Relationship Between Early Cleavage Time And Developmental Potential Of Pig Embryo

In order to explore the relationship between the time of primary cleavage and the development potential of pig parthenogenetic embryos,the experiment examined the late embryo development at different time points of cleavage.The results showed that the pig parthenogenetic activated embryos had cleavage from 16h after activation,and basically completed the two-cell division activity from 48h.Of 0 to 16 h,16 to 18 h,18-20 h,20 to 22 h,22 and 24 h,24-26 h,26-28 h,28 to 30 h,30 to 32 h,32-42 h and 42-48 h fir-st cleavage time a total of 11 patients were observed,results showed that between 20 to 26 h,produce the number of embryo cleavage accounted for 50%60%,cleavage embryos in 20 hours after 26 h interval,embryo splitting cells accounted for under 20%of the total number of cells.Parthenogenetic embryos after training to 168 h after activation,began to observe the blastula,found that the earlier embryo to carry on the first cleavage,the higher the probability of its development into a blastocyst,18 h cleavage embryo blastocyst rate is reached 79%,and observed from activated at the same time more than 42 h occurred first cleavage embryo usually can't development to the blastocyst stage the apoptosis.The total number of early cleavase embryos(16-22h)in the same batch of parthenogenetic activated embryos was much higher than that in the late cleavage embryos(26-32h)group(P<0.01).The blastocyst incidence in the 16-22h group was significantly higher than that in the 26-32h group(47.9±5.7%)(P<0.01).It was found that the probability of primary blastocyst,initial blastocyst,expanded blastocyst and incubated blastocyst in the first cleavage 16-22h test group was 44.5±4.2%,49.7±4.1%,74.8±6.4%and 65± 5.6%,respectively,which were significantly higher than that in the first cleavage 26-32h test group(P<0.05),and the late cleavage embryo in the 26-32h test group did not develop into incubated blastocyst at 168h.Blastocysts developed from early cleavage embryos(16-22h)and late cleavage embryos(26-32h)were collected,and the relative expression levels of pleuritic genes Oct4,Nanog,Sox2 and Klf4 were analyzed.It was found that Oct4,Sox2 and Klf4 genes in the early cleavage embryos(16-22h)test group were significantly higher than those in the late cleavage embryos(26-32h)test group(P<0.01).Nanog gene was significantly higher than that of the late cleavage blastocystis(26-32h)test group(P<0.05),in which the expression level of Klf4 gene in the early cleavage embryo(16-22h)was 147 times higher than that in the late cleavage embryo(26-32h).Further to the Bcl-xl apoptosis gene,Bax,Caspase-3 the relative expression level of testing,found early cleavage embryos(16-22 h)group Bax,Caspase 3 gene expression level was significantly lower than the late cleavage embryos(26-32 h)(P<0.01),the early cleavage embryos(16-22 h)apoptosis gene Bcl-x1 relative expression level was significantly higher than that of the late cleavage embryos(26-32 h)(P<0.05).Adopt the method of the second generation sequencing to detect early cleavage embryos(16-22 h)and late cleavage embryos(26-32 h)gene expression profiles,the Clean reads through TopHat and Bowtie software and pig genome(the version number Sus_scrofa.Sscrofa11.1.90.CRH),reads on than respectively 89.12%and 89.71%of the total the Clean reads,Clean reads of ear-ly cleavage embryos(16-22h)and late cleavage embryos(26-32h)were analyzed by single-cell transcriptome sequencing and 667 genes(FC?1,P ?0.05)with differential expression in the two groups were obtained,among which 393 genes were up-regulated and 274 genes were down-regulated.KEGG signaling pathway analysis was performed on 667 differentially expressed genes in early cleavage embryos(16-22h)and late cleavage embryos(26-32h),and the results showed that 667 genes were annotated into the signaling pathway,with a total of 275 signaling pathways enriched.The high correlation signaling pathways were apoptosis signaling pathway and endoplasmic reticulum protein synthesis pathway,and it was speculated that the high expression level of apoptosis genes in late embryo might be related to the misfolding of endoplasmic reticulum protein synthesis.In this study,high-throughput sequencing was used to sequence single-cell transcriptome of early-stage cleavage embryos(16-22h)and late-stage cleavage embryos(26-32h),and important genes and new genes related to embryo development ability were screened out,providing a basis for-fur-ther study on the association between early-stage cleavage and embryo development potential.The experimental results are of great significance for guiding the resear-ch of animal embryo transfer,especially for the improvement of pregnancy outcome by observing the first cleavage time in human assisted reproduction(ART)research.