Effects of palygorskite composites on growth performance and antioxidant status in broiler chickens

Effects of palygorskite composites on growth performance and antioxidant status in broiler chickens

∗

Key Laboratory of Clay Mineral Applied Research of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China; and † College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China ABSTRACT This work aimed to investigate the effects of the palygorskite (PAL) composites on the growth performance and antioxidant status in broiler chickens. A total of 192 one-day-old Ross 308 broilers were randomly divided into 3 treatment groups. Broilers were fed basal diets supplemented with either 50 mg/kg chlortetracycline (CTC group), 1 g/kg ZnO/PAL (ZnO/PAL group), or 1 g/kg chitooligosaccharides/ZnO/PAL (COS/ZnO/PAL group), respectively. The results showed that PAL composites were found to exhibit similar effects on growth performance as CTC (P > 0.05). ZnO/PAL and COS/ZnO/PAL enhanced the activity of serum glutathione peroxidase (GSH-Px) compared with CTC both at 21 and 42 d (P < 0.05). Compared with the CTC group, COS/ZnO/PAL enhanced serum catalase (CAT) activity at 21 d (P < 0.05), and decreased serum malondialdehyde (MDA) content at 42 d (P < 0.05). Compared with the CTC group, ZnO/PAL decreased duodenal mucous MDA content at 21 d, while ZnO/PAL did not affect

activities of superoxide dismutase (SOD) and GSH-Px in the duodenum (P > 0.05). The duodenal mucous activities of SOD and GSH-Px were the highest in the COS/ZnO/PAL group at 42 d (P < 0.05). At 21 d, broilers in the COS/ZnO/PAL group had the lowest MDA content and the highest total antioxidant capacity (T-AOC) in the jejunum (P < 0.05). Palygorskite composites decreased ileum mucous MDA content compared with CTC treated broilers at 21 d (P < 0.05). At 42 d, ileum mucous T-AOC was increased both in the ZnO/PAL and COS/ZnO/PAL groups compared with the CTC group (P < 0.05). The ileum mucous GSH-Px activities both in the ZnO/PAL and COS/ZnO/PAL groups were increased compared with the CTC group (P < 0.05). In conclusion, the broilers given the basal diet supplemented with the PAL composites exhibited similar growth performance to their counterparts in the AGP group. Additionally, the PAL composites improved the antioxidant status of broilers and the beneficial effects of COS/ZnO/PAL on the antioxidant status are more pronounced.

Key words: palygorskite composites, broilers, growth performance, antioxidant status 2019 Poultry Science 0:1–9 http://dx.doi.org/10.3382/ps/pez070

INTRODUCTION

increased use of antibiotics has given rise to antibioticresistant pathogenic bacteria (Wegener et al., 1998; Johnson et al., 2007) and residual contamination of the food chain with antibiotics (Kjeldgaard et al., 2012; Beyene, 2016); both problems are considered direct public health threats. Due to the ban on usage of antibiotics as growth promoters in the European Union (EU) and other countries since 2006, intensive research has focused on the development of efficient alternatives to AGP (Huyghebaert et al., 2011; Brown et al., 2017). Metal-oxide-loaded clay minerals have been identified to display excellent antibacterial activity (Hrenovic et al., 2012; Alswat et al., 2016, 2017). Currently, nonmetallic clay minerals are used for the fabrication of metal-oxide-loaded clay minerals, such as zeolite, palygorskite (PAL), and montmorillonite (Hu et al., 2013; Wang et al., 2014; Alswat et al., 2017). These non-metallic clay minerals have been permitted for the

Antibiotics have been widely used in animal production for several decades as growth promoters (Dibner and Richards, 2005). Antibiotic growth promoters (AGP) have been fed primarily to livestock to improve growth performance by reducing mortality, increasing feed intake and weight gain, enhancing immune function, and improving overall health of the animals (Dibner and Richards, 2005). In addition, a previous study reported that the enhancement of antioxidant status is also one of the mechanisms by which animal growth performance is promoted when using AGP (Kabploy et al., 2015, 2016). However, the

 C 2019 Poultry Science Association Inc. Received August 27, 2018. Accepted January 30, 2019. 1 Corresponding author: [email protected]

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Rui Yan,∗ Aiping Hui,∗ Yuru Kang,∗ Yanming Zhou,† and Aiqin Wang∗,1

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YAN ET AL.

zeolites demonstrated both antioxidant and antimicrobial properties (Tegl et al., 2018), thus we hypothesized that coating of quaternized COS on ZnO/PAL could enhance antioxidant activity. In the current study, ZnO/PAL was prepared and COS/ZnO/PAL was fabricated by coating ZnO/PAL with quaternized COS. As an alternative to AGP, the study on the effects of 2 PAL composites on growth performance and antioxidant status in broilers was conducted.

MATERIALS AND METHODS Materials Natural PAL was provided by Huida Mineral Technology Co. Ltd. (Jiangsu, China). The main chemical compositions of PAL determined by a Minipal 4Xray fluorescence spectrometer (PAN alytical, Almelo, Netherlands) were the following: SiO2 , 57.39%; Al2 O3 , 8.41%; MgO, 12.21%; Fe2 O3 , 5.05%; Na2 O, 2.03%; K2 O, 0.9%; and CaO, 1.35%. Chlortetracycline (CTC, feed grade, chlortetracycline was 15%) was provided by Charoen Pokphand Group (Henan, China). ZnO/PAL and COS/ZnO/PAL were prepared with the following procedures: briefly, ZnO/PAL was prepared by calcination method, where PAL and Zn(NO3 )2 ·6H2 O (Tianjin Kermel Chemical Regent Co., Ltd) were dissolved into deionized water, then an alkali source solution was added dropwise into this solution. The resulting mixture was aged and annealed in a muffle furnace. Chitooligosaccharides (Jiaxing Korui Biotech Co. Ltd) was modified by 3-chloro-2-hydroxy-N, N, N trimethylpropan-1-aminium chloride (Shanghai Macklin Biochemical Co., Ltd.) and also combined with ZnO/PAL of 1 wt. % content.

Experimental Design, Diets, and Management All procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University. A total of 192 one-day-old Ross 308 male broiler chicks (43.22 ± 0.18 g) were obtained from a commercial hatchery and randomly divided into 3 treatment groups of 8 replicates with 8 broilers per replicate for a 42 d trial. The 3 treatments were as follows: (1) CTC group (fed basal diet supplemented with 50 mg CTC per kilogram diet); (2) ZnO/PAL group (fed basal diet supplemented with ZnO/PAL at 1 g/kg); (3) COS/ZnO/PAL group (fed basal diet supplemented with COS/ZnO/PAL at 1 g/kg). The basal diet was formulated based on the NRC (1994) to meet the nutrient requirements of the broilers and was devoid of antibiotics. The composition and nutrient level of the basal diet is shown in Table 1.

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supplementation of livestock feed in China and EU, either as a raw material or additive (EU Commission 2003, 2011; Ministry of Agriculture of China, 2013). Palygorskite is a natural silicate clay mineral with 1-dimensional nanorod-like crystal structure and a theoretical formula of Si8 Mg5 O20 (OH)2 (H2 O)4 ·4H2 O (Galan, 1996; Chiari et al., 2003). Special micropores and channels, a fine particle size as well as fibrous habit, endow PAL with high surface area, ion-exchange and adsorption abilities, and rheological properties (Cao et al., 1996; Galan, 1996; Murray, 2000). Due to the characteristics mentioned above, PAL has been used as a carrier, pellet binder, adsorbent, or feed supplement in feed, exhibiting beneficial effects on pelleted feed quality as well as animal growth performance, nutrient utilization, antioxidant status, and immunity (Schell et al., 1993; Pappas et al., 2010; Chen et al., 2016; Cheng et al., 2016; Zhang et al., 2017). Zinc oxide has received considerable attention from both academic and industrial researchers due to its bactericidal activity, non-toxicity, stability, and extensive biological functions (Shi et al., 2014; Kumar et al., 2017). Previous studies have reported that zincoxide-loaded clay minerals display good antibacterial activity in vitro (Alswat et al., 2016; Javid et al., 2016). A previous study reported that supplementation with zinc-oxide-loaded zeolite in feed for weanling pigs was efficacious in alleviating postweaning diarrhea, improving intestinal microflora and barrier function, as well as improving growth performance (Hu et al., 2012). Similarly, supplementation with zinc-oxideloaded montmorillonite in the feed of broiler chickens was showed to improve growth performance, intestinal morphology, and barrier function, as well as the activities of digestive enzymes (Hu et al., 2013). However, the application of zinc-oxide-loaded PAL (ZnO/PAL) in livestock feed has not been previously reported. Another alternative compound of interest in animal nutrition is chitooligosaccharides (COS), which are the oligomers prepared by degraded chitosan or chitin either chemically or enzymatically (Aam et al., 2010). Generally, the average molecular weight of COS are <30 kDa with a degree of polymerization <20 (Mourya et al., 2011). Chitooligosaccharides have received considerable interest due to their antibacterial and antioxidant activities, as well as immune-enhancing effects (Swiatkiewicz et al., 2015). Chitooligosaccharides exhibit better biological properties than chitosan due to its better water solubility (Mourya et al., 2011; Laokuldilok et al., 2017). Although the surface charge of COS is positive, its zeta potential is relatively low. To further increase the solubility of COS in water, modification with quaternary ammonium is necessary. Composites based on zeolites fabricated by coating with 2 antioxidant agents (chitosan and caffeic acid) are reported to give the material antioxidant properties; and further immobilized the antimicrobial enzyme glucose oxidase, rendering the material’s antimicrobial activity (Tegl et al., 2018). In a combined approach, modified

PALYGORSKITE COMPOSITES AND BROILERS Table 1. The formulation and nutrient levels of basal diet. Items

Grower (22 to 42 d of age)

57.00 32.00 3.05 3.00 1.20 2.00 0.30 0.15 0.30 1.00 100.00

61.00 28.00 2.60 4.00 1.27 1.50 0.25 0.08 0.30 1.00 100.00

12.49 21.30 1.00 0.46 1.19 0.50 0.85

12.92 19.52 0.90 0.37 1.05 0.41 0.74

Ingredients (%, unless noted) Corn Soybean meal Corn gluten meal Soybean oil Limestone Dicalcium phosphate L-Lysine DL-Methionine Salt Premix1 Total Calculated nutrient levels AME (MJ/kg) CP Calcium Available phosphorus Lysine Methionine Methionine + cysteine 1

Premix provided the following per kilogram of diet: vitamin A (transretinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 3000 IU; vitamin E (all-rac-α -tocopherol acetate), 30 IU; menadione, 1.3 mg; thiamine, 2.2 mg; riboflavin, 8 mg; nicotinamide, 40 mg; choline chloride, 600 mg; calcium pantothenate, 10 mg; pyridoxine·HCl, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; vitamin B12 (cobalamin), 0.013 mg; Fe (from ferrous sulfate), 80 mg; Cu (from copper sulfate), 8 mg; Mn (from manganese sulfate), 110 mg; Zn (from zinc oxide), 60 mg; iodine (from calcium iodate), 1.1 mg; Se (from sodium selenite), 0.3 mg.

All birds were placed in 3-layer cages and housed in an environmentally controlled room maintained at 32 to 34◦ C for the first 3 d and then reduced by 2 to 3◦ C per week to a final temperature of 22◦ C. The birds were allowed ad libitum access to mash feed and water.

Preparation of Mucosal Homogenate and Protein Analysis Approximately, 0.2 g of mucosal sample were used to prepare mucosa homogenate. Mucosal samples were first diluted 1:9 (wt/vol) with 0.9% saline, and homogenized using an Ultra-Turrax homogenizer (Tekmar Co., Cincinnati, OH), and then centrifuged at 2800 × g at 4◦ C for 15 min. The supernatant was used for analysis. Protein concentrations of the mucosa homogenate were determined using a corresponding assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, P. R. China).

Total Antioxidant Capacity, Antioxidant Enzymes Activity, and Glutathione Content Analysis The serum and mucosa homogenates were used to measure total antioxidant capacity (T-AOC), glutathione (GSH) content, as well as the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) with corresponding assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, P. R. China).

Malondialdehyde Content Analysis Malondialdehyde (MDA) content of serum and mucosa homogenate was determined using a corresponding assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, P. R. China).

Sample Collection

Statistical Analysis

At 21 and 42 d of age, 1 bird was randomly selected from each replicate and weighed after feed deprivation for 12 h. Serum was obtained by centrifugation of a respective blood sample at 2000 × g for 15 min at 4◦ C, and was stored at −20◦ C until further analysis. After collection of blood samples, the broilers were sacrificed by cervical dislocation and necropsied immediately. The duodenum, jejunum, and ileum were isolated and rapidly excised. Intestinal mucosa was collected by scraping with a slide, and then stored at −80◦ C until further analysis.

A statistical analysis was performed by one-way ANOVA procedure of statistical software (SPSS 21.0, United States). Differences among means were tested by Duncan’s multiple comparison test, and they were considered significant at P < 0.05.

RESULTS Growth Performance As shown in Table 2, broilers exhibited similar growth performance among all groups (P > 0.05).

Growth Performance Broiler body weights were recorded for each replicate at 1, 21, and 42 d to calculate average daily gain (ADG). Feed was withdrawn for 12 h and water was provided for ad libitum before weighing at 21 and 42 d. Feed intake was recorded by replicate (cage) to calculate average daily feed intake (ADFI) and feed/gain (F/G) ratio. Mortality also was recorded and birds that died during experimentation were weighed, and the data were included in the calculation of F/G ratio.

Serum Antioxidant Status As shown in Table 3, at 21 d of age, there were no differences in serum T-AOC and CAT activities between the CTC and ZnO/PAL groups (P > 0.05). Compared with the CTC group, the serum T-AOC was decreased, while the CAT activity increased in the COS/ZnO/PAL group (P < 0.05). The CAT activity in the serum of broilers in the COS/ZnO/PAL group was higher than that of broilers in the ZnO/PAL group (P

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Starter (1 to 21 d of age)

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YAN ET AL. Table 2. Effects of palygorskite composites on growth performance of broilers.1 Treatment3 Item2

ZnO/PAL

COS/ZnO/PAL

S.E.M

P-value

43.19 778.61 2.45

43.25 745.07 2.32

43.24 770.26 2.43

0.18 8.55 0.03

0.992 0.258 0.261

48.84 35.02 1.40

45.57 33.42 1.37

48.39 34.62 1.40

0.65 0.41 0.01

0.080 0.257 0.570

145.67 79.47 1.83

139.93 75.10 1.87

150.86 78.91 1.92

2.33 1.34 0.02

0.163 0.368 0.314

97.26 57.25 1.70

92.75 54.26 1.71

99.62 56.77 1.76

1.33 0.78 0.01

0.099 0.256 0.230

1

Date represent the means of 8 replicates. Body weight (BW); Average daily feed intake (ADFI); Average daily gain (ADG); Feed/Gain ratio (F/G ratio). 3 CTC, broilers fed the basal diets supplemented with 50 mg/kg chlortetracycline; ZnO/PAL, broilers fed the basal diets supplemented with 1 g/kg ZnO/PAL; COS/ZnO/PAL, broilers fed the basal diets supplemented with 1 g/kg chitooligosaccharides/ZnO/PAL. 2

Table 3. Effects of palygorskite composites on serum antioxidant status in broilers.1 Treatment3 Item

2

21 d MDA, nmol/mL T-AOC, U/mL SOD, U/mL CAT, U/mL GSH-Px, U/μ L GSH, mg/L 42 d MDA, nmol/mL T-AOC, U/mL SOD, U/mL CAT, U/mL GSH-Px, U/μ L GSH, mg/L

CTC

ZnO/PAL

COS/ZnO/PAL

S.E.M

P-value

1.96 4.01a 426.66 1.19b 1.32b 8.16

2.09 3.96a 302.78 1.09b 1.73a 8.82

2.19 3.04b 447.06 1.62a 1.86a 9.07

0.11 0.16 30.76 0.08 0.08 0.31

0.673 0.014 0.114 0.012 0.004 0.485

3.19a 6.86a 474.53 2.47 1.60b 11.77a

2.82a,b 6.99a 625.83 2.57 2.15a 6.13b

2.17b 5.23b 645.23 1.98 2.45a 5.35b

0.16 0.29 37.17 0.16 0.12 0.66

0.021 0.015 0.119 0.298 0.008 < 0.001

1 Data represent the means of 8 replicates. Values in the same row with different superscripts were significantly different (P < 0.05). 2 Malondialdehyde (MDA); total antioxidant capacity (T-AOC); superoxide dismutase (SOD); catalase (CAT); glutathione peroxidase (GSH-Px); glutathione (GSH). 3 CTC, broilers fed the basal diets supplemented with 50 mg/kg chlortetracycline; ZnO/PAL, broilers fed the basal diets supplemented with 1 g/kg ZnO/PAL; COS/ZnO/PAL, broilers fed the basal diets supplemented with 1 g/kg chitooligosaccharides/ZnO/PAL.

< 0.05). GSH-Px activities in both the ZnO/PAL and COS/ZnO/PAL groups were increased compared with the CTC group (P < 0.05). No differences were found in SOD activity, content of MDA and GSH among all treatment groups (P > 0.05). At 42 d of age, the serum MDA content was decreased in the COS/ZnO/PAL group compared with the CTC group (P < 0.05), while there was no difference in MDA content between the ZnO/PAL and CTC groups (P > 0.05). The T-AOC of both the CTC and ZnO/PAL groups were higher than that in the COS/ZnO/PAL group (P < 0.05), while there was no difference in T-AOC between the ZnO/PAL and CTC groups (P > 0.05). Compared with the CTC group, the GSH-Px

activities were increased (P < 0.05), while GSH content was decreased in the ZnO/PAL or COS/ZnO/PAL groups (P > 0.05). No differences were found in the activities of SOD and CAT among all treatment groups (P > 0.05).

Duodenal Mucous Antioxidant Status As seen in Table 4, at 21 d, the duodenal mucous MDA content was decreased in the ZnO/PAL group compared with the CTC group (P < 0.05). Malondialdehyde content in the COS/ZnO/PAL group was similar to that of the ZnO/PAL group (P > 0.05). The

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1 d BW (g) 21 d BW (g) 42 d BW (Kg) 1 to21 d ADFI (g/bird) ADG (g/bird) F/G ratio (g/g) 21 to 42 d ADFI (g/bird) ADG (g/bird) F/G ratio (g/g) 1 to 42 d ADFI (g/bird) ADG (g/bird) F/G ratio (g/g)

CTC

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PALYGORSKITE COMPOSITES AND BROILERS Table 4. Effects of palygorskite composites on the duodenal mucous antioxidant capacity of broilers.1 Treatment3 Item2

ZnO/PAL

COS/ZnO/PAL

S.E.M

P-value

0.50a 1.93a 337.50 1.26 5.56 5.27a

0.34b 1.62b 315.51 1.06 4.95 3.91b

0.40a,b 1.60b 323.72 0.89 6.26 4.40a,b

0.03 0.04 4.54 0.10 0.25 0.21

0.027 0.001 0.135 0.311 0.089 0.022

2.47 0.61 280.55b 2.15a 8.15b 3.84a

2.76 0.70 265.64b 1.56b 6.68b 2.85b

2.74 0.60 423.42a 2.06a 10.32a 3.95a

0.12 0.04 16.49 0.10 0.42 0.14

0.553 0.472 < 0.001 0.006 < 0.001 < 0.001

1 Date represent the means of eight replicates. Values in the same row with different superscripts were significantly different (P< 0.05). 2 Malondialdehyde (MDA); total antioxidant capacity (T-AOC); superoxide dismutase (SOD); catalase (CAT); glutathione peroxidase (GSH-Px); glutathione (GSH). 3 CTC, broilers fed the basal diets supplemented with 50 mg/kg chlortetracycline; ZnO/PAL, broilers fed the basal diets supplemented with 1 g/kg ZnO/PAL; COS/ZnO/PAL, broilers fed the basal diets supplemented with 1 g/kg chitooligosaccharides/ZnO/PAL.

T-AOC was higher in the CTC group compared with both the ZnO/PAL and COS/ZnO/PAL groups (P < 0.05). Compared with the CTC group, the GSH content in the COS/ZnO/PAL group was not different (P > 0.05), while GSH content in the ZnO/PAL group was decreased (P < 0.05). There were no differences found in the activities of SOD, CAT, and GSH-Px among all groups (P > 0.05). At 42 d, the activities of SOD and GSH-Px in the duodenal mucous of the COS/ZnO/PAL group were the highest among all groups (P < 0.05). The activities of SOD and GSH-Px in the ZnO/PAL group were not different from that in the CTC group (P > 0.05). Compared with the CTC group, the CAT activity and GSH content in the COS/ZnO/PAL group was not different (P > 0.05), while the CAT activity and GSH content in the ZnO/PAL group was decreased (P < 0.05). No differences were observed in MDA content and T-AOC among all groups (P > 0.05).

Jejunal Mucous Antioxidant Status As indicated in Table 5, at 21 d of age, the jejunal mucous MDA content of the broilers was the lowest (P < 0.05) and the T-AOC was the highest in the COS/ZnO/PAL group among all groups (P < 0.05). There were no differences either in MDA content or in T-AOC between the ZnO/PAL and CTC groups (P > 0.05).There were no differences in the activities of SOD, CAT and GSHPx, and the content of GSH among all groups (P > 0.05). At 42 d, the SOD activity and GSH content in the CTC group was the highest among all groups (P < 0.05). There were no differences in the SOD

activity and GSH content between the ZnO/PAL and COS/ZnO/PAL groups (P > 0.05). Compared with the CTC group, GSH-Px activity in the COS/ZnO/PAL group was similar to that of the CTC group (P > 0.05), while GSH-Px activity in the ZnO/PAL groups was decreased (P < 0.05). There were no differences in the MDA content, T-AOC, and CAT activity among all groups (P > 0.05).

Ileal Mucous Antioxidant Status As shown in Table 6, at 21 d, the ileal mucous MDA content was decreased in both the ZnO/PAL and COS/ZnO/PAL groups compared with the CTC group (P < 0.05). The T-AOC, GSH content, and SOD as well as CAT activities were the highest in the CTC group (P < 0.05). There were no differences in the T-AOC, GSH content, and activities of SOD and CAT between the ZnO/PAL and COS/ZnO/PAL groups (P > 0.05). No difference was observed in GSH-Px activity among all groups (P > 0.05). At 42 d, the ileal mucous MDA content in the ZnO/PAL group was not different from that in the CTC group (P > 0.05), while the MDA content was increased in the COS/ZnO/PAL group compared with the CTC group (P < 0.05). The T-AOC were higher in both the ZnO/PAL and COS/ZnO/PAL groups compared with the CTC group (P < 0.05). Compared with CTC group, SOD activity in the COS/ZnO/PAL group was not unchanged, while SOD activity in the ZnO/PAL group was decreased (P < 0.05). Catalase activity in the ZnO/PAL group was not different from that in the CTC group (P > 0.05), while CAT activity in the COS/ZnO/PAL group was increased compared with the CTC group (P < 0.05). Glutathione peroxidase

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21 d MDA, nmol/mgprot T-AOC, U/mgprot SOD, U/mgprot CAT, U/mgprot GSH-Px, U/mgprot GSH, mg/gprot 42 d MDA, nmol/mgprot T-AOC, U/mgprot SOD, U/mgprot CAT, U/mgprot GSH-Px, U/mgprot GSH, mg/gprot

CTC

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YAN ET AL. Table 5. Effects of palygorskite composites on jejunal mucous antioxidant capacity of broilers.1 Treatment3 Item2

ZnO/PAL

COS/ZnO/PAL

S.E.M

P-value

0.64a,b 1.18b 199.19 2.69 4.20 6.71

0.76a 1.20b 203.46 2.54 3.53 7.06

0.47b 1.82a 204.40 2.38 4.85 8.08

0.04 0.08 4.13 0.10 0.28 0.60

0.012 < 0.001 0.871 0.450 0.160 0.647

1.89 0.76 296.21a 2.35 15.04a 8.05a

2.02 0.80 235.97b 2.45 5.48b 5.63b

1.82 0.86 249.41b 2.13 15.00a 4.38b

0.07 0.05 6.40 0.19 1.06 0.42

0.494 0.719 < 0.001 0.807 < 0.001 < 0.001

1 Data represent the means of 8 replicates. Values in the same row with different superscripts were significantly different (P < 0.05). 2 Malondialdehyde (MDA); total antioxidant capacity (T-AOC); superoxide dismutase (SOD); catalase (CAT); glutathione peroxidase (GSH-Px); glutathione (GSH). 3 CTC, broilers fed the basal diets supplemented with 50 mg/kg chlortetracycline; ZnO/PAL, broilers fed the basal diets supplemented with 1 g/kg ZnO/PAL; COS/ZnO/PAL, broilers fed the basal diets supplemented with 1 g/kg chitooligosaccharides/ZnO/PAL.

Table 6. Effects of palygorskite composites on ileum mucous antioxidant capacity of broilers.1 Treatment3 Item

2

21 d MDA, nmol/mgprot T-AOC, U/mgprot SOD, U/mgprot CAT, U/mgprot GSH-Px, U/mgprot GSH, mg/gprot 42 d MDA, nmol/mgprot T-AOC, U/mgprot SOD, U/mgprot CAT, U/mgprot GSH-Px, U/mgprot GSH, mg/gprot

CTC

ZnO/PAL

1.26a 1.60a 237.87a 2.07a 5.13 10.00a

0.94b 1.28b 205.20b 0.93b 5.15 6.54b

1.59b 0.44c 303.15a 1.03b 2.88b 7.44

1.80b 1.67a 264.98b 1.07b 7.66a 6.68

COS/ZnO/PAL

S.E.M

P-value

0.92b 1.20b 214.92b 0.94b 6.27 7.29b

0.05 0.05 4.02 0.15 0.43 0.41

0.005 < 0.001 0.001 < 0.001 0.487 < 0.001

2.14a 0.68b 281.91a,b 1.88a 7.92a 6.46

0.08 0.12 5.74 0.16 0.53 0.44

0.009 < 0.001 0.017 0.046 < 0.001 0.655

1 Data represent the means of 8 replicates. Values in the same row with different superscripts were significantly different (P < 0.05). 2 Malondialdehyde (MDA); total antioxidant capacity (T-AOC); superoxide dismutase (SOD); catalase (CAT); glutathione peroxidase (GSH-Px); glutathione (GSH). 3 CTC, broilers fed the basal diets supplemented with 50 mg/kg chlortetracycline; ZnO/PAL, broilers fed the basal diets supplemented with 1 g/kg ZnO/PAL; COS/ZnO/PAL, broilers fed the basal diets supplemented with 1 g/kg chitooligosaccharides/ZnO/PAL.

activities in both the ZnO/PAL and COS/ZnO/PAL groups were higher than that in the CTC group (P < 0.05). No difference was observed in GSH content among all groups (P > 0.05).

DISCUSSION In the current study, the growth performance of broilers fed diets supplemented with PAL composites was similar with broilers fed diets supplemented with AGP, which is in agreement with the results of Yan et al. (2016) and Yang et al. (2016). These findings indicate

that replacing CTC with ZnO/PAL or COS/ZnO/PAL from feed had no effects on the growth performance of broilers during 1 to 42 d. Previous studies have reported that PAL composites promoted growth performance through maintaining intestinal microflora balance, improving digestive tract health and nutritional utilization, as well as enhancing immune and antioxidant function (Hu et al., 2012; Tang et al., 2014a,b; Yang et al., 2016). Reactive oxygen and nitrogen species (RONS), formed as natural by-products of normal cell metabolism, can induce oxidative stress causing

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21 d MDA, nmol/mgprot T-AOC, U/mgprot SOD, U/mgprot CAT, U/mgprot GSH-Px, U/mgprot GSH, mg/gprot 42 d MDA, nmol/mgprot T-AOC, U/mgprot SOD, U/mgprot CAT, U/mgprot GSH-Px, U/mgprot GSH, mg/gprot

CTC

PALYGORSKITE COMPOSITES AND BROILERS

lipid antioxidation statues by ZnO/PAL may not be related to improving antioxidant enzymes activities. Interestingly, we found that dietary supplementation with COS/ZnO/PAL decreased mucous MDA content in the ileum and ileum of broilers at 21 d as compared with CTC group. Moreover, the T-AOC in the jejunal mucous of broilers at 21 d and the activities of SOD and GSH-Px in the duodenal mucous as well as the activities of CAT and GSH-Px in the ileum mucous of broilers at 42 d were higher as compared with CTC group. These findings indicate that the COS/ZnO/PAL exhibited the best antioxidant activity among the 3 treatments, which might have been caused by an enhancement of antioxidant activity by COS (Swiatkiewicz et al., 2015). Additionally, previous studies have found that the supplementation of PAL in feed also exhibits beneficial effects on animal intestinal antioxidant status, which may contribute to the antioxidant activity of COS/ZnO/PAL (Chen et al., 2016). Future studies will be necessary to better clarify which mechanisms mediate the observed effects of PAL composites on intestinal antioxidant status in broilers and to understand why there are differences in antioxidant activity between ZnO/PAL and COS/ZnO/PAL. In conclusion, the broilers given the basal diet supplemented with ZnO/PAL or COS/ZnO/PAL at 1 g/kg exhibited similar growth performance to their counterparts in the AGP group. Additionally, the antioxidant status of broilers was improved when supplementing the basal diet with ZnO/PAL or COS/ZnO/PAL, and the beneficial effects of COS/ZnO/PAL on the antioxidant status are more pronounced. These findings indicate that the PAL composites are a potential alternative to AGP in broiler chicken feed.

ACKNOWLEDGMENTS This work was financially supported by the Funds for Creative Research Groups of Gansu, China (17JR5RA306) and the Major Projects of the Natural Science Foundation of Gansu, China (18JR4RA001).

CONFLICT OF INTEREST The authors declare that there are no conflicts of interest.

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damage to unsaturated fatty acids, DNA, and proteins when RONS concentrations are above normal levels (Prisacaru, 2016). Importantly, RONS can be scavenged to control their concentrations by antioxidant systems in body. These systems play a role of neutralization of RONS, which protects the body from oxidative damage (Nanditha and Prabhasankar, 2009). The antioxidant system contains natural non-enzymatic antioxidants and antioxidant enzymes. The most familiar of antioxidant enzymes are SOD, CAT, as well as GSH-Px (Nanditha and Prabhasankar, 2009). Concentrations of oxidative products and activities of antioxidant enzymes are good indicators for evaluating the oxidant status of an organism (Prisacaru, 2016). In the current study, dietary ZnO/PAL and COS/ZnO/PAL increased the serum GSH-Px activity both at 21 and 42 d as compared with CTC, suggesting the ZnO/PAL and COS/ZnO/PAL could enhance serum antioxidant status of broiler chickens. Additionally, COS/ZnO/PAL increased the serum CAT activity at 21 d and decreased the MDA content at 42 d as compared with CTC, whereas the similar beneficial consequences were not observed in the broilers fed the basal diet supplemented with ZnO/PAL. These findings indicate that the beneficial effects on serum antioxidant status are more pronounced when supplementing COS/ZnO/PAL. Antioxidant function serves as an important component in maintaining intestinal barrier integrity against bacterial infection (Kelly et al., 2004). In the current study, supplementation with ZnO/PAL decreased MDA content in the duodenum and ileum of broilers at 21 d as compared with the CTC group, which was in agreement with the results of a study by Tang et al. (2014b). Intestinal mucosal MDA content results suggested that ZnO/PAL could improve intestinal lipid antioxidation statues. However, it was found that supplementation with ZnO/PAL did not enhance T-AOC, antioxidant enzymes activities, or GSH content in the duodenum and jejunum of broilers in the present study, which was in disagreement with Tang et al. (2014b), who found that supplementation with zinc-brearing clinoptilolite (ZnCP) increased T-AOC, SOD activity, and GSH content in jejunum of broilers at 21 d. The release of zinc ions may be a reason for the differences in the aforementioned results between ZnO/PAL and ZnCP. As a structural element of SOD enzymes (Natvig et al., 1996), zinc ions loaded on clinoptilolite may be released in the intestine, which would accelerate the regeneration of the mucosal cells and increases the levels of brush border enzymes (Coudray et al., 1992; Carlson et al., 2004). In addition, the release of zinc from ZnCP may increase zinc content in mucosal cells to maintain the GSH content, which is mainly related to zinc stores in the cell (Faa et al., 2008). However, the release of zinc ions may be decreased because zinc loaded on ZnO/PAL exists in the form of zinc oxidant (Franklin et al., 2007; Yang et al., 2008). The effects on MDA content and antioxidant enzymes activities indicated that the mechanism for improving intestinal

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