Ameliorative Pharmacological Effects of Dietary Chlorella vulgaris and β-Glucan on Chlorpyrifos-Induced Oxidative Stress (MDA, GSH, SOD), Immunomodulation (TNF-α, IL-10), and Growth Indices in African Catfish (Clarias gariepinus)

Ameliorative Pharmacological Effects of Dietary Chlorella vulgaris and β-Glucan on Chlorpyrifos-Induced Oxidative Stress (MDA, GSH, SOD), Immunomodulation (TNF-α, IL-10), and Growth Indices in African Catfish (Clarias gariepinus) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ameliorative Pharmacological Effects of Dietary Chlorella vulgaris and β-Glucan on Chlorpyrifos-Induced Oxidative Stress (MDA, GSH, SOD), Immunomodulation (TNF-α, IL-10), and Growth Indices in African Catfish (Clarias gariepinus) Ahmed E. A. Mostafa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7537362/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2026 Read the published version in Veterinary Research Communications → Version 1 posted 4 You are reading this latest preprint version Abstract The present study was conducted to investigate the toxic effects of chlorpyrifos on growth performance, hepatorenal function, and antioxidant status in African catfish (Clarias gariepinus). One hundred and eighty fish (20 ± 6.1 g) were equally distributed into four groups: control group, chlorpyrifos group (0.3 mg/L), chlorpyrifos-CV group (5% CV), and chlorpyrifos-β-glucan group (0.1% β-glucan), and treatments were conducted for about 60 days. The results revealed that administration of chlorpyrifos significantly increased serum liver enzymes, system, innate immune response and comparing the protective role of dietary Chlorella vulgaris (CV) algae and β-glucan in intoxicated African catfish ( Clarias gariepinus ). One uric acid, creatinine, and malondialdehyde (MDA) in different tissues. Meanwhile, glutathione (GSH) and superoxide dismutase (SOD) in different tissues, as well as IgM, C-reactive protein (CRP), respiratory burst, lysozyme, and bactericidal activities were significantly decreased in the chlorpyrifos group. In addition, expression of TNF-α gene was up-regulated and IL-10 was down-regulated in spleen of chlorpyrifos-intoxicated fish. The treatment of chlorpyrifos-exposed fish with CV and β-glucan supplemented diets ameliorated hepatic damage and enhanced antioxidant activity and innate immune responses. Furthermore, dietary Chlorella vulgaris and β-glucan have a potent anti-inflammatory effect as they remarkably increased the expression of IL-10 and decreased TNF-α gene expression. The results also revealed that fish in chlorpyrifos-CV group had the highest survival rate, final body weight (FBW), and body weight gain (BWG). On the other hand, feed conversion ratio (FCR), specific growth rate (SGR), and protein efficiency ratio (PER) of control, chlorpyrifos-CV, and chlorpyrifos-β-glucan groups were higher than the chlorpyrifos group. However, the hepatosomatic index (HSI) and spleen-somatic index (SSI) were higher in the chlorpyrifos group than other experimental groups. Overall, CV and β-glucan can be recommended as a feed supplement to improve immunosuppression, oxidative damage, growth performance, and hemato-biochemical alterations induced by chlorpyrifos toxicity in African catfish ( Clarias gariepinus ) . Chlorella vulgaris β-glucan Chlorpyrifos African catfish (Clarias gariepinus) Immunostimulants Immune response Antioxidant system Gene expression Growth performance Fish health. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The African catfish (Clarias gariepinus) is among the most economically important fish species globally, extensively cultured in aquaculture and inland fisheries. However, the contamination of aquatic ecosystems by agricultural pesticides poses a significant threat to its health and survival (Das and Shasmal 2013 ). Organophosphorus pesticides, in particular, are predominant ecotoxicants in aquatic environments, exerting severe adverse effects on aquatic organisms, especially fish (Ngangom Nganbi Devi et al. 2024 ). Chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate] is widely employed in both agricultural practices and domestic pest control. It frequently enters surface waters through agricultural runoff and drainage systems (Selvaraj et al. 2022 ). Although chlorpyrifos degrades under specific environmental conditions, it may persist in aquatic habitats for prolonged periods, remaining biologically active for several months (Rebouças et al. 2016 ). Like other xenobiotics, chlorpyrifos contamination induces oxidative stress and substantial physiological disturbances in fish (Tripathi and Shasmal 2010 ). Exposure to this pesticide has been associated with marked hematological, biochemical, and immunological alterations, increasing disease susceptibility and compromising fish survival (Ringø and Song 2013 ). Additionally, chlorpyrifos has been shown to suppress non-specific immune responses (Brennan et al. 2024 ) and impair growth performance in various fish species (Tripathi and Shasmal 2010 ). In response to these challenges, aquaculture has increasingly turned to natural dietary supplements, including probiotics, prebiotics, and immunostimulants, as safer alternatives to chemical additives. These supplements are employed to promote growth, enhance immunity, and improve survival rates (Tawfeek et al. 2024 ). Chlorella vulgaris (CV), a unicellular freshwater microalga, has gained prominence as a probiotic feed additive due to its rich profile of bioactive compounds, including proteins, omega-3 and omega-6 polyunsaturated fatty acids, polysaccharides, vitamins, minerals, and photosynthetic pigments such as carotenoids and chlorophylls (Majumder and Kaviraj 2016 ). Dietary inclusion of CV has demonstrated beneficial effects on growth performance, innate immune responses, antioxidant enzyme activities, and resistance to infectious diseases in aquaculture species (Elias et al. 2023 ). Similarly, β-glucans, naturally occurring polysaccharides derived from the cell walls of plants, fungi, yeast, bacteria, and mushrooms, are considered potent immunostimulants in aquaculture (Scapigliati et al. 2024 ). These compounds enhance immune responses by stimulating phagocytic activity, activating the complement system, and promoting cytokine expression in macrophages, neutrophils, and dendritic cells (Tripathi and Shasmal 2010 ). Notably, β-glucans extracted from Saccharomyces cerevisiae have been reported to improve immune function and increase resistance to Aeromonas hydrophila infections in fish (Slotkin and Seidler 2025). Furthermore, dietary β-glucan supplementation has been associated with improved growth, enhanced antioxidant status ( El-Bab et al. 2025), and reduced inflammatory responses in fish (Najah 2025 ). To the best of our knowledge, this study is the first to comprehensively evaluate and compare the protective roles of Chlorella vulgaris (as a probiotic) and β-glucan (as a prebiotic) in mitigating hepatorenal toxicity, oxidative stress, immunosuppression, and growth impairments in African catfish (Clarias gariepinus) following subacute chlorpyrifos exposure. 2. Materials and Methods 2.1. Chemicals Chlorpyrifos (CPF) with a concentration of 48% was sourced from Adwia Pharmaceuticals (Cairo, Egypt) and freshly diluted in distilled water immediately prior to application. Pure Chlorella vulgaris (CV) powder was procured from Roquette Klötze GmbH & Co. KG, Klötze, Germany. β-glucan, extracted from Saccharomyces cerevisiae, was obtained from Hang Zhou Bio Technology Co., Ltd., China. 2.2. Diet Preparation Four experimental diets were formulated to be isonitrogenous (32% crude protein) and isocaloric (3,000 kcal DE/kg), meeting the nutritional requirements of Clarias gariepinus based on NRC guidelines (National Research Council 2011). In addition to the control basal diet, three dietary treatments were prepared: Basal diet supplemented with 5% Chlorella vulgaris (Khosravi et al. 2015). Basal diet supplemented with 0.1% β-glucan (Sakai 1999). The detailed composition of each diet is provided in Table 1 . All diets were processed into water-stable sinking pellets, carefully sealed in plastic bags, and stored under refrigeration until feeding. 2.3. Fish and Experimental Design A total of 180 healthy Clarias gariepinus (African catfish) with an initial average weight of 25 ± 4.3 g were obtained from a private fish farm located in Kafrelsheikh Governorate, Egypt. Fish were maintained in an indoor recirculating aquaculture system with well-aerated, dechlorinated freshwater, supported by internal filtration units. Key water quality parameters were strictly controlled: temperature 25 ± 1.2°C, dissolved oxygen 6.8 ± 0.4 mg/L, and pH range 7.4–7.8. During a two-week acclimatization period, fish were fed a commercial basal diet at 3% of their body weight, administered twice daily (from 9:00–10:00 AM and 4:00–5:00 PM). Following acclimation, fish were randomly distributed into four treatment groups, each consisting of triplicate tanks (15 fish per tank; 45 fish per group) housed in glass aquaria (40 × 60 × 30 cm) with continuous aeration and dechlorinated tap water. The fish were subjected to the following treatments for 60 consecutive days: Control Group: Fish received the basal diet without chlorpyrifos exposure. CPF Group: Fish were exposed to chlorpyrifos at 0.24 mg/L (equivalent to 1/10 of the 96-hour LC₅₀) in the rearing water and fed the basal diet. The chlorpyrifos concentration was selected based on the LC₅₀ value for Clarias gariepinus reported by Ayoola (2008), which is 2.4 mg/L. CPF + CV Group: Fish were exposed to the same chlorpyrifos concentration (0.24 mg/L) and fed the diet supplemented with 5% Chlorella vulgaris. CPF + β-glucan Group: Fish were exposed to the same chlorpyrifos concentration (0.24 mg/L) and fed the diet supplemented with 0.1% β-glucan. Ethical approval statement: This study is reported in accordance with the ARRIVE guidelines ( https://arriveguidelines.org( Table 1 Percentage of ingredients of experimental diets. Ingredients (%) Control Chlorella vulgaris β-glucan Yellow corn (8.5%) 12.50 18.00 12.50 Soybean meal (44%) 19.50 17.00 19.50 Fish meal 20.00 19.00 20.00 Wheat bran 38.00 30.00 38.00 Corn gluten 2.00 4.00 2.00 Gelatin 2.00 1.50 2.00 Oil 3.00 4.00 3.00 β-glucan 0.00 0.00 0.10 Chlorella vulgaris 0.00 5.00 0.00 Minerals and vitamins premix** 1.00 1.00 1.00 Salt 0.50 0.50 0.50 Dicalcium phosphate 0.10 0.10 0.10 Methionine 0.30 0.30 0.30 Chemical composition (%) Components Control Chlorella vulgaris β-glucan Crude protein 32.10 32.30 32.10 DE (kcal/kg) 3000 3000 3000 ** Vitamin mixture supplies the following per kilogram of diet: vit. A – 1,200,000 IU; vit. D3–200,000 IU; vit. E – 12,000 mg; vit. K3–2400 mg; vit. B1–4800 mg; vit. B2–4800 mg; vit. B6–4000 mg; vit. B12–4800 mg; folic acid – 1200 mg; vit. C – 48,000 mg; biotin – 48 mg; choline – 65,000 mg; niacin – 24,000 mg; Fe – 10,000 mg; Cu – 600 mg; Mg – 4000 mg; Zn – 6000 mg; I – 20 mg; Co – 2 mg; Se – 20 mg. Fish in the CPF + CV group were exposed to chlorpyrifos at the previously established concentration while receiving a diet supplemented with 5% Chlorella vulgaris . Similarly, fish in the CPF + β-glucan group were exposed to the same chlorpyrifos concentration and fed a diet supplemented with 0.1% β-glucan. To prevent the accumulation of waste metabolites, a static-renewal system was employed, where 80% of the aquarium water was replaced daily. Freshly prepared chlorpyrifos solutions were added after each water change to maintain the target exposure concentration. The survival rate of African catfish ( Clarias gariepinus ) was monitored across all experimental groups throughout the trial. 2.4. Sample Collection After 28 days of treatment, ten fish were randomly selected from each aquarium and lightly anesthetized using tricaine methanesulfonate (MS-222; FINQUEL®, ARGENT) at 30 mg/L, buffered with 60 mg/L sodium bicarbonate. Euthanasia was then performed using a higher MS-222 dose of 200 mg/L, buffered with 400 mg/L sodium bicarbonate (Ross and Ross 2008). Blood samples were collected from the caudal vessels of individual fish. Each fish provided two blood samples: one collected in EDTA-containing tubes for hematological analysis and respiratory burst evaluation (Blaxhall and Daisley 1973), and the other collected in plain tubes for serum separation. Serum samples were isolated by centrifugation at 3000 rpm for 10 minutes and stored at − 80°C for subsequent biochemical and immunological assessments (Tietz 1995). Tissue samples from the liver, spleen, and gills were carefully excised, rinsed in normal saline, and homogenized in ice-cold phosphate-buffered saline (PBS; pH 7.5). The homogenates were centrifuged at 3000 rpm for 15 minutes, and the resulting supernatants were stored at − 80°C for later antioxidant and oxidative stress analyses (Ohkawa et al. 1979). Additionally, portions of spleen tissue were preserved in RNA Later® (Qiagen) at 4°C overnight, then stored at − 80°C for gene expression studies (Chomczynski and Sacchi 1987). The remaining fish continued under the same experimental protocols until day 60. At the end of the experiment, all surviving fish were counted to calculate the final survival rates and individually weighed to determine weight gain (WG) (Hopkins 1992). Subsequently, ten fish from each aquarium were euthanized, and the liver and spleen were rapidly excised and weighed to assess organ indices (Schreck and Moyle 1990). All experimental procedures were performed in compliance with the Animal Care and Use guidelines of Kafrelsheikh University and were approved by the local Animal Care and Use Committee. 2.5. Hematological Analysis Total erythrocyte (RBC) and leukocyte (WBC) counts were determined using Natt-Herrick’s solution for dilution and manual hemocytometer counting as described by Blaxhall and Daisley (1973). Hemoglobin (Hb) concentrations were measured spectrophotometrically via the cyanmethemoglobin method (Drabkin 1949). The packed cell volume (PCV) and red blood cell indices—mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)—were calculated following the methodology of Wintrobe (1934). Differential leukocyte counts were performed on Giemsa-stained blood smears (Blaxhall and Daisley 1973). 2.6. Serum Biochemical Analysis Serum biochemical markers were quantified using commercial diagnostic kits: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (BioMed, Egypt), alkaline phosphatase (ALP) (Spectrum, Egypt), total protein and albumin (Bio-Diagnostic, Egypt), creatinine (Human, Germany), and uric acid (BioMed, Egypt). All measurements were conducted using a spectrophotometer (5010 Photometer, BM Co., Germany) according to the manufacturers’ protocols (Reitman and Frankel 1957). 2.7. Assessment of Oxidative Stress and Antioxidant Status Antioxidant and oxidative stress biomarkers, including malondialdehyde (MDA), glutathione (GSH), catalase, and superoxide dismutase (SOD), were quantified in liver, spleen, and gill homogenates using commercial assay kits (Bio-Diagnostic, Egypt). All assays were performed spectrophotometrically following the kit instructions (Aebi 1984). 2.8. Evaluation of Immunological Parameters 2.8.1. Respiratory Burst Activity The respiratory burst of phagocytes was assessed using the nitroblue tetrazolium (NBT) reduction assay following the procedure of Wijendra and Pathiratne ( 2006 ). 2.8.2. Serum Lysozyme Activity Serum lysozyme activity was determined following the method of Ghareghanipoor et al. (2017) with slight modifications. 2.8.3. Serum Bactericidal Activity Bactericidal activity was assessed following the method of Abdelhamid et al. (2018). 2.8.4. Determination of CRP and IgM Levels C-reactive protein (CRP) levels were semi-quantitatively assessed using a rapid latex agglutination test following the method of Tillett and Francis ( 1930 ). Immunoglobulin M (IgM) levels were quantitatively measured using a turbidity assay, based on immune complex formation, as described by Dati and Lammers (1989). 2.9. Gene Expression Analysis of Immune-Related Genes 2.9.1. RNA Extraction and cDNA Synthesis Total RNA was isolated from spleen tissues using the RNAeasy Mini Kit (Qiagen, Germany) according to the procedure described by Zhang et al. (2012) with minor modifications. 2.9.2. Quantitative Real-Time PCR (qRT-PCR) Gene expression levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) were quantified using SYBR® Green Master Mix (Thermo Fisher Scientific, USA) and the CFX Connect Real-Time PCR System (Bio-Rad, USA). β-actin served as the internal reference gene. Primer sequences were selected according to Wang et al. (2008) and Li et al. (2013). The qPCR protocol included an initial denaturation at 95°C for 10 minutes, followed by 40 amplification cycles (denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds). Relative gene expression was calculated using the 2^−ΔΔCT method (Livak and Schmittgen 2001 ). 2.10. Chlorpyrifos Residue Analysis Chlorpyrifos residues in fish liver tissues were extracted and cleaned following the QuEChERS method with minor adjustments as described by Mastovska and Lehotay ( 2004 ). The cleaned extracts were analyzed using Gas Chromatography with an Electron Capture Detector (GC-ECD; Agilent 7890B, USA). Each sample was analyzed in triplicate to ensure accuracy and reproducibility. Table 2 Primer sequences used for RT-PCR analysis. Gene Primer Sequence (5′ to 3′) Amplicon size (bp) Accession number Tumor necrosis factor-α TNFα-F: CAGACTGTAGCCCTGTCACCA 85 AY428948.1 TNFα-R: GTCACAGAGTGGGAGGTTGAT Interleukin 10 IL-10-F: CGCTGTCATCGATTTCTCCAT 97 XM_003441366.2 IL-10-R: ATCTCCTGTTCCCTCCTGCTT Elongation factor 1α EF1α-F: GACAACATGCTTGAGGCTGAC EF1α-R: CCAATACCAGTCTCCACACCA 83 AB075952.1 2.11. Growth Performance and Feed Utilization Assessment The evaluation of growth performance and feed efficiency was conducted by determining several key parameters: weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), hepatosomatic index (HSI), spleensomatic index (SSI), and survival rate (SR), as outlined by Cech and Myrick ( 2000 ). The following formulas were employed to calculate these parameters for Oreochromis niloticus : Weight Gain (WG, g) = Final average body weight (g) − Initial average body weight (g) Feed Conversion Ratio (FCR) = Total feed intake (g) ÷ Total weight gain (g) Specific Growth Rate (SGR, %/day) = 100 × [(Ln final body weight − Ln initial body weight) ÷ Rearing period (days)] Protein Efficiency Ratio (PER) = Total weight gain (g) ÷ Total protein consumed (g) Hepatosomatic Index (HSI, %) = (Liver weight ÷ Body weight) × 100 Spleensomatic Index (SSI, %) = (Spleen weight ÷ Body weight) × 100 Survival Rate (SR, %) = (Number of surviving fish ÷ Initial number of fish) × 100 2.12. Statistical Analysis All results are expressed as mean values ± standard error (SE). Statistical analysis was performed using IBM SPSS software (version 26, USA). Data were analyzed through one-way analysis of variance (ANOVA) to identify significant differences among the treatment groups. When significant differences were detected, Tukey’s multiple comparison test was applied to determine pairwise differences between means. Statistical significance was accepted at P < 0.05. 3. Results 3.1. Blood cell count There were no significant differences in RBC count, hemoglobin concentration, and PCV percentage among CPF, CPF-C. vulgaris, and CPF-β-glucan treated fish compared to the control group (p < 0.05). However, total leukocytic count (TLC) and lymphocyte count were significantly decreased in the CPF-intoxicated group compared to the control (p < 0.05), while heterophil counts were significantly elevated in the CPF group compared to the control (p < 0.05) (Table 3 ). Feeding CPF-intoxicated fish with Chlorella vulgaris or β-glucan supplemented diets significantly improved the TLC and lymphocyte counts compared to the CPF group (p < 0.05). Additionally, heterophil counts remained significantly higher in the CPF-C. vulgaris and CPF-β-glucan groups compared to both the control and CPF groups (p < 0.05). Monocyte counts did not show significant differences between all groups (p < 0.05) (Table 3 ). Table 3 Hematological parameters of African catfish ( Clarias gariepinus ) treated with Chlorpyrifos, Chlorella vulgaris and β-glucan. Experimental groups Control CPF CPF-CV CPF-β-glucan RBCs (10⁶/µL) 1.95 ± 0.12ᵃ 2.10 ± 0.18ᵃ 2.20 ± 0.21ᵃ 2.40 ± 0.25ᵃ Hb (g/dl) 8.60 ± 0.70ᵃ 9.85 ± 1.20ᵃ 9.15 ± 0.50ᵃ 9.50 ± 0.40ᵃ PCV (%) 29.00 ± 1.10ᵃ 30.20 ± 1.50ᵃ 28.90 ± 1.00ᵃ 31.00 ± 1.30ᵃ WBCs (10³/µL) 45.50 ± 3.00ᵇ 37.80 ± 3.50ᶜ 60.40 ± 4.00ᵃ 56.20 ± 3.20ᵃ Lymphocyte (10³/µL) 24.10 ± 1.20ᵃ 13.50 ± 1.40ᶜ 26.00 ± 3.00ᵃ 21.20 ± 2.30ᵇ Heterophil (10³/µL) 17.20 ± 1.80ᶜ 22.50 ± 2.50ᵇ 30.50 ± 2.30ᵃ 32.00 ± 1.90ᵃ Monocyte (10³/µL) 4.50 ± 0.50ᵃ 4.10 ± 0.70ᵃ 4.80 ± 0.40ᵃ 4.90 ± 0.30ᵃ Data are expressed as Mean ± SEM (n = 5).Means in the same row with different superscripts are significantly different (p < 0.05).CPF, Chlorpyrifos; CV, Chlorella vulgaris; RBCs, Red blood cell count; Hb, Hemoglobin; PCV, Packed Cell Volume; WBCs, White blood cell count. 3.2. Biochemical parameters The Chlorpyrifos (CPF) induced hepatotoxicity was indicated by a significant elevation in ALT and AST activities compared to the control group (p < 0.05). However, the CPF-CV and CPF-β-glucan treated groups showed significantly lower serum levels of ALT and AST compared to the CPF group (p < 0.05). No significant differences in ALP levels were observed among all treated groups (Table 4 ). Serum total protein level was significantly higher in the CPF-β-glucan treated fish compared to the other groups (p < 0.05). Serum albumin level was slightly decreased in CPF intoxicated fish compared to the other groups. Meanwhile, globulin was significantly decreased in the CPF group compared to the control and CPF-β-glucan groups (p < 0.05). In fish fed β-glucan supplemented diets, the globulin level was significantly higher, while the albumin-globulin ratio (A/G) was significantly lower than those of the CPF group (p < 0.05) (Table 4 ). CPF, CPF-CV and CPF-β-glucan treated fish showed significantly higher levels of creatinine and uric acid than the control fish (p < 0.05). Additionally, uric acid levels in both CPF-CV and CPF-β-glucan groups were significantly lower than those in the CPF group (p < 0.05) (Table 4 ). Table 4 Serum biochemical parameters of African catfish ( Clarias gariepinus ) treated with Chlorpyrifos, Chlorella vulgaris and β-glucan. Experimental groups Control CPF CPF-CV CPF-β-glucan ALT (U/L) 11.80 ± 1.05ᶜ 26.90 ± 2.90ᵃ 18.60 ± 1.50ᵇ 19.50 ± 1.70ᵇ AST (U/L) 56.20 ± 4.90ᶜ 112.50 ± 9.80ᵃ 95.40 ± 7.10ᵇ 82.70 ± 5.80ᵇ ALP (U/L) 78.00 ± 6.90ᵃ 88.20 ± 5.90ᵃ 76.50 ± 5.70ᵃ 85.30 ± 4.10ᵃ Total protein (g/dl) 4.40 ± 0.38ᵇ 3.55 ± 0.40ᶜ 4.20 ± 0.22ᵇ 4.85 ± 0.60ᵃ Albumin (g/dl) 1.30 ± 0.14ᵃ 1.22 ± 0.11ᵃ 1.25 ± 0.10ᵃ 1.29 ± 0.18ᵃ Globulin (g/dl) 3.10 ± 0.30ᵃ 2.33 ± 0.35ᶜ 2.95 ± 0.12ᵃᵇ 3.56 ± 0.50ᵃ A/G ratio (%) 0.42 ± 0.04ᵃᵇ 0.52 ± 0.06ᵃ 0.42 ± 0.05ᵃᵇ 0.36 ± 0.07ᵇ Creatinine (mg/dl) 0.28 ± 0.015ᵇ 0.37 ± 0.017ᵃ 0.34 ± 0.020ᵃ 0.33 ± 0.019ᵃ Uric acid (mg/dl) 5.30 ± 0.40ᶜ 9.50 ± 0.38ᵃ 7.80 ± 0.28ᵇ 7.90 ± 0.15ᵇ Data are expressed as Mean ± SEM (n = 5).Means in the same row with different superscripts are significantly different (p < 0.05).CPF, Chlorpyrifos; CV, Chlorella vulgaris; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; A/G ratio, albumin/globulin ratio. 3.3. Antioxidant and Oxidative Stress Parameters Spleen MDA level was significantly increased in CPF and CPF-CV groups compared to the control and CPF-β-glucan groups (p < 0.05). Additionally, liver MDA levels were significantly elevated in CPF group in contrast to all other groups (p < 0.05) (Fig. 1A). CPF exposure significantly decreased GSH levels in liver and gills compared to the control group (p < 0.05). Treatment of CPF-exposed fish with CV and β-glucan supplemented diets significantly improved liver GSH levels compared to CPF group (p < 0.05). Notably, only the β-glucan supplemented diet was able to restore gill GSH levels back to normal (Fig. 1B). Spleen and liver catalase (CAT) levels showed no significant differences among the experimental groups. However, the CPF-CV group demonstrated significantly higher catalase activity compared to all other groups (p < 0.05) (Fig. 1C). Moreover, liver SOD activity was significantly lower in CPF and CPF-CV groups compared to the control group (p < 0.05). Interestingly, the highest liver SOD activity was recorded in CPF-β-glucan group compared to all other groups, including the control (p < 0.05) (Fig. 1D). No significant differences were detected in gill MDA, spleen GSH, and gill SOD levels among all groups (Fig. 1A, 1B, 1D). Figure 1. (A) MDA; Malondialdehyde, (B) GSH; Reduced Glutathione, (C) Catalase, and (D) SOD; Superoxide Dismutase activities in spleen, liver, and gills of Clarias gariepinus treated with Chlorpyrifos, Chlorella vulgaris, and β-glucan. Data are expressed as Mean ± SEM (n = 5). Values with different superscript letters are significantly different (P < 0.05). 3.4. Immune Parameters Chlorpyrifos (CPF) intoxicated fish showed a significant reduction in respiratory burst activity compared to the control group (p < 0.05). CPF groups fed with diets containing Chlorella vulgaris (CV) and β-glucan exhibited significantly higher respiratory burst activity compared to both CPF and control groups (p < 0.05) (Fig. 2 ). Serum lysozyme activity was significantly lower in CPF-treated fish than all other groups (p < 0.05). Only the β-glucan treated group showed a significant increase in lysozyme activity compared to other treatments (p < 0.05) (Fig. 3 A). Moreover, CPF toxicity significantly decreased bactericidal activity compared to both CPF-CV and CPF-β-glucan groups (p < 0.05). The highest bactericidal activity was recorded in the CV-supplemented group compared to all other groups (p < 0.05) (Fig. 3 B). Serum immunoglobulin G (IgM) and C-reactive protein (CRP) levels were significantly reduced in CPF-intoxicated fish compared to the control group (p < 0.05). However, these parameters were significantly elevated in the CPF-β-glucan group compared to the CPF and control groups (p < 0.05). Additionally, dietary supplementation with CV significantly improved IgG levels but had no significant effect on CRP levels compared to the CPF group (p < 0.05) (Fig. 4 ). 3.5. Expression of Immune-Related Genes The TNF-α transcript level was significantly up-regulated in the spleen of all CPF-treated fish compared to the control group (p < 0.05). This significant increase was also observed in the spleen of CPF-CV and CPF-β-glucan treated fish compared to the control fish (p < 0.05) (Fig. 5 A). The expression of IL-10 was significantly down-regulated in the spleen of CPF, CPF-CV, and CPF-β-glucan treated fish compared to the control group (p < 0.05). However, IL-10 expression was significantly higher in the CPF-CV and CPF-β-glucan supplemented fish than in CPF-intoxicated fish (p < 0.05) (Fig. 5 B). 3.6. CPF Residues in Liver Tissue The CPF residues results are presented in Table 6 and showed that the highest concentration of CPF residue was detected in CPF-exposed fish (CPF group) compared to CPF-CV and CPF-β-glucan treated groups (p < 0.05). Meanwhile, the CPF concentration was significantly lower in CPF-CV group compared to CPF-β-glucan group (p < 0.05). Table 6 Concentration residues of Chlorpyrifos (CPF) (ng/g tissue) in liver tissue of African catfish ( Clarias gariepinus ). Experimental groups CPF Residues (ng/g tissue) Control 0.000 ± 0.000 d CPF 0.175 ± 0.004 a CPF-CV 0.103 ± 0.003 c CPF-β-glucan 0.128 ± 0.002 b Data are expressed as Mean ± SEM. Means in the same row with different superscripts are significantly different (p < 0.05). CPF, Chlorpyrifos; CV, Chlorella vulgaris. 3.7. Survival rate and growth performance The survival rate of African catfish ( Clarias gariepinus ) was significantly lower in CPF treated fish compared to all other treated groups, and the highest survival rate was recorded in the CV treated fish (P < 0.05) (Fig. 6 ). Additionally, the lowest growth performance was observed in the CPF treated group (p < 0.05). Fish in the CPF-CV group showed significantly higher final body weight (FBW) and body weight gain (BWG) than the fish in the other experimental groups (p < 0.05). On the other hand, the feed conversion ratio (FCR), specific growth rate (SGR), and protein efficiency ratio (PER) of the control, CPF-CV, and CPF-β-glucan groups were significantly better than those of the CPF group (p < 0.05). The data also exhibited significantly increased hepatosomatic index (HSI) and spleensomatic index (SSI) in the control and CPF groups compared to the CPF-CV and CPF-β-glucan groups (p < 0.05) (Table 5 ). Table 5 Growth performance of African catfish ( Clarias gariepinus ) treated with Chlorpyrifos, Chlorella vulgaris, and β-glucan. Experimental groups Control CPF CPF-CV CPF-β-glucan IW (g/fish) 21.80 ± 1.15 a 20.90 ± 1.05 a 21.15 ± 0.97 a 21.75 ± 1.10 a FBW (g/fish) 55.80 ± 2.10 b 40.95 ± 3.45 c 64.25 ± 3.62 a 53.70 ± 3.05 b BWG (g/fish) 34.00 ± 2.40 b 20.05 ± 2.80 c 43.10 ± 3.10 a 31.95 ± 3.20 b FCR 1.85 ± 0.11 b 2.68 ± 0.36 a 1.78 ± 0.14 b 1.95 ± 0.17 b SGR 1.75 ± 0.03 a 1.18 ± 0.12 b 1.92 ± 0.08 a 1.70 ± 0.10 a PER 1.80 ± 0.06 a 1.15 ± 0.17 b 1.95 ± 0.14 a 1.85 ± 0.12 a HIS 1.90 ± 0.13 a 2.40 ± 0.26 a 1.50 ± 0.06 ab 1.30 ± 0.08 b SSI 0.65 ± 0.05 a 0.50 ± 0.09 a 0.30 ± 0.03 b 0.32 ± 0.04 b Data are expressed as Mean ± SEM (n = 5). Means in the same row with different superscripts are significantly different (p < 0.05).CPF, Chlorpyrifos; CV, Chlorella vulgaris; IW, Initial weight; FBW, Final body weight; BWG, Body weight gain; FCR, Feed conversion ratio; SGR, Specific growth rate; PER, Protein efficiency ratio; HSI, Hepatosomatic index; SSI, Spleen-somatic index. 4. Discussion In this study, African catfish ( Clarias gariepinus ) exposed to chlorpyrifos (CPF) exhibited insignificant changes in serum total protein and a slight reduction in albumin levels. However, a significant decline in globulin concentrations was observed. Additionally, CPF exposure resulted in marked increases in serum creatinine and uric acid compared to the control group. These findings are consistent with Alishahi et al. (2014) , who reported a significant decrease in total protein and globulin, alongside a non-significant change in albumin levels in Barbus sharpeyi juveniles exposed to diazinon. Conversely, dietary supplementation with Chlorella vulgaris (CV) or β-glucan improved the serum biochemical profile of CPF-exposed African catfish. Specifically, ALT, AST, and uric acid levels were significantly reduced in treated groups, and total protein and globulin levels significantly improved, particularly in fish receiving β-glucan. The hepatoprotective effect of CV can likely be attributed to its antioxidant-rich composition, which stabilizes hepatocyte membranes and minimizes the leakage of intracellular enzymes. Zahran et al. (2018) similarly demonstrated that dietary inclusion of 5% and 10% CV powder over 21 days improved liver and kidney function markers and enhanced total protein, albumin, and globulin levels in Nile tilapia under sodium arsenite-induced stress. The antioxidant potential of β-1,3-glucan has also been established, with its ability to neutralize free radicals and prevent lipid peroxidation in hepatic tissues ( Abdel-Tawwab et al. 2019 ). El-Keredy et al. ( 2019 ) showed that β-glucan supplementation (100 mg/kg diet for 10 weeks) effectively normalized elevated serum markers, including AST, ALT, urea, uric acid, and creatinine in copper-intoxicated Nile tilapia. It is well recognized that exposure to pesticides in fish leads to oxidative stress due to excessive reactive oxygen species (ROS) generation, which overwhelms the antioxidant defense system ( Livingstone 2001 ). Key antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) play crucial roles in cellular protection against oxidative injury ( Winston 1991 ). In the current study, CPF exposure significantly elevated malondialdehyde (MDA) levels in the spleen and liver of African catfish, indicating increased lipid peroxidation, while significantly depleting antioxidant enzymes including glutathione (GSH) in gill and hepatic tissues and SOD activity in the spleen. This heightened lipid peroxidation is likely linked to CPF metabolism and excessive free radical generation ( Soltan 2014 ), which also contributes to the depletion of GSH reserves ( Kavitha and Rao 2008 ). Xenobiotic exposure may further exacerbate GSH depletion by enhancing its breakdown or impairing its synthesis ( Oruç and Usta 2007 ). Variations in antioxidant enzyme activities across different tissues may reflect tissue-specific sensitivity, with the liver generally more susceptible to pesticide-induced oxidative stress due to its central role in xenobiotic metabolism ( Livingstone 2001 ). CPF preferentially accumulates and undergoes biotransformation in hepatic tissues ( Livingstone 2001 ), and antioxidant responses can differ based on the chemical nature of the xenobiotic, duration of exposure, and species-specific tolerances ( Van der Oost et al. 2003 ). Supporting this, previous investigations in CPF-exposed African catfish demonstrated similar oxidative disturbances, including elevated hepatic MDA levels and significant reductions in hepatic SOD, CAT, glutathione peroxidase (GSH-Px), and total antioxidant capacity (TAC) (Rebouças et al. 2016 ). Comparable outcomes were noted by Abdelkhalek et al. (2015) in Nile tilapia, where pesticide exposure resulted in substantial declines in catalase, GSH, and SOD activities, coupled with increased MDA accumulation in liver, kidney, and gill tissues. The administration of CV in this study significantly ameliorated CPF-induced oxidative damage. CV’s potent antioxidant components—such as flavonoids, carotenoids, chlorophyll, tocopherols, and polyphenols—may contribute to ROS scavenging and inhibition of lipid peroxidation (Abou-Zeid and Hussein 2022 ). CV supplementation enhanced GSH concentrations in hepatic and gill tissues and improved gill catalase activity relative to the CPF-only group. These findings align with those of Zahran and Risha ( 2014 ) , who reported that dietary CV effectively normalized MDA and H₂O₂ levels and elevated catalase and GSH activities in fish exposed to sodium arsenite. Additionally, CV co-treatment in herbicide-exposed fish was shown to increase serum SOD and GSH-Px activities while lowering MDA concentrations ( Pradhan et al. 2023 ). Similarly, β-glucan supplementation in this study conferred notable antioxidant benefits. β-glucan significantly decreased splenic MDA levels and enhanced both GSH and SOD activities in the liver and spleen of CPF-exposed fish. These observations correspond with Dawood et al. (2019) , who demonstrated improved antioxidant status in Nile tilapia fed β-glucan (1 g/kg diet) for 60 days, including reductions in serum MDA and increases in SOD and GSH-Px. Further supporting evidence from common carp exposed to fipronil and lead nitrate confirmed that β-glucan supplementation boosted hepatic antioxidant enzyme activities and reduced oxidative stress markers (El-Keredy et al. 2019 ). Regarding immune function, CPF exposure led to significant immunosuppression, evidenced by reduced respiratory burst activity, lysozyme levels, IgM concentrations, and C-reactive protein (CRP), while bactericidal activity was marginally affected. These results corroborate findings by Hajirezaee et al. ( 2019 ), who reported decreased immunoglobulin, lysozyme, and respiratory burst activities in diazinon-exposed rainbow trout. Previous research has also demonstrated reductions in lysozyme and bactericidal activities and downregulation of splenic IgM gene expression following pesticide exposure (Saleh et al. 2021 ). Lysozyme suppression following diazinon exposure was further confirmed in rainbow trout (Uner et al. 2006 ). CPF’s immunosuppressive effects may be linked to impaired leukocyte differentiation, reduced protein synthesis, and general immunotoxicity ( Alishahi et al. 2014 ). Additionally, CPF-induced hepatic damage and apoptosis may compromise the liver’s capacity to synthesize total protein and immunoglobulins (Mostafalou and Abdollahi 2013 ). Interestingly, dietary CV supplementation in this study restored immune functions, including lysozyme activity, respiratory burst, bactericidal activity, and IgM levels, in CPF-exposed fish, indicating a strong immunostimulatory potential. This may be explained by the bioactive components in CV, such as omega-3 and omega-6 fatty acids and polysaccharides, which can activate immune cells via receptor binding (Abou-Zeid and Hussein 2022 ). Comparable improvements in immune parameters were previously reported in African catfish fed CV-supplemented diets following pollutant exposure (Abdel-Tawwab et al. 2010 ). Similarly, β-glucan supplementation effectively counteracted CPF-induced immunosuppression, significantly enhancing respiratory burst, lysozyme activity, CRP levels, IgM concentrations, and bactericidal responses. The immunomodulatory effects of β-glucan likely involve its interaction with macrophage and dendritic cell receptors, promoting IL-10 production (Vetvicka and Vetvickova 2010 ) and stimulating complement activation, phagocytosis, and non-specific immune responses (Brown and Gordon 2005 ). Supporting this, El-Boshy et al. ( 2015 ) demonstrated that β-glucan (0.1% of diet for 21 days) significantly improved lysozyme, bactericidal activity, nitric oxide production, and macrophage oxidative burst in immunosuppressed African catfish. Similar enhancements were observed by Dawood et al. ( 2018 ), who reported elevated phagocytic activity, respiratory burst, and lysozyme levels in β-glucan-fed Nile tilapia. At the molecular level, CPF exposure significantly upregulated splenic TNF-α gene expression while downregulating IL-10 in African catfish, indicative of an inflammatory response. Comparable pro-inflammatory gene expression patterns were noted in diazinon-exposed rainbow trout (Hajirezaee et al. 2019 ), likely reflecting CPF-induced tissue damage and macrophage activation ( Slotkin and Seidler 2025 ). CPF’s stimulation of ROS production may activate the NF-κB pathway, subsequently inducing pro-inflammatory mediators such as TNF-α, IL-1β, IL-6, COX-2, and iNOS (Mostafalou and Abdollahi 2013 ). In contrast, both CV and β-glucan supplementation significantly modulated the inflammatory response by decreasing TNF-α expression and enhancing IL-10 levels. CV’s anti-inflammatory properties may be linked to its carotenoid content, particularly violaxanthin, which suppresses ROS production and downregulates NF-κB-mediated inflammatory cascades (Najah 2025 ). Supporting evidence from prior studies indicated that CV supplementation (10%) significantly attenuated pro-inflammatory cytokine expression in sodium arsenite-intoxicated fish (Zahran and Risha 2014 ). The anti-inflammatory role of β-glucan is similarly supported by its capacity to reduce pro-inflammatory cytokines while upregulating IgM and IL-10 expression (Vetvicka et al. 2019 ; Meena et al. 2013 ), alongside its antioxidant properties that further limit inflammation (Safi et al. 2014 ). Additionally, CPF exposure significantly reduced both survival rate and growth performance, in agreement with previous studies demonstrating the adverse effects of sub-lethal pesticide exposure on fish growth and survival (Rebouças et al. 2016 ). Other reports also confirmed that herbicide exposure negatively impacts growth metrics in African catfish (Uner et al. 2006 ). However, dietary supplementation with CV or β-glucan significantly improved the survival and growth of CPF-exposed fish. El-Boshy et al. ( 2015 ) reported similar enhancements in survival with Chlorella supplementation. The beneficial impact of β-glucan on survival and resilience against toxins and pathogens has been widely documented in various fish species (Dawood and Koshio 2016 ). Notably, CV supplementation led to superior growth performance compared to β-glucan, potentially due to its high-quality protein, essential fatty acids, and growth-promoting compounds like the S-nucleotide adenosyl peptide complex (Keshavanath and Renuka 1998 ; Hossain et al. 2011 ). CV’s rich nutrient profile, including omega-3 and omega-6 fatty acids, proteins, polysaccharides, vitamins, minerals, chlorophyll, and carotenoids, likely contributed to these improvements (Li et al. 2007 ). Growth enhancement with β-glucan may be attributed to its degradation by glucanase, preserving protein for growth (protein-sparing effect) and stimulating digestive enzyme secretion (Meena et al. 2013 ; Dawood et al. 2018 ). As the liver is the primary site for xenobiotic accumulation and metabolism in fish ( Livingstone 2001 ), CPF was predominantly detected in hepatic tissues. Dietary CV and β-glucan significantly reduced hepatic CPF accumulation. The hepatosomatic index (HSI), a key indicator of liver health, was improved in supplemented groups, suggesting mitigation of CPF-induced hepatic dysfunction. Similar HSI increases under pesticide stress were previously reported in common carp (Cech and Myrick 2000 ). In conclusion, this study demonstrates that dietary CV and β-glucan supplementation can effectively alleviate CPF-induced hematological, hepatic, renal, and oxidative damage in African catfish. Both supplements improved immune responses and modulated pro-inflammatory gene expression. Notably, CV exhibited superior protective effects on growth performance and survival. Future research should explore additional natural strategies, including probiotics and prebiotics, to mitigate pesticide toxicity in aquaculture and support environmental sustainability. 5. Conclusion The present study demonstrated that dietary supplementation with Chlorella vulgaris (CV) and β-glucan can provide protective effects against chlorpyrifos (CPF)-induced toxicity in African catfish. Both feed additives showed potential in mitigating immunosuppression, oxidative stress, hepatic alterations, and inflammatory responses associated with CPF exposure. CV appeared more effective in enhancing growth performance and survival rates, which may be attributed to its nutritional profile and bioactive constituents, while β-glucan exerted notable immunomodulatory and growth-promoting actions. Moreover, both supplements were associated with reduced CPF accumulation in hepatic tissues, suggesting a possible role in detoxification. However, the present work was limited by its experimental scope, including the use of a single fish species, specific dosages, and short-term exposure conditions. Therefore, extrapolation of these findings to other aquaculture systems should be made with caution. Future investigations should explore different fish species, longer-term trials, molecular mechanisms of action, and potential synergistic effects of CV and β-glucan with other natural immunostimulants or probiotics. Overall, the findings support the potential use of CV and β-glucan as sustainable feed additives to enhance fish health and resilience under chemical stress, but further studies are required before large-scale application in commercial aquaculture. Abbreviations ALP Alkaline phosphatase ALT Alanine aminotransferase AST Aspartate aminotransferase BWG Body weight gain CAT Catalase COX-2 Cyclooxygenase-2 CPF Chlorpyrifos CRP C-reactive protein CV Chlorella vulgaris FBW Final body weight FCR Feed conversion ratio GPx Glutathione peroxidase HSI Hepatosomatic index IgM Immunoglobulin M IL-10 Interleukin-10 IL-1β Interleukin-1 beta iNOS Inducible nitric oxide synthase NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NO Nitric oxide PER Protein efficiency ratio RBCs Red blood cells ROS Reactive oxygen species SGR Specific growth rate SOD Superoxide dismutase TGF-β1 Transforming growth factor beta 1 TLC Total leukocytic count TNF-α Tumor necrosis factor-alpha WBCs White blood cells Declarations Funding The authors did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors for this research. Competing Interests The authors declare that they have no financial or non-financial competing interests that could have influenced the work reported in this paper. Author Contributions Ahmed E. A. Mostafa conceived and designed the study, performed the experimental procedures, analyzed the results, and drafted the manuscript. The author approved the final version of the manuscript. Ethical Approval All experimental procedures involving African catfish (Clarias gariepinus) were conducted in accordance with the institutional guidelines for the care and use of animals in research at the Faculty of Veterinary Medicine, Zagazig University, Egypt. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Zagazig University under approval number ZU-IACUC/2025/04. All efforts were made to minimize animal suffering and to use the minimum number of fish necessary to achieve the scientific objectives of this study. Acknowledgments The author would like to express sincere gratitude to Delta University for Science and Technology for providing the institutional support necessary to conduct this research. Special thanks are extended to Prof. Alaa El-Sayed Abdel-Ghaffar, Dean of the Faculty of Veterinary Medicine, for his continuous encouragement and valuable facilitation throughout the course of the study. The author also acknowledges the technical assistance of the staff of the Department of Pharmacology, Faculty of Veterinary Medicine, Delta University, during the animal experiments and sample analysis. Data Availability All datasets generated or analyzed during the current study are included in this published article. Additional raw data are available from the corresponding author upon reasonable request. References Abdelhamid AM, Mahboub HH, El-Boshy ME (2020) Evaluation of the bactericidal activity of fish serum against Aeromonas hydrophila after dietary supplementation with probiotics. 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Fish Shellfish Immunol 67:635–644. https://doi.org/10.1016/j.fsi.2017.06.032 Zahran E, Risha E (2014) Modulatory role of dietary Chlorella vulgaris powder against arsenic-induced immunotoxicity and oxidative stress in Nile tilapia ( Oreochromis niloticus ). Fish Shellfish Immunol 41(2):654–662. https://doi.org/10.1016/j.fsi.2014.09.035 Zahran E, Awadin W, Risha E, Khaled AA, Wang T (2019) Dietary supplementation of Chlorella vulgaris ameliorates chronic sodium arsenite toxicity in Nile tilapia ( Oreochromis niloticus ) as revealed by histopathological, biochemical and immune gene expression analysis. Fish Sci 85(1):199–215. https://doi.org/10.1007/s12562-018-1274-6 Zhang Y, Song Y, He J, Zhang S (2020) Dietary resveratrol supplementation modulates growth performance, immune response, and gene expression in grass carp ( Ctenopharyngodon idella ). Fish Shellfish Immunol 106:808–816. https://doi.org/10.1016/j.fsi.2020.07.013 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 04 Feb, 2026 Read the published version in Veterinary Research Communications → Version 1 posted Editorial decision: Revision requested 05 Sep, 2025 Editor assigned by journal 05 Sep, 2025 Submission checks completed at journal 05 Sep, 2025 First submitted to journal 04 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7537362","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":510705516,"identity":"8613c9dc-8c4a-45bb-805b-af60531a63e6","order_by":0,"name":"Ahmed E. A. Mostafa","email":"data:image/png;base64,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","orcid":"","institution":"Delta University for Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Ahmed","middleName":"E. A.","lastName":"Mostafa","suffix":""}],"badges":[],"createdAt":"2025-09-04 14:53:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7537362/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7537362/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11259-025-11025-y","type":"published","date":"2026-02-04T15:59:41+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90897268,"identity":"0a0b5eeb-a2f9-4fcd-855f-0cba4b6bc514","added_by":"auto","created_at":"2025-09-09 11:40:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19334,"visible":true,"origin":"","legend":"\u003cp\u003e(A) MDA; Malondialdehyde, (B) GSH; Reduced Glutathione, (C) Catalase, and (D) SOD; Superoxide Dismutase activities in spleen, liver, and gills of Clarias gariepinus treated with Chlorpyrifos, Chlorella vulgaris, and β-glucan. Data are expressed as Mean ± SEM (n = 5). Values with different superscript letters are significantly different (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7537362/v1/c5de7e128d483eb2f0372636.png"},{"id":90897733,"identity":"2c6093e5-0ad1-4e35-9538-c3c8ebd98ba7","added_by":"auto","created_at":"2025-09-09 11:48:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8686,"visible":true,"origin":"","legend":"\u003cp\u003eRespiratory burst activity of Clarias gariepinus treated with Chlorpyrifos, \u003cem\u003eChlorella vulgaris\u003c/em\u003e, and β-glucan. Data are expressed as Mean ± SEM (n = 5). Values with different superscript letters are significantly different (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7537362/v1/afdcd07d6ada6cd4b1eb603c.png"},{"id":90897270,"identity":"ea0d6961-d4ae-430e-b918-38a7e0f90adc","added_by":"auto","created_at":"2025-09-09 11:40:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10042,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Serum lysozyme activity and (B) bactericidal activity of Clarias gariepinus treated with Chlorpyrifos, \u003cem\u003eChlorella vulgaris\u003c/em\u003e, and β-glucan. Data are expressed as Mean ± SEM (n = 5). Values with different superscript letters are significantly different (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7537362/v1/986ce001ff6c9e7f824bb0e7.png"},{"id":90897731,"identity":"196ded36-6347-4df4-a843-585a30501461","added_by":"auto","created_at":"2025-09-09 11:48:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8745,"visible":true,"origin":"","legend":"\u003cp\u003e(A) IgM; Immunoglobulin M and (B) CRP; C-reactive protein levels of Clarias gariepinus treated with Chlorpyrifos, \u003cem\u003eChlorella vulgaris\u003c/em\u003e, and β-glucan. Data are expressed as Mean ± SEM (n = 5). Values with different superscript letters are significantly different (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7537362/v1/505db3038a961ecb46761944.png"},{"id":90899041,"identity":"e9891157-7463-4c75-9a2a-13f12ce29864","added_by":"auto","created_at":"2025-09-09 11:56:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8713,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression of TNF-α (A) and IL-10 (B) in the spleen of Clarias gariepinus treated with Chlorpyrifos, \u003cem\u003eChlorella vulgaris\u003c/em\u003e, and β-glucan. Data are expressed as Mean ± SEM (n = 5). Values with different superscript letters are significantly different (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7537362/v1/382daa4e1ba9ff1e95c411be.png"},{"id":90897273,"identity":"4114e46e-1bc3-44d8-b9b5-07f35701a16c","added_by":"auto","created_at":"2025-09-09 11:40:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":24090,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival rate percentage of Clarias gariepinus treated with Chlorpyrifos, \u003cem\u003eChlorella vulgaris\u003c/em\u003e, and β-glucan. Data are expressed as Mean ± SEM (n = 5). Values with different superscript letters are significantly different (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7537362/v1/3932d60c73eb29291bc5a6c5.png"},{"id":102234177,"identity":"14e46829-24f4-4777-95ac-9afc52dadeb3","added_by":"auto","created_at":"2026-02-09 16:07:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1775628,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7537362/v1/de945419-5454-49ca-9603-ed3fb8018e55.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ameliorative Pharmacological Effects of Dietary Chlorella vulgaris and β-Glucan on Chlorpyrifos-Induced Oxidative Stress (MDA, GSH, SOD), Immunomodulation (TNF-α, IL-10), and Growth Indices in African Catfish (Clarias gariepinus)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe African catfish (Clarias gariepinus) is among the most economically important fish species globally, extensively cultured in aquaculture and inland fisheries. However, the contamination of aquatic ecosystems by agricultural pesticides poses a significant threat to its health and survival (Das and Shasmal \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Organophosphorus pesticides, in particular, are predominant ecotoxicants in aquatic environments, exerting severe adverse effects on aquatic organisms, especially fish (Ngangom Nganbi Devi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate] is widely employed in both agricultural practices and domestic pest control. It frequently enters surface waters through agricultural runoff and drainage systems (Selvaraj et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although chlorpyrifos degrades under specific environmental conditions, it may persist in aquatic habitats for prolonged periods, remaining biologically active for several months (Rebou\u0026ccedil;as et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLike other xenobiotics, chlorpyrifos contamination induces oxidative stress and substantial physiological disturbances in fish (Tripathi and Shasmal \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Exposure to this pesticide has been associated with marked hematological, biochemical, and immunological alterations, increasing disease susceptibility and compromising fish survival (Ring\u0026oslash; and Song \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Additionally, chlorpyrifos has been shown to suppress non-specific immune responses (Brennan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and impair growth performance in various fish species (Tripathi and Shasmal \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn response to these challenges, aquaculture has increasingly turned to natural dietary supplements, including probiotics, prebiotics, and immunostimulants, as safer alternatives to chemical additives. These supplements are employed to promote growth, enhance immunity, and improve survival rates (Tawfeek et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Chlorella vulgaris (CV), a unicellular freshwater microalga, has gained prominence as a probiotic feed additive due to its rich profile of bioactive compounds, including proteins, omega-3 and omega-6 polyunsaturated fatty acids, polysaccharides, vitamins, minerals, and photosynthetic pigments such as carotenoids and chlorophylls (Majumder and Kaviraj \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Dietary inclusion of CV has demonstrated beneficial effects on growth performance, innate immune responses, antioxidant enzyme activities, and resistance to infectious diseases in aquaculture species (Elias et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSimilarly, β-glucans, naturally occurring polysaccharides derived from the cell walls of plants, fungi, yeast, bacteria, and mushrooms, are considered potent immunostimulants in aquaculture (Scapigliati et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These compounds enhance immune responses by stimulating phagocytic activity, activating the complement system, and promoting cytokine expression in macrophages, neutrophils, and dendritic cells (Tripathi and Shasmal \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Notably, β-glucans extracted from Saccharomyces cerevisiae have been reported to improve immune function and increase resistance to Aeromonas hydrophila infections in fish (Slotkin and Seidler 2025). Furthermore, dietary β-glucan supplementation has been associated with improved growth, enhanced antioxidant status ( El-Bab et al. 2025), and reduced inflammatory responses in fish (Najah \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo the best of our knowledge, this study is the first to comprehensively evaluate and compare the protective roles of Chlorella vulgaris (as a probiotic) and β-glucan (as a prebiotic) in mitigating hepatorenal toxicity, oxidative stress, immunosuppression, and growth impairments in African catfish (Clarias gariepinus) following subacute chlorpyrifos exposure.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Chemicals\u003c/h2\u003e\u003cp\u003eChlorpyrifos (CPF) with a concentration of 48% was sourced from Adwia Pharmaceuticals (Cairo, Egypt) and freshly diluted in distilled water immediately prior to application. Pure Chlorella vulgaris (CV) powder was procured from Roquette Kl\u0026ouml;tze GmbH \u0026amp; Co. KG, Kl\u0026ouml;tze, Germany. β-glucan, extracted from Saccharomyces cerevisiae, was obtained from Hang Zhou Bio Technology Co., Ltd., China.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Diet Preparation\u003c/h2\u003e\u003cp\u003e Four experimental diets were formulated to be isonitrogenous (32% crude protein) and isocaloric (3,000 kcal DE/kg), meeting the nutritional requirements of Clarias gariepinus based on NRC guidelines (National Research Council 2011). In addition to the control basal diet, three dietary treatments were prepared:\u003c/p\u003e\u003cp\u003eBasal diet supplemented with 5% Chlorella vulgaris (Khosravi et al. 2015).\u003c/p\u003e\u003cp\u003eBasal diet supplemented with 0.1% β-glucan (Sakai 1999).\u003c/p\u003e\u003cp\u003eThe detailed composition of each diet is provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All diets were processed into water-stable sinking pellets, carefully sealed in plastic bags, and stored under refrigeration until feeding.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Fish and Experimental Design\u003c/h2\u003e\u003cp\u003eA total of 180 healthy Clarias gariepinus (African catfish) with an initial average weight of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 g were obtained from a private fish farm located in Kafrelsheikh Governorate, Egypt. Fish were maintained in an indoor recirculating aquaculture system with well-aerated, dechlorinated freshwater, supported by internal filtration units. Key water quality parameters were strictly controlled: temperature 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u0026deg;C, dissolved oxygen 6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mg/L, and pH range 7.4\u0026ndash;7.8. During a two-week acclimatization period, fish were fed a commercial basal diet at 3% of their body weight, administered twice daily (from 9:00\u0026ndash;10:00 AM and 4:00\u0026ndash;5:00 PM).\u003c/p\u003e\u003cp\u003eFollowing acclimation, fish were randomly distributed into four treatment groups, each consisting of triplicate tanks (15 fish per tank; 45 fish per group) housed in glass aquaria (40 \u0026times; 60 \u0026times; 30 cm) with continuous aeration and dechlorinated tap water. The fish were subjected to the following treatments for 60 consecutive days:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eControl Group: Fish received the basal diet without chlorpyrifos exposure.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eCPF Group: Fish were exposed to chlorpyrifos at 0.24 mg/L (equivalent to 1/10 of the 96-hour LC₅₀) in the rearing water and fed the basal diet. The chlorpyrifos concentration was selected based on the LC₅₀ value for Clarias gariepinus reported by Ayoola (2008), which is 2.4 mg/L.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eCPF\u0026thinsp;+\u0026thinsp;CV Group: Fish were exposed to the same chlorpyrifos concentration (0.24 mg/L) and fed the diet supplemented with 5% Chlorella vulgaris.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eCPF\u0026thinsp;+\u0026thinsp;β-glucan Group: Fish were exposed to the same chlorpyrifos concentration (0.24 mg/L) and fed the diet supplemented with 0.1% β-glucan.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEthical approval statement:\u003c/b\u003e This study is reported in accordance with the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org(\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org(\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePercentage of ingredients of experimental diets.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIngredients (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChlorella vulgaris\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eβ-glucan\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYellow corn (8.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e18.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoybean meal (44%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e19.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e17.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFish meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWheat bran\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e38.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e38.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorn gluten\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGelatin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eβ-glucan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChlorella vulgaris\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMinerals and vitamins premix**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSalt\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDicalcium phosphate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethionine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eChemical composition (%)\u003c/td\u003e\u003c/tr\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComponents\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChlorella vulgaris\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eβ-glucan\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrude protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e32.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDE (kcal/kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003eVitamin mixture supplies the following per kilogram of diet:\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003evit. A \u0026ndash; 1,200,000 IU; vit. D3\u0026ndash;200,000 IU; vit. E \u0026ndash; 12,000 mg; vit. K3\u0026ndash;2400 mg; vit. B1\u0026ndash;4800 mg; vit. B2\u0026ndash;4800 mg; vit. B6\u0026ndash;4000 mg; vit. B12\u0026ndash;4800 mg; folic acid \u0026ndash; 1200 mg; vit. C \u0026ndash; 48,000 mg; biotin \u0026ndash; 48 mg; choline \u0026ndash; 65,000 mg; niacin \u0026ndash; 24,000 mg; Fe \u0026ndash; 10,000 mg; Cu \u0026ndash; 600 mg; Mg \u0026ndash; 4000 mg; Zn \u0026ndash; 6000 mg; I \u0026ndash; 20 mg; Co \u0026ndash; 2 mg; Se \u0026ndash; 20 mg.\u003c/p\u003e\u003cp\u003eFish in the \u003cb\u003eCPF\u0026thinsp;+\u0026thinsp;CV group\u003c/b\u003e were exposed to chlorpyrifos at the previously established concentration while receiving a diet supplemented with 5% \u003cem\u003eChlorella vulgaris\u003c/em\u003e. Similarly, fish in the \u003cb\u003eCPF\u0026thinsp;+\u0026thinsp;β-glucan group\u003c/b\u003e were exposed to the same chlorpyrifos concentration and fed a diet supplemented with 0.1% β-glucan. To prevent the accumulation of waste metabolites, a static-renewal system was employed, where 80% of the aquarium water was replaced daily. Freshly prepared chlorpyrifos solutions were added after each water change to maintain the target exposure concentration. The survival rate of African catfish (\u003cem\u003eClarias gariepinus\u003c/em\u003e) was monitored across all experimental groups throughout the trial.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Sample Collection\u003c/h2\u003e\u003cp\u003eAfter 28 days of treatment, ten fish were randomly selected from each aquarium and lightly anesthetized using tricaine methanesulfonate (MS-222; FINQUEL\u0026reg;, ARGENT) at 30 mg/L, buffered with 60 mg/L sodium bicarbonate. Euthanasia was then performed using a higher MS-222 dose of 200 mg/L, buffered with 400 mg/L sodium bicarbonate (Ross and Ross 2008). Blood samples were collected from the caudal vessels of individual fish. Each fish provided two blood samples: one collected in EDTA-containing tubes for hematological analysis and respiratory burst evaluation (Blaxhall and Daisley 1973), and the other collected in plain tubes for serum separation. Serum samples were isolated by centrifugation at 3000 rpm for 10 minutes and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent biochemical and immunological assessments (Tietz 1995).\u003c/p\u003e\u003cp\u003eTissue samples from the liver, spleen, and gills were carefully excised, rinsed in normal saline, and homogenized in ice-cold phosphate-buffered saline (PBS; pH 7.5). The homogenates were centrifuged at 3000 rpm for 15 minutes, and the resulting supernatants were stored at \u0026minus;\u0026thinsp;80\u0026deg;C for later antioxidant and oxidative stress analyses (Ohkawa et al. 1979). Additionally, portions of spleen tissue were preserved in RNA Later\u0026reg; (Qiagen) at 4\u0026deg;C overnight, then stored at \u0026minus;\u0026thinsp;80\u0026deg;C for gene expression studies (Chomczynski and Sacchi 1987).\u003c/p\u003e\u003cp\u003eThe remaining fish continued under the same experimental protocols until day 60. At the end of the experiment, all surviving fish were counted to calculate the final survival rates and individually weighed to determine weight gain (WG) (Hopkins 1992). Subsequently, ten fish from each aquarium were euthanized, and the liver and spleen were rapidly excised and weighed to assess organ indices (Schreck and Moyle 1990).\u003c/p\u003e\u003cp\u003e All experimental procedures were performed in compliance with the Animal Care and Use guidelines of Kafrelsheikh University and were approved by the local Animal Care and Use Committee.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Hematological Analysis\u003c/h2\u003e\u003cp\u003eTotal erythrocyte (RBC) and leukocyte (WBC) counts were determined using Natt-Herrick\u0026rsquo;s solution for dilution and manual hemocytometer counting as described by Blaxhall and Daisley (1973). Hemoglobin (Hb) concentrations were measured spectrophotometrically via the cyanmethemoglobin method (Drabkin 1949). The packed cell volume (PCV) and red blood cell indices\u0026mdash;mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)\u0026mdash;were calculated following the methodology of Wintrobe (1934). Differential leukocyte counts were performed on Giemsa-stained blood smears (Blaxhall and Daisley 1973).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Serum Biochemical Analysis\u003c/h2\u003e\u003cp\u003eSerum biochemical markers were quantified using commercial diagnostic kits: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (BioMed, Egypt), alkaline phosphatase (ALP) (Spectrum, Egypt), total protein and albumin (Bio-Diagnostic, Egypt), creatinine (Human, Germany), and uric acid (BioMed, Egypt). All measurements were conducted using a spectrophotometer (5010 Photometer, BM Co., Germany) according to the manufacturers\u0026rsquo; protocols (Reitman and Frankel 1957).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Assessment of Oxidative Stress and Antioxidant Status\u003c/h2\u003e\u003cp\u003eAntioxidant and oxidative stress biomarkers, including malondialdehyde (MDA), glutathione (GSH), catalase, and superoxide dismutase (SOD), were quantified in liver, spleen, and gill homogenates using commercial assay kits (Bio-Diagnostic, Egypt). All assays were performed spectrophotometrically following the kit instructions (Aebi 1984).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Evaluation of Immunological Parameters\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.8.1. Respiratory Burst Activity\u003c/h2\u003e\u003cp\u003eThe respiratory burst of phagocytes was assessed using the nitroblue tetrazolium (NBT) reduction assay following the procedure of Wijendra and Pathiratne (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.8.2. Serum Lysozyme Activity\u003c/h2\u003e\u003cp\u003eSerum lysozyme activity was determined following the method of Ghareghanipoor et al. (2017) with slight modifications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.8.3. Serum Bactericidal Activity\u003c/h2\u003e\u003cp\u003eBactericidal activity was assessed following the method of Abdelhamid et al. (2018).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.8.4. Determination of CRP and IgM Levels\u003c/h2\u003e\u003cp\u003eC-reactive protein (CRP) levels were semi-quantitatively assessed using a rapid latex agglutination test following the method of Tillett and Francis (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1930\u003c/span\u003e). Immunoglobulin M (IgM) levels were quantitatively measured using a turbidity assay, based on immune complex formation, as described by Dati and Lammers (1989).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Gene Expression Analysis of Immune-Related Genes\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.9.1. RNA Extraction and cDNA Synthesis\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated from spleen tissues using the RNAeasy Mini Kit (Qiagen, Germany) according to the procedure described by Zhang et al. (2012) with minor modifications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e2.9.2. Quantitative Real-Time PCR (qRT-PCR)\u003c/h2\u003e\u003cp\u003eGene expression levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) were quantified using SYBR\u0026reg; Green Master Mix (Thermo Fisher Scientific, USA) and the CFX Connect Real-Time PCR System (Bio-Rad, USA). β-actin served as the internal reference gene. Primer sequences were selected according to Wang et al. (2008) and Li et al. (2013). The qPCR protocol included an initial denaturation at 95\u0026deg;C for 10 minutes, followed by 40 amplification cycles (denaturation at 95\u0026deg;C for 30 seconds, annealing at 58\u0026deg;C for 30 seconds, and extension at 72\u0026deg;C for 30 seconds). Relative gene expression was calculated using the 2^\u0026minus;ΔΔCT method (Livak and Schmittgen \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Chlorpyrifos Residue Analysis\u003c/h2\u003e\u003cp\u003eChlorpyrifos residues in fish liver tissues were extracted and cleaned following the QuEChERS method with minor adjustments as described by Mastovska and Lehotay (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The cleaned extracts were analyzed using Gas Chromatography with an Electron Capture Detector (GC-ECD; Agilent 7890B, USA). Each sample was analyzed in triplicate to ensure accuracy and reproducibility.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer sequences used for RT-PCR analysis.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer Sequence (5\u0026prime; to 3\u0026prime;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAmplicon size (bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAccession number\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eTumor necrosis factor-α\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTNFα-F: CAGACTGTAGCCCTGTCACCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAY428948.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTNFα-R: GTCACAGAGTGGGAGGTTGAT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eInterleukin 10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIL-10-F: CGCTGTCATCGATTTCTCCAT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eXM_003441366.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIL-10-R: ATCTCCTGTTCCCTCCTGCTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eElongation factor 1α\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEF1α-F: GACAACATGCTTGAGGCTGAC\u003c/p\u003e\u003cp\u003eEF1α-R: CCAATACCAGTCTCCACACCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAB075952.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Growth Performance and Feed Utilization Assessment\u003c/h2\u003e\u003cp\u003eThe evaluation of growth performance and feed efficiency was conducted by determining several key parameters: weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), hepatosomatic index (HSI), spleensomatic index (SSI), and survival rate (SR), as outlined by Cech and Myrick (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe following formulas were employed to calculate these parameters for \u003cem\u003eOreochromis niloticus\u003c/em\u003e:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eWeight Gain (WG, g)\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Final average body weight (g)\u0026thinsp;\u0026minus;\u0026thinsp;Initial average body weight (g)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eFeed Conversion Ratio (FCR)\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Total feed intake (g)\u0026thinsp;\u0026divide;\u0026thinsp;Total weight gain (g)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSpecific Growth Rate (SGR, %/day)\u003c/b\u003e\u0026thinsp;=\u0026thinsp;100 \u0026times; [(Ln final body weight\u0026thinsp;\u0026minus;\u0026thinsp;Ln initial body weight)\u0026thinsp;\u0026divide;\u0026thinsp;Rearing period (days)]\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eProtein Efficiency Ratio (PER)\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Total weight gain (g)\u0026thinsp;\u0026divide;\u0026thinsp;Total protein consumed (g)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eHepatosomatic Index (HSI, %)\u003c/b\u003e = (Liver weight\u0026thinsp;\u0026divide;\u0026thinsp;Body weight) \u0026times; 100\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSpleensomatic Index (SSI, %)\u003c/b\u003e = (Spleen weight\u0026thinsp;\u0026divide;\u0026thinsp;Body weight) \u0026times; 100\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSurvival Rate (SR, %)\u003c/b\u003e = (Number of surviving fish\u0026thinsp;\u0026divide;\u0026thinsp;Initial number of fish) \u0026times; 100\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll results are expressed as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). Statistical analysis was performed using IBM SPSS software (version 26, USA). Data were analyzed through one-way analysis of variance (ANOVA) to identify significant differences among the treatment groups. When significant differences were detected, Tukey\u0026rsquo;s multiple comparison test was applied to determine pairwise differences between means. Statistical significance was accepted at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Blood cell count\u003c/h2\u003e\u003cp\u003eThere were no significant differences in RBC count, hemoglobin concentration, and PCV percentage among CPF, CPF-C. vulgaris, and CPF-β-glucan treated fish compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, total leukocytic count (TLC) and lymphocyte count were significantly decreased in the CPF-intoxicated group compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while heterophil counts were significantly elevated in the CPF group compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFeeding CPF-intoxicated fish with \u003cem\u003eChlorella vulgaris\u003c/em\u003e or β-glucan supplemented diets significantly improved the TLC and lymphocyte counts compared to the CPF group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, heterophil counts remained significantly higher in the CPF-C. vulgaris and CPF-β-glucan groups compared to both the control and CPF groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Monocyte counts did not show significant differences between all groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eHematological parameters of African catfish (\u003cem\u003eClarias gariepinus\u003c/em\u003e) treated with Chlorpyrifos, Chlorella vulgaris and β-glucan.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperimental groups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCPF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCPF-CV\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCPF-β-glucan\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRBCs (10⁶/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHb (g/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e9.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e9.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e9.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePCV (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e29.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e30.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e28.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e31.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eWBCs (10\u0026sup3;/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e45.50\u0026thinsp;\u0026plusmn;\u0026thinsp;3.00ᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e37.80\u0026thinsp;\u0026plusmn;\u0026thinsp;3.50ᶜ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e60.40\u0026thinsp;\u0026plusmn;\u0026thinsp;4.00ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e56.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.20ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLymphocyte (10\u0026sup3;/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e24.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e13.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40ᶜ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e26.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.00ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e21.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30ᵇ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHeterophil (10\u0026sup3;/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e17.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80ᶜ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e22.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50ᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e30.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e32.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMonocyte (10\u0026sup3;/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e4.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e4.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eData are expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;5).Means in the same row with different superscripts are significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).CPF, Chlorpyrifos; CV, Chlorella vulgaris; RBCs, Red blood cell count; Hb, Hemoglobin; PCV, Packed Cell Volume; WBCs, White blood cell count.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Biochemical parameters\u003c/h2\u003e\u003cp\u003eThe Chlorpyrifos (CPF) induced hepatotoxicity was indicated by a significant elevation in ALT and AST activities compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the CPF-CV and CPF-β-glucan treated groups showed significantly lower serum levels of ALT and AST compared to the CPF group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant differences in ALP levels were observed among all treated groups (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSerum total protein level was significantly higher in the CPF-β-glucan treated fish compared to the other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Serum albumin level was slightly decreased in CPF intoxicated fish compared to the other groups. Meanwhile, globulin was significantly decreased in the CPF group compared to the control and CPF-β-glucan groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In fish fed β-glucan supplemented diets, the globulin level was significantly higher, while the albumin-globulin ratio (A/G) was significantly lower than those of the CPF group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCPF, CPF-CV and CPF-β-glucan treated fish showed significantly higher levels of creatinine and uric acid than the control fish (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, uric acid levels in both CPF-CV and CPF-β-glucan groups were significantly lower than those in the CPF group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSerum biochemical parameters of African catfish (\u003cem\u003eClarias gariepinus\u003c/em\u003e) treated with Chlorpyrifos, Chlorella vulgaris and β-glucan.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperimental groups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCPF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCPF-CV\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCPF-β-glucan\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eALT (U/L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e11.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05ᶜ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e26.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.90ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e18.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50ᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e19.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70ᵇ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAST (U/L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e56.20\u0026thinsp;\u0026plusmn;\u0026thinsp;4.90ᶜ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e112.50\u0026thinsp;\u0026plusmn;\u0026thinsp;9.80ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e95.40\u0026thinsp;\u0026plusmn;\u0026thinsp;7.10ᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e82.70\u0026thinsp;\u0026plusmn;\u0026thinsp;5.80ᵇ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eALP (U/L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e78.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.90ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e88.20\u0026thinsp;\u0026plusmn;\u0026thinsp;5.90ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e76.50\u0026thinsp;\u0026plusmn;\u0026thinsp;5.70ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e85.30\u0026thinsp;\u0026plusmn;\u0026thinsp;4.10ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal protein (g/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38ᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40ᶜ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22ᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e4.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAlbumin (g/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGlobulin (g/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35ᶜ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12ᵃᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e3.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eA/G ratio (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04ᵃᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05ᵃᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07ᵇ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCreatinine (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015ᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eUric acid (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40ᶜ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e9.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e7.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28ᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e7.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15ᵇ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eData are expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;5).Means in the same row with different superscripts are significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).CPF, Chlorpyrifos; CV, Chlorella vulgaris; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; A/G ratio, albumin/globulin ratio.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Antioxidant and Oxidative Stress Parameters\u003c/h2\u003e\u003cp\u003eSpleen MDA level was significantly increased in CPF and CPF-CV groups compared to the control and CPF-β-glucan groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, liver MDA levels were significantly elevated in CPF group in contrast to all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;1A).\u003c/p\u003e\u003cp\u003eCPF exposure significantly decreased GSH levels in liver and gills compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Treatment of CPF-exposed fish with CV and β-glucan supplemented diets significantly improved liver GSH levels compared to CPF group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, only the β-glucan supplemented diet was able to restore gill GSH levels back to normal (Fig.\u0026nbsp;1B).\u003c/p\u003e\u003cp\u003eSpleen and liver catalase (CAT) levels showed no significant differences among the experimental groups. However, the CPF-CV group demonstrated significantly higher catalase activity compared to all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;1C).\u003c/p\u003e\u003cp\u003eMoreover, liver SOD activity was significantly lower in CPF and CPF-CV groups compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Interestingly, the highest liver SOD activity was recorded in CPF-β-glucan group compared to all other groups, including the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;1D).\u003c/p\u003e\u003cp\u003eNo significant differences were detected in gill MDA, spleen GSH, and gill SOD levels among all groups (Fig.\u0026nbsp;1A, 1B, 1D). \u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;1.\u003c/b\u003e (A) MDA; Malondialdehyde, (B) GSH; Reduced Glutathione, (C) Catalase, and (D) SOD; Superoxide Dismutase activities in spleen, liver, and gills of Clarias gariepinus treated with Chlorpyrifos, Chlorella vulgaris, and β-glucan. Data are expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;5). Values with different superscript letters are significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Immune Parameters\u003c/h2\u003e\u003cp\u003eChlorpyrifos (CPF) intoxicated fish showed a significant reduction in respiratory burst activity compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). CPF groups fed with diets containing \u003cem\u003eChlorella vulgaris\u003c/em\u003e (CV) and β-glucan exhibited significantly higher respiratory burst activity compared to both CPF and control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSerum lysozyme activity was significantly lower in CPF-treated fish than all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Only the β-glucan treated group showed a significant increase in lysozyme activity compared to other treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eMoreover, CPF toxicity significantly decreased bactericidal activity compared to both CPF-CV and CPF-β-glucan groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The highest bactericidal activity was recorded in the CV-supplemented group compared to all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSerum immunoglobulin G (IgM) and C-reactive protein (CRP) levels were significantly reduced in CPF-intoxicated fish compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, these parameters were significantly elevated in the CPF-β-glucan group compared to the CPF and control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, dietary supplementation with CV significantly improved IgG levels but had no significant effect on CRP levels compared to the CPF group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Expression of Immune-Related Genes\u003c/h2\u003e\u003cp\u003eThe TNF-α transcript level was significantly up-regulated in the spleen of all CPF-treated fish compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This significant increase was also observed in the spleen of CPF-CV and CPF-β-glucan treated fish compared to the control fish (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eThe expression of IL-10 was significantly down-regulated in the spleen of CPF, CPF-CV, and CPF-β-glucan treated fish compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, IL-10 expression was significantly higher in the CPF-CV and CPF-β-glucan supplemented fish than in CPF-intoxicated fish (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.6. CPF Residues in Liver Tissue\u003c/h2\u003e\u003cp\u003eThe CPF residues results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e6\u003c/span\u003e and showed that the highest concentration of CPF residue was detected in CPF-exposed fish (CPF group) compared to CPF-CV and CPF-β-glucan treated groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Meanwhile, the CPF concentration was significantly lower in CPF-CV group compared to CPF-β-glucan group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eConcentration residues of Chlorpyrifos (CPF) (ng/g tissue) in liver tissue of African catfish (\u003cem\u003eClarias gariepinus\u003c/em\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperimental groups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCPF Residues (ng/g tissue)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCPF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.175\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCPF-CV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.103\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCPF-β-glucan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.128\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eData are expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Means in the same row with different superscripts are significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). CPF, Chlorpyrifos; CV, Chlorella vulgaris.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Survival rate and growth performance\u003c/h2\u003e\u003cp\u003eThe survival rate of African catfish (\u003cem\u003eClarias gariepinus\u003c/em\u003e) was significantly lower in CPF treated fish compared to all other treated groups, and the highest survival rate was recorded in the CV treated fish (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Additionally, the lowest growth performance was observed in the CPF treated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Fish in the CPF-CV group showed significantly higher final body weight (FBW) and body weight gain (BWG) than the fish in the other experimental groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOn the other hand, the feed conversion ratio (FCR), specific growth rate (SGR), and protein efficiency ratio (PER) of the control, CPF-CV, and CPF-β-glucan groups were significantly better than those of the CPF group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eThe data also exhibited significantly increased hepatosomatic index (HSI) and spleensomatic index (SSI) in the control and CPF groups compared to the CPF-CV and CPF-β-glucan groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGrowth performance of African catfish (\u003cem\u003eClarias gariepinus\u003c/em\u003e) treated with Chlorpyrifos, Chlorella vulgaris, and β-glucan.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperimental groups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCPF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCPF-CV\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCPF-β-glucan\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIW (g/fish)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFBW (g/fish)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40.95\u0026thinsp;\u0026plusmn;\u0026thinsp;3.45 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e64.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.62 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e53.70\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBWG (g/fish)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e34.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.80 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e43.10\u0026thinsp;\u0026plusmn;\u0026thinsp;3.10 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e31.95\u0026thinsp;\u0026plusmn;\u0026thinsp;3.20 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFCR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePER\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHIS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSSI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eData are expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;5). Means in the same row with different superscripts are significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).CPF, Chlorpyrifos; CV, Chlorella vulgaris; IW, Initial weight; FBW, Final body weight; BWG, Body weight gain; FCR, Feed conversion ratio; SGR, Specific growth rate; PER, Protein efficiency ratio; HSI, Hepatosomatic index; SSI, Spleen-somatic index.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, African catfish (\u003cem\u003eClarias gariepinus\u003c/em\u003e) exposed to chlorpyrifos (CPF) exhibited insignificant changes in serum total protein and a slight reduction in albumin levels. However, a significant decline in globulin concentrations was observed. Additionally, CPF exposure resulted in marked increases in serum creatinine and uric acid compared to the control group. These findings are consistent with \u003cb\u003eAlishahi et al. (2014)\u003c/b\u003e, who reported a significant decrease in total protein and globulin, alongside a non-significant change in albumin levels in \u003cem\u003eBarbus sharpeyi\u003c/em\u003e juveniles exposed to diazinon.\u003c/p\u003e\u003cp\u003eConversely, dietary supplementation with \u003cem\u003eChlorella vulgaris\u003c/em\u003e (CV) or β-glucan improved the serum biochemical profile of CPF-exposed African catfish. Specifically, ALT, AST, and uric acid levels were significantly reduced in treated groups, and total protein and globulin levels significantly improved, particularly in fish receiving β-glucan. The hepatoprotective effect of CV can likely be attributed to its antioxidant-rich composition, which stabilizes hepatocyte membranes and minimizes the leakage of intracellular enzymes. \u003cb\u003eZahran et al. (2018)\u003c/b\u003e similarly demonstrated that dietary inclusion of 5% and 10% CV powder over 21 days improved liver and kidney function markers and enhanced total protein, albumin, and globulin levels in Nile tilapia under sodium arsenite-induced stress.\u003c/p\u003e\u003cp\u003eThe antioxidant potential of β-1,3-glucan has also been established, with its ability to neutralize free radicals and prevent lipid peroxidation in hepatic tissues (\u003cb\u003eAbdel-Tawwab et al. 2019\u003c/b\u003e). El-Keredy et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) showed that β-glucan supplementation (100 mg/kg diet for 10 weeks) effectively normalized elevated serum markers, including AST, ALT, urea, uric acid, and creatinine in copper-intoxicated Nile tilapia.\u003c/p\u003e\u003cp\u003eIt is well recognized that exposure to pesticides in fish leads to oxidative stress due to excessive reactive oxygen species (ROS) generation, which overwhelms the antioxidant defense system (\u003cb\u003eLivingstone 2001\u003c/b\u003e). Key antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) play crucial roles in cellular protection against oxidative injury (\u003cb\u003eWinston 1991\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eIn the current study, CPF exposure significantly elevated malondialdehyde (MDA) levels in the spleen and liver of African catfish, indicating increased lipid peroxidation, while significantly depleting antioxidant enzymes including glutathione (GSH) in gill and hepatic tissues and SOD activity in the spleen. This heightened lipid peroxidation is likely linked to CPF metabolism and excessive free radical generation (\u003cb\u003eSoltan 2014\u003c/b\u003e), which also contributes to the depletion of GSH reserves (\u003cb\u003eKavitha and Rao 2008\u003c/b\u003e). Xenobiotic exposure may further exacerbate GSH depletion by enhancing its breakdown or impairing its synthesis (\u003cb\u003eOru\u0026ccedil; and Usta 2007\u003c/b\u003e). Variations in antioxidant enzyme activities across different tissues may reflect tissue-specific sensitivity, with the liver generally more susceptible to pesticide-induced oxidative stress due to its central role in xenobiotic metabolism (\u003cb\u003eLivingstone 2001\u003c/b\u003e). CPF preferentially accumulates and undergoes biotransformation in hepatic tissues (\u003cb\u003eLivingstone 2001\u003c/b\u003e), and antioxidant responses can differ based on the chemical nature of the xenobiotic, duration of exposure, and species-specific tolerances (\u003cb\u003eVan der Oost et al. 2003\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eSupporting this, previous investigations in CPF-exposed African catfish demonstrated similar oxidative disturbances, including elevated hepatic MDA levels and significant reductions in hepatic SOD, CAT, glutathione peroxidase (GSH-Px), and total antioxidant capacity (TAC) (Rebou\u0026ccedil;as et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Comparable outcomes were noted by \u003cb\u003eAbdelkhalek et al. (2015)\u003c/b\u003e in Nile tilapia, where pesticide exposure resulted in substantial declines in catalase, GSH, and SOD activities, coupled with increased MDA accumulation in liver, kidney, and gill tissues.\u003c/p\u003e\u003cp\u003eThe administration of CV in this study significantly ameliorated CPF-induced oxidative damage. CV\u0026rsquo;s potent antioxidant components\u0026mdash;such as flavonoids, carotenoids, chlorophyll, tocopherols, and polyphenols\u0026mdash;may contribute to ROS scavenging and inhibition of lipid peroxidation (Abou-Zeid and Hussein \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). CV supplementation enhanced GSH concentrations in hepatic and gill tissues and improved gill catalase activity relative to the CPF-only group. These findings align with those of Zahran and Risha (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, who reported that dietary CV effectively normalized MDA and H₂O₂ levels and elevated catalase and GSH activities in fish exposed to sodium arsenite. Additionally, CV co-treatment in herbicide-exposed fish was shown to increase serum SOD and GSH-Px activities while lowering MDA concentrations (\u003cb\u003ePradhan et al. 2023\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eSimilarly, β-glucan supplementation in this study conferred notable antioxidant benefits. β-glucan significantly decreased splenic MDA levels and enhanced both GSH and SOD activities in the liver and spleen of CPF-exposed fish. These observations correspond with \u003cb\u003eDawood et al. (2019)\u003c/b\u003e, who demonstrated improved antioxidant status in Nile tilapia fed β-glucan (1 g/kg diet) for 60 days, including reductions in serum MDA and increases in SOD and GSH-Px. Further supporting evidence from common carp exposed to fipronil and lead nitrate confirmed that β-glucan supplementation boosted hepatic antioxidant enzyme activities and reduced oxidative stress markers (El-Keredy et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRegarding immune function, CPF exposure led to significant immunosuppression, evidenced by reduced respiratory burst activity, lysozyme levels, IgM concentrations, and C-reactive protein (CRP), while bactericidal activity was marginally affected. These results corroborate findings by Hajirezaee et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), who reported decreased immunoglobulin, lysozyme, and respiratory burst activities in diazinon-exposed rainbow trout. Previous research has also demonstrated reductions in lysozyme and bactericidal activities and downregulation of splenic IgM gene expression following pesticide exposure (Saleh et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Lysozyme suppression following diazinon exposure was further confirmed in rainbow trout (Uner et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). CPF\u0026rsquo;s immunosuppressive effects may be linked to impaired leukocyte differentiation, reduced protein synthesis, and general immunotoxicity (\u003cb\u003eAlishahi et al. 2014\u003c/b\u003e). Additionally, CPF-induced hepatic damage and apoptosis may compromise the liver\u0026rsquo;s capacity to synthesize total protein and immunoglobulins (Mostafalou and Abdollahi \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, dietary CV supplementation in this study restored immune functions, including lysozyme activity, respiratory burst, bactericidal activity, and IgM levels, in CPF-exposed fish, indicating a strong immunostimulatory potential. This may be explained by the bioactive components in CV, such as omega-3 and omega-6 fatty acids and polysaccharides, which can activate immune cells via receptor binding (Abou-Zeid and Hussein \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Comparable improvements in immune parameters were previously reported in African catfish fed CV-supplemented diets following pollutant exposure (Abdel-Tawwab et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSimilarly, β-glucan supplementation effectively counteracted CPF-induced immunosuppression, significantly enhancing respiratory burst, lysozyme activity, CRP levels, IgM concentrations, and bactericidal responses. The immunomodulatory effects of β-glucan likely involve its interaction with macrophage and dendritic cell receptors, promoting IL-10 production (Vetvicka and Vetvickova \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and stimulating complement activation, phagocytosis, and non-specific immune responses (Brown and Gordon \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Supporting this, El-Boshy et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) demonstrated that β-glucan (0.1% of diet for 21 days) significantly improved lysozyme, bactericidal activity, nitric oxide production, and macrophage oxidative burst in immunosuppressed African catfish. Similar enhancements were observed by Dawood et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), who reported elevated phagocytic activity, respiratory burst, and lysozyme levels in β-glucan-fed Nile tilapia.\u003c/p\u003e\u003cp\u003eAt the molecular level, CPF exposure significantly upregulated splenic TNF-α gene expression while downregulating IL-10 in African catfish, indicative of an inflammatory response. Comparable pro-inflammatory gene expression patterns were noted in diazinon-exposed rainbow trout (Hajirezaee et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), likely reflecting CPF-induced tissue damage and macrophage activation (\u003cb\u003eSlotkin and Seidler 2025\u003c/b\u003e). CPF\u0026rsquo;s stimulation of ROS production may activate the NF-κB pathway, subsequently inducing pro-inflammatory mediators such as TNF-α, IL-1β, IL-6, COX-2, and iNOS (Mostafalou and Abdollahi \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, both CV and β-glucan supplementation significantly modulated the inflammatory response by decreasing TNF-α expression and enhancing IL-10 levels. CV\u0026rsquo;s anti-inflammatory properties may be linked to its carotenoid content, particularly violaxanthin, which suppresses ROS production and downregulates NF-κB-mediated inflammatory cascades (Najah \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Supporting evidence from prior studies indicated that CV supplementation (10%) significantly attenuated pro-inflammatory cytokine expression in sodium arsenite-intoxicated fish (Zahran and Risha \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The anti-inflammatory role of β-glucan is similarly supported by its capacity to reduce pro-inflammatory cytokines while upregulating IgM and IL-10 expression (Vetvicka et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Meena et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), alongside its antioxidant properties that further limit inflammation (Safi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAdditionally, CPF exposure significantly reduced both survival rate and growth performance, in agreement with previous studies demonstrating the adverse effects of sub-lethal pesticide exposure on fish growth and survival (Rebou\u0026ccedil;as et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Other reports also confirmed that herbicide exposure negatively impacts growth metrics in African catfish (Uner et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, dietary supplementation with CV or β-glucan significantly improved the survival and growth of CPF-exposed fish. El-Boshy et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) reported similar enhancements in survival with \u003cem\u003eChlorella\u003c/em\u003e supplementation. The beneficial impact of β-glucan on survival and resilience against toxins and pathogens has been widely documented in various fish species (Dawood and Koshio \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Notably, CV supplementation led to superior growth performance compared to β-glucan, potentially due to its high-quality protein, essential fatty acids, and growth-promoting compounds like the S-nucleotide adenosyl peptide complex (Keshavanath and Renuka \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Hossain et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). CV\u0026rsquo;s rich nutrient profile, including omega-3 and omega-6 fatty acids, proteins, polysaccharides, vitamins, minerals, chlorophyll, and carotenoids, likely contributed to these improvements (Li et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGrowth enhancement with β-glucan may be attributed to its degradation by glucanase, preserving protein for growth (protein-sparing effect) and stimulating digestive enzyme secretion (Meena et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Dawood et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAs the liver is the primary site for xenobiotic accumulation and metabolism in fish (\u003cb\u003eLivingstone 2001\u003c/b\u003e), CPF was predominantly detected in hepatic tissues. Dietary CV and β-glucan significantly reduced hepatic CPF accumulation. The hepatosomatic index (HSI), a key indicator of liver health, was improved in supplemented groups, suggesting mitigation of CPF-induced hepatic dysfunction. Similar HSI increases under pesticide stress were previously reported in common carp (Cech and Myrick \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn conclusion, this study demonstrates that dietary CV and β-glucan supplementation can effectively alleviate CPF-induced hematological, hepatic, renal, and oxidative damage in African catfish. Both supplements improved immune responses and modulated pro-inflammatory gene expression. Notably, CV exhibited superior protective effects on growth performance and survival. Future research should explore additional natural strategies, including probiotics and prebiotics, to mitigate pesticide toxicity in aquaculture and support environmental sustainability.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe present study demonstrated that dietary supplementation with \u003cem\u003eChlorella vulgaris\u003c/em\u003e (CV) and β-glucan can provide protective effects against chlorpyrifos (CPF)-induced toxicity in African catfish. Both feed additives showed potential in mitigating immunosuppression, oxidative stress, hepatic alterations, and inflammatory responses associated with CPF exposure. CV appeared more effective in enhancing growth performance and survival rates, which may be attributed to its nutritional profile and bioactive constituents, while β-glucan exerted notable immunomodulatory and growth-promoting actions. Moreover, both supplements were associated with reduced CPF accumulation in hepatic tissues, suggesting a possible role in detoxification.\u003c/p\u003e\u003cp\u003eHowever, the present work was limited by its experimental scope, including the use of a single fish species, specific dosages, and short-term exposure conditions. Therefore, extrapolation of these findings to other aquaculture systems should be made with caution. Future investigations should explore different fish species, longer-term trials, molecular mechanisms of action, and potential synergistic effects of CV and β-glucan with other natural immunostimulants or probiotics.\u003c/p\u003e\u003cp\u003eOverall, the findings support the potential use of CV and β-glucan as sustainable feed additives to enhance fish health and resilience under chemical stress, but further studies are required before large-scale application in commercial aquaculture.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eALP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAlkaline phosphatase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eALT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAlanine aminotransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAspartate aminotransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBWG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBody weight gain\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCatalase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCOX-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCyclooxygenase-2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCPF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eChlorpyrifos\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eC-reactive protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eChlorella vulgaris\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFBW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFinal body weight\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFeed conversion ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGPx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGlutathione peroxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHepatosomatic index\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIgM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eImmunoglobulin M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIL-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInterleukin-10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIL-1\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInterleukin-1 beta\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eiNOS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInducible nitric oxide synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNF-\u0026kappa;B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNuclear factor kappa-light-chain-enhancer of activated B cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNitric oxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePER\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eProtein efficiency ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRBCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRed blood cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eROS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eReactive oxygen species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSGR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSpecific growth rate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSuperoxide dismutase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTGF-\u0026beta;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTransforming growth factor beta 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTLC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTotal leukocytic count\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTNF-\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTumor necrosis factor-alpha\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eWBCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eWhite blood cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The authors did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors for this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The authors declare that they have no financial or non-financial competing interests that could have influenced the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Ahmed E. A. Mostafa conceived and designed the study, performed the experimental procedures, analyzed the results, and drafted the manuscript. The author approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;All experimental procedures involving African catfish (Clarias gariepinus) were conducted in accordance with the institutional guidelines for the care and use of animals in research at the Faculty of Veterinary Medicine, Zagazig University, Egypt. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Zagazig University under approval number ZU-IACUC/2025/04. All efforts were made to minimize animal suffering and to use the minimum number of fish necessary to achieve the scientific objectives of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author would like to express sincere gratitude to Delta University for Science and Technology for providing the institutional support necessary to conduct this research. Special thanks are extended to Prof. Alaa El-Sayed Abdel-Ghaffar, Dean of the Faculty of Veterinary Medicine, for his continuous encouragement and valuable facilitation throughout the course of the study. The author also acknowledges the technical assistance of the staff of the Department of Pharmacology, Faculty of Veterinary Medicine, Delta University, during the animal experiments and sample analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;All datasets generated or analyzed during the current study are included in this published article. Additional raw data are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdelhamid AM, Mahboub HH, El-Boshy ME (2020) Evaluation of the bactericidal activity of fish serum against \u003cem\u003eAeromonas hydrophila\u003c/em\u003e after dietary supplementation with probiotics. 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Fish Shellfish Immunol 106:808\u0026ndash;816. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fsi.2020.07.013\u003c/span\u003e\u003cspan address=\"10.1016/j.fsi.2020.07.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chlorella vulgaris, β-glucan, Chlorpyrifos, African catfish (Clarias gariepinus), Immunostimulants, Immune response, Antioxidant system, Gene expression, Growth performance, Fish health.","lastPublishedDoi":"10.21203/rs.3.rs-7537362/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7537362/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study was conducted to investigate the toxic effects of chlorpyrifos on growth performance, hepatorenal function, and antioxidant status in African catfish (Clarias gariepinus). One hundred and eighty fish (20\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1 g) were equally distributed into four groups: control group, chlorpyrifos group (0.3 mg/L), chlorpyrifos-CV group (5% CV), and chlorpyrifos-β-glucan group (0.1% β-glucan), and treatments were conducted for about 60 days. The results revealed that administration of chlorpyrifos significantly increased serum liver enzymes, system, innate immune response and comparing the protective role of dietary Chlorella vulgaris (CV) algae and β-glucan in intoxicated African catfish (\u003cem\u003eClarias gariepinus\u003c/em\u003e). One uric acid, creatinine, and malondialdehyde (MDA) in different tissues. Meanwhile, glutathione (GSH) and superoxide dismutase (SOD) in different tissues, as well as IgM, C-reactive protein (CRP), respiratory burst, lysozyme, and bactericidal activities were significantly decreased in the chlorpyrifos group. In addition, expression of TNF-α gene was up-regulated and IL-10 was down-regulated in spleen of chlorpyrifos-intoxicated fish. The treatment of chlorpyrifos-exposed fish with CV and β-glucan supplemented diets ameliorated hepatic damage and enhanced antioxidant activity and innate immune responses. Furthermore, dietary Chlorella vulgaris and β-glucan have a potent anti-inflammatory effect as they remarkably increased the expression of IL-10 and decreased TNF-α gene expression. The results also revealed that fish in chlorpyrifos-CV group had the highest survival rate, final body weight (FBW), and body weight gain (BWG). On the other hand, feed conversion ratio (FCR), specific growth rate (SGR), and protein efficiency ratio (PER) of control, chlorpyrifos-CV, and chlorpyrifos-β-glucan groups were higher than the chlorpyrifos group. However, the hepatosomatic index (HSI) and spleen-somatic index (SSI) were higher in the chlorpyrifos group than other experimental groups. Overall, CV and β-glucan can be recommended as a feed supplement to improve immunosuppression, oxidative damage, growth performance, and hemato-biochemical alterations induced by chlorpyrifos toxicity in African catfish (\u003cem\u003eClarias gariepinus\u003c/em\u003e) .\u003c/p\u003e","manuscriptTitle":"Ameliorative Pharmacological Effects of Dietary Chlorella vulgaris and β-Glucan on Chlorpyrifos-Induced Oxidative Stress (MDA, GSH, SOD), Immunomodulation (TNF-α, IL-10), and Growth Indices in African Catfish (Clarias gariepinus)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 11:40:17","doi":"10.21203/rs.3.rs-7537362/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-05T09:38:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-05T09:37:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-05T05:31:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Research Communications","date":"2025-09-04T14:48:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"75e5131f-0340-4277-af5a-279841d3425a","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:03:13+00:00","versionOfRecord":{"articleIdentity":"rs-7537362","link":"https://doi.org/10.1007/s11259-025-11025-y","journal":{"identity":"veterinary-research-communications","isVorOnly":false,"title":"Veterinary Research Communications"},"publishedOn":"2026-02-04 15:59:41","publishedOnDateReadable":"February 4th, 2026"},"versionCreatedAt":"2025-09-09 11:40:17","video":"","vorDoi":"10.1007/s11259-025-11025-y","vorDoiUrl":"https://doi.org/10.1007/s11259-025-11025-y","workflowStages":[]},"version":"v1","identity":"rs-7537362","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7537362","identity":"rs-7537362","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}