| The developmental competence of in vitro cultured(IVC)embryos is markedly lower than that of their in vivo counterparts,suggesting the need for optimization of IVC protocols.Embryo culture medium is routinely replaced three days after initial culture in bovine,however,whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear.Exosomes in embryo culture medium were extracted by ultracentrifugation.The exosomes were identified by electron microscopy and immunofluorescence staining with exosome specific protein CD9.The results showed that the exosomes could be secreted by the embryo during in vitro culture,and the exosomes would be lost by changing embryo culture medium.The exosomes extracted from 10 mL embryo culture medium on day 3.5 were suspended in 30 uL fresh culture medium.Adding 5 uL and 10 uL groups showed significantly higher blastocyst rates than the control group and 1uL.Also embryos were divided into three groups,renewal group(renewed on 3.5 days),non-renewal group(without renewal during IVC)and supplementary group(supplemented exosomes on 3.5 days during renewal).The results of gene expression showed that renewal affected endoplasmic reticulum stress,apoptosis and differentiation of inner cell mass and trophoblast cells.The expression level of Oct4 gene in the supplementary group was significantly higher than that in renewal group,suggesting that exosomes secreted by embryos play an important role in the development of inner cell mass and trophoblast cells.In this study,we found that renewal induced endoplasmic reticulum stress and apoptosis,which was not conducive to embryonic development;during the process of renewal,the exosomes secreted by embryos were lost,which affected the differentiation of inner cell mass and trophoblast cells.This study will provide technical support for optimizing embryo culture in vitro,improving cloning efficiency and improving reproductive and breeding efficiency of cattle. |
PDF Full Text Request