Introduction
Meagre is one of the most promising species for the diversification of European aquaculture. However, nutritional requirements for early larval stages are not sufficiently known. Due to their fast growth rate, high demand for essential fatty acids (EFA) is expected 1. Among them, Docosahexaenoic acid (DHA) is crucial for marine fish larval growth and development. Dietary deficiencies have been associated with increasing skeletal malformations, whereas high DHA levels have produced controversial results. The oxidative potential of elevated DHA levels has been related to deleterious results when inadequate amounts of antioxidants are included. 2. To study optimal levels of DHA supplementation and the most optimal level of antioxidant, meagre larvae were fed diets presenting increasing levels of DHA. In addition, the impact of the antioxidant level was examined by employing a diet with a deficient level of vitamin E. Furthermore, a commercial diet, presenting the lowest level of DHA was used as Control to compare larval culture success. The results of this comparative study including assessment of growth, skeletal development, and presence of skeletal deformities will be discussed together with the analysis of antioxidants enzymes activity. These insights will contribute to our understanding of the biochemical mechanisms employed by DHA in controlling skeletal metabolism, offering important knowledge to improve dietary formulations.
Material and methods
Fish larvae of A. regius from the same spawn were distributed by 9 rearing tanks of 300 L from 0 DAH to 42 DAH covering the period from the beginning of skeletogenesis, until the end of skeletal ossification. The larvae were fed with three different experimental diets (live preys and inert micro-diets) which presented increasing levels of DHA: CONTROL, MD with 2.6, and HD with 3.6 (% DW) with the adequate level of antioxidant for each level of DHA respectively. Furthermore, a fourth diet (MD-VE) with 2.6 of DHA (% DW) but a deficient level of antioxidant Vitamin E. All the emulsions and diets were produced by SPAROS, including the diet employed as a control for culture performance named WINFAST. Fish larvae were periodically sampled (at 12, 22, 32, and 42 DAH), photographed to assess larval growth, and stained with Alcian Blue and Alizarin S to assess the coefficient of mineralization and frequency of anomalies. Culture performance was further characterized by calculating daily growth (DGI, %), and specific growth rate (SGR, %). Besides, at 14, 28, and 42 DAH larvae were sampled for biochemical analysis including antioxidants enzymes activity (CAT, SOD, and GPX) and lipid composition of larval tissues. All the remaining larvae at the end of the trial were counted to determine the survival rate.
Results and Discussion
Fish larvae promptly accepted the experimental diets supplemented with ω3 fatty acids and survival was not affected by dietary treatment. However, growth performance in larval cultures seemed to improve by the supplementation with n-3 PUFAs, being the larvae fed with the HD diet exhibited the highest growth at the end of the trial. However, no linearity was observed between growth and DHA dietary level since meagre larvae fed MD diet with the intermedium level of n-3 supplementation exhibited lower growth at 42 DAH than the CONTROL. Similar results were also observed regarding DW. However, no significant differences were observed among treatments regarding SGR, and only HD presented a significantly higher DGI than the rest of the treatments. When growth performance was compared among the two intermedium diets with different content of Vitamin E, no differences were observed in TL whereas the lower level of Vitamin E presented in diet MD-VE improved DW significantly at the end of the trial.
Regarding the progress of ossification, the results reflected a similar tendency to growth, where the diet with the highest content of ω3 fatty acids presented the fastest results. However, contrary to growth the MD presented a faster rate of mineralization than the CONTROL diet, denoting that the improvement in the ossification was linearly correlated to the n-3 DHA dietary content. Moreover, no differences were observed between the diet MD and MD-VE denoting that Vitamin E levels didn’t affect mineralization progress. However, regarding the activity of the antioxidant enzymes in larval tissues, some differences in GPX total and GPX Se-Dependent were observed when treatments were compared regarding both conditions, the level of DHA in diet and the level of Vitamin E.
Despite no differences were observed a 42 DAH regarding the frequency of malformed larvae, some differences were observed during the trial regarding the type of malformities and the frequency of anomalies by body region. Therefore, we can conclude that the supplementation of high levels of DHA n-3 fatty acids improved growth and accelerated larval skeletal development in this specie without increase significantly the percentage of malformed individuals. However, the different dietary levels of DHA and the level of Vitamin E in the diet affected differently the type, the region, or the severity of malformations during the trial.
References
1. El Kertaoui, N. et al. The importance of dietary HUFA for meagre larvae (Argyrosomus regius; Asso, 1801) and its relation with antioxidant vitamins E and C. Aquac. Res. 48, 419–433 (2017).
2. Izquierdo, M. S. et al. Effects of dietary DHA and α-tocopherol on bone development, early mineralisation and oxidative stress in Sparus aurata (Linnaeus, 1758) larvae. Br. J. Nutr. 109, 1796–1805 (2013).
Acknowlegments
This work is part of a project that has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 766347.