Animals
The Institutional Animal Care and Use Committee of Huazhong Agricultural University approved all the animal procedures and all methods were performed in accordance with the relevant guidelines and regulations.
Newly hatched chickens were reared under similar husbandry conditions and were fed a corn-soybean, pathogen free diet in pellet form in a poultry farming with no medication or vaccination. The birds had ad libitum access of water and feed. When they were 7 weeks old, male and female chickens (n=10) with high or low body weight chickens were selected for next study. For fecal microbiota transplantation, adult high body weight chickens were selected as donors and one-day-old chickens (n=30) with the same genetic background were selected as recipients. The recipients were randomly divided into two groups: saline control group (group C) and fecal microbiota transplantation group (group FMT). Fecal bacteria were transplanted every day by oral administration for four weeks.
Samples collection
After being fasted 12 hours, the chickens were sacrificed. Blood, liver and abdominal adipose were harvested. Chest muscle and leg muscle were collected and weighed. To ensure the comparability of research results, the same part of each organ was chosen for next analysis. For gut microbiota analysis, the contents of the ceca from the selected twenty chickens were snap frozen in liquid nitrogen and stored at -80°C. For histo-morphological analysis, freshly harvested chest muscle, leg muscle, and liver tissues were fixed in 4% paraformaldehyde solution; abdominal adipose tissues from each chicken were fixed in optimal cutting temperature (OCT). For the molecular studies and gene expression analysis, the parts of freshly harvested muscle, adipose and liver tissues were snap frozen in liquid nitrogen and then stored at -80°C. For blood biochemical parameters analysis, blood samples from birds were centrifuged at 1500 × g for 15 min and serum was snap frozen in liquid nitrogen and stored at -80°C.
Muscle index calculation
The muscle index was calculated using the following formula: muscle index=muscle weight (g)/body weight (g).
Microbial genomic DNA extraction and 16S rRNA gene sequencing
16S rRNA sequencing was used to compare the microbial composition between high and low body weight chickens. Total bacterial genomic DNA samples were extracted using Fast DNA SPIN extraction kits (MP Biomedicals, Santa Ana, CA, USA), following the manufacturer’s instructions, and stored at -20°C prior to further analysis. The quantity and quality of extracted DNA fragments were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively.
PCR amplification of the bacterial 16S rRNA gene V3-V4 region was performed using the forward primer 338F (5’-ACTCCTACGGGAGGCAGCA-3’) and the reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’). Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The PCR components contained 5 μl of Q5 reaction buffer (5×), 5 μl of Q5 High-Fidelity GC buffer (5×), 0.25 μl of Q5 High-Fidelity DNA Polymerase (5 U/μl), 2 μl (2.5 mM) of dNTPs, 1 μl (10 μM) of each forward and reverse primer, 2 μl of DNA Template, and 8.75 μl of dd H2O. Thermal cycling consisted of initial denaturation at 98 °C for 2 min, followed by 25 cycles consisting of denaturation at 98 °C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final extension of 5 min at 72 °C. PCR amplicons were purified with Agencourt AMPure Beads (Beckman Coulter, Indianapolis, IN) and quantified using the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts, and paired-end 2×300 bp sequencing was performed using the Illumina MiSeq platform with the MiSeq Reagent Kit v3 at Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).
Sequencing data analysis
The Quantitative Insights Into Microbial Ecology (QIIME, v1.8.0) pipeline was employed to process the sequencing data, as previously described [56]. Briefly, raw sequencing reads with exact matches to the barcodes were assigned to respective samples and identified as valid sequences. The low-quality sequences were filtered through the following criteria [57, 58]: sequences that had a length of <150 bp, sequences that had average Phred scores of <20, sequences that contained ambiguous bases, and sequences that contained mononucleotide repeats of >8 bp. Paired-end reads were assembled using FLASH [59]. After chimera detection, the remaining high-quality sequences were clustered into operational taxonomic units (OTUs) at 97% sequence identity by UCLUST [60]. A representative sequence was selected from each OTU using default parameters. OTU taxonomic classification was conducted by BLAST searching the representative sequences set against the Greengenes Database [61] using the best hit [62]. An OTU table was further generated to record the abundance of each OTU in each sample and the taxonomy of these OTUs. OTUs containing less than 0.001% of total sequences across all samples were discarded. To minimize the difference in sequencing depth across samples, an averaged, rounded rarefied OTU table was generated by averaging 100 evenly resampled OTU subsets under 90% of the minimum sequencing depth for further analysis.
Blood biochemical parameters analysis
To test the fat metabolism level in blood, the concentrations of serum TG, TC, HDL-C and LDL-C were determined using a Rayto Chemistry Analyzer Chemray 240 (Chemray 240, China) according to the commercial diagnostic kits’ instructions.
Hematoxylin and eosin staining
To compare the morphological changes, liver, chest muscle, and leg muscle tissue samples embedded in paraffin were cut into 3-μm-thick sections. Abdominal adipose tissue samples fixed in OCT were cut into 10-μm-thick sections. HE staining was performed using a routine protocol, and the examination of stained tissue sections was accomplished by light microscopy (Olympus BX51, Tokyo, Japan) with a digital camera (DP72; Olympus). The average diameter of adipocytes and the average areas of chest muscle cells and leg muscle cells were quantitated using Image Pro Plus 6.0.
Immunohistochemical staining
To test the distribution and protein expression of P-AMPK, immunohistochemical staining was performed following the same steps as described in earlier studies [63, 64]. Briefly, the tissue sections were deparaffinized twice in xylene and rehydrated in a graded series of ethanol. A microwave oven (MYA-2270M, Haier, Qingdao, China) was used for heat antigen retrieval in citrate acid buffer solution (pH 6.0) for 20 minutes (5 minutes at high level, i.e., 700 W, and 15 minutes at low level, i.e., 116 W). After cooling at room temperature for 2-3 h, 3% H2O2 was used to block endogenous peroxidase. For blocking of nonspecific antibody binding, the tissue sections were incubated with 5% bovine serum albumin (BSA) at 37 °C for 30 minutes. Sections were then incubated with primary antibodies using rabbit anti-P-AMPK antibody (1:100) (Cell Signaling Technology, Inc., USA). Subsequently, tissue sections were incubated at 37 °C with suitable horseradish peroxidase (HRP)-conjugated secondary antibodies (Boster, Wuhan, China) for 30 minutes. Immunostaining for all the tissue sections was accomplished using the chromogenic marker diaminobenzidine (DAB) (Boster, Wuhan, China), and counterstaining was performed using hematoxylin. Finally, the sections were washed, dried, dehydrated, cleared, and finally mounted with a coverslip.
Serial sections were examined under a light microscope (BH-2; Olympus, Japan) with a digital camera (DP72; Olympus), and the fields of vision were chosen according to different regions of the liver and muscle tissue in each section. The distributions and expression levels of different proteins were measured in high-power fields selected at random. All of the images were taken using the same microscope and camera set. Image-Pro Plus (IPP) 6.0 software (Media Cybernetics, USA) was used to calculate the mean density for positive staining.
Western blotting
To test the protein expression of P-AMPK, western blotting was performed following previously described methods [65]. Briefly, the frozen specimens were powdered in liquid nitrogen and homogenized in lysis buffer with a protease inhibitor. The supernatants were vortexed, incubated on ice and centrifuged at 12,000 × g for 5 min. Protein concentrations were measured using the BCA protein quantification kit (Beyotime, Jiangsu, China). Equal amounts of total proteins (40 μg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (30 min at 80 volts, and after that 80 min at 120 volts). Then, the separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, USA). The membranes were incubated with rabbit anti-P-AMPK (1:1000) (Cell Signaling Technology, Inc., USA), rabbit anti-AMPK (1:1000) (ABclonal, China), mouse anti-GAPDH (1:10000) (Proteintech Group, Inc., USA) and rabbit anti-β-actin (1:5000) (ABclonal, China) antibodies for 12 h. After washing in 1X TBST buffer three times, samples were incubated with peroxidase-conjugated secondary antibody (1:5000) for 120 min (Boster, China). The blots were developed with Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and visualized using ChemiDoc-It™ Imaging System. Western blot results were analyzed using IPP 6.0 software.
Real-time quantitative polymerase chain reaction (Q-PCR)
To measure the expression of fat metabolism-related genes at the mRNA level, total RNA was isolated from the liver, abdominal adipose tissue, chest muscle and leg muscle with Trizol reagent (Takara, Japan) according to the manufacturer’s instructions. The cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Takara, Japan). The reaction mixture (10 μl) for qPCR contained 5 μL of SYBR Select Master Mix for CFX (Takara, Japan), 0.4 μL of each forward and reverse primer, 3.2 μL of ddH2O and 1 μL of template cDNA. The Q-PCR reactions were performed on a Bio-Rad CFX Connect real-time Q-PCR detection system.
Table 1
Primers used for real-time Q-PCR
Gene | Primer sequences (5’ to 3’) | Accession No. |
β-actin | f-TTGTTGACAATGGCTCCGGT | NM_205518.1 |
r-TCTGGGCTTCATCACCAACG | |
ACC | f-TCCAGCAGAACCGCATTGACAC | NM_205505.1 |
r-GTATGAGCAGGCAGGACTTGGC | |
FAS | f-GCTCTGCGTCTGCTTCAGTCTAC | NM_001199487.1 |
r-GGTACAGGACTCTGCCATCAATGC | |
FADS1 | f-CCGTGCCACTGTGGAGAAGATG | LC061145.1 |
r-GCCTAGAAGCAACGCAGAGAAGAG | |
CYP2C45 | f-AACAAGCACCACCACACGATACG | AJ430583.1 |
r-GGTCAGCCACGCAAGGTCTTC | |
ACSL1 | f-GACTAATGGTCACAGGAGCAGCAC | NM_001012578.1 |
r-CCAGGCATTGACAGTGAGCATCC | |
PPARα | f-TGCTGTGGAGATCGTCCTGGTC | AF163809.1 |
r-CTGTGACAAGTTGCCGGAGGTC | |
CPT-1 | f-GCCAAGTCGCTCGCTGATGAC | DQ314726.1 |
r-ACGCCTCGTAGGTCAGACAGAAC | |
fiaf | f-AGATCAAGCAGCAGCAGTACAAGC | XM_001232283.5 |
r-ACGCTCACATTATGGCTCTGGTTG | |
A-FABP | f-ACAATGGCACACTGAAGCAGG | FJ493543.1 |
r-AGCAGGTTCCCATCCACCAC | |
SREBP1 | f-GGTCCGGGCCATGTTGA | AJ310768.1 |
r-CAGGTTGGTGCGGGTGA | |
PPARG | f-GAATGCCACAAGCGGAGAAGGAG | NM_001001460.1 |
r-GCTCGCAGATCAGCAGATTCAGG | |
AP2 | f-ACTGAAGCAGGTGCAGAAGTGG | NM_204290.1 |
r-TGCATTCCACCAGCAGGTTCC | |
Adiponectin | f-TACGTGTACCGCTCCGCCTTC | KP729052.1 |
r-GTGCTGCTGTCGTAGTGGTTCTG | |
(Bio-Rad, Hercules, CA, USA). The Q-PCR conditions were as follows: predenaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and elongation at 72 °C for 20 s. The primer sequences are listed in Table 1. β-Actin was chosen as a reference for Q-PCR. All samples were run in triplicate, and gene expression levels were quantified using the ΔΔCt method.
Statistical analysis
Sequence data analyses were mainly performed using QIIME and R packages (v3.2.0). OTU-level alpha diversity indexes, such as the Chao richness index and Shannon diversity index, were calculated using the OTU table in QIIME. The taxonomy compositions and abundances were visualized using Excel. LEfSe was performed to detect differentially abundant taxa across groups using the default parameters (LDA>2) [66]. Based on high-quality sequences, microbial functions were predicted by PICRUSt, [67]. The significant differences between pairs of samples or among multiple groups of KEGG pathways were visualized using the STAMP software package. Spearman’s correlations between the gut microbiota and fat metabolism were determined using the R software package. All data are presented as the means ± standard error of mean (SEM). All analyses and graphic representations were performed with Prism software 5.01 (GraphPad Software, Inc., San Diego, USA). The statistical significance of the mean values in two-group comparisons was determined using Student’s t-test. A p value <0.05 was considered statistically significant.