An Exploratory Study On The Phenotypes And Genetic Factors Of Duration Of Fertility In Xinhua Chicken

Duration of fertility(DF)is the length of time that sperm stored in the sperm storage tube and fertilized eggs produced continuously by hens after sperm entering the oviduct,it is a crucial reproductive trait for hens.If the hen’s duration of fertility become higher,the interval of insemination will be increased,the stress of insemination to the hens will be reduced,and the breeding cost will be reduced.In this research,three lines including Xinhua chicken B,C and D(889 in total)were used to reveal the variation of DF in Xinhua chicken and the physiological differences in hens with different DF.Firstly,the DF of the three lines were measured,the progeny of hens with high DF were selected and the differences of DF in the different lines and the same line were both analyzed.Secondly,the uterine-vaginal junction(UVJ)tissue of the hens with the highest and the lowest DF were collected in line D,the proteome analysis was conducted with label-free proteomics technology,and the differentially expression proteins were explored between the high and low DF groups.Finally,the expression of candidate proteins at transcription was detected.And the expression of HSPA4 at protein levels was detected.Meanwhile,the SNP(Single Nucleotide Polymorphism)in the HSPA4 were detected and the association study was performed between them and DF.The results were as follows:1.In order to explore the DF in the different lines,some DF traits including the time of the last fertile egg in 17 days(DN),the fertile egg number in 17 days(FN),the effective fertilization interval in 17 days(EFD),the maximum number of consecutive fertilized eggs(MCF),fertility rate 1(fertility rate within 17 days,FR1),fertility rate 2(fertility rate within days of max fertilization,FR2),fertility rate 3(fertility rate within days of effective fertilization,FR3)were measured and the offspring of hens with high DF were selected according to them.Except for FR2 and FR3,there were significant differences in B,C,and D in other indicators(D>C>B,P<0.05).2.The repeatability of every indictor was calculated to study the stability and genetic law of them.We found that the repeatability of them were ranged from 0.20 to 0.60.In them,the repeatability of FN and MCF was the highest(close to 0.60),indicating that they could be used to judge the DF.At the same time,differences of DF traits were dectected in three lines.Thus,we can improve the DF traits through genetic breeding methods.3.The differential proteins expression profiles of D-line hens with high and low DF were constructed to find out the difference proteins,by using label-free proteomics.153 differential expression proteins were obtained,in which 60 were up-regulated in the low DF group and 93 were up-regulated in the high DF group.Through GO analysis,we found that the differentially expression proteins were mainly enriched in the biological processes including response to stimulus,cellular response to stimulus,organic substance catabolic process,cellular catabolic processls and protein catabolic process,and some pathways including PPAR signaling pathway,cellular senescence,cell adhesion molecules,peroxisome and drug metabolism-other enzymes.Finally,HSPA4,IFIT5,BF2,TMEM173,ACSL4,ANXA7 and LGALS2 were considered as the potential candidate proteins,which may be related to sperm preservation by SST.4.To identify whether the dfference of the selected candidate proteins exsist in different groups at the transcriptional level,the expression of candidate genes in the UVJ tissues were detected.The results showed that the expression differences of their m RNA in hens with high DF was consistent with the proteomic results.The protein expression of HSPA4 was also detected,the relative expression level of HSPA4 protein in high DF group tends to be higher than that in low DF group,but it did not reach the significant level(P=0.062).5.The gene body and upstream 2k bp sequence of IFIT5 and HSPA4 were amplified by primer design method,and SNPs were detected by mixed-pool sequencing method,and there were 81 SNPs in the HSPA4 gene and 20 SNPs in the IFIT5 gene.Five SNPs in HSPA4 were selected to perform association analysis on DF traits,among them g.17853192A>C and g.17864249A>G were associated with the MCF(P<0.05).In summary,in this study,we used different indexes to evaluatethe DF traits of Xinhua Chicken B,C,and D(889 in total).Most of those indexes have medium repeatability(0.3～0.6),indicating that this trait has the genetic selection and breeding value.At the same time,candidate proteins related to DF were screened through Label-free proteomic analysis,and seven genes such as HSPA4 and IFIT5 were detected and verified at the transcriptome level.Finally,two SNPs in HSPA4 were detected significantly associated with MCF.This study provides valuable reference materials for the basic research and genetic improvement of DF traits.