lipid peroxidation (LPO), antioxidant enzyme activities (SOD, CAT, GPx, GST, and GR) and gene expression analyses. Pyloric caeca presented larger unhealthy changes in oxidative status than proximal intestine. Although SL-fed fish showed the highest antioxidant activities, they were unable to cope with LPO that in pyloric caeca was 31.4 times higher than in the other groups. Instead, SLP fish presented the best oxidative status, with low LPO levels, antioxidant enzyme activities, and gene expression. In summary, between the vegetable oils dietary mixtures tested, SPL would maintain better intestinal health.

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.