Fish were kept for six weeks at three different initial stocking densities and water O₂ concentrations (low-LD, 8.5 kg/m³ and 95–70% O₂ saturation; medium-MD, 17 kg/m³ and 55–75% O₂ saturation; high-HD, 25 kg/m³ and 60–45% O₂ saturation), with water temperature increasing from 19 °C to 26–27 °C. The improvement in growth performance with the decrease in stocking density was related to changes in skin and intestinal mucosal microbiomes. Changes in microbiome composition were higher in skin, with an increased abundance of Alteromonas and Massilia in HD fish. However, these bacteria genera were mutually exclusive, and Alteromonas abundance was related to a reactive behavior and systemic growth regulation via the liver Gh/Igf system, while Massilia was correlated to a proactive behavior and a growth regulatory transition towards muscle rather than liver. At the intestinal level, microbial abundance showed an opposite trend for two bacteria taxa, rendering in a low abundance of Reyranella and a high abundance of Prauserella in HD fish. This trend was correlated with up-regulated host gene expression, affecting the immune response, epithelial cell turnover, and abiotic stress response. Most of the observed responses are adaptive in nature, and they would serve to infer new welfare indicators for increased stress resilience.

A confinement stress test with 75% tank space reduction and behavioural monitoring through tri-axial accelerometers externally attached to the operculum was designed. This procedure was validated by demonstrating the less pronounced stress response in gilthead sea bream than in European sea bass (950–1200 g). Our study aimed to assess habituation to high stocking densities with such procedure in gilthead sea bream. Animals (420–450 g) were reared (June–August) in a flow-through system at two stocking densities (CTRL: 10–15 kg/m³; HD: 18–24 kg/m³), with natural photoperiod and temperature (21–29 °C), and oxygen levels at 5.2–4.2 (CTRL) and 4.2–3.2 ppm (HD). At the end, blood and muscle were sampled for haematology and transcriptomic analyses, and external tissue damage was assessed by image-based scoring. Four days later, fish underwent a 45 min confinement stress test over two consecutive days. HD fish showed reduced feed intake, growth rates and haematopoietic activity. Muscle transcriptome changes indicated a shift from systemic to local growth regulation and a primed muscle regeneration over protein accretion in HD animals with slight external injuries. After stress testing, HD fish exhibited a decreased recovery time in activity and respiration rates, which was shorter after a second stressor exposure, confirming habituation to high densities.

The revalorization of animal by-products, such as porcine blood, is a key strategy for sustainable aquaculture and circular economy practices. This study aimed to fill the existing knowledge gap on the effects of spray-dried porcine blood hydrolysate (PBSH), assessing its potential as a functional feed ingredient for gilthead sea bream. Two practical diets were formulated: a control diet containing 5% blood meal, and a PBSH diet including 5% PBSH previously characterized in vitro. The results indicated that the PBSH diet promoted lower hepatosomatic index, a down-regulation of key hepatic lipogenic enzymes (scd1b, hl, lpl), and a better stress condition with lower circulating levels of glucose and cortisol and a reduction in aggressive attacks. Positive findings were also achieved in energy management, obtaining lower metabolic rates along with an enhanced swimming performance (20% increase in the critical speed) and a quicker weigh recovery after a fasting period. The PBSH diet also shaped the intestinal bacterial composition, determining a redistribution of abundant genera including Aureimonas and Halomonas. Ultimately, this study demonstrated that PBSH would act as a functional ingredient capable of enhancing fish energy management and resilience in the face of stressful events, exhibiting a transient transcriptional modulation, yet persistent physiological and welfare benefits.

The microalgae Nannochloropsis oceanica cultivated from brewery waste was considered a sustainable feed ingredient for gilthead sea bream ( Sparus aurata ), evaluating physiological, molecular and microbial responses. Over 96 days, fish were fed four isoproteic and isolipidic diets: a commercial‐based diet (D1), and three fishmeal‐free diets with increasing Nannochloropsis inclusion levels (0%, D2; 2%, D3; 14%, D4). The D4 diet was additionally supplemented with docosahexaenoic acid (DHA)‐rich algae oil instead of fish oil (FO). Growth performance remained almost equal in fish fed all the fishmeal‐free diets with only a slight and final improvement (<4%) in the control D1 fish, although an enhanced expression with a discriminant capacity was reported for hepatic growth‐related markers ( igf1 and ghr1 ) in D4 fish. Gene expression profiling also revealed a down‐regulation of fatty acid synthesis in combination with the up‐regulation of tissue lipid‐uptake genes, mostly linked to the dietary DHA enrichment. Antioxidant capacity was increased in both D3–D4 fish, suggesting a progressive activation of enzymatic and non‐enzymatic defences with the dietary microalgae diets (D3–D4). Moreover, intestinal transcriptomic profiling revealed a tightly regulated immune milieu, with coordinated modulation of pro‐ and anti‐inflammatory pathways indicative of an immune surveillance state induced by dietary microalgae. Water microbiota also shifted with diet composition, indicating a link between feeding strategy and environmental microbial communities. Taking all together, Nannochloropsis inclusion, particularly when combined with DHA‐rich microalgae oil, enhances fish health, immune and antioxidant status and beneficial gut microbiota markers. Altogether makes the circularly‐produced Nannochloropsis oceanica a valuable resource for the sustainable development of aquaculture feeds.

In fish gut microbiome studies, there are no standardized protocols regarding sampling region or post-feeding time, nor clear consensus on whether analyses should target resident (autochthonous) or transient (allochthonous) bacteria. This study examined the dynamics and interactions of both microbial communities in the anterior and posterior intestine of farmed gilthead sea bream and evaluated the resident microbiome at 24 and 48 h post-feeding. Microbial DNA was sequenced using the Oxford Nanopore Technology platform. Data were analyzed through statistical and discriminant approaches, as well as a Bayesian network framework to assess bacterial interactions. Transient communities showed higher richness and diversity, regardless of intestinal section, suggesting a more specialized and stable microbial environment in the mucus layer. The two communities differed markedly in structure and composition. Variations associated with intestinal region were less pronounced, particularly for autochthonous bacteria, and post-feeding fluctuations in the resident microbiome were minimal. Functionally, results indicated relevant synergies between communities. Protein metabolism pathways were enriched in autochthonous bacteria, whereas allochthonous microorganisms contributed mainly to bile acid and carbohydrate metabolism. Overall, resident and transient bacteria constitute distinct communities in the gut of gilthead sea bream, with numerous genera present in both but most being differentially represented and interconnected.