Edited by: Kesia Palma-Rigo, Universidade Estadual de Maringá, Brazil
Reviewed by: Rodrigo Mello Gomes, Universidade Federal de Goiás, Brazil; Jurandir F. Comar, Universidade Estadual de Maringá, Brazil
This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology
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The establishment of a stable bacterial flora in early life is associated with host metabolism. Studies of fecal microbiota transplantation (FMT) and antibiotics on neonatal pig mainly focused on intestinal development and mucosal immunity, but the information on metabolism is lacking. The objective of this study was to investigate the responses of metabolome and transcriptome in the livers of neonatal piglets that were orally inoculated with maternal fecal bacteria suspension and amoxicillin (AM) solution. Five litters of Duroc × Landrace × Yorkshire neonatal piglets were used as five replicates and nine piglets in each litter were randomly assigned to the control (CO), AM or FMT groups. Neonatal piglets in three groups were fed with 3 mL saline (0.9%), AM solution (6.94 mg/mL) or fecal bacteria suspension (>109/mL), respectively, on days 1–6. At the age of 7 and 21 days, one piglet from each group in each litter was sacrificed, and the serum and liver were collected for analysis. The RNA sequencing analysis showed that the mRNA expressions of
Animal intestinal microbiota has a great influence on the health and growth of its host, because they can provide nutrition, improve immune system and modulate gastrointestinal development for host (
Amoxicillin (AM) is a broad-spectrum antibiotic that has a bactericidal effect through the inhibition of bacterial cell wall synthesis. Now antibiotics are widely concerned because of their resistance. A previous study showed that early antibiotic intervention could reduce
Fecal microbiota transplantation (FMT) is a type of intervention therapy in which the functional flora of a healthy body is transplanted into a patient’s gastrointestinal tract and the intestinal microflora with normal function are reconstructed. FMT have been treated as an effective treatment for enteritis. Studies showed the significant therapeutic effect of FMT on pseudomembranous enterocolitis (
As the metabolic center, the liver is able to reflect the regulating effect of AM and fecal bacterial suspensions on the organism metabolism. In the present study, we hypothesized that the early intervention of AM and fecal bacteria suspension could affect the liver metabolism of new-born piglets, thereby affecting the metabolism of the whole body. Because pigs have a high similarity with man in physiology and other clinical research fields (
The experiment was approved and conducted under the supervision of the Animal Care and Use Committee of Nanjing Agricultural University (Nanjing, Jiangsu province, China). All pigs were raised and maintained on a local commercial farm under the care of the Animal Care and Use Guidelines of Nanjing Agricultural University.
The preparation of FMT was adapted from a previous method (
Five litters of healthy neonatal pigs (Duroc × Landrace × Yorkshire) from a commercial farm were used as five replicates. Nine piglets in each litter were randomly assigned into the AM, FMT or CO groups, with three piglets in each group. On days 1–6, the piglets in the AM group and CO group were orally administrated once a day with 3 mL AM solution (6.94 mg/mL), and physiological saline (0.9% NaCl), respectively, while the same volume of fecal bacteria suspension (>109/mL) was offered to the piglets in the FMT group. All pigs had access to breast milk and water
Glucose, cholesterol, triglyceride, low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), alanine aminotransferase, aspartate aminotransferase, total protein, serum albumin, globulin and alkaline phosphatase in the serum of piglets were measured with an Olympus AU400 Automatic Biochemical Analyzer (Tokyo, Japan) according to the manufacturer’s instructions.
In order to reduce the cost of the experiment, three biological replicates were randomly selected from the samples for sequencing. Total RNA was extracted from liver tissue with the RNAiso Plus Total RNA extraction reagent (Takara) following the manufacturer’s instructions, and the RNA integrity was detected with Agilent Bioanalyzer 2100 (Agilent Technologies, United States). After purification, mRNA was enriched through removing ribosomal RNA (rRNA) with Ribo-Zero rRNA removal beads, and then RNA was split into fragments using divalent cations. The fragments were converted into the first strand cDNA using SuperScript II reverse transcriptase, and the synthesis of second stand cDNA was executed with DNA polymerase I and RNase H. After terminal repair, addition of base and connection of adapters, the fragments were amplified to create final cDNA libraries by PCR.
The sequencing was performed applying Illumina HiSeq 2500 according to the manufacturer’s instructions. About 6 G reads were produced in each sample, and the Q20 value of each sample was higher than 90%. Thus, the samples were qualified for further analysis. The quality results of RNA and sequencing is presented in
About 100 mg liver tissues were taken in the 2 mL centrifuge tube and added with 1 mL of 80% methanol which pre-cooled at −20°C and five steel balls. The tubes were placed in a high-flux organization grinding apparatus at 70 Hz for 1 min, added with 60 μL of 2-chloro-L-phenylalanine (0.2 mg/mL stock in methanol) and 60 μL of heptadecanoic acid (0.2 mg/mL stock in methanol) as an internal quantitative standard and vortexed for 30 s. The tubes were put into an ultrasonic machine for 30 min at room temperature, and then stew for 30 min on the ice. The tubes were centrifuged for 10 min at 14,000 rpm (4°C), and 0.8 mL of the supernatant was transferred into a new centrifuge tube for blow-drying by vacuum concentration. The samples were added with 60 μL of methoxyamine pyridine solution (15 mg/mL), vortexed for 30 s, and reacted for 120 min at 37°C. About 60 μL of BSTFA reagent (containing 1% chlorotrimethylsilane) was added into the mixture, and then reacted for 90 min at 37°C. The supernatant that was obtained by centrifuging mixture for 10 min at 12,000 rpm (4°C) was transferred to inspect bottle.
Each derivative sample (1 μL) was injected by the Agilent 7683 autosampler (Agilent Technologies, Atlanta, GA, United States) into the Agilent 6890 GC system equipped with a fused-silica capillary column (10 m × 0.18 mm i.d.) and a chemically bonded 0.18 μm stationary phase (DB-5; J&W Scientific, Folsom, CA, United States). The carrier gas (helium) passed through column at a speed of 1.0 mL/min. The column temperature is kept at 70°C for two min, and then increased to 310°C at a speed of 30°C/min and kept for two min. The column effluent was guided by a transmission route into the ion source of the mass spectrometer. The temperature of the transmission line and the ion source was 250 and 200°C, respectively. With a rate of 30 spectra/s, the mass spectra were generated in mass range of 50–800 m/z.
The RNA was extracted from liver tissue with the RNAiso Plus Total RNA extraction reagent (Takara), and then 1 μg RNA of each sample was converted into cDNA with the Reverse Transcriptase Kit (Takara, Japan) following the manufacturer’s instructions. Real time PCR was performed on ABI PRISM 7300 sequence detection system (SDS, Foster City, CA, United States) using SYBR Premix DimerEraserTM Kit (Takara, Japan) following the manufacturer’s instructions. The mRNA expressions of
The date of serum measurement and real-time PCR were analyzed by SPSS v. 20 as a completely randomized design, and one litter was regarded as one experiment unit (
The raw data of RNA-seq were converted into clean sequences by removing the sequencing adapter and the low-complexity sequences with Seqtk
DAVID Bioinformatics Resources 6.8
The raw date obtained by GC-MS was converted into netCDF format by Agilent MSD Chemstation (
On day 7, compared with the CO group, AM increased the concentrations of aspartate aminotransferase and alanine aminotransferase and decreased the concentrations of triglyceride and LCL-C (
On day 7, AM enriched 7 type of fatty acids and 3 type of amino acids (
Enrichment analysis of selected metabolites. The metabolites discriminated among the amoxicillin (AM), fecal microbiota transplantation (FMT) and control (CO) groups on day 7
Differential metabolites in pig livers among the amoxicillin (AM), fecal microbiota transplantation (FMT) and control (CO) groups on day 7.
Metabolites | Biological roles | Metabolic subpathway | FC1 | VIP2 | ||
---|---|---|---|---|---|---|
AM/CO | Glutamine | Amino acid | Arginine biosynthesis | 3.34 | 1.464 | 0.007 |
Asparagine | Amino acid | Alanine, aspartate and glutamate metabolism | 1.81 | 1.355 | 0.016 | |
Ornithine | Amino acid | Ornithine cycle | 1.61 | 1.417 | 0.011 | |
9,12-(Z,Z)-Octadecadienoic acid | Unsaturated fatty acids | Linoleic acid metabolism | 1.38 | 1.73 | <0.001 | |
9-(Z)-Octadecenoic acid | Unsaturated fatty acids | Fatty acid biosynthesis | 1.35 | 1.77 | <0.001 | |
Arachidonic acid | Unsaturated fatty acids | Arachidonic acid metabolism | 1.23 | 1.618 | 0.003 | |
9-(Z)-Hexadecenoic acid | Unsaturated fatty acids | Fatty acid biosynthesis | 1.17 | 1.416 | 0.012 | |
Hexadecanoic acid | Saturated fatty acids | Fatty acid biosynthesis | 1.24 | 1.667 | 0.001 | |
Octadecanoic acid | Saturated fatty acids | Fatty acid biosynthesis | 1.19 | 1.699 | <0.001 | |
Heptanoic acid | Saturated fatty acids | Others | 1.15 | 1.493 | 0.004 | |
1-Monohexadecanoylglycerol | lipid | Others | 1.24 | 1.772 | <0.001 | |
1-Monooctadecanoylglycerol | lipid | Others | 1.12 | 1.517 | 0.009 | |
Pyruvic acid | Organates/carboxylates | Pyruvate metabolism | 0.788 | 1.75 | <0.001 | |
Malic acid | Organates/carboxylates | TCA cycle | 1.19 | 1.609 | 0.003 | |
Fumaric acid | Organates/carboxylates | TCA cycle | 1.25 | 1.625 | 0.001 | |
Uridine | nucleoside | Pyrimidine metabolism | 0.644 | 1.469 | 0.009 | |
Adenosine-5-monophosphate | nucleoside | Purine metabolism | 0.505 | 1.727 | <0.001 | |
Uridine-5-monophosphate | nucleoside | Pyrimidine metabolism | 3.93 | 1.605 | 0.001 | |
myo-Inositol-1-phosphate | Carbohydrate | Inositol phosphate metabolism | 0.877 | 1.695 | <0.001 | |
Fructose | Carbohydrate | Fructose and mannose metabolism | 0.834 | 1.284 | 0.026 | |
Phosphoric acid | Oxidative phosphorylation relatives | Oxidative phosphorylation | 0.931 | 1.283 | 0.024 | |
FMT/CO | Uridine-5-monophosphate | nucleoside | Pyrimidine metabolism | 4.86 | 1.964 | <0.01 |
Adenosine-5-monophosphate | nucleoside | Purine metabolism | 0.277 | 1.936 | <0.001 | |
Glutamine | Amino acid | Arginine biosynthesis | 4.83 | 1.69 | 0.001 | |
Asparagine | Amino acid | Alanine, aspartate and glutamate metabolism | 2.29 | 1.662 | 0.003 | |
S-methyl-Cysteine | Amino acid | Others | 1.46 | 1.317 | 0.035 | |
Octadecanoic acid | Saturated fatty acids | Fatty acid biosynthesis | 1.27 | 1.944 | <0.001 | |
Hexadecanoic acid | Saturated fatty acids | Fatty acid biosynthesis | 1.22 | 1.871 | 0.001 | |
9,12-(Z,Z)-Octadecadienoic acid | Unsaturated fatty acids | Linoleic acid metabolism | 1.22 | 1.612 | 0.011 | |
9-(Z)-Octadecenoic acid | Unsaturated fatty acids | Fatty acid biosynthesis | 1.19 | 1.536 | 0.018 | |
1-Monooctadecanoylglycerol | lipid | Others | 1.14 | 1.33 | 0.023 | |
1-Monohexadecanoylglycerol | lipid | Others | 1.1 | 1.418 | 0.031 | |
Xylose | Carbohydrate | Amino sugar and nucleotide sugar metabolism | 0.877 | 1.412 | 0.026 | |
Galactose | Carbohydrate | Galactose metabolism | 0.867 | 1.336 | 0.043 | |
Glucose | Carbohydrate | Glycolysis or Gluconeogenesis | 0.867 | 1.336 | 0.043 | |
myo-Inositol-1-Phosphate | Carbohydrates | Inositol phosphate metabolism | 0.801 | 1.92 | <0.001 | |
1,3-Di-tert-butylbenzene | Aromatic compounds | Others | 0.915 | 1.433 | 0.028 | |
Phosphoric acid | Oxidative phosphorylation relatives | Oxidative phosphorylation | 0.908 | 1.493 | 0.021 | |
Pyruvic acid | Organates/carboxylates | Pyruvate metabolism | 0.828 | 1.773 | 0.002 | |
FMT/AM | Adenosine | nucleoside | Purine metabolism | 1.65 | 1.985 | 0.001 |
Adenosine-5-monophosphate | nucleoside | Purine metabolism | 0.548 | 1.975 | 0.001 | |
Glyceric acid-3-phosphate | lipid | Glycolysis or Gluconeogenesis | 1.32 | 1.712 | 0.014 | |
1-Monohexadecanoylglycerol | lipid | Others | 0.885 | 1.825 | 0.006 | |
myo-Inositol-1-Phosphate | Carbohydrate | Inositol phosphate metabolism | 0.914 | 1.691 | 0.015 | |
myo-Inositol | Carbohydrate | Galactose metabolism | 0.729 | 1.457 | 0.049 | |
9-(Z)-Hexadecenoic acid | Unsaturated fatty acids | Fatty acid biosynthesis | 0.892 | 1.535 | 0.040 | |
9-(Z)-Octadecenoic acid | Unsaturated fatty acids | Fatty acid biosynthesis | 0.88 | 1.484 | 0.050 | |
Oxalic acid | carboxylates | Glyoxylate and dicarboxylate metabolism | 0.945 | 1.465 | 0.046 | |
1,3-Di-tert-butylbenzene | Aromatic compounds | Others | 0.935 | 1.588 | 0.022 | |
Heptanoic acid | Saturated fatty acids | Others | 0.877 | 1.852 | 0.004 |
On day 21, AM enriched 5 type of fatty acids and 3 type of amino acids (
Differential metabolites in pig livers in the AM, FMT, and CO groups on day 21.
Metabolites | Biological roles | Metabolic subpathway | FC1 | VIP2 | ||
---|---|---|---|---|---|---|
AM/CO | Glutamine | Amino acid | Arginine biosynthesis | 3.9 | 2.089 | 0.001 |
S-methyl-Cysteine | Amino acid | Others | 1.25 | 1.475 | 0.035 | |
Asparagine | Amino acid | Alanine, aspartate and glutamate metabolism | 1.77 | 2.102 | 0.001 | |
Pyroglutamic acid | Amino acid | Glutathione metabolism | 0.822 | 1.883 | 0.006 | |
Uridine-5-monophosphate | nucleoside | Pyrimidine metabolism | 3.09 | 1.857 | 0.004 | |
Adenosine-5-monophosphate | nucleoside | Purine metabolism | 0.305 | 1.843 | 0.009 | |
Arachidonic acid | Unsaturated fatty acids | Arachidonic acid metabolism | 1.38 | 2.044 | 0.001 | |
9,12-(Z,Z)-Octadecadienoic acid | Unsaturated fatty acids | Linoleic acid metabolism | 1.3 | 1.853 | 0.007 | |
9-(Z)-Octadecenoic acid | Unsaturated fatty acids | Fatty acid biosynthesis | 1.25 | 1.739 | 0.014 | |
Octadecanoic acid | Saturated fatty acids | Fatty acid biosynthesis | 1.24 | 1.849 | 0.007 | |
Hexadecanoic acid | Saturated fatty acids | Fatty acid biosynthesis | 1.24 | 1.7 | 0.026 | |
2-Hydroxyglutaric acid | Organates/carboxylates | Others | 0.789 | 1.72 | 0.013 | |
Pyruvic acid | Organates/carboxylates | Pyruvate metabolism | 0.727 | 1.917 | 0.006 | |
Sorbitol-6-phosphate | Carbohydrate | Fructose and mannose metabolism | 1.22 | 2.041 | 0.001 | |
Phosphoric acid | Oxidative phosphorylation relatives | Oxidative phosphorylation | 0.893 | 1.508 | 0.048 | |
FMT/CO | Glutamine | Amino acid | Arginine biosynthesis | 8.6 | 1.865 | <0.001 |
Asparagine | Amino acid | Alanine, aspartate and glutamate metabolism | 2.96 | 1.947 | <0.001 | |
Methionine | Amino acid | Cysteine and methionine metabolism | 1.59 | 1.566 | 0.017 | |
Ornithine | Amino acid | Arginine biosynthesis | 1.56 | 1.429 | 0.033 | |
S-methyl-Cysteine | Amino acid | Others | 1.38 | 1.48 | 0.019 | |
Arachidonic acid | Unsaturated fatty acids | Arachidonic acid metabolism | 1.56 | 1.737 | 0.003 | |
9,12-(Z,Z)-Octadecadienoic acid | Unsaturated fatty acids | Linoleic acid metabolism | 1.49 | 1.672 | 0.006 | |
9-(Z)-Octadecenoic acid | Unsaturated fatty acids | Fatty acid biosynthesis | 1.44 | 1.594 | 0.013 | |
Hexadecanoic acid | Saturated fatty acids | Fatty acid biosynthesis | 1.44 | 1.758 | 0.003 | |
Octadecanoic acid | Saturated fatty acids | Fatty acid biosynthesis | 1.4 | 1.917 | <0.001 | |
1-Monooctadecanoylglycerol | Lipid | Others | 1.33 | 1.608 | 0.008 | |
Glyceric acid-3-phosphate | Lipid | Glycolysis or Gluconeogenesis | 0.768 | 1.375 | 0.023 | |
Hypoxanthine | Nucleoside | Purine metabolism | 2.04 | 1.57 | 0.013 | |
Adenosine-5-monophosphate | Nucleoside | Purine metabolism | 0.122 | 1.679 | 0.002 | |
Sorbitol-6-phosphate | Carbohydrate | Fructose and mannose metabolism | 1.24 | 1.853 | <0.001 | |
Phosphoric acid | Oxidative phosphorylation relatives | Oxidative phosphorylation | 0.864 | 1.407 | 0.018 | |
Pyruvic acid | Organates/carboxylates | Pyruvate metabolism | 0.704 | 1.613 | 0.005 | |
FMT/AM | Glutamine | Amino acid | Arginine biosynthesis | 2.2 | 1.823 | 0.00426 |
Asparagine | Amino acid | Alanine, aspartate and glutamate metabolism | 1.68 | 1.92 | 0.00143 | |
Glycine | Amino acid | Glycine, serine and threonine metabolism | 1.49 | 1.453 | 0.0348 | |
Methionine | Amino acid | Cysteine and methionine metabolism | 1.45 | 1.635 | 0.0165 | |
Alanine | Amino acid | Alanine, aspartate and glutamate metabolism | 1.28 | 1.506 | 0.0312 | |
Ribose | Carbohydrate | Pentose phosphate pathway | 1.17 | 1.539 | 0.0287 | |
Maltose | Carbohydrate | Starch and sucrose metabolism | 2.07 | 1.721 | 0.00998 | |
Galactose | Carbohydrate | Galactose metabolism | 0.94 | 1.599 | 0.023 | |
Glucose | Carbohydrate | Glycolysis or Gluconeogenesis | 0.94 | 1.599 | 0.023 | |
Adenosine | nucleoside | Purine metabolism | 0.666 | 1.55 | 0.028 | |
Uridine-5-monophosphate | Nucleoside | Pyrimidine metabolism | 0.662 | 1.674 | 0.0125 | |
Adenosine-5-monophosphate | Nucleoside | Purine metabolism | 0.402 | 1.654 | 0.0183 | |
Octadecanoic acid | Saturated fatty acids | Fatty acid biosynthesis | 1.13 | 1.518 | 0.0321 | |
1-Monooctadecanoylglycerol | Lipid | Others | 1.17 | 1.548 | 0.0285 |
On day 7, a total of 632 DEGs (
Number of the total differentially expressed genes (DEGs) as well as the up- and downregulated genes in the livers of pigs among the AM, FMT and CO groups on days 7 and 21.
Expression of fatty acid metabolism-related genes in the liver of piglets in the AM, FMT, and CO groups on days 7 and 211.
Gene | Day 7 |
Day 21 |
||||
---|---|---|---|---|---|---|
AM/CO | FMT/CO | AM/FMT | AM/CO | FMT/CO | AM/FMT | |
CYP2C42 | 1.656(0.042) | 1.955(0.000) | 0.847(0.361) | 1.174(0.529) | 1.092(0.901) | 1.075(0.702) |
ALOX15 | 0.933(0.723) | 0.330(0.001) | 2.829(0.010) | 3.564(0.025) | 2.552(0.193) | 1.397(0.395) |
CYP1A2 | 0.457(0.005) | 0.513(0.019) | 0.890(0.297) | 1.081(0.722) | 1.075(0.835) | 1.006(0.962) |
ALOX12 | 0.603(0.002) | 0.788(0.058) | 0.765(0.124) | 0.923(0.616) | 0.952(0.624) | 0.970(1) |
ACAA2 | 0.600(0.007) | 0.739(0.104) | 0.813(0.105) | 0.810(0.436) | 0.926(0.514) | 0.874(0.798) |
Expression of amino acid metabolism-related genes in the liver of piglets in the AM, FMT, and CO groups on days 7 and 211.
Gene | Day 7 |
Day 21 |
||||
---|---|---|---|---|---|---|
AM/CO | FMT/CO | AM/FMT | AM/CO | FMT/CO | AM/FMT | |
GPT2 | 0.406(0.000) | 0.566(0.007) | 0.717(0.076) | 1.200(0.389) | 1.432(0.153) | 0.838(0.494) |
ARG1 | 0.541(0.000) | 0.881(0.306) | 0.614(0.005) | 1.127(0.587) | 1.162(0.613) | 0.970(0.921) |
TAT | 0.599(0.003) | 0.713(0.040) | 0.840(0.242) | 1.222(0.280) | 0.905(0.422) | 1.350(0.091) |
ASS1 | 0.659(0.020) | 0.771(0.165) | 0.855(0.330) | 1.051(0.859) | 1.188(0.583) | 0.884(0.707) |
AOC2 | 0.585(0.004) | 0.879(0.315) | 0.665(0.119) | 1.369(0.238) | 1.279(0.357) | 1.071(0.663) |
ALAS2 | 0.609(0.017) | 0.726(0.127) | 0.838(0.326) | 1.509(0.005) | 1.895(0.040) | 0.796(0.593) |
GATM | 0.618(0.035) | 0.710(0.016) | 0.870(0.488) | 0.948(0.817) | 0.669(0.105) | 1.418(0.073) |
OAT | 0.455(0.000) | 0.730(0.075) | 0.624(0.016) | 0.991(0.812) | 1.160(0.666) | 0.854(0.487) |
The result of the GO enrichment analysis is presented in
Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that on day 7, arachidonic acid metabolism and steroid hormone biosynthesis were significantly affected by AM and FMT treatment (
Kyoto Encyclopedia of Genes and Genomes pathway analysis of the DEGs in the livers among the AM, FMT and CO groups on day 7
To validate the gene expression profile from RNA-seq, we validated six genes (
The qPCR validation of the RNA-seq. The results are displayed as the values of fold changes in the CO group on day 7
Antibiotics are the frontline therapy against microbial infectious diseases, but most antibiotics can cause side effects in humans (
A previous study reported that subtherapeutic antibiotic treatment (penicillin VK, vancomycin, penicillin VK plus vancomycin and chlortetracycline) could significantly increase fat mass in mice and alter the fatty acid metabolism and lipid metabolic processes of the liver (
Arachidonate 15-lipoxygenase encoded by
The medium chain 3-ketoacyl-CoA thiolase encoded by
Furthermore, previous studies revealed that antibiotics increased the serum concentration of most amino acids and decreased most amino acids of the small intestine, and that they supplemented the upregulation of the mRNA expression levels for key amino acid receptors and transporters of the small intestine (
Glutamate pyruvate transaminase 2 encoded by
Argininosuccinate synthetase encoded by
The other genes related to amino acid metabolism, such as
In summary, our study investigated the transcriptome and metabolome responses induced by the short-term oral administration of AM and FMT in the early life of piglets. We found that both FMT and antibiotics could reduce fatty acid oxidative catabolism and amino acid biosynthesis in the liver, and that antibiotics had a more significant effect on reducing alanine biosynthesis and amino acid metabolism than FMT. Our finding provides a reference for regulation host metabolism through early intervention in animal production and even human health. However, more studies are needed for further understanding the impact and regulating mechanism of early intervention with AM and FMT on host metabolism in the later life.
YS and W-YZ conceived and designed the experiments. J-JW, C-HL, E-DR, and YS performed the experiments and analyzed the data. J-JW, C-HL, and YS wrote the paper.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: