Animals and feeding
Approximately one hundred Hy-line Gray laying hens and one hundred Lohmann Pink laying hens were hatched and fed together at a local hatchery. To eliminate the confounding effects that might be caused by diet, age, weight and feeding environment. Thirty Hy-line Gray laying hens and thirty Lohmann Pink laying hens at 28 weeks of age with similar weights (1.70±0.02kg and 1.71±0.02kg, respectively for Hy-line and Lohmann) were selected and moved into twelve respiration chambers in an environmentally controlled room for a daily H2S production measurement for the two breeds . Water and the commercial-type laying hen diet were fed to birds ad libitum (Table S3), and a 12-h light cycle at 24°C room temperature management schedule was used. At the end of the experiment, all birds were euthanized by cervical dislocation, and then the cecum was ligated at both sides and removed from the gastrointestinal tract. The contents were aseptically collected into an Eppendorf tube containing Bacterial Protect RNA reagent (Qiagen, Hilden, Germany) at an approximate 1:1 ratio (w/v), and immediately frozen at liquid nitrogen and stored at -80°C until analysis.
Animal ethics statement
All animal experiments were approved by the Animal Experimental Committee of South China Agricultural University (SYXK2014-0136). All experimental steps were performed to decrease animal suffering as much as possible. After the experiment, the bodies of laying hens were incinerated.
The determination of exosomes in the cecum contents
The exsomes purification referenced Liu, briefly, cecal contents from laying hens were suspended in PBS to 30 mg/ml, spun down at 10,000 ×g for 5 min to remove debris and then filtered through a 0.2 µm filter and the filtrates were observed by Thermo Fisher Talos L120C transmission electron microscope (Thermo Fisher Scientific, MA, US) .
Extraction and analysis of miRNA in the cecum of laying hens
Total miRNA was extracted by using mirVana™ miRNA Isolation Kit (Austin, TX, USA) according to Liu . Briefly, approximately 100 mg cecal content was mixed adequately with 600μL 1×DPBS, and the mixture was left at room temperature for 30 min, and then mashed to complete suspension. Then 600 μL acid-phenol: chloroform was added, and the samples were vortexed for 60 sec and then centrifuged for 15 min at 10,000 ×g to separate organic phases. The aqueous phase was recovered, and 1.25 volumes of 100% ethanol was added to the aqueous phase for final miRNA isolation. For each sample, a filter cartridge was placed into one of the collection tubes (supplied by the kit), and the sample was pipetted onto a filter and centrifuged for 90 sec at 10,000 ×g, and then the flow-through was discarded. The filter was washed with 700 μL miRNA Wash Solution 1 and then washed three time with 700/500/250 μL Wash Solution 2/3 (supplied by the kit). Finally, the filter was transferred into a fresh collection tube, and 50 μL nuclease-free water was applied to the center of the filter. The filter was incubated at room temperature for 10 min, then centrifuged for 5 min at 8000 ×g to recover miRNA and then stored at -80℃. Pooled miRNA was prepared by combining equal amounts of extracted miRNA from five birds of the same breed, which means that each breed was represented by six pooled miRNA samples. miRNA libraries were constructed according to the TruSeq Small RNA Sample Preparation protocol. The raw sequence reads were obtained with an Illumina HiSeqTM 2500 instrument (Illumina, San Diego, USA). FastQC was applied to obtain clean reads from the raw data by removing the joint sequences, low-quality fragments, and sequences <18 nucleotides (nt) in length. miRDeep2 was used to align the clean sequences to the miRBase database sequences (http://www.mirbase.org/).
The extraction and analysis of RNA of cecal microbiota
Cecal content aliquots (200 mg) were used for RNA extraction by using RNeasy® PowerMicrobiome Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Briefly, 200mg sample was placed into PowerBead Tubes. Cells were lysed according to the kit manual by adding 650µL Solution PM1 with β-mercaptoethanol and 100µL phenol/chloroform/isoamylalcohol, followed by vortexing for 10 minutes at maximum speed using a 24-sample vortex adapter (Kelly Bell, Jiangsu, China), Centrifuged at 13,000×g for 1 min at room temperature (15-25°C). Transfer the supernatant to a clean 2 ml Collection Tube. The following RNA purification was referenced the protocol of the manufacturer. The integrity and quantity of extracted RNA was measured with a NanoDrop 2000 spectrophotometer (Thermo Scientific, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA). Pooled RNA was prepared by homogenization of equal amounts extracted RNA from five birds of the same breed; thus, each breed was represented by six pooled RNA samples. RNA was subjected to standard Illumina library preparation with the TruSeq RNA Sample Prep Kit (Illumina, San Diego, USA), and rRNA was depleted with the Ribo-ZeroTM rRNA Removal Kit (Epicenter Biotechnologies, Madison, WI). Sequencing was performed on an Illumina HiSeq 2500 sequencer (Illumina, San Diego, USA). Sequences were quality filtered and poor-quality bases of raw reads were removed by using Cutadapt (v1.9.1) software. A 10 bp window was moved across each sequence, and nucleotides in windows with a mean quality score < 20 were removed; reads with “N” bases (>10%) and lengths below 75 bp were discarded; primer sequences and adaptor sequences were also removed. Next, rRNA, tRNA and host reads were filtered using BWA (v 0.7.5). Putative mRNA reads were then assembled using the Trinity (v2.1.1) de novo assembler. Gene annotation was performed by searching against a protein non-redundant database (NR database), and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was conducted for gene function classification. After the comparation of transcript profiles between the two breeds, we focused on three pathways related to H2S production, including cysteine and methionine metabolism, sulfur metabolism and butyrate metabolism, and the expression of microbial genes in these pathways between the two breeds was compared.
Target prediction of differentially expressed miRNAs
After the exploration of miRNA profiles in the cecal content of laying hens, the significantly different expression miRNAs between the two breeds were used for the target prediction analysis of microbial significantly different expression genes which related to H2S production. The target relationship between Bacterial mRNAs and miRNAs were identified by using miRanda (http://www.microrna.org). Furthermore, a prediction of host genome genes which was targeted by the 10 different expression miRNAs were also conducted by miRanda, and all the target genes were determined to be enriched by KEGG analysis.
In vitro fermentation experiment
The in vitro was referenced Menke and Steingass , briefly, thirty Hy-line Gray and thirty Lohmann Pink laying hens at age of 28 weeks were sacrificed respectively, and the caeca were ligated immediately. The cecal contents in the same breed group were pooled, then thoroughly mixed with the fermentation buffer solution which was pre-heat at 39°C as the fermentation broth. The fermentation buffer solution was homogenization by 474mL deionized water, 237mL macro element solution (per 1000mL, Na2HPO4 5.7 g, KH2PO4 6.2 g, MgSO4·7H2O 0.6 g), 237mL buffer solution (per 1000mL, 35.0 g NaHCO3, 4.0 g NH4HCO3), 0.12 trace element solution (per 100mL, CaCl2·2H2O 13.2 g, MnCl2·4H2O 10.0 g, CoCl2·6H2O 1.0 g, FeCl2·6H2O 0.8 g), 1.22mL resazurin and 50mL reductant (per 50mL, 1mol/L NaOH 2.0 mL, Na2S·9H2O 335 mg). The intestinal content-buffer mixture was blended for 60 sec in a blender after the solution was filtered through four layers of surgical gauze, and then mixed with the fermentation buffer solution at a 1:2 ratio (V/V), flushed with CO2 at 40℃ in order to eliminate all the O2 in the solution. A corn-soybean basal laying hen diet was used as a substrate for fermentation.
After the air in syringe was eliminated from the head-space, approximately 10mL fermentation broth (FB) was added to a 100 mL gas syringe with 0.2g of the substrate. Three groups with different treatments for each breed were designed, the blank group (10mL FB+0.2g substrate+1mL pure water), control group (10mL FB+0.2g substrate+1mL control mimic at a final concentration of 2µM) and treatment group (10mL FB+0.2g substrate+1mL gga-miR-222a mimic at a final concentration of 2µM) (Table. S4). The miRNAs applied in present study were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Then, these syringes were sealed with clips and placed in incubator and rotated at 42°C, 60rpm for 24h. At the end of incubation, the syringes were put on the ice to stop the fermentation, the gas production was recorded as the volume of head-space of syringe and the gas was also injected into a gas collection bag for H2S analysis. Ten milliliters of fermentation broth was sampled and stored at -80℃ for chemical analysis. The quantity of H2S of the gas sample and the concentrations of soluble sulfide (S2-) of the fermentation broth were determined using the methylene-blue colorimetric method, briefly, the adsorption liquid (per 1000mL, 3CdSO4·8H2O 4.3g, NaOH 0.3g, ammonium polyvinyl phosphate 10g) was mixed with gas (10mL adsorption liqiud) and fermentation broth (adsorption liquid : fermentation broth, V/V=9:1), then followed the steps mentioned in previous studies [43, 44]. The pH value was determined using a pH meter (INESA Scientific Instrument, Shanghai, China) . The concentration of sulfate radicals (SO42-) was determined using the turbidimetric method . The concentrations of VFAs were determined using high-performance liquid chromatography . The concentrations of methionine in the fermentation broth were tested by using automatic amino acid analyzer (Sykam, Munich, Germany), briefly, 1mL fermentation broth was mixed with 10mL hydrochloric acid solution (6mol/L), 3-4 drops of phenol liquid were added to the above mixture solution, then put the tube which contained the mixture solution on ice for 3-5min. The tube which contained the mixture solution was oxygen-free by the treatment of nitrogen flushing, then put the tube into air oven at 110℃ for 22h. After cooling the mixture solution at room temperature, the mixture solution was constant volume as 50mL after filtering by using filter paper. The 15mL filtering solution was dried at 40-50℃, and the deposition was washed with the deionized water twice and dried again. Then the deposition was diluted by 1mL 0.02mol/L hydrochloric acid solution, after filtering 0.22µm membrane the concentration of methionine was tested by automatic amino acid analyzer.
Bacterial abundance and gene expression in fermentation broth
DNA was extracted from fermentation broth by using a QIAamp PowerFecal DNA Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions, briefly 1mL fermentation broth was centrifuged at 20,000×g for 1 min at 4℃ to collect the precipitate (approximately 200mg) for the DNA extraction, and the DNA purification following steps was referenced the protocol of manufacturer. RNA extraction of fermentation broth followed the protocol of RNeasy® PowerMicrobiomeTM Kit (Qiagen, Hilden, Germany), briefly, 1-2mL fermentation broth was centrifuged at 20,000×g for 1 min at 4℃ to collect the precipitate (approximately 200-250mg) for the RNA extraction, and the RNA purification following steps was referenced the protocol of manufacturer.
The DNA was used to quantify the relative abundance of Odoribacter splanchnicus and Bacteroides fragilis NCTC 9343. Briefly, the primers of these two bacteria was designed by using the software Primer 3, the sequences of the two bacteria was referenced the 16S rRNA sequencing in the web of NCBI, the details of the primers were showed in Table S5, the primer of bacterial16S rRNA was referenced previous study . q-PCR was used to confirm the relative abundance of the two bacteria, the q-PCR reaction steps were followed the protocol of SYBR® Green PCR Kit (SYBR, Japan) (Table S6). The relative abundance of the two bacteria was calculated as 2△Ct, where △Ct represents the difference in the Ct value for the 16S rRNA gene minus that for the genes .
Extracted RNA was reverse transcribed into cDNA by using the PrimeScriptTM RT reagent kit (TaKaRa, Kusatsu, Japan). The cDNA was used to quantify the expression of Odosp_3416 and BF9343_2953. The primers were designed using the NCBI website with the total bacterial 16S rRNA gene as the reference gene (Table S5). q-PCR was used the confirm the relative expression level of the two genes, the q-PCR reaction steps were followed the protocol of SYBR® Green PCR Kit (SYBR, Japan) with a little modification (Table S7). The relative expression level of the two genes was calculated as 2△Ct, where △Ct represents the difference in the Ct value for the 16S rRNA gene minus that for the genes .
In vitro bacterial growth measurements
The anaerobic bacterium Bacteroides fragilis NCTC9343 were cultured at 37ºC by inoculating 40 mL aliquots of anaerobic basal medium (Becton Dickinson and Company, Lincoln Park, USA) and then grown anaerobically in an anaerobic chamber (Mitsubishi Gas Chemical Company, Inc. Tokyo, Japan). gga-miR-222a and the control mimic were supplied in the culture at a concentration of 2 µM. (RiboBio, Guangzhou, China). Growth was monitored as absorbance at 600 nm once per hour for up to 24 h with a spectrophotometer. The cultured bacterial cells were collected at 10h and used for BF9343_2953 gene expression measurement with the Bacteroides fragilis 16S rRNA gene as the reference gene. The concentrations of methionine in culture medium at 10h were tested as above mentioned.
In situ hybridization detection of the uptake of gga-miR-222a
The bacterial cells of Bacteroides fragilis NCTC9343 were centrifuged at 12,000×g and washed twice with ice cold PBS. Then, the cells were fixed in 4% PFA/0.25% glutaraldehyde. A 5’-DIG and 3’-DIG dual labeled probe for gga-miR-222a was used for in situ hybridization. The detection of the uptake of gga-miR-222a by bacteria was imaged using a Thermo Fisher Talos L120C transmission electron microscope Thermo (Fisher Scientific, MA, US).
The data of the comparison of fermentation incubation indexes, the comparison of the relative abundance of miRNA, bacteria and genes, gas production, H2S production and growth curve were examined by analysis of variance (ANOVA) with Statistical Package for the Social Sciences (SPSS) software, version 22.0. Significant differences between the means were determined by Tukey's test. Differences were considered significant at P＜0.05.
The metatranscriptomic results of each sample was analyzed by HTSeq software, and the model used was union, the number of genes in different expression levels and the expression level of individual genes were statistically analysed. In general, the value of FPKM is 0.1 or 1 as the threshold for determining whether genes are expressed. The software DESeq was used for normalization of the read counts from analysis of genes expression levels .
The expression level of miRNA was calculated by the TPM formula (normalization read counts= (readCount*1,000,000)/libsize)), libsize was the sum of the read count of all miRNAs.