Antagonistic Effect of Zinc Oxide Nanoparticles Dietary Supplementation Against Chronic Copper Waterborne Exposure on Growth, Behavioral, Biochemical, and Gene Expression Alterations of African Catfish, Clarias gariepinus (Burchell, 1822).

The harmful impact of waterborne copper (Cu) as a common abiotic stressor in aquatic environments has gained much more interest. The present study aimed to investigate the utilization of zinc oxide nanoparticles (ZnONPs) dietary supplementation to mitigate the chronic toxicity of Cu in African catfish (Clarias gariepinus). Two hundred and forty fish (92.94 ± 0.13 g) were assigned into six groups for 60 days. Control (C), ZnONPs20, and ZnONPs30 groups were fed on basal diets fortified with 0, 20, and 30 mg kg-1 ZnONPs without Cu exposure. Cu, Cu + ZnONPs20, and Cu + ZnONPs30 groups were exposed to Cu at a dose of 10 mg L-1 and fed on basal diets fortified with 0, 20, and 30 mg kg-1 ZnONPs, respectively. The results revealed that the Cu-exposed fish experienced abnormal clinical signs and behavioral changes. The growth indices and acetylcholine esterase activity were significantly decreased (P < 0.05) in the Cu group. Meanwhile, hepatorenal and serum stress indices (P < 0.05) were significantly elevated with chronic Cu exposure. In addition, a higher expression of stress (P < 0.05) (heat shock protein 60 and hypoxia-inducible factor-1 alpha) and apoptotic-related genes (C/EBP homologous protein, caspase-3, and Bcl-2 Associated X-protein) with down-regulation (P < 0.05) of the anti-apoptotic-related genes (B-cell lymphoma 2 and proliferating cell nuclear antigen) was noticed in the Cu-exposed fish. Histopathological alterations in the gills, liver, kidney, and spleen were markedly reported in the Cu-exposed group. The dietary supplementation with ZnONPs significantly alleviated the negative impacts of chronic waterborne-Cu exposure on growth performance, physiological changes, gene expression, and tissue architecture, especially at 30 mg kg-1 diet level. In particular, the inclusion of ZnONPs at the 30 mg kg-1 diet level produced better outcomes than the 20 mg kg-1 diet. Overall, ZnONPs could be added as a feed supplement in the C. gariepinus diet to boost the fish's health and productivity and alleviate the stress condition brought on by Cu exposure.

Immune-antioxidant trait, growth, splenic cytokines expression, apoptosis, and histopathological alterations of Oreochromis niloticus exposed to sub-lethal copper toxicity and fed thyme and/or basil essential oils enriched diets.

Two experiments were conducted in this study, using 250 Oreochromis niloticus (O. niloticus) (average weight 30.28 ± 0.27 g). The first experiment was conducted to investigate the 96-h lethal concentration 50 (LC50) of copper chloride (CuCl2) using the probit analysis, seventy fish was divided into seven different concentration of CuCl2 (0, 22, 23, 24, 25, 26, and 27 mg/L), the accurate Cu concentrations were (1.23, 5.36, 6.02, 6.98, 7.05, 7.93, 8.12 mg/L Cu). The second experiment was conducted for investigating the effect of dietary supplementation with thyme (Thymus vulgaris, T. vulgaris) and sweet basil (Ocimum basilicum, O. basilicum) essential oils (TEO and BEO respectively) against sub-lethal Cu exposure (1/10 96-h LC50 of CuCl2). About 180 fish was divided into six groups in triplicate (10 fish/replicate, 30 fish/group). Group 1 (C) was kept as a control group with no Cu exposure and was fed the control basal diet. Group 2 (C-Cu) was fed the control basal diet and simultaneously exposed to 1/10 of the 96 h LC50 of CuCl2 (2.574 mg/L) as a sub-lethal concentration of Cu, where the realistic Cu concentration was 3.976 mg/L. Group 3 (TEO) and group 4 (BEO) were fed the diets fortified with 1%TEO and BEO, respectively without exposure to Cu. Group 5 (TEO-Cu) and group 6 (BEO-Cu) were fed the diets fortified with 1%TEO and 1%BEO, respectively, and simultaneously exposed to 1/10 of the 96 h LC50 of CuCl2 (2.574 mg/L). The growth and behavioral performance, immunological response and its related gene expression, antioxidant status, stress biomarker indicators, apoptosis biomarkers, and histopathological alteration were investigated. The results of the first experiment showed that the 96-h LC50 of CuCl2 in O. niloticus was 25.740 mg/L with lower and upper confidence limits of 25.152 and 26.356 mg/L, respectively. The results of the second experiment showed that sub-lethal Cu exposure induced growth retardation (lowered final body weight, total weight gain, and specific growth rate %), behavioral abnormalities (slower swimming activity and feeding performance), immunosuppression (lowered nitric oxide, complement-3, lysozyme, total proteins, albumin, and globulin), and lowering the hepatic antioxidant functions (higher MDA, and lower SOD, CAT, and GPx) in the exposed fish. Furthermore, alteration in the immune-related genes expression (down-regulation of IL-10 and TGF-ß and up-regulation of IL-1ß, IL-6, IL-8, and TRL-4), hepato-renal dysfunction (elevated ALT, AST, urea, and creatinine), and high levels of serum stress indicators (cortisol and glucose) were markedly evident. sub-lethal Cu toxicity induced significant up-regulation of apoptosis biomarkers involving, nuclear factor-κß (NF-κß), Bcl-2 Associated X-protein (BAX), meanwhile, the expression of B-cell lymphoma 2 (BCL2) and Proliferating cell nuclear antigen (PCNA) was remarkably down-regulated. In addition, apoptosis was also evident by histopathological investigation of branchial, hepatic, and renal sections. TEO and/or BEO dietary supplementation mitigate the destructive impacts of sub-lethal Cu exposure in O. niloticus, depending on the results of our study, it could be concluded that TEO and BEO with a 1% dietary level could be a promising antioxidant, immunostimulant, anti-stress factors, and anti-apoptosis mediators against heavy metal contaminants (Cu) in O. niloticus, providing a solution to the problem of aquatic bodies pollution, consequently aiding in the development of aquaculture industry.

Efficacy of nano zinc oxide dietary supplements on growth performance, immunomodulation and disease resistance of African catfish Clarias gariepinus.

Zinc (Zn) is an important trace element in fish diets that is required for growth, immunity and antioxidant defense mechanisms. The current study assessed the effects of both organic and nanoparticle zinc oxide (ZnO and ZnO-NPs, respectively) on growth performance, immune response and the antimicrobial effect against Pseudomonas aeruginosa in African catfish Clarias gariepinus. Fish were fed either a control diet or diets supplemented with organic ZnO at concentrations of 20 and 30 mg kg-1 or ZnO-NPs at concentrations of 20 and 30 mg kg-1. After 60 d, a subset of the fish was injected intraperitoneally with 3 × 107 CFU ml-1 of P. aeruginosa. Results showed that body weight gain, feed conversion ratio and specific growth rates were significantly increased in ZnO-NPs20 compared to all other groups. The dietary supplementation with 20 mg kg-1 of ZnO-NPs improved the antioxidant status of fish. Moreover, IgM, lysozyme and nitric oxide showed a significant increase in the fish which received the ZnO-NPs20-supplemented diet. A significant upregulation of growth and stress-related genes was seen in the ZnO-NPs20-supplemented group compared to other groups. However, there was no significant difference in the expression of immune-related genes among ZnO-NPs20, ZnO-NPs30 and ZnO30 groups. These findings highlight the potential use of nano-ZnO for improving growth performance, antioxidant status, immunological status and antibacterial activity against P. aeruginosa in African catfish.

Influence of Feeding Quinoa (Chenopodium quinoa) Seeds and Prickly Pear Fruit (Opuntia ficus indica) Peel on the Immune Response and Resistance to Aeromonas sobria Infection in Nile Tilapia (Oreochromis niloticus).

In recent times, nutraceuticals have been used extensively to identify promising feed additives for the improvement of the aquaculture industry through the enhancement of growth and survival rates, potentiation of the immune responses, and fortification of the resistance against infectious bacterial diseases. In this study, Nile tilapia (Oreochromis niloticus) were fed with diets supplemented with quinoa seeds (QU) or prickly pear fruit peel (PP) at the dose levels of 10% or 20% of the diet. After 45 days of the feeding trial, the fish were exposed to Aeromonas sobria (A. sobria) challenge. The pre-challenge indices indicated that both supplements mediated a significant improvement in most of the estimated parameters, including survival rate, antioxidant status, hematological and immunological indices, and hepatoprotective potential. These effects were recorded in the groups fed with high doses of the supplements (20%). The least changes were observed in the QU10-supplemented fish. In the spleen tissue, the TGF-ß gene was upregulated in the PP10-, PP20- and QU20-supplemented groups, while the expression of the IFN-γ gene remained unaffected in all the supplemented groups, except for the PP20-supplemented group, which showed an upregulation. After the challenge with A. sobria, the relative survival percentage was improved by the supplementation of PP and QU, particularly in the PP20-supplemented group, possibly via the promotion of immunological responses, hepatoprotective potency, and modulation of the studied genes. Moreover, the morphological structure of the tissues showed marked recovery. The findings suggest that Nile tilapia fed with different levels of PP peel and QU seeds, particularly at the level of 20%, enhanced the immune response in fish and improved their resistance against A. sobria infection.

The Effect of Dietary Replacement of Fish Meal with Whey Protein Concentrate on the Growth Performance, Fish Health, and Immune Status of Nile Tilapia Fingerlings, Oreochromis niloticus.

The present study was conducted to assess the effect of replacing fish meal with whey protein concentrate (WPC) on the growth performance, histopathological condition of organs, economic efficiency, disease resistance to intraperitoneal inoculation of Aeromonas hydrophila, and the immune response of Oreochromis niloticus. The toxicity of WPC was tested by measuring the activity of caspase 3 as an indicator of cellular apoptosis. Oreochromis niloticus fingerlings with average initial weight 18.65 ± 0.05 gm/fish (n = 225) for a 10-week feeding trial. The fish were randomly allocated to five experimental groups, having five replacement percentages of fish meal with WPC: 0%, 13.8%, 27.7%, 41.6%, and 55.5% (WPC0, WPC13.8, WPC27.7, WPC41.6, and WPC55.5); zero percentage represented the control group. The results show that the fish fed WPC had the same growth performance as the WPC0. The total weight of bacterially challenged surviving fish increased linearly and quadratically (p ≤ 0.05) by increasing the replacement percentage of fish meal with WPC. The growth hormone, nitric oxide, IgM, complement 3, and lysozyme activity were seen to increase significantly in WPC27.7, especially after a bacterial challenge. The phagocytic percentage and phagocytic index increased significantly in WPC27.7, WPC41.6, and WPC55.5 groups. Histopathological examination of liver sections was badly affected by high replacement in WPC41.6-55.5. The activity of caspase 3 in the immunohistochemical stained sections of the intestine was increased significantly by increasing the inclusion level of WPC. Economically, the total return of the total surviving fish after the bacterial challenge was increased significantly by fish meal replacement with WPC. It could be concluded that WPC could replace the fish meal in Nile tilapia diets up to 27.7%, with improving the gut health, the total weight of survival fish, and immune status of fish challenged with A. hydrophila. High inclusion levels of WPC are not recommended in fish diets, since they negatively affected the intestinal and liver tissues and increased the level of cellular apoptosis, as indicated by the increased caspase 3 activity. Further researches are recommended to evaluate the effect of fish meal replacement with WPC on the histopathological examination of the kidney and to test the capacity of serum IgM to clot the bacteria used for the challenge.

Assessment of hematological, hepato-renal, antioxidant, and hormonal responses of Clarias gariepinus exposed to sub-lethal concentrations of oxyfluorfen.

Little is known about the effects of oxyfluorfen, a diphenyl ether herbicide, exposure on the African catfish (Clarias gariepinus) health. Consequently, the existing investigation was designed to highlight the impacts of oxyfluorfen exposure on C. gariepinus hematological indices, liver and kidney functions, reproductive hormones, and oxidative status. Furthermore, a consequent 10-day depuration period was adopted to evaluate the recovery of the disturbed indicators to normal values. In the first experiment, the 96-h lethal concentration 50 (LC50) of oxyfluorfen for C. gariepinus was determined using probit analysis. Next, in a second experiment, 180 healthy fish (average initial body weight: 164.23 ±â€¯0.24) were randomly assigned to 4 experimental groups exposed to 0, 1/10, 1/8, or 1/5 96-h LC50 of oxyfluorfen. The hematological profile, hepatic enzymes, kidney damage byproducts, reproductive hormones, oxidative stress, and lipid peroxidation indicators together with acetylcholinesterase (AChE) content were assessed. A histopathological examination of the hepatic, renal, brain, and testicular tissues was accomplished. Moreover, the expression of the oxidative stress-related gene was carried out. The results showed that 96-h LC50 of oxyfluorfen for C. gariepinus was 11.698 mg/L. Exposure to sublethal levels of oxyfluorfen induced macrocytic hypochromic anemia, leukopenia, lymphopenia, monocytopenia, and eosinopenia. Also, a concentration-dependent increase in alanine transaminase, alkaline phosphatase, aspartate transaminase, urea, creatinine, catalase, and malondialdehyde was detected following oxyfluorfen exposure together with upregulation of catalase gene. But, significant concentration-dependent reductions in AChE, glutathione transferase, reduced to oxidized glutathione ratio, estradiol, and testosterone activities were recorded. These biochemical alterations were accompanied by pathological perturbations in hepatic, renal, brain, and testicular tissues. Following 10 days of recovery, only the hematological impairments were abolished. Conclusively, the herbicides oxyfluorfen could induce multiple negative impacts on C. gariepinus with oxidative stress as a probable underlying mechanism. Additionally, a recovery period of 10 days was not enough to restore these impairments.

Transcriptomic profile change, immunological response and disease resistance of Oreochromis niloticus fed with conventional and Nano-Zinc oxide dietary supplements.

The present investigation was performed to evaluate the efficiency of Zinc oxide (ZnO) as a fish feed additive in immunomodulation of Oreochromis niloticus. Fish were fed on ZnO nano-particles (nZnO) and conventional (ZnO) in two concentrations (30 and 60 mg/kg diet), in addition to the control fish which was fed on Zn free diet. After 6° days, the highest survival rate was recorded in the nZnO30 -supplemented group. The total antioxidant capacity (TAC) and antioxidant enzymes were improved in different dietary Zn supplementation, obviously in the nZnO30 -supplemented group, while the lowest antioxidant status was noticed nZnO60 supplemented fish. The lipid peroxides (MDA) level was diminished upon Zn supplementation, particularly in nZnO30-supplemented group but showed a significant elevation in the nZnO60-supplemented group. Furthermore, the immune parameters examined, lysozyme activity, bactericidal activity, and IgM were significantly higher in ZnO60, and nZnO30 supplemented groups. The C-reactive protein (CRP) level showed no significant increase in response to Zn supplementation in the both forms at level of 30 mg/kg diet, but showed marked elevation in nZnO60- supplemented group. The mRNA expression profile of both interleukin 8 (IL-8), interleukin 1, beta (IL-1ß) encoding genes showed an up-regulation that was found in all Zn- supplemented groups, but more pronounced in nZnO60-supplemented group. On the other hand, the expression pattern of myxovirus resistance (Mx)-encoding gene showed no remarkable difference between the Zn- supplemented and control fish. The expression level of CXC-chemokine, toll-like receptor 7 (TLR-7), immunoglobulin M heavy chain (IgM heavy chain) and interferon gamma (IFN-γ) gene was upregulated in Zn-supplemented groups particularly in the nZnO30- supplemented group. While, the lowest expression was found in nZnO60- and ZnO30-supplemented groups. Here, Zn supplementation promoted the immune and antioxidant strength in fish mainly in nano form at the level of 30 mg/kg diet but not at 60 mg/kg diet that disrupt the immune and antioxidant status and promote inflammatory response.