Study On Early Cleavage Behaviors And Effect Of Uneven Division 2-cell On Embryos Development In Pig

Since pig have great high application value as a kind of economic animals in animal husbandry,biomedical research,it is very important to improve the efficiency and quality of porcine embryo produced in vitro.The quality of preimplantation embryos could affect the efficiency of transplantable pig embryos,but the assessment of preimplantation embryos was unsatisfied so far especially for pig embryos.In human,the transplantation efficiency of in-vitro fertilization(IVF)embryos had been improved by using quality assess parameters.Different cleavage behaviors,such as uneven division,odd blastomere embryos and cytoplasmic fragments have been defined as abnormal cleavage behaviors that are unfavorable to subsequent embryo development in human IVF embryo.However,no systematic and standardized parameters have been assessed for porcine embryo quality assessment.In the present study,the handmade cloning(HMC)embryos,parthenogenetic activation embryos without zona pellucida(PAZF)and with zona pellucida(PAZI)were produced to invest an available and applicable parameter to evaluate the development potential and quality of porcine preimplantation embryos.1.Observation of Cleavage Pattern,Division BehaviorThe whole development processes of the HMC,PAZF and PAZI embryos were observed in this study.After the first mitosis,2-cell embryo presented two phenotypes,two blastomere with equal volume and two blastomere with unequal volume.The results showed that the proportion of UD 2-cell in PAZI and PAZF embryos was similar to that of ED 2-cell,while the proportion of UD 2-cell in HMC embryos was significantly higher than that of PAZF and PAZI(72.56±4.56%vs.47.13±10.04%vs.47.80±3.63%).In addition,the cytoplasmic fragments have been observed after the first cleavage.However,no significant difference was observed on the cytoplasmic fragments rate in HMC,PAZF and PAZI(18.93±4.34%vs.14.81±4.90%vs.10.29±4.86%).Subsequently,at the second mitosis,the asynchronously division of each blastomere was observed,which resulted in 3-cell embryos.The same asynchronously division was observed with formation of 5-cell embryos.Therefore,the mainly in vitro development patterns of pig embryos can be classified into three routes,which were Type-1(2-cell→3-cell→4-cell→≥5-cell→Morula→Blastocyst),Type-2(2-cell→3-cell→4-cell→Morula→Blastocyst),Type-3(2-cell→3-cell/4-cell→Morula→Blastocyst).The proportion of blastocysts formed through Type 2 is the highest,followed by Type 3,and Type 1 was the lowest.2.Effect of uneven division on embryo development abilityThe morphology and location of spindles in 1-cell were determined to explore the cause of UD 2-cell formation.The result showed that there are two type of spindle localization in the cytoplasm,which are in the cytoplasmic center forming ED 2-cell or migrated away from the center forming UD 2-cell.According to the degree of spindle off center,it can be further divided into slightly off center and serious off center.Subsequently,the UD 2-cell and ED 2-cell were divided into two groups as UD group and ED group.The cleavage,blastocyst formation and distribution of organelles were explored.The results showed that 3-cell rate was significantly higher than that in ED group at 36 h.And 4-cell rate in UD group was significantly lower than that in ED group at 48 h.However,no significant difference was observed on 3cell rate and 4-cell rate between the two groups at 48 h.Also,UD group showed significantly lower blastocyst on 3-cell rate and the number of cells in blastocyst.At the same time,the mitochondria and lipid droplets were distributed unevenly in two blastomeres of ED 2-cell.In conclusion,the present study confirmed that the uneven division event was a common behavior in vitro produced porcine embryo(HMC,and PA)with unevenly distribution of organelles(mitochondria and lipid droplet)to the two sister blastomeres.We confirmed that the uneven division is related to spindle migrates along the long axis of the cell,which resulted that the cleavage furrow is not formed in the center of the cytoplasm.Additionally,the uneven 2-cell has the tendency of development slow as well as lower blastocyst rate and quality.