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The present study investigated the effects of a feed additive (FA) containing a probiotic consortium, fructooligosaccharide and yeast extract on growth performance, humoral immune responses, hepatic antioxidant parameters and intestine digestive enzymes, morphology and transcripts in rainbow trout, Oncorhynchus mykiss. The fish were reared for 8 weeks, feeding on diets containing 0 (CNT), 0.3 (0.3FA), 1 (1FA) and 2 (2FA) g/kg FA. The results showed that fish growth parameters were significantly and quadratically related to FA levels and FA treatments had better growth performance than CNT treatment. Intestinal amylase activity significantly increased in 2FA, whereas, intestinal protease activity increased in all FA treatments. Intestinal villus length and muscular layer thickness significantly increased in 0.3FA treatment. Blood leucocyte and lymphocyte counts, plasma lysozyme activity and hepatic glutathione content significantly increased in 0.3FA and 1FA treatments; whereas hepatic malondialdehyde significantly decreased in these treatments. Blood neutrophil and monocyte counts significantly increased in 0.3FA treatment, while plasma alternative complement activity significantly increased in 1FA treatments. Plasma bactericidal activities against Aeromonas hydrophila, Yersinia ruckeri and Streptococcus iniae, and intestinal expression of heat shock protein 70 and beta-defensin significantly increased in all FA treatments. The abundance of A. hydrophila, Y. ruckeri and S. iniae in fish gut significantly decreased in 0.3FA treatment; these bacteria were absent in the intestines of 1FA and 2FA treatments. The present results suggest that dietary 0.3-1 g/kg of FA can significantly improve growth performance, immune response, intestinal health and hepatic antioxidant capacity in rainbow trout.

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This study examined the impact of dietary limonene treatment on the growth performance, immune response, and disease resistance of common carp, Cyprinus carpio. The fish were fed with either a control diet (CTL; no limonene supplementation) or four experimental diets containing 50 (50 L), 100 (100 L), 200 (200 L), and 400 (400 L) mg/kg limonene over a 70-day period, followed by Aeromonas hydrophila challenge. The 200 L treatment resulted in a significant decrease in FCR compared to the CTL treatment. The highest post-challenge mortality was associated with the CTL treatment (62.7%), while the 200 L treatment had the lowest mortality (30.7%). Before the challenge, dietary limonene significantly increased humoral and skin mucosal immune parameters compared to the CTL treatment. The highest leukocyte, lymphocyte counts, skin mucosal protease activity, and intestinal lactic acid bacteria were observed in the 200 L treatment before the challenge. The highest plasma lysozyme activity was observed in the 400 L treatment, whereas the highest skin mucosal lysozyme and peroxidase activities were observed in the 100 L and 200 L treatments before the challenge. There were no significant differences in the blood neutrophil, monocyte, and eosinophil counts, humoral alternative complement activity, skin mucosal alkaline phosphatase activity, and the intestinal total viable bacteria among the treatments before the challenge. After the challenge, the 200 L treatment exhibited the highest leukocyte, neutrophil, and monocyte count, skin mucosal immune parameters, and intestinal lactic acid bacteria, whereas the highest blood eosinophil count was observed in the 100 L, 200 L, and 400 L treatments. At this time, the lowest blood lymphocyte counts were observed in the 100 L and 200 L, but the lowest intestinal total viable bacteria were observed in the 100 L, 200 L, and 400 L treatments. Based on these findings, dietary limonene at 200 mg/kg is ideal for common carp to promote feed efficiency, innate immunity boosting, and resistance against A. hydrophila.

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The present study investigated the effects of dietary lactic acid (LA) supplementation on the growth performance, intestinal digestive/antioxidant enzymes' activities, gene expression, and bacterial communities in common carp, Cyprinus carpio. Four diets were formulated to contain 0 g/kg LA (control), at 2.5 g/kg LA (2.5LAC), 5 g/kg LA (5LAC), and 10 g/kg LA (10LAC) and offered to the fish over a period of 56 days. The results showed that dietary 5 g/kg LA supplementation improved growth performance and feed efficiency in the fish. All LA treatments exhibited significant elevations in the intestinal trypsin and chymotrypsin activities, whereas the intestinal lipase, amylase, and alkaline phosphatase activities exhibited significant elevations in the 5LAC and 10LAC treatments. All LA treatments exhibited significant elevations in the intestinal heat shock protein 70, tumor necrosis factor-alpha, interleukin-1 beta, and defensin gene expressions, and the highest expression was observed in the 5LAC treatment. Additionally, dietary LA treatment significantly increased the lysozyme expression and Lactobacillus sp. population in the intestine of the fish, and the highest values were observed in the 5LAC and 10LAC treatments. Aeromonas sp. and Vibrio sp. populations decreased in the LA treatments, and the lowest Aeromonas sp. population was observed in the 10LAC treatment. The intestinal mucin2 and mucin5 expressions, and the hepatic reduced glutathione content, significantly increased, whereas hepatic glutathione peroxidase, glutathione reductase, and malondialdehyde significantly decreased in the 5LAC and 10LAC treatments. In conclusion, dietary 5 g/kg LA is recommended for common carp feeding to improve growth rate, antioxidant capacity, and intestinal health.

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The effects of dietary glycine supplementation, 0 (control), 5 (5 GL), and 10 (10 GL) g/kg, have been investigated on growth performance, hematological parameters, erythrocyte antioxidant capacity, humoral and mucosal immunity in common carp, Cyprinus carpio. After eight weeks feeding, the 5 GL treatment exhibited significant improvement in growth performance and feed efficacy, compared to the control treatment. Red blood cell (RBC) and white blood cell (WBC) counts, hemoglobin, hematocrit, neutrophil and monocyte counts/percentages, RBC reduced glutathione (GSH) content, and skin mucosal alkaline phosphatase, peroxidase, protease, and lysozyme activities were similar in the glycine-treated fish and significantly higher than the control treatment. Blood lymphocyte percentage decreased in the glycine-treated fish, but lymphocyte count increased, compared to the control fish. RBC glutathione reductase activities in the glycine-treated fish were similar and significantly lower than the control treatment. The highest plasma lysozyme and alternative complement activities were observed in GL treatment. The glycine-treated fish, particularly 5 GL, exhibited significant improvement in RBC osmotic fragility resistance. Dietary glycine had no significant effects on RBC glutathione peroxidase activity, plasma immunoglobulin, eosinophil percentage/count, and hematological indices. In conclusion, most of the benefits of dietary glycine supplementation may be mediated by increased glutathione synthesis and antioxidant power.

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The present study aimed at assessing the effects of dietary Hyssop, Hyssopus officinalis, extract on rainbow trout, Oncorhynchus mykiss, responses to thermal stress. The juveniles (69.8 ± 0.38 g) were stocked in 12 through-flow tanks at a density of 12 fish per tank. Methanolic extract of Hyssop (HME) was added to diet at 0, 100, 250, and 500 mg/kg and the fish were fed (3% of biomass) over a 70-d period: 62 d at 13.3 ± 0.08°C and 7 d at 21-22°C. At the end of the trial, the plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), triiodothyronine (T3), thyroxin (T4), cortisol, glucose, lactate, total antioxidant capacity (TAC), ascorbate, and the gill glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), and malondialdehyde (MDA). The results showed that HME had no significant effects on fish growth performance, survival, and feed efficiency. Dietary 250 mg/kg HME significantly decreased plasma ALT activity (P < 0.001), but showed no significant effects on plasma AST) (P = 0.106) activity, T3 (P = 0.992), and T4 (P = 0.070) levels. Thermal stress significantly (P < 0.001) increased plasma ALT and AST activities, but lowered plasma T3 and T4 levels. Dietary HME and thermal stress had interaction effects on plasma cortisol (P < 0.001), glucose (P = 0.007), lactate (P = 0.010), LDH (P = 0.005), TAC (P = 0.038), ascorbate (P < 0.001), and the gill GPx (P = 0.001), GR (P < 0.001), GST (P < 0.001), and MDA (P = 0.001). Thermal stress significantly increased plasma cortisol, glucose, lactate, and LDH, the gill GPX, GR, and GST, but dietary HME supplementation significantly reduced such elevations, particularly at 250 mg/kg level. Dietary HME significantly increased plasma TAC before the thermal stress and mitigated the stress-induced decreased in TAC, particularly at 250 mg/kg level. Dietary HME significantly decreased the gill MDA before and after the thermal stress, and lowest MDA was observed in 250 mg/kg HME level. Based on the present results, 250 mg/kg HME is recommended as suitable dose to improve antioxidative responses and hepatoprotection in rainbow trout under heat stress.

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The present study was conducted to find the effects of dietary lactic acid (LA) supplementation on growth performance, hematological characteristics, humoral antioxidant and immunological factors, intestinal gene expression and bacteriological parameters in rainbow trout, Oncorhynchus mykiss. The fish (104 ± 0.90 g) were randomly distributed in twelve tanks (150 L water) as four treatments, receiving diets supplemented with 0, 5, 10, and 20 g/kg LA for 70 days. At the end of the feeding trial, no significant differences were observed in the fish growth performance and survival among the treatments. Blood/plasma red blood cell (5-20 g/kg LA), white blood cell (10 g/kg LA), hematocrit (5 and 10 g/kg LA), hemoglobin (5 and 10 g/kg LA), superoxide dismutase (10 g/kg LA), glutathione peroxidase (10 g/kg LA), total antioxidant capacity (10 g/kg LA), lysozyme (5-20 g/kg LA), and alternative complement activity (10 g/kg LA) significantly increased, as malondialdehyde (10 g/kg LA) significantly decreased in the LA-treated fish. Dietary LA supplementation induced no significant changes in the intestinal defensin gene expression, but significantly up-regulated the expression of toll-like receptor-3 (5-20 g/kg LA), Mx-9 (5-20 g/kg LA), and heat shock protein-70 (5 g/kg LA) genes; the highest expressions were observed at 5 g/kg LA supplementation. Dietary LA supplementation significantly increased total bacterial count (5 and 10 g/kg LA), Lactobacillus sp. (5-20 g/kg LA), and Bacteroides sp. (5-20 g/kg LA), but decreased Streptococcus iniae (5-20 g/kg LA) in the fish intestine; the highest Lactobacillus sp. And Bacteroides sp. Were observed in 10 and 5 g/kg LA supplementation, respectively. Overall, dietary LA supplementation, although has no significant effects on the fish growth performance, is beneficial to improve humoral antioxidant parameters, humoral and intestinal immunological parameters and intestinal microflora. According to the results, dietary 5-10 g/kg LA is recommended for the formulation of rainbow trout feed.

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The present study aimed at assessing the effects of exposure to silver nanoparticle (AgNP) and a subsequent acute stress on the expression of various genes involved in the hypothalamus-pituitary-interrenal (HPI) axis in zebrafish, Danio rerio. The fish were exposed to 0 (Control), 0.1 (LC), 0.4 (MC), and 1.2 (HC) mg Ag/L (as AgNP) over a 2-week period, followed by an acute air exposure stress. The whole body cortisol and the expression of selected genes in the fish brain and kidney were analyzed, before and after the acute stress. The results showed that AgNP increased basal cortisol levels and the expression of corticotropin releasing factor, prohormone convertase 1, pro-opiomelanocortin, and melanocortin 2 receptor; however, it suppressed/inhibited whole body cortisol, brain corticotropin releasing factor responses, pro-opiomelanocortin, and the kidney melanocortin 2 receptor responses to the acute stress. AgNP down-regulated the expression of the steroidogenic acute regulatory protein, but it intensified the gene expression in response to the acute stress. Before the acute stress, LC treatment exhibited an up-regulation in Cytochrome P450-11A-1 expression, but MC and HC treatments induced down-regulation. After the acute stress, the AgNP-exposed fish exhibited decreased Cytochrome P450-11A-1 expressions, compared with the Control. Exposure to AgNP significantly increased Cytochrome P450-11B expression. However, after the acute stress, LC treatment exhibited an up-regulation, but MC and HC treatments exhibited down-regulation in the Cytochrome P450-11B gene expression. In conclusion, AgNP suppressed cortisol response to stress, which appears to be a consequence of alterations in the HPI axis at the transcriptomic levels.

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The combined effects of copper and polyvinyl chloride (PVC) microparticles were investigated on the metal accumulation, histopathological biomarkers, and targeted transcriptomics in Cyprinus carpio liver. The fish were exposed to 0.25 mg/L copper and/or 0.5 mg/L PVC microparticles over a 14-d period. The results showed that hepatic copper accumulation is facilitated by the PVC microparticles presence in water. All treatments induced significant hepatic stress and inflammation; however, the transcriptional responses involving in detoxification pathways and apoptotic mechanisms were mixed and often down-regulated in the fish exposed to copper and/or PVC microparticles. Exposure to copper and/or PVC microparticles induced hypermeia, leukocyte infiltration and increase in melanomacrophage centers number and area. Generally, the severity of the lesions was in the following order: PVC microparticles < copper < copper+ PVC microparticles. In conclusion, PVC MPs act as a copper vector, facilitating accumulation of copper in the fish liver and increasing the tissue damage.

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The aim of this study was to examine the combined effects of thyme essential oil (TEO) and prebiotic (Immunogen ®) on growth performance, hematological parameters, innate immunity, and oxidative status of rainbow trout, Oncorhynchus mykiss. For this purpose, the fish (11.92 ± 0.06 g) were fed (3% of biomass) experimental diets that contained 0 (Control; without Immunogen and/or TEO); 1% TEO + 0.1% Immunogen (T1); 1% TEO + 0.2% Immunogen (T2); 2% TEO + 0.1% Immunogen (T3) and 2% TEO + 0.2% Immunogen (T4) for 60 days. According to results, all experimental treatments exhibited similar final weight, weight gain, specific growth rate (SGR), and survival rate (SR), which were significantly higher than those of the control treatment. Although all treatments reduced the feed conversion ratio (FCR) compared to the control group, the lowest value was observed in T4. All experimental treatments showed a significant increase in amylase and protease activity compared to the control group. Moreover, the fish fed on T4 and T1 diets showed the highest and lowest lipase activity. Dietary TEO and Immunogen supplementations significantly increased WBC count and Hb level compared to the control group. Fish fed on control and T2 diets displayed lower hematocrit than fish fed on other experimental diets. The highest and lowest MCH index were recorded in T3 and control groups, respectively. The fish fed diets supplemented with T3 diet presented significantly higher MCV index compared to the control and T2 treatment. All experimental treatments exhibited similar AST, and ALP activities, which were significantly lower than those of the control group. Also, the lowest ALT activity was observed in T2 and T4 treatments compared to other groups. Dietary TEO and Immunogen supplementations significantly enhanced skin mucus total Ig, total protein level, and ACH50, protease, and lysozyme. All experimental treatments exhibited enhanced intestine total Ig, ACH50, and lysozyme level. Dietary thyme essential oil and Immunogen supplementations significantly enhanced liver antioxidant parameters including catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD) and decreased MDA production compared to fish fed on control diet. As a result, it can be suggested that the combination of thyme essential oil and Immunogen (specially 2% TEO + 0.2% Immunogen) is useful for enhancing the yield and well-being of farmed rainbow trout.

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The aim of this study was to assess the anesthetic efficacy and biochemical effects of citronellal on rainbow trout, Oncorhynchus mykiss. The fish were exposed to 200-800 µl/L citronellal to find the anesthesia induction and recovery times. The deep anesthesia stage was reached within 117-613 s, using 800-200 µl/L citronellal, respectively, and all fish recovered within 240-420 s. To assess biochemical responses, the fish were exposed to 200, 400, 600, and 800 µl/L of citronellal and blood-sampled after deep anesthesia. The results showed that anesthesia led to significant elevations in blood erythrocytes, hematocrit, hemoglobin, mean corpuscular volume, plasma lactate, and lactate dehydrogenase; these parameters exhibited increasing trends as citronellal concentration increased. These results along with significantly higher plasma lysozyme activity, cortisol, and glucose levels in 200 µl/L treatment suggest that an increase in citronellal concentration significantly mitigates hypoxia/stress responses in the fish. Significant elevations in plasma alkaline phosphatase activity and malondialdehyde level were observed in the 200 µl/L treatment, which suggests induction of hemolysis and oxidative stress in this treatment. There was a tendency to higher ammonia levels along with the increase in citronellal concentration, which might be due to early ammonia accumulation because of lower opercular movement in the fish. Except for blood hematocrit and mean corpuscular volume, there were no significant differences between the 600 and 800 µl/L citronellal treatments. In conclusion, high concentrations of citronellal are more suitable for trout anesthesia and blood sampling than low concentrations. Based on the present study, 600-800 µl/L citronellal is recommended for trout anesthesia.

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The present study aimed at assessing the singular and combined effects of water copper and polyvinyl chloride microplastic (MPVC) on intestinal copper accumulation, histopathological damage, and stress-/immune-related genes' expression in common carp, Cyprinus carpio. Four groups of fish were maintained in triplicate: control (kept in clean water), Cu (exposed to 0.25 mg/L of copper), MPVC (exposed to 0.5 mg/L of MPVC), and Cu-MPVC (exposed to 0.25 mg/L of copper + 0.5 mg/L of MPVC). After 14-day exposure, the fish of Cu and Cu-MPVC treatments exhibited significantly higher intestinal copper contents, compared to the fish of control and MPVC treatments. In this regard, the Cu-MPVC fish had significantly higher copper content than the Cu fish. Exposure to copper and/or MPVC significantly upregulated the intestinal heat shock protein 70 (hsp70), cytochrome P450 family 1 subfamily A member 1 (cyp1a1), lysozyme (lys), defensin (def), mucin 2 (muc2), and mucin 5 (muc5) expression. The highest expression of hsp70, cyp1a1, lys, and def was related to Cu-MPVC treatment; whereas, the highest expression of muc2 and muc5 was observed in Cu and MPVC treatments. Exposure to copper and/or MPVC induced intestinal damage, which Cu-MPVC fish exhibited the highest severity. The present study revealed that exposure to copper and/or MPVC causes intestinal histopathological damage and upregulation in stress- and immune-related genes' expression. The most serious effects were observed in Cu-MPVC treatment that might be due to additive effects of copper and MPVC and/or higher copper accumulation in this treatment.

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In this research, bioactive nano-hybrids based on the nano-fibrillar chitosan-ZnO (NF-CS-ZnO) were synthesized to diminish the toxicity of ZnO-NPs. The successful formation of nano-hybrids was confirmed by FT-IR, UV-Vis, and FE-SEM analyses, showing a uniform spherical ZnO-NPs with an average diameter of 20-30 nm, homogeneously dispersed on NF-CS. The obtained results demonstrated a remarkable antibacterial activity of NF-CS-ZnO-0.6 nano-hybrid against E. coli and S. aureus and, interestingly, no cytotoxic on normal cells (even at a high concentration of 100 µg/mL). Furthermore, NF-CS hybridization efficiently decreased the up-regulation in Cas3, Cas9, and Il6 of inspected fishes compared to the ZnO-NPs. Histopathological examination revealed hepatocyte necrosis in the fish exposed to ZnO-NPs and hyperemia exposed to NF-CS-ZnO-0.6 nano-hybrid. Finally, NF-CS efficiently improved the bio-safety and bactericidal activity of ZnO-NPs; therefore, NF-CS-ZnO nano-hybrid is prominently recommended as a talented low-toxicity antibacterial agent replacement of conventional ZnO-NPs for use in different applications.

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Effects of dietary hydroalcoholic extract of Zataria multiflora (ZE) on growth performance, plasma and hepatic antioxidant capacities, and humoral and skin mucus immune parameters were evaluated in rainbow trout (Oncorhynchus mykiss) juveniles. in vitro tests showed that ZE had antioxidant property comparable to butylated hydroxytoluene (BHT) at 100-200 µg/mL concentrations, although its antioxidant property was lower than BHT at concentration below 100 µg/mL. Moreover, ZE had anti-bacterial activity against Aeromonas hydrophila, which was 30-50% lower than that of tetracycline. After feeding the fish with diets supplemented with 0 (CT, 1 (ZE1), 2 (ZE2), and 3 (ZE3) g/kg ZE for eight weeks, there were no significant differences in growth performance and feed efficiency among the treatments; however, the fish in ZE2 and ZE3 treatments showed significantly higher survival than the fish in CT treatment. Blood leukocyte counts, plasma globulin, total immunoglobulin, lysozyme and bactericidal activity against A. hydrophila in ZE2 and ZE3 groups were significantly higher than that of CT group. All the ZE-treated groups had higher plasma complement activity compared to the CT group. Mucosal lysozyme and bactericidal activities of the ZE2 fish were significantly higher than the other treatments. Expression of tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, and lysozyme genes increased in head kidney of the fish treated with ZE; the highest increases were related to the ZE2 treatment. Plasma total antioxidant (TA) activities of ZE2 and ZE3 treatments were significantly higher than that of the CT treatment. Plasma and hepatic superoxide dismutase (SOD) and catalase (CAT) activities of ZE2 group were significantly higher than the other treatments. Plasma malondialdehyde (MDA) levels were significantly lower in ZE2 treatment, compared to the other treatments. However, hepatic MDA level of ZE2 treatment was significantly lower than those of the ZE1 and CT treatments. In conclusion, dietary ZE supplementation level of 2 g/kg is suggested for rainbow trout feed supplementation to augment fish survival, antioxidant and immune strength.

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The present study assessed the potential benefits of myrcene administration to suppress negative effects of copper exposure on immune-, antioxidant-, tight junction-, stress- and osmoregulatory-related gene expressions in common carp (Cyprinus carpio) gill. Fish were fed with diets containing 0% (control), 0.5% (M0.5) and 1% (M1) myrcene for 6 weeks, and then, exposed to 0.25 mg/L copper for further two weeks. The fish gill samples were taken for gene expression assays after six and eight weeks. The results showed that there were interaction effects of myrcene levels and copper exposure on superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione-s-transferase (gst), glutathione reductase (gr), heat shock protein-70 (hsp70), interleukin 1-beta (il1b), interleukin 10 (il10), tumor necrosis factor-alpha (tnfa), occludin (occl), caludin 3 (cld3), caludin 7 (cld7), and Na+-K+-ATPase (nka) genes expressions. Overall, the M0.5 treatment had significantly lower antioxidant genes expression, and higher hsp70, cytokines, tight-junction proteins, and nka genes expression, compared to the control treatment, before copper exposure. Copper exposure significantly down-regulated most of the tested genes (except il10), however, the M0.5 treatment had significantly higher antioxidant (except gpx), hsp70, cld7, and nka gene expression compared to the control treatment. The M1 treatment showed fluctuated antioxidant gene expressions, down-regulated gene expression of the pro-inflammatory cytokines, and occl, and up-regulation of cld3 gene expressions, before copper exposure. After copper exposure, this treatment had significantly higher gr and cat expression compared to the control; moreover, there was a marked up-regulation in il10 gene expression in this treatment, which was the highest value among all treatment combinations. In conclusion, copper exposure significantly down-regulates antioxidant-, inflammatory-, and tight junction-related along with hsp70 and nka genes expression in common carp gills. Pre-treatment with 0.5% myrcene is beneficial to suppress such negative effects, probably due to its antioxidant properties. However, myrcene administration must be done with caution, as higher levels may interfere with antioxidant and immune defenses.

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The aim of this study was to investigate plasma proteins, total immunoglobulin (Ig), lysozyme and complement (ACH50) levels, and gill superoxide dismutase (SOD) and catalase (CAT) gene expression in common carp (Cyprinus carpio), following exposure to lufenuron (LUF) and flonicamide (FL). Fish were distributed in 12 tanks as three quadrupled treatments: control (fish were kept in pesticide-free water), LUF [fish exposed to 10% of LUF LC50 (4.3 mg/L)], and FL [fish exposed to 10% of FL LC50 (0.1 mg/L)]. The plasma parameters were assessed after 7 and 21 days exposure to pesticides, whereas the gene expressions were assessed after 21 days. The results showed that LUF exposure significantly decreased plasma total protein and globulin levels compared to the control group. Both pesticide significantly decreased plasma total Ig levels compared to the control group; however, LUF exhibited a greater effect. There were no significant effects of pesticides or sampling time on plasma ACH50 activity. Pesticides and sampling time interacted to affect plasma lysozyme activity. Seven days after exposure, both pesticides significantly increased lysozyme activity, and the effect of FL was greater than LUF. Nevertheless, there was no significant difference in plasma lysozyme activities among the pesticides, 21 days after the exposure. Both pesticides significantly decreased SOD and CAT gene expression, nevertheless, FL exhibited greater effects than LUF. In conclusion, both pesticides induced immunosuppression in the fish, though such effects were more severe in LUF group, compared to the FL. These pesticides negatively affect expression of gill antioxidant genes, and the FL effects were greater than the LUF.

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Intestine in fish is a complex multifunctional organ, not only plays roles in digestion and absorption of nutrient, but also has critical role in immunity. The present study evaluated the effects of different levels of dietary sodium butyrate [Butirex® C4 (Butirex)] on intestinal immune-,antioxidant-and tight junction-related gene expression injuvenile rainbow trout(Oncorhynchusmykiss). 240 healthy rainbow trout were dispensed in 12 fiberglass tanks appointed to four treatments [0 (control), 1.5 (B1.5), 2.5 (B2.5) and 5 (B5)g Butirex per kg diet]. After a 45-day feeding trial, the fish fed with the Butirex-supplemented diets showed higher intestinal lysozyme (LYZ), complement(ACH50) and bactericidal activities; the elevations in ACH50 and bactericidal activities depended on Butirex levels (P < 0.05). The Butirex-supplemented groups, particularly the B2.5 group, had significantly higher LYZ gene expression compared to the control group (P < 0.05). Butirex at 2.5 and 5 g/kg levels led to significantly higher IL-1ß gene expression. B2.5 and B5 had significantly lower and higher TNF-α gene expression compared to the control group (P < 0.05). The B2.5 group had significantly higher TGF-B, and significantly lower IL-8 compared to the control group (P < 0.05). The B1.5 and B2.5 group had significantly higher IL-10 gene expression compared to the control group (P < 0.05). The B2.5 and B5 groups had significantly higher SOD gene expression compared to the other groups; the highest expression was related to the B2.5 group (P < 0.05). Dietary Butirex supplementation significantly up-regulated CAT and GPx genes expression compared to the control group; the highest expression as related to the B2.5 and B5 groups (P < 0.05). The B2.5 group had significantly lower CLD12 gene expression compared to the control group (P < 0.05). The B2.5 and B5 groups had significantly higher CLD3, OCLD and ZO-1 gene expression compared to the control. The highest CLD3, ZO-1 gene expressions was related to the B2.5, and B5 groups respectively (P < 0.05). After challenge with Streptococcus iniae, B2.5 and B5 had significantly higher survival compared to the control group (55.6 ±â€¯7.70 and 68.9 ±â€¯10.2 vs. 33.3 ±â€¯6.67). In conclusion, Butirex is efficient immune stimulant and health booster in rainbow trout, which augments the fish resistance to disease. Modulation of immune components, cytokines, antioxidant system and intestinal integrity might involve in improving disease resistance in Butirex-treated fish. Although most of the examined genes were modulated by 2.5 g/kg Butirex under normal conditions, 5 g/kg level is recommended under pathogenic state to mitigate mortality.

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The aim of the present study was to investigate beneficial effects of dietary 1,8-cineole (cineole) supplementation on physiological, immunological and antioxidant responses of rainbow trout (Oncorhynchus mykiss) to crowding stress. The fish were fed for 50 days with diets containing 0 (control), 0.05, 0.1, 0.25, 0.5 and 1% cineole prior to exposure to a 14-day crowding stress. Serum stress markers (cortisol, glucose, lactate, T4 and T3), antioxidant responses [catalase (CAT) and superoxide dismutase (SOD) activities and total antioxidant capacity (TAC) and malondyaldehyde (MDA) levels] and immune responses [lysozyme and alternative complement (ACH50) activity, and total immunoglobulin (Ig) levels], and blood leukocyte (WBC) and differential counts were measured before and after crowding stress. Results showed that 1% cineole was capable to reduce the basal and stress-induced cortisol elevation and increase the serum T3 levels after stress. Increase in dietary cineole levels significantly decreased serum cortisol, glucose and lactate levels. Increase in dietary cineole levels significantly increased serum CAT, SOD, lysozyme and ACH50 activities, and TAC and eosinophil levels, and decreased MDA and monocyte levels. After the stress, there was no significant difference in the control group CAT and SOD activities compared to the basal values; however, CAT activities decreased and SOD activities increased in the cineole-treated groups compared to the basal values. Nevertheless, the control group had significantly lower CAT and SOD activities compared to the fish treated with 0.1-1% cineole. Cineole significantly increased blood WBC and serum lysozyme, ACH50 and total Ig. Moreover, cineole administration significantly mitigated the stress-induced decrease in total Ig levels as well as increase in leukocyte count. The cineole-treated fish had higher survival and growth performance compared to the control group. Although all levels of cineole (0.05-1%) showed beneficial effects on different tested factors, 0.5 and 1% levels had beneficial effects on most of the tested factors; thus, are recommended for dietary inclusion to suppress adverse effects of stress in trout.

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The aim of the present study was to investigate anesthetic efficacy of myrcene in rainbow trout (Oncorhynchus mykiss) along with the fish biochemical response to anesthesia in comparison with eugenol. In the first experiment, 240 fish were stocked in 12 tanks and acclimatized to experimental conditions for 2 weeks. Then, the fish of each tank were subjected to one concentration of either eugenol (12, 20, 30, 50, 80, and 130 µL/L) or myrcene (100, 150, 200, 300, 400, and 500 µL/L) concentrations. Induction time of and recovery time from anesthesia were recorded for each fish separately. Using these results, desired concentrations to induce anesthesia within 60, 180, 300, and 600 s were determined, being 81, 30, 19, and 10 µL/L eugenol and 531, 251, 177, and 111 µL/L myrcene. In the second experiment, 96 fish were stocked in 8 tanks. Six fish were netted from each tank and exposed to the calculated eugenol or myrcene concentrations. Blood samples were taken after the fish reached anesthesia. The results showed that there was no significant difference in serum lactate, alanine transaminase, and alkaline phosphatase. Increase in the induction time of anesthesia resulted in increased serum glucose with no significant difference between the anesthetics. Increase in induction time of anesthesia led to increase in serum lactate dehydrogenase activity in the eugenol-anesthetized fish and aspartate transaminase activity in myrcene-anesthetized fish. In conclusion, myrcene is capable to anesthetize rainbow trout, but at higher concentrations compared to eugenol. In addition, biochemical analysis showed that increase in induction time of anesthesia leads to hyperglycemia and increase in AST and LDH activities depending on anesthetic type.

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The present study aimed to determine hemolysis interference in measurement of plasma biochemical parameters in fish specimens. For this purpose, blood samples were harvested from 24 Huso huso juveniles. After centrifugation, each plasma sample was divided into seven portions to make seven levels of hemolysis. Hemolysis was induced by addition of different percentages of the corresponding whole blood [0 (non-hemolyzed control group), 1, 3, 5, 10, 15, and 20% of whole blood]. Albumin, total protein, calcium, phosphorus, glucose, sodium, potassium, chloride, alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT) were measured in different samples. Results showed that plasma albumin, ALP, potassium, and AST significantly increased (more than 200% at the highest hemolysis level) in the hemolyzed samples. Also, plasma total protein and phosphorus showed significant elevation in the hemolyzed samples (more than 70% at the highest hemolysis level). Plasma glucose, calcium, chloride, and ALT showed narrow but significant increase in hemolyzed samples (11.8-35.2% at the highest hemolysis level). Plasma sodium showed no significant changes in the hemolyzed samples. In conclusion, the present results show that hemolysis markedly affects plasma parameters levels, which interferes with plasma results interpretation. Therefore, analysis of hemolyzed samples should be avoided or the results should be interpreted with caution. This study encourages further investigations to develop methods for omitting the effects of hemolysis by sample blanking and/or presenting correction coefficients for measurement of plasma parameters in samples with different levels of hemolysis.

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Differences in toxicological effects of dissolved copper and copper nanoparticles were studied in common carp, Cyprinus carpio. The fish were exposed to 0.25mgL-1 copper as copper sulfate (0.25Cu), 0.25mgL-1 copper as copper oxide nanoparticles (0.25NCu) and 25mgL-1 copper as copper oxide nanoparticles (25NCu) over 14days. Plasma biochemical, enzymatic and hormonal characteristics, and liver and kidney histopathology were examined at the end of the experiment. The results showed that both forms of copper had no significant effects on plasma calcium levels, however, significantly increased plasma phosphorous levels, compared to control group (no added copper). Plasma alanine transaminase (ALT) activity increased in 0.25Cu and 25NCu treatments compared to the control and 0.25NCu treatments. Nanoparticle copper exposure significantly decreased plasma alkaline phosphatase (ALP) activity compared to the control and 0.25Cu treatments. Only copper sulfate exposure caused plasma aspartate transaminase (AST) elevation. Both copper forms increased plasma T4 and free T4 (FT4); however, copper sulfate effect was higher than nanoparticle copper. Copper sulfate exposure increased plasma albumin fraction, whereas, 25mgL-1 copper nanoparticle exposure increased plasma α2-globulin fraction compared to the control. Both copper forms damaged the fish liver and kidney, however, copper sulfate caused more severe damages compared to nanoparticle copper. Overall, except for plasma ALP and α2-globulin fraction, dissolved copper seems to be more toxic than nanoparticle copper in common carp.

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