1. Effect of Some Local Immunostimulants on the Survival, Growth Performance and challenge infection of Oreochromis niloticus By Ahmed Said Diab*, Salah Mesalhy Aly, George John, *Yasser Mohamed Abde- Hadi* and Mohamed Fathi Mohamed

2. Introduction Fish culture is an important industry where the production of fish worldwide increases every year. Intensification of fish adversely affects fish health and tends to produce poor environment for fish and increase their susceptibility to infections (Sakai, 1999). Disease outbreaks are encountered in the rapidly developing aquaculture industry and affecting the economic development of this sector (Yunxia et al., 2001). Various chemotherapeutics have been used to treat bacterial infections in cultured fish for the last 20 years. However, the incidence of drug-resistant bacteria has become a major problem in fish culture (Aoki, 1992). Vaccination is a useful prophylaxis for infectious diseases of fish but the development of vaccine against intracellular pathogens has not so far been successful. Therefore, the immediate control of all fish diseases using only vaccines is impossible (Sakai, 1999). The most effective method, in our opinion, may be the development of natural disease resistance in fish, where, the use of immunostimulants may be an effective mean of increasing the immune competency and disease resistance of fish. The present study was aimed to evaluate the effect of some immunostimulants on the survival, growth performance, immune response and disease resistance of O. niloticus, in addition to their tolerance to the cold stress through field experiments.

3. Materials and methods

4. I- Fish: II- Immunostimulants: III- Experiment: A- First phase (summer season): B- Second phase (Winter season): IV- Statistical analysis:

5. Results

6. Table (1) Analysis and ingredient of the basal diet used along the experiment. ME* in Feed ME* (in Ing.) % protein in feed % protein (in ing.) % in dietIngredients (Ing.) 3200040005.760.728.00Fish meal 151823287025.3920.4852.9Soybean meal 3608412403.17190.10929.1Ground corn 1350027000.670.1345.00Wheat flour 1820091000.00 2.00Vegetable oil 1820091000.00 2.00Cod liver oil 00000.00 1.00Dicalcium phosphate 00000.00 0.07Mineral mix. 00000.00 0.05Vitamin mix. 00000.00 0.03Vitamin C 2698070.0034.99390.00100.15Total 28000035Dietary requirement *ME= metabolic energy.

7. Table (2) showing the random distribution of the 4 replicates in all treatments and the control within the 4 race ways. Raceway 4 Raceway 3 Raceway 2 Raceway 1 B2B1G3G1 Bs 1 CB3Bs3 B1G3C Bs 2 G2 Bs 3 B2G3 G1B3 Bs 1 B2 B3 Bs 3 C G2B1G2 CB2G2B3 Bs 2 G1 Bs 1 G3 Bs 2 G1B1

8. Table (3) Effect of immunostimulants on the growth performance and mortality of O. niloticus fingerlings during the first phase (summer season ) (Mean ± SE). Mortality(%) Growth performance Immunostimulants Condition factor Specific growth rate Body gain (gm) Dose (%) Type 20.83 A ±3.00 1.86 A ± 0.02 4.66 AB ± 0.25 16.46 AB ± 0.89 0.0Control 5.83 B ± 1.86 1.86 A ± 0.04 4.06 BC ± 0.18 14.32 BC ± 0.62 1.0 Black seed 7.50 B ± 1.86 1.50 B ± 0.08 3.99 C ± 0.19 14.10 C ± 0.65 2.0 9.17 B ± 3.00 1.81 A ± 0.20 4.04 BC ± 0.22 14.27 BC ± 0.73 3.0 5.84 B ± 1.37 1.87 A ± 0.04 4.03 BC ± 0.18 14.22 BC ± 0.60 1.0 Garlic 5.84 B ± 1.37 1.76 A ± 0.03 4.4 ABC ± 0.21 15.52 ABC ± 0.68 2.0 5.00 B ± 1.41 1.88 A ± 0.02 4.72 A ± 0.24 16.68 A ± 0.78 3.0 10.00 B ±2.36 1.81 A ± 0.05 4.34 ABC ± 0.19 15.33 ABC ± 0.63 0.5 Biogen 5.00 B ± 1.41 1.79 A ± 0.02 3.31 D ± 0.21 14.02 C ± 0.65 1.0 7.50 B ±1.86 1.88 A ± 0.01 4.51 ABC ± 0.23 15.91 ABC ± 0.83 1.5 * The number of fish in each treatment was 120.

9. Table (4) Effect of immunostimulants on some hematological and serum biochemical parameters of experimented O. niloticus during the first phase (summer season ) (Mean ± SE). Serum parameters (gm/dl) Hematocrit value (%) Immunostimulants γβαAlbumin Total protein NBT (mg/ml) Dose % Type 0.15 C ±0.02 0.19 C ±0.03 0.19 D ±0.08 0.52 C ±0.1 1.05 B ±0.13 0.28 D ±0.04 15.07 DE ±1.67 0.0Control 0.8 A ±0.01 0.56 AB ±0.22 0.79 A ±0.28 1.32 AB ±0.21 3.47 A ±0.66 0.25 D ±0.06 14.24 E ±2.39 1.0 Black seed 0.54 AB ±0.12 0.35 BC ±0.18 0.59 ABC ±0.06 1.41 AB ±0.34 2.83 A ±0.68 0.31 CD ±0.05 8.93 E ±1.59 2.0 0.55 AB ±0.04 0.63 A ±0.14 0.79 A ±0.21 1.93 A ±0.8 3.89 A ±1.05 0.33 CD ±0.05 27.83 BCD ±6.25 3.0 0.52 B ±0.02 0.56 AB ±0.08 0.69 ABC ±0.11 1.69 AB ±0.41 3.46 A ±0.3 0.47 AB ±0.06 35.01 AB ±3.37 1.0 Garlic 0.76 AB ±0.27 0.47 AB ±0.14 0.54 ABC ±0.22 1.53 AB ±0.3 3.28 A ±0.66 0.38 ABCD ±0.04 42.7 A ±11.49 2.0 0.75 AB ±0.26 0.44 AB ±0.11 0.47 CD ±0.29 1.2 B ±0.33 2.86 A ±0.94 0.51 A ±0.02 36.07 AB ±1.33 3.0 0.6 AB ±0.19 0.57 AB ±0.19 0.57 ABC ±0.17 1.56 AB ±0.46 3.3 A ±0.94 0.28 D ±0.35 31.15 ABC ±6.52 0.5 Biogen 0.67 AB ±0.15 0.49 AB ±0.17 0.48 BCD ±0.15 1.29 AB ±0.17 2.92 A ±0.4 0.36 BCD ±0.01 16.54 DE ±2.06 1.0 0.65 AB ±0.17 0.47 AB ±0.13 0.42 CD ±0.16 1.69 AB ±0.52 3.25 A ±0.51 0.43 ABC ±0.03 18.92 CDE ±1.99 1.5 * The number of samples per each group was 12 out of total number of 120 fish.

10. Table (5): Mortality rate among experimented fish after challenge infection. BiogenGarlic Black seed ControlTreatment 1.51.00.53.02.01.03.02.01.00.0 Dose (%) 25.041.616.603.825.025.033.341.641.675.0 Mortality (%)

11. Table (6) Effect of immunostimulants used on growth performance and mortality of O. niloticus during the second phase (Winter season) (Mean ± SE). Mortality (%) Growth performance Immunostimulants Condition factor Specific growth rate Body gain GroupType 60.42 A ±1.36 1.65 C ±0.03 0.18 BC ±0.03 2.13 B ±0.42 CControl 16.67 C ±5.46 1.67 BC ±0.04 0.12 C ±0.02 1.82 B ±0.33 Bs 1 Black seed 14.59 C ±6.45 1.68 ABC ±0.03 0.09 C ±0.06 1.17 B ±0.76 Bs 2 19.44 C ±6.33 1.72 ABC ±0.004 0.28 ABC ±0.11 3.50 AB ±1.19 Bs 3 25.00 C ±7.04 1.75 ABC ±0.02 0.40 AB ±0.11 5.10 A ±1.35 G1G1G1G1 Garlic 29.18 BC ±9.31 1.77 AB ±0.04 0.19 BC ±0.05 2.78 AB ±0.72 G2G2G2G2 27.09 C ±4.09 1.72 ABC ±0.03 0.19 BC ±0.01 3.18 AB ±0.19 G3G3G3G3 45.84 AB ±7.22 1.72 ABC ±0.04 0.45 A ±0.17 2.81 AB ±0.65 B1B1B1B1 Biogen 31.25 BC ±2.73 1.72 ABC ±0.02 0.19 BC ±0.04 2.69 B ±0.47 B2B2B2B2 20.84 C ±2.73 1.78 A ±0.03 0.23 ABC ±0.05 3.00 AB ±0.68 B3 B3 B3 B3

12. Conclusion It could be concluded that, the used immunostimulants could increase the resistance of fish to infectious diseases by enhancing non-specific defense mechanism. Immunostimulants can reduce the losses caused by most not all diseases in aquaculture and could enhance the growth performance especially after prolonged application. Moreover, garlic seemed more effective than black seeds and Biogen for initiating the disease resistance based on its stimulatory effect on hematocrit and NBT test. Immunostimulants are recommended to be used by fish farmers in the regional countries and some questions about their efficacy need more clarification. However, timing, dosages, method of administration and the physiological condition of the fish need to be taken into consideration.

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