Histological Observation Of The Primary Follicle In Different Maturity Of Feathers And Associated With VEGF Gene And VEGFR-2 Gene In Chicken

In this research,Qingyuan partridge chicken,Guangxi Three-yellow chicken and Princess chicken were selected as research material to investigate the development of feathers at 120 days by observation feather morphogenesis.Paraffin embedded tissue section was stained by H.E dyeing technology for studying follicle changes in the microstructure in different phases of feather growth cycle in Qingyuan partridge chickens.At the same time,candidate genes of VEGF and VEGFR-2 were selected to screen the nucleotide variation by direct sequencing and PCR-SSCP/RFLP,which aimed to study the association between different genotypes and the feather maturity,respectively.Meanwhile,the expressions of them were dedected in various kinds of tissues during different feather growth cycle in Qingyuan partridge chickens by real-time quantitative PCR(RT-PCR).The results were as follows:(1)The results of phenotypic observation showed that the growth-feather was stayed at growth phase of feather cycle basically,the rachis was big,gray-black,and pulp were full of blood vessels,the calamus were deeply embedded with the skin tissues.The mature-feather always stayed at resting phase(telogen),the rachis showed contracting thin and without feather pulp,hollow and transparent,which were caused by vasoconstriction in the calamus and showed loosely packed with skin tissues.By comparing with three different breeds chicken,they were showed some differences in feather development.The mature-feather-score in Qingyuan partridge chicken and Guangxi Three-yellow chicken were higher than that in Princess chicken,of which score was 86.932,87.917 and41.917(P<0.01),respectively.In Qingyuan partridge chicken,the primary follicle wall for non-mature was thicker than that in mature-feather by H.E staining method and histological observation,meanwhile,the feather sheath and pulp was clearly visible in non-mature-feather follicles.By observation of longitudinal-section,the dermal papilla became maturity and full of blood vessels in non-mature-feather follicles.Statistic results showed that the average follicle diameter in nonmature-feather follicles was 222.24 μ m,it was two-fold greather than that in mature-feather follicles.The average depth in nonmature-feather follicles was 178.05 μ m higher than that in mature-feather follicles,however,the average follicle density in mature-feather was more 2.34 /cm2 than that in growth-feather,which was about 6.88 /cm2.(2)According to the sequence of VEGF gene and VEGFR-2 gene,six primers and nine primers were designed,respectively.No mutation was found for primers P1~P6.Four SNPs(exon5-A+7009G,intron7-C+9869G,exon9-G+10224A、exon10-T+11163C)were found for primers R3,R5 and R6.Statistic results showed that the sites intron7-C+9869G and exon9-G+10224A deviated from Hardy-Weinberg Equilibrium(P < 0.01)in Qingyuan partridge chicken and Princess chicken.As well as the site exon10-T+11163C was the same in Qingyuan partridge chicken(P<0.05).The mutations were moderate polymorphism except the exon5-A+7009G and exon9-G+10224A locus in Princess chicken.The Least Square Means analysis showed DD had higher mature-feather-score than CD(P < 0.01)in Qingyuan partridge chicken,while CD had higher mature-feather-score than CC and DD in Princess chicken and also EF in exon9-G+10224A site,which was shown significantly higher than FF(P<0.05)and EE(P<0.01).There was no significant differences in all genotypes of Guangxi Three-yellow chicken.The analysis of haplotype showed that there was one stable haplotype block,which including 3 major kinds haplotype in Guangxi Three-yellow chicken.The haplotype frequency of AC,AG and GC was 50%,44.2% and 5.8%,respectively.The association of different combined-genotypes with mature-feather-score were showed that AACDEFGG had lower values than those in Qingyuan partridge chicken.However,AADDFFGH and AADDFFHH had the high mature-feather-score in Princess chicken.AACDFFGH was the advantageous combined-genotype in Guangxi Three-yellow chicken,of which the mature-feather-score was 98.33.(3)The expression of VEGF gene and VEGFR-2 had a significant change in different tissues between growth-feather and mature-feather in Qingyuan partridge chicken.Compared to the control group,a significant increased expression of VEGF gene was observed,and reached the maximum of 3.62-fold in the lung;nevertheless,it was down-regulated in heart in comparison with that in control group.There were showed that much higher levels of the VEGFR-2 expression in tissues such as skin,kidney,spleen,lung,ovary,and pituitary;however,a significantly down-regulated expression was found in heart.In skin,significantly difference of the VEGF and VEGFR-2 m RNA was found between growth phase and resting phase chickens(P<0.05).The level of growth-feather chicken was 2.62 and 1.65 times higher than that of mature-feather chicken.