Clove essential oil nanoencapsulated in β-cyclodextrins improves the welfare at slaughter in different farm fish speciesparameters of stress and innate immune response

Abstract

Clove essential oil nanoencapsulated in ?-cyclodextrins improves the welfare at slaughter in different farm fish species: parameters of stress and innate immune response. Amanda Esperanza López Canovas, 2020. The main objective of this thesis was to improve the welfare of five farmed fish species (gilthead seabream, Atlantic salmon, Nile tilapia, European seabass and rainbow trout) at slaughtering time through the use of clove essential oil nanoencapsulated in ?-cyclodextrins (CEO+?-CD). The anaesthetic complex was compared with the stunning method most used in aquaculture (cooling in crushed ice and seawater). A range of CEO+?-CD concentrations (from 5 to 60 mg/kg), temperatures (from -8.0 to 29.0 °C), types of ice (liquid and crushed) and farm conditions (experimental and industrial) were tested. Also, an innovative management of gilthead under industrial farm condition, using a low dose of CEO+?-CD in farmed fish that had never been anesthetized in their productive cycle as their size had always been calculated with underwater cameras, was studied. In addition, a low dose of eugenol nanoencapsulated in ?-CD in seawater was compared with eugenol or clove oil alone. The first aim was to reduce the induction times of anaesthesia during the total loss of equilibrium and the loss of reflex activity. In all farmed fish studied, the CEO+?-CD allowed a shorter induction time of anaesthesia, using low doses of CEO under experimental and industrial farm conditions at different temperatures in sea and freshwater. The nanoencapsulation of eugenol in ?-CD also reduced the induction times of anaesthesia, compared to eugenol or CEO alone. The second aim was to determine the stress level induced by the anaesthetic complex during the stunning process. For that, the levels of glucose, lactate, cortisol, pO2, pCO2, HCO3, TCO2, SO2, base excess and pH in plasma and skin mucus were tested in all the species studied. The level of expression of RyR3 gene in the skeletal muscle of gilthead seabream, were also studied. We concluded that the less stressful stunning method for gilthead seabream is with crushed ice (-0.2 °C) containing 15 mg CEO+?-CD/kg in sea water. Interestingly, similar conclusion for gilthead seabream specimens that had never been anesthetised during their productive cycle was obtained. In addition, in all the species studied we found that a low dose of CEO+?-CD used to keep under control or reduce the metabolic alteration induced by the stress response was enough to assume that fish would not suffer irreversible lesions. Thus, 40 mg CEO+?-CD/L water, 60 mg CEO+?-CD/L water, 5 mg CEO+?-CD/kg ice, 5 mg CEO+?-CD/kg ice are the optimal doses that triggered the lowest stress response in Atlantic salmon, Nile tilapia, European seabass and rainbow trout, respectively. When EU+?-CD was tested in gilthead seabream, the data suggest a strong stress response that leads to physiological alterations. The third aim was to study the innate immune response induced by the anaesthetic complex during the stunning process. For this, the expression level of the genes coding for two cytokines (il-1? and tfg-?) and a heat sock protein (Hsp70) in the head-kidney (the main haematopoietic tissue in fish) of gilthead seabream, were previously analysed. Then, we analysed several innate immune activities (peroxidase, bactericidal, anti-protease and protease activity) in the plasma and skin mucus of all the species. Our data revealed that the doses of CEO+?-CD mentioned above for each species altered some innate immune parameters in plasma and skin mucus. However, impairment of the immune response can be discounted as the bactericidal activity was not affected in most of the species. The exception was Nile tilapia in which we observed a level of innate activities that pointed to an immune-suppression effect at the optimal dose of CEO+?-CD. Similarly, gilthead seabream specimens treated with EU alone or EU+?-CD showed immune-suppression, which was not observed in specimens treated with CEO alone or CEO+?-CD.