Effects of pre-encapsulated and pro-encapsulated Enterococcus faecalis on growth performance, blood characteristics, and cecal microflora in broiler chickens

Effects of pre-encapsulated and pro-encapsulated Enterococcus faecalis on growth performance, blood characteristics, and cecal microflora in broiler chickens L. Zhang,∗ J. Li,∗ T. T. Yun,† W. T. Qi,†,1 X. X. Liang,† Y. W. Wang,† and A. K. Li†,1 ∗

Northeast Agricultural University, Harbin, Heilongjiang 150030, P. R. China; and † Cereals & Oils Nutrtion Research Group, Academy of Sci. & Tech. of State Administration of Grain, Xicheng, Beijing 100037, P. R. China

Key words: pre-microencapsulation, pro-microencapsulation, Enterococcus faecalis, broiler chicken, growth performance 2015 Poultry Science 00:1–10 http://dx.doi.org/10.3382/ps/pev262

INTRODUCTION

ity, high pelleting temperature and pressure, drying, and other processes can lead to decreased probiotic activity in feed processing by the animals (Saarela et al., 2000). Furthermore, many reports have indicated that there is poor survival of bacteria in products containing free probiotic cells during passage through the upper gastrointestinal system (De Vos et al., 2010). Several approaches that increase the resistance of these sensitive microorganisms to adverse environmental conditions have been reported and include the appropriate selection of acid- and bile-resistant strains, stress adaptation, and microencapsulation (Gismondo et al., 1999). Microencapsulation has been suggested as an effective approach and has been suggested for use in targeted delivery to the gastrointestinal tract (Gerez et al., 2012). The vast majority of applications of microcapsules to living cells can be divided into 2 general types: pre-encapsulation and pro-encapsulation. In preencapsulation, a small volume of cells is mixed with

Residues of antibiotic growth promoters (AGP) in animal products and in the environment have brought about bans on in-feed AGP in many countries (Van den Bogaard and Stobberingh, 2000). Probiotics serve as alternatives to AGP in animal production. Recently, there has been an explosion of probiotic, health-based products, and the addition of probiotics to diets has improved growth performance, intestinal microflora and morphology, modulated immunity, and prevented some cancers (Kailasapathy and Chin, 2000; Mountzouris et al., 2010; Lee et al., 2014; Zhang and Kim 2014). However, adverse conditions that include high humid C 2015 Poultry Science Association Inc. Received June 2, 2015. Accepted July 18, 2015. 1 Corresponding author: E-mail: [email protected] [email protected]

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than the CON group and their FCR was decreased significantly (P < 0.05). During the entire feeding period, only the PRE group showed greater (P < 0.05) ADG and lower (P < 0.05) FCR. On day 21, only birds in the PRE group had greater (P < 0.05) total antioxidant capacity and number of Lactobacillus than the CON group. On day 42, The PRE group showed greater (P < 0.05) superoxide dismutase than the CON group. Serum IgA and IgM concentrations were increased (P < 0.05) in the PRE group. Serum IL-6 in the PRE group was greater (P < 0.05) than in the other groups with the exception of ANT. At the phylum level, Firmicutes was enriched (P < 0.05) and Proteobacteria was depleted (P < 0.05) only in the PRE group. At the genus level, only the PRE diets increased (P < 0.05) the number of both Lactobacillus and Enterococcus. The results indicate that pre-encapsulation assists the efficient functioning of probiotics in broilers.

ABSTRACT The effects of microencapsulation of Enterococcus faecalis on the growth performance, antioxidant activity, immune function, and cecal microbiota in broilers were investigated. Broilers (1-day-old) were assigned randomly as follows: 5 treatments, 5 replicate pens per treatment, and 20 broilers per pen. Treatments included (1) a basal diet (CON), (2) CON + Aureomycin (1 g/kg of diet) (ANT), (3) CON + free non-encapsulated probiotics (1 × 109 cfu/kg of diet) (FREE), (4) CON + proencapsulated probiotics (1 × 109 cfu/kg of diet) (PRO), and (5) CON + pre-encapsulated probiotics (1 × 109 cfu/kg of diet) (PRE). Feedings included starter (1 to 21 d) and grower (21 to 42 d) phases. In the starter phase, the ANT and the PRE groups had greater (P < 0.05) ADG than the CON groups, and the feed conversion ratio (FCR) for these 2 groups was decreased (P < 0.05). In the finisher phase, the PRE and PRO groups had greater (P < 0.05) ADG

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ZHANG ET AL.

MATERIALS AND METHODS All procedures were performed in accordance with the guidelines set forth by the Animal Welfare Committee of Northeast Agricultural University (Harbin, China).

Bacterial Strains Enterococcus faecalis (CGMCC No. 9353) was cultured under pre-encapsulated and free nonencapsulated conditions in man rogosa sharpe (MRS) broth at 37◦ C under anaerobic conditions for 18 h. The bacterial cells were counted by plating onto MRS agar plates and collected by centrifuge. Then the cells were washed with sterilized water and resuspended in fresh MRS broth in the targeted numbers.

Preparation of Microcapsules The pre-encapsulation of the probiotics was performed based on a modification of the emulsion method described by Krasaekoopt et al. (2003). Briefly, 100 mL of sterile 1.8% (w/v) sodium alginate and 5 mL of washed and concentrated probiotic bacteria (1 × 108 cfu/mL) were mixed with 0.45 g of calcium carbonate dissolved in 300 μL sterile water. After homogenization, the mixture was dispersed into a paraffin oil phase, which contained 0.2% (w/v) Span 80, and emulsified for 5 min by stirring at 400 rpm. Then, 900 μL of

glacial acetic acid dissolved in 10 mL of paraffin oil was added, and stirring continued for 10 min. Clean, sterile water (100 mL) was added to the emulsification system to draw the microbeads into the water phase. The oil layer on the top phase was harvested by aspiration, and centrifuged for the next use. After being washed with sterile water 3 times, the microencapsulated bacteria were transferred to the new MRS medium to continue growing. Finally, the growth of the microencapsulated probiotics were harvested and dried using a fluidized bed. The pro-encapsulation of the probiotics was performed as follows. The bacteria were collected and mixed with glycerol at a mass ratio of 10:6 after which 0.4% whey protein isolate, 8% modified starch, and 10% glucose were added. The number of cells was approximately 5 × 1010 cfu/g. The mixture to be microencapsulated by spray drying was microencapsulated using a laboratory spray dryer to apply aqueous solutions of maltodextrin mixed with gelatin. The microcapsule powder was collected at the bottom of the dryer’s cyclone. The free, non-encapsulated cultured bacteria were harvested and dried in a fluidized bed for use as a control.

Birds, Diets, and Experimental Design Five hundred 1-day-old Arbor Acre male broilers were randomly allotted to 5 treatments with 5 replicate pens per treatment and 20 chickens per pen. All birds were raised in stainless steel pens (1.75 × 1.55 m) under continuous light in a controlled room. The room temperature was maintained at 35◦ C for the first 3 d, after which the temperature was reduced to 24◦ C until the end of the experiment. A standard management procedure was used throughout the experiment. The broilers were allowed ad libitum access to feed and drinking water. All birds were offered the same antibiotic-free basal diet. The dietary treatments included (1) a basal diet (CON), (2) CON + aureomycin (1 g/kg of diet) (ANT), (3) CON + free non-encapsulated probiotics (1 × 109 cfu/kg of diet) (FREE), (4) CON + proencapsulated probiotics (1 × 109 cfu/kg of diet) (PRO), and (5) CON + pre-encapsulated probiotics (1 × 109 cfu/kg of diet) (PRE). Feeding was divided into 2 phases: the starter phase from 1 to 21 d and the grower phase from 21 to 42 d. The basal diet was formulated to meet the nutritional requirements suggested by the NRC (NRC, 1994; Tables 1 and 2).

Growth Performance On days 21 and 42, BW and feed intake for each pen were measured after a 12 h fast. ADG, ADFI, and feed conversion ratio (FCR) were calculated during the starter (1 to 21 d), grower (21 to 42 d), and overall (1 to 42 d) phases.

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the encapsulation materials to produce the microcapsules, after which the capsules loaded with the probiotics are cultured by adding new media. When the microbial cells are mature and contain the microcapsules, the pre-encapsulated products were prepared. For pre-encapsulation, the most suitable method is emulsion, which is a method first developed by Nilsson et al. (1983). In pro-encapsulation, fully-grown probiotic cells are collected and mixtures of cell concentrates are dried with aqueous solutions of various polymers, such as modified starch, gelatin, whey protein isolate, maltodextrin mixed with gum arabic, ß-cyclodextrin mixed with gum arabic, etc. to produce the capsulated products (Thomas et al., 2012). For the pro-encapsulation, the most popular method is spray drying. In recent years, spray drying has been utilized to encapsulate probiotic cells as an alternative to the encapsulation methods based on emulsion. Enterococcus faecalis, a group of lactic acid bacteria that occur naturally in the human intestine, has been developed for commercial use as a probiotic for animals (Pollmann et al., 2005). Therefore, E. faecalis was pre- and pro-encapsulated by emulsion and spray drying, respectively, in this study, and the effects of these treatments were evaluated according to the growth performance, immune function, antioxidant system, and cecal microflora in broiler chickens.

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EFFECTS OF ENTEROCOCCUS FAECALIS IN BROILERS Table 1. Composition and nutrient contents of diets in the starter phase (1 to 21 d).

Table 2. Composition and nutrient contents of diets in the grower phase (21 to 42 d).

Treatment1

Treatment1

CON

ANT

FREE

PRO

PRE

Item

CON

ANT

FREE

PRO

PRE

Ingredients (%) Corn Soybean meal Cottonseed meal Corn gluten meal Wheat middlings Soybean oil Limestone NaCl Dicalcium phosphate DL- Methionine L- Lysine HCl Antioxygen Vitamin premix2 Choline chloride 2 Trace mineral premix3

53.33 22.65 4.62 10.00 3.00 2.37 1.27 0.35 1.75 0.20 0.02 0.02 0.02 0.20 0.20

53.33 22.65 4.62 10.00 3.00 2.37 1.27 0.35 1.75 0.20 0.02 0.02 0.02 0.20 0.20

53.33 22.65 4.62 10.00 3.00 2.37 1.27 0.35 1.75 0.20 0.02 0.02 0.02 0.20 0.20

53.33 22.65 4.62 10.00 3.00 2.37 1.27 0.35 1.75 0.20 0.02 0.02 0.02 0.20 0.20

53.33 22.65 4.62 10.00 3.00 2.37 1.27 0.35 1.75 0.20 0.02 0.02 0.02 0.20 0.20

Ingredients (%) Corn Soybean meal Cottonseed meal Corn gluten meal Wheat middlings Soybean oil Limestone NaCl CaHPO4 DL-Met L-Lys·HCl Antioxygen Vitamin premix2 Choline chloride2 Trace mineral premix3

60.37 15.58 7.30 5.00 5.00 3.13 1.21 0.35 1.49 0.12 0.01 0.02 0.02 0.20 0.20

60.37 15.58 7.30 5.00 5.00 3.13 1.21 0.35 1.49 0.12 0.01 0.02 0.02 0.20 0.20

60.37 15.58 7.30 5.00 5.00 3.13 1.21 0.35 1.49 0.12 0.01 0.02 0.02 0.20 0.20

60.37 15.58 7.30 5.00 5.00 3.13 1.21 0.35 1.49 0.12 0.01 0.02 0.02 0.20 0.20

60.37 15.58 7.30 5.00 5.00 3.13 1.21 0.35 1.49 0.12 0.01 0.02 0.02 0.20 0.20

Total

100.00

100.00

100.00

100.00

100.00

Total

100.00

100.00

100.00

100.00

100.00

2.91

2.91

2.91

2.91

19.85 5.02 3.07 1.02 0.41 0.92 0.59

19.84 5.01 3.08 1.03 0.42 0.91 0.59

19.86 5.02 3.07 1.03 0.42 0.92 0.59

19.86 5.02 3.07 1.02 0.42 0.91 0.60

Calculated composition ME, Mcal/kg 2.80 Analyzed composition4 (%) CP 21.34 Crude fat 4.81 Crude fiber 3.47 Lysine 1.13 Methionine 0.50 Calcium 1.01 Total phosphorus 0.62

2.80

2.80

2.80

2.80

21.35 4.82 3.47 1.13 0.51 1.02 0.62

21.34 4.83 3.47 1.14 0.50 1.02 0.62

21.34 4.84 3.46 1.14 0.52 1.03 0.63

21.35 4.83 3.48 1.15 0.51 1.03 0.63

1 Treatments: CON = a basal diet; ANT = CON + 1 g of Aureomycin per kg diet; FREE = CON + 1 × 109 cfu of free non-encapsulated probiotics/kg of diet; PRO = CON + 1 × 109 cfu of pro-encapsulated probiotics/kg of diet; PRE = CON + 1 × 109 cfu of pre-encapsulated probiotics/kg of diet. 2 Vitamin premix provided per kilogram of diet: vitamin A, 9,500 IU; vitamin D3 , 62.5 μ g; vitamin K3 , 2.65 mg; vitamin B12 , 0.025 mg; vitamin B2 , 6 mg; vitamin E, 30 IU; biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg; nicotinic acid, and 50 mg. 3 The multimineral provided the following per kilogram of diet: Cu, 8 mg; Zn, 75 mg; Fe, 80 mg; Mn, 100 mg; Se, 0.15 mg; and I, 0.35 mg. 4 CP, crude fat, crude fiber, calcium, total phosphorus were determined by routine methods. Lysine and Methionine were detected by automatic amino acid analyzer. Each diet was measured for 6 replicates.

Blood Characteristics At 21 and 42 days of age, fifteen broiler chickens (3 from each replicate pen) were randomly selected from each treatment, and blood samples were collected from the wing vein. Blood samples (10 mL) were collected in silica vacutainer tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) and were then centrifuged at 3,000 × g for 15 min at 4◦ C; then the serum was separated from each sample, and these samples were stored at −20◦ C until they could be analyzed (Becton Dickinson Vacutainer Systems). Reagent kits for malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, catalase (CAT), IgG, IgM, IgA, IL-2, and IL-6 were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The MDA level was analyzed with 2-thiobarbituric acid, SOD activity was measured by the WST-1 method, T-AOC capacity was determined by the ABTS method, and CAT activity was detected

Calculated composition ME (Mcal/kg) 2.91 Analyzed composition4 (%) CP 19.85 Crude fat 5.02 Crude fiber 3.08 Lysine 1.02 Methionine 0.41 Calcium 0.91 Total phosphorus 0.59

1 Treatments: CON = a basal diet; ANT = CON + 1 g of Aureomycin per kg diet; FREE = CON + 1 × 109 cfu of free non-encapsulated probiotics /kg of diet; PRO = CON + 1 × 109 cfu of pro-encapsulated probiotics/kg of diet; and PRE = CON + 1 × 109 cfu of pre-encapsulated probiotics/kg of diet. 2 Vitamin premix provided per kilogram of diet: vitamin A, 9,500 IU; vitamin D3 , 62.5 μ g; vitamin K3 , 2.65 mg; vitamin B12 , 0.025 mg; vitamin B2 , 6 mg; vitamin E, 30 IU; biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg; and nicotinic acid, 50 mg. 3 The multimineral provided the following per kilogram of diet: Cu, 8 mg; Zn, 75 mg; Fe, 80 mg; Mn, 100 mg; Se, 0.15 mg; and I, 0.35 mg. 4 CP, crude fat, crude fiber, calcium, total phosphprus were determined by routine methods. Lysine and Methionine were detected by automatic amino acid analyzer. Each diet was measured for 6 replicates.

with visible spectroscopy. Serum IgG, IgM, IgA, IL-2, and IL-6 were determined by double-antibody sandwich ELISA.

Cecal Microbiome Twenty-five samples of cecal content (5 from each treatment group) were randomly selected on days 21 and 42 respectively. Samples were collected in sterile containers, packed carefully, and then frozen by immersion in liquid N2 and stored at -80◦ C. The cecal microbiota was analyzed using high throughput sequencing, including DNA extraction, PCRamplified 16S rRNA, amplicon sequencing, and sequence data processing. Microbial genomic DNA was extracted from the cecal contents using a Power Soil DNA Isolation Kit (Mobio Laboratories Inc., Carlsbad, CA) following the manufacturer’s recommendation. Using barcoded fusion primers, we PCR-amplified

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Item

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ZHANG ET AL. Table 3. Growth performance of broilers in 5 experimental feeding treatments. Treatment1 Item ADG (g) d 1–21 d 21–42 d 1–42 ADFI (g) d 1–21 d 21–42 d 1–42 FCR3 d 1–21 d 21–42 d 1–42

CON

ANT

FREE

PRO

PRE

SEM2

P-value2

24.00c 65.27b 48.95b

26.87a,b 68.39a,b 51.28a,b

24.14b,c 67.67a,b 49.98a,b

25.26a,b,c 70.87a 50.64a,b

27.34a 71.19a 52.95a

0.36 0.39 0.29

0.045 0.022 0.035

45.20 151.31 101.58

45.04 150.48 101.47

44.33 151.35 101.12

45.35 152.45 102.33

44.99 152.10 101.45

0.36 0.41 0.30

0.930 0.787 0.962

1.90a 2.32a 2.08a

1.68b 2.21a,b 1.98a,b

1.84a,b 2.24a,b 2.03a,b

1.80a,b 2.15b 2.01a,b

1.66b 2.14b 1.93b

0.02 0.03 0.02

0.032 0.027 0.033

1

the V3 hypervariable region of the 16S rRNA gene from the microbial genomic DNA that was harvested from the cecal contents. The primers used were 5 CCTACGGGAGGCAGCAG-3 with adapter A (forward primer) and 5 -ATTACCGCGGCTGCTGG-3 with adapter B (reverse primer). The PCR conditions were 94◦ C for 5 min; 94◦ C for 30 s, 48◦ C for 30 s, and 72◦ C for 30 s, repeated for 25 cycles; and 72◦ C for 10 min. The PCR product was excised from a 2% agarose gel and purified with a QIAGEN MinElute Gel Extraction Kit (Qiagen, Hilden, Germany). The final sequencing library was prepared by mixing equal amounts of purified PCR products and then adding poly (A) to repair the ends. Thereafter, the amplicons were connected with sequencing adapters. Following agarose gel electrophoresis, suitable fragments were selected as templates for PCR amplification. Finally, the library was sequenced by the Illumina MiSeq (San Diego, CA). Following sequencing, all barcodes were sorted, removed, and reads were assessed for quality. To reduce random sequencing errors, those sequences with lengths shorter than 100 bp, mismatches in PCR primers, more than one undetermined nucleotide, and an average phred quality of ≤25 were eliminated. Barcode and sequencing primers were trimmed from the assembled sequences. Trimmed sequences were uploaded to Mothur and QIIME for further study.

Statistical Analysis Significance tests and principal component analyses (PCA) were performed using SPSS for Windows version 17.0 (SPSS, Chicago, IL). The data were assessed with an ANOVA, and significant differences were determined by Duncan’s new multiple range test. Statements of statistical significance were based on P < 0.05.

RESULTS Growth Performance In the starter phase from day 1 to 21, the ANT and the PRE diet groups had greater (P < 0.05) ADG than the CON groups (Table 3). In the finisher phase from day 21 to 42, the PRE and PRO diet groups had greater (P < 0.05) ADG than the CON groups, whereas only the PRE diet groups showed greater (P < 0.05) ADG in both phases from day 1 to 42 compared with the CON group. There were no significant differences in the ADFI among all groups in both phases. The FCR of the ANT and PRE groups were significantly decreased (P < 0.05) compared with the CON group in the starter phase. In the finisher phase, the FCR of the PRE and PRO groups were lower (P < 0.05) than the CON group, while only the PRE groups had decreased (P < 0.05) FCR in both the starter and finisher phases compared with the CON group.

Serum Antioxidant Activities The results for the levels of antioxidants in the serum are presented in Table 4. On day 21, the PRE and PRO groups had lower (P < 0.05) MDA than all CON, ANT, and FREE groups. Furthermore, the PRE group had the lowest MDA of all groups. There were no significant differences in MDA among the CON, ANT, and FREE groups. The same results were observed for MDA on day 42. There were no significant differences in SOD on day 21. However, on day 42, the PRE and FREE groups were found to have greater (P < 0.05) SOD than the CON group, in addition, SOD in PRE group was greater (P < 0.05) than those in both ANT and PRO groups. On day 21, birds in the PRE group had the greatest (P < 0.05) T-AOC among all the groups. On

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Means within a row with different superscripts differ (P < 0.05). Significance was analyzed by the new multiple range method (Duncan) with ANOVA in SPSS 17.0 (IBM). Treatments: CON = a basal diet; ANT = CON + 1 g of Aureomycin per kg diet; FREE = CON + 1 × 109 cfu of free non-encapsulated probiotics/kg of diet; PRO = CON + 1 × 109 cfu of pro-encapsulated probiotics/kg of diet; and PRE = CON + 1 × 109 cfu of pre-encapsulated probiotics/kg of diet. 2 Each mean represents 5 replicate pens with 20 broilers per pen. 3 FCR = feed conversion ratio. a–c

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EFFECTS OF ENTEROCOCCUS FAECALIS IN BROILERS Table 4. Serum antioxidant activity of broilers in the 5 experimental feeding treatments. Treatment1 Item

CON

MDA (nmol/mL) d 21 6.40a d 42 5.41a SOD (U/mL) d 21 52.75 d 42 56.78c T-AOC (U/mL) d 21 14.41c d 42 14.59 CAT (U/mL) d 21 7.30 d 42 7.31

ANT

FREE

PRO

PRE

SEM2

P-value2

6.53a 5.56a

6.70a 5.82a

5.25b 4.13b

4.26c 3.42c

0.20 0.20

< 0.001 < 0.001

61.17 61.78b,c

66.12 66.78a,b

64.22 62.42b,c

58.57 69.93a

1.55 1.27

0.078 0.005

14.77c 15.63

14.71c 15.83

15.35b,c 15.12

17.30a 15.30

0.59 0.20

0.021 0.332

9.63 7.92

8.68 7.92

7.88 8.53

9.67 9.65

0.13 0.25

0.054 0.358

1

day 42, no significant differences were found in T-AOC among all the groups. Finally, no significant differences were found in CAT among any groups during the entire feeding period.

the serum IL-2 concentration was greater (P < 0.05) in the PRE group than in the FREE or PRO groups, and serum IL-6 concentrations in the PRE group were higher (P < 0.05) than those in all the other groups except ANT group.

Serum Immunoglobulins and Cytokine On day 21, no significant differences were observed in the serum IgA and IgM among all the groups (Table 5). On day 42, the serum IgA concentrations were increased (P < 0.05) in both the PRE and PRO groups, and IgM was increased (P < 0.05) only in the PRE group compared with the CON group. No significant differences were found in the serum IgG levels among all the treatments on day 21 and 42. The results of the serum cytokine concentrations are shown in Table 6. Both IL-2 and IL-6 showed no significant differences among all the groups on day 21. However, on day 42,

Cecal Microbiome In total, 1,541,502 V3 16S rRNA amplicon sequence reads were analyzed from the 25 samples collected on day 21. Furthermore, a total of 1,368,304 of the V3 16S rRNA amplicon sequences reads were analyzed from the 25 samples collected on day 42. However, no significant differences were found among all groups. The sample richness and diversity were also reflected by the operational taxonomic units, Shannon index, Chao1, and Simpson index, and no differences (P > 0.05) were observed between any of groups (Table 7). The taxon

Table 5. Serum immunoglobulins of broilers in the 5 experimental feeding treatments. Treatment1 Item (mg/mL) IgA d 21 d 42 IgM d 21 d 42 IgG d 21 d 42

CON

ANT

FREE

PRO

PRE

SEM2

P-value2

1.33 1.34b

1.47 1.55a,b

1.50 1.55a,b

1.50 1.66a

1.48 1.68a

0.03 0.03

0.456 0.025

0.69 0.66b

0.77 0.73a,b

0.74 0.70b

0.78 0.74a,b

0.79 0.78a

0.01 0.01

0.139 0.017

4.32 4.66

5.67 4.07

4.34 4.97

4.67 5.22

5.91 4.86

0.19 0.18

0.258 0.828

Means within a row with different superscripts differ (P < 0.05). Significance was analyzed by the new multiple range method (Duncan) with ANOVA in SPSS 17.0 (IBM). Treatments: CON = a basal diet; ANT = CON + 1 g of Aureomycin per kg diet; FREE = CON + 1 × 109 cfu of free non-encapsulated probiotics/kg of diet; PRO = CON + 1 × 109 cfu of proencapsulated probiotics/kg of diet; and PRE = CON + 1 × 109 cfu of pre-encapsulated probiotics/kg of diet. 2 Each mean represents 5 replicate pens with 3 broilers per pen. a–b 1

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Means within a row with different superscripts differ (P < 0.05). Significance was analyzed by the new multiple range method (Duncan) with ANOVA in SPSS 17.0 (IBM). Treatments: CON = a basal diet; ANT = CON + 1 g of Aureomycin per kg diet; FREE = CON + 1 × 109 cfu of free non-encapsulated probiotics/kg of diet; PRO = CON + 1 × 109 cfu of proencapsulated probiotics/kg of diet; and PRE = CON + 1 × 109 cfu of pre-encapsulated probiotics/kg of diet. 2 Each mean represents 5 replicate pens with 3 broilers per pen. a–c

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ZHANG ET AL. Table 6. Serum cytokines of broilers in the 5 experimental feeding treatments. Treatment1 Item (ng/L) IL-2 d 21 d 42 IL-6 d 21 d 42

CON

ANT

FREE

PRO

PRE

SEM2

P-value2

10.54 9.62a,b

10.61 10.56a,b

9.46 9.19b

10.49 9.28b

10.17 11.29a

0.18 0.23

0.245 0.031

4.72 3.74c

5.32 4.35b,c

4.71 4.06c

2.96 3.22c

3.15 6.16a,b

0.29 0.30

0.277 0.006

Means within a row with different superscripts differ (P < 0.05). Significance was analyzed by the new multiple range method (Duncan) with ANOVA in SPSS 17.0 (IBM). Treatments: CON = a basal diet; ANT = CON + 1 g of Aureomycin per kg diet; FREE = CON + 1 × 109 cfu of free non-encapsulated probiotics/kg of diet; PRO = CON + 1 × 109 cfu of proencapsulated probiotics/kg of diet; and PRE = CON + 1 × 109 cfu of pre-encapsulated probiotics/kg of diet. 2 Each mean represents 5 replicate pens with 3 broilers per pen. a–c 1

Treatment1 Item

CON

# of Sequences d 21 50,820 d 42 55,469 # of OTU d 21 867 d 42 2,438 Chao1 (richness) d 21 11,072 d 42 1,238 Shannon (diversity) d 21 6.48 d 42 6.22 Simpson (diversity:1-D) d 21 0.94 d 42 0.92

ANT

FREE

PRO

PRE

69,912 93,483

33,998 107,112

28,836 26,440

88,550 75,203

1530 1,628

726 1,704

464 350

1,488 2,247

11,631 10,959

7,824 11,264

6,161 5,210

10,178 15,038

6.79 6.24

6.93 5.85

5.36 5.01

5.96 6.53

0.95 0.93

0.95 0.89

0.93 0.79

0.88 0.91

1 Treatments: CON = a basal diet; ANT = CON + 1 g of Aureomycin per kg diet; FREE = CON + 1 × 109 cfu of free non-encapsulated probiotics/kg of diet; PRO = CON + 1 × 109 cfu of pro-encapsulated probiotics/kg of diet; and PRE = CON + 1 × 109 cfu of pre-encapsulated probiotics/kg of diet.

abundance of each sample was further analyzed at the level of phylum and genus using mainly the database of the Ribosomal Database Project with a bootstrap confidence threshold of 80%. In both phases, the dominant phylum was Firmicutes, which comprised 54.22 to 82.48% of the samples on day 21, and 74.14 to 83.14% of the samples on day 42. On day 42, there were a number of Firmicutes that were enriched (P < 0.05) in the PRE group compared with the CON group, whereas the Proteobacteria were depleted (P < 0.05) in both the PRE and the PRO groups compared with the CON, ANT, and FREE groups. Furthermore, no differences were found in either the Bacteroidetes or the Tenericutes throughout the entire growing period of the broilers (Table 8). At the genus level, the PRE diets increased (P < 0.05) the number of Lactobacillus in the finisher phase and the number of Enterococcus in both the starter and finisher phases. No differences in quantities were found in other bacterial genera among any of the groups in either phase (Table 9).

DISCUSSION Microencapsulation is defined as the technology of packaging solids, liquids, or gaseous materials in miniature, sealed capsules that can release their contents at controlled rates under specific conditions (Anal and Stevens, 2005). Microencapsulation of probiotics is primarily used to protect the cells against an adverse environment rather than for controlled release. This technology increases the survival of the probiotic bacteria to as much as 80 to 95% (Kebary et al., 1998). The motivation for microencapsulating bacteria, rather than simply using free non-encapsulated cultured microorganisms, is to improve the growth rate; dilution rate without washout in continuous processes; collection; biocatalytic stability (Wang et al., 1997); and tolerance against high temperature, high humidity, drying, and other animal feed processing activities (Ortakci et al., 2012). Furthermore, probiotic microcapsules can survive passage through the upper

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Table 7. Number of operational taxonomic units (OTU) per group and estimators of sequence diversity and richness.

7

EFFECTS OF ENTEROCOCCUS FAECALIS IN BROILERS Table 8. Bacterial phyla distributions in the 5 experimental feeding treatments. Treatment1 Phyla (%) Firmicutes d 21 d 42 Bacteroidetes d 21 d 42 Proteobacteria d 21 d 42 Tenericutes d 21 d 42

CON

ANT

FREE

PRO

PRE

SEM2

P-value2

67.18 76.19b

71.63 76.92b

82.48 74.14b

54.22 74.86b

61.27 83.14a

2.44 0.99

0.303 0.006

21.91 11.86

19.97 13.17

9.72 14.11

14.96 4.74

6.98 19.73

1.26 1.98

0.383 0.086

7.33 4.81a

5.10 4.10a

4.04 3.62a

3.18 1.14b

3.61 0.69b

0.72 0.30

0.824 0.003

1.60 0.92

0.94 0.60

1.56 1.78

0.68 0.68

1.15 1.48

0.03 0.02

0.421 0.402

1

Table 9. Bacterial genera distribution in the 5 experimental feeding treatments. Treatment1 Genus (%) Faecalibacterium d 21 d 42 Ruminococcus d 21 d 42 Oscillospira d 21 d 42 Coprococcus d 21 d 42 Clostridium d 21 d 42 Lactobacillus d 21 d 42 Enterococcus d 21 d 42

CON

ANT

FREE

PRO

PRE

SEM2

P-value2

1.31 17.88

1.66 12.01

1.61 20.71

2.13 33.82

6.17 17.82

0.66 3.71

0.293 0.479

2.63 5.66

3.40 3.91

6.70 5.24

2.72 2.67

3.39 3.21

0.58 0.52

0.625 0.306

5.07 5.78

5.11 3.96

6.40 5.99

3.30 2.31

4.63 3.49

0.21 0.29

0.625 0.718

1.18 0.38

1.12 0.37

0.98 0.66

0.72 0.38

0.58 0.37

0.01 0.05

0.293 0.405

0.15 1.43

0.19 1.43

0.21 0.47

0.24 0.71

0.23 0.32

0.02 0.04

0.741 0.301

0.67 0.10b

0.33 0.02b

0.21 0.05b

0.58 0.02b

0.83 0.49a

0.09 < 0.01

0.142 < 0.001

0.08b 0.05b

0.07b 0.03b

0.04b 0.05b

0.06b 0.04b

0.27a 0.21a

< 0.01 < 0.01

< 0.001 0.016

Means within a row with different superscripts differ (P < 0.05). Significance was analyzed by the new multiple range method (Duncan) with ANOVA in SPSS 17.0 (IBM). Treatments: CON = a basal diet; ANT = CON + 1 g of Aureomycin per kg diet; FREE = CON + 1 × 109 cfu of free non-encapsulated probiotics/kg of diet; PRO = CON + 1 × 109 cfu of proencapsulated probiotics/kg of diet; and PRE = CON + 1 × 109 cfu of pre-encapsulated probiotics/kg of diet. 2 Each treatment was measured for 5 replicates. a–b 1

gastrointestinal tract and arrive alive at the site of action and are thereby able to function in the gut environment (Mattila-Sandholm et al., 2002). In this paper, we have divided the microencapsulation into 2 types: pro-encapsulation and pre-encapsulation. In addition to the general advantages offered by pro-encapsulation, pre-encapsulation also has the following characteristics: (1) this technique has been used successfully to encapsulate small microorganisms for batch and continuous fermentation and thus, pre-encapsulation ensures that

the microbial cells have higher productivity than the pro-encapsulated microbial cells; (2) pre-encapsulation also simplifies the purification process of the microcapsule products; (3) unlike pro-encapsulation, the microbial activity is not stressed by the high temperature and high pressure that are normally used in the proencapsulation process. Evidence has been presented that probiotics can promote growth performance, enhance immunomodulation, and modulate the intestinal microflora in broiler

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Means within a row with different superscripts differ (P < 0.05). Significance was analyzed by the new multiple range method (Duncan) with ANOVA in SPSS 17.0 (IBM). Treatments: CON = a basal diet; ANT = CON + 1 g of Aureomycin per kg diet; FREE = CON + 1 × 109 cfu of free non-encapsulated probiotics/kg of diet; PRO = CON + 1 × 109 cfu of proencapsulated probiotics/kg of diet; and PRE = CON + 1 × 109 cfu of pre-encapsulated probiotics/kg of diet. 2 Each treatment was measured for 5 replicates. a–b

8

ZHANG ET AL.

study, the PRE and PRO probiotics increased the IgA levels and the PRE probiotics further increased the IgM level in the finisher phase, while no significant improvements in any of the immunoglobulins by the ANT and FREE additions were found. These indicated an efficient enhancement by the PRE probiotics on the broilers immunity. The immune response to infection is controlled by a complex interplay among the various cytokines (Kelso and Metcalf, 1990). In this study, the addition of the PRE probiotics enhanced both the IL-2 and IL-6 levels of the broiler chickens on day 42. The cytokine IL-2 is one of the most important T cell growth factors and is a potent immune system modulator, affecting nearly every facet of the host immune response (Choi and Lillehoj, 2000). IL-6 is a multifunctional cytokine that plays an important role in regulating immune responses, hematopoiesis, and acute phase reactions (Hoene and Weigert, 2008). Thus, coupled with the immunoglobulin and cytokine, our results suggests there was a more beneficial effect on the immune system is provided by the pre-encapsulated probiotics than the antibiotic or probiotics with the free and proencapsulated forms. The cecum is the most heavily populated gastrointestinal region, the main site of fermentation in the gastrointestinal tract, and harbors a diverse microbial community (Mead, 2000). Probiotics play an important role in stabilizing the intestinal ecosystem of animals by enhancing the growth of beneficial bacteria and competing with harmful bacteria in the intestine (Higgins et al., 2008; Vicente et al., 2008). Beneficial bacteria such as Lactobacillus are known to produce digestive enzymes that can help improve feed conversion and enhance digestion in host animals (Ramaswami et al., 2005). Lactobacilli are also able to inhibit the growth of pathogens such as Salmonella, Escherichia coli, and Clostridium by the competitive exclusion of pathogens (Kawai et al., 2004). Li et al. (2009) reported that supplementation with probiotics increased Lactobacilli and Bifidobacterium numbers and decreased E. coli in the cecum of chickens. In the present study, probiotics with different forms did not change the diversity and richness of the intestinal ecosystem; however, we can confirm that the dietary inclusion of PRE probiotics significantly increased the cecal Lactobacillus and Enterococcus counts compared with those of the CON and other treatments. The greater survival of the Enterococcus confirmed that pre-encapsulation offered better protection against gastrointestinal disorders. Furthermore, increased Enterococcus led to an increase in the number of Lactobacilli in the cecum. These results also showed that the microencapsulated biotics, especially the pre-encapsulated probiotics, significantly decrease the number of Proteobacteria, which is well known for the wide variety of pathogens it comprises, including E. coli and Salmonella. These results might help to explain why the use of the pre-encapsulated E. faecalis results in improved growth performance in broiler chickens.

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chickens (Apata, 2008; Zhou et al., 2010). Many recent studies have addressed the beneficial properties of E. faecalis as a probiotic and as an alternative to antibiotics in animal feed (Strompfov´ a et al., 2006). Several studies did not report beneficial effects of probiotics on the growth performance of broiler chickens (Li et al., 2008; Lee et al., 2010; Zhang et al., 2011), but other researchers have reported positive effects of the probiotics on the growth performance of broilers (Talebi et al., 2008; Zhou et al., 2010; Zhang and Kim, 2014). This inconsistency might be attributable to the strains of the probiotics, administration dosage, or to the forms of the probiotics (Zhang et al., 2012). In the current experiment, E. faecalis, including free nonencapsulated (FREE), pro-encapsulated (PRO), and pre-encapsulated (PRE) forms, was added into the diet at 1 × 109 cfu/kg in broiler chickens. Our data showed that the addition of the FREE and PRO probiotics did not improve the ADG of the birds significantly; however, the PRE probiotics did demonstrate a significant increase in ADG. Moreover, the PRE group decreased the FCR significantly when compared with the CON. These results indicate that the probiotics potentially had similar effects to the antibiotic in the growth performance of chickens, and pre-encapsulation can protect the probiotics and improve the beneficial effects efficiently in the rearing period. In agreement with our results, Han et al. (2012) reported higher ADG and lower FCR in broilers when the basal diet was supplemented with similar pre-microcapsule probiotics. T-AOC, SOD, and CAT activity are the main parameters to assess oxidative status. Contents of MDA in blood can generally be used as a biomarker for radical-induced damage and endogenous lipid peroxidation (Wang et al., 2008). In the present study, both the PRE and PRO probiotics decreased the MDA levels, which indicates an antioxidant effect of the PRE and PRO probiotics in the broilers. The PRE group had lower MDA than the PRO group during the entire feeding period, indicating that the PRE showed more effective protection to the microbial cells than the PRO process. PRE probiotics also increased the SOD level in the finisher phase and the T-AOC in the starter phase. These results confirmed the enhancement on the total antioxidant capacity of the body by the PRE probiotics. We did not find any significant improvement on antioxidant capacity by the ANT and FREE additions. Thus, the treatments with PRE probiotics were the best ones that showed effective protection through improvements in the antioxidant capacity of the broiler chickens. Because of their important roles in immune function, plasma immunoglobulin and cellular cytokine concentrations can be used as parameters to evaluate the immune status of birds. Numerous studies have shown that probiotics supplementation results in an enhanced broiler humoral immune response by increasing the number of immunoglobulin (Yang et al., 2012; Amerah et al., 2013; Salim et al., 2013). In the present

EFFECTS OF ENTEROCOCCUS FAECALIS IN BROILERS

In conclusion, probiotics such as E. faecalis can be a potential substitute for antibiotics in broiler feeds. However, a suitable form is needed to guarantee that the probiotics will be able to function efficiently. The results of the present experiment indicated that preencapsulation is a more appropriate method compared with pro-encapsulation to enable the probiotics to improve the intestinal microbes, immune function, and growth performance of broilers.

ACKNOWLEDGMENTS

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This work was supported by a grant from State Key Laboratory of Animal Nutrition (2004DA125184F1306) and with funds from the National Twelfth Five-Year Science and Technology Plan Program of Rural Areas in China (2013BAD10B02). We also acknowledge the support of China Agriculture Research System Poultryrelated Science and Technology Innovation Team of Peking (CARS-PSTP).

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