In recent years microalgal biomass has become one of the most promising sources of bioactive compounds in aquafeeds, especially in terms of lipids and fatty acids (Soto-Sánchez et al. 2023) . Among the several species Microchloropsis gaditana , Phaeodactylum tricornutum , Schizochytrium sp. and Ti sochrysis lutea are of great interest to aquaculture with the first two well known as source of EPA and the last rich in DHA. That being the case, a dietary combination of these four species could potentially substitute fish oil in fish diets satisfying the essential fatty acids requirements . Ergo, the aim of this study was to evaluate the fish oil substitution by different blends of these four microalgal species i n the diet of Gilthead seabream (Sparus aurata).
Materials and Methods
Juvenile seabreams of 8.77 ±0.01 g initial mean weight were obtained from a commercial fish hatchery and distributed after an acclimatization period of 15 days in triplicate to 18 closed seawater circulation system tanks (125 L) (27 individuals/tank, 3 reps/dietary group). The groups were fed six different isoenergetic (21 MJ/Kg), isonitrogenous (48% CP) and isolipidic (15.5 %) diets that satisfied the EPA+DHA requirements of the species (>1.8% of diet) . The control diet (C) contained 8% fish oil, 4% soybean oil and 25% fishmeal resembling a commercially available seabream diet . Four other diets were formulated replacing 50% of the dietary fish oil of the control diet by a blend of microalgae biomasses of the species : Schizochytrium sp. and M. gaditana (SM), Schizochytrium sp. and P. tricornutum (SP), P. tricornutum and T. lutea ( PT) and M. gaditana and T. lutea (M T). A sixth diet was also used as a reference containing 12% of fish oil as the sole dietary oil (FO) . The inclusion level of each microalgae contributed a certain amount of proteins in the diet and as such fishmeal protein was also subsequently substituted. Fish were hand-fed to apparent satiation twice a day for 11 weeks.
Results and Discussion
The SM, SP and MT groups of fish had similar final body weight, weight gain, SGR and FCR with both control groups (C, FO) (Table 1) indicating that dietary fish oil can be partially replaced by the blends of M. gaditana and Schizochytrium sp., Schizochytrium sp. and P. tricornutum , M. gaditana and T . lutea without impairing the growth of S. aurata nor feed efficiency. A growth retardation occurring in the PT-fed fish was due to their lower feed intake, which denotes a lower acceptability of either P. tricornutum or T . lutea by S. aurata . A lower feed intake was also obvious in the SP group but not in the MT group, implying that the inclusion of P. tricornutum was probably the responsible factor for this decrease.
Up to date, the strategy of mixing different microalgae species to balance dietary fatty acids and to replace dietary fish oil has been scarcely studied. The blend of Microchloropsis sp. and Schizochytrium sp. has been previously proven successful as a fish oil replacement for S. aurata (Karapanagiotidis et al. 2022) as well as for other species (Qiao et al. 2014; Seong et al., 2021. , Sarker et al., 2020a). A blend of Tisochrysis lutea with Tetraselmis suecica successfully replaced 36% of dietary fish oil in Dicentrarchus labrax without adversely affecting fish growth performance (Cardinaletti et al. 2018). Sarker et al. (2020) using different combinations of Microchloropsis sp., Isochrysis sp., and Schizochytrium sp. in the diet of Oncorhynchus mykiss reported that Schizochytrium sp. and Isochrysis sp. are good candidates for DHA supplementation and that the latter is better than Nannochloropsis sp. as a substitute for fish oil.
The present study showed that blends of specific microalgae species is a promising strategy for further fish oil replacement in the diet of S. aurata, that in turn can promote a more sustainable aquaculture production. Certainly, the effectiveness of such dietary manipulation for increasing the n-3 fatty acids in fish tissues should be further investigated.
This research has been co-financed by the European Union and Greek national funds through the Operational Programme Competitiveness, Entrepreneurship and Innovation- EPAnEK 2014-2020 ( project code: MIS 5045804).
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