Dietary supplementation with nano-composite of copper and carbon on growth performance, immunity, and antioxidant ability of yellow-feathered broilers

Abstract

New feed additives as antibiotics substitutes are in urgent need in poultry production. Nano-composite of copper and carbon (NCCC), a novel copper donor with stronger antibacterial properties, is expected to promote broiler growth and diminish the negative effects of excess copper (Cu). Hence, the purpose of this study is to investigate the effects of NCCC on growth performance, immunity, and antioxidant ability of yellow-feathered broilers. A total of 240 1-d-old male yellow-feathered broilers were selected and randomly divided into four groups, with five replications per group and 12 birds per replication. The CON group was fed corn-soybean basal diets, while the N50, N100, and N200 groups were supplemented with 50, 100, and 200 mg/kg of NCCC in basal diets, respectively. The trial lasted for 63 d. The results demonstrated that only 200 mg/kg NCCC addition significantly increased the Cu content in serum and feces, and liver Cu content linearly increased with NCCC dosage increment (P < 0.05). Meanwhile, NCCC supplementation did not alter the growth performance, slaughter performance, immune organ indexes, and liver antioxidant ability of broilers (P > 0.05), but optimized the serum cytokine pattern by elevating the level of serum IL-10 (P < 0.05), and there were linear and quadratic increases in serum IL-4 with NCCC dosage increment (P < 0.05). On the whole, in spite of no impact on growth performance, 50 mg/kg NCCC was optimal to supplement in chicken diets due to the rise of serum IL-10 level and no extra environmental pollution and tissue residues.

This study found that dietary nano-composite of copper and carbon supplementation increased the serum level of anti-inflammatory factor IL-10 in yellow-feathered broilers, despite no impact on the growth performance of chickens.

Introduction

Copper (Cu) is an essential trace mineral for biochemical processes such as cellular respiration, tissue pigmentation, and hemoglobin formation, which were impaired when Cu was deficient (Kaya et al., 2006; Sharif et al., 2021). In livestock and poultry production, Cu was commonly added to diets at pharmacological levels, defined as levels above the nutritional requirement, for its growth-promoting effect, which is likely a result of its bactericidal property, and increases in lipase activity, growth hormone secretion, and expression of genes involved in postabsorptive metabolism of lipids (Espinosa and Stein, 2021; Sharif et al., 2021; Tsang et al., 2021). However, Cu in the bulk form (mostly inorganic Cu) would interfere with the absorption and utilization of other metal elements, and cause environmental pollution due to heavy metal residue (Scott et al., 2018). Moreover, excess Cu in pig diets led to greater acquired antibiotic resistance (Solioz et al., 2010). Therefore, the European Union, China, and other countries have reduced the authorized maximum concentration of Cu in animal feed (Lin et al., 2020).

Organic Cu has been investigated to replace inorganic Cu due to better bioavailability and digestibility (Zhao et al., 2010), but inconsistent results and cost of the organic Cu hinder its widespread use in poultry diets (Pineda et al., 2013). To our delight, the application of nanotechnology made the metal size adjustable to fundamentally change the physical and chemical properties of Cu. Reduced size and high surface area of Cu nanoparticles (Cu-NP) endow them with high bioavailability compared to conventional Cu suppliers, thus making low-dose dietary Cu possible in animal production. Aminullah et al. (2021) found that CuSO₄ (the commonly used inorganic form of Cu) can be replaced with 50% of Cu proteinate (organic Cu) or 25% of Cu-NP without negative impact on productivity, hatchability, progeny, and egg quality of Swarnadhara breeder hens. Some studies showed that Cu-NP were related to improving the antioxidant and immunologic functions of animals (Wang et al., 2011; Ognik et al., 2018). The serous activities of superoxide dismutase, catalase (CAT), and ceruloplasmin were reported to increase with increasing dosage of Cu-NP in broilers’ diet (0.5, 1, and 1.5 mg/kg per day) (Ognik et al., 2018). 100 mg/kg copper-loaded chitosan nanoparticle supplementation could elevate thymus, spleen, and bursa of Fabricius indexes and the populations of Lactobacillus and Bifidobacterium in cecal digesta of broilers (Wang et al., 2011). After supplementing 200 mg/kg nano-copper for 30 d in the diet of Wumeng semi-fine wool sheep, the levels of serous IL-2, IL-6, IL-1β were declined (Song et al., 2020).

Biochar has small particle and membrane pore size, high porosity, and specific surface area, and fits perfectly for constructing functional nanomaterials (Gulzar et al., 2017). Meanwhile, biochar is also used in animal husbandry to improve animal health, intestinal flora and increase the efficiency in milk production as well as reproduction (Kutlu et al., 2001; Kana et al., 2011).

Hence, in our experiment, a nano-composite of copper and carbon (NCCC), prepared by using biofiber as a template and copper ion as the copper source, was selected as a novel Cu donor. The Cu-Cu₂O-Cu cycle of NCCC in an aerobic and non-aerobic environment can disturb the physiological functions related to oxidation-reduction balance in microbes, leading to strong antimicrobial effect. Coupled with a high number of active catalytic sites per unit volume and potential promising benefits of redundant copper (Espinosa and Stein, 2021), low-dose NCCC supplementation was expected to exert a growth-promoting effect on chickens without the adverse impact of excess Cu.

Thus, the purpose of this study was to explore the effects of dietary NCCC supplementation on growth performance, immunity, and antioxidant function of yellow-feathered broilers, and to provide a scientific basis and theoretical reference for NCCC as a potential functional additive in the poultry industry.

Material and Methods

All animal experimental procedures used in this experiment have been approved by the Animal Protection and Use Committee of Fujian Agriculture and Forestry University.

Experimental materials

NCCC (12% copper and 88% carbon) was obtained from Myron Biotechnology Co., Ltd. (Zhangzhou, China). NCCC has a copper core (diameter: 20 to 50 nm) surrounded by a carbon shell (diameter: about 15 μm). The process comprises loading copper ions into fibers of biological origin and then carbonizing the fibers. 1-d-old male broilers were offered by WENS Foodstuff Group Co., Ltd. (Yunfu, China). Feedstuffs for broiler diets were purchased from Fujian Jinhualong Feed Co., Ltd. (Fuzhou, China).

Experimental design and bird management

A total of 240 healthy 1-d-old yellow-feathered male broilers (32.03 ± 1.35 g) were selected and randomly divided into four groups. Each group has five replicates, with 12 broilers per replicate. The control group (CON) was fed basal diets. The NCCC treatment groups N50, N100, and N200 were fed basal diets supplemented with 50, 100, and 200 mg/kg NCCC, respectively. The experimental period was 63 d, including three feeding stages: the Starter stage, from days 1 to 21; the Grower stage, from days 22 to 42; the Finisher stage, from days 43 to 63. The basal diets were formulated according to the China Chicken feeding standard (NY/T 33-2004), and the dietary composition and nutrient levels were presented in Table 1. This experiment was conducted at the Poultry Experimental Station of Fujian Agriculture and Forestry University. Broilers were reared in cage (1,562.5 cm²/bird) with 23 h of light, ad libitum feeding, and water intake. The temperature of the bird room was controlled at 35 °C for the first week and then lowered by 2 °C to 3 °C per wk until 22 °C. The humidity was kept between 50% and 70%.

Table 1.

Composition and nutrient levels of basal diets (as-fed basis, %)

Items	Starter phase (days 1 to 21)	Grower phase (days 22 to 42)	Finisher phase (days 43 to 63)
Ingredients 1
Corn	57.00	64.00	72.00
Soybean meal	32.00	26.40	20.00
Expanded soybean	6.60	6.00	4.50
Limestone powder	1.30	1.00	1.00
Dicalcium phosphate	1.60	1.20	1.10
DL-Met	0.20	0.10	0.10
NaCl	0.30	0.30	0.30
Premix2	1.00	1.00	1.00
Total	100.0	100.00	100.00
Nutrient levels
ME (MJ/kg)	11.95	12.36	12.89
Crude protein	21.00	18.90	15.98
Ca	0.80	0.77	0.60
Total P	0.55	0.54	0.50
Available P	0.31	0.33	0.30
Lys	1.10	0.96	0.75
Met + Cys	0.97	0.93	0.67

¹Feedstuffs for broiler diets were purchased from Fujian Jinhualong Feed Co., Ltd. (Fuzhou, China).

²Nutrient levels of premix in the Starter phase (per kg diet): VA 5 000 IU, VB₁ 1.3 mg, VB₂ 3.6 mg, VB₆ 2.5 mg, VB₁₂ 0.01 mg, VD₃ 1 000 IU, VE 10 IU, VK₃ 0.5 mg, biotin 0.15 mg, folic acid 0.55 mg, pantothenic acid 10 mg, nicotinic acid 35 mg, choline chloride 1 000 mg, Cu (as copper sulfate) 8 mg, Fe (as ferrous sulfate) 80 mg, Mn (as manganese sulfate) 80 mg, Zn (as zinc sulfate) 60 mg, I (as potassium iodide) 0.35 mg, and Se (as sodium selenite) 0.15 mg.

Nutrient levels of premix in the Grower phase (per kg diet): VA 5 000 IU, VB₁ 1.3 mg, VB₂ 3.6 mg, VB₆ 2.5 mg, VB₁₂ 0.01 mg, VD₃ 1 000 IU, VE 10 IU, VK₃ 0.5 mg, biotin 0.15 mg, folic acid 0.55 mg, pantothenic acid 10 mg, nicotinic acid 30 mg, choline chloride 750 mg, Cu (as copper sulfate) 8 mg, Fe (as ferrous sulfate) 80 mg, Mn (as manganese sulfate) 80 mg, Zn (as zinc sulfate) 60 mg, I (as potassium iodide) 0.35 mg, and Se (as sodium selenite) 0.15 mg.

Nutrient levels of premix in the Finisher phase (per kg diet): VA 5 000 IU, VB₁ 1.3 mg, VB₂ 3.0 mg, VB₆ 2.5 mg, VB₁₂ 0.01 mg, VD₃ 1 000 IU, VE 10 IU, VK₃ 0.5 mg, biotin 0.15 mg, folic acid 0.55 mg, pantothenic acid 10 mg, nicotinic acid 25 mg, choline chloride 500 mg, Cu (as copper sulfate) 8 mg, Fe (as ferrous sulfate) 80 mg, Mn (as manganese sulfate) 80 mg, Zn (as zinc sulfate) 60 mg, I (as potassium iodide) 0.35 mg, and Se (as sodium selenite) 0.15 mg.

Growth performance detection and sample collection

Birds were weighed on days 1 and 63, and feed intake was measured every day for calculation of average daily gain, average daily feed intake, and gain:feed (G:F) to assess growth performance. The death and cull were monitored daily and used to adjust feed intake and G:F. The feces samples were continuously collected from each cage for 1 to 3 d before slaughter, then weighed and mixed separately by the cage for copper content detection. Two broilers for each replicate (close to the average weight of each replicate) were selected for slaughter and sampling on day 63 (after fasting 12 h). Ten milliliters of blood were collected from the basilic vein, left at room temperature for 2 h, and then centrifuged at 1,450 × g for 10 min to collect serum samples, which were stored at −20 °C until analysis. The liver tissue in the middle part was isolated on an ice tray and put into 2-mL freezing tubes, which were first frozen in a liquid nitrogen tank and then stored at −80 °C for antioxidant parameters analysis.

Slaughter performance

At the end of the experiment (63 d of age), two birds with an average body weight from each cage (replicate) were selected, individually weighed, and sacrificed after fasting. The sacrificed birds were de-feathered, eviscerated, and manually dissected. The semi-eviscerated carcass, eviscerated carcass, breast muscle, and thigh muscle were weighed. The dressing percentage, semi-eviscerated slaughter ratio, eviscerated slaughter ratio, breast muscle rate, and thigh muscle rate were calculated as follows:

• Dressing percentage (%) = slaughter weight (g)/ live weight (g) × 100%;

• Semi-eviscerated slaughter ratio (%) = semi-eviscerated weight (g)/ live weight (g) × 100%;

• Eviscerated slaughter ratio (%) = eviscerated weight (g)/ live weight (g) × 100%;

• Breast muscle rate (%) = bilateral breast muscle weight (g)/ eviscerated weight (g) × 100%;

• Thigh muscle rate (%) = bilateral thigh muscle weight (g)/ eviscerated weight (g) × 100%.

Immune organ index

The immune organs of sacrificed birds, such as the spleen, thymus, and bursa of Fabricius, were collected and weighed. The calculation formula of the immune organ index was as follows: Immune organ indexes (g/kg) = immune organ weight (g)/ live weight (kg).

Determination of copper content

The Cu contents in diets, feces, liver, and serum were determined by atomic absorption spectrophotometry (AA6701F; Shimadzu, Kyoto, Japan) with wet digestion (Jiao et al., 2018). One mL of serum was added to a mixture of 4 mL nitric acid and 1 mL perchloric acid for wet digestion, then dissolved with deionized water and analyzed for Cu content using atomic absorption spectrophotometry. Two grams of liver tissue and 1 g of diet were pulverized, digested with mixed acid, and filtered through cheesecloth to separate solid and liquid fractions. The liquid fraction was centrifuged at 15,000 × g for 10 min at 25 °C, and the supernatant was then dissolved with deionized water for Cu determination using atomic absorption spectrophotometry. One gram of Feces was dried in an oven, then pulverized, digested with mixed acid, and dissolved with deionized water for Cu detection.

Serum immunologic and liver antioxidant parameters

The liver was isolated on an ice tray, fully homogenized with cold PBS (pH = 7.4), with a weight-to-volume ratio of 1:80, and then centrifuged at 1000 × g for 20 min at 4 °C. Finally, the supernatant was collected for measuring the concentration of total protein using a BCA protein quantitative kit (GK10009; GlpBio, Montclair, USA). The contents of liver CuZn superoxide dismutase (CuZn-SOD), glutathione peroxidase (GSH-Px), CAT and malondialdehyde (MDA), and the levels of serum interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-10 (IL-10), were measured by Enzyme-Linked Immunosorbent Assay with commercial kits (ML002350, ML002333, ML002342, ML002362, ML062692, ML062690, ML042736, ML042739, and ML059830, respectively; Shanghai Enzyme-Linked Biotechnology Co., Ltd, Shanghai, China), and the absorbance was detected by a Microplate Reader (iMark; Bio-Rad, California, USA).

Data analysis

The data were collated and counted by Excel 2019 and analyzed using ANOVA in Statistical Product and Service Solutions (SPSS 25.0). Differences between the groups were tested by Duncan’s tests. In all cases, one-way analysis, linear and quadratic regression analysis of variance with the diet as the fixed factor were used. All differences were considered significant at P < 0.05. Testing of significant differences was carried out according to the following mathematical-statistical models:

$${\displaystyle \begin{array}{l}{\displaystyle Y_{ij}=\mu +{\alpha }_i+e_{ij},}\end{array}}$$

Where Y_ij is the value of the trait; μ is the overall mean; α_i is the diet effect and e_ij is the random residual error.

$${\displaystyle \begin{array}{l}{\displaystyle Y_i=\alpha +\beta X_i+e_{ij},}\end{array}}$$

Where Y_i is the dependent variable; α is the intercept of the y-axis; β is the regression coefficient of Y on X; X_i is the independent variable and e_ij is the random residual error.

$${\displaystyle \begin{array}{l}{\displaystyle Y=b_0+b_{1}X+b_2{X}^2+e,}\end{array}}$$

Where Y is the dependent variable; b₀ is the constant term; b₁ and b₂ are the partial regression coefficients; X is the independent variable and e is the random residual error.

Results and Discussion

Cu is a vital trace element for animal growth. High-dose Cu was often supplemented in the diets of piglets and chickens because of its bactericidal and growth-promoting effects (Ma and Guo, 2008; Scott et al., 2018). However, heavy metal pollution and antibiotic resistance emerged and ­deteriorated due to long-term excess CuSO₄ intake (Sharif et al., 2021). Previous studies have shown that chelated copper and nano-copper could reduce Cu excretion and elevate Cu utilization efficiency (Veum et al., 2004; Sawosz et al., 2018). Hence, in our study, NCCC, as a high-biocompatibility nano-copper supplier, was chosen to explore its safety and if there is a growth-stimulation impact on chickens with its low-dose addition. Simultaneously, slaughter performance, antioxidant, and immunologic function are also evaluated.

Cu residues

Firstly, we detected the Cu contents in diets at different growth stages. As shown in Table 2, the actual Cu contents in basal diets were about 11~14 mg/kg, mostly provided by CuSO₄, and the Cu increments in N50, N100, and N200 groups were consistent with the theoretical values. Then, the Cu concentrations in the liver, serum, and feces were measured and the results were presented in Table 3. We discovered that only 200 mg/kg NCCC addition significantly increased the Cu content in serum and feces, while liver Cu content was linearly decreased with NCCC dosage increment (P < 0.05). The result is consistent with a previous study, which has shown that only when diet Cu levels are > 200 mg of Cu/kg of diet did liver or plasma Cu levels change (Miles et al. 1998). Therefore, no more than 100 mg/kg NCCC addition would not cause Cu residues in liver, serum, and environment.

Table 2.

The measured Cu content in yellow-feathered broiler’s diets at different phases (as-fed basis)¹

Diets	Groups2
	CON	N50	N100	N200
Starter phase (days 1 to 21), mg/kg	11.70 ± 0.68	19.78 ± 0.35	24.75 ± 1.65	33.87 ± 2.30
Grower phase (days 22 to 42), mg/kg	13.29 ± 0.43	22.38 ± 1.24	26.23 ± 1.62	38.60 ± 0.54
Finisher phase (days 43 to 63), mg/kg	11.30 ± 1.05	19.28 ± 1.73	24.47 ± 1.82	38.24 ± 1.84

¹The results are presented as mean ± SD (n = 2).

²Group CON: basal diet; Group N50: basal diet added with 50 mg/kg nano-composite of copper and carbon (NCCC); Group N100: basal diet added with 100 mg/kg NCCC; Group N200: basal diet added with 200 mg/kg NCCC.

Table 3.

Effects of nano-composite of copper and carbon supplementation on Cu residues in yellow-feathered broilers¹

Items	Groups2				SEM	P-value
	CON	N50	N100	N200		Diet	Linear	Quadratic
Liver, mg/kg DM	9.47	9.33	8.47	7.73	0.297	0.138	0.020	0.062
Serum, mg/L	0.14b	0.52b	0.31b	5.10a	0.553	<0.001	<0.001	<0.001
Feces, mg/kg DM	156.33b	273.47b	261.01b	451.58a	29.43	<0.001	<0.001	<0.001

¹ n = 5.

²Group CON: basal diet; Group N50: basal diet added with 50 mg/kg nano-composite of copper and carbon (NCCC); Group N100: basal diet added with 100 mg/kg NCCC; Group N200: basal diet added with 200 mg/kg NCCC.

^a-

^bMeans in the same row without the same superscript differ significantly (P < 0.05).

Growth performance

Next, we measured the growth performance of broilers in each group, and found that NCCC supplementation didn’t affect the average daily gain, average daily feed intake, and G:F of broilers (P > 0.05) (Table 4). The result is in agreement with a study conducted by Servestani et al. (Scott et al., 2016). However, several reports revealed that Cu-NP could improve the growth performance of broilers and pigs (Gonzales-Eguia et al., 2009; Wang et al., 2012; Sharif et al., 2021). It is well established that Cu-NP possesses antibacterial properties, and three mechanisms of action were hypothesized, namely changing the permeability of the bacterial membrane, generating reactive oxygen species to cause subsequent oxidative damage and depleting intracellular ATP, and disrupting DNA replication after uptake into cells (Amro et al., 2000; Sondi and Salopek-Sondi, 2004; Fang et al., 2007). Besides, the growth-promoting role of Cu-NP probably benefits from its relation to the growth hormone axis and hypothalamic appetite-regulating genes (Scott et al., 2018; Sharif et al., 2021). Heretofore, other efforts to reduce the amount of dietary Cu were made by researchers, like using montmorillonite and chitosan nanoparticles as Cu carriers (Ma and Guo, 2008; Wang et al., 2012, 2022). Montmorillonite is an aluminum silicate mineral clay with a huge surface area, strong absorptive capacity, and ion exchange capabilities, and Cu²⁺-loaded montmorillonite supplementation could elevate body weight and feed efficiency of broilers, along with the increment of the maltase, aminopeptidase N and alkaline phosphatase activities in small intestinal mucosa and intestinal morphology improvement (Ma and Guo, 2008). Similar growth-promoting effects are also present in studies carried out in weaned piglets (Jiao et al., 2018; Li et al., 2021). Therefore, the reason why there was no effect of NCCC on growth performance in our study might be due to the unique size, structure, and concentration of NCCC, which results in discrepant biological activity compared to other products.

Table 4.

Effects of NCCC supplementation on growth performance of yellow-feathered broilers¹

Items2	Groups3				SEM	P-value
	CON	N50	N100	N200		Diet	Linear	Quadratic
ADG, g	27.41	28.75	27.78	27.65	0.215	0.122	0.898	0.243
ADFI, g	62.70	65.34	63.85	63.14	0.534	0.345	0.861	0.322
G:F	0.444	0.440	0.435	0.438	0.002	0.376	0.154	0.233

¹Values are means of five replicates per treatment with 12 broilers each.

²ADG, average daily gain; ADFI, average daily feed intake; G:F, gain:feed.

³Group CON: basal diet; Group N50: basal diet added with 50 mg/kg nano-composite of copper and carbon (NCCC); Group N100: basal diet added with 100 mg/kg NCCC; Group N200: basal diet added with 200 mg/kg NCCC.

Slaughter performance

Slaughter performance is a comprehensive index to measure the growth performance and economic value of animals. Cu acts on collagen and elastin synthesis as it is a cofactor of lysyl oxidase, an enzyme involved in collagen cross-linking (Rucker et al., 1998), thus contributing to muscle growth and development (Sharif et al., 2021). Several previous studies demonstrated that in ovo injection of 50 ppm Cu-NP to chicken eggs could increase the percentages of breast and thigh muscles (Mroczek-Sosnowska et al., 2015), and the greatest breast muscle percentage was also discovered with an injection of 12 μL Cu-NP/egg (Joshua et al., 2016). However, as shown in Table 5, adding different levels of NCCC to the diet had no impact on the slaughter performance of yellow-feathered broilers in our experiment (P > 0.05). Similar to our result, Yen et al. (Yen and Pond, 1993) reported that 250 ppm Cu (in CuSO₄ form) in the diet also did not affect the dressing percentage of pigs. We speculate that the different results shown here might be caused by the discrepant dosage, adding form, and physicochemical characteristics of Cu suppliers.

Table 5.

Effects of NCCC supplementation on slaughter performance of yellow-feathered broilers¹

Items	Groups2				SEM	P-value
	CON	N50	N100	N200		Diet	Linear	Quadratic
Dressing percentage, %	90.96	91.15	90.35	90.42	0.231	0.556	0.251	0.521
Semi-eviscerated slaughter ratio, %	86.07	86.11	85.44	85.30	0.261	0.605	0.207	0.451
Eviscerated slaughter ratio, %	70.52	71.58	70.29	70.75	0.339	0.596	0.861	0.916
Thigh muscle rate, %	19.81	20.45	19.19	19.98	0.280	0.492	0.930	0.842
Breast muscle rate, %	13.27	13.88	13.89	13.77	0.271	0.840	0.654	0.569

¹ n = 5.

²Group CON: basal diet; Group N50: basal diet added with 50 mg/kg nano-composite of copper and carbon (NCCC); Group N100: basal diet added with 100 mg/kg NCCC; Group N200: basal diet added with 200 mg/kg NCCC.

Immune organ index and serum cytokine pattern

The spleen, thymus, and bursa of Fabricius are the main immune organs of broilers, which are central zones for immunologic response, and immune organ indexes were evaluated to reflect the immune organ development and general immune status of broilers (Alagawany et al., 2019). The administration of 10 mg/L Cu-NP to the drinking water of broilers was reported to elevate lymphoid organ indexes compared to the control and CuSO₄ (10 mg/L) treated group. (Ognik et al., 2018) However, Zhou et al. (Zhou et al., 2021) discovered that the high-dose copper-amino acid complex supplementation had no effect on the spleen index of laying hens. Similarly, dietary NCCC supplementation did not affect the immune organ indexes of broilers in our study (P > 0.05) (Table 6).

Table 6.

Effects of NCCC supplementation on immune organ indexes of yellow-feathered broilers¹

Items	Groups2				SEM	P-value
	CON	N50	N100	N200		Diet	Linear	Quadratic
Spleen index, g/kg	1.27	1.30	1.46	1.51	0.057	0.357	0.078	0.214
Thymus index, g/kg	2.54	2.21	2.70	3.01	0.166	0.400	0.198	0.281
Bursa of Fabricius index, g/kg	1.58	1.98	2.01	2.15	0.099	0.188	0.420	0.103

¹ n = 5.

²Group CON: basal diet; Group N50: basal diet added with 50 mg/kg nano-composite of copper and carbon (NCCC); Group N100: basal diet added with 100 mg/kg NCCC; Group N200: basal diet added with 200 mg/kg NCCC.

The immune system is a network of biological processes that protect broilers from pathogen invasion. Hence, we further evaluated the serum concentrations of several representative cytokines involved in immune responses. Our results revealed that 200 mg/kg NCCC treatment notably increased the content of serum IL-10, and there were ­linear and quadratic increases in serum IL-4 with the increase of NCCC dosage (P < 0.05), despite no changes in the contents of IFN-γ, TNF-α, and IL-2 (Table 7). IL-10 is a crucial anti-inflammatory cytokine and is primarily produced by monocytes. IL-10 can down-regulate the expression of Th1 cytokines such as IFN-γ, IL-2, and TNFα, and block the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, which is a major signal pathway that regulates genes responsible for both the innate and adaptive immune responses (Ferruzza et al., 2002; Ouyang and O’Garra, 2019). IL-4 is a cytokine that induces the differentiation of naive helper T cells (Th0 cells) to Th2 cells and possesses vast biological roles, like stimulating the proliferation of activated B cells and T cells, and the differentiation of B cells into plasma cells (Gandhi et al., 2016). Accordingly, 200 mg/kg NCCC supplementation in our experiment optimized the cytokine pattern by affecting IL10 and IL4 secretion. In accordance with our result, Cu-loaded montmorillonite supplementation was reported to down-regulate the mRNA expression of interleukin 1β, interleukin 6, and TNF-α, and upregulate the mRNA expression of IL-4 and IL-10 in jejunum and increase the levels of serum ­immunoglobulins of broilers (Wang et al., 2022). A total of 100 mg/kg Cu-loaded chitosan nanoparticles also could elevate the levels of serum immunoglobulin A (IgA), IgG, and IgM (Wang et al., 2011). Up to now, although several researchers claimed some adverse effects of Cu-NP on immune function in mice, rats, and fish (Chen et al., 2006; Doudi and Setorki, 2014; Wang et al., 2015), no negative impacts have been found in chickens. The immunoregulatory role of NCCC may be partly attributed to the fact that Cu can negatively regulate a potassium channel KCa3.1, which plays a role in the activation of T cells, B cells, and mast cells. More specifically, Cu can be coordinated onto histidine residues to block the phosphorylation modification on the histidine, thus preventing potassium channel KCa3.1 activation, and subsequently reducing cytokine production from immune cells (Tsang et al., 2021). Meanwhile, Cu has been found to regulate an E3 ubiquitin ligase, X-linked inhibitor of apoptosis protein, which is implicated in the NF-κB pathway. Additionally, the vital role of Cu in redox system also indirectly affects the immunologic function of broilers. The function, performance, and survival of immune cells are strongly regulated by redox mechanisms, and most immunologic transcription factors, like hypoxia-inducible factor-1α, NF-κB, and signal transducer and activator of transcription six, are redox targets too (Mullen et al., 2020; Morris et al., 2022). Hence, we then investigated the alteration of antioxidant function after NCCC supplementation.

Table 7.

Effects of NCCC supplementation on serum immune factor of yellow-feathered broilers¹

Items2	Groups3				SEM	P-value
	CON	N50	N100	N200		Diet	Linear	Quadratic
IFN-γ, pg/mL	55.94	56.82	57.02	56.89	0.248	0.396	0.158	0.217
TNF-α, pg/mL	50.36	51.50	50.73	51.56	0.318	0.513	0.337	0.637
IL-2, pg/mL	246.58	246.32	247.98	266.23	4.412	0.326	0.128	0.181
IL-4, pg/mL	116.79	117.91	122.50	133.01	2.484	0.069	0.012	0.026
IL-10, pg/mL	46.64c	59.69b	57.23b	65.52a	1.728	<0.001	<0.001	<0.001

¹ n = 5.

²INF-γ, interferon-γ; TNF-α, tumor necrosis factor-α; IL-2, interleukin-2; IL-4, interleukin-4; IL-10, interleukin-10.

³Group CON: basal diet; Group N50: basal diet added with 50 mg/kg nano-composite of copper and carbon (NCCC); Group N100: basal diet added with 100 mg/kg NCCC; Group N200: basal diet added with 200 mg/kg NCCC.

^a-cMeans in the same row without the same superscript differ significantly (P < 0.05).

Antioxidant function

Cu has diverse roles in biological electron transport and oxygen transportation, processes that exploit the easy interconversion of Cu(I) and Cu(II) (Vest et al., 2013). The antioxidant function of glutathione and CuZn-SOD is Cu-dependent (Tsang et al., 2021), and Cu deficiency would weaken the activity of CAT (Uriu-Adams and Keen, 2005). So far, so many studies demonstrated better antioxidant capacity in chickens and pigs after Cu-NP, CuO-NP, or Cu-loaded montmorillonite supplementation (Zhang et al., 2017; Kozłowski et al., 2018; Wang et al., 2022). Other ­mineral ­nanoparticles, like nano-selenium or nano-silver, were also reported to enhance the expression of antioxidase genes in muscles of broilers (Saleh and El-Magd, 2018; Saleh and Ebeid, 2019). However, no obvious alternation was found in the contents of liver CuZn-SOD, CAT, GSH-Px, and MDA after NCCC supplementation in our study (P > 0.05, Table 8). It is reasonable to assume that the advantage in antioxidant ability might be offset by potential toxicities, like oxidative stress and tissue damage. Cu-NP were reported to possibly cause ion channels blockage, deactivate enzymes, and induce DNA damage, further causing damage to tissues by generating oxidative stress, due to the physiochemical properties such as small size and high surface area, solubility, and reactivity (Wapnir, 1998; Jia et al., 2009). Nevertheless, according to our experimental results, despite no impact on growth performance and liver antioxidant function, dietary NCCC could act as a promising immunomodulator for the better health status of broilers.

Table 8.

Effects of NCCC supplementation on liver antioxidant enzyme content in 63-d-old yellow-feathered broiler¹

Items2	Groups3				SEM	P-value
	CON	N50	N100	N200		Diet	Linear	Quadratic
CuZn-SOD, ng/mg prot	85.52	83.40	87.81	87.52	2.076	0.886	0.588	0.848
GSH-Px, ng/mg prot	193.09	194.59	198.88	201.53	2.111	0.486	0.112	0.291
CAT, pg/mg prot	877.71	870.60	907.64	886.29	8.270	0.448	0.411	0.660
MDA, nmol/mg prot	12.36	11.99	12.77	12.31	0.136	0.253	0.622	0.877

¹ n = 5.

²CuZn-SOD, copper-zinc superoxide dismutase; GSH-Px, glutathione peroxidase; CAT, catalase; MDA, malondialdehyde.

³Group CON: basal diet; Group N50: basal diet added with 50 mg/kg nano-composite of copper and carbon (NCCC); Group N100: basal diet added with 100 mg/kg NCCC; Group N200: basal diet added with 200 mg/kg NCCC.

Conclusion

NCCC supplementation did not affect the growth performance, slaughter performance, immune organ indexes, and liver antioxidant ability, and caused no extra environmental pollution and tissue residues, but optimized the serum cytokine pattern of yellow-feathered broiler via the rise of IL-10 level.

Glossary

Abbreviations

CAT

catalase

CON

control group

Cu

copper

Cu-NP

Cu nanoparticles

CuZn

CuZn superoxide dismutase

G:F

gain:feed

GSH-Px

glutathione peroxidase

Ig

immunoglobulin

IL-2

interleukin-2

IL-4

interleukin-4

IL-10

interleukin-10

INF-γ

interferon-γ

MDA

malondialdehyde

NCCC

nano-composite of copper and carbon

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

TNF-α

tumor necrosis factor-α

Conflict of Interest Statement

In this study, the authors asserted that they had no conflict of interest.