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 ± 0.02 and 1.71 ± 0.02, 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 . A commercial-type laying hen diet was fed to birds ad libitum, water was available at all times (Table S1), 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 -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
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 with the mirVana™ miRNA Isolation Kit (Austin, TX, USA) according to Liu et al. (2016). Briefly, approximately 100 mg cecal content was mixed adequately with 600 μL 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, centrifuged for 5 min at 8000 ×g to recover miRNA and then stored it 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 with the RNeasy® PowerMicrobiome Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 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 combining equal amounts of 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, significantly differentially expressed miRNAs between the two breeds were used for target prediction analysis for microbial significantly differentially expressed genes related to H2S production. Bacterial mRNAs potentially targeted by miRNAs were identified by miRanda (http://www.microrna.org). Furthermore, a prediction of host genome genes targeted by of 10 differentially expressed miRNAs was also conducted by miRanda, and all the target genes were determined to be enriched by KEGG analysis.
In vitro fermentation experiment
Thirty Hy-line Gray and thirty Lohmann Pink laying hens at 28 weeks of age were sacrificed, and the ceca were ligated immediately. The cecal contents in the same breed group were pooled, thoroughly mixed and diluted in sodium and ammonia bicarbonate buffer solution (35.0 g NaHCO3+4.0 g NH4HCO3 for a 1 L volume) at a ratio of 1:3 (W/V) . The intestinal content–buffer mixture was blended for 60 sec in a blender after which the solution was squeezed through four layers of surgical gauze, and then mixed with a buffer mineral solution at a 1:2 ratio (V/V) at 40℃ under continuous flushing with CO2. A corn-soybean basal laying hen diet was used as a substrate for fermentation.
Approximately 10 mL of the inoculum was added to a 100 mL gas syringe with 0.2 g of the substrate. Six groups with different treatments were designed, including the blank group (10 mL inoculum+ 0.2 g substrate+ 1 mL pure water), control group (10 mL inoculum+ 0.2 g substrate+ 1 mL control mimic at a final concentration of 2 µM) and treatment group (10 mL inoculum+ 0.2 g substrate+ 1 mL gga-miR-222a mimic at a final concentration of 2 µM) for each of the types of hens, Hy-line and Lohmann (Fig. 1). The miRNAs were supplied by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). After removing the air from the head-space of the syringe, the syringe was sealed with a clip and placed in a 42°C incubator, where it was rotated at 60 rpm for 24 h. At the end of incubation, the syringes were put into an ice box to stop further fermentation, the volume of gas produced in the head-space was recorded and the gas was transferred by syringes 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 in the syringes and the concentrations of soluble sulfide (S2-) in the fermentation broth were determined using the methylene-blue colorimetric method. 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 fermentation broth were detected by an automatic amino acid analyzer (Sykam, Munich, Germany).
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. The DNA was used to quantify the abundance of Odoribacter splanchnicus and Bacteroides fragilis NCTC 9343. RNA was extracted from fermentation broth using the RNeasy® PowerMicrobiomeTM Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Extracted mRNA was reverse transcribed into cDNA 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 S2).
In vitro bacterial growth measurements
To further verify the effect of gga-mir-222a on the growth and metabolism of bacteria in a pure culture environment and considering that Odoribacter splanchnicus could not be cultured in vitro, we selected Bacteroides fragilis NCTC9343 cultured in anaerobic medium to investigate the effect of gga-miR-222a on the growth and metabolism of bacteria. 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. 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 10 h 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 10 h were detected by an automatic amino acid analyzer (Sykam, Munich, Germany).
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).
Comparisons among groups were performed using SPSS 22.0 for statistical analysis. Data are presented as the means with standard errors of the mean. Differences were considered significant if P<0.05.