Preliminary Study On The Early Cleavage Time Of Porcine IVF Embryo And Its Developmental Potential

Early cleavage(EC)refers to the first division of an embryo,that is,from a fertilized egg to a 2-cell embryo.Early cleavage is important for screening high-quality embryos,whereas early embryo quality not only vital for research and application value but also influence the outcomes of human assisted reproductive pregnancy,pregnancy efficiency of cloned embryo transfer,the life efficiency of in vitro fertilized embryo transfer in human assisted reproduction and animal in vitro embryo production.This research aimed to discover the relationship between the early embryo cleavage time and the developmental potential of porcine embryos after IVF.The specific research contents are as follows:1.Observation of the first cleavage time of swine IVF embryos: the different times of the first cleavage of pig IVF embryos were divided into 10 time periods,namely 18 h,18-20 h,20-22 h,22-24 h,24-26 h,26-28 h,28-30 h,30-32 h,32-42 h and 42-48 h.Then,their development status were tested and it was found that pig IVF embryos began to have cleavage after fertilization for 18 h.By 48 h,the two-cell division activity was basically completed.Between 20-32 h,the number of cleaved embryos accounted for 60 %-70 % of the total number of embryos.Meanwhile,after 32 h,the number of cells dividing embryosaccounted for less than 30 % of the total number of embryos.The general trend was basically in accordance with the normal distribution.The time of the first cleavage was closely related to the chance of developing into a blastocyst.24.25 % of the embryos whose cleavage happened in 20-22 h after fertilization developed into blastocysts.However,embryos that had undergone the first cleavage after 42 h mostly withered during development,which were difficult to develop into the blastocyst stage,thus it can be preliminarily proved that the embryos that were undergone the first cleavage in the early stage have a higher development potential.2.The effect of cleavage time on the relative expression level of candidate genes: this experiment was divided into two groups,pig IVF early cleavage embryo group(?24 h)and pig IVF late cleavage embryo group(30-36 h).The early and late cleavage embryos were collected to analyze the differential expression of candidate genes.The results presented that the relative expression levels of Oct4,Sox2 and Klf4 genes in pig IVF early cleavage embryo group(?24 h)were significantly higher than those in pig IVF late cleavage embryo group(30-36 h)(P<0.05).The difference in the expression of Sox2.pig IVF early cleavage embryo group(?24 h)and pig IVF late cleavage embryo group(30-36 h)was more significant(P<0.01).Furth more QRT-PCR analysis detected the relative expression levels of apoptotic genes Bcl-xl and Caspase-3,the expression of apoptotic genes in pig IVF early cleavage embryo group(?24h)was significantly lower than that of pig IVF late cleavage embryos group(30-36 h)(P<0.01).As a protective factor,expression level of Bcl-xl gene was significantly up-regulated compared to the pig IVF late cleavage embryo group(30-36 h)(P<0.05).3.Construction of differentially expressed gene profiles of pig IVF early cleavage embryo group and late cleavage embryo group.The single cell transcriptome PE150 was sequenced in the early cleavage group(n=2)and the late cleavage group(n=2)and 6 G high-quality(Q30?90.76 %)sequencing datawas obtained in each trial.According to Cuffdiff analysis,there were 123 differential genes(log2?1,P?0.05)in 4 transcriptomes of the early cleavage embryo group(?24 h)and the late cleavage embryo group(30-36 h),including88 up-regulated genes and 35 down-regulated genes.The results of GO enrichment analysis of differentially expressed genes showed that the enrichment of the expressed genes was significant in Golgi protein glycosylation,glycoprotein anabolic process regulation,mRNA and RNA transport,and nucleic acid transport.Further annotation analysis of related genes enriched by GO function revealed 14 genes that may be involved in the regulation of early embryonic development,namely TMEM59,ITGB1,QKI,SAR1 A,HEXB,EFNA1,MRPS26,SLC16A7,MTMR8,YBX3,GLCE,ANKRA2,LCMT2,LDHB.Among them,TMEM59 belongs to type I transmembrane protein,participates in Golgi regulation and protein anabolism,and has a pro-apoptotic effect.ITGB1 is related to the regulation of proteins and the process of collagen catabolism.In terms of cells,it mainly acts on cell membranes and is related to cell proliferation and apoptosis.The results of this experiment showed that the developmental potential of pig IVF embryos with early cleavage time was significantly higher than that of late cleavage.Quantitative PCR and single-cell transcriptome sequencing revealed that some genes had higher relative expression levels in early embryonic development stage,which were involved in regulation of glycosylation of body proteins,glycoprotein anabolic processes,mRNA and RNA transport and nucleic acid transport.This study will contribute to the establishment of a technique for selecting pig IVF embryos with higher development potential based on early cleavage time.It also has certain reference value for guiding the selection of other animal embryos and improving pregnancy outcomes.Furthermore,in the production of animal husbandry,it can greatly improve the reproductive capacity and sowing rate of sows.