Mechanism Of Cecal Secreted MiRNA Regulating The Dissimilar Hydrogen Sulfide Production In Different Layer Breeds

In this study,Hy-line grey and Lohmann pink laying hens were used as experimental subjects to explore the differences of hydrogen sulfide emission between different breeds,as well as the effect and mechanism of cecal secreted mi RNA on the production of hydrogen sulfide.Part one:Comparison of hydrogen sulfide production between Lohmann Pink and Hy-line GreyIn order to explore the production of hydrogen sulfide between Lohmann Pink and Hy-line Grey,the following two experiments were designed.Respiratory chamber experiment:30 28-week-old Lohmann Pink and Hy-line Grey from the same layer farm were selected,and placed in 12 respiratory chambers and grouped by breed.The experiment lasted 21 days and gas production measure process was performed last 3 days.Laying hens were slaughtered and samples of the cecum contents were collected after the experiment.In vitro fermentation:The contents of the cecum from 20 28-week-old Lohmann Pink and Hy-line Grey were sampled to make the source of the fermentation bacteria and fermented at 40°C for 12 h,the fermentation gas and fermentation incubation were collected.The average daily hydrogen sulfide production of Hy-line and the concentration of soluble sulfide in the contents of Hy-line cecum was significantly lower than that of Lohmann（P<0.05）,but the concentration of butyric acid was significantly higher than that of Lohmann（P<0.05）;Hydrogen sulfide production of Hy-line and the concentrations of soluble sulfide and sulfate ions in Hy-line fermentation incubation was significantly lower than Lohmann（P<0.05）,but the content of butyric acid and total volatile fatty acids was significantly higher than Lohmann（P<0.05）.The results showed that Hy-line had a lower hydrogen sulfide emission compared to Lohmann.Part two:Expression of secreted mi RNAs in cecal contents of laying hensIn order to investigate the expression characteristic of cecum secreted mi RNA and its relationship with hydrogen sulfide production.The morphology and expression information of secreted mi RNAs in cecum contents of laying hens were investigated by transmission electron microscopy and metatranscriptome.Transmission electron microscopy confirmed the presence of microvesicles in the cecum content of laying hens.10 mi RNAs were significantly different between the two breeds by sequencing.Correlation analysis found that gga-mi R-222a was significantly negatively correlated with hydrogen sulfide production（P<0.05）,while gga-mi R-10a-5p and gga-mi R-6651-5p were significantly positively correlated with hydrogen sulfide production（P<0.05）.The results showed that mi RNA existed in the cecum contents of two breeds,among which gga-mir-222a was significantly related to the hydrogen sulfide production.Part three:Cecal microbiota structure of Lohmann and Hy-lineIn order to explore the microbial structure of dissimilar hydrogen sulfide emission between the two breeds.The abundance of the hydrogen sulfide-producing bacteria in the contents and fermentation incubation were determined by 16S r RNA sequencing,and the relationship between mi RNA and cecal bacteria was also explored.It found that the abundance of Helicobacter,Campylobacter and Sutterella,which had strong protein fermentation ability,and sulfate-reducing bacteria such as Desulfovibrio,Mailhella and Lawsonia was significantly lower than that of Lohmann（P<0.05）,whereas the abundance of some acid-producing bacteria like Bacteroides,Butyricicoccus and Butyricimonas was significantly higher than that of Lohmann（P<0.05）.The above indicated that the difference in the abundance of hydrogen sulfide related bacteria resulted in the lower hydrogen sulfide emission of Hy-line compared to Lohmann.The correlation analysis found that there was a significant correlation between differentially expressed mi RNA with hydrogen sulfide related and acid producing bacteria（P<0.05）,suggesting that mi RNA may play a regulatory role in the genesis of hydrogen sulfide by affecting the abundance and function of cecal bacteria.Part four:Target analysis of hydrogen sulfide-related microbiol genesIn order to explore target relationship between the mi RNA and the bacterial m RNA.The expression of hydrogen sulfide genesis genes in cecal microbiota of laying hens was studied by metatranscriptome sequencing,and the target relationship was selected.It found that It was found that the expression of genes related to the synthesis of cysteine,methionine and butyric acid in the cecal microbiota of Hy-line was significantly higher than that in Lohmann（P<0.05）,while the expression of genes related to the degradation of cysteine,methionine and sulfate reduction in the cecal microbiota of Hy-line was significantly lower than that in Lohmann（P<0.05）.The results showed that the expression of genes related to hydrogen sulfide reduction was higher but the expression of genes related to hydrogen sulfide emission was lower in Hy-line cecum than that of Lohmann.The target relationship between gga-mi R-222a and the genes of methionine synthetase Odosp＿3416（expressed by Odoribacter splanchnicus）and BF9343＿2953（expressed by Bacteroides fragilis NCTC 9343）was found.Part five:Verification of gga-mi R-222a on hydrogen sulfide production of laying hensIn order to verify the effect of gga-mi R-222a on hydrogen sulfide production.Cecal contents from 28-week-old Lohmann and Hy-line were used to make a source of fermentation bacteria with synthetic gga-mi R-222a was added.It found that gga-mi R-222a had a reduction effect on hydrogen sulfide generation,and could significantly reduce the concentration of soluble sulfide in the fermentation incubation（P<0.05）,significantly increased the expression of Odosp＿3416 and BF9343＿2953 gene（P<0.05）,as well as increased the concentration of methionine in the fermentation incubation（P<0.05）.The co-cultured of gga-mi R-222a and Bacteroides fragilis NCTC 9343 showed that gga-mi R-222a could be absorbed into the cell by Bacteroides fragile and bound with its nucleic acid to exert its regulatory function,increased the abundance of Bacteroides fragile in logarithmic phase,and up-regulated the expression of the methionine gene BF9343＿2953,increased the methionine content in germ solution,reduced the production of hydrogen sulfide.In summary:（1）The production of hydrogen sulfide of Hy-line was significantly lower than that of Lohmann,and there were mi RNAs in the cecum of layer which were related to the H₂S emission and the abundance of cecal bacteria,and there was a target relationship between these mi RNAs and microbial genes.（2）gga-mi R-222a could increase the abundance of Bacteroides fragilis NCTC 9343,increase the content of methionine and decrease the concentration of soluble sulfide in the fermentation incubation and germ solution,to realize the emission reduction of H₂S.