PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

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formance of animals, but also result in the development ... egg quality, antioxidant enzyme activities, and intestinal barrier function ... such as protease, amylase, and lipase (Hagedorn et al., ... about reacting with oxygen that inhibits the growth.

PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION Influence of dietary inclusion of Bacillus licheniformis on laying performance, egg quality, antioxidant enzyme activities, and intestinal barrier function of laying hens K. Lei,1 Y. L. Li,1 D. Y. Yu,1 I. R. Rajput,1 and W. F. Li1,2 Key Laboratory of Molecular Feed Sciences, Institute of Animal Nutrition and Feed Sciences, College of Animal Science, Zhejiang University, Zhejiang, China 310058 ABSTRACT This experiment was conducted to evaluate the effects of dietary inclusion of Bacillus licheniformis on laying performance, egg quality, antioxidant enzyme activities, and intestinal barrier function of laying hens. Hy-Line Variety W-36 hens (n = 540; 28 wk of age) were randomized into 6 groups, each group with 6 replications (n = 15). The control group received the basal diet formulated with maize and soybean meal. The treatment groups received the same basal diets supplemented with 0.01, 0.02, 0.03, 0.06, and 0.09% Bacillus licheniformis powder (2 × 1010 cfu/g) for an 8-wk trial. The results showed that dietary supplementation with 0.01 and 0.03% B. licheniformis significantly increased egg production and egg mass. However, no significant differences were observed in egg weight, feed consumption, and feed conversion efficiency among

the 6 groups. Supplementation with different levels of B. licheniformis was found to be effective in improvement of egg quality by increasing egg shell thickness and strength. Compared with control, d-lactate content, diamine oxidase activity, and adrenocorticotropic hormone level in serum decreased significantly, and the level of estradiol and follicle-stimulating hormone increased significantly in plasma of all the experimental groups. Dietary supplementation with B. licheniformis increased the intestinal villus height and reduced the crypt depth. In conclusion, dietary inclusion of B. licheniformis could improve laying performance and egg quality significantly in a dose-dependent manner by decreasing the stress response, upregulating the growth hormone, and improving intestinal health.

Key words: Bacillus licheniformis, laying performance, egg quality, hormone 2013 Poultry Science 92:2389–2395 http://dx.doi.org/10.3382/ps.2012-02686

INTRODUCTION Antibiotics can improve health and productive performance of animals, but also result in the development of drug-resistant microorganisms, which can then pass the resistance on to infectious microorganisms in humans (Li et al., 2006). The European Union has banned the use of antibiotics as growth-promoting agents in the poultry industry, and many countries are restricting the use of antibiotic as growth-promoting agents. Therefore, it is essential to find possible alternatives to antibiotics for growth promotion and improvement in poultry production. Since Tortuero first applied a probiotic (Lactobacillus acidophilus) as an alternative to antibiotics in poultry (Tortuero, 1973), application of probiotics as feed additives has been persistently tested and used in a variety of poultry production settings. ©2013 Poultry Science Association Inc. Received August 15, 2012. Accepted May 10, 2013. 1 All the authors of this research contributed equally. 2 Corresponding author: [email protected]

Probiotics are nonpathogenic bacteria that exert a beneficial influence on health, physiology, or both of the host (Rajput et al., 2013), which can improve intestinal structure, aid in development of immunity to defend against pathogens, and subsequently improve growth performance. It also has been shown that application of probiotics could improve weight gain and feed conversion rate and reduce mortality in broiler chickens (Tortuero, 1973; Jin et al., 1998; Kurtoglu et al., 2004; Lutful Kabir, 2009). Supplementation of probiotics in a basal diet has been shown to be useful for ameliorating the adverse influence of stress (Deng et al., 2012), promoting the activities of antioxidant enzymes (Rajput et al., 2012), and improving the health of the host beyond their inherent basic nutrition (Fuller, 1989). Recently, studies have shown that supplementation of probiotics has the ability to inhibit the adhesion of pathogenic bacteria to the intestinal wall and to enhance immune potency (Balevi et al., 2001), which suggests that probiotics have a significant role in normalization of colonic physiologic function and barrier integrity of conjunctions of the cells with a reduction in mucosal

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pro-inflammatory cytokine levels (Mack et al., 1999; Madsen et al., 2001). Several selected probiotics have been applied in poultry production including Lactobacillus, Streptococcus, Saccharomyces, Aspergillus, and Bacillus species (Tannock, 2001). Bacillus species are ideally suited as feed additives because of their stability as spore-forming bacteria and ability to produce a variety of enzymes such as protease, amylase, and lipase (Hagedorn et al., 1985). Diets supplemented with Bacillus subtilis, Bacillus subtilis fermented product, or probiotic powder can improve weight gain and feed efficiency (Santoso et al., 1995; Li et al., 2006). Bacillus licheniformis has a strong ability to produce protease, lipase, and amylase, which facilitates the degradation of feed for nutrient, absorption, and utilization of feed (Rozs et al., 2001). It is reported that B. licheniformis can produce antimicrobial active substances and has a unique mechanism about reacting with oxygen that inhibits the growth and reproduction of pathogens (Kim et al., 2004), and promotes the growth and homeostasis of the intestine to adjust the intestinal flora for the recovery of bowel functions. However, little research has been conducted on the use of B. licheniformis in laying hens; therefore, a dearth of information about the effects of probiotics in vivo on laying hens inspired us to focus on the issue. Thus, the objectives of this study were to investigate the effect of dietary supplemental B. licheniformis on laying performance and egg quality, serum hormone, antioxidant enzyme activities, and intestinal barrier functions.

MATERIALS AND METHODS Birds and Management A total of 540 Hy-Line Variety W-36 hens, 28 wk of age, were randomly divided into 6 groups, each of which had 6 replicates of 15 hens. Three hens were housed per cage under the same management conditions in a windowed poultry house. This trial lasted from 28 to 38 wk of age, including a 2-wk acclimatization period and 8-wk experimental period. During the experimental period, birds were fed the diets ad libitum twice daily at 0800 and 1600 h and allowed free access to water with a photoperiod of 16L:8D. The average ambient RH inside the barn was 55 ± 5% and the mean daily temperature was 23 ± 2°C. The experiment was carried out in accordance with the Chinese guidelines for animal welfare and approved by the animal welfare committee of Animal Science College, Zhejiang University.

Diets and Bacterial Strains All hens were fed the same basal diet to which Bacillus cultures were added to derive treatments (Table 1). Bacillus licheniformis cells were suspended in skim milk powder (2 × 1010 cfu/g) by the Laboratory of

Table 1. Composition and nutrition of the basal experimental diet (%) to which Bacillus was added1 Item Ingredient  Maize   Soybean meal   Wheat bran   Fish meal  CaHPO4  Limestone  Salt   dl-Methionine   l-Lysine   Soybean oil  Premix2 Calculated value   ME (MJ/kg)   CP (g/kg)   Calcium (%)   Total phosphorus (%)   Available phosphorus (%)   Lysine (%)   Methionine + cysteine (%)

Value 60.20 24.00 3.11 2.24 1.00 7.00 0.33 0.11 0.01 1.00 1.00 11.50 187 3.34 0.59 0.38 1.04 0.75

1Bacillus licheniformis cells were suspended in skim milk powder (2 × 1010 cfu/g); B. licheniformis powders (2 × 1010 cfu/g) were added to the basal diet at 0 (control), 0.01% (2 × 106 cfu/g of diet), 0.02% (4 × 106 cfu/g of diet), 0.03% (6 × 106 cfu/g of diet), 0.06% (1.2 × 107 cfu/g of diet), and 0.09% (1.8 × 107 cfu/g of diet) level. 2Premix provided per kilogram of diet: retinyl palmitate, 3.96 mg; cholecalciferol, 0.07 mg; dl-α-tocopheryl acetate, 20 mg; menadione sodium bisulfite, 4 mg; thiamine mononitrate, 1.63 mg; riboflavin, 5 mg; niacin, 30 mg; pantothenic acid, 10 mg; folic acid, 0.5 mg; biotin, 0.22 mg; choline chloride, 250 mg; cyanocobalamin, 0.012 mg; Cu, 8 mg; Fe, 30 mg; I, 0.6 mg; Mn, 50 mg; Se, 0.12 mg; Zn, 40 mg.

Microbiology, Institute of Feed Sciences, Zhejiang University, PR China. Bacillus licheniformis powders (2 × 1010 cfu/g) were added to the basal diet at levels of 0 (control), 0.01% (2 × 106 cfu/g of diet), 0.02% (4 × 106 cfu/g of diet), 0.03% (6 × 106 cfu/g of diet), 0.06% (1.2 × 107 cfu/g of diet), and 0.09% (1.8 × 107 cfu/g of diet). The experimental diet was stored in a dry and well-ventilated storeroom.

Laying Performance and Egg Quality Hen-day egg production, feed consumption, egg weight, and hen mortality were recorded daily. Feed conversion ratio was calculated as grams of feed intake per gram of egg mass. At 38 wk of age, 12 eggs from each replicate were randomly collected to assess egg quality parameters. Albumen height, Haugh units, yolk color, eggshell thickness, and eggshell strength were measured with a digital egg tester after eggs were weighed and cracked open.

Blood Sampling At the end of the experiment, after 12 h of feed withdrawal, blood samples of 12 hens (2 birds per replicate) were drawn from the axillary vein into vacuum tubes (5 mL) containing coagulant and centrifuged for 10 min (3,000 × g) at 4°C. Pure serum samples were aspirated

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by pipette, stored in sterilized 1.5-mL Eppendorf tubes at −80°C, and thawed at 4°C before analysis.

Antioxidant Enzyme Estimation Assay kits for superoxide dismutases (SOD), glutathione (GSH), catalse (CAT), total antioxidant capacity (T-AOC), malondialdehyde (MDA), glutathione reductase (GR), glutathione S-transferase (GST), and thioredoxin reductase protein (TrxR) were obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The GR, GST, TrxR, d-lactate, and diamine oxidase levels were measured by ELISA with commercial kits provided by Hangzhou Nuoyang Biotechnology Co. Ltd. (Hangzhou, China), whereas SOD (Winterbourn et al., 1975), GSH (Zakowski and Tappel, 1978), CAT (Beers and Sizer, 1952), T-AOC (Erel, 2004), and MDA (Gomez et al., 1998) levels were measured by spectrophotometric methods using a spectrophotometer.

Serum Hormone Determination Concentrations of serum luteinizing hormone (LH), progesterone (P), follicle-stimulating hormone (FSH), estradiol (E2), adrenocorticotrophic hormone (ACTH), and corticosterone (CORT) were measured by ELISA with commercial kits provided by Hangzhou Nuoyang Biotechnology Co. Ltd. (Hangzhou, China).

Histological Structure and Assessment of Intestinal Function One-centimeter lengths from the medial portions of jejunum were washed in physiological saline solution and fixed in 10% buffered formalin. Tissue samples were later embedded in paraffin, and a 20-μm section of each sample was placed on a glass slide and stained with hematoxylin and eosin (Thompson and Richter, 1960). The villus was observed under a Nikon microscope (Nikon Corp., Tokyo, Japan). Villus height was measured from the top of the villus to the villus crypt junction and crypt depth measured as the depth of the invagination between adjacent villus (total of 5 samples for intestinal segments and 30 measurements of each intestinal segment per group).

Statistical Analysis Data were statistically analyzed by one-way ANOVA procedure of SPSS 16.0 for Windows (SPSS Inc., Chicago, IL). When significant differences were found (P < 0.05), Tukey’s test was further performed. Statements of significance were based on P < 0.05. The data were expressed as the means ± SEM.

RESULTS AND DISCUSSION Laying Performance and Egg Quality Dietary supplementation with different levels of B. licheniformis did not show any adverse effects on laying performance of hens. The results clearly showed that all treatment groups had higher egg production and egg mass output compared with the control through a 56-d trial period (Table 2). Hens receiving B. licheniformis at 0.01 and 0.06% had improved egg production over hens fed the basal diet devoid of Bacillus. The highest egg production (98.4%) was from the 0.01% B. licheniformis group, and the lowest (94.0%) from the control group. Although diets containing 0.02, 0.03, and 0.09% B. licheniformis resulted in intermediate hen egg production, it has been shown that hens fed 250, 500, and 750 mg of probiotic (3.2 × 109 cfu/g)/kg of diet had improved egg production (Kurtoglu et al., 2004). However, the impact of probiotics on hen egg production varies as Li et al. (2006) and Yalçin et al. (2010) showed no effect of probiotics on hen egg production. Similar to egg production, hens fed 0.01 and 0.06% B. licheniformis had higher egg mass than hens fed the control diet, and hens fed diets containing 0.02, 0.03, and 0.09% B. licheniformis had intermediate hen egg mass. However, differences among treatments for egg weight did not occur. There were also no significant differences for feed consumption and feed-conversion efficiency among all 6 groups, which were in agreement with previous findings (Goodling et al., 1987; Nahashon et al., 1994; Mohan et al., 1995). In contrast, Nahashon et al. (1994) showed that addition of probiotics could improve feed consumption and decrease feed conversion efficiency, whereas Thanh et al. (2009) showed that feeding metabolite combinations produced by Lactobacillus plantarum decreased feed conversion ratio.

Table 2. Laying performance of the hens fed diets without or with Bacillus licheniformis B. licheniformis (%) 0 0.01 0.02 0.03 0.06 0.09 a,bDifferent

Egg production (%) 94.0 98.4 96.6 96.3 97.9 96.1

± ± ± ± ± ±

0.4b 0.6a 0.6ab 0.3ab 0.2a 0.7ab

Egg weight (g)

Egg mass (g/hen per d)

57.8 57.9 57.9 58.3 58.2 58.1

54.3 57.0 55.7 56.1 57.0 55.8

± ± ± ± ± ±

0.8 0.5 0.6 0.3 0.4 0.1

± ± ± ± ± ±

0.6b 0.8a 0.5ab 0.1ab 0.5a 0.5ab

Feed consumption (g/hen per d) 112.8 113.1 116.6 116.0 112.8 113.9

± ± ± ± ± ±

0.7 4.1 2.0 2.0 1.9 0.1

superscripts in the same column indicate values significantly different (P < 0.05) among the groups.

Feed conversion (g/g) 2.06 1.99 2.09 2.06 1.97 2.04

± ± ± ± ± ±

0.03 0.10 0.03 0.04 0.03 0.02

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Table 3. Internal and external quality of the eggs from hens fed diets without or with Bacillus licheniformis at different levels B. licheniformis (%)

Albumen height (mm)

0 0.01 0.02 0.03 0.06 0.09 a–dDifferent

6.50 6.78 6.64 6.95 6.93 6.67

± ± ± ± ± ±

Yolk color

0.15b 0.16ab 0.12ab 0.12a 0.13a 0.11ab

6.86 7.00 6.95 7.20 6.93 6.51

± ± ± ± ± ±

Eggshell thickness (mm)

Haugh units

0.09b 0.11ab 0.10ab 0.10a 0.11ab 0.08c

79.6 82.0 82.1 82.7 86.5 81.6

± ± ± ± ± ±

1.4b 1.4b 0.7b 1.0b 1.5a 0.7b

0.303 0.332 0.324 0.342 0.327 0.319

± ± ± ± ± ±

Eggshell strength (N)

0.004d 0.004ab 0.003bc 0.005a 0.004bc 0.003c

33.91 38.12 37.44 38.51 38.51 36.85

± ± ± ± ± ±

0.08c 0.08ab 0.08ab 0.09a 0.10a 0.06ab

superscripts in the same column indicate values significantly different (P < 0.05) among the groups.

Some dietary levels of B. licheniformis improved egg quality, but responses were not dose dependent (Table 3). Compared with the control, albumen height was significantly increased in 0.03 and 0.06% B. licheniformis group, whereas Haugh units increased only in the 0.06% group. This effect may be related to increased protein synthesis and the transfer of water from yolk. Our results differ from those of Aghaii et al. (2010), who demonstrated that the addition of probiotics had no effects on albumen height and Haugh units. The thickness and strength of egg shell were significantly increased in all B. licheniformis treated groups compared with the control. The 0.09% B. licheniformis group, among all the 5 treatments, showed the least improvement in thickness and strength of eggshell. There were no significant differences in the Haugh units and eggshell strength among all 5 treatment groups. That may be associated with the ability of probiotics to decrease pH and improve intestinal barrier function (Resta-Lenert and Barrett, 2003). Lower pH and intestinal epithelium functions both are required for the dissolution of calcium and phosphorus to promote absorption and utilization (Li et al., 2006). Another study reported that calcium and phosphorus retention were improved in layers when the diet was supplemented with Lactobacillus (Nahashon et al., 1994). The darkest yolk color score was from the 0.03% B. licheniformis group, whereas the lightest was from the control. The findings are in agreement with those of Nahashon et al. (1994) and Mohan et al. (1995), who reported that a slight improvement in yolk color was observed when hens were supplemented with probiotics during the peak period of laying. Because our treatments varied greatly, we conclude that the impact of probiotics on egg yolk color is unclear. The difference in laying performance and egg quality among the various studies might be due to

the different probiotic species, trial length, and environmental conditions.

Intestinal Barrier Function d-Lactate is a byproduct of bacterial metabolism; it is neither produced nor metabolized by mammalian cells. The resident microflora in an ischemic segment of bowel rapidly proliferate and overpopulate; consequently, the mucosal barrier of the gut begins to break down (Murray et al., 1993). Plasma d-lactate levels may be a useful marker to evaluate the degree of intestinal injury and gut barrier dysfunction. Diamine oxidase (DAO) is an enzyme found in high concentration in the intestinal mucosa of humans and other mammalian species, and its activity serves as a marker of mucosal maturation and integrity (Luk et al., 1980). The DAO activity in serum is a marker of the total mass of functional to enterocytes; a decreased level during gastroenteritis is reflected in a decrease of serum DAO activity (Forget et al., 1985). This study showed that B. licheniformis supplementation in the diet significantly decreased the d-lactate level and diamine oxidase activity (Table 4). The lowest values for d-lactate and diamine oxidase were from the 0.01 and 0.03% B. licheniformis groups, respectively. These changes are due to the ability of probiotics to modulate the intestinal microbial environment in favor of the host. The structure of the intestinal mucosa can reveal some information on gut health. Increased villus height indicates increased surface area for nutrient absorption, and deeper crypt indicates fast tissue turnover and a high demand for new tissue. In the present study, compared with the control, the B. licheniformis treated groups showed better intestinal integrity (Figure 1). Hence, the addition of different levels of B. lichenifor-

Table 4. Intestinal barrier function of laying hens fed diets without or with Bacillus licheniformis at different levels B. licheniformis (%)

d-Lactate (μmol/L)

0 0.01 0.03 0.06

5.3 2.2 2.6 2.7

a–dDifferent

± ± ± ±

0.7b 0.3a 0.6a 0.3a

Diamine oxidase (ng/L) 761.6 337.1 320.3 483.6

± ± ± ±

24.5c 25.6a 49.7a 32.1b

Villus height (μm) 211.2 228.8 232.5 286.0

± ± ± ±

1.9c 1.2b 2.4b 2.0a

Crypt depth (μm) 45.7 31.1 41.7 49.6

± ± ± ±

1.1b 0.6d 0.9c 1.0a

superscripts in the same column indicate values significantly different (P < 0.05) among the groups.

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Figure 1. Structure of the intestinal mucosa of hens fed control versus hens fed various Bacillus licheniformis levels. Color version available in the online PDF.

mis significantly increased villus height and decreased the crypt depth (Table 4). Similarly, feeding of the metabolite combination produced by Lactobacillus plantarum increased small intestinal villus height (Thanh et al., 2009). The improvement of the structure of the intestinal mucosa in B. licheniformis-treatment groups might lead to prolific nutrient absorption, and increase disease resistance and decrease diarrhea-producing factors (Xu et al., 2003).

in the probiotic-treated groups were increased significantly, whereas ACTH level was significantly decreased compared with the control. The level of P was significantly decreased in 0.01 and 0.06% B. licheniformis supplemented groups compared with the control, but the level of P was numerically improved in 0.03% B. licheniformis group to the control level. The ACTH is often produced in response to biological stress (Raikhinstein and Hanukoglu, 1993). Follicle-stimulating hormone is synthesized and secreted by gonadotrophs of the anterior pituitary gland, which regulates the development, growth, pubertal maturation, and reproductive processes and stimulates the growth and recruitment of immature ovarian follicles in the ovary (Radu et al.,

Serum Hormone No differences were observed among the 6 groups for CORT and LH (Table 5). The levels of E2 and FSH

Table 5. Serum hormone levels of laying hens fed diets without or with Bacillus licheniformis at different levels1 B. licheniformis (%) 0 0.01 0.03 0.06 a–cDifferent

ACTH (ng/L) 136.9 87.2 75.9 95.4

± ± ± ±

10.5b 10.0a 5.9a 6.0a

CORT (ng/L) 363.3 218.8 327.0 205.5

± ± ± ±

12.2 13.6 17.7 10.9

E2 (ng/L) 21.0 78.1 94.1 81.0

± ± ± ±

7.2b 8.8a 20.7a 17.0a

FSH (mIU/mL) 2.7 5.3 5.1 5.2

± ± ± ±

0.1b 0.6a 0.6a 0.2a

LH (mIU/mL) 5.6 5.4 6.4 5.3

± ± ± ±

2.0 0.4 1.9 1.2

P (ng/mL) 218.3 106.4 235.4 119.5

± ± ± ±

40.0ab 25.1c 46.3a 28.2bc

superscripts in the same column indicate values significantly different (P < 0.05) among the groups. of serum luteinizing hormone (LH), progesterone (P), follicle-stimulating hormone (FSH), estradiol (E2), adrenal cortical hormone (ACTH), and corticosterone (CORT) were measured by ELISA with commercial kits provided by Hangzhou Nuoyang Biotechnollgy Co. Ltd. (Hangzhou, China). 1Concentrations

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Table 6. Antioxidant enzyme activities of laying hens fed diets without or with Bacillus licheniformis at different levels1 B. licheniformis (%) 0 0.01 0.03 0.06

CAT (U/mL) 19.8 15.7 19.2 15.7

± ± ± ±

3.3 0.7 2.2 2.1

GR (ng/mL) 2.3 1.8 2.6 2.1

± ± ± ±

0.5 0.1 0.4 0.2

GST (ng/mL) 2.8 3.3 5.5 4.5

± ± ± ±

0.7c 0.2bc 0.7a 0.3ab

TrxR (U/L) 42.8 45.9 57.4 48.8

± ± ± ±

7.9 5.7 9.4 5.2

T-AOC (U/mL) 4.3 4.3 3.0 4.2

± ± ± ±

0.5 0.6 0.4 0.7

SOD (U/mL) 462.9 457.9 366.6 392.3

± ± ± ±

19.0 32.4 21.7 22.6

GSH (μmol/L) 375.0 394.1 535.0 365.5

± ± ± ±

49.6ab 5.1ab 70.1a 40.0b

MDA (nmol/mL) 10.3 8.1 11.4 9.9

± ± ± ±

1.3 1.3 0.4 0.4

a–cDifferent

superscripts in the same column indicate values significantly different (P < 0.05) among the groups. (CAT), glutathione reductase (GR), glutathione S-transferase (GST), thioredoxin reductase protein (TrxR), total antioxidant capacity (T-AOC), superoxide dismutases (SOD), glutathione (GSH), and malondialdehyde (MDA). 1Catalase

2010). The lower level of ACTH implied that addition of B. licheniformis could alleviate biological stress. The increased levels of E2 and FSH resulting from addition of B. licheniformis may be related to lower serum ACTH, which reduces inhibition of the reproductive hormone secretion (Matteri et al., 1984). It was also reported that increased serum ACTH inhibits gonadotropin secretion (Dobson and Smith, 1995).

mance of hens by improving villus structure, sustaining a balanced intestinal barrier function, decreasing the stress response, and regulating hormone secretion. Therefore, the probiotic B. licheniformis may be useful for ameliorating certain adverse influences on production and gut health of laying hens.

Activity of Antioxidant Enzymes

This study was supported by the Key Science and Technology Program of Zhejiang Province, China (no. 2006C12086). The research was designed by W. F. Li; Kai Lei, Y. L. Li, and I. R. Rajput conducted the research; D. Y. Yu analyzed the data; and Kai Lei completed writing the paper.

The antioxidant defenses include antioxidants (vitamin C, vitamin E, and uric acid) and antioxidant enzymes present in the biological system (Sies, 1991). Supplementation with 0.03 and 0.06% B. licheniformis improved GST activity (Table 6) over the control, and there were no differences for other antioxidant enzyme activities among all groups. The GST is a soluble protein that catalyzes the conjugation of GSH with many xenobiotics, and their reactive metabolites could form more water-soluble compounds (Cai and Harrison, 2000). The finding of Matés (2000) showed that expression of GST was regulated by a common antioxidant response element, which suggested that their gene products play a protective role against oxidative damage in various tissues by neutralizing reactive oxygen species. Consistent with the antioxidant enzyme activity of serum, there was no significant effect of treatment on T-AOC and MDA among groups, but a slight numerical decrease was observed in MDA contents. The MDA endogenously reflects the lipid peroxidation (Yang et al., 2008), which is the consequence of diminished antioxidant protection when levels of reactive oxygen species increase (Yang et al., 2008). The findings of Capcarova et al. (2010) revealed that T-AOC in broiler chickens was significantly increased in both Lactobacillus fermentum and Enterococcus faecium supplemented groups, but the activities of antioxidant enzymes were not determined. The discrepancy among these studies is likely due to the different animals, physiological status, diet compositions, the probiotic source, and their application levels. All together, our results demonstrated that supplementation of B. licheniformis has no impact on improving the antioxidant enzymes activities in laying hens except for GST. In conclusion, inclusion of B. licheniformis in the hen diet was effective in increasing egg production perfor-

ACKNOWLEDGMENTS

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