Corrigendum: Future impact of thymoquinone-loaded nanoemulsion in rabbits: prospects for enhancing growth, immunity, antioxidant potential and resistance against Pasteurella multocida.

[This corrects the article DOI: 10.3389/fvets.2023.1340964.].

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.

Effect of dietary intervention with Capsicum annuum extract on growth performance, physiological status, innate immune response, and related gene expression in Nile tilapia.

The red pepper (Capsicum annuum) has gained great attention recently because of its biological and pharmacological characteristics. The present approach aimed to evaluate the effects of C. annuum alcoholic extract (CAE) supplementation on Nile tilapia (Oreochromis niloticus) growth performance, physiological status, some metabolic, immune, and regulatory genes expression, and resistance against Streptococcus agalactiae infection. Fish (22.26 ± 0.19 g) were assigned to four treatments (five replicates, each with 10 fish replicate-1) and fed tested diets for 60 days. The experimental diets were supplemented with CAE at 0, 0.4, 0.8, and 1.6 g kg-1, expressed as CAE0, CAE0.4, CAE0.8, and CAE1.6, respectively. The findings exhibited that CAE dietary supplementation improved growth performance, feed utilization, elevated growth hormone level, and digestive enzyme activities (amylase and protease), and lowered leptin hormone in a level-dependent manner. Boosting the mRNA expression of the transporter proteins (solute carrier family 15 member 2 and solute carrier family 26 member 6) and insulin-like growth factor-1 genes with a decrease in the myostatin gene expression was noticed in the CAE-fed groups. The innate immune (serum bactericidal activity %, complement 3, and phagocytic activity %) and antioxidant (glutathione peroxidase and total antioxidant capacity) parameters were significantly (p < 0.05) improved, and the serum malondialdehyde level was significantly decreased by CAE dietary inclusion. A marked upregulation in the mRNA expression of interleukins (il-1ß, il-6, il-8, and il-10), transforming growth factor-ß, glutathione peroxidase, and glutathione synthetase genes were observed in CAE-fed groups. Dietary CAE decreased the cumulative mortalities after the challenge with S. agalactiae by 20, 13.33, and 10% in CAE0.4, CAE0.8, and CAE1.6, respectively, compared to the control (40%). Overall, dietary supplementation with CAE could improve growth performance and physiological status, and modulate the expression of several regulatory genes in Nile tilapia. The recommended level of CAE is 1.6 g kg-1 to augment growth and health status.

Nelumbo nucifera synthesized selenium nanoparticles modulate the immune-antioxidants, biochemical indices, and pro/anti-inflammatory cytokines pathways in Oreochromis niloticus infected with Aeromonas veronii.

Bacterial infection is considered one of the major issues in fish culturing that results in economic losses. Metal nanoparticles are a cutting-edge and effective disease management and preventive strategy because of their antibacterial ability. In this investigation, the selenium nanoparticles were prepared by a biological method using Nelumbo nucifera leaves extract. The in-vitro antibacterial activity of N. nucifera synthesized selenium nanoparticles (NN-SeNPs) was tested against Aeromonas veronii. A treatment assay was conducted on 210 Oreochromis niloticus (average body weight: 27 ± 2.00 g). A preliminary approach was conducted on 90 fish for determination of the therapeutic concentration of NN-SeNPs which was found to be 4 mg/L. Fish (n = 120) were categorized into four groups for 10 days; G1 (control) and G2 (NN-SeNPs) were non-challenged and treated with 0 and 4 mg/L NN-SeNPs, respectively. While, G3 and G4 were infected with 2 × 106 CFU/mL of A. veronii and treated with 0 and 4 mg/L NN-SeNPs, respectively. NN-SeNPs exhibited an inhibition zone against A. veronii with a diameter of 16 ± 1.25 mm. The A. veronii infection increased the hepato-renal biomarkers (alanine and aspartate aminotransferases and creatinine) than the control group. An oxidative stress was the consequence of A. veronii infection (higher malondialdehyde and hydrogen peroxide levels with lower glutathione peroxidase superoxide, dismutase, and catalase activity). A. veronii infection resulted in lower immunological biomarker values (immunoglobulin M, lysozyme, and complement 3) with higher expression of the inflammatory cytokines (interleukin-1ß and tumor necrosis factor-ɑ) as well as lower expression of the anti-inflammatory cytokines (interleukin-10 and transforming growth factor-ß). Therapeutic application with 4 mg/L NN-SeNPs prevented the disease progression; and modulated the hepato-renal function disruptions, oxidant-immune dysfunction, as well as the pro/anti-inflammatory cytokines pathway in the A. veronii-infected fish. These findings suggest that NN-SeNPs, employed as a water therapy, can safeguard fish from the harmful effects of A. veronii and serve as a promising antibacterial agent for sustainable aquaculture.

Dietary Curcumin Modulating Effect on Performance, Antioxidant Status, and Immune-Related Response of Broiler Chickens Exposed to Imidacloprid Insecticide.

Birds appear to be especially vulnerable to adverse impacts from insecticides. This is especially true for imidacloprid (IMI), which is considered the most toxic to avian species. Recently, prospective studies aimed at including natural alternative products to alleviate the toxic impact that comes from insecticides have been increased. Focusing on herbal growth promoters and antioxidative medicament for the poultry industry, this ongoing experiment was conducted to examine the curcumin role (CUR) in mitigating IMI-prompted detrimental effects on broilers' performance, immunity, and antioxidant status. A total number of one hundred and fifty commercial meat-type Ross 308 broilers chicks (one-day-old) were randomly allocated into equal five groups (30 chicks/group and 10 birds/replicate). The first group (C) was the control; the second group (CUR) was fed a diet containing CUR at the level of 450 mg/kg; the third group (IMI) was fed control diet for 14 days and then was fed a diet containing IMI at the level of 50 mg/kg; the fourth group (CUR+IMI co-treated) was fed a diet containing CUR+IMI; and the fifth group (CUR+IMI pro/co-treated) was fed a diet containing CUR for 14 days as protective and then a diet containing CUR+IMI for the rest of the trial. CUR supplementation either in the (CUR pro/co-treated) or (CUR co-treated) groups significantly (p < 0.05) improved final body weight and total body weight gain while decreasing the total feed intake and feed conversion ratio when compared to the IMI-exposed and non-treated birds. CUR induced a significant (p < 0.05) enhancement in hematological indices, phagocytosis %, phagocytic index, intracellular killing capacity, total proteins, globulin, liver function enzymes, lysozyme activity, and immunoglobulin-G levels compared to IMI-exposed and non-treated birds. In addition, dietary supplementation of CUR significantly (p < 0.05) modulated oxidative stress-related biomarkers in splenic tissues (total antioxidant capacity, superoxide dismutase, catalase, and glutathione peroxidase) and decreased malondialdehyde levels (p < 0.05) when compared to IMI-exposed and non-treated birds. CUR significantly down-regulated mRNA levels expression of IL-1ß, TNF-α, and TLR4 and up-regulated IL-10 mRNA expression levels in spleens of birds when compared to those exposed to IMI-and non-treated. Finally, our results provided new insight into IMI-induced immuno-toxicity in broiler chickens. Furthermore, for the first time, our study informed that CUR can cause an in vivo protective effect against IMI toxicity, principally as a protective and/or as concurrent supplementation during the exposure to IMI toxicity.

Potential role of dietary Boswellia serrata resin against mancozeb fungicide-induced immune-antioxidant suppression, histopathological alterations, and genotoxicity in Nile tilapia, Oreochromis niloticus.

This study was established to look into the toxicological consequences of chronic exposure to a fungicide (mancozeb; MAZ) on the immune-antioxidant response, gene expressions, hepato-renal functions, and histological pictures of Nile tilapia (Oreochromis niloticus). Additionally, the effectiveness of Indian frankincense resin extract (IFRE) to mitigate their toxicity was taken into account. Fish (n =240; average body weight: 22.45 ± 2.21 g) were randomized into four groups for eight weeks in six replicates (control, IFRE, MAZ, and IFRE + MAZ), where ten fish were kept per replicate. The control and IFRE groups received basal diets that included 0.0 and 5 g/kg of IFRE without MAZ exposure. The MAZ and IFRE+MAZ groups received the same diets and were exposed to 1/10 of the 96-h of LC50 of MAZ (1.15 mg/L). The outcomes displayed that MAZ exposure resulted in a lower survival rate (56.67 %) and significantly decreased levels of immune-antioxidant variables (antiprotease, complement3, phagocytic activity, lysozyme, glutathione peroxidase, superoxide dismutase, and total antioxidant capacity) compared to the control group. The MAZ-exposed fish showed the greatest levels of lipid peroxide (malondialdehyde), alkaline phosphatase, alanine amino-transferase, and stress indicators (cortisol and glucose). Additionally, histopathological alterations, including vacuolation, severe necrosis, degeneration, and mononuclear cell infiltrations in the hepatic, renal, and splenic tissues resulted, besides a reduction in the melanomacrophage center in the spleen. A down-regulation of immune-antioxidant-associated genes [toll-like receptors (TLR-2 and TLR-7), nuclear factor kappa beta (NF-κß), transforming growth factor-beta (TGF-ß), phosphoinositide-3-kinase regulatory subunit 3 gamma b (pik3r3b), interleukins (IL-1ß and IL-8), glutathione synthetase (GSS), glutathione peroxidase (GPx), and superoxide dismutase (SOD)] were the consequences of the MAZ exposure. Remarkably, the dietary inclusion of IFRE in MAZ-exposed fish augmented the immune-antioxidant parameters, including their associated genes, decreased stress response, and increased survival rate (85 %) compared with the MAZ-exposed fish. Moreover, dietary IFRE improved hepato-renal function indices by preserving the histological architecture of the hepatic, renal, and splenic tissues. The insights of this study advocate the use of an IFRE-dietary addition to protect Nile tilapia from MAZ toxicity, which provides perspectives for future implementations in enhancing fish health for sustainable aquaculture.

Impacts of Solid-State Fermented Barley with Fibrolytic Exogenous Enzymes on Feed Utilization, and Antioxidant Status of Broiler Chickens.

The present and future high demand of common cereals as corn and wheat encourage the development of feed processing technology that allows for the dietary inclusion of other cereals of low nutritional value in poultry feeding. Barley grains contain anti-nutritional factors that limit their dietary inclusion in the poultry industry. The treatment of barley with solid-state fermentation and exogenous enzymes (FBEs) provides a good alternative to common cereals. In this study, barley grains were subjected to solid-state microbial fermentation using Lactobacillus plantarum, Bacillus subtilis and exogenous fibrolytic enzymes. This study aimed to assess the impact of FBEs on growth, feed utilization efficiency, immune modulation, antioxidant status and the expression of intestinal barrier and nutrient transporter-related genes. One-day-old broiler chicks (Ross 308, n = 400) comprised four representative groups with ten replicates (10 chicks/replicate) and were fed corn-soybean meal basal diets with inclusions of FBEs at 0, 5, 10 and 15% for 38 days. Solid-state fermentation of barley grains with fibrolytic enzymes increased protein content, lowered crude fiber and reduced sugars compared to non-fermented barley gains. In consequence, the group fed FBEs10% had the superior feed utilization efficiency and body weight gain (increased by 4.7%) with higher levels of nutrient metabolizability, pancreatic digestive enzyme activities and low digesta viscosity. Notably, the group fed FBEs10% showed an increased villi height and a decreased crypt depth with a remarkable hyperactivity of duodenal glands. In addition, higher inclusion levels of FBEs boosted serum immune-related parameters and intestinal and breast muscle antioxidants status. Intestinal nutrient transporters encoding genes (GLUT-1, CAAT-1, LAT1 and PepT-1) and intestinal barriers encoding genes (MUC-2, JAM-2, occludin, claudins-1 and ß-defensin 1) were upregulated with higher dietary FBEs levels. In conclusion, feeding on FBEs10% positively enhanced broiler chickens' performance, feed efficiency and antioxidant status, and boosted intestinal barrier nutrient transporters encoding genes.

Chitosan neem nanocapsule enhances immunity and disease resistance in nile tilapia (Oreochromis niloticus).

Finding eco-friendly alternatives for antibiotics in treating bacterial diseases affecting the aquaculture sector is essential. Herbal plants are promising alternatives, especially when combined with nanomaterials. Neem (Azadirachta indica) leaves extract was synthesized using a chitosan nanocapsule. Chitosan neem nanocapsule (CNNC) was tested in-vitro and in-vivo against the Aeromonas sobria (A. sobria) challenge in Nile tilapia. A preliminary experiment with 120 Nile tilapia was conducted to determine the therapeutic dose of CNNC, which was established to be 1 mg/L. A treatment study was applied for seven days using 200 fish categorized into four groups (10 fish/replicate: 50 fish/group). The first (control) and second (CNNC) groups were treated with 0 and 1 mg/L CNNC in water without being challenged. The third (A. sobria) and fourth (CNNC + A. sobria) groups were treated with 0 and 1 mg/L CNNC, respectively, and challenged with A. sobria (1 × 107 CFU/mL). Interestingly, CNNC had an in-vitro antibacterial activity against A. sobria; the minimum inhibitory concentration and minimum bactericidal concentration of CNNC against A. sobria were 6.25 and 12.5 mg/mL, respectively. A. sobria challenge caused behavioral alterations, skin hemorrhage, fin rot, and reduced survivability (60%). The infected fish suffered a noticeable elevation in the malondialdehyde level and hepato-renal function markers (aspartate aminotransferase, alanine aminotransferase, and creatinine). Moreover, a clear depletion in the level of the antioxidant and immune indicators (catalase, reduced glutathione, lysozymes, nitric oxide, and complement 3) was obvious in the A. sobria group. Treatment of the A. sobria-challenged fish with 1 mg/L CNNC recovered these parameters and enhanced fish survivability. Overall, CNNC can be used as a new versatile tool at 1 mg/L as a water treatment for combating the A. sobria challenge for sustainable aquaculture production.

Using Azadirachta indica protein hydrolysate as a plant protein in Nile tilapia (Oreochromis niloticus) diet: Effects on the growth, economic efficiency, antioxidant-immune response and resistance to Streptococcus agalactiae.

A feeding trial for 90 days was conducted on Nile tilapia (Oreochromis niloticus) (average weight: 25.50 ± 0.05 g) to evaluate the effect of dietary inclusion of Azadirachta indica seed protein hydrolysate (AIPH). The evaluation included the impact on the growth metrics, economic efficiency, antioxidant potential, hemato-biochemical indices, immune response, and histological architectures. A total of 250 fish were randomly distributed in five treatments (n = 50) and received diets included with five levels of AIPH (%): 0 (control diet, AIPH0), 2 (AIPH2), 4 (AIPH4), 6 (AIPH6) or 8 (AIPH8), where AIPH partially replace fish meal by 0, 8.7%, 17.4%, 26.1%, and 34.8%, respectively. After the feeding trial, a pathogenic bacterium (Streptococcus agalactiae, 1.5 × 108 CFU/mL) was intraperitoneally injected into the fish and the survival rate was recorded. The results elucidated that AIPH-included diets significantly (p < 0.05) enhanced the growth indices (final body weight, total feed intake, total body weight gain, and specific growth rate) and intestinal morpho-metrics (villous width, length, muscular coat thickness, and goblet cells count) in comparison to the control diet, with the AIPH8 diet recording the highest values. Dietary AIPH inclusion significantly improved (p < 0.05) the economic efficacy indicated by reduced feed cost/kg gain and increased performance index. The fish fed on the AIPH diets had noticeably significantly higher (p < 0.05) protein profile variables (total proteins and globulin) and antioxidant capabilities (superoxide dismutase and total antioxidant capacity) than the AIPH0 group. The dietary inclusion of AIPH significantly (p < 0.05) boosted the haematological parameters (haemoglobin, packed cell volume %, and counts of red blood cells and white blood cells) and immune indices (serum bactericidal activity %, antiprotease activity, and immunoglobulin M level) in a concentration-dependent manner. The blood glucose and malondialdehyde levels were significantly (p < 0.05) lowered by dietary AIPH (2%-8%). The albumin level and hepatorenal functioning parameters (aspartate aminotransferase, alanine aminotransferase, and creatinine) were not significantly (p > 0.05) altered by AIPH diets. Additionally, AIPH diets did not adversely alter the histology of the hepatic, renal or splenic tissues with moderately activated melano-macrophage centres. The mortality rate among S. agalactiae-infected fish declined as dietary AIPH levels rose, where the highest survival rate (86.67%) was found in the AIPH8 group (p < 0.05). Based on the broken line regression model, our study suggests using dietary AIPH at the optimal level of 6%. Overall, dietary AIPH inclusion enhanced the growth rate, economic efficiency, health status, and resistance of Nile tilapia to the S. agalactiae challenge. These beneficial impacts can help the aquaculture sector to be more sustainable.

Acute mancozeb-fungicide exposure induces neuro-ethology disruption, health disorders, and immune-oxidative dysfunction in Nile tilapia (Oreochromis niloticus).

An acute exposure study of mancozeb (MAZ) fungicide was applied on Oreochromis niloticus for 96-h duration. Three hundred fish (20.50 ± 1.60 g) were assigned into six groups (50 fish/ group; 10 fish/replicate) and exposed to different six concentrations (0, 4, 8, 12, 16, and 20 mg L-1) of MAZ for 96-h. The Probit analysis program was used to compute the 96-h lethal concentration 50 (96-h LC50) of MAZ. During the exposure duration, the fish's behavior, clinical symptoms, and mortalities were recorded daily. After the exposure period was ended, the hematological, biochemical, immunological, and oxidant/antioxidant parameters were evaluated. The results of this study recorded the 96-h LC50 of MAZ for O. niloticus to be 11.49 mg L-1. Acute MAZ exposure badly affected the fish's behavior in the form of increased the breath gasping and swimming activity with aggressive mode. The exposed fish showed excessive body hemorrhages and fin rot. The survival rate of the exposed fish to MAZ was 100, 80, 66, 50, 38, and 30% in 0, 4, 8, 12, 16, and 20 mg L-1 MAZ, respectively. The hematological indices (red blood cell count, hemoglobin, packed cell volume%, and white blood cell count) were significantly decreased by increasing the MAZ exposure concentration (8-20 mg L-1). The acetylcholine esterase activity and immune indices (lysozyme, nitric oxide, immunoglobulin M, complement 3) were decreased by MAZ exposure (4-20 mg L-1). Acute MAZ exposure induced hepato-renal dysfunction and elevated stress-related parameter (cortisol) by increasing the MAZ concentration. A significant reduction in the antioxidant parameters (total antioxidant activity, catalase, and superoxide dismutase) with increasing the lipid peroxidation marker (malondialdehyde) was noticed by acute MAZ exposure (4 -20 mg L-1) in O. niloticus. Based on these outcomes, the MAZ exposure induced toxicity to the fish evident in changes in fish behavior, neurological activity, hepato-renal functioning, and immune-antioxidant responses which suggest physiological disruption.

Future impact of thymoquinone-loaded nanoemulsion in rabbits: prospects for enhancing growth, immunity, antioxidant potential and resistance against Pasteurella multocida.

Phytochemical nanoemulsions, such as thymoquinone nanoemulsions (TQN), are regarded as innovative alternatives to antimicrobials that significantly improve the performance, digestion, antioxidant potential and immunity of rabbits. Thus, the potential effects of TQN on growth, digestibility, antioxidant potential, immunity and resistance against Pasteurella multocida (P. multocida) in rabbits were assessed. Herein, 240 rabbits were offered either a basal diet or diets fortified with three TQN-graded concentrations. At 60 days of age, rabbits were challenged with multidrug-resistant (MDR) virulent P. multocida strain. Our outcomes described that dietary inclusion of TQN, especially at higher concentrations, significantly enhanced the growth performance of rabbits, which was supported by increasing the levels of jejunal lipase, amylase and trypsin enzymes. Of note, the levels of muscle and jejunal antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and total antioxidant capacity (T-AOC)], serum immunological markers (IgG, IgG, IgM and total Igs) and blood phagocytic percentage were significantly provoked after TQN fortification; meanwhile, the levels of muscle and jejunal MDA, serum biochemical parameters (total cholesterol, TG and LDL), abdominal fat percentage, breast and thigh cholesterol were significantly decreased following TQN supplementations. Our findings showed that TQN protected rabbits against P. multocida experimental challenge as evidenced by reducing P. multocida counts in rabbits' lungs, downregulating the transcription levels of P. multocida virulence-related genes (ptfA, toxA and nanB) at 48 and 96 h post-infection and ameliorating the expression levels of cytokines-related genes (IL-1ß, IL-10, IL-8, IL-6, DEFB1, TNF-α, TLR-4 and TLR-2) at 96 h post-infection. Our findings suggest the utilization of TQN in rabbits' diets due to their stimulating effects on digestibility as well as their growth-promoting, anti-inflammatory, antioxidant, antibacterial, anti-virulence and immunostimulant properties, which enhance the rabbits' P. multocida resistance.

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.