| Xinjiang Yili goose is the only goose species in China that originated from the gray goose,and it is also one of the few goose species in China that can fly over short distances.The biological characteristics and reproductive performance of the Yili goose are similar to those of its ancestors,the gray goose.The low fertilization rate of its eggs is one of the important reasons that affect the development of the population base of the Yili goose.The quality of male poultry semen is an important factor affecting the fertilization rate of breeding eggs.Sperm motility in semen quality is a reflection of sperm motility and metabolic ability,and is highly positively correlated with fertilization rate.Genetic factors are one of the important factors affecting the motility traits of poultry sperm.At present,research on the molecular mechanisms of sperm motility traits in poultry is relatively scarce,especially in the regulation of epigenetic level,which still needs further research.Therefore,this study focuses on 3-year-old male Yili geese under the same feeding and management conditions,in good health,and during the breeding period(February to June).The semen quality data of 100 Yili goose male geese were measured,and they were divided into high sperm motility group and low sperm motility group based on sperm motility.The fertilization rate of six batches of Yili goose eggs was continuously monitored.Based on sperm motility and egg fertilization rate,5 Yili geese were selected from the high and low sperm motility groups for slaughter.Testicular samples were taken to measure testicular development related indicators,and tissue sections were observed.At the same time,transcriptome sequencing technology was used to perform full transcriptome sequencing on the testicles of Yili geese with high and low sperm motility.By combining bioinformatics analysis,in vitro cultivation of testicular sertoli cells,RT-qPCR,dual luciferase assay,CCK-8 and other detection techniques,we aim to deeply explore and identify the genes and ncRNA functions that affect sperm motility in Yili geese,in order to analyze the genetic basis of sperm motility in Yili geese from the perspective of ncRNA and provide a theoretical basis for genetic breeding improvement in Yili geese.The main results of this study are as follows:(1)A comparative analysis was conducted on the semen quality and testicular development of Yili geese with high and low sperm motility.The results showed that in terms of semen quality,the high sperm motility group(HFR group)had higher sperm motility,sperm motility rate,and sperm density than the low sperm motility group(LFR group)(P<0.01).In terms of reproductive performance,the fertilization rate and hatching rate of the HFR group were higher than those of the LFR group(P<0.01).In terms of correlation analysis,sperm density showed a highly significant positive correlation with the hatching rate of incubated eggs(P<0.01),and a significant positive correlation with the hatching rate of fertilized eggs(P<0.05);There is a significant positive correlation between semen volume and fertilization rate(P<0.05).In terms of testicular development,both the HFR group and the LFR group had slightly larger left testicles than the right testicles,and the HFR group had slightly larger bilateral testicles than the LFR group(P ?0.05).In terms of testicular sections,the HFR group of male geese showed better development of convoluted tubules,with neatly and tightly arranged spermatogenic cells at all levels,and a large number of mature sperm visible;The lumen of the fine tubules in the LFR group of male geese is smaller than that in the HFR group,and mature sperm are rare.(2)This study identified a total of 1575 known miRNAs and 68 novel miRNAs from10 testicular tissues of Yili geese.A total of 71 differentially expressed miRNAs and 660 differentially expressed genes were screened.GO functional analysis shows that miRNAs target genes are mainly involved in binding,kinase activity,structural components of the cytoskeleton,and intermediate filament cytoskeleton.KEGG is mainly significantly enriched in arginine and proline metabolism,glycolysis/gluconeogenesis,fructose and mannose metabolism,and β-Pathways such as alanine metabolism.The miRNAs mRNAs interaction network suggests that cfa-miR-140/gga miR-140-3p/ola-miR-140-3p-NKAIN3,cfa-let-7d-BTG1,and dre-miR-145-5p/mmu miR-145a-5p-Clec2 e may play important roles in testicular development and spermatogenesis.(3)In this study,a total of 41308 new lncRNAs were identified in the testicular tissue of Yili geese.Compared with the low sperm motility group,a total of 2760 differentially expressed lncRNAs were screened,of which 1565 were upregulated and1195 were downregulated.The general characteristics of lncRNA are: the average length of lncRNAs is shorter than mRNAs,the average number of exons of lncRNAs is lower than mRNAs,and the average ORF length of lncRNAs is lower than mRNAs.Enrichment analysis of the target genes predicted by lncRNAs for differences in testicular motility between high and low sperm showed that they were concentrated in biological processes such as ion channel activity,metabolic processes,and immune processes.By constructing a differential lncRNAs mRNAs regulatory network,core regulatory factors were identified as RGS17,CCDC122,PDE6 C,Grxcr1,TGM6,GTF2E2,evm.model.chr33.134,CYB561D2,Cep83,pol,and GNG7.(4)In this study,a total of 26311 circRNAs were obtained from the testicular tissue of Yili geese,and 173 differential circRNAs were screened between the two groups,of which 82 were upregulated and 91 were downregulated.The source genes of circRNAs mainly participate in biological processes and are enriched in signaling pathways such as autophagy animals,ubiquinone and other terpenoid quinone biosynthesis,progesterone mediated oocyte maturation,and regulation of actin cytoskeleton.The constructed differential circRNAs lncRNAs miRNAs mRNAs visual regulatory network consists of 20 circRNAs and 18 miRNAs,and the key regulatory factor selected is novel Circle 0017590,LINC4993,LINC6211,miR-140-3p or miRNA140,NKAIN3,etc.(5)Through acridine orange staining,oil red O staining,AKP staining,and RT-PCR assay,it can be comprehensively identified that the cultured testicular sertoli cells after purification in vitro.Through the dual luciferase activity detection test,it was found that LINC9137 has a targeted binding site of miR-140-3p with NKAIN3.In addition,this study found that overexpression of miR-140-3p significantly inhibited the expression of LINC9137 and NKAIN3 in goose testicular sertoli cells,and the expression of both was significantly increased after interference with miR-140-3p.By measuring cell proliferation activity and apoptosis related gene expression,it was found that overexpression of LINC9137 decreased cell proliferation activity(P>0.05),while the expression level of apoptosis factor Bax/Bcl increased(P>0.05).On the contrary,when interfering with LINC9137,the cell proliferation activity of testicular sertoli cells was extremely significantly increased(P<0.01),and the expression level of apoptosis factor Bax/Bcl was significantly reduced(P<0.05);The effect of miR-140-3p on the proliferation and apoptosis of granulosa cells is opposite to that of LINC9137.Meanwhile,this study co transfected the overexpression of LINC9137 and miR-140-3p plasmids into testicular sertoli cells and found that the proliferation effect of sertoli cells induced by LINC9137 overexpression was weakened by miR-140-3p.This study constructed a full transcriptome transcription factor expression profile of testicular tissues with different sperm motility in Yili geese,and preliminarily screened differentially expressed mRNAs and ncRNAs involved in the development and regulation of sperm motility traits in Yili geese.From the mRNA-ncRNA regulatory network,LINC9137 was screened and verified to regulate the expression of NKAIN3 as a ceRNA of miR-140-3p,affecting the proliferation and apoptosis of goose testicular support cells,thereby indirectly regulating sperm motility traits.This study aims to elucidate the interaction mechanism between mRNA-ncRNAs that affect sperm motility in Yili geese,and thus lay a theoretical foundation for improving goose reproductive performance. |
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