Interactive Effects of Curcumin and Silver Nanoparticles on Growth, Hemato-Biochemical Parameters, Digestive Enzymes Activity and Histology of Common Carp (Cyprinus Carpio)


 Background: Promising physicochemical characteristics of nanoparticles (NPs) have encouraged their increasing synthesis and application in various industries which is accompanied with their leakage to the environment with inevitable effects on non-target animals including fish. Accordingly, developing strategies to protect fish against NPs contamination has gained attention. The aims of this study were to (a) explore the effects of feed born silver nanoparticles (AgNPs) in common carp (Cyprinus carpio), and (b) to examine whether dietary curcumin supplementation can ameliorate the impacts of AgNPs on growth, hemato-biochemical parameters, digestive enzymes activity and organ histology. Methods: Nine experimental diets were prepared with three different levels of AgNPs (0, 0.05 and 0.15 g/kg) and curcumin (0, 0.75 and 1.5 g/kg) and fed to triplicate groups of common carp (4.82 ± 0.41 g) for 60 days. Results: The results showed that AgNPs significantly (P < 0.05) reduces growth performance and enhances feed conversion ratio in a dose dependent manner. Supplementing 0.75 g/kg curcumin at lower AgNPs level improved growth rate while its inclusion at higher AgNPs level further hampered fish growth. A similar trend was observed for survival rate. The highest hematocrit and hemoglobin concentrations and white blood cells count were recorded in the group received 0.75 g/kg curcumin, and inclusion of the same dose of curcumin in the diet containing 0.05 g/kg AgNPs retrieved the reduction of these parameters. Serum aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase activities, and glucose, cholesterol and triglyceride concentrations were enhanced by increasing AgNPs level, and curcumin inclusion particularly at lower level of AgNPs significantly decreased their values. Activity of digestive enzymes including alkaline protease and lipase were progressively decreased by increasing AgNPs level and significant improvements were found by curcumin application at lower AgNPs level. In addition, severe pathological changes in liver and intestine were observed at high concentrations of AgNPs and curcumin. Conclusions: The findings in this study demonstrated that dietary supplementation of curcumin at lower dose of AgNPs can restrain the toxic effects, however, its inclusion at higher dose of AgNPs exacerbated the negative impacts.

body weight (BW) once a day with the basal diet [30]. The feeding trial was lasted for 60 days under natural photoperiod. In the course of the experiment, sh were fed three times a day at the rate of 3 % BW [31]. The water pH (7.3), dissolved oxygen content (7.6 mg L -1 ) and temperature (23 °C) of tanks were monitored daily. In addition, daily water exchange rate was 50 %.

Sample collection
The sh were fasted for 24 h before sampling and were anaesthetized with 200 ppm clove oil solution to minimize stress on sh. All the sh in each tank were counted and weighed collectively for calculation of survival and growth parameters. Blood was withdrawn from the caudal vein using 2-mL sterilized hypodermic syringes. The collected blood samples were divided into two portions. One portion was transferred into Eppendorf tubes containing heparin and immediately used for hematological examination. The second portion was transferred into Eppendorf tubes, left to clot at room temperature for 30 min, and centrifuged at 1500 ×g for 15 min. The collected serum was stored at -80 ºC for further biochemical analyses.

Hematology assays
The red blood cell (RBC) and white blood cell (WBC) counts were determined using Neubaeur hemocytometer [32]. Hematocrit (Ht) was measured using the standard microhematocrit method and hemoglobin concentration (Hb) was measured by the cyanomethemoglobin spectrophotometry method [33].
Blood biochemistry assays Albumin, cholesterol, glucose and triglyceride concentrations were measured using commercial kits (Pars Azmoon, Tehran, Iran) using an autoanalyzer (Labsystemphotic100, Japan) [36]. Serum total protein level was quanti ed using a BioRad Protein Assay Kit (No. 500 -0006, Bio-Rad Laboratories, New Orleans, LA, USA) using bovine serum albumin as the standard, and following the method described by Bradford [37]. The activity of in ammatory enzymes including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured according to the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), alkaline phosphatase (ALP) according to the Deutsche Gesellschaft fur Klinische Chemie (DGKC) method and lactate dehydrogenase (LDH) in compliance with DGKC (P-L) [38].

Digestive enzymes activity
After removing visceral fat on ice, intestine was dissected out and washed by cold normal saline solution and stored at −80 °C until extraction [39]. The samples were homogenized in 1:3 (w/v) cold 50 mM tris-HCl buffer (pH = 7.5), using Polytron PT 1300 D homogenizer with a 7 mm generator at a setting of 10 for 3 × 30 s. The homogenate was centrifuged at 10,000 ×g for 20 min at 4 °C and the supernatant was collected and stored at −80 °C until analyses [40].
Amylase activity was assayed by means of a starch-iodine detection following Metais and Bieth [41]. Brie y, 50 µL of enzymatic extract was mixed with the substrate (3 g L -1 starch in 66 mM Na 2 PO 4 ) and incubated for 20 min at 25 °C. The reaction was stopped with 20 µL of 1 N HCl, and after addition of 2 mL of 0.33 mM iodine solution the absorbance was read at 580 nm. One unit of α-amylase activity was de ned as the mg starch hydrolyzed per min at 25 °C.
Lipase activity was determined by hydrolysis of nnitrophenyl myristate. Each assay (0.5 ml) contained 0.53 mM nnitrophenyl myristate, 0.25 mM 2methoxyethanol, 5 mM sodium cholate and 0.25 M Tris-HCl (pH 9.0). Incubation was carried out for 15 min at 30 °C, and the reaction was terminated by adding 0.7 ml of acetone/n-heptane (5:2, v/v). The reaction mixture was vigorously mixed and centrifuged at 6080 g for 2 min. The absorbance at 405 nm in the resulting lower aqueous layer was measured. The extinction coe cient of n-nitrophenol was 16,500 M −1 cm −1 l −1 . One unit of enzyme activity was de ned as 1 μmol of n-nitrophenol released per min [42].
Alkaline protease activity was assayed as described by García-Carreño and Haard [43] using Azocasein 2% in Tris-HCl, pH = 7.5 as substrate. The speci c enzyme activity was reported as unit activity per mg protein per min.

Histological studies
The intestinal, hepatic and gill tissue samples were xed in Bouin's solution for 72 h. Afterwards, samples were submitted to standard tissue passage procedures for histological examinations by 70%, 80%, 90% and 100% ethanol (Razi, Iran) and nally were embedded in para n blocks (Merck, Germany). Sections of 4 µm thickness were prepared and stained with hematoxylin and eosin (Merck, Germany). The slides were analyzed under light microscope (Olympus, Germany). Tissue pathological changes including atrophic or elongated nuclei of hepatocells, morphologically abnormal lamellar body, necrosis and hyperplasic lamellas of gill tissue along with increased goblet cell population and epithelial hyperplasia of intestinal tissue were observed and graded as normal (0), mild (+1), moderate (+2), severe (+3) and very severe (+4).

Statistical analysis
The homoscedasticity of variance of the dependent variables was checked by Levene's test. Standard normality test of Kolmogorov-Smirnov was applied to determine normality of data set. Two-way ANOVA was used to elucidate whether or not there were signi cant differences among various experimental groups. Tukey's HSD test at P <0.05 was used to assess signi cant differences among treatments. All statistical analyses were carried out using SPSS statistical software (version 19, SPSS, Inc., Chicago, IL).

Growth performance
Two-way ANOVA results revealed that all growth indices were affected by two-way interaction of dietary AgNPs and curcumin supplementation ( Table 3, P < 0.05). Dietary inclusion of AgNPs led to the signi cant reduction (P < 0.05) of nal body weight, weight gain and speci c growth rate in a dose dependent manner. Curcumin supplementation in diets contacting 0.05 g/kg AgNPs improved growth performance but its inclusion at higher level of AgNPs aggravated the growth depression. A progressive increase in feed conversion ratio (FCR) was observed by increasing AgNPs level and supplementing 0.75 g/kg curcumin at lower level of AgNPs decreased FCR. A similar trend was observed for sh survival rate. Curcumin supplementation did not in uence the abovementioned parameters compared to the control group.

Hemato-biochemical Parameters
All hemato-biochemical parameters were interactively affected by dietary AgNPs and curcumin contents (P < 0.05). Ht and Hb concentrations, and WBC and RBC counts were signi cantly decreased by AgNPs level, and curcumin inclusion in diets containing 0.05 g/kg AgNPs signi cantly enhanced their values (Table 4). Moreover, the highest Ht, Hb, WBC and RBC values were detected in sh fed the diet containing 0.75 g/kg curcumin. Higher MCV values were found in groups received AgNPs containing diets and curcumin supplementation reduced its value. MCHC was remarkably decreased by AgNPs inclusion and a signi cant improvement was found by supplementing 0.75 g/kg curcumin in the diet containing lower level of AgNPs.
Serum glucose, cholesterol and triglyceride concentrations, and activities of AST, ALT and LDH were notably increased by AgNPs inclusion and curcumin supplementation at lower AgNPs level decreased their values. Serum protein content signi cantly decreased at both levels of AgNPs and inclusion of curcumin in diets containing 0.05 g/kg AgNPs signi cantly improved the protein concentration. The signi cant reduction of ALP activity at lower level of AgNPs was retrieved by curcumin application (Table 5).

Histology
Hepatocyte nucleus narrowing and atrophy were observed in sh fed diets containing different concentrations of AgNPs and curcumin (Fig. 1). The highest liver tissue destruction was detected in treatments containing high concentrations of AgNPs and curcumin (Table 7). No lesions were observed in liver tissue of sh fed with 0.75 g/kg curcumin. Gill blades deformation, necrosis and hyperplasia as well as sticking of the end of the gill bers were observed in sh fed diets containing different concentrations of AgNPs and curcumin (Fig. 2). The highest gill tissue destruction was detected in treatments containing high concentrations of AgNPs and curcumin (Table 7). No lesions were observed in gill tissue of sh fed with 0.75 g/kg curcumin. Increased number of goblet cells and intestinal epithelial hyperplasia were observed in sh fed diets containing different concentrations of AgNPs and curcumin (Fig. 3). The severe pathological changes in intestine tissue were observed in treatments containing high concentrations of AgNPs and curcumin (Table 7). Similarly, no changes or lesions were observed in intestine of sh fed diet contained 0.75 g /kg of curcumin.

Discussion
Pollutants including nano-sized materials discharged into water bodies affect aquatic ecosystems via their uptake by the aquatic organisms and their accumulation in animal tissues over time leading to subsequent gastrointestinal toxicity [44]. Our results showed that feed born AgNPs hampers growth performance of common carp, and curcumin supplementation at lower inclusion level could partially prevent suppressive effect of AgNPs. Moreover, no adverse effect of curcumin was found on sh growth performance which is consistent with the results of studies by Lee et al. [45], Jang et al.
[46] and Imani et al. [14] indicating no impacts of plant extracts on animal feed intake, weight gain and feed conversion ratio. Dose dependent reduction of growth performance by AgNPs in this study agrees with depression of growth performance in Clarias batrachus [47] and Epinephelus coioides [48] by AgNPs and copper nanoparticles, respectively. However, Ramsden et al. [49] showed that titanium dioxide nanoparticles had no effects on growth of rainbow trout (Oncorhynchus mykiss) but resulted in subtle biochemical disturbances in the brain.
All hematological parameters (with exception of hematocrit) were bene cially in uenced by application of curcumin in AgNPs containing diets. Our results are in accordance with ndings of Shaluei et al. [50] in silver carp (Hypophthalmichthys molitrix) and Laban et al. [51] in fathead minnow (Pimephales promelas). The reduction of RBC count, and Hb and Ht levels by AgNPs in this study could be due to following reasons: (a) erythropoiesis disorder and the formation of RBC, (b) the conditions of con nement or stress induced by the lack of food and (c) lysing of RBC due to toxicant stress [52,53].
Blood parameters (biochemical and hematology) are used as useful diagnostic tools for monitoring health status, detecting illnesses, and following the progress of disease and response to therapy in organisms [54]. For instance, blood enzymes (e.g., ALT, AST, LDH) are often used in diagnosis of sh diseases and detection of tissue damages caused by environmental pollutants [55]. Large quantities of ALT and AST are released into blood stream mostly during hepatocytes damage, so analysis of their concentrations in serum can contribute to the monitoring of liver function and health status [56]. In the present study, increased AST, ALT and LDH activities were detected in sh fed AgNPs containing diets probably indicating cellular membrane damage and increased enzymes leakage. This is consistent with the results of previous studies on rainbow trout [54] and Caspian kutum (Rutilus kutum) [57]. Furthermore, our results showed that curcumin inclusion in AgNPs containing diets can remarkably reduce serum AST, ALT and LDH activities indicating that its application can protect hepatocytes against AgNPs induced damages. The protective effects of curcumin on blood biochemical parameters could be attributed to the presence of various compounds found in plant extracts (e.g., antioxidants and avonoids) which bene cially in uence cellular physiological functions (e.g., improving antioxidant capacity and membrane stability, and preventing leakage of intracellular enzymes into the blood during oxidative stress) [7,58].
Digestive enzymes have been widely used for assessment of physiological status including growth phase of sh, and been implicated in monitoring water bodies [59]. It has been shown that sh experiencing different environmental conditions would express different digestive enzymes activity and they can regain their digestive capacity after removal of constraints [60-62]. In the present study, dietary inclusion of AgNPs suppressed lipase and alkaline protease activities and increased the amylase activity. Likewise, Le Bihan et al. [63] showed that silver exposure results in lower protease activity in cuttle sh (Sepia o cinalis). Also, the results of another study by Wang et al. [48] revealed that exposure to copper nanoparticles and copper sulphate in grouper (E. coioides) results in depressed digestive enzymes activity (protease, amylase and lipase). In contrast, Samanta et al. [64] reported enhancement of digestive enzymes activity in three teleost species including Anabas testudineus, Heteropneustes fossilis and Oreochromis niloticus following herbicide Almix exposure. These authors suggested that sh might respond to increased energy requirement through regulating digestive enzymes pro le. However, such elevation in digestive enzymes activity might be also an indication of pancreatitis [65, 66] which remains to be elucidated in the future. In the present study, the sole curcumin supplementation led to signi cant reduction of lipase and amylase activities compared to the control. Furthermore, its inclusion in diets containing 0.05 g/kg AgNPs retrieved the reduction of lipase and protease activities. Similarly, Imani et al. [14] showed that cinnamon oil supplementation in diets for rainbow trout lowers digestive enzymes activity. Also, Nazdar et al. [13] showed that dietary silymarin inclusion could partially prevent toxic effects of NiO-NPs in rainbow trout. However, in this study curcumin application could not provide enough protection at higher concentration of AgNPs. This nding implies that supplemental effect of plant metabolites is variable depending on the duration of feeding trial, sampling time, feed composition, and toxicant moiety and type. Their results showed that the supplementation of a mixture of vitamin C and E has a bene cial effect against the ZnONPs toxicity, which leads to reduction in abnormal tissues changes. Hajirezaee et al.
[71] also reported that supplementation of diet with vitamin C at a level of 500 to 1000 mg/kg of feed could effectively prevent undesirable effects of TiO 2 -NPs in liver of C. carpio. In addition, histological analysis revealed the altered morphology in studied tissues following the exposure of sh to NPs. These histological changes might occur due to various biochemical and molecular level changes as a result of NPs induced stress. Therefore, further studies especially at the molecular level are needed.

Conclusions
In summary, the results of this research suggested that curcumin supplementation in diet containing lower concentration of AgNPs can retrieve the adverse effects of AgNPs on sh growth performance, feed utilization and digestive enzymes activity.

Ethics approval
The experimental sh were used after approval of the experimental protocol by the ethics committee of Faculty of Veterinary Medicine, Urmia University.

Consent for publication
Not applicable.

Availability of data and materials
The datasets produced and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
There are no con icts to declare.