Nano Copper Supplementation Increases Superoxide Dismutase and Catalase Gene Expression Profiles and Concentration of Antioxidants and Immune Variables in Sahiwal Heifers

This study was conducted to evaluate the effect of inorganic and nano copper (nanoCu) supplementation on superoxide dismutase (SOD) and catalase (CAT) gene expression, antioxidant status, and immune response in growing Sahiwal heifers. Twenty-four Sahiwal heifers were allocated at random into four groups of six heifers in each groups and fed for 120 days. Feeding regimen was similar in all the groups except that treatment groups were supplemented with 0.0 mg Cu, 10.0 mg inorganic copper (inCu), and 5.0 and 10.0 mg of nanoCu per kg dry matter (DM) in four respective groups. Feed intake and growth performance were similar in growing Sahiwal heifers fed on basal diet with or without supplemental Cu. Antioxidative variables like SOD, CAT, ceruloplasmin (Cp), total antioxidant status (TAS), and glutathione peroxidase (GSH-Px) were found higher in Cu-supplemented groups than control. Variables like malondialdehyde (MDA) and lipid peroxidation (LPO) were found lower in treatment groups than control. Total immunoglobulins (total Ig) and immunoglobulin G (IgG) were higher in treatment groups than control, although interleukin-6 (IL-6) was similar in all groups. There were upregulation of mRNA expression of SOD and CAT genes in experimental animals fed on Cu-supplemented diet while mRNA expression of interleukin-6 (IL-6) and interleukin-10 (IL-10) genes was not altered by dietary treatment. The results suggest that the level of 5-ppm nanoCu can be selected for feeding in growing cattle as it exerts similar effects as showed by 10-ppm inorganic Cu.


Introduction
Minerals are necessary for growth and reproduction and are involved in different processes like digestive, physiological, and biosynthetic processes within the body [1]. They therefore fulfill several important functions for the maintenance of animal growth and reproduction [2]. Cu is an essential trace element of animals. It can effectively maintain the stability of the internal environment, and is closely related with hematopoiesis, metabolism, growth, reproduction, and other important life activities [3]. Cu entered to the body in the form of sulfate is dissociated in the digestive tract to ion form, which is then absorbed in the small intestine [4]. Due to its ability to easily accept and donate electrons, Cu is involved in numerous biochemical processes [5,6].
Feed ingredients are normally deficient in Cu; so, the commercially prepared diet should provide the crucial amount of Cu in a biologically dynamic form, that depends on the physical and chemical attributes of the form of the supplement in which the Cu is provided in the diet. It can ensure the growth and decline in the homeostasis and peroxides damage to the body [7,8]. Cu added at levels higher than normal requirement has a growth promoting effect because Cu inhibits intestinal harmful microbes [9]. Cu sulfate is the main Cu source in the diet of livestock; which shows poor bioavailability caused by the presence of ingredients that can inhibit absorption. The digestibility of inorganic salts of Cu is low, and about 80% of Cu is eliminated via feces [10], and so its effect on soil microorganisms, plants, and aquatic species is currently one of the crucial environmental concerns causing the environmental pollution [11].
Cu nano particle (Cu NP) is one of the nano metal which is recently prepared for application in various fields, so if the Cu absorption is enhanced, the Cu supplementation and excretion amount may be reduced. The activity of nano particles depends upon huge surface area, exposing their atoms to direct contact with target cells. Since Cu NPs have the same effect on animal health and performance as other Cu source and because of their high physical activity, the quantity of Cu applied to animal diets and its consequent contamination of the environment can be notably reduced. It has been documented that Cu NPs has beneficial effects on the animal performance and could be used to replace Cu sulfate [12,13]. NanoCu has different biological, chemical, and physical properties compared to the inorganic form. The unique bioactivity of nanoCu is mainly due to the particle size and the large surface area. The size is important for penetration through for which a pore structure of a cellular membrane is required, thus affecting the number of proliferating cells [14].
Nano-sized Cu particles have been widely used in various chemical, physical, and biological fields including breeding, feed industry, animal husbandry, and veterinary field. This study aims to investigate the effect of dietary nanoCu supplementation on SOD and CAT gene expression, antioxidant status, and immune response of indigenous heifers. Furthermore, to determine the effective level of nanoCu compare with Cu sulfate for Sahiwal heifers.

Animals, Diets, and Experimental Design
A total of 24 growing Sahiwal heifers were selected from Livestock Farm Complex (LFC), DUVASU, Mathura and randomly assigned into four dietary treatments on body weight (100.20 ± 4.84 kg) and age (12-15 months) basis. Heifers either received a basal diet devoid of supplemental Cu (control) or were supplemented with 10 ppm of inorganic Cu as copper sulfate pentahydrate (CuSO 4 .5H 2 O, molecular weight 249.68, minimum assay purity 99%, Central Drug House Pvt. Ltd. New Delhi), 5 ppm and 10 ppm of nanoCu as cupric oxide nanopowder (CuO, molecular weight 79.54, APS: 40 nm, SSA: 80m 2 /g, minimum assay purity 99%, Sisco Research Laboratories Pvt. Ltd. Maharashtra, India). The dietary requirements of experimental animals were met by offering total mixed ration (TMR) consisted of concentrate: green berseem fodder: wheat straw in the proportion of 50:35:15 following NRC [15] guidelines.
To ensure that each animal consumed the calculated amount of Cu, the calculated amount of CuSO 4 .5H 2 O and CuO nanopowder was mixed with barley flour and prepared the premix @ 2 ppm Cu/g of barley flour and offered prior to providing the ration. TMR was prepared daily by hand mixing and was offered at 09:00 h and 18:00 h. The animals were provided with fresh and clean drinking water free of choice daily. Experimental animals were housed in a proper ventilated shed having the good arrangement for individual feeding and watering without having access to the other animal's diet. Deworming of all the experimental animals was done before the start of the experiment.

Observation Recorded and Analytical Procedures
Feeds and fodders intake were observed daily by weighing feedstuffs offered and leftover residue, and dry matter intake (DMI) was enumerated daily according to the DM content of the diet. Body weight (BW) of the experimental heifers was recorded at the starting of experiment and then at 15,30,45,60,75,90,105, and 120 days of experiment by using computerized weighing machine (Leotronic Scales Pvt. Ltd., India). Heifers were weighed for 2 consecutive days in the morning at 06:00 h before offering feeds, fodders, and water. The average of consecutive 2 days was considered as BW for that fortnight and considered for average daily gain (ADG), feed conversion ratio (FCR), and feed conversion efficiency (FCE). Samples of feeds and fodders offered, and leftover residue was dried in a hot air oven at 60 °C up to a constant weight was achieved and then ground to pass a 1-mm sieve in a Wiley mill. The samples were analyzed for DM (Method 973.18c), CP (Method 4.2.08), ether extract (EE; Method 920.85), and total ash (TA; Method 923.03) [16]. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to the procedures described by Van Soest et al. [17]. Calcium (Ca) and phosphorus (P) in feeds, fodders, leftover residue, and fecal samples were evaluated by Talapatra et al. [18] and volumetric method, respectively. The concentration of trace minerals like Cu, Zn, and Fe in different samples of feeds and fodders and orts left were evaluated by atomic absorption spectrophotometer (AAS; Perkins, USA). Ingredients and chemical composition of the basal diet fed during the experimental period are depicted in Table 1.
Peripheral blood samples were collected from jugular vein before feeding and watering of heifers at 07:00 h in a heparinized vacutainer tubes (BD Franklin, USA) at 0, 30, 60, 90, and 120 days post Cu supplementation. Collected blood samples were centrifuged at 3000 rpm for 30 min to remove the plasma from packed erythrocytes. Samples of blood plasma were kept at − 20 °C until further analysis of SOD, CAT, GSH-Px, MDA, LPO, Cp, TAS, total Ig, IgG, and IL-6 content. SOD, CAT, GSH-Px, MDA, LPO, Cp, IgG, and IL-6 were estimated in blood plasma by using ELISA test kits (Catalog No. E0003Bo; E0025Bo; E0006Bo; E0198Bo; E0561Bo; E0395Bo; E0010Bo; E0001Bo, respectively) from Bioassay Technologies, China. TAS measured as ferric-reducing antioxidant power (FRAP) assay procedure described by Benzie and Strain [19]. The total Ig was estimated by zinc turbidity method [20].

mRNA Expression Study
The immunity and antioxidant status-related genes (namely IL-6, IL-10, SOD, and CAT) were estimated in peripheral blood mononuclear cells (PBMCs) isolated from fresh blood samples.
Blood samples were collected from jugular vein using sodium heparinized vacutainer under sterile conditions. About 4 ml of blood was collected in the sterile fresh tube. Peripheral blood mononuclear cells (PBMCs) were separated from blood samples with the use of histopaque-1077 (Sigma). RNA was isolated from PBMCs by Trizol method. The purity and concentration of total RNA were examined with the use of the Biophotometer (Eppendorf, Germany). RNA samples of A 260 /A 280 value more than 1.8 were used for cDNA synthesis. cDNA synthesis was carried out from the mRNA found in the total RNA using Revertaid® First strand cDNA synthesis kit (Thermo Scientific, USA) with the use of moloney murine leukemia viral reverse transcriptase enzyme by using the manufacturer's guidelines. Real-time RT-PCR (qRT-PCR) was carried out after checking each primer for its specificity. The cDNA was checked for quality by performing RT-PCR with standard GAPDH primers. After that, amplified DNA was exposed to agarose gel electrophoresis as per guidelines given by Sambrook and Russel [21]. The size of the PCR products amplified was calculated from the standard 50 bp or 100 bp DNA ladder (Invitrogen, USA) ( Table 2).

Statistical Analyses
The data for measured variables were subjected to analysis of variance using the mixed model repeated measure procedure of the Statistical Software Package (SPSS for windows, V21.0; Inc., Chicago, IL, USA). The effect of treatments, days in trial, and their interaction on variables like antioxidants and immune response were analyzed by using the following model: where Yijk is the dependent variable, µ is the overall mean of the population, Ti is the mean effect of the Cu, Dj is the mean effect of days of sampling (j = 0, 30, 60, 90, and 120 days of dietary treatment), (T × D)ij is the effect of the interaction between treatment and days of trial and eijk is unexplained residual element assumed to be independent and normally distributed. Individual animal was used as the experimental unit for all data. The statistical difference between the means was determined by using ''Tukey's honest significant difference (HSD) test.''.

Feed Intake and Growth Performance
Dietary supplementation of inCu and nanoCu in heifers had no significant effect on DMI. DMI increased with the increase in the body weight of the experimental heifers. Percent DMI (kg/100 kg BW) in different groups showed similar effect of inCu and nanoCu supplementation. Statistical analysis of data showed that variation between the groups for mean BW change as well as fortnightly BW change was not significant (P > 0.05). The variation between the groups for mean fortnightly BW gain was not significant (P > 0.05). The variation between the groups for mean ADG (g/day) was not significantly different (P > 0.05). In the present study, FCR and FCE were used as feed efficiency measures. Statistical analysis of data showed that variation between the groups for mean FCR was not significant (P > 0.05). The mean values for FCE showed non-significant effect (P > 0.05) of inCu and nanoCu supplementation ( Table 3).

Biomarkers of Antioxidant Status
SOD was significantly higher (P < 0.05) in heifers receiving either 10-ppm inCu or 5-and 10-ppm nanoCu-supplemented diets than control at 90th day and 120th day of experimental period. Mean values for CAT were significantly higher (P < 0.05) in inorganic-and nanoCu-supplemented groups than control on 90th days of sampling. GSH-Px was significantly higher (P < 0.05) in heifers receiving either 10-ppm inorganic Cu or 5-and 10-ppm nanoCu-supplemented diets than control at 60th day and 120th day of experimental period. TAS was significantly higher (P < 0.05) in inorganic-and nanoCu-supplemented groups than control on 90th and 120th days of sampling. Mean values for Cp were significantly higher (P < 0.05) in inorganic-and nanoCusupplemented groups than control on 90th and 120th days of sampling. Plasma MDA showed significant difference (P < 0.05) between control and inorganic-and nanoCusupplemented experimental heifers but values were found within normal physiological level. Plasma LPO at 120th day was significantly lower (P < 0.05) in heifers receiving either inorganic-or nanoCu-supplemented diets than control.

Biomarkers of Immune Response
Plasma total Ig concentration in inorganic-and nanoCusupplemented groups were significantly higher (P < 0.05) than non-supplemented group. Plasma IgG at 120th day was significantly higher (P < 0.05) in heifers receiving either inorganic-or nanoCu-supplemented diets than control. Plasma IL-6 concentrations were in physiological range and reported similar trend in inCu, nanoCu supplemented and unsupplemented group (Table 4). To study the relative change in gene expression, the 2 −∆∆C T method was used as described previously by Livak and Schmittgen [25].

IL-6 mRNA Expression
The gel electrophoresis image and fold changes in the expression pattern of IL-6 gene in Cu supplemented and control groups during the experiment have shown in Figs. 1-2. The fold changes in the expression of IL-6 gene among the treatment groups were ranges from 0.26 to 2.38. Value showed non-significant changes among all four groups.

IL-10 mRNA Expression
The gel electrophoresis image and fold changes in the expression pattern of IL-10 gene in unsupplemented and Cu-added groups are depicted in Figs. 3-4. The fold changes in the expression of IL-10 gene among the treatment groups were ranges from 0.27 to 3.68 among the all four treatment groups. Similar to the IL-6 mRNA gene expression, mRNA expression of IL-10 gene was also showed non-significant effect among all the groups.

SOD mRNA Expression
The gel electrophoresis image and fold changes in the expression of SOD gene in Cu supplemented and control groups during the experiment have shown in Figs. 5-6. Cu supplementation showed significant effect (P < 0.05) on the mRNA expression of SOD gene. The mRNA expression was observed higher in Cu-supplemented groups compared to the control groups. However, sources of the Cu did not exert any significant effect. The upregulation of mRNA SOD expression began 90 days post Cu supplementation. The fold changes in the expression of SOD gene among the treatment groups were ranges from 0.60 to 3.49 among the all four

Feed Intake and Growth Performance
Results obtained in this study demonstrate that fortnightly daily DMI and body weight gain pattern between the control, inCu 10 , nanoCu 5 , and nanoCu 10 groups were similar. DMI increased with the increase in the body weight of the experimental heifers. Similar to our results, Cheng et al. [26] showed that Cu supplementation at 10 or 20 mg Cu/ kg DM and source had no effects on ADG and average daily feed intake (DFI) in lambs. Mullis et al. [27] reported that there were no changes in DMI on supplementation of 7-or 14-ppm Cu in heifers. Similar to present findings, Waghmare et al. [28] showed no effects on ADG and FCR on supplementation of Cu as CuSO 4 and Cu-methionine in kids diet. Vaswani et al. [29] also reported the similar observation that supplementing 8.0 mg Cu/kg DM either in the form of Cu-proteinate, Cu-propionate, or Cu sulfate did not affect feed intake, daily gain, feed:gain ratio, and BCS in growing heifers. The results of the present study are similar to the observations of Dezfoulian et al. [30] who reported that there were no significant differences between treatments for DFI or ADG in lambs. In contrary to the findings of the present study, Mondal and Biswas [31] reported that supplementation of Cu from different sources and different dose levels in a concentratebased diet may enhance performance, nutrient utilization, and plane of nutrition in castrated goat kids. Moreover, Slyvchyk et al. [32] observed that the administration of Cu NPs in the dose of 50 μg/kg of body weight has positive effects on the reproductive system of female rabbits and the processes of development of embryo. In opposite to the results of the present study, Zhang et al. [33] reported that supplementation of the basal diet with 10-mg Cu/kg DM improved growth performance and nutrient digestibility and plasma Cu status of Cashmere goats. Younan et al. [34] also showed that rabbit bucks fed diet with nanoCu had numerically higher values for live BW and BW gain, followed by those fed a diet having organic and metallic Cu than a control group. Engle and Spears [35] also found that supplementation of either 10-or 40-ppm Cu in Simmental steers showed no effect in feed:gain ratio. There was a study conducted on rabbits fed diets supplemented with 50-or 75-mg Cu/kg diet as nanoCu recorded significantly higher final body weight and performance index and has better feed conversion ratio during all growth periods than the control group [36]. Some studies compared the inorganic forms of Cu with Cu NP and the latter showed an improvement in the growth performance of piglets [37]. Chang et al. [38] also reported that dietary supplementation of nanoCu enhances performance of weaning piglets.

Biomarkers of Antioxidant Status
In the present study, SOD, CAT, GSH-Px, TAS, and Cp were used as biomarkers of antioxidant status, whereas plasma MDA and LPO levels were used as biomarker of oxidative stress. Plasma SOD, CAT, GSH-Px, TAS, and Cp were significantly higher (P < 0.05) in heifers receiving either 10-ppm inCu or 5-and 10-ppm nanoCu-supplemented diets than control. The present study showed that plasma MDA and LPO levels were significantly lower (P < 0.05) in heifers receiving either inorganic-or nanoCu-supplemented groups than control. Increased peroxidation of lipids in intracellular and extracellular membranes causes damage to the cells, tissues, and organs. SOD is major antioxidant enzyme, which can eliminate excess free radicals in the body and reduce the degree of nucleic acid damage [39,40]. CAT is another important antioxidant enzyme, which can catalyze the decomposition of H 2 O 2 , thereby playing an antioxidant role [41]. GSH-Px is important antioxidant enzyme that can catalyze the reduction of reduced glutathione to hydrogen peroxide and thus help to protect the integrity of the cell membrane structure and function [42,43]. This was concurrence with the finding of Shen et al. [41] who showed that when compared with the Cu-deprived goats, serum SOD, GSH-Px, CAT, and TAS in nanoCu-and CuSO 4 -fed groups were significantly higher, while serum MDA content was significantly lower. Limited work has been conducted on the effect of nanoCu on antioxidant status of ruminants. Therefore, findings of the present study have been discussed with findings of monogastric animals. SOD activity was significantly improved in nanoCu-fed piglets [37]. TAS, SOD, and GSH-Px were more in the birds fed a diet with nanoCuO than other treatments, and lowest MDA level was observed [44]. Senthilkumar et al. [45] found that SOD activity was improved in Cu-supplemented lambs. The replacement of Cu-sulfate with Cu NPs differentially modulated the redox status of selected tissues, i.e., enhanced SOD activity in small intestinal tissue and decreased total glutathione levels in the bursa of fabricius of turkeys [46]. The nanoCu supplementation in rabbit significantly increased the activity of SOD compared with the control group; however, CAT was unaffected [36]. Cu NP treatment significantly enhanced SOD and reduced MDA levels in broilers when compared to the control and CuSO 4 -treated birds [47]. Likewise, Vaswani et al. [29] found that TAS as antioxidant activity (FRAP value) was higher (P < 0.05) in heifers receiving Cu-supplemented diets in growing heifers. Dezfoulian et al. [30] also reported Cu source had a significant effect on Cp concentration (P < 0.05) in lambs. In accordance with the present results, Arthington et al. [48] showed increased plasma Cp concentrations (P < 0.05) independent of Cu source in Cu-supplemented heifers. In comparison to feed-Cu sources, administration of the CuO bolus (one-time   dose of 20 g of Cu) resulted in a higher mean plasma ceruloplasmin concentration in heifers [49]. On the contrary, Dezfoulian et al. [30] observed that Cu supplementation had no significant effect on SOD activity in lambs. Excessive MDA produced during the lipid peroxidation will damage the cell membrane structure, resulting in the loss of cell function by apoptosis. So, variation in MDA concentration can reflect the degree of lipid peroxidation and also the degree of damage to the body. MDA is the most common product of lipid peroxidation, and its level can directly reflect the degree of lipid oxidative injury [50,51]. If the MDA concentration is higher, then there is more damage to body tissue [52].

Biomarkers of Immune Response
In the current study, plasma total Ig and IgG concentration in inorganic-and nanoCu-supplemented groups were significantly higher (P < 0.05) than non-supplemented group. A non-significant (P > 0.05) effect of inorganic and nanoCu supplementation on plasma IL-6 concentration in four different groups across 120 days study was observed. Immunity of the animals is a defense system engaged in immune response and immune function, which maintains the relative stability of the internal environment [43]. The content of serum Ig is an important indicator of the immune function of animals [53]. In accordance with the present results, Gonzales-Eguia et al.
[37] showed significant improvements in the IgG and γ-globulin levels of the nanoCu group of piglets. Injection of Cu NPs in layer embryo did not change the plasma concentration of IgG, indicating that it did not relate with humoral system [54]. Ognik et al. [55] also reported increased immune defense of chickens by supplementing their diets with nanoCu. It may be explained by the fact that the increase in Ig may be the result of the activation of phagocytes which indicates an enhanced immune status of broilers after nanoCu treatment. IL-6 revealed a non-significant but numerical change in their values between the three groups of broiler chickens [47]. A study showed that a Cu deficiency in the diet of rats decreases plasma level of IL-6, whereas, reducing the level of Cu decreases the plasma content of IL-6. Replacing CuCO 3 with Cu NP in rat diets affects their metabolism, as indicated by decreases in IL-6 [56].

mRNA Expression of Biomarkers of Immunity and Antioxidants Status
The upregulation of SOD and CAT gene expression may be correlated with the defense mechanism against oxidative stress caused by Cu in growing heifers. Thus, Cu NPs stimulated a significant upregulation of SOD and CAT gene expression levels. The drift for greater mRNA expression of SOD in the treatment groups may be related to anti-oxidative damage. Enhanced SOD and CAT mRNA expression levels and enzyme activities (P < 0.05), with a definite linear association among the gene expression level and enzyme activity. El-Kassas et al. [57] reported that CuO NP supplementation at 100% and 50% of the Cu requirement to heat stressed chickens stimulated a significant upregulation of the SOD mRNA expression level, with the level being higher with supplementation of CuO NPs at 50% of the recommended Cu concentration. Supplementation of CuO NPs at 100 or 50% to the diet of heat-stressed chickens similarly upregulated the CAT gene expression level. In the present experiment, the expression of immune-related genes (IL-6  and IL-10) was not affected by the treatments. In a study on broiler birds, the immune-related genes were not affected by the treatments in relation to the control, probably due to the biocompatible properties of Cu NP and CuSO 4 [58]. In an experiment, the expression of immune-related genes (NF-kB and TNF-α) was not affected, indicating the absence of the pro-inflammatory property of CuO NP [59]. Our results also indicate that nanoCu does not have pro-inflammatory properties and does not interact with humoral responses in growing heifers.

Conclusion
Supplementation of Cu either from inorganic or by nano source did not exert any adverse effect on growth performance. There were significant improvements in antioxidant status and immune response in Cu-supplemented groups. Cu supplementation improves mRNA expression of SOD and CAT genes while expression of IL-6 and IL-10 genes was similar among all groups. Findings of the present study revealed that the level of 5-ppm nanoCu can be selected for feeding in growing cattle as it exerts similar effects as showed by 10-ppm inorganic Cu.