A total of 120 1-day-old male, Arbor Acres (AA) broilers, were purchased from the Huadu Broiler Breeding Co. (Beijing, China), and housed in the Nankou experimental farm of the Feed Research Institute, CAAS, Beijing, China. The day-old chicks (body weight, 47.2+ 0.31 g) were randomly divided into two groups, control and treatment. Each group had 6 cages (replicates) with 10 birds per cage. The chickens were reared in two stages, starter (1–21 days) and grower (22–42 days), and fed a basal (control) corn-soybean meal diet (Table 1), to which 6.75×109 cfu/g of E. faecium was added for the treatment group. Microcapsules of E. faecium CGMCC 2516[17, 18] (viable count ≥15×1010 cfu/g; Challenge Group, Beijing, China) were used in this study.
Table 1
Ingredient and nutrient composition of basal broiler diets
Ingredient
|
Starter (1–21 days) (g/kg)
|
Grower (22–42 days) (g/kg)
|
Corn
|
593.1
|
604.2
|
Soybean meal
|
298.8
|
288.7
|
Cotton seed meal
|
50.0
|
30.0
|
Soybean oil
|
15.1
|
39.8
|
L-Lys
|
1.5
|
0.9
|
DL-Met
|
1.4
|
1.6
|
Limestone
|
12.7
|
10.2
|
CaHPO4
|
19.4
|
16.6
|
NaCl
|
3.0
|
3.0
|
Choline chloride
|
2.0
|
2.0
|
Vitamin premix
|
0.3
|
0.3
|
Mineral premix1)
|
1.0
|
1.0
|
Zeolite powder
|
1.7
|
1.7
|
Total
|
1000
|
1000
|
Nutrient levels2)
|
|
|
ME (MJ/kg)
|
12.35
|
13.02
|
CP
|
211.8
|
198.4
|
Ca
|
10.1
|
8.5
|
AP
|
4.5
|
4.0
|
TP
|
6.9
|
6.3
|
Lys
|
11.4
|
10.5
|
Met
|
4.9
|
4.8
|
Met+Cys
|
8.3
|
8.1
|
Thr
|
7.7
|
2.2
|
1) The premix provided the following per kg diet: VA 10, 000 IU, VD3 2000 IU, VE 10 IU, VK3 2.5 mg, VB1 1 mg, VB2 6 mg, VB3 10 mg, VB5 40 mg, VB6 3 mg, VB11 0.3 mg, VB12 0.01 mg, biotin 0.12 mg, Cu (as copper sulfate) 8 mg, Fe (as ferrous sulfate) 80 mg, Mn (as manganese sulfate) 60 mg, Zn (as zinc sulfate) 40 mg, Se (as sodium selenite) 0.15 mg, I (as potassium iodide) 0.35 mg.
2) Calculated values.
Bird management
Birds were raised in accordance with the AA Broiler Management Guide. Chicks were vaccinated for Marek’s Disease at day-old and for Newcastle Disease and Infectious Bronchitis at 7 days post-hatching. Room temperature was maintained at 33 °C for days 0~3 and gradually reduced to 24 °C and maintained at 24 ℃ till the end of the study. Photoperiod was controlled to 23 hours of light and 1 hour of darkness. Relative humidity was set at 60 %~70 % during the first week and then at 50 %~60 % for the rest of the experiment.
Sample collection and parameter determination
From day 18 to 21 and day 39 to 42 of the experiment, excreta from each replicate was collected, mixed and dried in an oven at 105 °C to a constant weight. The dried excreta was ashed in a muffle furnace at 550 °C for 4 hours. The P content of the ash samples was determined using the vanadate-molybdate method[19].
On day 21 and day 42, body weight (BW) and feed intake were measured to calculate the average daily gain (ADG), average daily feed intake (ADFI), and the ratio of feed/gain (F/G). On those days, one broiler close to the cage average body weight was randomly selected from each replicate. The chosen birds were electrically stunned, and manually slaughtered within 5 min[20]. Blood was collected from the jugular vein, and serum was obtained after centrifuging at 3000 g for 10 min at 4 ℃ and stored at -20 ℃ for further analysis. Serum alkaline phosphatase (ALP) and P were determined with a Hitachi 7600 automatic biochemical analyzer, using kits were purchased from Nanjing Jiancheng Biological Engineering Institute.
After the blood sampling on day 42, the duodenum (about 10 cm distal to the pylorus), jejunum (about 10 cm preceding the Meckel’s diverticulum) and ileum (about 10 cm preceding the ileocecal junction) were separated[21], and flushed gently with saline solution. The mucosa samples were scraped with a coverslip and snap-frozen in liquid nitrogen for analysis of mRNA.
The right tibiae was cleaned and dried for determination of tibia weight and tibia breaking strength[22]. The bones were then ashed in a muffle furnace at 550 °C for 16 h[19]. After that, the tibia P content was measured using the vanadate-molybdate method.
RNA extraction, reverse transcription and real-time quantitative PCR
Total RNA was extracted using TRNzol-A+ (TIANGEN, Beijing, China). The concentration of total RNA was estimated by spectrophotometer (Ultrospec 2100 pro, GE Healthcare), and the purity was determined by agarose gel electrophoresis. 500 ng of total RNA was reversely transcribed into cDNA using the Fast Quant RT Kit (with gDNase) (TIANGEN). qPCR was conducted using the iCycler iQ5 system. The specific primers for NaP-IIb, PiT-1, PiT-2, and β-actin were listed in Table 2. β-actin, was used as internal reference gene. Relative gene expression was calculated using the 2-ΔΔCt method[23]. All the samples were analyzed in triplicate and operational program for qPCR strictly followed the MIQE[24].
Table 2
Primer sequences of chicken NaP-IIb, PiT-1, 2, and β-actin
Gene
|
Primer sequence (5’-3’)
|
Accession number
|
NaP-IIb
|
F: CTGGATGCACTCCCTAGAGC
R: TTATCTTTGGCACCCTCCTG
|
NM_204474.1
|
PiT-1
|
F: GCTCGTGGCTTCGTTCTTG
R: GACCATTTGACGCCTTTCT
|
XM_015297502.1
|
PiT-2
|
F: GCAGCAGATACATCAACTC
R: ATTTCCACTCCACCCTC
|
NM_001305398·1
|
β-actin
|
F: GAGAAATTGTGCGTGACATCA
R: CCTGAACCTCTCATTGCCA
|
NM_205518.1
|
Illumina sequencing analysis
The fecal samples were collected on day 42 and snap-frozen in liquid N2 prior to further processing. Gene sequencing (16S rDNA) was performed by OE Biotech Co., Ltd (Shanghai, China). Total genomic DNA from frozen fecal samples was isolated using the GenElute™ Stool DNA Isolation Kit (Sigma-Aldrich, St. Louis, USA), then the V3-V4 hypervariable region of the 16S rDNA genes was amplified. The PCR products were collected and sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA). High-quality reads were clustered into operational taxonomic units (OTUs) based on sequences with ≥ 97 % similarity and then analyzed using the QIIME platform.
Statistical analysis
The statistical analyses were performed using SPSS 17.0. The data were statistically analyzed by one-way or two-way ANOVA, and the F-test was used for factor significance. For the indexes with significant main effect, Duncan’s method was used to compare the mean values among groups. A P-value less than 0.05 was considered significant.