Effects Of HSP70-2 Gene Polymorphism On Semen Quality And Its Function Verification For Heat Stress Resistance In Beijing-you Chicken

Taking HSP70-2 as the candidate gene, this experiment searched SNPs thoughout the 5'flanking region and coding region of the whole genes in Beijing-You male chickens and analyzed the relation between these SNPs and sperm quality traits. Effects of heat stress on reproductive performance and expression of HSP70-2 gens in different genotypes of Beijing-You male chickens were studied in the end.Test 1: The polymorphisms of gene HSP70-2 in 220 Beijing-You male chickens were studied, and the associations between the genotypes and sperm quality traits were analyzed. Test 2: Eighty four birds at 30 wk age, which selected by genotype combinations from test 1, were assigned to two groups. One group of chicken grew under the condition of heat stress (37oC, 10h; 30oC, 14h) and the other is set as a control (23oC, 24h). Chickens from each group were sampled to assess semen quality, fertilization rate, hatching rate, healthy chick rate and expression of candidate genes. The results are as follows:PCR-SSCP, PCR-RFLP and DNA sequencing were used to investigate mutations in the 5′flanking region and coding region of HSP70-2 gene. The A→G mutation at -68 bp(P5 locus), G→C mutation at 286 bp (P7 locus) and A→G mutation at 1054 bp(P8 locus) were found. Both of the mutations at coding region were silent mutations which didn't cause alteration of amino acid sequence. The genotype AG (P5 locus), GG (P7 locus) and AG (P8 locus) affected sperm motility and sperm abnormality rate significantly (P<0.05).Among nine different genotype combinationss of any two sites, AGCC (P5P7 locus) and AGAG (P5P8 locus) genotypes affect sperm vitality and sperm abnormality rate significantly (P<0.05).Five kinds of P5P8 genotype combinations were selected for heat stress experiments. Results showed that the heat stress significantly decreased sperm motility and improved abnormality (P<0.05). The semen quality of AGAG genotype combinations was improved most.The mRNA expressions of gene HSP70-2 of different genotype combinationss were conducted by Real-time PCR. The results showed that, the gene expression of different genotype combinationss were significant different under the same condition, the genotype combinations AAAA was highest and the AGGG was lowest under heat press. The gene expression of same genotype combinations was significant differences under different conditions, the genotype combinations AAAA was highest in the stress group. The gene expression of AGAG genotype combinations was affected greatest by the heat press.The fertilization rate, hatching rate and healthy chick rate of genotype combinations GGGG (P5P8 locus) were higher than AGAG (P5P8 locus) genotype under heat press. The fertilization rate, hatching rate and healthy chick rate of both genotype were decreased due to the heat press and didn't recovery to normal levels until one or two weeks after the end of heating. The fertilization rate, hatching rate and healthy chick rate of genotype combinations AGAG was affected greatest by the heat press.To sum up, our study suggested that HSP70-2 gene could be a candidate locus around the major gene affecting semen quality in Beijing-You chicken. To reject the genotype combinations AGAG (P5P8 locus) from the colony, reproductive traits of Beijing-You male chickens would be improved possibly. Meanwhile, this study validate the heat resistance function of HSP70-2 gen from reproductive traits of chicken for the first time.