DOI: https://doi.org/10.21203/rs.3.rs-1638131/v1
This experiment was conducted to study the effects of copper sulfate and cupric citrate on growth performance, nutrient utilization, antioxidant capacity and intestinal microbiota of broilers. A total of 360 one-day-old Ross 308 broilers were randomly divided into 5 groups with 6 replicates in each group and 15 broilers per replicate. Broilers in the control group were fed a basal diet, and animals in other four groups were fed basal diets supplemented with 2 sources (copper sulfate and cupric citrate) and 2 levels (50 and 100 mg/kg dry matter). The experiment lasted for 42 days. The results showed that dietary cupric citrate supplementation increased the average daily gain (P = 0.0313). The average daily feed intake and feed gain ratio, however, were not affected by dietary copper sulfate or cupric citrate (P > 0.10). Additionally, dietary copper sulfate or cupric citrate supplementation increased the digestibility of crude protein (P = 0.0554) and energy (P = 0.0191). For intestinal microflora, dietary cupric citrate supplementation decreased the concentration of Lactobacillus and Escherichia coli (P < 0.05) in the ileal digesta or cecal digesta. In addition, dietary Cu supplementation increased the pH in duodenum (P = 0.0008) and jejunum (P = 0.0589). The activities of serum Cu-Zn superoxide dismutase (P = 0.0899), and ceruloplasmin (P = 0.0269) were increased by Cu addition. The present study demonstrated cupric citrate fed to broilers has a positive effect on growth and nutrient utilization. Our results also show that moderately high Cu in the diet increases the pH in duodenum and jejunum, and reduced the concentration of Lactobacillus and Escherichia coli in the ileal digesta or cecal digesta.
Copper is a trace element widely distributed in organisms. It is involved in the composition of plasma ceruloplasmin (CP), copper zinc superoxide dismutase (Cu/Zn SOD), cytochrome c oxidase, monoamine oxidase and other enzymes. Copper citrate can be used as a new copper source to replace copper sulfate in feed because of its reducing the destruction of nutrients and high biological potency. Since Braude [1] found that adding high copper (100-250mg / kg) to feed can improve the growth performance of piglets in 1945, many studies have confirmed that feeding high copper can improve the average daily gain, average daily feed intake and feed conversion efficiency of animals, reduce the pH value of intestinal chyme and improve the activities of proteinase and phospholipase[2,3].
Copper has a wide range of bactericidal spectrum and is effective for bacteria, molds and fungi. Dietary copper supplementation could reduce the infection of broilers with Eimeria[4] and and inhibit the growth of Escherichia coli (E. coli). [5]. Peng et al. [6] found that adding 60 mg / kg copper citrate to piglet diet can improve growth performance, reduce copper residue in the body, improve antioxidant function, reduce the number of cecal E. coli and increase the number of Lactobacillus. Yan et al. [7] found that adding 20 mg / kg copper citrate to piglet diet can increase the expression of bone marrow antimicrobial peptides and significantly reduce the diarrhea rate of piglets.
Citric acid is an important bioactive compound and a chelating ligand with strong coordination ability [8]. Citric acid chelates have the advantages of high bioavailability, compared with other organic acid chelates, the price is cheaper, more in line with the market demand [9,10]. Copper citrate is a new feed additive approved by Ministry of Agriculture and Rural Affairs of China in 2019. The research on copper citrate in diet mainly focuses on piglets, while the research on the application of copper citrate in broiler diet is rarely reported. To this end, this experiment used Ross 308 broilers as the object to study the effects of adding copper citrate on growth performance, nutrient utilization, antioxidant capacity and intestinal microbiota of broilers. It aims to provide a basis for the scientific application of copper citrate in broiler production.
The animal protocol for this experiment was approved by the Animal Care Committee of the Anhui Science and Technology University. Animals were maintained and processed in accordance with the Anhui Science and Technology University Guide for the Care and Use of Laboratory Animals.
Animals, diets, and management
Three hundred and sixty 1-day-old healthy broiler chicks (Ross 308) were allotted to 5 groups with 6 replicates in each group and 12 broilers in each replicate. Dietary treatments were as follows: (1) basal diet without supplemental Cu (control); (2)basal diet+50 mg Cu/kg dry matter (DM) as copper sulphate (CS-50); (3) basal diet+100 mg Cu/kg DM as copper sulphate (CS-100); (4) basal diet+50 mg Cu/kg DM as copper citrate (CC-50) and (5) basal diet+100 mg Cu/kg DM as copper citrate (CC-100). The experiment lasted for 42 days, and diets were formulated in two stages (1 ~ 21 days old and 22 ~ 42 days old). Ingredients and chemical composition of the basal diets are presented in Table 1. The final Cu content of the diets are presented in Table 2.
The single-layer cage (200 cm long × 100 cm wide × 40 cm high, with concrete floors of 0.167 m2/bird) was adopted in the experiment. The chicken house and related appliances were cleaned and disinfected before the experiment. The temperature of the chicken house was controlled by human industrial temperature control three days before entering the chicken house. Diets and water (less than 0.01 mg/L Cu by analysis) were available ad libitum.
Sample collections
On the d 1, d 21 and d 42, the weight was measured (stop feeding for 12 hours before weighing and drink freely), and the feed intake was recorded at the same time. Average daily gain (ADG), average daily feed intake (ADFI) and the feed/gain ratio (F/G) were calculated based on the recorded data.
On d 42, 60 birds (2 chicks per replicate) were randomly selected and weighed after feed deprivation for 12 h. Blood samples were collected from the wing vein and centrifuged at 3,500×g for 10 min. Serum was separated and stored at −80°C for further analyses.
After blood sampling, birds were killed by cervical dislocation immediately and the pectorals and liver were excised in situ. The pH of the digesta in the jejunum and ceca was subsequently measured with a calibrated digital pH meter with a glass-tipped probe (model IQ120, IQ Scientific Instruments Inc., Carlsbad, CA). Two independent pH readings were taken in situ in each location along the digestive tract. The cecal samples and the content samples of the ileal (Meckel’s diverticulum up to 40 mm above the ileo-cecal junc-tion) digesta were aseptically collected and placed on ice for transportation to the laboratory, where fresh cecal samples were diluted 10-fold by weight in buffered peptone wate and homogenized using a stomacher. Viable counts of bacteria in the samples were then determined by plating serial 10-fold dilutions (in 10 g/L peptone solution) onto MacConkey agar, Lactobacilli MRS agar, and Bifidobacterium agar plates (Beijing Luqiao technology Limited by Share Ltd) to verify the E. coli i, Lactobacillus, and Bifidobacterium, respectively. Agar plates were incubated at 37°C for 36 or 48 h, after which bacterial colonies were counted. Concentration of microflora was finally expressed as log10 colony-forming units per gram of intestinal content.
At the 39th to 42nd day of the experiment, the feed intake was recorded and all excreta was collected for 4 consecutive days. Excreta were collected from plastic trays placed under the cages 6 times per day (06:00, 9:00, 12:00, 15:00, 18:00 and 21:00 h) and stored at −20℃. Finally, feces from each cage were collected, mixed and weighed. Fecal samples were dried in an oven at 65°C for 48 h. Feed and excreta samples were ground through a 0.45 mm screen and stored for further analysis.
Chemical analysis
The nutrient contents of feed were analyzed by the methods of the Association of Official Analytical Chemists (AOAC)[11]. Copper concentrations of feed, tissue and plasma were analyzed by Flame Atomic Absorption Spectroscopy (Shimadzu Scientific Instruments, Kyoto, Japan). The activities of serum Cu-Zn superoxide dismutase (Cu/Zn SOD), and ceruloplasmin were determined by using respective assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, P. R. China).
Statistical analysis
All data were analyzed as a 2 × 2 + 1 factorial experiment based on a completely randomized design using the General Linear Model procedure of the SAS software (Statistical Analysis System, Version 9.13, 2002). Data were analyzed as repeated measures with a model containing source, level, and source × level. Means were compared using Duncan’s multiple range test and P < 0.05 was considered as the significant level, and P > 0.05 and < 0.10 was considered a trend.
Growth performance
The effect of copper sulfate and cupric citrate on growth performance of broilers in Table 3. Copper sulfate or copper citrate at 50 mg/kg Cu or 100 mg/kg Cu of the diet had no effect on broiler ADFI in any period. However, the ADG was increased by Cu supplementation in the periods of 1 to 21 d (P = 0.0313) and the overall period of 1 to 42 d (P = 0.0812). Broilers in the CC-100 groups had higher ADG than those in the control and CS-50 group (P < 0.05). Neither copper sulfate nor cupric citrate affected F/G in any period. Nevertheless, the lowest F/G was seen in the CC-100 group.
Nutrient Utilization
The effect of copper sulfate and cupric citrate on apparent nutrients digestibility of broilers in Table 4. Neither copper sulfate nor cupric citrate affected the apparent digestibility of DM, Ca, or P. However, dietary Cu supplementation increased the digestibility of CP (P = 0.0554) and energy (P = 0.0191). Broilers supplemented copper (as copper citrate) with 100 mg/kg had greater (P< 0.05) CP digestibility than those receiving 0 or 50 mg/kg copper (as copper sulfate). Copper sulfate or copper citrate at 50 mg/kg Cu or 100 mg/kg Cu of the diet had higher (P < 0.05) energy digestibility than those in the control group.
Intestinal and Excreta Microflora
The effect of copper sulfate and cupric citrate on intestinal microflora of broilers in Table 5. In the ileal digesta or cecal digesta, adding copper sulfate and cupric citrate decreased (P < 0.05) the concentration of E. coli. Similarly, the concentration of Lactobacillus also showed the same trend. In the ileal digesta, copper sulfate or copper citrate at 100 mg/kg Cu of the diet had lower (P < 0.05) the concentration of Lactobacillus than those in the control group, and broilers in the CC-100 groups had lower (P < 0.05) the concentration of E. coli than those in the CS-50, CC-50 and control group. In the cecal digesta, broilers in the CC-100, CC-50, CS-100 groups had lower (P < 0.05) the concentration of Lactobacillus andE. coli than those in the CS-50 and control group.
Gastrointestinal pH
Neither copper sulfate and cupric citrate affected pH of glandular stomach, muscular stomach, ileum and cecum. However, dietary Cu supplementation increased the pH in duodenum (P = 0.0008) and jejunum (P = 0.0589). In the duodenum and jejunum, broilers in the CC-100 groups had higher (P < 0.05) the pH than those in the CS-50 and control group.
Antioxidant defenses
Table 7 presents the influence of dietary copper sulfate and cupric citrate on serum antioxidant defenses parameters in broilers. Neither Cu-Zn SOD (P = 0.0899) nor ceruloplasmin (P = 0.0269) were increased by Cu addition. Broilers in the CC-100 groups had higher (P < 0.05) the activities of serum Cu-Zn SOD than those in the control group. Broilers in the CC-100, CC-50, CS-100 groups had higher (P < 0.05) ceruloplasmin concentration than those in the control group.
Many studies have shown that the effect of adding chelated copper to feed is significantly better than that of adding copper sulfate [12, 13]. The results showed that there were no significant differences in ADFI, ADG and F/G of the diets supplemented with copper citrate and copper sulfate. However, dietary supplemented with 50 mg/kg or 100 mg/kg copper citrate has a higher ADG, indicating that copper citrate had a certain effect on promoting the growth of broilers. The increase of feed intake was an important reason that copper citrate promoted the growth of broilers. However, the mechanism of copper citrate induced increase in feed intake of broilers is still unknown and needs to be further studied. The results showed that adding 50 mg / kg copper citrate in the diet could improve the growth performance of broilers, and there was no difference in the growth performance of broilers compared with adding 100 mg / kg copper sulfate. This provides a new idea for adding low-dose copper citrate instead of high-dose copper sulfate to provide copper for broilers in practical production.
Adding copper to feed can improve the activity of enzymes, thus improving the nutrient digestion efficiency [14, 15]. Kirchgessner et al. [16] found that appropriate copper ions can activate pepsin activity and promote protein absorption. Luo, Dove [17] showed that high copper can improve the activities of lipase and phospholipase A in the small intestine, thus improving the absorption of essential fatty acids. Our experiment found that dietary copper supplementation had no significant difference in the digestibility of calcium and phosphorus, while the digestibility of dry matter, energy and crude protein were significantly increased.
Jejunum is the main place for the digestion and absorption of nutrients in broilers, and its pH has an important effect on the activity of digestive enzymes[18]. The optimum pH of trypsin is about 8 [19], and the pH of intestinal tract near duodenum is almost neutral [20]. No relevant studies have been found on the effect of copper on pH of intestinal contents. We found that dietary supplementation of 50 mg/kg or 100 mg/kg copper, whether copper sulfate or citrate, slightly increased pH of jejunum and duodenum contents in broilers. This may be why copper promotes growth and nutrient utilization in animals.
Peng et al. [6] showed that copper citrate could reduce cecal E. coli and increase lactobacillus, thus reducing diarrhea rate and mortality rate of animals. The results showed that dietary copper citrate had a significant inhibitory effect on E. coli in ileum and cecum of broilers, and the sensitivity of E. coli to copper citrate increased with the increase of copper citrate concentration. Warnes et al. [21] showed that copper substantially reduces bacterial growth and limits bacterial infectious behavior. Tan et al. [22] found that copper inhibits iron-mediated DNA oxidative damage in E. coli. In addition, copper can inhibit the growth of wild-type and mutant E. coli, and the addition of branched chain amino acids restored the growth, indicating that copper hinders their biosynthesis [23]. High concentrations of copper significantly increased the number of Dehalobacterium, Coprococcus, and Spirochaetales in the rectum, while the number of Salinicoccus, Bacillales, Staphylococcus, and Lactobacillales decreased dramatically, interrupting the dynamic balance of the microbiota [24]. The precise mechanism of how copper kills microorganisms may be controlled by many factors, and there are different mechanisms for different microorganisms, which need to be further studied.
Copper is an important component of ceruloplasmin and Cu-Zn superoxide dismutase, which are important antioxidant enzymes in animals and play an important role in maintaining animal health [25]. Ceruloplasmin inhibits the free radical production of iron ions through its ferrous oxidase activity [26]. Cu-Zn superoxide dismutase can inhibit the production of superoxide anion free radicals and protect the structure and function of cell membrane [27]. Many studies have shown that high copper diet can significantly increase the activities of ceruloplasmin and cu-Zn superoxide dismutase in animal serum [28, 29]. The results of this study showed that dietary supplementation of copper citrate or copper sulfate can increase the activities of ceruloplasmin and copper zinc superoxide dismutase in serum of broilers.
The result of this feeding trial demonstrated that cupric citrate fed to broilers has a positive effect on growth and nutrient utilization. Additionally, this study also indicated that moderately high Cu in the diet increases the pH in duodenum and jejunum, and reduced the concentration of Lactobacillus and Escherichia coli in the ileal digesta or cecal digesta of broiler chickens.
Funding
The funding for this study was from Anhui Provincial Natural Science Foundation (2108085MC114), Key research and development Program of Anhui Province (202004f06020048), Major project of Shandong Agricultural Improved Varieties Project (2019LZGC019), High-level Talents Introduction Project of Anhui Institute of Science and Technology (DKYJ201701).
Contributions
All the authors wrote the manuscript and revised the manuscript, reviewed, and agreed on the final manuscript before submission. Mingxia Zhu, Qingkui Jiang contributed to the conception of the study. Xuezhuang Wu performed the experiment. Xuezhuang Wu Aiyou Wen performed the data analyses and wrote the manuscript. Yahao Zhou, Qingkui Jiang helped perform the analysis with constructive discussions.
Data Availability
Not applicable
Ethics approval and consent to participate
The protocol for the present experiment was approved by the Animal Care Committee of the Anhui Science and Technology University (2021012).
Competing interests
The authors declare no competing interests.
| Item | Starter | Finisher |
|---|---|---|
| Ingredient, % | ||
| Corn | 55.2 | 57.6 |
| Soybean meal (48% CP) | 37.0 | 34.0 |
| Soybean oil | 3.0 | 4 |
| Dicalcium phosphate | 2.0 | 1.4 |
| Ground limestone | 0.8 | 0.9 |
| Salt | 0.4 | 0.4 |
| DL–Methionine | 0.3 | 0.3 |
| L-Lysine | 0.3 | 0.4 |
| Premix1 | 1 | 1 |
| Analyzed composition | ||
| Metabolisable energy2 [MJ/kg DM] | 12.40 | 12.76 |
| Dry matter [g/kg] | 912.1 | 903.4 |
| Crude protein [g/kg DM] | 220.3 | 207.8 |
| Contents of animo acid [ %] | ||
| Lysine | 1.53 | 1.55 |
| Methionine | 0.63 | 0.62 |
| Methionine + cyseine | 0.99 | 0.95 |
| Contents of mineral elements [mg/kg] | ||
| Calcium | 10,016 | 8,641 |
| Available phosphorus | 5,476 | 4,239 |
| Copper | 7.81 | 7.03 |
| 1Provided per kilogram of diet: 11,000 IU vitamin A; 1,800 IU vitamin D3; 32 mg vitamin E; 0.5 mg vitamin K3; 2.5 mg vitamin B1; 8 mg riboflavin; 4.5 mg vitamin B6; 26 mg vitamin B12; 65 mg niacin; 1.5 mg folic acid; 0.2 mg biotin; and 18 mg pantothenic acid; 60 mg Zn (as ZnSO4); 95 mg Mn (as MnO2); 65 mg Fe (as FeSO4∙7H2O); 0.7 mg I (as KI); and 0.2 mg Se (as Na2SeO3∙5H2O). | ||
| 2Calculated values, others were analyzed values based on dry matte samples. | ||
| Items | Additive Cu does | Actually Cu does (Starter) | Actually Cu does (Finisher) |
|---|---|---|---|
| Control | 0 | 7.81 | 7.03 |
| CS-50 | 50 | 57.74 | 57.12 |
| CS-100 | 100 | 107.32 | 107.57 |
| CC-50 | 50 | 58.22 | 57.12 |
| CC-50 | 100 | 108.25 | 107.69 |
| Items | Treatment | SEM | P-values | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Control | CS-50 | CS-100 | CC-50 | CC-100 | Treat | Sources | Level | Sources × Level | ||
| Day 1 to 21 | ||||||||||
| ADG, g/d | 36.40c | 36.67bc | 38.23ab | 38.15ab | 38.56a | 0.51 | 0.0313 | 0.0260 | 0.0900 | 0.3155 |
| ADFI, g/d | 49.72 | 49.33 | 50.51 | 49.83 | 51.32 | 0.90 | 0.6399 | 0.7164 | 0.1854 | 0.8801 |
| F/G | 1.37 | 1.34 | 1.32 | 1.31 | 1.33 | 0.02 | 0.4516 | 0.2740 | 0.9542 | 0.3109 |
| Day 22 to 42 | ||||||||||
| ADG, g/d | 85.47 | 86.73 | 87.25 | 89.34 | 89.45 | 1.40 | 0.3110 | 0.1029 | 0.8377 | 0.8940 |
| ADFI, g/d | 170.82 | 169.55 | 171.03 | 175.14 | 171.70 | 2.89 | 0.7774 | 0.5941 | 0.7601 | 0.4431 |
| F/G | 2.00 | 1.96 | 1.96 | 1.96 | 1.92 | 0.03 | 0.6237 | 0.4020 | 0.6043 | 0.4925 |
| Day 1 to 42 | ||||||||||
| ADG, g/d | 60.93b | 61.70ab | 62.74ab | 63.75a | 64.01a | 0.78 | 0.0812 | 0.0245 | 0.4540 | 0.6534 |
| ADFI, g/d | 110.27 | 109.44 | 110.77 | 112.48 | 111.51 | 1.62 | 0.7830 | 0.5314 | 0.9193 | 0.5193 |
| F/G | 1.81 | 1.77 | 1.77 | 1.77 | 1.74 | 0.02 | 0.4389 | 0.1989 | 0.5469 | 0.7819 |
| a−bMeans in a row without common superscripts differ (P < 0.05). | ||||||||||
| CS copper sulfate, CC cupric citrate, ADG average daily gain, ADFI average daily feed intake, F/G feed gain ratio. | ||||||||||
| Items | Treatment | SEM | P-values | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Control | CS-50 | CS-100 | CC-50 | CC-100 | Treat | Sources | Level | Sources × Level | ||
| DM, % | 73.27 | 73.84 | 75.28 | 74.66 | 75.74 | 0.95 | 0.4567 | 0.3371 | 0.2381 | 0.8642 |
| Energy, % | 75.34b | 78.22a | 79.01a | 79.20a | 79.91a | 0.86 | 0.0191 | 0.0042 | 0.4322 | 0.9627 |
| CP, % | 50.99b | 51.44b | 53.29ab | 52.61ab | 53.91a | 0.69 | 0.0554 | 0.0641 | 0.0459 | 0.7180 |
| Ca, % | 37.98 | 38.45 | 38.95 | 37.66 | 38.42 | 0.82 | 0.8744 | 0.7114 | 0.4890 | 0.8859 |
| P, % | 26.16 | 26.48 | 26.50 | 26.07 | 26.95 | 0.67 | 0.9252 | 0.9196 | 0.5456 | 0.5656 |
| a−bMeans in a row without common superscripts differ (P < 0.05). | ||||||||||
| CS copper sulfate, CC cupric citrate, DM dry matter, CP crude protein, Ca calcium, P phosphorus. | ||||||||||
| Items | Treatment | SEM | P-values | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Control | CS-50 | CS-100 | CC-50 | CC-100 | Treat | Sources | Level | Sources × Level | ||
| Ileum, log10 cfu/g | ||||||||||
| Lactobacillus spp. | 7.35a | 7.05ab | 6.43b | 6.93ab | 6.55b | 0.23 | 0.0892 | 0.1095 | 0.0552 | 0.6377 |
| Escherichia coli | 5.39a | 4.72b | 4.36bc | 4.56b | 3.98c | 0.16 | 0.0001 | 0.0001 | 0.0126 | 0.5267 |
| Cecum, log10 cfu/g | ||||||||||
| Lactobacillus spp. | 8.40a | 7.94a | 7.27b | 7.28b | 6.83b | 0.20 | 0.0002 | 0.0001 | 0.0163 | 0.6262 |
| Escherichia coli | 7.31a | 6.86a | 5.86b | 6.09b | 5.36c | 0.16 | 0.0001 | 0.0001 | 0.0001 | 0.4330 |
| a−cMeans in a row without common superscripts differ (P < 0.05). | ||||||||||
| CS copper sulfate, CC cupric citrate. | ||||||||||
| Items | Treatment | SEM | P-values | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Control | CS-50 | CS-100 | CC-50 | CC-100 | Treat | Sources | Level | Sources × Level | ||
| Glandular stomach | 2.96 | 2.92 | 2.92 | 2.99 | 2.92 | 0.04 | 0.7499 | 0.6996 | 0.4035 | 0.4973 |
| Muscular stomach | 3.78 | 3.75 | 3.73 | 3.82 | 3.78 | 0.05 | 0.8120 | 0.5244 | 0.6503 | 0.8545 |
| Duodenum | 5.86c | 6.02bc | 6.19ab | 6.09ab | 6.25a | 0.05 | 0.0008 | 0.0007 | 0.0112 | 0.9208 |
| Jejunum | 6.03b | 6.06b | 6.18ab | 6.13ab | 6.28a | 0.06 | 0.0589 | 0.0765 | 0.0393 | 0.8279 |
| Ileum | 6.00 | 6.10 | 5.92 | 6.09 | 5.94 | 0.09 | 0.5547 | 0.9837 | 0.0936 | 0.9205 |
| Cecum | 7.10 | 7.08 | 6.93 | 7.13 | 6.97 | 0.09 | 0.5108 | 0.7107 | 0.1147 | 0.9157 |
| a−cMeans in a row without common superscripts differ (P < 0.05). | ||||||||||
| CS copper sulfate, CC cupric citrate. | ||||||||||
| Items | Treatment | SEM | P-values | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Control | CS-50 | CS-100 | CC-50 | CC-100 | Treat | Sources | Level | Sources × Level | ||
| Ceruloplasmin (U/L) | 1.93b | 2.00ab | 2.07a | 2.05a | 2.09a | 0.03 | 0.0269 | 0.0142 | 0.1126 | 0.5794 |
| Cu/Zn-SOD (U/mL) | 90.75b | 95.98ab | 98.63ab | 103.29ab | 106.69a | 3.77 | 0.0899 | 0.0254 | 0.4708 | 0.9273 |
| a−bMeans in a row without common superscripts differ (P < 0.05). | ||||||||||
| CS copper sulfate, CC cupric citrate, Cu-Zn SOD Cu-Zn superoxide dismutase,. | ||||||||||