Kinetics of reactive oxygen species produced by rainbow trout (Oncorhynchus mykiss) leukocytes and the effect of the antioxidant astaxanthin

Abstract

Reactive oxygen species contribute to kill of microorganisms. Their activity is usually measured by their capacity to reduce nitroblue tetrazolium into formazan. The incubation time to allow nitroblue tetrazolium reduction by reactive oxygen species usually ranges from 30 to 60 min. The aim of our study was to determine the kinetics of formazan formation, to determine the shortest incubation time possible, and to find if astaxanthin negatively affects the availability of reactive oxygen species (and defense mechanisms of fish). The blood/nitroblue tetrazolium method is based on nitroblue tetrazolium reduction into formazan by reactive oxygen species present in blood. Formazan can be spectrophotometrically measured, allowing quantification of reactive oxygen species. Reactive oxygen species were measured in blood samples from 30 trout intramuscularly injected with astaxanthin (0.3 mg/100 g of fish) (experimental group) and 30 non-injected trout (controls). Results indicated that in trout non-treated with astaxanthin, the plateau of formazan production was reached after 20 min of incubation. Trout intramuscularly injected with astaxanthin showed the following: on Day 1 after astaxanthin injection, the kinetics were slower but finally reached a plateau similar to astaxanthin-free trouts, and by Day 11 the plateau was significantly higher after 60 min incubation. In conclusion, the kinetics curves here reported allow reducing incubation time of the method to only 20 min in antioxidant-free trout and, on the other hand, our results also revealed that astaxanthin can be used to improve flesh colour in salmonids without affecting reactive oxygen species availability and therefore the defense mechanisms of trout.