Molecular Mechanisms Of Sperm Quality And Natural Resistance To Disease And Transport Stress

Reproduction, stress and disease are important factors that restricting dairy production and economic benefits, the estimated heritability values of these traits tend to be relatively low, and it is difficult to make great genetic progress through direct selection. In order to breed disease resistance individuals, we studied the polymorphisms of toll like receptor family and analyzed its association with mastitis, bovine tuberculosis and bovine brucellosis; we further analyzed the variants of BMP15with sperm quality traits, expecting to conduct resistance selection by molecular breeding and improve fertility and disease resistance of animals. Transport, as an frequent and inevitably behavior in animal production, would cause big stress to animals, for example, significant increase in disease incidence, however, up to now, there is little literature about the impact of transport stress on animal reproduction. Therefore, in this experiment, we use mice as model, investigated the mRNA and protein levels of LHR, AR and Hsp70in mice testis after long road transport; Second, screened differentially expressed genes in pituitary, uterus and placenta of different pregnancy phase by use of Agilent mouse microarray, and further functional annotation to some of the differentially expressed genes. To provide reference or basis for the study of transport stress on cattle reproductive physiology and product development of anti-transport stress.1. BMP15gene polymorphisms and its association with sperm quality traits in Chinese Holstein cattleWe analyzed the association between polymorphism of BMP15gene and sperm quality traits. One SNP in intron (C5697T) was detected. Age and genotype had significant effect on sperm quality trait of fresh sperm motility, however, the interaction of age and genotype had no effect on any sperm quality traits. We further analyzed the effect of different genotype on fresh sperm motility and found that fresh sperm motility of CT genotype was significantly lower than that of CC or TT genotype. These results indicate that BMP15may be candidate genes for sperm quality traits.2. Identification of candidate genes associated with bovine disease resistanceIn this study, we first chose TLR1、TLR2、TLR、TLR6and TLR9as candidate genes to study their variants with mastitis, bovine tuberculosis and bovine brucellosis by PCR-SSCP, PCR-RFLP combined with sequencing. The main results are as follows: (1) Five SNPs C632T, G1409A, A1475C, G1550A and G1596A were detected in TLR1gene. The allele and genotype frequencies of C632T, G1409A and G1550A did not vary significantly between bovine tuberculosis (bTB) infected and non-infected cattle. In G1596A site, genotype frequencies differed significantly between bTB-infected and non-infected cattle, and relative risk of bovine tuberculosis incidence of GH is2.43times than that of GG genotype; In A1475C site, there was a trend toward an increased relative risk of tuberculosis incidence in CD genotype (P=0.056).(2) Three SNPs T385G, G398A and C1828T were detected in TLR2gene, they can be recognized by restriction enzyme EcoRV, HaeⅢ and Dral respectively. In T385G site, GG genotype had significantly higher SCS than GT and TT genotype, and its relative risk of bovine brucellosis incidence is2.084times than that of TT genotype and GG may be disease susceptibility genotype; No significant differences were observed between G398A and C1828T site in SCS and bovine brucellosis incidence.(3) There were no significant differences between different genotypes of TLR4and bovine tuberculosis, and this may indicate TLR4is not a candidate gene in controlling bovine tuberculosis.(4) Four SNPs G1793A, C1859A、G1934A and A1980G were detected in TLR6gene. There were significant differences between different genotypes of C1859A and A1980G and bovine tuberculosis. In C1859A, cattle with AA or CA genotype had greater relative risk of disease incidence (OR=2.73,95%CI0.869-8.573; OR=1.547,95%CI0.803-2.982) than those with CC genotype, and CC maybe bovine tuberculosis resistance genotype; In A1980G, cattle with GG or GA genotype had greater relative risk of disease incidence (OR=2.986,95%CI1.245-7.165; OR=1.582,95%CI0.734-3.409) than those of AA genotype, and AA maybe bovine tuberculosis resistance genotype.(5) There were no significant differences between different genotypes of TLR9and bovine tuberculosis or bovine brucellosis, and this may indicate TLR9is not a candidate gene in controlling bovine tuberculosis or bovine brucellosis.3. Effect of transport stress on animal reproductionIn order to study the effect of transport stress on animal reproduction, we chose Kunming mice as model to study its role in male and pregnant for about15h (1000km) long road transport.(1) Long road transport significantly reduced body weight, epididymis index and relative sperm count in epididymis. In order to analyze the possible mechanism, the mRNA and protein expression of LHR, AR and Hsp70were conducted. Results showed that expression of AR was significantly lower than before transport, LHR and Hsp70expression were somewhat higher after transport, however, not significant.(2) Compared with control group, there are no significant differences in living birth rate, dead birth rate and absorbed fetus rate on long road transport during the last trimester of pregnancy, while declined living birth rate and increased dead birth rate after transport during the second trimester of pregnancy.(3)133genes,586genes,146genes and562genes were scanned out in E10pituitary, E10uterus, E15pituitary and E15placenta respectively. These differential expression genes, according to biological process of Go analysis, mainly focused on the cellular process, biological regulation, metabolic process and regulation of biological process.(4) We use pituitary and gonadal tissue, which play main roles in reproduction, as our taget, screeded several pathways and genes that may play major role in reproduction during transportation.