Screening Of Key MRNAs And Non-coding RNAs Related To Sperm Motility Based On Testis Transcriptome Sequencing In Pigeons(Columba Livia)

Sperm motility is one of the most important indicators in assessing semen quality and,it is often used to evaluate poultry fertility.At present,the research on male reproductive performance in livestock and poultry production mainly focuses on mammals,poultry mainly focusing on chickens.The research on pigeon reproductive performance is relatively scarce,especially in non-coding RNA research.Sperm motility trait has high heritability,but the molecular mechanism of its regulation is still unclear.To explore the molecular regulation mechanism of poultry sperm motility,pigeons were selected as the research object to compare their sperm motility and egg production and fertilization rate index.High-throughput sequencing technology was used to analyze the lncRNA,mRNA and miRNA expression profiling in pigeon testie,and the significantly differentially expressed lncRNAs,mRNAs and miRNAs were screened to identify genes,non-coding RNAs and signaling pathways related to sperm motility.The main research results are as follows:The sample groups of pigeons with high and low sperm motility was established by measuring the semen quality of 200 male pigeons.Then,five individuals from each group were selected for transcriptome sequencing according to the recorded data of egg production and fertilization rate.LncRNA sequencing of pigeon testes with high and low sperm motility was performed.A total of 100 M（million）double-ended original data with 150 nt read length was obtained,and 139.0 GB（gigabase）sequencing quantity was obtained.After quality control,13.93G of high-quality clean data were obtained,of which about 84%could be compared to the pigeon genome.A total of 17463 lncRNAs were screened,including 3383 intronic lncRNAs,3201 bidirectional lncRNAs,1039 sense lncRNAs,8438 intergenic lncRNAs and 1402 antisense lncRNAs.Compared with pigeon coding genes,the lncRNA identified in this study has the characteristics of shorter transcript,fewer exons,and shorter ORF length.Differential expression analysis revealed that 2673 mRNAs and 229 lncRNAs were significantly differentially expressed between the high and low sperm motility groups.The functional enrichment analysis of GO and KEGG showed that the target genes of differentially expressed lncRNAs and differentially expressed mRNAs were mainly enriched in calcium ion binding,ATP binding,and spermatogenesis pathways.In the target gene prediction of lncRNA,it was found that lncRNAMSTRG.7787.5 may be involved in the regulation process of pigeon sperm motility by regulating the expression of target gene UBB.A total of 133M raw data were obtained by small RNA sequencing,and 8M valid data were obtained after quality control,of which 61%valid data could be compared in the pigeon genome.As a result,a total of 705 known miRNAs and 385 new miRNAs were predicted in pigeon testes.Differential expression analysis showed that 6 miRNAs were significantly differentially expressed in the high and low sperm motility groups,including 4 known miRNAs and 2 new miRNAs.Bioinformatics analysis found that the target genes of differentially expressed miRNAs were mainly regulate pigeon sperm motility by participating in serine/threonine kinase activity,ATP binding,Wnt and MAPK signaling pathway.A total of 3567 target genes were predicted by differential expression of miRNAs.Combined with the results of differentially expressed mRNA in the second chapter,four target mRNAs were selected to validate by qPCR.The miRNA-mRNA interaction network analysis,indicated that mmu-miR-183-5p/FOXO1 and PC-3p-244994₃1/CHDH pairs might affect sperm motility.In conclusion,this study screened and studied the differentially expressed mRNA,lncRNAs and miRNAs in the high and low sperm motility group of pigeons,revealing the lncRNAs and miRNAs involved in the regulation of sperm motility in pigeons.In addition,the research results will provide a theoretical basis for further revealing the regulation mechanism of sperm motility of pigeon.