Mechanism Study On Chicken Sperm Motility Related MRNA-miRNA-lncRNA Regulatory Network Based On Testis Transcriptome Sequencing

Sperm motility is the most important indicator in evaluating roosters' fecundity.It has important influence on poultry production,reproduction,and breeding.It is revealed from several studies and investigations that a higer proportion breeder roosters of indigenous breeds have the problem of low sperm motility(Asthenospermia).Sperm motility is heritable,while the genetic mechanisms underlying this trait is not yet clear.Therefore,a powerful approach should be used to identify biomarkers related to sperm motility trait in chicken,which can be used to eliminate low sperm motility birds.It will provide important clues for revealing the molecular mechanism of sperm motility regulation,and offer a theoretical basis for breeding of high fecundity chicken by molecular techniques.In this study,Beijing-you cocks with extreme sperm motility phenotype were selectedfor high-throughput sequencing.The differentially expressed mRNAs,lncRNAs,and miRNAs in the testis of high and low sperm motility cocks were identified.The underlying signaling pathways were also identified by KEGG analysis.mRNA-miRNA-lncRNA regulatory network was constructed to identify the key transcription regulation mechanism of sperm motility.The main results of this study were as follows.High sperm motility group and low sperm motility group,each containing 20 birds,were selected from 200 beijing-you cocks by semen quality evaluation.The comparison of two groups showed that most reproductive indicators of high sperm motility cocks was better than those of low sperm motility birds.and the correlation between the egg fertilization rate and sperm motility was the highest.Histological observation showed the less integrity of testicular seminiferous in low sperm motility cocks.Testicular IncRNA sequencing yielded about 56.2 Gb high-quality sequence data reads from 3 cocks of high sperm motility and 3 low sperm motility.In total,2597 lncRNAs were identified in the chicken testis,including 1267 lincRNAs,975 anti-sense lncRNAs,and 355 intronic lncRNAs.The identified IncRNAs were shorter in transcript length,fewer in exon number,and less expression level than mRNAs.In the differential expression analysis,544 mRNAs and 124 lncRNA were differentially expressed between two groups.GO annotation analysis revealed these genes were involved in ATP binding,cilium assembly,and oxidation-reduction processes.Integrating analysis of lncRNA and mRNA profiles predicted 10 IncRNA-gene pairs.LncRNA MSTRG.3652 and MSTRG.4081 may play roles in regulating the sperm motility by governing their potential target genes CDK13 and LOC428510,respectively.These results were further confirmed by qPCR.Testicular small RNA sequencing yielded about 6.8 Gb high-quality sequence data reads from 2 cocks of high sperm motility and 2 low sperm motility.Length distribution analysis showed that the dominant size of the small RNAs detected in the testis was 24-28 nt.In total,518 known and 106 novel miRNAs were identified.A number of 23 miRNAs were found differentially expressed between the HS and LS,including 18 known and five novel miRNAs.Function enrichment of predicted target genes of the differential miRNAs indicated that the differentially expressed miRNAs were involved in the pathways of GnRH signaling,MAPK signaling,and Wnt signaling.The miRNA-gene interaction network revealed two key candidate miRNA-gene pairs that may affect chicken sperm motility viz gga-miR-155/KCNA1 and gga-miR-7480-5p/AHI1.qRCR was also used to further validate their expression.By constructing the chicken sperm motility mRNA-miRNA-lncRNA regulatory network,a key bunch of 35 IncRNA,119 mRNA,and 18 miRNA were identified.Combined the existed evidences,MSTRG.4081-LOC428510-gga-miR-155,MSTRG.3652-CDK13-gga-miR-6631-5p and gga-miR-155-KCNA1-MSTRG.4692 might play important roles in sperm motility regulation,which requires further investigation.In conclusion,chicken sperm motility is a complex trait which could be influenced by genes and non-coding RNAs.CDK13,LOC428510,gga-miR-155,MSTRG.4081 and MSTRG.3652 played important roles in chicken sperm motility regulation through their interactions.