2.5. Experimental design and challenge study
A. sebae were divided into five groups of 20 each in three replicates (5 × 20 × 3 = 300) as: 1) uninfected control fed with basal control diet (without extract), 2) infected fish fed with: basal control diet (without extract), 3) 1% extract supplementation diet, 4) 5% extract supplementation diet, and 5) 10% extract supplementation diet. After 2 weeks of feeding, all groups except the uninfected control were individually injected with 0.1 ml of PBS containing V. alginolyticus at a dose 1.5 × 107 cells ml−1 intramuscularly. Similarly, the control fish was injected with same volume of PBS alone. The experimental fish were provided the corresponding diets twice a day until the end of experiment.
The LD50 value obtained from previous work (Dhayanithi et al., 2012) was applied. To study the cumulative mortality and post survival rate, 20 fish from each experimental group was maintained as above for 30 days.