Genetic Analysis Of Variations In SPATA 7,SPATA 16,and SPEF 2 Genes Inrams As Well As The Effects Of GAA Supplement On Their Semen Quality In Sheep

The breeding of stud rams is becoming more and more important for the breeding of mutton sheep with the extension and application of artificial insemination.At present,there are few reports on the population genetics of the semen quality traits of rams for the limitations of difficulty in the construction of the resource group of stud rams.No reports on the associations of the genetic polymorphisms on the SPATA7,SPATA16 and SPEF2 genes with the semen quality traits of rams have been found.Fortunately,our research group has successfully established a group of several breed of mutton rams in recent years with the cooperation of Henan Zhonghe animal husbandry Co.,Ltd.,and several studies on the population genetics of some candidate genes related to the quality of ram semen have been conducted.On this basis,the present study firstly focused on the genetic polymorphisms of SPATA7,SPATA16 and SPEF2 genes and their association with the semen quality of the rams from the breeds analyzed.The purposes were to select the SNPs loci related to the semen quality,and to reveal the population genetic characteristics of these loci,and to take a useful reference for the application of molecular marker-assisted breeding.Secondly,GAA was added to the diet to improve the quality of the semen,and to overcome the widespread male sterility in the production of mutton sheep in the Central Plains.the main results of this study were as follows:(1)Identification of variation loci: the genetic polymorphisms of SPATA7,SPATA16 and SPEF2 genes were identified by pooled DNA sequencing method,and43 polymorphic loci were certified.In details,11 polymorphic loci were verified in the SPATA7 gene,9 of which located in the introns,1 UTR mutation as well as a 16 bp insertion/deletion mutation,and 1 missense mutation.In addition,3 synonymous mutations and 9 intron mutations were detected in the SPATA16 gene,while 2 UTR mutations,3 missense mutations and 15 intron mutations were certified in the SPEF2 gene.(2)Analysis of population genetic characteristics: the individual genotypings and population analysis were carried out employing the Sequenom Mass Array method,the main results showed that:(1)SPATA7 gene: Analysis of 8 loci showed that the rs161954996G>A mutation was not present in the populations studied.All other loci were in low polymorphic and heterozygous except rs595936513A>G locus,which was found to be at middle degree of polymorphism and heterozygosity.Moreover,the locus rs595936513A>G did not match the Hardy-Weinberg equilibrium,while the remaing 7 loci were in accordance with the Hardy-Weinberg law.(2)SPATA16 gene: Analysis of 10 loci demonstrated that the other SNP loci were in low degree of polymorphism and heterozygosity,but rs422481856A>G,rs427627457A>G and rs400591110C>T were middle polymorphic and heterozygous.Except for rs422481856A>G,rs427627457A>G and rs400591110C>T sites,all other loci did not match Hardy-Weinberg equilibrium.(3)SPEF2 gene: Analysis of the 18 loci in this gene revealed that most of them were middle polymorphic and heterozygoous with a exception of rs409270318A>G in low polymorphic and heterozygoous.Except rs400981214C>T,rs405869686A>G,rs409270318A>G and rs420463053T>C,the other loci were all consistent with the equilibrium of Hardy-Weinberg.(3)Association analysis of SNPs with the semen quality traits:(1)SPATA7 gene: the semen volume of AA genotype of rs415416547A>G locus seemed to be higher than those of AG and the GG genotypes(P=0.089),while the path velocity(VAP)and the linear velocity(VSL)of the AA genotype was significantly higher than that of GG genotype(P<0.05).For rs418082626A>G locus,GG genotype was significantly higher than those of AA and AG genotypes(P<0.01),and VSL of GG genotype was also significantly higher than that of AA genotype(P<0.05).AS the indel locus rs599404794,it was found that-/-genotype seemed to be higher than the genotype-/+ for the VSL indice(P=0.063).(2)SPATA16gene: the 3 loci rs408318574C>T,rs425333178C>T,and rs428462879C>T of the SPATA16 gene seemed to have close associations with the acrosome integrity rate(P=0.058)and the sperm motility(P=0.093).(3)SPEF2 gene:it seemed that the rs399938665A>G and rs400991588A>G loci of the SPEF2 gene were closely related to sperm motility(P=0.072 and P=0.07,respectively).For rs400991588A>G locus,the sperm viability of GG genotype was significantly lower than those of AA and AG genotypes(P<0.05).For rs407363126T>G locus,the TT genotype was significantly lower than GT and GGgenotypes in Sperm viability(P<0.05).For rs419749948A>G locus,the AA genotype was significantly lower than AG and GG genotypes in Sperm viability(P<0.05).For rs407524184A>G locus,it seemed that that that this locus had a close relationship with the acrosome integrity(P=0.088).(4)Associations of the haplotypes with the semen quality traits:(1)SPATA7 gene: there were 8 putative haplotypes in the 3 loci rs418082626,rs422662838 and rs428090770 of SPATA7 gene.The hapotype A-G-G was a favorable haplotype(P<0.05)for the integrity of sperm acrosome(P<0.05).(2)SPATA16 gene : there were 7 putative haplotypes in the 3 loci rs398311646,rs400591110 and rs408318574 of SPATA16 gene.The hapotype A-C-C was a favorable haplotype(P<0.05)for the integrity of sperm acrosome(P<0.05).(3)SPEF2 gene : there were 3 putative haplotypes in the 3 loci rs407363126,rs407524184 and rs407899691 of SPATA16 gene.The hapotype G-G-T was a favorable haplotype(P<0.05)for the integrity of sperm acrosome(P<0.05).(5)Associations of the SNPs in the 3 candidate genes with the physiological and biochemical indexes of rams.(1)SPATA7 gene: For rs409407645A>G site,AA genotype was significantly higher than AG genotype in the absolute value of lymphocyte(LYM),lymphocyte ratio(LYM%),neutrophils percentage(Gran%)and absolute neutrophil absolute value(Gran),but the AA genotype was lower than AG genotype in the platelet count(PLT);in addition,the platelet aggregation volume(PCT)of GG genotype was significantly higher than that of AA genotype(P<0.05).For the indel locus rs599404794 and SNP rs428090770A>G,there was a significant difference in serum urea among the 3genotypes(P<0.05).Finally,the serum glucose levels of AG and GG genotype in rs409407645A>G locus were significantly higher than that in AA genotype(P<0.01).(2)SPATA16 gene: there were significant differences in platelet counts PLT of 3genotypes of rs408318574C>T and rs425333178C>T loci in PATA16 gene(P<0.05).AS rs410515272T>G locus,there were significantly differences among the 3genotypes for the absolute neutrophils Gran,hemoglobin HGB,the total number of red blood cells(RBC)and the average hemoglobin content MCH(P<0.01 or P<0.05).The significant differences were also found among the different genotypes in the rs604103514A>G locus for the Gran and HGB(P<0.01 or P<0.05);the serum urea of AA genotype in this site was also higher than that of GG genotype(P<0.05).(3)SPEF2 gene: As thers400981214C>T locus,the CC genotype was the dominantgenotype for LYM%,RBC and serum Mg level(P<0.05).As the rs405869686A>G locus,the GG genotype was significantly higher than the AA and AG genotypes for the ratio of intermediate cell(Mid%)(P<0.05),and the genotype AA was also the dominant genotype for PLT and platelet distribution width(PDW)(P<0.01 or P<0.05).As the rs413789102C>T locus,the CC genotype was significantly higher than the CT and TT for RBC and serum Mg level(P<0.05).As the rs421390194C>T locus,the TT genotype was significantly higher than CC and CT genotypes for PLT and PDW(P<0.05),and the PLT and PDW of CC genotype were significantly different from those of CT and TT(P<0.01,P<0.05).As the rs406155807C>T locus,the TT genotype was significantly higher than the CT and TT genotypes for serum ALT(P<0.05).Finally,for the rs406155807C>T locus,the CT genotype was significantly lower than CC and TT genotypes for serum P(P<0.05).(6)Effect of the addition of guanidic acetic acid(GAA)in the ration of ram feed on the semen quality indices: to examine the effect of guanidinic acetic acid on the quality of the semen of rams,we added the 0.1% and 0.2% GAA respectively into the ram diet.The results showed that guanidic acetic acid could significantly improve the semen motility of rams,and the sperm motility of the rams could come up to normal required levels after the addition of 1 to 2 months.it was better to use GAA at a dose of 0.1%GAA in the ram diet.To sum up: the genetic polymorphisms of 43 SNPs in ovine SPATA7,SPATA16 and SPEF2 were analyzed by pooled DNA sequencing.The rs418082626A>G locus of the SPATA7 gene was closely related to the amount of semen(P<0.05),while the rs400991588A>G,rs407363126T>G and rs419749948A>G loci of the SPEF2 gene were all closely related to the sperm viability(P<0.05).The hapotype A(rs398311646)-C(rs400591110)-C(rs408318574)A in ovine SPATA7 gene and the A(rs418082626)-G(rs422662838)-G(rs428090770)in ovine SPATA16 gene intimately associated with the sperm acrosome integrity.A lot of SNPs in these 3 candidate genes were also found to have intimate relationships with many serum physiological and biochemical indexes of rams.Finally.the addition of 0.1% GAA in the ram diet significantly improved semen quality(P<0.05).The results may provide a better foundations for the improvement of semen quality in stud rams.therefore,it is concluded that ovine SPATA7,SPATA16 and SPEF2 genes have abundant polymorphism,and some loci may be linked to the quality traits of ram Semen,which is expected to be used as molecular marker in mutton sheep breeding.In addtion,the addition of GAA to the ram diet can effectively improve the semen quality of rams.