Study On Degeneration Of Early Porcine Embryos Derived From Fertilization In Vitro

The low development rate and quality of blastocysts have been the main obstacle to produce porcine embryos in vitro.Huge number of abnormal development of embryonic cells underwent during early stage embryo development,particularly to great extent existing during culture in vitro.Apoptosis is prevalent in preimplantation embryos,even the embryo is morphologically normal.The large ratio of apoptosis happened in embryos may influence the viability of embryos.The death of preimplantation embryos was paid more and more attention.Observation of bovine and porcine embryos had been conducted and the fragmented blastomere, developmental arrest,nonhomogeneous cytoplasm together with soften zona pellucida were found in embryos with the prolonged culture time during early development. Some embryos were degenerate or died.This research studied on the anaplasia of early porcine embryos,discussed the relationship between morphologic index and development potential,in order to set up a scoring system to predict the late development.We use the PI staining and comet assay,to analysis the change of nuclear and the damage of DNA,and to discuss the apoptosis rate of normal and abnormal embryos.In addition,the outcomes of the study could be regarded as one part of research and application basics in exploiting and obtaining high quality of blastocyst.The results derived from present experiments were showed as follows:1.During development of early porcine embryos derived from fertilization in vitro, we found the death rate increased gradually on Day 3,Day 4 and Day 5(Respectively 3.6%, 5.7%and 9.6%),and then on Day 6 and Day 7,the death rate increased suddenly(Possiblely exceeding 50%),because most of arrest embryos didn't died until Day 7,while before Day 5, senescence of the embryos without cleavage.2.During development of early porcine embryo,we found the morphologic change of embryos occurred,including decreased development velocity,increased the number of cytoplasmic fragmentation etc.On Day 3,the number of embryos of normal development rate of 59.1%(39/66) was higher when compared with embryos on Day 5 and Day 6(5.3%(5/94) and 7.7%(5/65).In addition,the ratio of embryos without fragmentation decreased gradually. The ratio of fragmentation in embryos on Day 3 was less than 20%,but on Day 6,the embryos increased(over 13.8%) in which the ratio of fragmentation was more than 20%.3.The development velocity,the ratio and type of cytoplasmic fragmentation could influence the late embryo development potential.Accordaing to the scoring criteria of human embryo,we set up a scoring system with these three indexes to predict the late porcine embryo development potential.The result showed that compared to embryos with lower score,the quality of the embryo who obtained higher score was much better.The developmental rate to morula of those embryos scored 46-60 was 32.8%,while other embryos scored 30-45 only yielded 10.4%of developmental rate to morula.4.The cellular nucleus of embryos when degenerated would condense and disrupt. Their DNA were damaged and fragmented,showing different extent apoptosic morphology by comet assay.The nuclear morphological analysis showed that there were number of cellular nucleus with relatively normal structure in embryos cultured for 3 days. When embryos were cultured for 6 days,partial cellular nucleus of embryos became to be condensed and behaved dark staining.To the 9^th day of culture,most embryos appeared to be negative for the nuclear staining,occasionally some condensed nucleus could be found on the edge of the embryo.The commet assay showed that the longer embryos were cultured the shorter commet tail be together with the higher content of DNA.It indicated that degree of the damage of DNA went worse with the longer culture and the apoptosic percentage would increase also.There existed no significant relationship between the embryonic morphology and the damage of embryonic DNA.