l.S. Azad et al. Fish Shellfish Immunology 23(2007)154-163 —21db TI Fig. 3. Protective response of vaccinated juveniles of Chanos chanos fed vitamin(C and E)enriched diets to Vibrio vulnificus(plot points o treatment means sharing common labels are not significantly (P 0.05) different from one another. RPS(C-1): protective response unvaccinated control. RPS( C-ll): protective response relative to the vaccinated control ooster vaccination was rendered highly efficient in milkfish juveniles fed high Vitamin C (T2)and low Vitamin E (T3)indicating a probable interaction between the two vitamins. It is widely accepted that Vitamin C, in the water phase, spares Vitamin E and helps in its regeneration from the radical form [32]. Lower antibody titres and protective responses as reported in rainbow trout from a feeding trial with varying combinations of vitamins C and E(2 O response of milkfish juveniles fed higher Vitamin E (T4)are probably due to the reduced lymphoprolifera Protective response of milkfish juveniles followed closely the results of antibody production with high levels of Vitamin C not differing significantly from that of low levels of Vitamin E fed fish. Rainbow trout fed double deficient or double low vitamin(vitamins C/E)diets recorded high mortalities upon challenge with Yersinia ruckeri [28].Low protective response of T5 is probably due to the negligible levels of vitamins C and E(90 mg kg and 1.2 mg kg of diet, respectively) in the control diet and these levels were not enough to make the fish overcome the vaccination stress.It has been very well shown by previous researchers that sampling and confinement stress can be managed with Vitamin C supplementation [20, 21]. Stress is known to reduce the immune response and disease resistance in fish [33; hence, low levels of vitamins C and E (T5)resulted not only in the reduced antibody production but also in protective response upon challenge with live V. vulnificus. High protective response of T2 and T3 in the present tudy is probably due to both enhanced specific immune response and non-specific immune response. Results of the present study open up new avenues of making milkfish an alternative for mixed crop species in hrimp aquaculture ponds utilizing the high nutrient feed and supplementing additionally to keep the immune system fit to fight diseases Table 3c comparisons(Tukey's HSD)of average percentage of mortality in different treatments following challenge with live Vibrio vuinificr Calculated difference between means 0.00 8.33 .Difference betweenBooster vaccination was rendered highly efficient in milkfish juveniles fed high Vitamin C (T2) and low Vitamin E (T3) indicating a probable interaction between the two vitamins. It is widely accepted that Vitamin C, in the water phase, spares Vitamin E and helps in its regeneration from the radical form [32]. Lower antibody titres and protective response of milkfish juveniles fed higher Vitamin E (T4) are probably due to the reduced lymphoproliferation responses as reported in rainbow trout from a feeding trial with varying combinations of vitamins C and E [28]. Protective response of milkfish juveniles followed closely the results of antibody production with high levels of Vitamin C not differing significantly from that of low levels of Vitamin E fed fish. Rainbow trout fed double deficient or double low vitamin (vitamins C/E) diets recorded high mortalities upon challenge with Yersinia ruckeri [28]. Low protective response of T5 is probably due to the negligible levels of vitamins C and E (90 mg kg1 and 1.2 mg kg1 of diet, respectively) in the control diet and these levels were not enough to make the fish overcome the vaccination stress. It has been very well shown by previous researchers that sampling and confinement stress can be managed with Vitamin C supplementation [20,21]. Stress is known to reduce the immune response and disease resistance in fish [33]; hence, low levels of vitamins C and E (T5) resulted not only in the reduced antibody production but also in protective response upon challenge with live V. vulnificus. High protective response of T2 and T3 in the present study is probably due to both enhanced specific immune response and non-specific immune response. Results of the present study open up new avenues of making milkfish an alternative for mixed crop species in shrimp aquaculture ponds utilizing the high nutrient feed and supplementing additionally to keep the immune system fit to fight diseases. Fig. 3. Protective response of vaccinated juveniles of Chanos chanos fed vitamin (C and E) enriched diets to Vibrio vulnificus (plot points of treatment means sharing common labels are not significantly (P ¼ 0.05) different from one another. RPS (C-I): protective response relative to unvaccinated control. RPS (C-II): protective response relative to the vaccinated control. Table 3c Multiple comparisons (Tukey’s HSD) of average percentage of mortality in different treatments following challenge with live Vibrio vulnificus Treatments Calculated difference between means T1 T2 T3 T4 T5 T1 0.00 25.00* 27.78* 5.56 13.89 T2 0.00 2.78 30.56* 38.89* T3 0.00 33.33* 41.67* T4 0.00 8.33 T5 0.00 Tukey’s HSD at a ¼ 0.05 16.48 *Difference between means significant at P < 0.05. I.S. Azad et al. / Fish & Shellfish Immunology 23 (2007) 154e163 161