IntroductionAnimal welfare has become an increasingly important issue in the aquaculture sector, and improving it is essential for maintaining the health of farmed fish. Therefore, the aim of the present study was to evaluate the potential effect of dietary cannabidiol (CBD) supplementation (0.001%) environmental enrichment (EE) through modified aeration or their combination over 5 weeks to assess their suitability as strategies for improving welfare in aquaculture.MethodsMethods: After this period, fish were subjected to a confinement stress challenge. Welfare indicators such as survival, selected stress and immunological parameters, lipid mediators, and the expression of several stress-response and appetite-regulating related genes in different brain regions (including telencephalon, hypothalamus, and pituitary gland) were analysed in European sea bass.ResultsThe results showed that survival rates were significantly higher in all welfare-promoting groups; EE improved production parameters and increased dissolved oxygen levels; CBD treatments did not negatively affect growth performance, reduced plasma cortisol during both acute and adaptive stress responses, enhanced basal serum lysozyme activity, and promoted a faster post-stress stabilization; and the combined treatment supported a synergistic interaction between EE and CBD, in which lipid mediators suggested modulations of inflammatory responses under stress and helped to mitigate the effects of the stress response-related genes and a potential involvement in appetite regulation and energy balance strategies.DiscussionThese results highlight the potential of microbubble curtain enrichment and CBD for improving welfare of juvenile sea bass, enhancing growth, stress resilience, and physiological regulation, with the combined treatment showing the strongest and most effective improvements.

In the 21st century, the emergence of multidrug-resistant bacteria has become a major concern in human and animal health, promoting the search for sustainable alternatives to antibiotics in aquaculture. Among these, probiotics have shown promising potential to improve fish health and disease resistance. This study evaluates the probiotic potential of Paenibacillus polymyxa SABE1 for application in European seabass (Dicentrarchus labrax) aquaculture. Its functional properties were assessed through in vitro assays, a challenge test against Vibrio parahaemolyticus, and whole-genome sequencing to determine safety and potential risks. Results indicate that P. polymyxa SABE1 exhibits strong antimicrobial activity, adhesion capacity, digestive resilience, and no hemolytic or pathogenic traits. Notably, the challenge assay showed a survival rate of 56.67% in treated fish, confirming its protective effect. Genomic analysis further supported its safety, revealing no relevant antibiotic resistance or virulence factors. Overall, P. polymyxa SABE1 demonstrates strong potential as a safe and effective probiotic candidate, supporting its use as a sustainable alternative to antibiotics in aquaculture.

The identification of safe and functionally robust probiotics is a key challenge for sustainable aquaculture and requires genome-based validation of candidate strains. Here, we performed an integrative genomic characterization of Bacillus velezensis D-18, a strain previously shown to enhance disease resistance in European seabass. Whole-genome sequencing generated a draft genome of approximately 4.06 Mb containing 4,178 protein-coding genes and 84 RNA genes. Comparative genomics confirmed the taxonomic placement of the strain within the B. velezensis lineage and its clear separation from pathogenic members of the Bacillus cereus group. Genome-wide screening detected no acquired antimicrobial resistance genes or virulence determinants, supporting the biosafety of the strain. Functional annotation revealed genetic determinants associated with quorum quenching, biofilm formation, stress tolerance, and antimicrobial activity. AntiSMASH analysis further identified seven biosynthetic gene clusters encoding bioactive metabolites including surfactin, fengycin, bacilysin, and macrolactin. In addition, molecular docking analyses suggested potential interactions between bacterial proteins and mucin glycoproteins, consistent with previously reported mucus adhesion. Together, these findings provide a genomic framework supporting the probiotic potential of B. velezensis D-18 for aquaculture applications.

The expansion of aquaculture and the drive toward more sustainable ingredients have promoted the incorporation of alternative and novel raw materials as alternatives to traditional marine raw materials, which can provide bioactive functions in addition to fulfill fish nutritional requirements. In this context, agro-industrial by-products and low-value marine biomass emerge as promising sources of antibacterial, immunomodulatory, and antioxidant bioactive compounds. Valorizing these raw materials within a circular economy framework offers the dual benefits of reducing waste and improving fish resilience. This study evaluated nine natural extracts of terrestrial and marine origin as potential functional ingredients for aquaculture. Terrestrial by-product extracts (TE) included pomegranate peel (rich in punicalagin or ellagic acid), citrus fruits, and grape seeds, whereas marine included marine macro- and micro-algal extracts (ME) (Rhodomonas lens, Desmodesmus sp., Osmundea pinnatifida, Gracilaria sp., and Dictyota sp.). Extracts were characterized by determining their total phenolic and flavonoid contents. Antioxidant activity was evaluated using two methods: 2, 2 '-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and Ferric Reducing Antioxidant Power (FRAP) assays. Antibacterial activity was assessed against Vibrio anguillarum, V. harveyi, and Photobacterium damselae subsp. piscicida. To assess the effects of extracts at cellular level, ex vivo assays were performed on head kidney leukocytes from gilthead seabream (Sparus aurata), evaluating cytotoxicity, respiratory burst, phagocytic activity, and peroxidase activity. TE showed higher levels of both phenolic compounds and flavonoids than ME, which are usually related to higher antioxidant activity. In addition, TE showed stronger antibacterial effects against the three pathogenic bacteria tested. However, ME in general terms, presented higher immunomodulatory potential, causing respiratory burst activation or higher peroxidase activity in leukocytes. These findings highlight distinct bioactivities depending on extract origin, suggesting that future in vivo studies evaluating the combined use of terrestrial and marine extracts may be of interest to explore potential complementary effects in aquaculture species such as Sparus aurata.

Background: Adopting novel feed ingredients and aligning feeding strategies with these formulations are key to improving aquaculture sustainability. This study assessed the combined effects of alternative protein and lipid sources and feeding regime on growth, nutrient utilization, and body composition of European sea bass (Dicentrarchus labrax) juveniles. Methods: Two isoenergetic and identical digestible protein diets (39%) were formulated: a control (conventional fishmeal/fish oil (FM/FO) and plant proteins, containing 20% FM and 6% FO) and an alternative diet replacing 50% of FM and 25% of vegetable proteins with a blend of poultry by-products, insect meal, and single-cell protein (Corynebacterium glutamicum) and totally replacing fish oil with alternative lipid sources (microalgae and by-product oils). Fish (28 g of initial body weight) were fed for 210 days either to apparent satiety (AS) or under moderate restriction (85% and 65% of AS). The number of fish used was 65 fish per 500 L tank (triplicate for each experimental group). Growth performance, feed conversion, nutrient efficiency ratios, protein retention, and proximate and fatty acid composition were measured. Results: The alternative diet significantly improved growth, feed and nutrient efficiency, and protein retention compared with the control. Whole-body fatty acid profiles of fish fed the alternative diet showed higher contents of nutritionally important fatty acids, including DHA. Restricted feeding at 65% of AS enhanced nutrient efficiency ratios and protein retention relative to 85% and AS, but reduced growth. Feeding to AS produced the highest feed intake and growth but poorer feed conversion and nutrient efficiency. No significant interaction between diet and feeding strategy was observed. Conclusions: Incorporating novel protein and lipid sources can improve sea bass performance and product nutritional value while supporting sustainability. Feeding at ~85% of AS may offer a practical compromise between growth and efficient nutrient utilization.

As aquaculture adopts more sustainable feed formulations, interest in functional feed additives has grown to help mitigate the health and performance challenges associated with low-marine-ingredient diets. This study evaluated the effects of dietary supplementation with a commercial blend of mono-, di-, and triglycerides of short- and medium-chain fatty acids (SCFAs and MCFAs; BalanGUT (TM) AQ P, BASF) on growth, health, and disease resistance to Vibrio anguillarum in juvenile gilthead sea bream (Sparus aurata) fed practical low fishmeal and fish oil diets. Over an 8-week trial, fish were fed diets containing 0.3%, 0.5%, or 1% of a glyceride blend of SCFAs and MCFAs (BalanGUT (TM) AQ P) or a Control diet without functional additive supplementation. Growth performance and feed utilization were not affected by the supplementation of SCFAs/MCFAs glycerides, although non-significant trends (p > 0.05) toward improved specific growth rate (up to 12%) and reduced feed conversion ratio (up to 17%) were observed in sea bream fed supplemented diets, particularly during the 4 initial weeks and at the highest inclusion level (1%). Moderate (0.5%) and high (1%) supplementation levels of SCFAs and MCFAs significantly improved survival following Vibrio anguillarum challenge, despite no significant changes being observed in general systemic innate immune markers, such as serum lysozyme or ACH50 activities. SCFAs/MCFAs supplementation, particularly at 0.3% or 0.5%, also modulated intestinal morphology, including thinner submucosa and smaller goblet cell area in the posterior intestine, suggestive of a more homeostatic mucosa and reduced basal inflammation when feeding a low-FM/FO-based diet. These results suggest that the protective effects of this SCFAs/MCFAs glyceride blend are mediated primarily through local rather than systemic immune modulation. Overall, this study supports the use of functional SCFAs and MCFAs glyceride blends as a functional strategy to promote resilience and health in fish fed sustainable, low-marine-ingredient diets.

Gilthead seabream (Sparus aurata) is a prominent aquaculture species in Europe; however, the repercussions of growth-oriented selective breeding on reproductive performance under industrial conditions have not been adequately characterised. The present study evaluated the influence of divergent Best Liner Unbiased Prediction (BLUP)-based selection for low growth (LG) (n = 49; mean weigh ± SD = 842 ± 189 g) and high growth (HG) (n = 50; mean weight ± SD = 1127 ± 407 g) on spawning quality throughout the commercial mass-spawning season. A number of significant differences were detected between the genetic lines. The LG broodstock produced substantially higher oocyte yields and numbers of fertilised eggs (26% and 25% increases, respectively), indicating greater quantitative reproductive output. In contrast, the HG line exhibited marginally higher fertilisation, egg viability, and hatching rates, indicative of enhanced early developmental efficiency. Despite these contrasting patterns, both lines exhibited similar numbers of viable eggs, larvae, and comparable larval survival. These findings demonstrate that selection for growth impacts reproductive traits through different pathways: The selection of HG results in an enhancement of developmental performance, while the selection of LG leads to an optimisation of egg production. Across the spawning period, oocyte yield was identified as the primary driver of overall spawn quality. The findings of this study offer pertinent insights into the optimisation of broodstock management and the enhancement of sustainability and efficiency in gilthead seabream aquaculture.

Vitamin D3 is the typical vitamin D supplement in aquafeeds, but this form requires further metabolization to the active form. Calcifediol is used in livestock as an alternative, however it isn't common in aquaculture. This trial shows the effects of calcifediol in gilthead seabream. 600 seabream (12.8 +/- 1.5 g) were fed 5 levels of calcifediol (<2.0, 99.7, 167.4, 298.6 or 825.4 ppb) for 15 weeks. 99.7 ppb of calcifediol improved growth, while 99.7-167.4 ppb reduced the FCR. 99.7 ppb of calcifediol increased the deposition of DHA and omega 3. The level of calcium in the vertebrae increased quadratically up to 99.7 and 167.4 ppb. No effect of the diet was observed on the prevalence of skeletal abnormalities. Mid-intestine fold length increased with 99.7 and 167.4 ppb of calcifediol compared to 825.4 ppb, while mid-intestine lamina propria width was thinner in fish fed 99.7 ppb than those without supplementation. 167.4 ppb calcifediol increased 50 % the percentage of goblet cells per surface area in the anterior intestine compared to those without supplementation. The results indicate that 99.7 ppb improved growth, FCR, whole-body omega3/omega6 ratio and calcium level in the vertebrae, while the highest levels of supplementation reduced growth, vertebrae calcium and mid-intestine fold length, suggesting a toxic effect of high levels of calcifediol in gilthead seabream juveniles.

Krill meal (KM) emerges as a promising sustainable marine ingredient in aquafeeds, providing a rich source of protein, amino acids, phospholipids, omega-3 fatty acids, and bioactive compounds. This study aimed to investigate the effects of including KM (3, 5 and 7 % of the diet) on growth performance, nutrient utilization, and antioxidant defenses in juvenile gilthead sea bream ( Sparus aurata) exposed to a crowding stress challenge. The dietary inclusion of 7 % KM could effectively replace up to 47 % FM in the diet (8 % FM in 7 % KM in comparison to 15 % FM in control diet), without compromising growth or feed conversion. Although not statistically different, dietary supplementation with 5 and 7 % KM showed a tendency to further optimize feed conversion ratio and nutrient efficiency ratios compared to the control FM diet. Under stressful conditions, a significant interaction between diet and time was observed in fish blood omega-3 index (O3I). At 24 h after the stress challenge, all dietary treatments except KM3 presented a significant increase in n-3 PUFA, EPA, DHA as well as OI3, whereas a decrease in MUFA. At 7d (168 h) post-stress, fish fed the control diet presented a significant reduction in O3I down to the basal levels. On the contrary, those fish fed KM5 and KM7 diets kept increased O3I levels as well as n-3 PUFA content to the end of the stress challenge. Indeed, 24 h after stress, fish fed KM5 and KM7 showed a lower increase of cat and sod gene expression in head kidney, which was further inversely correlated with fish blood OI3. Therefore, these results show that KM modulates red blood cells fatty acid profile by increasing fish OI3 after stress as well as potentially functioning as an antioxidant modulator in fish feeds for mitigating stressful conditions. Hence, KM is a valuable functional ingredient in aquafeeds, aiming to expand the basket of raw materials with functional properties to be used in aquafeed formulation to enhance fish robustness.

The orchid dottyback Pseudochromis fridmani is considered one of the most attractive species in the marine ornamental fish trade because of its bright colour, resilience, and relatively small size. Orchid dottyback aquaculture faces bottlenecks related to broodstock management and spawning conditioning, which make large-scale production challenging but can be addressed through targeted improvements. The present study addresses the knowledge gaps with regard to the management of P. fridmani under artificial conditions, describing the pair formation process with the associated behaviours, and the histological maturation process of gonads. The effects of low (25 °C) and high (28 °C) temperature on spawning activity and filial cannibalism were studied by monitoring six pairs for a period of 6 months. Water temperature strongly influenced broodstock spawning activity and filial cannibalism. The spawning rate increased significantly at 28 °C, but concurrent filial cannibalism also increased, leading to spawn losses. Notably, the total monthly number of viable spawns that successfully developed until hatching did not differ significantly between the 28 and 25 °C treatments. Examination of the size and shape of specimens sexed by histology (n = 6) suggested that these criteria may allow rapid visual sex identification in this species. Males tended to be longer and exhibited a more slender body shape, while females were comparatively shorter and showed a rounder body shape with a more pronounced belly. However, the accuracy of this method needs to be evaluated using larger sample sizes. In summary, maintaining the broodstock temperature around 25 °C facilitates the management of spawns and improves the performance of this highly demanded species.

Abstract Bacillus velezensis D-18 isolated from a wastewater sample collected from a fish farm, In a previous experiment, Bacillus velezensis D-18 had demonstrated its ability to be a good candidate as probiotic in aquaculture, However, information on its genomic content is lacking. This is the complete genome assembly of Bacillus velezensis D18 using Illumina paired-ends sequencing, which resulted in a 21 contigs assembly of 3.9 Mb. About 4,179 protein-coding genes, 84 encode RNAs were predicted from this assembly.

In a time of unprecedented coral reef decline, improved aquaculture protocols and high-quality live feeds may contribute to the sustainability of the ornamentals industry producing new valuable and stronger specimens bred under controlled conditions, mitigating the pressure on wild natural stocks. In the present study, the effect of microalgae enriched live feed diets (15 ind. mL−1), the copepod Acartia tonsa and the rotifer Brachionus plicatilis, were tested on early larvae stages (0–15 days post hatch) of the orchid dottyback Pseudochromis fridmani. Enriched A. tonsa significantly improved P. fridmani larval growth, survival, biochemical composition, as well as the morphological development of liver, intestine and skeletal system, compared to rotifers treatment. The results obtained showed that is feasible to totally replace the enriched rotifer B. plicatilis with the enriched copepod A. tonsa during first larval feeding of orchid dottybacks, resulting in a more robust larvae with better culture performance. Indeed, feeding larvae with enriched A. tonsa avoid the different negative effects when used enriched rotifers diets, which led to liver steatosis and nuclear pyknosis, delays in intestine and skeletal development, altered proximate composition and FA content. This study provided valuable information about rearing and breeding under controlled conditions of P. fridmani, contributing to its mass scale production and to the implementation of new hatchery protocols employing high quality live feeds.

Genetic selection and novel raw materials for aquafeeds are current key tools in the ongoing effort to increase the productivity, efficiency, and sustainability of the aquaculture sector. Selective breeding could also improve the utilization of novel dietary formulations with emergent ingredients. Gilthead sea bream juveniles, either coming from a selective breeding program based on growth traits, or a non-selected population, were nutritionally challenged with two novel dietary formulations that were compared with a Control diet based on 15% FM and 6% FO dietary commercial levels for this species. The novel formulations included an insect meal diet (INS) at 5% of the diet to replace 33.3% of the dietary FM, or a single-cell protein diet (SCP) at 10% of the diet and to replace 66.7% of the dietary FM. Fish oil was also totally replaced in these diets by a blend of poultry oil and Veramaris algal oil. Better growth and feed utilization of the selected genotype compared to non-selected fish was observed, at any of the diets assayed. INS and SCP novel diets reduced general performance of fish by reducing feed intake. However, selected fish fed novel diets showed very similar growth and lower feed conversion ratio compared with non-selected fish fed a control diet. The novel formulations increased n-3 LC-PUFA in fish tissues, particularly DHA, irrespective of the genotype, as a result of the dietary inclusion of the DHA-rich microalgal oil. Neither genetic selection nor the use of novel raw materials affected fillet proximate composition and consequently, sea bream fillet quality in terms of texture and sensorial perception of consumers. Overall, the results reaffirm the positive effects of selective breeding programs in improving sea bream key productive indicators, as well as support the use of novel dietary formulations, using insect meal from H. illucens, single-cell protein from M. capsulatus as partial replacers of FM in diets for gilthead sea bream (33 and 66% of replacement, respectively), and a blend of DHA-rich microalgal and poultry oils as total replacer of FO.

The impacts of tire wear particles and their associated chemicals on the aquatic systems are a major environmental concern. In this study, we investigated the acute toxicity of two pollutants derived from tire rubber, 6PPD-quinone and 4-tert-octylphenol, on marine plankton. Specifically, we determined the acute effects of these pollutants on various organisms within the plankton food web: the microalgae Rhodomonas salina, the adult copepod Acartia tonsa, and the early life stages of the echinoderms Arbacia lixula and Paracentrotus lividus and the fish Sparus aurata. Exposure to 6PPD-quinone did not affect the microalgae growth, copepod survival, or fish embryo viability after 48 h of exposure at concentrations up to 1000 µgL-1. However, 6PPD-quinone significantly inhibited the growth of early developmental stages of both echinoderm species, with median effective concentrations of 7 and 8 µgL-1. Conversely, 4-tert-octylphenol was toxic to all studied organisms, with median lethal and effective concentrations ranging from 21 to 79 µgL-1 depending on the species and endpoints. The most sensitive planktonic organisms to 4-tert-octylphenol were echinoderm embryos and copepods, which exhibited negative effects at concentrations as low as 1 and 25 µgL-1, respectively. Our results demonstrate that acute exposure to 6PPD-quinone and 4-tert-octylphenol can cause harmful effects on key planktonic organisms at environmentally relevant concentrations. Overall, our findings highlight the need for develop ecologically safer tire rubber additives and reduce traffic-related tire particle emissions to mitigate their entry and potential impacts on aquatic ecosystems.

In vitro cell culture systems serve as instrumental platforms for probing biological phenomena and elucidating intricate cellular mechanisms. These systems afford researchers the opportunity to scrutinize cellular responses within a regulated environment, thereby circumventing the ethical and logistical challenges associated with in vivo experimentation. Three-dimensional (3D) cell cultures have emerged as a viable alternative to mimic in vivo environments. Within this context, spheroids are recognized as one of the most straightforward and efficacious models, presenting a promising substitute for conventional monolayer cultures. The application of 3D cultures of fish cells remains limited, focusing mainly on physiological and morphological characterization studies. However, given the capacity of spheroids to emulate in vivo conditions, researchers are exploring diverse applications of these 3D cultures. These include eco-toxicology, immunology, drug screening, endocrinology, and metabolism studies, employing a variety of cell types such as fibroblasts, hepatocytes, embryonic cells, gonadal cells, gastrointestinal cells, and pituitary cells. This review provides a succinct overview, concentrating on the most frequently employed methods for generating fish cell spheroids and their applications to date. The aim is to compile and highlight the significant contributions of these methods to the field and their potential for future research.

Optimizing growth and feed conversion ratios by improving gut function and health is critial to ensuring cost-effective production in aquaculture, especially in the current context of low fishmeal and low fish oil-based diets. However, the use of practical diets based on high levels of plant-based raw materials as an alternative to traditional marine ingredients has been associated with negative effects on fish performance, feed utilization, and health. Organic acids and their salts have been widely used as functional ingredients not only for their antimicrobial and immunomodulatory properties, but also for their potential to promote animal digestive capacity and gut health. The aim of the present study was to evaluate the effects of a mixture of formic acid and sodium formate (Amasil NA®, BASF, Germany- AMA diet) at a dietary level of 0.3%, on key performance indicators, gut morphology and disease resistance to Vibrio anguillarum in juvenile gilthead sea bream (Sparus aurata). The results of the present study showed that fish fed the AMA diet for 8 weeks, performed similar to fish fed the control diet in terms of growth but presented an optimization of 8% in the utilization of a low in fishmeal and high in plant proteins based diet. The AMA-diet also increased the folds length of gilthead sea bream anterior gut, increasing intestinal absorption area, and decreasing the submucosa width and goblet cell size in the posterior gut when included in a high dietary plant protein content diet. Furthermore, dietary supplementation with Amasil NA® at 0.3% increased gilthead sea bream disease resistance against V. anguillarum compared with fish fed the unsupplemented diet. These results highlight the potential of this combination of formic acid and sodium formate based product as a feed efficiency enhancer, and as a gut health promoter in gilthead sea bream plant protein-based diets.

When necessary, sea turtles are held captive for veterinarian care and research purposes. Protocols and basic guidelines have been described for husbandry of sea turtles with veterinarian needs but not considering physiological indicators of animal welfare. Because all sea turtle are imperiled species, monitoring their welfare is important. The aim of this study was to standardize husbandry protocols for loggerhead (Caretta caretta) juveniles held under seminatural conditions, based on circulating concentration of plasma corticosterone (Cort) and behavior. Two experiments were performed to analyze physiological and behavioral responses of the animals facing changes in stocking density and different dry-docking times. Cort analyses suggested that the number of animals per tank can be modified occasionally, without affecting their health and welfare. However, dry-docking time should be < 30 min, as indicated by the significant elevation of circulating Cort at ≥ 30 min, rising from 1.51- ng/ml to 5.28-ng/ml. Protocols tested did not affect behavioral responses, except for the breaths per move, which increased while Cort increased, despite differences exhibited by experimental animals in behavioral responses according to daily times (morning vs afternoon) and the sex of the animals.

Amid growing concerns about antibiotic resistance, innovative strategies are imperative in addressing bacterial infections in aquaculture. Quorum quenching (QQ), the enzymatic inhibition of quorum sensing (QS), has emerged as a promising solution. This study delves into the QQ capabilities of the probiotic strain Bacillus velezensis D-18 and its products, particularly in Vibrio anguillarum 507 communication and biofilm formation. Chromobacterium violaceum MK was used as a biomarker in this study, and the results confirmed that B. velezensis D-18 effectively inhibits QS. Further exploration into the QQ mechanism revealed the presence of lactonase activity by B. velezensis D-18 that degraded both long- and short-chain acyl homoserine lactones (AHLs). PCR analysis demonstrated the presence of a homologous lactonase-producing gene, ytnP, in the genome of B. velezensis D-18. The study evaluated the impact of B. velezensis D-18 on V. anguillarum 507 growth and biofilm formation. The probiotic not only controls the biofilm formation of V. anguillarum but also significantly restrains pathogen growth. Therefore, B. velezensis D-18 demonstrates substantial potential for preventing V. anguillarum diseases in aquaculture through its QQ capacity. The ability to disrupt bacterial communication and control biofilm formation positions B. velezensis D-18 as a promising eco-friendly alternative to conventional antibiotics in managing bacterial diseases in aquaculture.

Disease prevention is pivotal in aquaculture, and while vaccines offer protective immunity, challenges such as cost and low efficacy persist. The present study investigated the potential of plant-derived compounds, known as phytogenics, to bolster the effectiveness of vaccines against vibriosis in European seabass. Two phytogenic blends, namely PHYTO1 (terpenes) and PHYTO2 (terpenes and flavonoids) were supplemented to a commercial diet to obtain three experimental diets: a non-supplemented control diet, PHYTO1 (a 200-ppm blend of garlic and Lamiaceae oils with 87.5 mg kg(-1) terpenes), and PHYTO2 (a 1000 ppm blend containing citrus fruits, Asteraceae and Lamiaceae oils with 57 mg kg(-1) terpenes and 55 mg kg(-1) flavonoids). Following vaccination by bath immersion, juvenile European seabass were divided into groups and fed one of the three diets for 30 days. After this feeding period, fish were anesthetized and boosted with a single dose of vaccine through intraperitoneal injection. They continued to be fed their respective diets for another 30 days. At day 60, after the priming vaccination, fish were challenged with Vibrio anguillarum via intraperitoneal injection. Various parameters were measured at different time points post each vaccination, including total weight, circulating plasma cortisol and glucose levels, serum immunoglobulin M (IgM) titers, antioxidant power of leucocytes, and the expression of several antioxidant and immune-related genes. The results showed that fish fed with phytogenic supplements did not differ in weight compared to the control group. However, they exhibited lower plasma cortisol and glucose levels, increased IgM titers, and enhanced antioxidant protection and antioxidant power of head kidney leucocytes. In addition, phytogenics upregulated several immune-related genes in the gills and head kidney immediately after each vaccination. Notably, PHYTO2, enriched with flavonoids and terpenes, exhibited an even more pronounced positive effect on boosted fish by reducing vaccine-associated stress while improving antioxidant protection and modulating the vaccine-induced immune response. This synergistic effect of vaccination combined with phytogenics introduces new pathways for enhancing fish health in aquaculture.

The synergies between selective breeding and feed additives remain under -explored in farmed fish, despite their sustainability. Reference (REF) and selected gilthead sea bream for growth (GS) were fed with the control (CTRL) diet during 14 days. CTRL diet was oil -coated with three functional additives (PHY: phytogenic based on garlic and medium chain fatty acid; OA: organic acid mixture with a 70% of butyric acid sodium salt; PROB: probiotic based on Bacillus subtilis, pumillus and licheniformes species). These experimental diets were then sequentially administered at high (PHY/OA = 7.5 g/kg, PROB = 2 x 10 11 CFU/kg; 2 weeks) and low (PHY = 5 g/kg, OA = 3 g/kg, PROB = 4 x 10 10 CFU/kg; 10 weeks) additive doses. The capacity of a given genotype and additive to modify the fish growth performance, gut health and the host interaction with its anterior intestine (AI) microbiota was evaluated as a whole population or individually (9 fish/diet/genetics). GS fish showed a better growth and feed conversion ratio, linked to a reduced individual variability of gut microbial composition. The PHY additive had a major impact upon the intestinal transcriptome of GS-PHY fish, with the up -regulation of markers of epithelial integrity, sphingolipid and cholesterol/bile salt metabolism. With the OA additive, impaired growth performance, reduced AI goblet cell area and enhanced AI granulocyte infiltration were concomitant with a down -regulation of neutrophil degranulation markers associated with a decrease of pathogenic genera ( Staph- ylococcus / Streptococcus / Neisseria ), and an over -representation of acetone/butanol/ethanol fermentation and vitamin K biosynthesis inferred pathways. Bacillus establishment and lack of AI inflammation were parallel in PROB fish of both genetic backgrounds. However, GS fish grew and utilized feed better with the additive, whereas a worsening appeared in REF fish. This amelioration was related with a higher abundance of the nitratereducer Kocuria , an up -regulation of markers of epithelial cell maintenance and proliferation, and a downregulation of microbiota-correlated protein synthesis and ubiquitination markers, supporting a reduced epithelial turnover and improved intestinal barrier function. Overall, the success of nutritional innovations in gilthead sea bream is largely dependent on the host genome predisposition, but also on the intestinal microbiota according to the hologenome theory.