Identification And Mechanism Of Major Genes Associated With Caprine Litter Size As Well As Bovine Semen Quality And Follicular Development Traits

Being as two important grain-saving livestocks,cattle and goat can provide abundant regular products for human being,such as meat,milk,fur and cashmere.However,low prolificacy(monotocous) is the main reason limiting the economic profit of these two species.To improve the fecundity,it is very important to clarify the basic mechanisms of reproduction in cattle and goat to explore the new breeding/reproductive techniques.As we all know,it is an efficient way to promote the bovine and caprine fecundity by breeding of high prolific lines or breeds.Analyzing the genetic characteristics and regulatory mechanisms of genes importment for reproduction traits and exploring the molecular marker-assisted selection(MAS) are the bases of breeding new breeds with high fecundity in cattle and goat.The reproductive traits represent both important economic traits and complex quantitative traits,which are controlled by polygenes and one/several major genes.Therefore,it is necessary to screen and identify the major genes of reproduction and further clarify the roles and regulatory mechanisms during reproduction,such as folliculogenesis,which are very important to explore the new reproductive techniques(such as embryo engineering and reproduction control),build up molecular breeding techniques(such as MAS and transgenic breeding),to faster improve the fecundity and reproductive traits in cattle and goat.This study was designed to analyze and identify the major genes for bovine and caprine reproductive traits and further explore their roles and regulatory mechanisms in reproduction.In order to identify those major genes,we first detected the polymorphisms of several candidate genes,including reproductive hormones and some cytokines,and then analyzed the associations between the polymorphisms of these genes and the reproductive traits such as litter size,follicular development and semen quality.Several major genes were confirmed and selected for further study.RNA interference was applied to explore the functions and regulatory mechanisms of these major genes in ovary,which could provide the basic theory to explore the new techniques for improving the bovine and caprine fecundities.The main results are as follows:1.Identification of major genes associated with caprine litter size,bovine semen quality and follicular development traitsPolymorphisms of candidate genes for caprine litter size such as FecB,FecX(including FecXI,FecXH,FecXB and FecXG),5 microsatellite loci(BMS2508,OARHH35,BM143, BM1329 and TGLA68) linked with FecB and FecX,GH and INHA in 614 individuals from 6 goat breeds including Boer goat,Haimen goat,Huanghuai goat,Matou goat,Nubi goat and first generation of Boer goat crossed with Huanghuai goat were detected with PCR-SSCP,PCR-RFLP and forced PCR-RFLP.Based on the associations between those polymorphisms and caprine litter size,several molecular markers were selected for caprine MAS,and several genes were confirmed as major genes controlling caprine litter size and growth traits.Furthermore,the INHβA was identified as the major gene controlling Holstein bulls' semen quality by polymorphisms detection and association analysis through PCR-RFLP.The major points were as follows.(1) The polymorphisms of FecB mutation in BMPR-IB gene and loci FecXI,FecXH, FecXG,FecXB in BMP15 gene were analyzed by forced PCR-RFLP in 614 individuals from 6 flocks or breeds of goats including Boer,Haimen,first generation of Boer goat crossed with Huanghuai goat,Huanghuai,Nubi and Matou goat.None of mutations were detected in these goat breeds and their crossbreed.These results suggested that fecundity of goat was not linked to the same loci in BMPR-IB and BMP15 as sheep.(2) There are abundant polymorphisms in 4 microsatellite loci(BMS2508,OARHH35, BM143 and BM1329) linked with FecB(BMPR-IB mutant) and locus(TGLA68) linked with FecX(BMP15 mutants) in goat.The caprine mean litter size was differed significantly among different genotypes.These results suggested that BMPR-IB and BMP15 were major genes controlling caprine litter size.There may be other loci in these genes linked with caprine litter size traits excluding FecB and FecX.(3) Two single nucleotide polymorphisms(SNPs)-A781G(Ser/Gly35) and A1575G (Leu147) were identified in gGH gene.Neither BB homozygote genotype nor DD was observed in A781G and A1575G loci,respectively.There are different effects on growth traits with different GH genotypes between Boer bucks and does,as the regulation of GH was more significantly in bucks than in does.These results implied that GH was the major gene controlling growth traits in Boer goat bucks.The litter size of Boer goat does with different genotypes was not shown a significant difference.Whether or not there is a direct regulation of GH on caprine litter size still needs to be further studied.(4) Eleven SNPs were identified in caprine INHA such as -506-＞G,-446C＞T,-155C＞T, -65G＞C,129G＞A,567G＞A,651A＞G,792T＞C,906C＞T and the non-synonymous polymorphisms 911T/C,946A/C resulting 299Val＞Ala and 311Thr＞Pro amino acid mutations,repectively.Among these 11 SNPs,3 loci including -446C＞T,651A＞G and 946A＞C were associated significantly with caprine litter size.These results strongly suggested that INHA was major gene controlling caprine litter size. (5) Sty I-RFLP was identified in INHβA in Holstein bulls with AA,AB(dominant genotype) and BB genotypes.The association analysis indicated that the bovine semen quality differed significantly among different genotypes with effects AB＞AA＞BB.Thus, INHβA was identified as major gene controlling bovine semen quality.2.Regulatory mechanism of major gene INHA on bovine follicular developmentOn the bases of the researches on candidate genes,the effects and regulatory mechanism of INHA on bovine granulose cells(GCs) proliferation,apoptosis,steroid secretion were explored,as well as the local regulatory mechanism on follicular development and ovulation.The results were as follows.(1) The relative expression of INHA in bovine GCs can be significantly reduced by RNAi with psiRNA1,psiRNA2,psiRNA3 and psiRNA4 recombined plasmids.The suppression rate of psiRNA3 was the highest among these 4 plasmids.(2) The relative expressions of bcl-2,bcl-xl,IGF-Ⅰand IGF-Ⅱwere increased, however,the relative expression of Bax was reduced by INHA RNAi in bovine GCs.In addition,the apoptosis was suppressed in GCs.The cell cycle was changed,with a decreased percentage of cells at G0/G1 stage and an increased percentage of cells at both S and G2/M stages.Besides,the secretions of estradiol and progesterone were increased with a decreased rate of estradiol/progesterone.In conclusion,these results suggested that Inhibin can function as a local regulatory mediator,promoting the apoptosis and suppressing the cell proliferation,which might be mediated by regulating the expression of bcl-2 family members and cytokines(IGFs family),and/or the secretion of steriod hormones in GCs,and finally controlling the follicluar develpopment and ovulation.