Introduction
Functional diets play a pivotal role in aquaculture by enhancing fish health, resilience, and performance under the various stressors encountered throughout the production cycle, such as handling, transport, or high stocking densities. The integration of functional additives (e.g. prebiotics, probiotics, amino acids, and bioactive compounds from micro- and macroalgae) into aquafeeds has shown potential to mitigate oxidative stress and support immune responses. Among these additives, algae-derived compounds have gained particular attention for their richness in natural pigments, antioxidants, and polysaccharides. These ingredients may contribute to health status and help maintain gut integrity, which is essential for nutrient absorption and overall resilience. Species such as gilthead seabream (Sparus aurata), a key species in Southern European aquaculture, can particularly benefit from these dietary strategies. However, the successful implementation of functional diets requires a rigorous process of fine-tuning through controlled trials to determine optimal formulations and dosages. These trials are essential to ensure efficacy, cost-effectiveness, and compatibility with existing farming practices, while supporting the development of a range of reliable and sustainable aquafeed products for the market. As part of this effort, the Vertical Algae project aims to develop algae-derived products for a range of applications. Specifically, sub-project 5 is dedicated to aquaculture, focusing on the formulation of functional diets that incorporate algal biomass or extracts, with the goal of enhancing fish resilience through improved nutritional support.
Materials and Methods
Trial 1: A 3-week feeding trial was conducted to evaluate the effect of microalgal extracts on the resilience of juvenile gilthead seabream (± 18 g) to stress and bacterial challenge. Fish were fed one of three diets: a commercial-like premium control diet supplemented with immunostimulants (CTRL+), or the same diet further supplemented with either a low (LOW) or high (HIGH) dose of Tetraselmis chui extract. At the end of the feeding period, fish were sampled to assess physiological and molecular biomarkers related to antioxidant status, gut health, and immune function. Subsequently, fish were exposed to an acute stress challenge involving handling, crowding, and transport. Six hours after the onset of the stressor, additional samples were collected to evaluate the fish response to the challenge. Following this, fish were infected with Photobacterium damselae subsp. piscicida, and mortality was monitored for 10 days.
Trial 2: In a second trial, early juvenile seabream (± 6.5 g) were fed for 7 days. The dietary treatments included the same commercial-like premium diet used in Trial 1 (CTRL+), a control diet without immunostimulants (CTRL−), and two additional diets based on the CTRL− formulation, supplemented with a low dose of Tetraselmis chui extract combined with either Ulva extract (Low+UL) or processed Euglena biomass (Low+EU). At the end of the feeding period, fish were sampled as before. Afterwards, they were subjected to the same stress protocol, followed by infection with P. damselae subsp. piscicida. Additional samples were taken 6 hours post-infection to assess the early fish response to this challenge, and mortality was monitored over the subsequent 10 days.
Results and Discussion
Trial 1: Following the stress challenge, fish fed the LOW diet exhibited significantly higher hepatic total antioxidant status (TAS) than those fed CTRL+ or HIGH, suggesting a protective effect of the lower Tetraselmis dose against oxidative stress. Both LOW and HIGH diets appeared to buffer oxidative stress effectively, as lipid peroxidation (LPO) levels remained stable post-stress, unlike in the CTRL+ group, which showed a significant increase. Despite these physiological effects, no significant differences in mortality were observed between groups following the P. damselae subsp. piscicida challenge.
Trial 2: Following the stress and bacterial challenge, liver catalase (CAT) activity was significantly higher in fish fed the LOW+UL and LOW+EU diets compared to the CTRL− but not the CTRL+ groups, indicating enhanced antioxidant responses with algal supplementation. In the intestine, fish fed the LOW+EU diet showed upregulated expression of il1b and cldn12 compared to those fed the CTRL+ diet. The elevated expression of il1b, a key pro-inflammatory cytokine, suggests an activated local immune response, potentially modulated by the algal supplementation. Simultaneous upregulation of cldn12, a tight junction protein, may reflect a strengthening of the gut barrier, which is critical during infection. These findings point to a potential synergistic effect between Tetraselmis and Euglena in supporting intestinal immune responses and maintaining barrier integrity under stress and pathogen challenge. As in Trial 1, no differences in mortality were observed between treatment groups during the post-infection period.
Conclusion
Both trials highlight the potential and the challenges of using functional diets to improve fish resilience. While some algal extracts showed positive effects on antioxidant status and gut health, these changes did not lead to improved survival after bacterial infection. Functional diets can produce specific effects in different tissues, and results often depend on context, making it difficult to rely on single biomarkers. This reinforces the need for broad datasets and multivariate analyses to fully understand the impact of these diets. In the end, developing effective functional feeds requires a careful, data-driven approach that considers species, ingredients, and farming conditions.
Acknowledgements
This work was financially supported by “Pacto da Bioeconomia Azul” (Project No C644915664-00000026) within the WP5 Algae Vertical, funded by Next Generation EU European Fund and the Portuguese Recovery and Resilience Plan (PRR), under the scope of the incentive line “Agendas for Business and Innovation” through the funding scheme C5 – Capitalization and Business Innovation; and by FCT – Foundation for Science and Technology through projects UIDB/04326/2020 (DOI:10.54499/UIDB/04326/2020), UIDP/04326/2020 (DOI:10.54499/UIDP/04326/2020), and LA/P/0101/2020 (DOI:10.54499/LA/P/0101/2020) to CCMAR and SAUDE&AQUA II (MAR-021.1.3-FEAMPA-00018) to IPMA. The authors thank the company Necton SA for supplying the microalgae.