Publicaciones técnicas de European Natural Additives

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Torrecillas, S.1*, Martin, M.2, Astorga, F.2, Román, L. 1 and Montero, D.1

1Grupo de Investigación en Acuicultura (GIA), IU-ECOAQUA, Universidad de Las Palmas de Gran Canaria, Crta. Taliarte s/n, 35214 Telde, Spain; 2 European Natural Additives (ENA), Julián Camarillo, 10, Oficina 317, 28037 Madrid, Spain.
*Presenter (Esta dirección de correo electrónico está siendo protegida contra los robots de spam. Necesita tener JavaScript habilitado para poder verlo.).


Physical stressors associated to intensive fish production (injuries, manipulations and handling) are low but continuous. Those processes usually derive in a chronic stress situation involving suppressive effects on the fish immune system, disease resistance and energetic metabolism as result of the allostatic load imposed to the organism (Tort, 2011). These adverse effects have been mainly associated with increased secretion of cortisol by interrenal cells via activation of the HPI axis (Vijayanet al., 2005).

Glucocorticoids have a potent immunosuppressive effect on vertebrates immune system cells via: induction of apoptosis, change of differentiation patterns and inhibition of cytokine release and leucocyte migration (Baschant and Tuckermann, 2010; Tort, 2011). In this regard, plant-derived natural compounds may potentially be beneficial in fish production as preventive agents or/and relieving the consequences of intensive fish rearing. Terpenoids are plant organic acids with antioxidant, anxiolytic and sedative properties (Nam et al., 2013) and flavonoids are plant polyphenolic compounds with strongantioxidant and anti-inflammatory activities(Tripoli et al., 2007), which for example may help fish to reduce chronic stress-side effects by reducing circulating plasma glucocorticoid concentration and/or to recover after physical injuries derived from handling and manipulation practices.

Thus, the objective of this study was to evaluate the effect of a dietary supplementation of a mixture of plant terpenoids and flavonoids in European sea bass (Dicentrarchus labrax) stress resistance by a series of experimental and commercial trials.

Materials and methods

For the whole study, a mixture of vegetal flavonoids and terpenoids standardized by high-performance liquid chromatography (HPLC) (ENARECOX2®) at 100 ppm was included in European sea bass commercial diets. The study was carried out by a series of experiments finalizing with a commercial scale trial.

For the first experiment, European sea bass juveniles (average weight 30 g) were randomly distributed in 12 indoor cylindroconical 500 L fiberglass tanks (initial stocking density of 3.1 kg·m-3; 6 tanks/diet) and fed control or ENARECOX® diets (45% Protein, 19% Lipid, 10 % Ash, 2% Fiber) for 7 days. Afterwards, fish were subjected to a confinement stressor by increasing the stocking density by x10 times (30 kg·m-3). Samples of plasma were extracted at 0h, 2h (acute response) and 3 days (adaptive response) post stress for cortisol analyses. Mortality was recorded daily.

For the second experiment, two cages (1 control and 1 ENARECOX2®) of adult European sea bass (average weight of 500 g; stocking density of 10 kg·m-3) were fed control or ENARECOX® diets (43% Protein, 18% Lipid, 9 % Ash, 2% Fiber) for 7 days before routine net change. Samples of scales of dead fish trapped in the nets were recollected for chronic cortisol measurements. A third experiment based on the results obtained in Experiments I and II was designed at commercial scale. For that purpose, 3 cages (1 control and 2 ENARECOX2®) of adult European sea bass (average weight of 500 g) were fed control or ENARECOX® diets (43% Protein, 18% Lipid, 9 % Ash, 2% Fiber) for 15 days before harvesting. Samples of plasma (1h after starting to harvest) and scales were recollected to analyze plasmatic and chronic cortisol levels.


In experiment I, fish fed ENARECOX2® diet presented lower (P<0.05) basal cortisol levels (0h) than fish fed control diet after 7 days of feeding (Figure 1; n=6 tanks/diet). However, no differences (P>0.05) were found for plasma cortisol levels along the stress panel between fish fed control and ENARECOX2® diet, presenting all fish evaluated the typical pattern of response to acute stress by confinement (2h) and recovering basal levels after 3 days of challenge. European sea bass fed ENARECOX2® diet presented a reduced (P<0.05) cumulative mortality percentage compared to fish fed control diet at the end of the stress challenge (Figure 2; n=3 tanks/diet). In experiment II, scales of fish fed ENARECOX2® diet presented lower accumulation of cortisol than fish fed control diet (7.70±4.40 and 1.00±0.38 μg/kg respectively). In experiment III, no differences in plasmatic cortisol (P>0.05) between adult European sea bass fed control and ENARECOX2® diet were found, presenting all fish evaluated a similar pattern of response to stress by manipulation (commercial harvesting) in agreement with the results found in Experiment I. Besides, scales chronic cortisol measurements were also correlated with the results obtained in Experiment II.

Cortisol basal levels for European sea bass (Dicentrarchus labrax) fed the different dietary treatments

Fig 1. Cortisol basal levels for European sea bass (Dicentrarchus labrax) fed the different dietary treatments. Data presented in mean±SD. P < 0.05; n=6 tanks/diet.

Cumulative mortality percentages for European sea bass (Dicentrarchus labrax) fed the different dietary treatments

Fig 2. Cumulative mortality percentages for European sea bass (Dicentrarchus labrax) fed the different dietary treatments. P < 0.05, K-M.


• Tort, Ll., 2011. Stress and immune modulation in fish. Developmental and Comparative Immunology, 35: 1366-75.
• Vijayan, M.M., Prunet, P., Boone, A.N. 2005. Xenobiotic impact of corticosteroid signaling. 6:365-394. In: Biochemical and Molecular Biology of Fishes Environmental Toxicology Moon, T.W., Mommsen, T.P. (Editors). Elsevier B.V.
• Baschant, U., Tuckermann, J., 2010. The role of the glucocorticoid receptor in inflammation and immunity. Journal Steroid Biochemistry Molecular Biology, 120:69-75.
• Nam, S.M., Choi, J.H., Yoo, D.Y., Kim, W., Young, H., et al. 2013. Valeriana officinalis extract and its main component, valerenic acid, ameliorate D-galactose-induced reductions in memory, cell proliferation, and neuroblast differentiation by reducing corticosterone levels and lipid peroxidation Experimental Gerontology, 48:1369-77.
• Tripoli, E., Guardia, M.L., Giammanco, S., Majo, D.D., Giammanco M. 2007. Citrus flavonoids: molecular structure, biological activity and nutritional properties: a review. Food Chemistry 104:466-79.

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