Aquaculture is the last frontier to solve the problem of protein deficiency globally. Several studies report that nanoparticles have great potential in controlling pathogens, improving immune function and growth in aquaculture. The present investigation was carried out to evaluate nano zinc oxide (nano ZnO) compared with its conventional form as a fish feed additive in growth promotion and immunostimulation of Nile tilapia. (Oreochromis niloticus). Nano zinc oxide are chemically prepared and mixed with fish feed. 405 Oreochromis niloticus were fed for 120 days on conventional (ZnO) and nanograde (nano ZnO) zinc oxide supplements at different concentrations (15, 30, 45 and 60 mg/kg feed) in addition The control was fed on a free ZnO feed. nanoZnO (15mg/kg) achieved the same specific growth rate as higher concentrations of bulk ZnO (60mg/kg). Nano ZnO 60mg/kg gave the highest rate of Specific Growth Rate (4 times higher than the control). growth hormone was higher in the serum of fish fed with ZnO nano-supplemented feed compared with the bulk form. Immunity was assessed through measurement of total protein and IgM titers and IL1-beta (IL-1β) gene expression. Total protein and IgM titres showed high values that increased with increasing concentrations of ZnO feed additive in the two forms compared with controls, but nano ZnO gave better results than conventional ZnO. IL1-beta gene expression analysis showed that 60mg/kg of conventional ZnO was the best concentration to upregulate IL1-beta, followed by a concentration of 30mg/kg in two forms; nano and regular form. Inorganic conventional ZnO regulates IL1β better than the nanoform ZnO. Zinc concentration was higher in the muscle of fish fed with ZnO nano supplement than in fish fed with normal ZnO but still within acceptable limits. A statistical analysis ANOVA was used in all analyzes with p < 0.05.
Fish feed is an essential component of the input in any fish farm. Feed ingredients must ensure growth promoting factors, immunity and health to achieve a large impact on the net profit of the farm. Minerals are nutrients necessary for normal body processes; Mineral requirements vary depending on the form, interactions with other factors, water quality and the fish itself (age, size and species). Minerals are required in lesser amounts than other dietary essential nutrients, e.g. protein, carbohydrates and fats. Since they are essential, they are otherwise toxic. Feed additives in the nanoform form have been evaluated for effects ranging from growth enhancement and immunity through antioxidant effects to their use in lesser quantities. compared with bulk additives that enhanced dietary criteria (Rather et al., 2011; Rajendran, 2013). Nanoparticles have great potential in controlling pathogens in aquaculture. Various metal and metal oxide nanoparticles have been screened for their antibacterial activities against a wide range of bacterial and fungal agents including some freshwater cyanobacteria (Swain et al., 2014). ). Among the different nanoparticles, synthetic copper oxide (CuO), zinc oxide (ZnO), silver (Ag) and silver-doped titanium dioxide (Ag-TiO2) showed broad-spectrum antibacterial activity (Swain et al. , 2014). As CuO, ZnO and Ag nanoparticles showed higher antibacterial activity, they could be explored for use in aquaculture. Zinc oxide nanoparticles are one of the very versatile metal oxides as they are involved in a wide variety of applications ranging from sensing, catalysis, energy storage, electronics and biomedical applications. Chemically, zinc oxide (ZnO) and nano zinc oxide (nZnO) with the same chemical formula show similar ratios of Zinc and oxygen, but at the nanoscale the atoms are arranged with energy levels wider and smaller Zn size, which can lead to more reactive atoms as the surface increases (Zhong, 2004). Interleukin-1 (IL-1) is a multi-directional endocrine endocrine signaling molecule and is produced by many cell types. Phagocytosis is an important source for synthesis and release of IL-1 to stimulate T-cell activation suggesting that in fish; These molecules can be quite different. IL1-β is the most important of the IL-1 group. The main functions of IL1-β are to trigger proliferation of lymphocytes such as T cells and B cells, to activate cytotoxic activity in macrophages and natural killer (NK) cells. , and induction of immunoglobulin (Ig) secretion. IL1-β is therefore an important member of the immune system (Sebastián et al., 2012). This study investigated the evaluation of Zinc oxide (ZnO) as an animal feed additive in two forms; conventional and nanoformulas in enhancing growth and immune processes in tilapia (Oreochromis niloticus). Growth was assessed through several growth parameters and immunity was assessed after infection with fluorescent bacteria Pseudomonas (challenge) through measurement of total protein, immunoglobulin M, and gene expression of Pseudomonas. one of the most important proinflammatory cytokines; Interleukin-1-beta (IL1-β).
Materials and methods
Fish: 405 mixed sex, 35-45 gm, healthy-looking tilapia (Oreochromis niloticus) taken from Rahil Aquaculture, Fayoum, Egypt, June 2014. Fish were weighed upon arrival. and treated with 4 mg/L potassium permanganate for 10 min before dispensing in eighteen plastic tanks provided with dechlorinated water and automatic aerators. Fish were reared for 120 days and sampled in November 2014.
Nano zinc oxide (nZnO)
nZnO was prepared by chemical co-precipitation according to Li et al. (In 2006). XRD and TEM were used for characterization of nZnO.
Four concentrations (15,30,45 and 60mg/kg) of conventional nZnO and ZnO were added to the feed in two different treatments in three additions outside the control (no treatment). All groups were copied. Fish were fed twice daily with 3% body weight for 120 days.
Estimated growth performance
Growth performance is calculated through two tests; aWeight gain (WG%), b- Specific growth rate (SGR)
Estimation of Growth Hormone in the Blood
Growth hormone (GH) was measured by ELISA kit
Catalog No: MBS701414_48T (MyBiosource, Vancouver, British Columbia)
Determination of Zn concentration in muscle
Zinc concentrations in muscle samples were analyzed according to FAO digital document 212 (FAO, 1983).
Non-specific immune function assessment
A challenge test was performed. Fluorescent bacteria Pseudomonas was used to immunize Oreochromis niloticus fish at a dose of 0.2 ml below lethal (1 × 106 CFU/ml) to test the nonspecific immune response of tilapia through tilapia. quantification of total protein, immunoglobulin M and interleukin 1 beta gene expression by real-time PCR assay. Samples were collected 3 and 5 days after intraperitoneal bacterial injection.
The serum of the fish studied was subjected to calorimetric analysis for total protein content using a total protein quantifier (Spectrum, Egypt), a colorimetric method (Biuret reagent).
Serum immunoglobulin M (IgM):
IgM values were determined using the IgM ELISA kit (Catalog No: MBS700823, MyBiosource, Vancouver, British Columbia).
IL1-β gene expression and immune response
Fish liver was isolated and quick frozen immediately in liquid nitrogen and stored at -70°C for further RNA extraction, RNA was extracted using Gene Jet RNA Purification Kit, Two µg RNA was transcribed. In contrast to Revert Aid First Strand cDNA Maxime RT PreMix Kit uses hexanucleotides and is used as a template for real-time PCR. The reaction was performed using a Bio-Rad heat press; at 45 oC for 60 min then stop the reaction by increasing the temperature to 70 oC. Primers and probes were designed using NCBI Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and GenScript
(http://www.genscript.com/). Real-time PCR (Rotor-Gene Q – QIAGEN) was performed using methods previously described by Zhang et al. (2008) used TaqMan Overall Spectrum II, with UNG to quantify gene expression in both target and housekeeping genes. Serial dilutions for primers, probes and cDNA were performed. Concentrations of 50 picomol primer, 5 picomol prope, 1/100 for the HK gene and 1/10 for the IL1b gene gave the best results (standard concentration).
Characterization of Nano zinc oxide
The calculated crystal sizes of nZnO range from 32-57 nm in diameter as revealed by XRD images and 34.4-50.5 nm by TEM analyzer. Figure 1 (A&B).
Specific weight gain and growth rate (SGR)
The weight gain of tilapia increased gradually and had a proportional relationship with the increase in ZnO concentration, it was observed that the addition of nano zinc oxide increased the growth rate more than that of conventional ZnO, sometimes twice as much. double weight gain. The two treatments showed more rapid changes in weight gain and enhanced growth than controls. In this study, the SGRs for ZnO-fed fish (15 and 30mg/kg) were 0.31 and 0.37% per day compared to the control (0.27% day) with a difference of 1.15 and 0.37%, respectively. 1.37. The magnitude of the difference increased to 2.37 and 2.4 with concentrations of 45 and 60 mg/kg ZnO, respectively. With regard to nZnO, the degree of difference between the test and control fish from 15 mg/kg to 45 mg/kg was almost constant and close to the values achieved in ZnO 45 and 60 mg/kg (2.5, 2.2 and 2.5), respectively. nZnO introduced a fold difference that was 4 times larger than that of the control (Table 2). Growth hormone in serum Both groups of fish fed ZnO and nZnO supplemented diets showed higher serum growth hormone levels than controls. In the conventional ZnO group, as the ZnO treatment concentration increased, the growth hormone level was increasing (respectively 0.16, 0.19, 0.25 and 0.27ng/ml) with the highest value of the hormone Growth was obtained at a concentration of 60mg/kg of ZnO added to the feed. Similarly, in the nZnO groups there was a positive correlation between blood growth hormone levels and treatment concentrations (mg/kg), where the blood growth hormone concentrations for the treatments The different nZnO values were (0.2, 0.24, 0.398 and 0.402 ng/ml, respectively).
Zinc content in muscles
In all treatments, muscle Zn concentrations showed higher values than controls, but were still within the allowable limit (40ppm) higher than those of FAO (1983). Figure 3.
Nonspecific immune response
Total serum protein and total immunoglobulin
The serum total protein concentration of fish fed with conventional ZnO-supplemented diet was significantly lower than that of fish fed with nZnO-supplemented with the observation that its values and Immunoglobulin values M(IgM) has a positive direct relationship with additive concentration (Table 3).
IL1-β . gene expression
Immunization with a low dose lethal to Pseudomonas fluorescent bacteria showed an increase in IL1-β expression three and five days after experimental infection. Figure 4 (A-D) illustrates the expression levels of the IL1-β gene compared with the 18sRNA internal control in all investigated groups. Real-time threshold cycle (Ct) raw data show increased in-test expression compared with control and internal control 18sRNA.
The expression of the IL1-β gene was consistently higher than that of the control when four concentrations of ZnO were used, but the increase in expression varied over time (3 & 5 days) sporadically. Alterations in gene expression between different groups were expressed as a value of 2^-ΔΔCt in tilapia grown for 4 months on ZnO and nZnO supplemented diets and infected with bacteria, respectively. The results of the fold change indicated that the lowest concentration of nZnO (15mg/kg) in the tested diet increased the expression of the target gene compared with the conventional form of ZnO after 3 and 5 days of testing. challenge, but more after 5 days. but still lower than other concentrations. (Figure 6- A&B). It is clear that Zn is essential for animal growth, immunity and development in certain amounts (Hao et al., 2013). Nano zinc oxide ZnO-NPs have been reported to enhance growth performance, improve feed utility and provide economic benefits in piglets and weaned poultry (Yang and Sun, 2006 and Mishra et al. events, 2014); Encouraging results in terms of average daily growth were obtained when basal diets supplemented with 200, 400, 600 mg/kg nZnO or 3,000 mg/kg ZnO (Hongfu, 2008). Lina and colleagues. (2009) in her study indicated that ZnO-NP zinc oxide nanoparticle was found to improve production performance and dressing performance of broiler chickens after 42 days of feeding at 40 mg/kg in Ration. Buentello et al. (2009) in his study reported that differences exist in growth rates in response to different dietary Zn sources and that different chemical forms of Zn indicate bioavailability. different in fish. In hybrid sea bass, Zn proteinate was used approximately 1.7 times more efficiently than ZnSO4. Faiz et al. (2015) reported that the growth performance of juvenile C. idellain in response to different inorganic sources of Zn showed that the highest %WG, SGR and FCE were obtained in fish fed the diet containing ZnO-NPs (various concentrations), while diets supplemented with zinc at both sulfate and oxide levels at lower levels showed impaired growth, that is, nZnO promoted growth more than other conventional inorganic forms and the growth performance of fish fed the ZnO-1 diet was statistically compared with the control group of fish. This study also showed that fish fed diets supplemented with Zn in nano form at the rate of 30 mg Zn/kg diet (Zn-NP) showed significantly high %WG (p < 0.05). This level is within the range reported by many investigators for different fish species (Clearwater et al., 2002) but slightly below that reported for hybrid striped bass (Buentello et al., 2009), juvenile abalone (Tan and Mai, 2001). 90-day trial of feeding young grass carp with Zinc oxide (nano and conventional) and Zinc Sulphate, with two doses each; lower and higher levels (30 and 60mg/kg diets). Nano zinc oxide were found to promote growth (%WG, SGR and FCR) at its two levels (lower > higher) and raise erythrocyte count and MCHC values significantly (p). <0.05) at lower levels than other inorganic forms. However, Zinc Sulphate at both levels and conventional Zinc Oxide at higher levels had negative effects on both growth performance and hematological parameters (RBCs & MCHC) (Faiz et al., 2015).
Our study showed that the weight gain of Oreochromis niloticus increased gradually and had a positive relationship with the increase in the concentration of ZnO in its two forms, with the observation that the addition of nano ZnO increased the growth rate. growth than conventional ZnO, sometimes twice the weight gain. The highest WG and SGR (41.0 and 1.1%, respectively) were observed in the group of fish fed a diet supplemented with 60mg/kg ZnO-NP compared to the fish fed with the same normal concentration of ZnO. (17.3 and 0.65), the degree of difference in growth rate from the control increased with the increase of bulk ZnO concentration in the feed., With respect to nZnO, the magnitude of the difference between test and control fish from concentrations of 15mg/kg to 45 mg/kg were almost stable (2.5, 2.2 and 2.5, respectively) and close to the values achieved in ZnO 45 and 60mg/kg . The nano-ZnO concentration of 60mg/kg gave a difference of 4 times greater than that of the control. Overall; observed that low concentrations of nZnO added (15mg/kg) to fish feed achieved the same specific growth rates as higher concentrations of conventional ZnO (60 mg/kg) and 60mg/kg ZnO nano for the highest SGR; 4 times than control. This result may be because reducing macromolecules to the nanoscale changed their properties and increased their application (Rather et al., 2011). Significantly higher % WG and SGR (p < 0.05) were observed (p < 0.05) in fish fed the nZnO-fortified diet compared with the conventional ZnO-enriched diet. at the same concentration possibly due to the small particle size (32 to 57 nm) of nZnO, the intestinal tract is higher than the absorption, bioavailability and catalytic activity as reported by Alishahi et al. (2011). Alternatively, it can be attributed to somatic growth by stimulating DNA and RNA synthesis and growth hormone protein synthesis (Siklar et al., 2003). Serum growth hormone measurements showed that both ZnO and nZnO supplements produced higher serum growth hormone levels than controls, with the highest and comparable values for concentrations of 45 and 60mg/kg. This may be due to the natural function of the endocrine system in physiological processes, since when the body is saturated with a hormone produced under specific stimuli, the induction will begin to decrease because of the DNA. will stop transcription with consequent translation and synthesis of some proteins. However, tilapia growth hormone values were found to be increased more in serum in the case of fish fed with nZnO compared with normal zinc oxide in the same concentration. These results are consistent with the study of Hina et al. (2015), who found that nZnO promoted the growth performance of juvenile C. idellain more than other conventional inorganic forms. The physicochemical properties of the nanoparticles were found to influence the immunogenic effects of the nanoparticles (Rather et al., 2011). Luo et al. (2015) reported that nanoparticles can stimulate innate and adaptive immune responses depending on their physicochemical properties; however, it remains unclear how nanoparticles affect the immune response. Furthermore, it has been well documented that Zn deficiency reduces immune responses and disease resistance in humans and animals (Chesters, 1997).
Nano zinc oxide are being used in the food industry as additives and in packaging processes due to their antimicrobial properties (Gerloff et al., 2009 and Jin et al., 2009), demand The sudden increase in zinc oxide nanoparticles (ZnO NPs) is largely attributed to better antibacterial properties than conventional ZnO (Padmavathy and Vijayaraghavan, 2016). Antibacterial activity has been demonstrated using nZnO against L. monocytogenes, S. enteritidis and E. coli O157:H7 (Jin et al., 2009; Costa et al., 2011; Incoronato et al., 2011). ; Singh et al., 2014) was attributed to the disruption of bacterial cell membranes according to these studies. In this study, total serum protein and IgM were affected by dietary ZnO content in its two forms, but it was clear that nZnO enhanced total protein and IgM, possibly due to increased synthesis. protein in the liver; An important function of serum proteins is to maintain osmotic balance between the blood and the tissue space as these proteins are very sensitive to metal poisoning (Sakr et al., 2005). The parallel increases in serum protein and immunoglobulin M with ZnO in the diet suggest an important role for protein in Zn transport. This is consistent with Gopal et al. (1997), who stated that analysis of the serum globulin level of Cyprinus carpio fish showed a strong increase during different periods of nZnO treatment.
Several studies report that, when nanoparticles enter the body, they can interact with immune cells and trigger an inflammatory response, accompanied by the secretion of signaling molecules (cytokines, chemokines) provide communication between immune cells and coordinate molecular events. Interlekin 1 beta (IL1-β) is a member of the interleukin 1 family of cytokines, which are produced by activated macrophages as a proprotein, and are proteolytically processed to their active form by caspase 1 (CASP1/ICE). This cytokine is an important mediator of the inflammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. The study of nanoparticles that induce IL-1b through inflammatory signaling pathway mechanisms is an emerging topic (Reisetter et al., 2011) The more knowledge we have about the cytokine profiles due to nanoparticle, the better we can use IL1-β a biomarker for the immune response. In this study, the results of RT -PCR showed that; in the groups of fish fed with conventional ZnO supplement, the lowest ZnO concentration (15mg/kg) increased IL1-β gene expression was highest after 3 days of challenge, decreased after 5 days but still higher than the control group. control (no treatment) and 18sRNA internal control. Regarding the groups of fish fed with nZnO supplement, the concentration of 45mg/kg nZnO in the fish feed increased the expression of the most studied target gene (34,24), followed by concentration of 15mg/kg feed supplement (33.7). All concentrations of nZnO induced IL1-β expression after 3 days, after which it was clear that after 5 days of vaccination, this expression was reduced at the final concentration (60mg/kg). With regards to the sequential change of gene expression, this means how many times the expression of the target gene is increased compared to that of the internal conformational regulatory gene (18sRNA) Figures 13 and 14 show that, after 3 and 5 days of vaccination, there are 4 important factors observed points that can be reported here; First, in the nZnO group, the lowest concentration (15mg/kg) of nZnO addition to the feed caused more changes in IL1-β gene expression in the feed than the same concentration of conventional ZnO added. into the ZnO addition group. Thus, the same picture of the immune response shown in the IL1β gene fold expression appears when ZnO and nZnO are compared at the lowest concentration of the additive; nZno was the higher inducer of this low concentration with the observation that two types of ZnO; normal and nZnO induced higher expression 5 days after vaccination. The second point is that, at the other three concentrations (30, 45 and 60mg/kg), conventional inorganic ZnO was reported to induce and modulate a greater change in IL1-β folding expression than the nZnO form in same concentration (Figures 13 & 14). The third observation point is that the concentration that results in good and better target gene induction is 30mg/kg in two forms; conventional and nanoscale than other treatments, but the results also indicate that conventional ZnO at this concentration (30mg/kg) outperforms nano ZnO in the induction and regulation of IL1-β gene expression. . The fourth and final observation was that after 5 days of experimental infection, the expression of the target gene was better after 3 days, so the immune response contained in this gene was improved after 5 days.
In general, it can be concluded that the traditional inorganic zinc oxide induces the target gene better than the nanoform at higher concentrations and the persistence to induce gene expression is at the concentration of 30 mg/kg after 3 and 5. days, therefore this concentration may be considered the best for inducing immune function in the form of IL1-beta. Reisetter et al. (2011) stated that, the study of nanoparticles that induce IL1-β through the signaling pathway of inflammatory disease is an emerging topic. In their in vitro experiment, Lucarelli et al. (2004) exposed human macrophages to different nontoxic concentrations of SiO2, TiO2, ZrO2 and Co nanoparticles and observed an increase in the expression of TLR receptors and the production of inflammatory cytokines. inflammation, experiments show that different nanoparticles trigger the inflammatory response in different ways. SiO2 nanoparticles induce the production of inflammatory cytokines IL-1β and TNF-α. Yang et al. (2012) found that silver nanoparticles induce acne formation and trigger the subsequent release of IL1-β and activation of caspase-1. Affects IL1-β production associated with dendritic cell in vivo activation (Sharp et al., 2009). These studies support our results regarding the effect of nZnO on IL1-β gene expression.
Concerning the muscle zinc concentration, it was found that the zinc concentration showed a higher value in all groups of fish fed with ZnO supplement than the control, but still within the allowable limit (40ppm) set by FAO (1983). It was found that as ZnO concentration increased, Zn concentration in muscle increased. Among all the fish fed ZnO (Bulk and Nano), in the group of fish fed with 60mg/kg nZnO supplement, the value of Zn in muscle was the highest. This can be explained by the fact that the nanoparticles are smaller in size allowing for higher absorption and higher bioavailability (Zaboli et al., 2013). Feng et al., (2009) also pointed out the same suggestion that because of their small size, nano-minerals are more easily absorbed by the body, these suggestions are consistent with our results.
From this study, it can be concluded that the addition of nano zinc oxide to fish feed can improve the growth rate shown here as weight gain, specific growth rate and growth hormone in fish feed. blood. This substance can be better than common zinc oxide, so it can be used in fish farms and aquaculture in low concentrations and this can improve the economics of agriculture.
Regarding immunity, the results indicated that the total protein as well as immunoglobulin M were increased in the groups of fish fed with ZnO supplement with the indication that nZnO enhanced synthesis of immune products. This translation is more than the block form. The results also indicated that the usual concentration of 60mg/kg ZnO was the best concentration to upregulate IL1beta, followed by a concentration of 30mg/kg in two forms; nano and regular form. However, it was observed that purely conventional inorganic ZnO was reported to induce and regulate IL-1β better than the nanoZnO form. stimulant feed additives. The higher value of zinc in muscle of fish fed with zinc nanoparticle compared with conventional ZnO supplements may be due to the higher absorption and bioavailability of the small sized nZnO particles.