Recent studies have demonstrated that minerals and vitamins such as Cu and VC are required for normal cell functioning, improvement of growth performance, and physiological and immunological process of aquatic species (El Basuini et al. 2016; Dawood and Koshio 2016; Chen et al. 2015; Ai et al. 2006; Watanabe et al. 1997). Also, it has been reported that nanominerals such as Cu nanoparticles have novel features such as high surface activity, larger specific surface area, high catalytic efficiency, high surface active centers, and stronger adsorption capacity, making them more capable of crossing biological barriers so that they are rapidly absorbed by cells and exhibit higher bioavailability than mineral salts (Gharaei et al. 2020a; Izquierdo et al. 2016; Rather et al. 2011).
The present study showed that growth indices including WG, FCR, SGR, and PER were significantly affected by Cu-NPs and VC supplementations, which is in parallel with previous studies according to which dietary Cu and CV supplementations improved growth indices, immune response, and antioxidant status in several species of fish (El Basuini et al. 2016, Mohseni et al. 2014; Tang et al. 2013; Faramarzi 2012; Sabatini et al. 2009; Wang et al. 2009). The growth-promoting effects of Cu-NPs may be explained by the fact that optimal dietary copper induces growth by improving metabolism, activities of the brush border, and preventing lipid peroxidation and protein oxidation in the hepatopancreas and intestine (Tang et al. 2013). Although it has been demonstrated that growth performance is significantly increased in grass carp (Ctenopharyngodon idella), beluga (Huso huso), red sea bream (Pagrus major), and freshwater prawn (Macrobrachium rosenbergii) under the effect of dietary Cu supplementation (Tang et al. 2013; Mohseni et al. 2014; El Basuini et al. 2016; Muralisankar et al. 2015). However, unlike this study, Gatlin and Wilson (1986) and Lorentzen et al. (1998) reported no significant effect of Cu supplementation on WG and feed efficiency of channel catfish (Ictalurus punctatus) and Atlantic salmon (Salmo salar), respectively. Therefore, the severity of the response to copper supplementation and its different impact on growth performance may be attributed to species, age, Cu dosage, and Cu chemical forms.
Crustaceans and fish species are limitedly capable of VC synthesis, so adding it to the diet of farmed species is crucial for improving growth performance and maintaining normal physiological functions (Dawood and Kushio 2016; Chen et al. 2015). Improved growth performance through the nutrition of VC often appears as a result of the increased feed efficiency of the diet as proven by the previous studies on red seabream (P. major) (Dawood et al. 2016), Caspianrouch (Rutilus rutilus caspicus) (Roosta et al. 2014), common carp (C. carpio) (Liu et al. 2011), cobia (Rachycentron canadum) (Zhou et al. 2012), Oreochromis spilurus (Al-Armoudi et al. 1992), and Korean rockfish (Sebastes schegelii) (Kim and Kang 2015). In the present study, the growth performance reached a significantly higher level in the fish fed on the T5 diet (2 mg kg− 1 Cu-NPs mixed with 500 mg kg− 1 VC) compared with the control group. Adel and Khara (2016) presented that the highest WG and SGR and lowest FCR in rainbow trout fingerlings were observed in those fed on 250 mg kg− 1 VC. Yousefi et al. (2013) evidenced that the growth performance of Barbus sharpeyi was improved by VC supplementation. Similarly, Faramarzi (2012) indicated that the growth performance of common carp was increased in the fish fed on dietary VC supplementation at a rate of 800–2000 mg kg− 1 diet. Unlike this study, it was reported that dietary VC supplementation did not influence the growth performance in large yellow croaker (Pseudosciaena crocea) juveniles (Ai et al. 2006). However, our results in this study revealed that Cu-NPs combined with VC have a synergism effect on the growth indices and the physiological status of rainbow trout.
Lysozyme and ACH50 widely participate as humoral components in the innate defense system, so they are important for fish protection against diseases (Kaya et al. 2016). The antibacterial activity of the complement system, reported in various fish, has been suggested as one of the most important mechanisms of bacterial killing and clearing in fish, which can be activated by various immune stimuli (Srivastava & Pandey 2015). One of the triggers of complement activation is cytokines, some of which are effective in regulating proteins involved in iron metabolism, such as ceruloplasmin (Di Bella et al. 2017). In our study, lysozyme activity enzyme and ACH50 value were enhanced significantly (p < 0.05) in fish fed on the Cu-NPs and/or CV supplemented diet. This incremental fluctuation may be due to the immune suppressive effects of Cu-NPs (Kaya et al. 2016). This result coincides with the investigation of El Basuini et al. (2016) and Mohseni et al. (2014) who reported increasing lysozyme level in P. major and H. huso fed on dietary Cu-NPs and inorganic copper supplementations, respectively. In addition, Gharaei et al. (2020a) indicated that the lysozyme and ACH50 values were increased in H. huso fed on the dietary Chitosan-Zn NPs supplemented diet. Unfortunately, there is a lack of knowledge on the effect of nanoparticles on the ACH50 level in fish. However, it has been demonstrated that VC is a strong inducer of the immune system, especially non-specific immunity as it results in enhanced lysozyme activity level reported in various fish species including P. major (El Basuini et al. 2016); O. mykiss (Adel and Khara 2016); Pseudosciaena crocea (Ai et al. 2004, 2006); Takifugu rubripes (Eo and Lee 2008); Scophthalmus maximus (Lin and Shiau 2005), Pangasianodon gigas (Pimpimol et al. 2012). Qinghui et al. (2004) observed increased fish lysozyme and ACH50 values when the dietary VC supplementation was enhanced up to 489.0 mg kg− 1. Similarly, Chen et al. (2003) demonstrated that the ACH50 level in golden shiner (Notemigonus crysoleucas) was increased under the effects of dietary VC supplementation.
The antioxidant defense system is highly correlated with the health and safety of fish, and its major enzymes (SOD, CAT, GPX, and MDA) decompose reactive oxygen species (ROS) into a less reactive form (Sheikh Asadi et al. 2018; Dekani et al. 2018). The role of SOD is to stimulate the oxidation and reduction of superoxide anions to hydrogen peroxides, which are then used as a substrate by the CAT and GPX enzymes (Saffari et al. 2016). As shown in Table 5, maximum activities of the SOD, CAT, and GPX enzyme are observed in the fish fed on the T5 (2 mg kg− 1 Cu-NPs mixed with 500 mg kg− 1 VC) diet compared with the control group. Previous studies have shown that Cu-containing diet, Cu/Zn-SOD enzyme activities in hepatocyte of rainbow trout (Oncorhynchus mykiss) (Osredkar and Sustar, 2011; Trenzado et al. 2009) and grass carp (Ctenopharyngodon idella) (Tang et al. 2013) and GPX increases in the plasma of goldfish (Carassius auratus gibelio) (Shao et al. 2010). In fact, copper is positively associated with the antioxidant defense system (Fang et al. 2013) and the effect of dietary copper on stopping oxidative damage may be related to the reaction with ROS such as anion superoxides and hydroxyl radicals (Tang et al. 2013). On the other hand, ceruloplasmin is a Cu-containing protein whose activity increases with appropriate levels of dietary Cu (Shaiu and Ning 2003). This Cu-containing protein is capable of stopping superoxide radical production (Valko et al. 2007) and hydroxyl radical formation (Zhang et al. 2013). One of the major antioxidant additives in the fish diet and food industry is VC, which alleviates oxidative stress (Dawood et al. 2016; Geo et al. 2013). VC plays an important role in scavenging free radicals (ROS and reactive nitrogen species) by acting as an early electron donor and reducing the agent (Dawood et al. 2016). Many previous studies have stated that dietary VC supplementation increases the SOD, CAT, and GPX activities in yellow catfish (Pelteobarus fulvidaco) (Liang et al. 2015), Siberian sturgeon (Acipenser baerii) (Xie et al. 2006), and black carp (Mylopharyngodon piceus) (Hu et al. 2013). Therefore, the results suggested that dietary Cu-NPs + VC supplementation are probably able to increase the antioxidant level and they have a synergistic interactive effect on inducing the antioxidant system. Our results showed no significant variations in the MDA value in all treatments. On the contrary, Jankowski et al. (2020) reported a reduction of MDA concentration under the effects of various forms of Cu (mineral and nanoparticle) in turkeys.
Hematological assessments can provide an indication of the physiological status of fish (Behera et al. 2013). In the present study, the Hb, Hct, and MCV values were increased more significantly in the fish fed on the T5 diet (2 mg kg− 1 Cu-NPs mixed with 500 mg kg− 1 VC) than the control fish, suggesting the positive effect of Cu-NPs + VC on physiological responses. The lower values of these hematological parameters in the control group indicate the necessity of adding Cu and VC to the diet to improve blood counts. The measured levels of blood variables in the normal range are for trout health, which confirms the effects of non-toxic Cu-NPs used under the present experimental conditions. It was confirmed that increased Hb indicates a stress response or increased hematopoiesis (Clauss et al. 2008). While the fish fed on Cu-NPs/VC were healthier than the control group, which was determined by the level of antioxidant, safety, and survival rates in the bacterial stress test. Thus, an increase in RBC, Hct, and Hb associated with hematopoiesis increased or decreased hemolysis (Hosseini et al. 2018). This may be due to the role of Cu as a combination of many enzymes and glycoproteins that aid in the synthesis of hemoglobin (Nordberg et al. 2015; Dawood et al. 2020). Ceruloplasmin (a liver-derived protein) is required to release iron and transfer it from cells and tissues to plasma. There are several copper molecules in the structure of this protein, and its synthesis in the liver requires the presence of copper. In fact, copper deficiency impairs the ability of iron absorption or release from tissues for hemoglobin synthesis (Haver and Hardy 2008). The same results were recorded by Adel and Khara (2016) and Zhou et al. (2012) for pirarucu (Arapaima gas) and cobia (R. canadum), respectively.
To investigate the effects of Cu-NPs and VC on inflammatory and antioxidant responses, we measured the expression of several gene biomarkers including three pro-inflammatory cytokines (TNF-α, IL-10, and IL-1ß) and three antioxidant systems (CAT, SOD, and GPX). The results of the present study showed that the expression levels of TNF-α, IL-10, and IL-1ß genes were decreased in the intestine of the fish in the T3, T4, and T5 treatment groups. TNF-α (tumor necrosis factor) is known as a multifunctional cytokine that plays a key role in cell-mediated inflammatory immunity responses (Lykouras et al. 2008; Mocellin et al. 2015). IL-1ß acts as a mediator of the inflammatory response and helps reduce inflammatory pain sensitivity in various cellular activities, including cell proliferation and apoptosis by inducing cyclooxygenase-2 (PTGS2 /COK2) in the central nervous system. TNF-α and IL-1ß are considered important indicators of phagocytic activity and they are the first cytokines produced in the early stages of inflammation in fish (Skadberg et al. 2015). IL-10 is known as a cytokine synthesis inhibitory factor that minimizes damage to target cells by suppressing the transcription of pro-inflammatory cytokine (Shafiei-Jahani et al. 2020). The significant reduction in the expression of pro-inflammatory cytokine genes in fish fed on the diets containing Cu-NPs/VC supplements can be interpreted as their significantly down-regulated synergistic effect on the immune response, which was also dose-dependent. IL-10 has also been reported to be capable of degrading pro-inflammatory cytokine mRNA, reducing TNF-α receptor expression, and regulating macrophage-derived TNF-α and IL-1 secretion (Opal et al. 1998; Opal and DePalo 2000). Suska et al. (2003) reported that cellular Cu sites induce the secretion of TNF-α and IL-1ß by inflammatory cells ex vivo and in vivo. On the other hand, TNF-α has been shown to increase phagocytosis of neutrophils under apoptosis. Thus, Cu-NPs may reduce the production of pro-inflammatory cytokine because Cu ion is closely related to RNA and DNA. Antioxidant vitamins, including VC, can increase immune function by increasing the proliferation of lymphocytes and macrophages (Jang et al. 2014). Changes in gene expression reported in this study suggest a low inflammatory potential of the Cu-NPs / VCs tested. In this regard and consistent with our results, Yun et al. (2012) and Jang et al. (2014) reported that dietary supplemental VC significantly reduced TNF-α, IL-1ß, and IL-6 mRNA levels in mice and broiler chick, respectively.
Despite the various benefits of copper nanoparticles in aquatic organisms, their toxic effects have also been reported in some cases. In Epinephelus coioides, adverse effects on gut, gill, and liver (Wang et al. 2015), Cyprinus carpio caused a sharp decrease in alkaline phosphatase and increased T4 and free T4 in blood plasma (Hoseini et al. 2016), Oncorhynchus mykiss reduced hematocrit percentage and the amount of potassium and sodium in the blood plasma (Shaw et al. 2012). Nowadays, the interaction between transition metals, e.g., Cu, with VC is well known (Akbıyık et al. 2012) as the rate of VC oxidation stability increases with the fixed concentration of Cu and prevents catalytic oxidation of VC in the presence of a stable Cu complex.
Various studies have shown that dietary VC can produce antioxidants (Biller et al. 2018). VC bonds to ROS in the body and retrieves free radicals through H+ donation. VC has also been shown to act as a reducing agent, primarily by reducing the transport of metals such as Cu and Fe ions, which react with H2O2 to form hydroxyl radicals (Babior 1997).
Despite reports of inducing increased expression of the SOD, CAT, and GPX genes due to the toxicity of Cu-NPs in aquatic organisms (Ramya et al. 2016; Muralisankar et al. 2019; Dawood et al. 2020). The results of this experiment suggested that the Cu-NP level of 2 mg kg− 1 had no adverse effect on fish and, when applied with VC, had the greatest effect on ROS production. SOD and CAT are related to stress management (Li et al. 2010) and are used as important indicators in the early detection of oxidative pollution. Decreased expression of the SOD, CAT, and GPX genes in fish exposed to Cu-NPs/VC could indicate oxidant eradication. The results of Nile tilapia (Oreochromis niloticus) exposed to ZnO-NPs and vitamins C and E (Abdelazim et al. 2018), O. niloticus exposed to Ag-NPs (Afifi et al. 2013), and Carassius auratus exposed to a mixture of Cu-NPs and ZnO-NPs and cerium oxide-NPs and pure NP (Xia et al. 2013) are in agreement with our findings. It is confirm that VC can destroy the superoxide anion by forming radical semidehydra ascorbate (Abdelazim et al. 2018). The results of our experiment showed significant neutralization in the antioxidant system so that the fish fed on a mixture of Cu-NPs and VC exhibited the lowest expression level of the SOD, CAT, and GPX genes. This ability of VCs to combat possible oxidative damage was caused by exposure to Cu-NPs. Othman et al. (2017) explained that they used VC to prepare ligands for cerium oxide nanoparticles as a tool to facilitate the detection of NP in tissues because they suggested that VC could bind tightly to NPs and showed its performance. It has also been reported that the presence of NPs themselves can increase the VC activity (Astete et al. 2011).
The maximum resistance to Y. ruckeri and survival rate in this study were recorded in the fish fed on the T5 diet. Many previous studies have shown that various dietary additives have increased the survival rate and resistance to the pathogen in rainbow trout (Gharaei et al. 2020b; Yilmaz et al. 2018; Aghamirkarimi et al. 2017). The significant enhancement in survival rate in this study may be related to the induction of non-specific immune defenses and antioxidant system by synergistic interaction of Cu-NPs and VC.
In the present study, histopathological alterations were not observed in gill, intestine, liver, and kidney tissues affected by Cu-NPs and Cu-NPs + VC. According to the previous studies, the gill, liver, and kidney are the most sensitive organs to Cu-NP exposure and respond by showing various degrees of necrosis and tissue damage (Ostaszewska et al. 2018). Gills are an important organ with several functions like respiratory osmoregulation and respiratory gas exchange, acid-base balance, and excretion of metabolites. Thus, they are the primary target for a high concentration of Cu-NPs (Ostaszewska et al. 2018).
The intestine is the site of absorption of a huge portion of the nutrients and non-nutrients digested. It has been shown that Cu-NPs are well oxidized to ionic forms in acidic environments and have high adsorption capacity (Pirarat et al. 2011). On the other hand, the height of intestinal villi is an important indicator of the efficiency of digestion and absorption in the gut (Ringoe et al. 2003). Rathore et al. (2019) has reported that dietary VC increases the height of villi in the gut of tilapia (Oreochromis niloticus). The combination of Cu-NPs and vitamin C is likely to increase the efficiency of nutrient absorption in the gut by providing a larger surface area as well as an acidic environment of the gut, which strengthens the immune system against the pathogens through the viscous mucin layers that cover the receptors for infectious agents (present on the intestinal mucosa) (Johari et al. 2015).