Chimera production by embryo aggregation method and cultured in vitro without zona pellucida in mice

Abstract

The objective of this research was to study the developmental competence of single and aggregated embryos develop in vitro and in vivo in relation to the phenotypically aspects of the chimeric mice. Chimeric embryos were produced by aggregation of early stage embryos (8-cell stage) collected from mice with different coat coloration (white and brown). Zona pellucida was removed by exposing the embryo into the medium contained 0.25% pronase. The aggregation of zona-free embryo was done by physically micromanipulation with different composition of embryos collected from white and brown mice. The developmental competence of single zona-free or aggregated embryo in in vitro culture was evaluated morphologically and by counting the total of the cell number of the embryos.The treatment of zona removal and aggregation did not affect the developmental competence of embryo to expanded blastocyst after culture in vitro (P>0.05). The development rate of embryo to expanded blastocyst was 86.49%, 75.00%, 72.22%, 81.82% and 76.92% for normal embryo, single zona-free embryo, aggregated two, aggregated three and aggregated four embryos, respecthely. The size of aggregated embryos was bigger (P<0.05) when more number of embryos was aggregated than less number of embryos. Similarly, the total cell number of the aggregated embryos developed to blastocyst was significantly different among the treatment (73.85, 120.50, 160.82 and 220.00 cells per embryo for the single zona-free, aggregated two, aggregated three and aggregated four embryos, respectively). The v•iability of aggregated embryos develop in vivo was examined by transfer of the aggregated embryos to the pseudopregnant female.Twenty four aggregated embryos were transferred and two chimeric mice were born.These results showed that chimeric mice could be produced by aggregation of early stage embryo and cultured in vitro without zona pellucida.