الفهرس 
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Abstract
Nile tilapia (O. niloticus) culture is widely practiced in many tropical and subtropical regions of the world. It is one of the most important commercial fish in Egypt, therefore it is extensively used in both laboratory and field studies. The present study highlights the role of EM as a probiotic-enhancing biological feature and biodegradation of some environmental pollutants exposed to Cu, Cd, and Pb toxicity on Nile tilapia (O. niloticus) with variable temperatures (24°C, 28°C, and 32°C) under experimental conditions.
Before the experiment begins, Cu, Cd and Pb toxicity test was carried out to determine their 96hr LC50. The study include three experiment “treated with CuSO4, CdCl2 and Pb(NO3)2” then each experiment exposed to three temperatures (24°C, 28°C, and 32°C).
For the water quality, Cu showed the highest dissolved oxygen (DO) levels ranged from 7.8-10.4 mg/l, while Pb showed the lowest one levels ranged from 5-8.2 mg/l. Analysis of total disoved solids (TDS) revealed a significant increase in EM/Cu group when compared to other fish groups (at 24°C and 28°C). Meanwhile, increase in the Cu group when compared to other experimental groups (at 32°C). TDS revealed a significant increase in Cd group when compared to other fish groups except in EM/Cd group at 24°C. Meanwhile, the significance increase in Cd group and EM/Cd group after exposure to 28°C. The highest value was recorded in EM/Cd at 32°C. Also, TDS revealed a significant increase in EM/Pb group (at 28°C and 32°C). The chemical oxygen demand (COD) levels indicated a remarkable fluctuation for Cu experiment with a slight decrease in the control at 28°C. Meanwhile, the levels reduced in the control and EM groups after exposed to 32°C. For Cd experiment COD levels decrease in the control group at 28°C, while, the levels reduced in the control and EM groups compared to other experimental groups. In the third experiment, that of Pb COD levels decrease in the control group at 24°C and 28°C. Meanwhile, the levels reduced in the control and EM groups after exposed to 32°C compared to other groups.
Regarding the hematological parameters, in the Cu experiment, WBCs revealed a significance increase in cell counts in Cu group when compared to other experimental groups subjected to (24°C and 32°C). In the same time, a significance increase in EM group exposed to 28°C. In Cd experiment WBCs significantly increase in Cd group when compared to other experimental groups except in EM/Cd group subjected to 24°C, but there is a significance decrease in control group exposed to 28 °C. The highest values were recorded in Cd group and EM/Cd group subjected to 32°C. WBCs results in Pb experiment showed a significance increase in cell counts in EM groups subjected to 28°C and 32°C. In the same time, the significance decrease in control group exposed to 24°C.
For Cu experiment, the highest value of hemoglobin levels were found significantly in EM fish group when compared to other groups except in control group exposed to 32°C. Meanwhile, the highest levels were recorded in EM group and control group exposed to 28°C. Cd experiment indicated non-significant results among all fish groups at 24°C, 28°C and 32°C. The highest value was recorded in EM/Pb grou when compared to other groups in Pb experiment except EM group exposed to 28°C.
In Cu experiment, the highest values of platelets count were significant in EM group exposed to 24°C and also in EM and control groups subjected to 28°C. Moreover, the highest value were significant in EM group when compared to Cu group exposed to 32°C. In Cd experiment, the highest values of platelets were significant in Cd group exposed to 32°C. While the highest value was significant in EM group in comparison to Cd group subjected to 24 °C. Whereas, the lowest value was significant in Cd group exposed to 28°C. The platelet was increased significantly in EM/Pb group exposed to 28°C and 32 °C.
For the biochemical analysis, the first experiment that of Cu, had the lowest activity of AST found in control group exposed to 24°C and 28°C. While at 32°C, the lowest activity of AST was recorded significantly in control group and EM group. On the other hand, ALT was recorded significantly in Cu group exposed to 24°C and 32°C. After exposure to 28°C the highest levels were found significantly in EM and EM/Cu groups. In Cd experiment, the lowest acivity of AST was markedly found in control group exposed to 24°C and 28°C and the maximum was recorded significantly in Cd group subjected to 32°C. The highest acivity of ALT was recorded significant in EM/Cd fish group exposed to 24°C. After exposure to 28°C the highest acivity was found in Cd and EM/Cd groups, but at 32°C the highest one was found significantly in Cd group only. In Pb experiment, the highest activity of AST was found in EM/Pb group exposed to 24°C, but the highest was recorded significantly in Pb group and EM/Pb group at 28°C. While at 32 °C the highest activity of AST was recorded significantly in Pb group. The highest activity of ALT was recorded significantly in Pb group exposed to 28°C and 32°C. The highest one was recorded in EM / Pb group exposed to 24°C.
In the analysis of uric acid, results showed that its highest level was recorded significantly in Cu group and EM/Cu group exposed to 28°C and 32°C for Cu experiment. Also, its results showed that its highest value in Cd group and EM/Cd group exposed to 28°C. While the highest level was found significantly in Cd fish group exposed to 32°C. In Pb experiment, the analysis of uric acid showed that its highest level was recorded in Pb group exposed to 32°C while the highest levels were found in EM/Pb group and Pb group exposed to 28°C.
To assess the antioxidant activities in Cu experiment, values of catalase activities showed a significant increase in EM group in comparison to other groups exposed to 24°C, while the values showed a significant decrease in Cu group compared to other groups exposed to 32°C. The effect of CdCl2 on CAT activity in liver of tilapia fish increased significantly in EM and control groups when exposed to 28°C and 32°C. While, the highest enzyme activity was recorded significantly in EM group exposed to 24°C. The impact of Pb(NO3)2 showed a significant increase in CAT activity in liver of tilapia fish in EM group exposed to 24°C and 32°C. The highest activity was recorded in control and EM groups subjected to 28°C.
GST assessment in Cu experiment showe an increased activity in EM group in comparison to other groups exposed to 24 °C and 28 °C. When fish exposed to a higher temperature at 32 °C the highest level was recorded significantly in Cu group. In Cd experiment, the GST activity increased significantly in EM group exposed to 24°C and 28°C. While the highest one was recorded in EM/Cd group exposed to 32°C. Pb experiment had increased in EM group exposed to all temperature levels; 24°C, 28°C and 32 °C.
Expression of heat shock protein genes in the first experiment of CuSO4, showed that expression of HSP27 and HSP90 genes in liver tissues increased significantly in fish groups treated with CuSO4 in all temperature levels while expression of HSP70 gene increased significantly in fish group treated with CuSO4 at 28 °C only, but after exposure to 32°C increased significantly in EM/Cu group in comparison to other groups. In Pb experiment expression of HSP27 increased significantly in control groups at 24°C, 28°C. While HSP27 levels increased significantly in EM group after exposure to 32°C, however expression levels of HSP70 reached highest levels were recorded significantly in EM/Cd groups at both 28°C and 32°C but increased significantly in EM group after exposure to 24°C. Concerning HSP90 expression levels waa increased significantly in EM and control groups at 24°C and 28 °C respectively, while at 32°C the expression increased significantly in EM and EM/Cd groups. In Pb(NO3)2 experiment, HSP27 high expression were seen in control groups only in all temperature levels, while HSP70 expression increased significantly in Pb groups at both 24°C and 32°C. The expression of HSP90 increased significantly in EM group at all temperature levels.
The immune responses was measured by B and T proliferation level in spleen and kidney. In the first experiment of CuSO4, B and T cell proliferation in spleen showed the highest value of B lymphocyte recorded significantly in EM group at 24°C, while the highest value of T lymphocyte recorded significantly in Cu group at 32°C. Results of T and B lymphocyte proliferation in kidney, T lymphocyte showed non-significant among all experimental groups, but the value of B lymphocyte showed a significant increase in EM/Cu group at 32°C. The effect of CdCl2 on immune responses during heat stress, results in increased stimulation index of B cell in the spleen of tilapia fish in EM group compared to other groups except in Cd group when exposed to 24°C. In the third experiment of treatment with Pb(NO3)2, stimulation index of B cell in spleen of tilapia fish increased significantly in EM group when compared to other groups except in EM/Pb group exposed to 24°C. The highest levels were recorded significantly in Pb and EM groups subjected to 32°C.
The level of complement activity showed a significant increase at 24 °C, 28°C and 32°C in Cu group when compared to other fish groups. The complement activity also showed a significant increase at 28°C and 32°C in Cd and Pb groups in their experiments when compared to other fish groups, but the level showed a significant increase at 24°C in EM/Cd and EM/Pb groups when compared to other fish groups.
EM showed a positive effect on fish immunity by modifying the applied stress condition of copper, cadmium and lead exposure in favor of body growth and anabolic processes rather than by reducing metal uptake by the fish. The current study recommends using beneficial microorganisms in fish culture to increase fish productivity and lessen the detrimental impacts of contaminants with changing temperatures.