Biomarkers of Immunity
In the present study, the experimental calves fed on the diet supplemented with the combination of nano Cu and nano Zn had better immunity which was evidenced from higher plasma concentrations of IgG, IgM, IgA and TIg and lower plasma TNF-α and cortisol concentrations innanoCu10+nanoZn32group. The concentrations of circulating immunoglobulins, especially IgG, IgM, and IgA, are important indicators of immune function. No work has been conducted to see the combined effect of nano Cu and nano Zn on immune response in animals. Therefore, the findings of the present study have been discussed with those of the others who used any source of Cu and Zn separately or in combination. Kushwaha et al. [32] observed a significant (p<0.05) improvement in immune response in nano Cu supplemented Sahiwal heifers. He indicated that nano Cu does not have pro-inflammatory properties and does not interact with humoral responses in growing heifers. Pineda et al. [33] found the same results that the expression of immune-related genes (TNF-α) was not affected, indicating the absence of the pro-inflammatory property of nano CuO. Gonzales-Eguia et al. [34] showed significant improvements in the IgG and γ-globulin levels of the nano Cu group of piglets. Ognik et al. [35] also reported increased immune defence of chickens by supplementing their diets with nano-Cu. Compared with the control group, the dietary supplementation with 100 mg/kg of nano-Cu significantly increased the concentrations of IgA, IgG, IgM and lysozyme in the serum of broilers [36]. It could be explained by the fact that the immunoglobulin’s enhancement may be the result of the activation of phagocytes, which indicates an improved immune status of broilers after nano Cu treatment [35]. Another study reported that nano Cu-loaded chitosan improved immune status, enhanced protein synthesis and was beneficial to the caecal microbiota of broiler chickens [36].
Zn plays a role in molecular and membrane stability. One of the first lines of defence the body has against an immunological attack is the skin. The most direct connection between Zn and immune function is its role in cell replication and proliferation, which is of great importance for maintaining the normal activity and integrity of immune cells and systems Wang et al. [37]. Sharish et al. [38] reported that plasma total immunoglobulin concentration was found higher in the nano Zn supplemented group than the control group at 30, 60, and 90 days, and total immunoglobulin concentration was found higher in the inorganic Zn supplemented group than the control group. He concluded that nano Zn supplementation at 25 and 50 ppm has better immunogenic effects and thus may replace inorganic Zn sources at a lower level of Zn i.e. 25ppm. Nagalakshmi et al. [39] demonstrated that administration of a low level of organic Zn improves growth performance and the immune response of calves. Consistent with these findings, they found that ZnO supplementation increased serum IgG and IgM concentrations above those of the control by 3.85 and 2.86 mg/mL, respectively, compared with Zn-methionine supplementation, indicating that the administration of a low level of ZnO is superior to Zn-methionine with respect to the immune function of dairy calves. Serum IgG, IgM and IgA can protect the extra vascular compartment against pathogenic viruses and microorganisms [40], so the increase in the levels of serum IgG and IgM indicates that weaned piglets fed on diets supplemented with nano ZnO may undergo improvements in immune function. Feng et al. [41] reported an improvement in IgA, IgM and IgG levels with the dietary replacement of 120 mg/kg of inorganic Zn with 90 or 120 mg/kg of organic Zn. In the study of Chang et al. [42], total serum IgG and IgM concentrations in the ZnO group were significantly higher than in the control group. Similarly, Nagalakshmi et al. [43] and Wang et al. [37] observed better immune responses with organic Zn supplementation compared to inorganic Zn supplementation in lambs and dairy cows, respectively. Engle et al. [44] implied that when cattle are in a Zn deficient state, cell-mediated immune responses are decreased, making calves more susceptible to infectious disease.
Biomarkers of Antioxidant Status
Antioxidant status is known to be a significant predictor of disease and mortality in infants, especially premature infants. In present study, plasma MDA level was used as biomarker of oxidative stress whereas, SOD, CAT, GSH-Px, Cp and TAS were used as the biomarkers of antioxidant status. Similar to the immune response, the calves supplemented with a combination of nano Cu and nano Zn showed better antioxidant status than calves in control, nano Cu or nano Zn alone groups. Better antioxidant status in the nanoCu10+nanoZn32group is evidenced by lower MDA concentrations and higher SOD, GSH-Px, Cp and TAS activity in this group. Pineda et al. [45] found that the nano Cu injection reduced lipid oxidation, which could be associated with the lower O2 consumption in broiler chicks. SOD is one of the main antioxidants (Cu-Zn linked metallo-enzymes), which can remove excess free radicals in the body and reduce the degree of nucleic acid damage [46]; Zhao et al. [47]. There was significant (p<0.05) improvement in antioxidant status in nano Cu supplemented groups in Sahiwal heifers, and nano Cu supplementation improves mRNA expression of SOD and CAT genes Kushwaha et al. [32]. Shen et al. [48] showed that when compared with the Cu deprived goats, serum SOD, GSH-Px, CAT, and total antioxidant capacity in nano Cu and CuSO4 groups were significantly higher, while serum MDA content was significantly lower. Likewise, Vaswani et al. [49] found that antioxidant activity (TAS) was higher in heifers receiving Cu-supplemented diets. Dezfoulian et al. [50] also reported that Cu source had a significant effect on Cp concentration (p<0.05) in lambs. Total antioxidant capacity, SOD and GSH-Px were more in the birds fed diet inclusion of 60 and 90 mg nano CuO than other treatments and lowest MDA level was observed [51]. Antioxidant mechanism of the blood, by reflected as elevated catalase and plasma FRAP, became more intensive during nano-Cu supplementation in wistar rats [52]. The replacement of inorganic Cu with nano Cu 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 [53]. The nano-Cu supplementation in the rabbit significantly increased the activity of the SOD enzyme compared with the control group, but catalase activity was unaffected [54]. On the contrary, Dezfoulian et al. [50] observed that Cu supplementation (regardless of source and level) had no significant effect on SOD activity in lambs.
The antioxidant effect of Zn may be mediated through direct action of Zn ion, its structural role in antioxidant proteins, and modulation of metallothionein induction. Direct antioxidant activity of Zn ions is associated with their binding to thiol groups, thus protecting them from oxidation [55, 56]. Sharish et al. [38] reported that plasma SOD concentration was found higher in the nano Zn supplemented group than the control group and inorganic Zn supplemented group at 30, 60, and 90 days, and TAS concentration increased within all groups over the time, and TAS concentration was higher in all treatment groups than the control group. Wang et al. [57] reported that the serum SOD levels increased and MDA levels decreased with the inclusion of ZnO and 0.4-0.6 mg/kg nano-ZnO in their diets in weaning piglets. These results are similar to the findings of Zhao et al. [58], who observed that dietary supplementation with 0.06 and 0.1 g/kg nano ZnO improved serum Cu-Zn-SOD activity but decreased serum MDA levels in broilers on days 28 and 35. Bakhshizade et al. [59] noticed in cows that the SOD concentration was higher in the nano Zn and Zn-Glycine supplemented groups than in the inorganic Zn supplemented group. Zn as Zn oxide nanoparticles in Japanese quails and broiler chickens improved the total antioxidant capacity and reduced the MDA concentrations compared to controls [58, 60]. The activity of GSH-Px increased in high Zn and coated nano ZnO fed pigs compared with the control group. Pigs fed on coated nano ZnO had a higher activity of serum SOD (p<0.05) compared with the control and high Zn groups [61]. Furthermore, the antioxidant capacity of growing pigs is fundamental for maintaining the normal metabolic state to protect a pig’s health; we hypothesized that the effects of dietary nano ZnO could promote growth by indirectly regulating the antioxidant capacity of pigs. High doses of ZnO (3000 mg/kg) supplementation reduced the serum MDA concentration and increased the SOD activity in piglets [62]. His findings also show that pigs fed a high dose of ZnO had an improved antioxidant capacity by increasing GSH-Px activity. Meanwhile, a low dose of coated nano ZnO could increase the activities of SOD and GSH-Px in the serum. Previous studies have shown that Zn has an antioxidant function [63], and supplementation with Zn methionine reportedly decreases the concentration of MDA but increases that of MT and T-AOC in the serum of ruminants [64].
Growth Performance and Health Status
Dietary supplementation of either nano Cu or nano Zn alone or their combination did not exert any impact on the growth performance of the experimental calves. Kushwaha et al. [32] found no effects of 10 ppm inorganic Cu, 5.0 and 10.0 ppm nano Cu on the growth performance in growing Sahiwal heifers, which was similar to the findings of our study. Vaswani et al. [49] reported a similar observation that supplementing 8.0 mg Cu/kg DM either in the form of Cu-proteinate, Cu-propionate and Cu sulphate did not affect ADG in growing heifers. Kim et al. [65] also found a similar effect on growth performance in nano Cu supplemented pigs compared with inorganic and organic Cu. The results of the present study are similar to the observations of Dezfoulian et al. [50] who reported that there was no significant effect of Cu supplementation on ADG in lambs. Waghmare et al. [66] observed that supplementation of Cu as CuSO4 and Cu-methionine did not alter ADG and feed: gain ratio in kids. However, in contrast to the findings of the present study, some studies compared the inorganic forms of Cu with nano Cu and the latter showed an improvement in the growth performance of piglets [34, 67]. Zhang et al. [68] reported that supplementation of the basal diet with 10 mg Cu/kg DM in the basal diet enhanced growth performance in Cashmere goats. Chang et al. [67] reported that dietary supplementation with 25 mg/kg body weight nano Cu improves the performance of weaning piglets.
No significant difference in body weight observed on supplementation of different levels and different sources of Zn has been observed in previous research, though overall body weight increases as the age of experimental animal advances. Zn supplementation above NRC (2001) recommended requirements did not consistently affect growth rate in cattle [69]. The results of the present study are similar to the observations of Zaboli et al. [70] reported that ADG in goat kids was not affected due to the supplementation of Zn from different sources at different levels. However, contrary to the findings of present study, Chang et al. [71] showed that supplementation with Zn-methionine but not ZnO, significantly increased the ADG of new-born calves in the first 2 week after birth. Anil et al. [72] observed significantly higher body weight gain and ADG in the 20 ppm nano Zn supplemented calves group followed by 10 ppm nano Zn, 5 ppm nano Zn supplemented groups and 25 ppm ZnSO4 group. Hongfu et al. [73] observed significant increase in ADG in nano-Zn oxide (200,400, and 600 mg/kg) supplemented group and higher ZnO (3000 mg/kg) supplemented piglet groups compared to the no Zn supplemented group. The discrepancy in growth performance in the findings of different studies may be a consequence of different sources and levels of Cu and Zn used, differing ages of animals used in the study, differences in study period, different genetics in various breeds, etc.
In the present study, frequency of diarrhea, incidence of diarrhea, FCS, attitude score, pneumonia occurrence, joint ill and navel ill and mortality were used to assess the health status of the experimental calves. The number of calves affected with diarrhea, frequency of diarrhea, time until resolution of diarrhea and incidence of diarrhea were lower in the nanoZn32 and nanoCu10+nanoZn32groups. FCS and attitude score were also better in nano Zn and combination groups. One calf from the control group was found affected with joint ill and two calves from the control group were found affected with navel ill. No any case of calf mortality and pneumonia were reported in all four groups. Calves having diarrhea were found affected by E. coli. The anti-diarrheal function of Cu and Zn may be associated with their role in immunity [74, 75]. The mechanisms of the anti-diarrheal effect of Zn are thought to involve the regulation of intestinal fluid transport and mucosal integrity, the promotion of immunity, and the modulation of oxidative stress [76, 77]. Wang et al. [78] reported that, the incidence of diarrhea in control calves fluctuated between 20 and 34.29% during the first 2 wk of life. However, supplementation with ZnO or Zn-Met helped to reduce the incidence of diarrhea in neonatal dairy calves during their early lives and no diarrhea was found in calves in the ZnO group during the first 3 d after birth, which is consistent with previous findings [78, 79]. In addition, supplementation with Zn reduced the incidence of diarrhea, which is consistent with the results obtained by Feldmann et al. [80], who showed that Zn-Met-treated calves had a 14.7% lower risk of diarrhea than placebo-treated calves. Hu et al. [81] reported that 0.3 g/kg Zn as nano ZnO inclusion in the diet decreased the incidence of diarrhea in early weaned piglets (5.7 kg), exhibiting a similar effect to 3.0 g/kg Zn as ZnO administration in a 14-day experiment. Dietary ZnO at therapeutic concentrations to 2,000 to 4,000 mg/kg could effectively prevent and treat post-weaning diarrhea [82]. Limited information is available on the role of Cu in controlling calf diarrhea. Within individual calves, the onset of diarrhoea, incidence of leg abnormalities, and anaemia were correlated with the onset of Cu deficiency and are indicative of relatively severe deficiency [83].