Aquaculture is one of the fastest growing food sectors worldwide, driven by the increasing demand for healthy and nutritious aquatic products (FAO, 2022; Verdegem et al., 2023). Despite significant investments in technological advances aimed at optimizing the production and value of farmed seafood, nowadays, aquaculture expansion continues to be defied by several environmental threats that hamper farmed animals’ welfare, growth performance, quality, and survival. Among these threats, disease outbreaks are a primary concern for the sector, causing substantial animal and economical losses (Cain, 2022). In many instances, disease outbreaks in aquaculture facilities are not the result of an isolated factor (exposure to a specific pathogen), but rather a combination of multiple variables, such as the occurrence of co-infections (viral, bacterial, parasitic or fungal infections), exposure to abiotic conditions outside the species’ optimal range, and inappropriate nutrition, leading to a compromised immune system and increased formation of free radicals responsible for cell oxidation and damage (Yarahmadi et al., 2016; FAO, 2018; Cain, 2022). This complex interplay between the various stressors at stake and their effects emphasizes the urgent need to develop strategies to improve the welfare and resilience of aquaculture animals to environmental stressors.

Finding eco-friendly and cost-efficient ways of enhancing farmed animals’ immune system and antioxidant responses has now become more important than ever, as disease outbreaks are expected to become more frequent and severe in the future due to the foreseen changes of the climate, resulting in even greater losses for the aquaculture sector (FAO, 2022). It is widely acknowledged that the overall performance and disease resistance of fish are highly reliant on proper nutrition (Oliva-Teles, 2012). In this sense, an increasingly popular and promising strategy that is gaining attention in the aquaculture sector involves the use of functional feeds enriched with natural immunostimulant ingredients. The purpose of these feeds is not only covering the specific nutritional requirements of farmed fish species, but also to promote animal well-being and optimal functionality by enhancing their immune status (Passos et al., 2021). Seaweeds play a crucial role in this approach, as they are natural products with great potential as aquafeed ingredients, being rich in nutrients and biological active compounds, specifically polysaccharides (e.g., alginates, carrageenans, agar, fucoidan, β-glucan and mannitol), polyphenols (e.g., phenolic acids, phlorotannins, flavonoids, and halogenated derivatives) and carotenoids (e.g., fucoxanthin and β-carotene) (Holdt and Kraan, 2011; Costa et al., 2021). These bioactive compounds have demonstrated anti-inflammatory, immunomodulatory, antioxidant and antibacterial properties, making seaweeds valuable allies against fish pathogens and promoting overall fish health (Morais et al., 2020; Silva et al., 2020; Cai et al., 2021). Previous studies have reported that incorporating a wide range of seaweed species into aquafeeds yields promising health and immune outcomes for different aquatic species (Peixoto et al., 2019; Shi et al., 2019; Passos et al., 2021; Thépot et al., 2021; Thépot et al., 2022). However, while a fair amount of information on red and green macroalgae species’ nutritive and functional potential is already available, literature on brown macroalgae is still scarce. Laminaria digitata, a brown seaweed belonging to the Laminaria genus (Laminariaceae, Ochrophyta) is a native Irish species that is globally harvested and cultured (Purcell-Meyerink et al., 2021). The use of this species has had diverse applications in the food, pharmaceutical, bioenergy and animal sectors (Purcell-Meyerink et al., 2021), and recent studies have investigated its potential as a feed ingredient for several terrestrial farmed animal species, including chickens (Costa et al., 2022), pigs (Ribeiro et al., 2022; Ribeiro et al., 2023) and ruminants (Belanche et al., 2016; De La Moneda et al., 2019). Within the aquaculture context, only a very limited number of studies had demonstrated the positive impact of L. digitata in marine aquafeeds (Ribeiro et al., 2015; Ferreira et al., 2022b), specifically regarding its bioactive properties and their effects on immune parameters (Cipriano-Maack et al., 2016; Palstra et al., 2018). Hence, more research is needed to fully understand the potential of L. digitata as functional feed ingredient and validate its use as an efficient non-chemically based approach to mitigate disease outbreaks in aquaculture. To this purpose, defining the effective doses of seaweeds’ inclusion in aquafeeds (i.e., the dose that can trigger an immune and/or overall health improvement, while maintaining optimal feed digestibility and animal growth) is a crucial and rather challenging step, especially in the rearing of carnivorous marine species (e.g., gilthead seabream, Sparus aurata) that have particular nutritional requirements (FAO, 2023).

The main objective of the present study was to assess the functional potential of L. digitata (at different inclusion levels) as an effective approach to improve the immune (antiprotease activity, peroxidase activity and immunoglobulin M), antioxidant responses (lipid peroxidation, catalase activity, glutathione S-transferase activity, superoxide dismutase activity and total antioxidant capacity) and overall physiological well-being of farmed marine fish (Sparus aurata). The results herein presented are expected to highlight the potential of L. digitata as a promising functional feed ingredient whose use may contribute to the implementation of smarter, tailor-made and more environmentally-friendly aquaculture practices.

The incorporation of novel and sustainably produced ingredients in aquafeed formulations has become one of the focal points in modern aquaculture research (FAO, 2022). Among these promising alternatives, macroalgae have emerged as noteworthy candidates, acquiring significant attention as potential feed ingredients to improve fish health due to their anti-inflammatory, antioxidant, and antimicrobial properties (Morais et al., 2020; Silva et al., 2020; Cai et al., 2021).

In the present study, we explored the effects of incorporating a brown macroalga, Laminaria digitata, as a functional ingredient in aquafeeds to bolster immunity and antioxidant responses in juvenile Sparus aurata. The results herein presented suggest that, overall, the inclusion of this seaweed in aquafeeds yields a positive effect on fish health.

Animal condition indexes serve as key indicators of the overall welfare, which is shaped by a complex interaction of factors such as feeding conditions, parasitic infections, physiological aspects, as well as physical and biological environment (Le Cren, 1951). Animals that remain in good condition are more likely to possess a robust and effective immune system, capable of defending themselves against pathogens and maintaining overall well-being (Zaccone, 2009). In our study, as the factor under analysis was the type of diet, we can state that the inclusion of L. digitata in the aquafeed formulation at the tested levels (1.5%, 3%, and 6%) did not compromise fish welfare. In other words, despite the differences observed throughout time, which are most likely related with the expected fish growth, animal condition (assessed through K and SSI indexes) remained consistently stable across all treatments. The maintenance of a consistent and appropriate (i.e., high K, WGR and SGR values) growth across all treatments suggests that the dietary inclusion of L. digitata did not hinder nutrient utilization or led to any adverse effects on the S. aurata’s metabolism, indicating that the seaweed was efficiently digested and its nutrients available to the fish. The fact that the FCR was consistently lower during the first 30 days of feeding trial shows that the fish were able to grow with a relatively lower amount of feed during this period, indicating a higher efficiency in converting feed into growth. Furthermore, this was also reflected in higher SGR values. The results of this study align with previous research that has demonstrated the potential of several species of brown macroalgae as a valuable eco-innovative aquafeed ingredient to enhance fish health and growth (Pham et al., 2006; Ribeiro et al., 2015; Kamunde et al., 2019; Shi et al., 2019; Negm et al., 2021; Maghawri et al., 2023).

The haematological profile evaluation of S. aurata indicated that the experimental feeds supplemented with L. digitata did not affect the type and number of blood cells produced by juvenile fish. In addition, the absence of significant alterations in the number of viable erythrocytes and/or erythrocyte nuclear abnormalities (ENAs) indicates that aquafeeds biofortified with L. digitata up to 6% did not have cytotoxic and/or genotoxic potential, a result also aligned with the high K values observed in all treatments. The consistent leucocyte percentages below 4.0% of the total cell counts across all treatments indicate that there were no signs of inflammation or acute immune responses in the fish. Leucocytes play a crucial role in the fish’s immune response. They are responsible for identifying and fighting off pathogens, such as bacteria and viruses, and play a role in inflammatory responses (Ellis, 1977; Fánge, 1992). An increase in leucocyte percentages could be an indication of an active immune response, usually in response to an infection, inflammation, or a stressful condition (Seibel et al., 2021). In the context of the present study, the low leucocyte percentages suggest that the fish’s immune system remained stable and indicate the absence of exposure to infectious agents or other stressors that could trigger an immune response. Furthermore, the high erythrocyte percentages observed in all treatments, accounting for over 96.0% of total cells, strongly suggest that the fish had a stable and efficient oxygen-carrying capacity, which is essential for overall metabolic activities (Nikinmaa et al., 2019; Esmaeili, 2021). Additionally, the finding that more than 96.1% of erythrocytes were viable, with a minimal percentage of nuclear abnormalities, indicates that the fish were in good haematological health and well-adapted to the experimental conditions (Witeska et al., 2022). The findings from our study are consistent with those of Passos et al. (2021), who explored the effects of diets supplemented with Gracilaria gracilis in Sparus aurata and found no significant differences in erythrocyte and leukocyte counts between the supplemented feeds and the control treatment. However, other studies have shown contrasting results, highlighting the significant impact of seaweed supplementation in aquafeeds on the haematological profile of fish. For example, Maghawri et al. (2023) incorporated brown algae Padina pavonica as a dietary supplement in Oreochromis niloticus, while Quezada-Rodríguez and Fajer-Ávila (2016) utilized extracts from the green alga Ulva clathrata for the same fish species. Additionally, Sattanathan et al. (2020) investigated the effects of dietary inclusion of Chaetomorpha aerea (Chlorophyta) in Labeo rohita against post-challenges of Aeromonas hydrophila, and all these three studies observed an increase in the percentage of erythrocytes and leucocytes compared to the control. Their findings suggest a protective response during times of stress, as dietary changes may trigger physiological responses, activating the immune system as a precautionary measure to maintain homeostasis and defend against potential stressors or pathogenic challenges associated with the new feeds. However, given the complexity of dietary interactions and the variability in outcomes observed across different fish species, types of seaweed, and experimental conditions, further research is needed to fully elucidate the underlying mechanisms responsible for these effects.

The analysis of immune humoral parameters (IgM, AP, and POD) among the different dietary treatments provided valuable insights into the potential immunomodulatory effects of L. digitata in S. aurata juveniles. Immunoglobulin M (IgM) is an essential antibody involved in both adaptive and innate immune responses, which recognises and neutralises invading pathogens, thereby enhancing fish ability to combat infections (Mashoof and Criscitiello, 2016). Additionally, POD are enzymes with an important microbicidal role, effectively utilizing hydrogen peroxide (H₂O₂) as a co-substrate to generate highly reactive species. These reactive species play a vital role in phagocytic activities, as they are responsible for damaging and killing pathogens and microbial invaders (Koeth et al., 2013; Guardiola et al., 2014). Meanwhile, AP plays a crucial role in neutralizing pathogen proteases by utilizing various protease inhibitors such as α1-antitrypsin (AAT) and α2-macroglobulin (α2M), which possess anti-enzyme activity. By controlling the activity of proteases, AP helps to maintain tissue integrity, preventing excessive tissue damage, and ensuring effective immune processes without harming surrounding tissues (Priyadarshini et al., 2017). Together, these three immune components contribute significantly to the fish’s defence against infections, promoting overall health and well-being.

In our study, a remarkable increase in IgM content and POD activity was observed in the fish that consumed the feed containing 1.5% L. digitata (1.5-LD). These results suggest that the inclusion of 1.5% L. digitata in the feed positively influenced the fish’s immune responses, enhancing their ability to combat pathogens and strengthening their immune defence mechanisms. Our findings are in line with previous studies that have established immunomodulatory roles of various macroalgae components, their impact on immunity parameters, and their contribution to maintaining overall health in different species (Peixoto et al., 2016; Hoseinifar et al., 2018; Shi et al., 2019; Negm et al., 2021). The inclusion of 3% L. digitata in aquafeeds only showed a significant increase in POD activity compared to the control, although with lower values than the 1.5-LD treatment. This suggests that a 3% inclusion of L. digitata also potentially contributed to an improved defence against pathogens, but never exceeding the effect observed with the 1.5-LD treatment. Conversely, the absence of significant differences in immune parameters (IgM, AP and POD) between the fish fed with 6% of L. digitata and those fed with a non-biofortified aquafeed (CTR treatment) indicates that macroalga inclusion levels above 3% do not lead to immunomodulatory improvements in juvenile S. aurata. These specific results contrast with those of Ferreira et al. (2022a), who observed a more pronounced immune response, in a dose-dependent manner, when using a blend of microalgae (Nannochloropsis oceanica - Eustigmatophyceae and Chlorella vulgaris - Chlorophyta) and macroalgae (Gracilaria gracilis - Rhodophyta and Ulva rigida - Chlorophyta) in European seabass (Dicentrarchus labrax). Some potential explanations for our observation include:

Regarding AP activity, overall, the inclusion of L. digitata in the feeds did not have a significant impact on the regulation of proteases activity in juvenile fish. Hence, the results obtained in this specific biomarker contrast somewhat the trends observed in the other studied immunity parameters and, thus, do not clearly point out to a lower or higher susceptibility to disease in fish biofortified with L. digitata. Yet, considering that the main role of these enzymes is to neutralize proteases produced by bacteria, viruses, fungi and protozoans, future studies focused on exploring the immunostimulatory potential of L. digitata should, perhaps, include pathogen challenge tests in the experimental design, as such an approach would likely yield more evident responses.

Another notable finding is that, between T30 and T60, immune parameters were stable in CTR, 3-LD, and 6-LD treatments, but a decrease in IgM content and POD activity was observed in the 1.5-LD treatment. This significant decrease is an intriguing finding that warrants further investigation, since several possible explanations exist for this pattern. An adaptive response of the S. aurata immune system to continuous feeding of diets containing L. digitata is one possibility. Initially, the inclusion of macroalga may have stimulated a more robust immune response; however, over time, the immune system may have adapted to the constant presence of the dietary component, leading to a reduction in IgM content and POD activity. Another potential influencing factor could have been changes in diet composition during the experimental period, as it is well-established that some ingredients, including bioactive compounds and nutrients, can undergo degradation or loss over time (Filipe et al., 2023). Therefore, it is premature to conclude that 30 days is inherently better than 60 days based only on this finding. To draw robust conclusions, further investigations are needed to explore the underlying mechanisms and the long-term effects of L. digitata inclusion on the immune response of juvenile S. aurata.

The interaction between the immune response and its antioxidant defence mechanisms is crucial for maintaining a healthy and functional immune system in fish. Spleen is the major secondary lymphoid organ in fish, thus, hosting a wide range of immunological (including the filtration of blood-borne pathogens and antigens), hematopoietic and red blood cell clearance mechanisms (Lewis et al., 2019; Mokhtar et al., 2023). Additionally, oxidative stress biomarkers are important indicators of physiological status and overall condition at the organ and whole-body levels.

In the present study, the activity of three antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST), along with two essential antioxidant parameters, total antioxidant capacity (T-AOC) and lipid peroxidation (LPO), were assessed in fish spleen. These antioxidant enzymes play a major role in antioxidant mechanisms, protecting the cells from oxidative damage: SOD is the first line of defence against reactive oxygen species (ROS), catalysing the conversion of superoxide radicals (O₂ ^-) into hydrogen peroxide (H₂O₂) and oxygen (O₂) (Lesser, 2006); then, CAT is responsible to neutralize hydrogen peroxide (H₂O₂) into less harmful compounds, namely water (H₂O) and oxygen (O₂) (Lesser, 2006); and GST catalyse the conjugation of glutathione (GSH) with several toxic compounds, including ROS that escape from the first line of defence (Domingues et al., 2010). This mechanism makes the conjugation products less toxic and more water-soluble, facilitating their excretion. LPO and T-AOC are also essential indicators of antioxidant responses, since LPO represents a complex chain reaction causing cellular damage through oxidative degradation of lipids (Gutteridge and Halliwell, 1990), while T-AOC reflects the organism’s overall ability to combat oxidative stress and neutralize ROS (Silvestrini et al., 2023).

In our study, the feed with 1.5% inclusion of L. digitata (1.5-LD) also emerged as the most effective in promoting antioxidant responses in juvenile S. aurata. The increase in CAT and GST activities, as well as T-AOC, alongside the corresponding decrease in LPO levels observed in 1.5-LD treatment indicates that L. digitata had a considerable impact on fish antioxidant defence mechanisms. Our results are consistent with previous studies reporting that seaweed supplementation modulates antioxidant status and oxidative stress in fish (Kamunde et al., 2019; Shi et al., 2019; Negm et al., 2021). The abundant presence of bioactive compounds in L. digitata provides a plausible explanation for the increased activity of antioxidant enzymes, since it is already known that this macroalga is rich in polyphenols (phenolic acids, phlorotannins, flavonoids, and halogenated derivatives), carotenoids (e.g., fucoxanthin and β-carotene) and polysaccharides (e.g., alginates, fucoidan, laminarin and mannitol) (Holdt and Kraan, 2011; Schiener et al., 2014; Bonfim-Mendonca et al., 2017; Vissers et al., 2017; Fernando et al., 2021). These compounds have demonstrated their remarkable ability to neutralize free radicals and reduce oxidative stress within cells, thus displaying antioxidant and anti-inflammatory properties. When incorporated into the fish diet, these antioxidants may have increased the activity of CAT and GST enzymes and raised the organism’s total antioxidant capacity (T-AOC). Consequently, this increased cellular protection against oxidative damage led to a decrease in LPO levels.

The inclusion of 3% and 6% of L. digitata also increased CAT activity (both percentages of inclusion), GST activity and T-AOC (only 6%). However, the magnitude of the increase in these biomarker activities was consistently lower for the higher inclusion levels of L. digitata. This pattern aligns with the findings observed in the immune responses (IgM and POD), indicating that increasing the macroalga concentration in the feeds does not add any benefit from the immune, antioxidant and overall animal condition points of view.

SOD activity showed no significant differences between the experimental feeds and the CTR treatment, meaning that the inclusion of L. digitata in the feeds did not have a substantial impact on this enzyme. This observation highlights the complexity of the fish’s antioxidant response, which involves multiple enzymes and pathways working in harmony to maintain redox balance and protect against oxidative stress.

In line with the pattern observed in IgM content and POD activity (immune responses), CAT activity and T-AOC showed a decrease between 30 and 60 days with the 1.5-LD feed. This decrease can be explained, once again, by the adaptation of the organism to the functional feed, as the fish adjusted to the new diet, leading to reduced antioxidant response needs. Alternatively, the effects of L. digitata bioactive compounds on CAT activity and T-AOC might be more pronounced at fish early life stages, decreasing over time due to metabolism or other regulatory mechanisms. This decrease may indicate that the fish’s redox balance has been achieved and maintained at a healthy level after the initial adaptation phase, suggesting that the antioxidants provided by the 1.5% L. digitata feed are sufficient to preserve redox homeostasis and protect cells against oxidative damage. Additionally, genetic regulation can also play a role in stabilizing antioxidant enzyme activity over time, ensuring overall antioxidant response homeostasis and adaptability to dietary changes for maintaining cellular health (Martin and Król, 2017; Mierziak et al., 2021).

The immune and antioxidant responses in organisms are highly complex and influenced by a wide array of factors. These factors include various aspects, such as diet composition, prevailing environmental conditions, exposure to pathogens, physiological changes, among others. Although this study focused specifically on S. aurata under optimal experimental conditions, it is essential to recognize that different fish species or varying environmental factors could yield different responses to the inclusion of L. digitata in aquafeeds.

The valuable findings from this study constitute a substantial contribution to the scientific understanding of fish health and underscore the potential utilization of L. digitata as a functional alternative feed ingredient in aquaculture. Further research on the interactions between dietary components, fish physiology, and immune-antioxidant responses will undoubtedly open new windows of opportunity in fish nutrition, contributing to improve animal welfare, quality and resilience to environmental stressors (e.g. pathogens, pollutants, climate change) that may challenge the survival of farmed animals and the economic viability of the sector.

This study highlights the promising potential of Laminaria digitata as a functional and eco-innovative aquafeed ingredient to improve immunity and antioxidant responses in juvenile Sparus aurata.

The results indicate that a dietary inclusion of 1.5% L. digitata is the most cost-effective inclusion percentage, significantly improving the antioxidant responses of S. aurata, through increased levels of CAT, GST and T-AOC, as well as reduced LPO levels. Additionally, the inclusion of 1.5% L. digitata showed to enhance immunity by inducing IgM synthesis and POD activity, while maintaining the growth performance and condition indexes of the fish.

Although aquafeeds biofortified with higher incorporation percentages of L. digitata (3% and 6%) showed no cytotoxic and/or genotoxic potential, the lack of consistent and clear beneficial effects in fish fed with these doses of macroalga suggests that future studies should prioritise inclusion percentages below 3%. Indeed, in this study it seemed evident that “less is more” when it comes to incorporating L. digitata in aquafeeds, as this approach appears to be more advantageous in terms of achieving optimal animal health status and considering cost-effectiveness.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The animal study was approved by IPMA’s Animal Welfare and Ethics Body (ORBEA). The study was conducted in accordance with the local legislation and institutional requirements.

IM: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. MD: Investigation, Writing – review & editing. AG: Investigation, Writing – review & editing. PP-F: Resources, Writing – review & editing. MD: Supervision, Writing – review & editing. AM: Resources, Supervision, Writing – review & editing. ALM: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing.