| Breeding roosters play an important role in the breeding system,and the quality of semen directly affects the fertilization rate and offspring production performance.Studies have shown that low sperm motility is the main manifestation of low fertility in poultry production.80% of low-fertility males have low sperm motility,and sperm motility is positively correlated with fertilization rate.In recent years,there are many studies on the influence of sperm motility by external environment,but it is difficult to reach a unified conclusion on the major genes that affect sperm motility.Therefore,in this experiment,by measuring the semen quality of Gushi roosters,the testicular tissue of high and low sperm motility roosters was sequenced,and the differential genes that may affect sperm motility were screened out,and the functional verification was carried out in Leydig cells.To reveal the regulatory mechanism of chicken sperm motility,lay a theoretical foundation for the reproductive regulation of poultry.The main findings of this study are as follows:1.Determination and analysis of semen quality in Gushi chickensIn this study,the semen quality of 79 healthy and uniformly weighted 28-32 weekold Gushi breeding hens was measured 8 times,and the ejaculate volume was 0.37±0.12 ml,p H was 7.70±0.20,sperm motility was 0.51±0.12,sperm viability was 0.76±0.05,sperm density was 20.00±4.60 million/ml,and sperm malformation rate was The ejaculate volume was significantly correlated with p H and sperm density(P < 0.01),p H was significantly correlated with sperm density(P < 0.05),and sperm density was significantly correlated with sperm malformation rate(P < 0.01).Statistical analysis of the determination of sperm motility in the Gushi breeder flock showed that sperm motility below 0.42 was considered as low sperm motility males and sperm motility above 0.61 was considered as high sperm motility males.The testicular tissues of three high sperm motility and three low sperm motility roosters were selected and observed.The testicular tissues of low sperm motility roosters showed higher deformity of the varicose vasculature,smaller official lumen,disorganized arrangement of spermatogenic cells at all levels and fewer mature spermatozoa in the center of the duct.2.Transcriptome sequencing of testes from high sperm motility and low sperm motility roostersIn this experiment,transcriptome sequencing was performed on testis tissues from three high and three low sperm motility roosters,and the results showed that there were 140 significantly different m RNAs,including 67 down-regulated genes and 73 upregulated genes.Among them,the genes related to spermatogenesis and sperm motility were KIFC1,REC8,DIO2,etc.GO and KEGG enrichment analysis obtained that pathways such as oxidative phosphorylation and metabolism of glycine,serine and threonine are involved in regulating sperm motility.And the candidate gene DIO2 was obtained by reviewing related literature and gene function.3.Identification and analysis of lncRNA in testes of high and low sperm motility roostersIn this experiment,7830 new lncRNAs were obtained by constructing a non-strand specific library based on experiment 2.103 lnc RNAs were screened for significant differences,of which 56 were significantly down-regulated,of which 31 were specifically expressed in the low sperm motility group;47 were significantly upregulated,of which 25 lnc RNAs were specifically expressed in the high sperm motility group.The GO enrichment results showed that most of these entries were focused on biological processes such as growth and development,reproduction,immunity and metabolism;KEGG analysis showed that DE-lncRNA cis-target genes and trans-target genes were significantly enriched to m TOR,phosphatidylinositol and other signaling pathways related to reproduction.And a network map MSTRG.15920.1-REC8-MSTRG.11860.2-VWC2,which may be related to sperm viability,was constructed.4、Validation of the function of DIO2 in testicular mesenchymal stromal cellsIn order to verify the role of DIO2 in testicular mesenchymal cells in this assay,the expression of DIO2 in chicken was firstly verified in all tissues.Bioinformatics analysis of it showed that this protein has protein interactions with DIO2,THRB and SLC16A2.The amino acid sequence of its protein was found to be the most related to turkey and quail and the most distant to zebrafish by constructing a phylogenetic tree.The pc DNA3.1-DIO2-EGFP vector was successfully constructed and transfected into F1 generation testicular mesenchymal cells for 48 h.The expression of STAR,3β-HSD,THRA and THRB was found to be significantly elevated. |
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