At the end of the feeding trial, all the fish in each tank were bulk-weighed and counted for the calculation of growth parameters and survival. Blood samples were collected from individual fish, and each sample was analyzed separately. Three fish per tank (nine fish
per dietary treatment) were randomly captured, and anesthetized with 2-phenoxyethanol solution (200 mg L−1) and blood samples were taken from the caudal vein with heparinized syringes for determination of hematocrit and hemoglobin concentration, and respiratory burst activity. After the above-mentioned measurements with whole blood, plasma was separated by centrifugation at 5000 ×g for 10 min and stored at −70 °C for the determination of total immunoglobulin
(Ig), glucose, total protein, total cholesterol and triglyceride levels.
Another set of blood samples was taken (three fish per tank, nine fish
per dietary treatment) without heparin and allowed to clot at room
temperature for 30 min. Then, the serum was separated by centrifugation
for 10 min at 5000 ×g and stored at −70 °C for the analysis of
innate immune response including lysozyme, superoxide dismutase
(SOD), myeloperoxidase (MPO) and antiprotease activities. Hematocrit
was determined by a microhematocrit technique (Brown, 1980).
Hemoglobin and plasma levels of glucose, total protein, total cholesterol
and triglyceride were determined by using an automated blood analyzer
(SLIM; SEAC Inc., Florence, Italy).