Introduction
In intensive aquaculture systems, routine practices including handling, air exposure, transport, and overcrowding impose recurrent physiological challenges that elicit stress responses in fish. These acute challenges can compromise fish health and welfare, predisposing individuals to disease and ultimately result in mortality and economic losses. Concurrently, the reduction of fishmeal (FM) in aquafeeds, driven by sustainability pressures, is altering the dietary supply of essential nutrients, particularly for carnivorous species (e.g., European seabass) and potentially limiting their metabolic capacity to cope with stressors. Animal by-products protein hydrolysates (PH) are emerging as protein-rich ingredient with functional properties, largely attributed to bioactive peptides with antioxidant activity and metabolic regulatory potential. This study aimed to investigate the potential of dietary PH inclusion to antioxidant capacity and metabolic responses in European seabass subjected to an acute stress challenge.
Materials and Methods
Juvenile European seabass were reared in triplicates tanks and fed for 89 days with four plant-based diets: CTRL diet (12.5% fishmeal, FM) and three other diets (SHARK, FISH and SWINE), each including 3% PH derived from blue shark (Prionace glauca) skin, fish by-products and swine processed animal protein, respectively, at the expense of FM. At the end of the trial, five fish per tank were immediately sampled, while another five fish were subjected to an acute stress protocol consisting of overcrowding (100 kg m-3 for 5 min) followed by air exposure (1 min), and subsequently sampled 1 h post-stress. Plasma and liver were collected to evaluate metabolic and oxidative stress responses.
Results
The diets equally promoted a normal fish growth and ensured high feed efficiency (Rodrigues-dos-Santos et al., 2025). The stress challenge elicited a clear systemic response, characterized by increased plasma cortisol, glucose and lactate levels, along with a reduction in plasma protein. Diet-specific effects were also observed, with the SWINE diet increasing plasma cholesterol levels, whereas the FISH diet resulted in higher plasma lactate concentrations, independently of the stress condition.
Stress also significantly modulated hepatic metabolic activity leading to an increase in the activities of pyruvate kinase (PK) and malic enzyme (ME) under stress conditions, independently of dietary treatment. Fish fed the SWINE diet exhibited even higher PK and ME activities compared to the CTRL group, despite the stress conditions.
In parallel with systemic changes, stress promoted a marked shift in hepatic energy metabolism. In stressed fish, hepatic glucose levels increased in the CTRL group but decreased in the SHARK group, compared to baseline. Moreover, the stress challenge significantly reduced hepatic glycogen and increased hepatic lactate levels only in the CTRL and FISH groups.
Regarding antioxidant responses, under basal conditions, GST activity was higher in fish fed the SWINE diet compared to the other groups, whereas no differences were detected among treatments under stress conditions. In contrast, under basal conditions, CAT activity did not differ among dietary groups, but was significantly increased in the SHARK group under stress. These metabolic and antioxidant responses were reflected in markers of oxidative damage. Lipid peroxidation (LPO) was reduced in fish fed the SWINE diet irrespective of stress, and was also decreased in the SHARK group under stress conditions. In turn, under stress a decrease in protein carbonyl content was only detected in the SWINE group.
Conclusion
Dietary protein hydrolysates modulated the metabolic and oxidative stress responses of European seabass to acute stress. SHARK and SWINE diets promoted improved metabolic stability, as reflected by better maintenance of hepatic energy reserves and reduced reliance on anaerobic metabolism compared to CTRL and FISH. These diets also enhanced antioxidant status, as indicated by LPO, with SWINE showing additional protection against protein oxidation. Overall, the results suggest that SHARK and SWINE hydrolysates can improve fish stress resilience through coordinated regulation of energy metabolism and oxidative balance, while contributing to reduced dietary FM reliance in plant-based diets for European seabass.
Acknowledgment
This work was financed by the Blue Bioeconomy Pact (project code C644915664-00000026), under the Pep4Fish project as part of activities carried out within WP6 FEED, with support from international funding provided by the European Union. Additional financial support was provided by the Foundation for Science and Technology (UIDB/04423/2020 and UIDP/04423/2020). Luciano Rodrigues-dos-Santos also gratefully acknowledges FCT for the doctoral scholarship (2023.00947.BDANA), accessible at https://doi.org/10.54499/2023.00947.BDANA
References
Rodrigues-Dos-Santos, L., Basto, A., Monteiro, M., António, C., Rodrigues, A. M., Sá, T., Velasco, C., Martins, R., Rosa, A., Pintado, M., Almeida, A., & Valente, L. M. P. (2025). Locally-Sourced Animal Protein Hydrolysates in High-Plant-Protein Diets Can Promote European Seabass Growth and Nutrient Utilization, Reducing Reliance on Fishmeal. Aquaculture nutrition, 2025, 3415083. https://doi.org/10.1155/anu/3415083