Study On The Regulation Mechanism Of Sperm Motility In Simmental Bulls Based On Seminal Plasma Transcription And Metabonomics

In recent years,the application of bovine artificial insemination technology has greatly promoted the development of the cattle industry.However,with the continuous promotion of this technology,the bottleneck restriction of the quality and output of frozen semen to the development of the cattle industry has become increasingly apparent.Studies have shown that sperm motility is one of the key factors affecting the preservation effect of bovine frozen semen,but the factors affecting sperm motility are complex and changeable,and traditional semen quality analysis cannot fully diagnose the causes of sperm motility changes.A key biomarker that provides a fast and accurate method for screening high-quality breeding bulls for the production of high-quality frozen semen.Semen is a complex body fluid consisting of sperm and seminal plasma.Among them,seminal plasma is rich in non-coding RNA,proteome,metabolites and chemical molecules,which provide sperm with nutrition,energy and a safe environment.As an essential mediator of spermatozoa,seminal plasma is a rich source of potential biomarkers related to male infertility and other reproductive disorders,but seminal plasma has been less studied by transcriptomic and metabolomic studies,and the correlation between miRNAs and metabolites in seminal plasma is unclear.In this study,we investigated the key biomarkers affecting sperm viability in seminal plasma of Simmental bulls through seminal plasma transcriptome and metabolome,which are important for screening high-quality sires and improving diluent formulation to establish an efficient bovine frozen semen system.The main findings were as follows.(1)By analyzing the culling data and frozen semen production records of Simmental bulls from core breeding farms and breeding bull stations in the past 10 years,it was found that age,frequency of sperm collection and season were the main factors affecting semen quality;the volume,total sperm count and fresh sperm viability of semen were significantly lower in the second consecutive ejaculation,but would not affect the sperm viability after thawing.(2)By comparing and analyzing the sperm morphology of normal and weak spermatozoa of Simmental bulls and replacing seminal plasma with different sperm vitality(adding a small amount of seminal plasma of "high" or "low" vitality bulls),it was found that seminal plasma of high vitality bulls could improve the sperm motility parameters of low vitality semen.motility parameters.(3)The miRNA expression profiles of seminal plasma in the high viability(SPH)and low viability(SPL)groups were comparatively analyzed at the transcriptome level by non-coding small RNA sequencing.59 differentially expressed miRNAs were identified in the seminal plasma of the SPH and SPL groups,of which 38 miRNAs were up-regulated in the SPL group and 21 miRNAs were down-regulated in the SPL group.A total of 59 differentially expressed miRNAs were found in the seminal plasma of SPH and SPL groups.The differentially expressed miRNAs were enriched in 55 KEGG pathways.The pathways with higher enrichment were Axon guidance,Tight junction and cell adhesion molecules.(4)The differences between the SPH and SPL groups of seminal plasma were examined at the metabolic level by metabolomics analysis,and 391 and 327 significantly different metabolites were identified from the positive and negative ion models,respectively.Among them,347 metabolites were up-regulated and 44 metabolites were down-regulated in the positive ion model.The number of upregulated and downregulated metabolites in the negative ion model was 229 and 88,respectively.These differentially expressed metabolites were mainly involved in metabolic pathways such as glycerophospholipid metabolism,α-linolenic acid,arachidonic acid metabolism,linoleic acid metabolism,and propionic acid metabolism.(5)The expression of bta-miR-1264 was found to be significantly associated with the abundance of three metabolites closely related to methionine metabolism,glutamyl-L-methionine,homocysteine and methyl-L-cysteine,by combined transcriptome-metabolome analysis.(6)By transfecting bta-miR-1264 into GC-1 cells and observing the effect of bta-miR-1264 on the biological function of GC-1 cells,we found that bta-miR-1264 had a regulatory effect on apoptosis of spermatogonia and a regulatory effect on the expression of CARMIL2 and RASAL2.(7)By studying the correlation between seminal plasma homocysteine(Hcy)and sperm parameters in Simmental bulls.It was found that seminal plasma homocysteine(Hcy)concentration was negatively correlated with sperm motility(P<0.05)in Simmental bulls,while seminal plasma Hcy concentration was not significantly correlated with other semen parameters.(8)By adding different concentrations of betaine(a drug for Hcy disease)to the dilution solution,it was found that betaine had better protective effects on bovine sperm cryopreservation,effectively improved the viability,vitality,plasma membrane integrity and acrosome integrity of bovine sperm after thawing,and reduced the sperm homocysteine content,and the best effect was achieved when 100 nM of betaine was added.In conclusion,this study used seminal plasma transcriptome sequencing technology and metabolome sequencing technology to establish seminal plasma miRNA expression profiles and seminal plasma metabolite expression profiles for different sperm viability,and analyzed their regulatory effects on sperm viability,and initially screened potential biomarkers in seminal plasma for regulating sperm viability,which provided a basis for future insight into the pathogenesis of weak spermatozoa in bulls,for improving the production and quality of bovine frozen sperm It also provides a theoretical basis for improving the production and quality of frozen semen in cattle and establishing a semen cryopreservation system for Simmental cattle.