Effects ofClostridium butyricum on growth performance, antioxidation, and immune function of broilers

Effects of Clostridium butyricum on growth performance, antioxidation, and immune function of broilers X. D. Liao, G. Ma,1 J. Cai, Y. Fu, X. Y. Yan, X. B. Wei, and R. J. Zhang2 Laboratory of Feed Biotechnology, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China ondialdehyde (MDA) concentration in duodenal mucosa than those in the control and CB1 groups (P < 0.05). Chicks fed the CB2, CB3, and antibiotic diets had a lower MDA concentration in ileal mucosa than those in the control and CB1 groups (P < 0.05). Broilers fed the CB3 diet had greater superoxide dismutase (SOD) activity in the ileal mucosa on d 21 and in jejunal mucosa on d 42 than those in the other groups (P < 0.05). Chicks fed the CB2, CB3, and antibiotic diets had a higher GSH concentration in duodenal and jejunal mucosa on d 42 than those in the control group (P < 0.05). Broilers fed the CB2 and CB3 diets had a lower MDA concentration in the jejunal mucosa on d 42 than those in the control and CB1 groups. Chicks fed diets supplemented with C. butyricum had a higher IgM concentration than those in the control group at 21 and 42 d of age (P < 0.05). The results indicate that C. butyricum improves broilers’ growth performance, antioxidation, and immune function.

Key words: Clostridium butyricum, antioxidation, immune function, broiler 2015 Poultry Science 00:1–6 http://dx.doi.org/10.3382/ps/pev038

INTRODUCTION

et al., 1973; Okamoto et al., 2000; Kong et al., 2011). Moreover, C. butyricum can produce short-chain fatty acids (e.g., butyric acid) and hydrogen (Vandak et al., 1995; Junghare et al., 2012). Increased butyrate production has often been hypothesized to be one of the beneficial effects of prebiotics. The effects of butyrate on colonic mucosal health and antioxidation have been widely studied (Courtois et al., 2003; Hamer et al., 2009). H2 metabolism, which reflects the balance between H2 -producing (hydrogenogenic) bacteria and H2 utilizing (hydrogenotrophic) microbes, has a primary influence on the final composition of colonic gases (Carbonero et al., 2012). Recent studies have revealed that molecular H2 mediates beneficial effects in different systems as an optimal antioxidant agent by selectively scavenging free hydroxyl radicals ( rOH). H2 gas has come to the forefront of research on therapeutic medical gas, which is an important physiological regulatory factor with antioxidant, anti-inflammatory, and antiapoptotic protective effects on cells and organs (Ohsawa et al., 2007; Huang et al., 2010). Because

During the past decade, some studies have supported the potential health benefits of probiotics, such as improved gastrointestinal microbiota ecosystems, stimulation of the immunological system, anticarcinogenic activities, and reduced oxidative stress (Takahashi et al., 2004; Martarelli et al., 2011; Yang et al., 2012; Amaretti et al., 2013). The most widely studied probiotics are Lactobacillus and Bifidobacterium. However, Clostridium butyricum can produce endospores, which have the ability to survive at lower pH and relatively higher bile concentrations and temperatures compared with Lactobacillus and Bifidobacterium and has been used in a wide range of human and veterinary intestinal diseases as an important symbiotic bacteria (Douglas  C 2015 Poultry Science Association Inc. Received August 25, 2014. Accepted December 2, 2014. 1 Both authors contributed equally to this study. 2 Corresponding author: [email protected]

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ABSTRACT To investigate the effects of Clostridium butyricum on growth performance, antioxidation, and immune function of broilers, 320 one-day-old Arbor Acres commercial male chicks were assigned to one of 5 treatments with 8 replicates in a completely randomized design for 42 d. The 5 treatments were basal diet (control), basal diet supplemented with 2.5 × 108 cfu C. butyricum/kg (CB1), basal diet supplemented with 5 × 108 cfu C. butyricum/kg (CB2), basal diet supplemented with 1 × 109 cfu C. butyricum/kg (CB3), and basal diet supplemented with 150 mg aureomycin/kg (antibiotic). The results showed that all C. butyricum– supplemented groups during d 1 to 21 and the CB2 group during d 22 to 42 had higher ADG compared with the control (P < 0.05). Chicks fed the CB3 diet had higher glutathione S-transferase (GST) activity (P < 0.05), and chicks fed the CB2 diet had a higher glutathione (GSH) concentration in duodenal and ileal mucosa at 21 d of age than those in the control group (P < 0.05). Chicks fed the CB3 diet had a lower mal-

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LIAO ET AL.

C. butyricum can produce both butyrate and H2 , it is reasonable to hypothesize that C. butyricum could regulate animal antioxidation. However, to our knowledge, information is lacking on the effects of C. butyricum on broiler antioxidation. Therefore, the present study was conducted to assess the effects of C. butyricum on growth performance, antioxidation, and immune function of broiler chicks.

MATERIALS AND METHODS Chicks, Diets, and Experimental Design

Table 1. Composition of broilers’ basal diets (as-fed basis). Item (% unless noted) Ingredient Corn Soybean meal Soybean oil Calcium monohydrogen phosphate Ground limestone Sodium chloride DL-Methione Lysine Choline chloride (50%) Mineral premix1 Vitamin premix2 Calculated nutrient composition ME (Mcal/kg) CP Lysine Methionine Calcium Nonphytate phosphorus 1

Starter (d 1 to 21)

Grower (d 22 to 42)

53.33 38.75 3.70 1.98 1.05 0.35 0.18 0.04 0.30 0.30 0.02

60.87 32.04 3.26 1.69 1.08 0.35 0.12 0.02 0.25 0.30 0.02

2.95 21.00 1.15 0.50 0.98 0.45

3.00 19.00 1.00 0.40 0.90 0.40

Supplied per kilogram of diet: Mn, 100 mg; Fe, 80 mg; Zn, 75 mg; Cu, 8 mg; I, 0.35 mg; Se, 0.15 mg. 2 Supplied per kilogram of diet: vitamin A, 12,500 IU; cholecalciferol, 2,500 IU; vitamin E, 30 IU; vitamin K3 , 2.65 mg; thiamin, 2 mg; riboflavin, 6 mg; pantothenic acid, 12 mg; vitamin B12 , 0.025 mg; niacin, 50 mg; folic acid, 1.25 mg; biotin, 0.0325 mg.

Sample Collections and Preparations Chicks were weighed, and 8 chicks (one bird per cage) were chosen from each treatment based on average BW following a 12-h fast on d 21 and 42 of the experiment. Blood samples were taken by cardiac puncture and centrifuged at 3,600 × g for 10 min at 4◦ C. The serum was collected and stored at −20◦ C until analysis. After the chicks were killed by cervical dislocation, the duodenal, jejunal, and ileal mucosa was scraped off with a glass microscope slide, respectively, and then was immediately frozen at −20◦ C for until analysis.

Sample Analyses The intestinal mucosa was homogenized with ice-cold saline in a ratio of 1:9, and then the homogenate was centrifuged at 2,500 × g for 10 min at 4◦ C. The activities of superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione peroxidase (GPX), glutathione (GSH), and malondialdehyde (MDA) in the intestinal mucosa were measured using SOD, GST, GPX, GSH, and MDA assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and automated spectrophotometric analyzer (Cobas FARA II, Roche, Palo Alto, CA), respectively. Serum immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) concentrations were measured with the commercial chicken-specific IgA, IgG, and IgM ELISA kits (Beijing Fangcheng Biotechnology Co., Ltd., Beijing, China). All the procedures were carried out according to the manufacturers’ instructions.

Statistical Analyses Data from the experiment wree analyzed by one-way ANOVA with the general liner model procedure of SAS (release 8.1, SAS Institute Inc., Cary, NC). Cage was the experimental unit. We considered P < 0.05 to be statistically significant. Differences among means were tested by the least significant difference method.

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A total of 320 one-day-old Arbor Acres male broilers (Huadu Broiler Breeding Corporation, Beijing, China) were maintained in accordance with appropriate guidelines for raising broilers (Yang and Diao, 1999). The animal care protocol was approved by the China Agricultural University Animal Care and Use Committee (Beijing, China). Birds were housed in electrically heated thermostatically controlled room with feeders, nipple drinkers, and steel cages. The temperature was maintained at 35◦ C during the first 3 d and gradually reduced to 30◦ C at 1 week of age, between 28 and 30◦ C during the subsequent couple of weeks and reduced to 25◦ C at 3 weeks of age, and at 25◦ C during the last 3 weeks of the experiment. Feed and tap water were available ad libitum. All birds were offered the same basal diet, which was formulated to meet or slightly exceed the NRC (1994) broilers requirements for all nutrients (Table 1). Birds were assigned randomly to one of 5 dietary treatments of 8 replicate cages, each with 8 birds per cage in a completely randomized design.

The 5 treatments were basal diet (control), basal diet supplemented with 2.5 × 108 cfu C. butyricum/kg diet (CB1), basal diet supplemented with 5 × 108 cfu C. butyricum/kg diet (CB2), basal diet supplemented with 1 × 109 cfu C. butyricum/kg diet (CB3), and basal diet supplemented with 150 mg of aureomycin/kg diet (antibiotic). The strain of C. butyricum used in this study was isolated from healthy animal intestine and deposited into China General Microbiological Culture Collection Center (CGMCC 8187). The C. butyricum was first mixed with premix that was subsequently mixed with other ingredients and then stored in covered containers prior to feeding. Mortality was recorded daily, and chick weight and feed intake per cage were measured on d 21 and 42 for determination of ADFI, ADG, and feed conversion rate (FCR).

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CLOSTRIDIUM BUTYRICUM IN BROILERS Table 2. Effect of C. butyricum on growth performance1 . Item

Control

d 1 to 21 ADFI (g/d) ADG (g/d) Feed conversion ratio (g/g) d 22 to 42 ADFI (g/d) ADG (g/d) Feed conversion ratio (g/g)

CB1

CB2

51.0 36.0b 1.42

52.2 37.5a 1.39

52.9 37.9a 1.40

146 76.0c 1.95

150 78.8b,c 1.90

156 83.9a 1.89

CB3

Antibiotic

Pooled SE

P-value

53.1 37.7a 1.41

52.8 37.2a,b 1.43

0.5 0.4 0.01

0.1166 0.0320 0.0817

153 81.4a,b,c 1.91

149 82.7a,b 1.85

3 1.8 0.05

0.1886 0.0343 0.6524

1 Each value represents the mean of 8 replicates; control = basal diet; CB1 = basal diet supplemented with 2.5 × 108 cfu C. butyricum/kg; CB2 = basal diet supplemented with 5 × 108 cfu C. butyricum/kg; CB3 = basal diet supplemented with 1 × 109 cfu C. butyricum/kg; antibiotic = basal diet supplemented with 150 mg aureomycin/kg. a,b,c Means with different superscripts within the same row differ (P < 0.05).

RESULTS

C. butyricum–supplemented groups had higher ADG than those in the control group during d 1 to 21 (P < 0.05), while no differences in ADG were observed between the antibiotic group and the C. butyricum– supplemented groups (P > 0.05) (Table 2). Broilers fed the CB2 diet had higher ADG than those in the control and CB1 groups during d 22 to 42 (P < 0.05). No differences in ADFI and FCR during either experimental period were detected among all the treatments (P > 0.05).

Antioxidation Broilers fed the diets supplemented with C. butyricum had a higher duodenal mucosa GST activity

Table 3. Effect of C. butyricum on antioxidation in intestinal mucosa at 21 d of age1 . Item Duodenal mucosa SOD (U/mg protein) GST (U/mg protein) GPX (U/mg protein) GSH (μ mol/g protein) MDA (nmol/mg protein) Jejunal mucosa SOD (U/mg protein) GST (U/mg protein) GPX (U/mg protein) GSH (μ mol/g protein) MDA (nmol/mg protein) Ileal mucosa SOD (U/mg protein) GST (U/mg protein) GPX (U/mg protein) GSH (μ mol/g protein) MDA (nmol/mg protein)

Control

CB1

CB2

CB3

Antibiotic

Pooled SE

P-value

73.5 29.0c 1.26 27.7b 5.69a

84.4 36.3a,b 1.42 45.3a 5.06a

86.0 36.6a,b 1.64 48.4a 3.65ab

79.4 38.3a 1.37 42.3a 2.90b

72.8 33.2b,c 1.28 43.0a 4.05a,b

4.4 1.6 0.19 2.8 0.66

0.0962 0.0016 0.7543 < 0.0001 0.0349

79.0 31.2b 5.09 22.7a,b 2.96

81.3 33.2b 4.71 24.6a,b 2.76

77.6 35.1a,b 4.90 23.1a 3.44

79.1 32.0b 3.87 18.5b,c 2.31

79.1 38.8a 4.69 17.3c 3.32

2.2 1.8 0.34 1.6 0.32

0.8669 0.0374 0.1337 0.0038 0.1312

72.6b,c 30.2b,c 4.84 19.4b 12.4a

67.2c 26.7c 4.19 17.9b 10.9a

74.8b 34.8a,b 4.48 22.8a 5.5b

84.6a 40.9a 4.05 17.7b 5.4b

67.8b,c 38.8a 3.95 17.1b 7.0b

2.7 2.1 0.25 0.8 0.8

0.0006 0.0003 0.1122 0.0003 < 0.0001

1 Each value represents the mean of 8 cages with one chick per cage; SOD = superoxide dismutase; GST = glutathione S-transferase; GPX = glutathione peroxidase; GSH = glutathione; MDA = malondialdehyde; control = basal diet; CB1 = basal diet supplemented with 2.5 × 108 cfu C. butyricum/kg; CB2 = basal diet supplemented with 5 × 108 cfu C. butyricum/kg; CB3 = basal diet supplemented with 1 × 109 cfu C. butyricum/kg; antibiotic = basal diet supplemented with 150 mg aureomycin/kg. a,b,c Means with different superscripts within the same row differ (P < 0.05).

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Growth Performance

and GSH concentration at 21 d of age compared with those in the control group (P < 0.05) (Table 3). Birds in the CB3 group had a lower duodenal mucosa MDA concentration at 21 d of age than those in the control and CB1 groups (P < 0.05). No differences in duodenal mucosa SOD and GPX activities at 21 d of age were detected among all the treatments (P > 0.05). Birds in the antibiotic group had a higher jejunal mucosa GST activity at 21 d of age than those in the control, CB1, and CB3 groups (P < 0.05); no differences in jejunal mucosa GST activity at 21 d of age were observed between antibiotic group and CB2 group (P > 0.05). Birds in the CB1 and CB2 groups had a higher jejunal mucosa GSH concentration at 21 d of age than those in the antibiotic group (P < 0.05). However, no differences in jejunal mucosa SOD and GPX activities and MDA concentrations at 21 d of age were detected among all the treatments (P > 0.05). Birds in the CB3 group had higher SOD activity in the ileal mucosa at 21 d of age than those in

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LIAO ET AL. Table 4. Effect of C. butyricum on antioxidation in intestinal mucosa at 42 d of age1 . Item Duodenal mucosa SOD (U/mg protein) GST (U/mg protein) GPX (U/mg protein) GSH (μ mol/g protein) MDA (nmol/mg protein) Jejunal mucosa SOD (U/mg protein) GST (U/mg protein) GPX (U/mg protein) GSH (μ mol/g protein) MDA (nmol/mg protein) Ileal mucosa SOD (U/mg protein) GST (U/mg protein) GPX (U/mg protein) GSH (μ mol/g protein) MDA (nmol/mg protein)

Control

CB1

CB2

CB3

Antibiotic

Pooled SE

P-value

80.7 31.5 2.22 19.3c 0.90

77.3 29.9 2.14 20.4b,c 0.78

77.1 24.9 2.28 30.1a 0.83

79.8 25.7 2.36 26.3a,b 0.77

74.3 27.2 2.34 26.9a 0.80

2.7 2.0 0.34 2.1 0.09

0.4998 0.2016 0.9857 0.0094 0.8455

80.3b 29.9 2.97 15.0c 0.98a

77.2b 29.6 2.73 20.7a,b 0.82b

81.6b 30.0 3.44 22.3a,b 0.62c

87.9a 32.2 3.40 24.3a 0.62c

79.3b 30.1 2.97 19.9b 0.70b,c

1.6 1.5 0.30 1.3 0.06

0.0010 0.7997 0.3923 0.0005 0.0003

80.1 29.0 3.04 16.1 0.62

81.6 29.6 3.41 17.6 0.63

79.9 27.1 3.02 15.1 0.67

79.7 30.3 4.05 17.4 0.73

77.1 33.4 3.74 15.6 0.77

3.4 1.4 0.64 1.5 0.06

0.9145 0.0782 0.7625 0.7070 0.4577

the other groups (P < 0.05). Birds in the CB3 and antibiotic groups had higher ileal mucosa GST activity at 21 d of age than those in the control and CB1 groups (P < 0.05). Birds in the CB2 group had higher ileal mucosa GSH concentration at 21 d of age than those in the other groups (P < 0.05). Birds in the CB2, CB3, and antibiotic groups had lower ileal mucosa MDA concentrations at 21 d of age than those in the control and CB1 groups (P < 0.05). No differences in ileal mucosa GPX activity at 21 d of age were detected among all the treatments (P > 0.05). Chicks fed the CB2, CB3, and antibiotic diets had greater duodenal mucosa GSH concentrations on d 42 than those in the control group (P < 0.05) (Table 4). No differences in duodenal mucosa SOD, GST, and GPX activities and MDA content on d 42 were detected among all the treatments (P > 0.05). Birds fed the CB3 diet had higher jejunal mucosa SOD activity on d 42 than those in the other groups (P < 0.05). Birds fed the CB1, CB2, and CB3 diets had higher jejunal mucosa GSH concentrations on d 42 than those in the control group (P < 0.05); moreover, birds fed the CB3 diet had higher jejunal mucosa GSH concentrations on d 42 than those in the antibiotic group (P < 0.05). Birds fed the CB2 and CB3 diets had lower jejunal mucosa MDA concentrations on d 42 than those in the control and CB1 groups (P < 0.05). No differences in jejunal mucosa GST and GPX activities in jejunum mucosa on d 42 were detected among all the treatments (P > 0.05). No differences in ileal mucosa SOD, GST, and GPX activities or GSH and MDA concentrations on d 42 were detected among all the treatments (P > 0.05).

Immune Function Broilers in the C. butyricum–supplemented and antibiotic groups had higher IgM concentrations at 21 d of age than those in the control group (P < 0.05) (Table 5). Birds in the C. butyricum–supplemented groups had higher IgM concentrations at 42 d of age than those in the control group (P < 0.05). No differences in IgM concentrations were observed at 42 d of age between the antibiotic group and the C. butyricum–supplemented groups (P > 0.05). No differences in IgA and IgG concentrations on d 21 and 42 were detected among all the treatments (P > 0.05).

DISCUSSION In recent years, probiotics have been shown to promote growth performance and improve nutrient utilization efficiency in chickens (Grimes et al., 2008; Mountzouris et al., 2010; Zhao et al., 2013). Yang et al. (2012) and Zhang et al. (2014) found that supplementation of C. butyricum significantly improved ADG in broilers, and no differences were observed between C. butyricum–supplemented groups and a group receiving antibiotics. Cao et al. (2012) also observed that dietary supplementation of C. butyricum improved broilers’ growth performance. In the present study, chicks fed diets supplemented with C. butyricum had higher ADG compared with those in the control group (P < 0.05); no differences in ADG were found between the antibiotic group and C. butyricum–supplemented groups (P > 0.05). Thus, both the present experiment and the above reports indicate that dietary supplementation of

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1 Each value represents the mean of 8 cages with one chick per cage; SOD = superoxide dismutase; GST = glutathione S-transferase; GPX = glutathione peroxidase; GSH = glutathione; MDA = malondialdehyde; control = basal diet; CB1 = basal diet supplemented with 2.5 × 108 cfu C. butyricum/kg; CB2 = basal diet supplemented with 5 × 108 cfu C. butyricum/kg; CB3 = basal diet supplemented with 1 × 109 cfu C. butyricum/kg; antibiotic = basal diet supplemented with 150 mg aureomycin/kg. a,b,c Means with different superscripts within the same row differ (P < 0.05).

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CLOSTRIDIUM BUTYRICUM IN BROILERS Table 5. Effect of C. butyricum on immune function1 . Item d 21 IgA (ng/ml) IgG (ng/ml) IgM (μ g/ml) d 42 IgA (ng/ml) IgG (ng/ml) IgM (μ g/ml)

Control

CB1

CB2

CB3

Antibiotic

Pooled SE

P-value

83.5 396 29.7b

75.0 425 33.6a

72.2 424 36.4a

76.3 410 36.5a

72.3 431 33.5a

3.3 15 1.4

0.1157 0.4476 0.0068

83.9 399 28.2b

82.8 430 32.0a

82.5 438 35.3a

86.5 437 34.8a

79.3 422 31.4a,b

3.2 13 1.2

0.5737 0.2110 0.0068

1 Each value represents the mean of 8 cages with one chick per cage; control = basal diet; CB1 = basal diet supplemented with 2.5 × 108 cfu C. butyricum/kg; CB2 = basal diet supplemented with 5 × 108 cfu C. butyricum/kg; CB3 = basal diet supplemented with 1 × 109 cfu C. butyricum/kg; antibiotic = basal diet supplemented with 150 mg aureomycin/kg. a,b Means with different superscripts within the same row differ (P < 0.05).

stimulating the immune system reduces inflammation, or inhibiting intestinal pathogens reduces inflammation and its associated oxidative damage (Martarelli et al., 2011; Chauhan et al., 2014). However, the exact mechanisms need to be further studied. Probiotics have been shown to benefit immune function and immune response–related diseases in a variety of animal models. These probiotics have been found to enhance innate immunity and modulate pathogeninduced inflammation via Toll-like receptor–regulated signaling pathways (Vanderpool et al., 2008). It is well known that immunoglobulins are usually used to evaluate immune status due to their important roles in immune function. Some studies have found that diets supplemented with C. butyricum promoted IgA, IgG, and IgM in mice (Wang et al., 1996), broilers (Yang et al., 2012), and Peyer’s patch cell (Murayama et al., 1995). The present study observed that dietary supplementation of C. butyricum could improve IgM concentrations in broilers (P < 0.05). Similar results reported by Wang et al. (1996) and Song et al. (2006), when taken together with the aforementioned results, demonstrate that C. butyricum beneficially affects the immune system. In conclusion, results from the current study indicate that dietary supplementation of C. butyricum enhances ADG, improves antioxidation in intestinal mucosa, and promotes immune function in broilers. It is recommended that the appropriate level of C. butyricum supplementation may be 5 × 108 cfu/kg or 1 × 109 cfu/kg.

ACKNOWLEDGMENTS This study was financed by the National Key Technology R & D Program during the Twelfth Five-year Plan Period (No. 2011BAD26B0403).

REFERENCES Amaretti, A., M. di Nunzio, A. Pompei, S. Raimondi, M. Rossi, and A. Bordoni. 2013. Antioxidant properties of potentially probiotic bacteria: In vitro and in vivo activities. Appl. Microbiol. Biotechnol. 97:809–817.

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C. butyricum could improve the growth performance of broilers and could be as an alternative to antibiotics. In contrast, (Zhang et al., 2011 observed that dietary supplementation of C. butyricum had no effect on broilers’ growth performance. These inconsistent results might be due to different basal diets, type of chicks, growth phases, or different supplementation. The mechanism by which C. butyricum improves growth performance may be via a healthy microecological environment that increases digestive enzyme activity and concentrations of short-chain fatty acids (Nakanishi et al., 2003; Zhao et al., 2013). The intestinal epithelium sits at the interface between an organism and its luminal environment, and thus it is constantly exposed to various toxic compounds originating from diet, bacterial metabolites, ingested drugs, and oxidants formed during metabolism (Circu and Aw, 2012). These compounds can generate free radicals on their own or during reaction with other compounds (Benard and Balasubramanian, 1993). Therefore, the intestine is prone to oxidative damage induced by reactive oxygen species (ROS). During the past decade, some studies have supported the potential of probiotics such as Lactobacillus and Bifidobacterium to reduce oxidative stress (Truusalu et al., 2004; Martarelli et al., 2011; Amaretti et al., 2013; Chauhan et al., 2014). However, to our knowledge, no study has been performed to investigate the antioxidative effects of C. butyricium in broilers. C. butyricum can produce both butyrate and H2 , which have been proved to modulate oxidative stress by increasing the activity of antioxidative enzymes and reducing reactive oxygen metabolites (Ohsawa et al., 2007; Hamer et al., 2009; Nakao et al., 2010; Jahns et al., 2014). Our study found that dietary supplementation of C. butyricum could increase antioxidative enzymes’ activities, main non-enzymatic antioxidant GSH concentration and decrease lipid peroxidation MDA concentration in intestinal mucosa of broilers (P < 0.05). The probiotic strain exerts antioxidant activity that may be attributed to producing digestive enzymes, butyrate and H2 to influence the expression of antioxidative enzymes and decrease ROS,

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