Skip to main content
NCBI home page
Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2020 Jun 28;58(3):834–843. doi: 10.1007/s13197-020-04597-x

A comparative study on the antioxidant status, meat quality, and mineral deposition in broiler chicken fed dietary nano zinc viz-a-viz inorganic zinc

Sagar Dukare 1, Nasir Akbar Mir 1,✉, Asit Baran Mandal 1, Kapil Dev 1, Jubeda Begum 2, J J Rokade 1, Avishek Biswas 1, Praveen K Tyagi 1, Pramod K Tyagi 1, S K Bhanja 1
PMCID: PMC7884527  PMID: 33678866

Abstract

Use of nano minerals in farm animal nutrition offers considerable advantages over inorganic or organic mineral sources. But, the conventional chemical synthesis of nano minerals suffers from disadvantage of possible environmental accumulation and pollution due to the non-biodegradable materials and chemicals. This study investigated the effects of green nano-zinc (GNZ) and market nano-zinc (MNZ) with respect to the inorganic zinc (IZ) on meat quality, antioxidant status, mineral deposition, and bone development in broiler chicken. Following a 3 × 3 factorial design, nine dietary treatments were formulated by employing three levels (40, 60, and 80 ppm) and three sources (inorganic, green nano, and market nano) of zinc viz. IZ-40, GNZ-40, MNZ-40, IZ-60, GNZ-60, MNZ-60, IZ-80, GNZ-80, MNZ-80. Six replicates of broiler chicken were assigned to each treatment with eight birds in each. The birds fed 80 ppm Zinc of either GNZ or MNZ source resulted in significantly higher serum SOD, glutathione peroxidase, catalase, zinc, calcium, and phosphorus levels; increased bone dimensions, weight, total ash, phosphorus, and zinc content along with higher liver and muscle zinc concentration. The meat of chicken fed 80 ppm zinc of MNZ source followed by GNZ source has shown significantly better antioxidant (DPPH and ABTS values) status and lower lipid peroxidation (free fatty acid and TBARS values). The 80 ppm zinc of either MNZ or GNZ source resulted in significantly lower fat and cholesterol content of chicken meat compared to lower Zn levels and IZ source. This study indicated that 80 ppm dietary zinc of either MNZ or GNZ source improved the antioxidant status, and reduced the meat cholesterol, fat content, and lipid peroxidation of chicken meat along with increased bone dimensions and mineralization.

Keywords: Nano zinc, Antioxidant status, Lipid peroxidation, Mineral deposition, Bone growth

Introduction

Zinc (Zn) is one of the essential trace minerals required for proper growth and functioning of physiological processes in the body system. Zn is required for the activity of over 300 enzymes involved in metabolic functions in the body of animals (Prasad and Kucuk 2002). Zn enzymes are associated with the synthesis and/or breaking down of carbohydrates, lipids, proteins, and nucleic acids, and encompass all known classes of enzymes (Classen et al. 2011). Zn is the structural component of wide variety of proteins and dependent enzymes like superoxide dismutase (SOD) which act as an essential component of antioxidant defense system (Bao and Choct 2009). The negative effects of lipid oxidation in meat can be alleviated by utilization of antioxidants (Galanakis 2018) such as Zn. There are reports of decreased serum malondialdehyde, an indicator of lipid peroxidation, with dietary Zn picolinate supplementation in heat-stressed quail (Sahin et al. 2005). Further, dietary Zn affects bone growth and development in broiler chicken which may be due to its direct impact on protein synthesis via hormonal growth mediators which positively affects the bone formation (Seo et al. 2010). For example, the anabolic effect of insulin-like growth factor 1 on osteoblasts is enhanced by Zn supplementation in broiler chicken (Wang et al. 2002). However, the use of nano particles as a source of minerals in animals including poultry is a new promising application of nanotechnology. The nano particles of mineral elements have higher bioavailability because of their greater surface area, higher surface activity, high catalytic efficiency, and stronger adsorbing ability (Zhang et al. 2001). Supplementation of nano-Zn to broiler chicken exhibited better bioavailability and less toxicity when compared with other Zn sources (Sahoo et al. 2014). But, conventional chemical synthesis of nano minerals suffers from the disadvantage of possible environmental accumulation and pollution due to the non-biodegradable materials and chemicals (Schmidt 2009). Thus, objective of the present study was to investigate the biological effects of green synthesised nano Zn (GNZ) viz-a-viz market nano Zn (MNZ) and inorganic Zn (IZ) in terms of meat quality, antioxidant status, mineral profile, and bone morphology and mineralization of broiler chicken.

Materials and methods

All the experimental procedures carried in this study were approved by the Institutional Animal Ethics Committee of Indian Veterinary Research Institute, Izatnagar.

Experimental diets, design, and nano-Zn synthesis

Basal diet of broiler chicken was formulated as broiler pre-starter, starter, and finisher to meet the nutrient requirement of birds at different ages of rearing (Table 1). Following a 3 ×  3 factorial design, nine dietary treatments were formulated by employing three levels (40, 60, and 80 ppm) and three sources (inorganic, green nano, and market nafno) of zinc oxide viz. IZ-40, GNZ-40, MNZ-40, IZ-60, GNZ-60, MNZ-60, IZ-80, GNZ-80, and MNZ-80. The MNZ and IZ used in this study were procured from Sisco Research Laboratories Pvt. Ltd. (SRL) Mumbai, India. The biological synthesis of zinc oxide nanoparticles (ZnO–NPs) was done by using Catharanthus roseus plant. The synthesis of ZnO-NPs was confirmed by UV-visible spectroscopy. The particle size of synthesized ZnO-NPs, determined by Transmission electron microscopy (TEM) (JEOL JEM-1400), ranged between 12 and 53 nm and were largely spherical and rod shaped. The concentration of Zn as determined by Atomic absorption spectrophotometry (AAS, Varian Spectra AA 220 Model) in different samples of the product ranged from 42.84 to 45.75% and recovery of Zn ranged from 27.83 to 30.54%.

Table 1.

Ingredients and nutrient composition of broiler chicken basal ration

Ingredients (%) Starter (0–14 d) Grower (14–24 d) Finisher (24–42 d)
Maize 51.94 55.11 60.82
Soybean 41.1 37.00 31.00
Guar korma 3.00 4.00 4.00
Oil 0.50 0.60 1.00
Limestone 0.90 0.90 0.90
Di-Calcium phosphate 1.70 1.60 1.50
Salt 0.30 0.30 0.30
DL-Methionine 0.20 0.13 0.12
Trace mineral premixa 0.10 0.10 0.10
Vitamin premixb 0.15 0.15 0.15
Vitamin B complexc 0.02 0.02 0.02
Choline chloride 0.05 0.05 0.05
Toxin binder 0.05 0.05 0.05
Nutrient composition (Calculated based on the analyzed values of ingredients)
 Crude protein 22.50 21.50 19.53
 ME (kcal/kg diet) 2856.2 2894.4 2973.7
 Lysine 1.36 1.28 1.13
 Methionine 0.60 0.52 0.48
 Threonine 0.97 0.91 0.81
 Calcium 1.02 0.98 0.94
 Available P 0.45 0.43 0.40
Open in a new tab

aTrace mineral mixture (100 g): FeSO4·7H2O—8 g, MnSO4·H2O—10 g, CuSO4.5H2O—1 g, KI—30 g, Zinc (variable)

bVitamin premix (1 g): vitamin A—82.5 IU, vitamin B2—50 mg, vitamin D3—12,000 unit, Vitamin K—10 mg

cVitamin B complex (1 g): Vitamin B1—8 mg, vitamin B6—16 mg, vitamin B12—80 mcg, niacin—120 mg, calcium panthotheonate—80 mg, Vitamin E 50%—160 mg, L-lysine—10 mg and DL-methionine—10 mg

ME metabolizable energy

Experimental birds and husbandry

A total of 432 broiler chicken (CARIBRO Vishal) of same hatch with uniform body weight were used in this experiment. The birds were randomly and equally distributed among nine dietary treatments having six replicates per treatment with eight birds per replicate (48 birds/treatment). The birds were housed in specially designed battery brooder cages with provision of ad libitum feed and water during the feeding trial of 42 days. Each battery of the cage housed eight birds providing a space of 1.0 ft2 per bird. The birds were provided 24 h light on day one followed by a decrease of 1 h per day till it reached 18 h light period which was continued till 42nd day.

Sample collection

At the end of experiment 12 birds from each dietary treatment (2 bird/replicate) were randomly selected and slaughtered after 12 h of fasting with ad libitum drinking water for the collection of meat samples. The meat samples of both thigh and breast were collected for evaluation of fat and cholesterol content, antioxidant status, lipid oxidation status, and mineral profile of broiler chicken meat. The blood samples from 12 birds per treatment were collected at 28 days and 42 days of age in sterile tubes without anticoagulant to harvest serum for the estimation of serum antioxidant enzymes, Zn, calcium, and phosphorus concentration. At the time of slaughter left tibia bones of all birds were collected for the study of bone morphology and mineralization.

Antioxidant and lipid oxidation status of meat

The antioxidant status of broiler chicken meat was analyzed by ABTS (2, 2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) and DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. The spectrophotometric (Perkin Elmer, Model: Lambda EZ 201) analysis of ABTS and DPPH radical scavenging activity of fresh meat was done by the methods of Shirwaikar et al (2006) and Kato et al. (1988), respectively. The lipid oxidation status was assessed by estimation of thiobarbituric acid reactive substances (TBARS) value (Witte et al. 1970) and free fatty acid value (Koniecko 1979) of meat. The TBARS value was calculated as mg malondialdehyde (MDA)/Kg of sample by multiplying O.D value with K-factor of 5.2. The fat percentage of breast and thigh meat samples were estimated by using Soxhlet extraction apparatus (AOAC 1995) and the cholesterol estimation was done by extraction of meat samples in chloroform–methanol mixture (2:1) (Wybenga et al. 1970).

Serum enzyme estimation

The body antioxidant defence system consists of mainly superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px). The estimation of these enzymes was done by using Cayman diagnostic kits and all samples were measured in duplicate (Wheeler et al. 1990).

Tissue and serum mineral profile

The serum, liver, and breast Zn concentrations were estimated by using atomic absorption spectrophotometer (AAS, Varian Spectra AA 220 Model). The serum calcium and phosphorus were estimated by using span diagnostic kits following manufacturer’s instructions. The calculations were done as following:

Calcium (mg/dl) = (Absorbance of test/Absorbance of standard) × 10.

Phosphorus (mg/dl) = (Absorbance of test/Absorbance of standard) × 5.

Bone morphology and mineralization

The tibia bones collected were cleared from all adhered soft tissues and weighed. The bone length, proximal width, mid shaft width, and distal width were measured with the help of Vernier callipers. In order to study the bone mineralization, the tibia bones were defatted for 18hrs by extraction with petroleum spirit using the Soxhlet apparatus and then oven dried overnight at 100 °C prior to ashing at 650 °C for 6 h in a muffle furnace. The bone calcium, phosphorus, total ash, and zinc were estimated as described by Mir et al. (2014).

Statistical analysis

For the data analysis each sampled bird was taken as an experimental unit. All the data were tested for normality and homogeneity of variances with the Shapiro–Wilk test and Levene’s test, respectively, prior to the analyses. The data were analysed by two way ANOVA for a completely randomized design, using the general linear model procedure (IBM SPSS Software-20). Tukey post-hoc analysis was done to test the significant mean differences between the groups with significance level defined at P < 0.05.

Results and discussion

Antioxidant and lipid oxidation status of meat

The results of antioxidant and lipid oxidation status of broiler chicken meat are given in Table 2. The 80 ppm Zn level resulted in significantly higher DPPH values in thigh (P = 0.001) and breast (P = 0.007) meat compared to 40 ppm level, whereas, 60 ppm level was statistically similar to both 80 and 40 ppm Zn levels. Among the Zn sources, MNZ resulted in significantly higher DDPH values in thigh (P = 0.003) and breast (P = 0.002) meat followed by statistically different GNZ compared to IZ. The interaction effect of Zn levels and sources have revealed significantly (P = 0.010) higher DDPH values of thigh meat from birds fed MNZ-80 or MNZ-60 followed by GNZ-80 compared to IZ-40, whereas, other combinations yielded intermediate DDPH values. Similarly, the ABTS values of thigh meat were significantly lower (P = 0.009) at 40 ppm Zn level compared to 60 and 80 ppm levels which did not differ significantly from each other. Among the Zn sources, the MNZ resulted in significantly higher ABTS values in thigh (P < 0.000) as well as breast (P = 0.007) meat followed by statistically different GNZ compared to IZ. The breast meat of birds fed MNZ-80, MNZ-60, and MNZ-40 has shown significantly (P = 0.010) higher ABTS values followed by statistically different GNZ-80, GNZ-60, and GNZ-40 compared to lower values in birds fed IZ-40, IZ-60, and IZ-80.

Table 2.

Effect of different levels and sources of zinc on antioxidant and lipid oxidation status of broiler chicken meat

Zn source Zn (ppm) DPPH (% inhibition) ABTS (% inhibition) Free fatty acid (%) TBARS (mg MDA/kg)
Thigh Breast Thigh Breast Thigh Breast Breast Thigh Liver
IZ 40 20.2a 17.2 89.2 73.2a 0.109 0.088 0.228 0.248 0.217
GNZ 23.5 cd 18.4 90.9 78.0b 0.102 0.081 0.157 0.164 0.148
MNZ 24.5de 19.2 91.3 83.6c 0.104 0.084 0.160 0.169 0.150
IZ 60 21.8b 17.6 89.4 73.6a 0.098 0.083 0.223 0.243 0.215
GNZ 23.9d 18.6 91.2 78.4b 0.092 0.077 0.154 0.157 0.145
MNZ 25.1ef 19.7 91.4 84.4c 0.093 0.075 0.153 0.159 0.144
IZ 80 22.7bc 17.8 89.7 73.9a 0.088 0.077 0.219 0.239 0.211
GNZ 24.3de 18.9 90.6 78.6b 0.080 0.075 0.147 0.152 0.141
MNZ 26.6f 19.9 91.8 84.6c 0.082 0.073 0.150 0.154 0.142
SEM 0.25 0.13 0.12 0.65 0.0089 0.0070 0.0041 0.0052 0.0072
Zinc levels (ppm)
 40 22.7a 18.3a 90.4a 78.2 0.105c 0.084c 0.181b 0.193b 0.171b
 60 23.6ab 18.6ab 90.7b 78.8 0.094b 0.078b 0.176ab 0.186ab 0.168ab
 80 24.5b 18.9b 90.7b 79.0 0.083a 0.075a 0.172a 0.181a 0.164a
Zinc source
 IZ 21.6a 17.5a 89.4a 73.5a 0.098b 0.082b 0.223b 0.243b 0.214b
 GNZ 23.9b 18.6b 93.2b 78.3b 0.092a 0.076a 0.153a 0.155a 0.140a
 MNZ 25.1c 19.6c 91.5c 84.2c 0.094a 0.077a 0.150a 0.159a 0.145a
ANOVA (P values)
 Interaction 0.010 0.097 0.151 0.010 0.081 0.051 0.079 0.113 0.082
 Zinc levels 0.001 0.007 0.009 0.070 0.002  <0.000 0.010 0.010 0.009
 Zinc source 0.003 0.002  <0.000 0.007 0.007 0.005  <0.000  <0.000 0.001
Open in a new tab

Values bearing different superscripts within the column differ significantly

TBARS Thio-barbituric acid reactive substances, MDA malondialdehyde, DPPH 1,1-diphenyl-2-picrylhydrazyl, ABTS 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid, SEM Standard error of mean , IZ Inorganic zinc, GNZ green nano zinc, MNZ market nano zinc

The 80 ppm Zn level resulted in significantly lower free fatty acid values of thigh (P = 0.002) and breast meat (P < 0.000) followed by statistically different 60 ppm level compared to higher values at 40 ppm level. Among the Zn source, significantly higher free fatty acid values were observed in thigh (P = 0.007) and breast (P = 0.005) meat of birds fed IZ compared to MNZ and GNZ which did not differ significantly from each other. Similarly, the 80 ppm Zn resulted in significantly lower TBARS values of breast (P = 0.010), thigh (P = 0.010), and liver (P = 0.009) compared to 40 ppm, whereas, 60 ppm level was statistically similar to both 80 and 40 ppm Zn level. The IZ resulted in significantly higher TBARS values of breast (P < 0.000), thigh (P < 0.000), and liver (P = 0.001) compared to GNZ or MNZ which did not differ significantly from each other.

The demand for functional meat products with higher natural antioxidants is increasing because they exert beneficial health effects (Bursac Kovacevic et al. 2018) and increase storage stability. Zn is known as an effective antioxidant trace mineral in biological system, but with no well-defined mechanism of action. However, it is reported that Zn increases the synthesis of metallothionein, a cystine-rich protein, which acts as a free radical scavenger (Oteiza et al. 1996). In support to this statement the present study revealed a significantly higher free radical scavenging capability (ABTS and DPPH values) of broiler chicken meat from birds fed 80 ppm Zn compared to 40 ppm level. Further, the MNZ source imparted superior free radical scavenging capability to broiler chicken meat compared to GNZ synthesised in this study, which in turn was superior to IZ source. However, notwithstanding the knowledge of authors, there is no literature available showing the direct correlation of DPPH and ABTS values of broiler chicken meat with different dietary Zn levels and sources. The antioxidant property of Zn has also been attributed to its role in fat metabolism and partly in fat absorption in the intestine that plays an important role in numerous biological processes in avian and mammalian species (Hunt 2003). The unsaturated lipids in body are the main targets of oxidants and increase in the lipid peroxidation and free radical formation increases the blood MDA levels in broiler chicken (Yahav et al. 1997). In the present study, free fatty acid and MDA levels representing the oxidation status of broiler chicken tissues were significantly lower in birds fed 80 ppm Zn of either MNZ or GNZ source. Similarly, it has been reported that the MDA content of thigh tissue at different time intervals was significantly reduced in birds supplemented with nano Zn of different sources (Mohammadi et al. 2015). The layer birds fed Zn and vitamin E enriched diet have shown significantly lower blood MDA levels because of lower MDA production in liver by increasing the levels of glutathione peroxidase enzyme that protects tissues from oxidative damage by removing the peroxides produced by free radicals (Naziroglu et al. 2000).

Fat and cholesterol content of meat

The results pertaining to meat fat and cholesterol content have been given in Table 3. The 80 ppm Zn level revealed significantly lower fat (%) in thigh (P = 0.010) and breast (P = 0.009) meat compared to 40 ppm level, whereas, the 60 ppm level was statistically similar to both 80 and 40 ppm levels. Similarly, IZ supplemented birds had significantly higher fat content in thigh (P < 0.000) and breast (P = 0.006) compared to MNZ or GNZ supplemented birds which did not differ significantly from each other. The interaction effect between levels and sources of Zn has shown significantly (P = 0.001) lower thigh fat content in birds fed GNZ-80 or MNZ-80 followed by statistically similar GNZ-60 and MNZ-60 compared to higher values in birds fed IZ-40 or IZ-60, whereas, other combinations of levels and sources yielded intermediate values. The 80 ppm Zn level yielded significantly lower cholesterol content in thigh (P = 0.031) as well as breast (P = 0.019) meat samples followed by statistically different 60 ppm level compared to higher values at 40 ppm level. Among Zn sources, IZ resulted in significantly higher cholesterol content of thigh (P = 0.004) and breast (P = 0.007) meat compared to GNZ or MNZ which did not differ significantly from each other.

Table 3.

Effect of different levels and sources of zinc on fat and cholesterol content of broiler chicken meats

Zn Source Zn (ppm) Fat (%) Cholesterol (mg/100 g)
Thigh Breast Thigh Breast
IZ 40 12.79c 6.84 86.41 93.47
GNZ 7.81b 4.54 71.45 80.56
MNZ 7.87b 4.47 73.44 82.50
IZ 60 12.27c 6.53 85.68 92.57
GNZ 7.64ab 4.23 72.67 83.53
MNZ 7.51ab 4.13 70.71 81.85
IZ 80 11.57c 6.21 84.90 91.56
GNZ 7.18a 3.93 69.49 82.44
MNZ 7.21a 3.81 71.57 80.72
SEM 0.251 0.132 0.294 0.153
Zinc levels (ppm)
 40 9.49b 5.28c 77.10c 85.98c
 60 9.14ab 4.96b 76.35b 85.51b
 80 8.65a 4.65a 75.32a 84.90a
Zinc source
 IZ 12.21c 6.53c 85.66b 92.53b
 GNZ 7.54a 4.23a 70.54a 79.51a
 MNZ 7.50a 4.10a 72.57a 81.69a
ANOVA (P values)
 Interaction 0.001 0.071 0.068 0.091
 Zinc levels 0.010 0.009 0.031 0.019
 Zinc source  <0.000 0.006 0.004 0.007
Open in a new tab

Values bearing different superscripts within the column differ significantly

SEM standard error of mean, IZ inorganic zinc, GNZ green nano zinc, MNZ market nano zinc

Thus, the present study reveals that 80 ppm Zn level of either GNZ or MNZ source resulted in significantly lower fat and cholesterol content of broiler chicken meat. This may be attributed to possible involvement of Zn in lipid metabolism. Ahmadi et al. (2013) reported decline of breast cholesterol content in broiler chicken by supplementing nano ZnO up to 120 ppm level. However, authors of present study could not find further reports dealing with the effects of Zn on fat and cholesterol content of broiler chicken meat.

Serum antioxidant status

The serum antioxidant enzyme profile as affected by different levels and sources of Zn is given in Table 4. The 80 ppm Zn level resulted in significantly higher SOD and catalase activity at 28th day (P = 0.010, P = 0.021) and 42nd day (P = 0.018, P = 0.026) compared to 40 ppm level, whereas, 60 ppm level yielded intermediated activity of SOD and catalase which were statistically similar to both 80 and 40 ppm Zn levels. Among the Zn sources, IZ yielded lower SOD and catalase activity at 28th day (P < 0.000, P < 0.000) and 42nd day (P = 0.001, P = 0.006) compared to GNZ and MNZ which did not differ significantly from each other. Only the Zn sources were found to have significant effect on the GSH-Px activity at 42nd day, where IZ resulted in lower (P = 0.017) values compared to MNZ or GNZ which were statistically similar to each other.

Table 4.

Effect of different levels and sources of zinc on serum antioxidant enzyme levels

Zn source Zn (ppm) SOD (U/ml) Glutathione peroxide (nmol/min/ml) Catalase (nmol/min/ml)
28 day 42 day 28 day 42 day 28 day 42 day
IZ 40 35.9 40.9 6.3 7.1 38.2 40.4
GNZ 55.8 61.9 6.5 7.7 41.3 43.5
MNZ 56.2 61.7 6.5 7.7 41.3 43.5
IZ 60 37.2 43.0 6.3 7.2 38.3 40.6
GNZ 57.1 63.6 6.5 7.8 41.5 43.7
MNZ 57.6 64.1 6.5 7.8 41.6 43.8
IZ 80 39.2 45.0 6.4 7.3 38.8 41.0
GNZ 59.7 65.8 6.6 7.8 41.8 44.0
MNZ 59.1 65.9 6.6 7.8 41.9 44.1
SEM 1.23 1.29 0.19 0.14 0.18 0.13
Zinc levels (ppm)
 40 49.3a 54.8a 6.4 7.5 40.3a 42.5a
 60 50.6ab 56.9ab 6.5 7.6 40.5ab 42.7ab
 80 52.6b 58.9b 6.5 7.6 40.9b 43.0b
Zinc source
 IZ 37.4a 43.0a 6.3 7.2a 38.5a 40.7a
 GNZ 59.5b 65.8b 6.6 7.9b 42.6b 45.7b
 MNZ 57.6b 63.9b 6.6 7.8b 41.6b 43.8b
ANOVA (P values)
 Interaction 0.057 0.052 0.137 0.082 0.067 0.083
 Zinc levels 0.010 0.018 0.071 0.078 0.021 0.026
 Zinc source  <0.000 0.001 0.054 0.017  <0.000 0.006
Open in a new tab

Values bearing different superscripts within the column differ significantly

SEM standard error of mean, SOD superoxide dismutase, IZ inorganic zinc, GNZ green nano zinc, MNZ market nano zinc

The SOD, GSH-Px, and catalase protect cells against damage caused by reactive oxygen species (ROS) and lipoperoxides. Although, Zn is supposed to have no antioxidant action by itself but is required for the activity of many antioxidant enzymes, such as SOD, which catalyses the breakdown of the superoxide anion into oxygen and hydrogen peroxide (Kataria et al. 2008) and are present in almost all aerobic cells and extracellular fluids. Zn has been reported as an antioxidant agent which boost activation of antioxidant enzymes in combination with vitamin E (Johnson and Giulivi 2005). In the present study supplementation of 80 ppm Zn of either MNZ or GNZ source significantly increased the serum SOD, GSH-Px, and catalase activity in broiler chicken compared to 40 ppm level and IZ source of Zn. Similarly, an increased activity of serum GSH-Px and catalase have been reported in broiler chicken by increasing the supplementation of nano Zn from 20 to 100 ppm level compared to 20 ppm ZnSO4 control (Zhao et al. 2014). Ahmadi et al. (2014) observed that supplementation of 60–90 ppm of zinc oxide nano particles in broiler chicken ration significantly increased SOD activity compared to control and 30 ppm level. However, they observed lowest SOD activity at 120 ppm level indicating the inhibitory effect of higher Zn levels. The nano Zn supplementation has also been reported to increase the SOD activity in Wistar rats (Raje 2016).

Tissue and serum mineral profile

The results pertaining to tissue and serum mineral profile are given in Table 5. The 80 ppm Zn significantly increased the Zn concentration of liver (P = 0.027) and serum at 28th day (P = 0.013) compared to 40 ppm level, whereas, 60 ppm level was statistically similar to both 40 ppm and 80 ppm levels. Among the Zn sources, IZ resulted in significantly lower Zn concentration of liver (P = 0.005), muscle (P = 0.007), and serum at 28th day (P = 0.006) and 42nd day (P = 0.001) compared to GNZ and MNZ which did not differ significantly from each other. The interaction effect observed on liver Zn revealed significantly (P = 0.017) higher concentration in birds fed MNZ-80, GNZ-80, and MNZ-60 compared to IZ-40 and IZ-60, whereas, other combinations of Zn levels and sources yielded intermediate values. In the present study the liver has shown much higher accumulation of Zn than the breast muscle. It has been reported that metallothionein is synthesized in tissues in response to dietary Zn and can bind 7 atoms of Zn per molecule of protein (Cousins and Lee-Ambrose 1992). The increase in this Zn binding protein may be responsible for greater Zn accumulation in the body tissues. The higher accumulation of Zn in tissues of the broiler chicken may also be due to the greater ability of nano minerals to pass through the small intestine and distribute in the body than the inorganic and organic minerals (Hillyer and Albrecht 2001). The increase of dietary Zn levels in rats has been reported to result in greater hepatic storage of Zn (Mwangi et al. 2017). Zinc concentration in breast muscle increased in a dose dependent manner of dietary Zn in broiler chicken (Saleh et al. 2018). Similar to the findings of the present study there are the reports of increased blood Zn levels in broiler chicken with increased dietary Zn levels (Mohanna and Nys 1999). The dietary nano Zn has been reported to increase the serum Zn concentration in Wistar rats (Raje 2016) compared to inorganic Zn. Also, the serum Zn level was significantly higher in nano-Zn supplemented groups than other zinc sources.

Table 5.

Effect of different levels and sources of zinc on serum and tissue mineral profile

Zn source Zn (ppm) Serum Zn (ppm) Meat Zn (ppm) Serum calcium (mg/dl) Serum phosphorus (mg/dl)
28 day 42 Day Liver Muscle 28 day 42 day 28 day 42 day
IZ 40 1.24 1.73 25.4a 14.6 10.2a 16.3a 4.33a 4.48a
GNZ 1.36 2.42 40.5c 30.4 12.4e 18.5e 5.28e 5.44e
MNZ 1.38 2.42 42.5cd 33.5 12.3de 18.4de 5.29e 5.45e
IZ 60 1.26 1.76 26.6a 15.7 10.5a 16.6a 4.64b 4.80b
GNZ 1.41 2.46 41.3c 33.6 13.3f 19.3f 5.49f 5.65f
MNZ 1.40 2.44 43.5d 34.5 13.5f 19.6f 5.50f 5.66f
IZ 80 1.26 1.78 27.8b 16.6 11.3b 17.4b 4.76cd 4.92cd
GNZ 1.42 2.47 44.5d 30.8 14.3g 20.4g 5.69g 5.84g
MNZ 1.43 2.47 43.3d 34.8 14.4g 20.5g 5.65g 5.80g
SEM 0.010 0.041 1.00 1.13 0.17 0.14 0.011 0.061
Zinc levels (ppm)
 40 1.32a 2.19 36.1a 26.2 11.6a 17.7a 4.96a 5.12a
 60 1.35ab 2.22 37.1ab 27.9 12.4b 18.5b 5.21b 5.37b
 80 1.37b 2.24 38.5b 27.4 13.3c 19.4c 5.36c 5.52c
Zinc source
 IZ 1.25a 1.75a 26.6a 15.6a 10.6a 16.7a 4.58a 4.73a
 GNZ 1.40b 2.45b 45.5b 35.3b 14.3b 20.4b 5.49b 5.64ab
 MNZ 1.37b 2.44b 43.1b 34.2b 13.4b 19.5b 5.44b 5.59b
ANOVA (P values)
 Interaction 0.113 0.094 0.017 0.151 0.018 0.003 0.031 0.010
 Zinc levels 0.013 0.078 0.027 0.087 0.024 0.012 0.004 0.009
 Zinc source 0.006 0.001 0.005 0.007 0.009  <0.000 0.001 0.007
Open in a new tab

Values bearing different superscripts within the column differ significantly

SEM standard error of mean, IZ inorganic zinc, GNZ:green nano zinc, MNZ market nano zinc

The 80 ppm Zn resulted in significantly higher serum calcium and phosphorus at 28th day (P = 0.024, P = 0.004) as well as 42nd day (P = 0.012, P = 0.009) followed by statistically different 60 ppm level compared to the lower values at 40 ppm level. Among the Zn sources, the IZ resulted in significantly lower serum calcium at 28th day (P = 0.009) as well as 42nd day (P < 0.000) and serum phosphorus at 28th day (P = 0.001) compared to GNZ and MNZ which were statistically similar to each other. The serum phosphorus at 42nd day was significantly (P = 0.007) higher in birds fed MNZ compared to IZ, whereas, GNZ was statistically similar to both sources. Among the interaction effects, in general significantly higher serum calcium at 28th day (P = 0.018) as well as 42nd day (P = 0.003) and serum phosphorus at 28th day (P = 0.031) and 42nd day (P = 0.010) values were observed in birds fed MNZ-80 and GNZ-80 compared to IZ-40, whereas, other combinations of Zn levels and sources yielded intermediate values. In the present study higher serum calcium, and phosphorus were observed in birds fed 80 ppm Zn level and MNZ or GNZ source compared to lower levels and IZ source. Zn is an integral part of the enzyme system involved in the synthesis of vitamin D3 which regulates the calcium and phosphorus absorption at the level of intestines, kidneys, and bones (Guyton and Hall 2006). The increase in calcium and phosphorus levels by increasing dietary Zn level may be attributed to the action of vitamin D3. Further, vitamin D3 may promote calcium absorption by the formation of calcium stimulated ATPase in the brush border of the epithelial cells and ALP in the epithelial cells (Guyton and Hall 2006). The lower serum calcium levels were reported in broiler chicken fed IZ compared to organic Zn (Zakaria et al. 2017).

Bone morphology and mineralization

The results of bone morphology and mineralization of broiler chicken are given in Table 6. The results have revealed significant interaction and main effects of different Zn levels and sources. The bone length (P = 0.002), proximal width (P = 0.008), mid shaft width (P = 0.010), distal width (P = 0.009), and bone weight (P = 0.006) were significantly higher at 80 ppm Zn compared to 40 ppm level, whereas, 60 ppm level was statistically similar to both. Among Zn source GNZ and MNZ resulted in higher values of bone length (P = 0.003), proximal width (P = 0.001), mid shaft width (P = 0.007), distal width (P = 0.008), and bone weight (P = 0.007) which were statistically similar to each other compared to IZ source. The interaction effect of Zn levels and sources revealed that higher bone length (P = 0.007), proximal width (P = 0.009), distal width (P = 0.009), and bone weight (P = 0.004) were observed at 80 ppm Zn level of either MNZ or GNZ source followed by 60 ppm level of either MNZ or GNZ source and lower values were observed at 40 ppm Zn of IZ source. The other dietary combinations of Zn levels and sources yielded intermediate values.

Table 6.

Effect of different levels and sources of zinc on bone morphology and mineralization

Zn source Zn (ppm) Bone length (mm) Proximal width (mm) Mid shaft width
(mm)		 Distal width (mm) Bone weight (g) Moisture (%) Total ash (%) Calcium (%) Phosphorus (%) Zinc (mg/kg)
IZ 40 92.9a 9.7a 7.09 7.69a 4.32a 45.9 40.7a 20.7 7.07 219.2
GNZ 93.1ab 10.3 cd 7.41 7.86b 4.88d 44.4 43.3bc 20.3 7.31 228.4
MNZ 93.1ab 10.3 cd 7.43 7.85b 4.87d 44.3 43.2bc 20.2 7.30 234.3
IZ 60 93.4b 9.9bc 7.14 7.73a 4.44b 45.5 43.0ab 20.2 7.33 234.6
GNZ 93.8bc 10.4de 7.49 7.90bc 4.91de 44.0 43.5cd 19.7 7.57 243.8
MNZ 94.8d 10.4de 7.51 7.88bc 4.95e 44.0 43.6cd 19.7 7.58 249.7
IZ 80 94.1c 9.9bc 7.17 7.74a 4.74c 45.2 43.7cd 19.7 7.46 251.6
GNZ 94.2c 10.5e 7.52 7.92bc 4.95e 43.8 43.9de 19.3 7.65 260.8
MNZ 94.8d 10.6e 7.65 7.97c 4.97e 43.8 44.1e 19.2 7.71 266.7
SEM 0.05 0.10 0.092 0.081 0.034 0.20 0.15 0.07 0.031 2.09
Zinc levels (ppm)
 40 93.0a 10.0a 7.31a 7.80a 4.69a 44.9c 42.4a 20.4c 7.23a 226.8a
 60 94.0ab 10.2ab 7.38ab 7.84ab 4.77b 44.5b 43.4b 19.9b 7.49b 242.1b
 80 94.4b 10.3b 7.45b 7.88b 4.89b 44.3a 43.9c 19.4a 7.61c 259.2c
 IZ 93.5a 9.8a 7.13a 7.72a 4.50a 45.5b 42.5a 20.2b 7.29a 235.1a
 GNZ 93.7ab 10.4b 7.47b 7.89b 4.91b 44.1a 43.6b 19.8a 7.51b 246.3b
 MNZ 94.2c 10.4b 7.53b 7.90b 4.93b 44.0a 43.6b 19.7a 7.53b 250.2b
ANOVA (P values)
 Interaction 0.007 0.009 0.074 0.009 0.004 0.078 0.010 0.081 0.085 0.064
 Zinc levels 0.002 0.008 0.010 0.009 0.006 0.008 0.005 0.008 0.003 0.001
 Zinc source 0.003 0.001 0.007 0.008 0.007 0.009 0.005 0.009 0.001 0.007
Open in a new tab

Values bearing different superscripts within the column differ significantly

SEM standard error of mean, IZ inorganic zinc, GNZ green nano zinc, MNZ market nano zinc

The significantly lower moisture and calcium (P = 0.008) contents were observed at 80 ppm Zn followed by 60 ppm compared to 40 ppm level. However, the reverse trend was observed in total ash (P = 0.005), phosphorus (P = 0.003), and zinc (P = 0.001) content of tibia. Among the Zn sources significantly lower moisture and calcium (P = 0.009) were observed due to GNZ or MNZ sources which were statistically similar to each other compared to IZ source. However, significantly total ash (P = 0.005), phosphorus (P = 0.001), and zinc (P = 0.007) were observed due to IZ source compared to GNZ or MNZ which did not differ significantly from each other. The significantly (P = 0.010) higher bone ash was observed at 80 ppm Zn level of IZ, GNZ, or MNZ source and lower bone ash was observed at 40 ppm Zn of IZ source, whereas, other combina150tions of levels and sources of Zn resulted in intermediate bone ash content.

The requirement of Zn for proper bone growth and development is well documented (Brandeo Neto et al. 1995). The effect of dietary Zn on the bone growth and development may be due to its direct impact on protein synthesis via hormonal growth mediators which positively affects the bone formation in broiler chicken (Seo et al. 2010). For example, the anabolic effect of insulin-like growth factor 1 on osteoblasts is enhanced by Zn supplementation in broiler chicken (Wang et al. 2002). The increase in the dimensions and weight of tibia bone in present study can be attributed to this enhanced osteoblast activity. Moreover, in the present study it is the higher bioavailability of nano Zn which might have resulted in its greater absorption in the gut which not only got deposited in bony tissue in greater concentration but also influenced cell function through its effects on hormones and growth factors to a greater extent. The longitudinal growth of bones in chicken seem to be highly sensitive to Zn status because the cellular changes, induced by reduced Zn availability from blood supply, are locally observed in areas located away from blood vessels (Wang et al. 2002). In line with the results of the present study significantly higher bone weight, total ash, bone Zn and lower calcium content were observed in broiler chicken at 80 ppm Zn level (Mir et al. 2014). Similarly, the tibia bone ash and Zn content increased linearly with increasing dietary Zn level and reach plateau at 75 ppm level (Mohanna and Nys 1999). However, in contrast to results of the present study no significant differences were observed on the bone dimensions and weight due to different Zn sources and levels in broiler chicken (Oliveira et al. 2015).

Conclusion

Different sources and levels of dietary zinc had significant impact on the antioxidant status, and mineral profile of broiler chicken. The 80 ppm dietary nano zinc resulted in significantly lower meat cholesterol and fat content, higher free radical scavenging capacity, lower lipid peroxidation of meat, higher serum antioxidant enzyme activities, and enhanced bone growth and development along with greater accumulation of Zn in the body of broiler chicken. However, the effects of green nano zinc were similar as that of market nano zinc.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Ahmadi F, Ebrahimnezjad Y, Ghalehkandi JG, Sis NM (2014) The effect of dietary zinc oxide nanoparticles on the antioxidant state and serum enzymes activity in broiler chickens during starter stage. International conference on biological, civil, and environmental engineering, March 17–18; Dubai, UAE
  2. Ahmadi F, Ebrrahimnezhad Y, Sis MN, Ghalehkandi JG. The effects of zinc oxide nanoparticles on performance, digestive organs and serum lipid concentrations in broiler chickens during starter period. Int J Biosci. 2013;3(7):23–29. doi: 10.12692/ijb/3.7.23-29. [DOI] [Google Scholar]
  3. AOAC . Official methods of analysis. 16. Washington, DC: Association of Analytical Chemists; 1995. [Google Scholar]
  4. Bao YM, Choct M. Trace mineral nutrition for broiler chickens and prospects of application of organically complexed trace minerals: a review. Anim Prod Sci. 2009;49:269–282. doi: 10.1071/EA08204. [DOI] [Google Scholar]
  5. Brandeo-Neto J, Stefan V, Mendonca B, Bloise W, Castro A. The essential role of zinc in growth. Nutr Res. 1995;15:736–738. [Google Scholar]
  6. Bursac Kovacevic D, Barba FJ, Granato D, Galanakis CM, Herceg Z, Dragovic-Uzelac V, Putnik P. Pressurized hot water extraction (PHWE) for the green recovery of bioactive compounds and steviol glycosides from Stevia rebaudiana Bertoni leaves. Food Chem. 2018;254:150–157. doi: 10.1016/j.foodchem.2018.01.192. [DOI] [PubMed] [Google Scholar]
  7. Classen HG, Grober U, Low D, Schmidt J, Stracke H. Zinc deficiency: Symptoms, causes, diagnosis and therapy. Med Monatsschr Pharm. 2011;34:87–95. [PubMed] [Google Scholar]
  8. Cousins RJ, Lee-Ambrose LM. Nuclear zinc uptake and interactions and metallothionein gene expression are influenced by dietary zinc in rats. J Nutr. 1992;122:56–64. doi: 10.1093/jn/122.1.56. [DOI] [PubMed] [Google Scholar]
  9. Galanakis CM. Phenols recovered from olive mill wastewater as additives in meat products. Trends Food Sci Technol. 2018;79:98–105. doi: 10.1016/j.tifs.2018.07.010. [DOI] [Google Scholar]
  10. Guyton AC, Hall JE. Text-book of medical physiology. 11. Amsterdam: Elsevier Saunders; 2006. [Google Scholar]
  11. Hillyer JF, Albrecht RM. Gastrointestinal persorption and tissue distribution of differently sized colloidal gold nanoparticles. J Pharm Sci. 2001;90(12):1927–1936. doi: 10.1002/jps.1143. [DOI] [PubMed] [Google Scholar]
  12. Hunt J. Bioavailability of iron, zinc, and other trace minerals from vegetarian diets. Am J Clin Nutr. 2003;78(3):633–639. doi: 10.1093/ajcn/78.3.633S. [DOI] [PubMed] [Google Scholar]
  13. Johnson F, Giulivi C. Superoxide dismutases and their impact upon human health. Mol Aspects Med. 2005;26:340–352. doi: 10.1016/j.mam.2005.07.006. [DOI] [PubMed] [Google Scholar]
  14. Kataria N, Kataria AN, Gahlot AK. Ambient temperature associated variation in serum hormones and—interrelated analysis of broiler chickens in arid tract. Slov Vet Res. 2008;45:127–134. [Google Scholar]
  15. Kato K, Terao S, Shimamoto N, Hirata M. Studies on scavengers of active oxygen species Synthesis and biological activity of 2-O-alkylascorbic acids. J Med Chem. 1988;31(4):793–798. doi: 10.1021/jm00399a019. [DOI] [PubMed] [Google Scholar]
  16. Koniecko EK. Handbook for meat chemists. Wayne: Avery Publishing Group Inc; 1979. pp. 68–69. [Google Scholar]
  17. Mir NA, Deo D, Mandal AB, Tyagi PK. Effect of feeding different levels of zinc and vitamin A on morphometry and mineralization of tibia bone in broiler chickens. Indian J Poult Sci. 2014;49(2):224–227. [Google Scholar]
  18. Mohammadi V, Ghazanfari S, Sangcheshmeh AM, Nazaran MH. Comparative effects of zinc-nano complexes, zinc-sulphate and zinc-methionine on performance in broiler chickens. Br Poult Sci. 2015;56(4):486–493. doi: 10.1080/00071668.2015.1064093. [DOI] [PubMed] [Google Scholar]
  19. Mohanna C, Nys Y. Effect of dietary zinc content and sources on the growth, body zinc deposition and retention, zinc excretion and immune response in chickens. Br Poult Sci. 1999;40:108–114. doi: 10.1080/00071669987926. [DOI] [PubMed] [Google Scholar]
  20. Mwangi S, Timmons J, Ao T, Paul M, Macalintal LC, Pescatore A, Cantor A, Ford M, Dawson KA. Effect of zinc imprinting and replacing inorganic zinc with organic zinc on early performance of broiler chicks. Poult Sci. 2017;96(4):861–868. doi: 10.3382/ps/pew312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Naziroglu M, Sahin K, Imek H, Aydilek N, Erta ON. The effect of food withdrawal and darkening on lipid peroxidation of laying hens in high ambient temperatures. Dtsch Tierarztl Wschr. 2000;107:199–202. [PubMed] [Google Scholar]
  22. Oliveira TFB, Bertechini AG, Bricka RM, Kim EJ, Gerard PD, Peebles ED. Effects of in ovo injection of organic zinc, manganese, and copper on the hatchability and bone parameters of broiler hatchlings. Poult Sci. 2015;7:240–248. doi: 10.3382/ps/pev248. [DOI] [PubMed] [Google Scholar]
  23. Oteiza PL, Olin KL, Fraga CG, Keen CL. Oxidant defense systems in testes from Zn deficient rats. Proc Soc Exp Biol Med. 1996;213:85–91. doi: 10.3181/00379727-213-44040. [DOI] [PubMed] [Google Scholar]
  24. Prasad AS, Kucuk O. Zinc in cancer prevention. Cancer Metastasis Rev. 2002;21:291–295. doi: 10.1023/A:1021215111729. [DOI] [PubMed] [Google Scholar]
  25. Raje K (2016) Effect of supplementation of zinc nano particles on growth and health status of wistar rats (Rattus norvegicus). MVsc Thesis, Deemed University, Indian Veterinary Research Institute, Izatnagar
  26. Sahin K, Smith MO, Onderci M, Sahin N, Gursu MF, Kucuk O. Supplementation of zinc from organic or inorganic source improves performance and antioxidant status of heat-distressed quail. Poult Sci. 2005;84:882–887. doi: 10.1093/ps/84.6.882. [DOI] [PubMed] [Google Scholar]
  27. Sahoo A, Swain RK, Mishra SK. Effect of inorganic, organic and nano zinc supplemented diets on bioavailability and immunity status of broilers. Int J Adv Res. 2014;2(11):828–837. [Google Scholar]
  28. Saleh AA, Ragab MM, Ahmed EAM, Abudabos AM, Ebeid TA. Effect of dietary zinc-methionine supplementation on growth performance, nutrient utilization, antioxidative properties and immune response in broiler chickens under high ambient temperature. J Appl Anim Res. 2018;46(1):820–827. doi: 10.1080/09712119.2017.1407768. [DOI] [Google Scholar]
  29. Schmidt CW. Nanotechnology-related environment, health and safety research: Examining the national strategy. Environ Health Perspect. 2009;117:158–161. doi: 10.1289/ehp.117-a158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Seo HJ, Cho YE, Kim T, Shin HI, Kwun IS. Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. Nutr Res Pract. 2010;4(5):356–361. doi: 10.4162/nrp.2010.4.5.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shirwaikar A, Kirti SP, Punitha SR. In vitro antioxidant studies of Sphaeranthus indicus (linn) Indian J Exp Biol. 2006;44:993–996. [PubMed] [Google Scholar]
  32. Wang X, Fosmire GJ, Gay CV, Leach RM. Short-term zinc deficiency inhibits chondrocyte proliferation and induces cell apoptosis in the epiphyseal growth plate of young chickens. J Nutr. 2002;132:665–673. doi: 10.1093/jn/132.4.665. [DOI] [PubMed] [Google Scholar]
  33. Wheeler CR, Salzman JA, Elsayed NM, Omaye ST, Korte DW., Jr Automated assays for superoxide dismutase, catalase, glutathione peroxide and glutathione reductase activity. Anal Biochem. 1990;184:193–199. doi: 10.1016/0003-2697(90)90668-Y. [DOI] [PubMed] [Google Scholar]
  34. Witte VC, Krauze GF, Bailey ME. A new extraction method for determining 2-thiobarbituric acid values of pork and beef during storage. J Food Sci. 1970;35:582–585. doi: 10.1111/j.1365-2621.1970.tb04815.x. [DOI] [Google Scholar]
  35. Wybenga DR, Pileggi VJ, Dirstine H, Giorgio JD. Direct manual determination of serum total cholesterol with a single stable reagent. Clin Chem. 1970;16:980–984. doi: 10.1093/clinchem/16.12.980. [DOI] [PubMed] [Google Scholar]
  36. Yahav S, Straschnow A, Plavnik I, Hurwitz S. Blood system response of chickens to changes in environmental temperature. Poult Sci. 1997;76:627–633. doi: 10.1093/ps/76.4.627. [DOI] [PubMed] [Google Scholar]
  37. Zakaria HA, Jalal M, AL-Titi HH, Souad A, (2017) Effect of sources and levels of dietary zinc on the performance, carcass traits and blood parameters of broilers. Braz J Poult Sci 19(3):519–526
  38. Zhang JS, Zhang LD, Gao XY, Zhang LD, Bao YP. Biological effects of nano red elemental selenium. BioFactors. 2001;15:27–38. doi: 10.1002/biof.5520150103. [DOI] [PubMed] [Google Scholar]
  39. Zhao CY, Tan SX, Xiao XY, Qiu XS, Pan JQ, Tang ZX. Effects of dietary zinc oxide nanoparticles on growth performance and antioxidative status in broilers. Biol Trace Elem Res. 2014;160(3):361–370. doi: 10.1007/s12011-014-0052-2. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Food Science and Technology are provided here courtesy of Springer

RESOURCES