In vivo Transfer of Reconstructed Goat Chimeric Embryos Produced by Microinjection of Embryonic Stem Cells into Tetraploid IVF Embryos - Abstract
The aim of this study to observe in vivo development of a chimeric embryo (4n/2n) produced by tetraploid complementation. Cumulus oocyte complexes (COC’s, 585) were collected and matured in maturation media. Out of which 524 matured oocyes were randomly divided into two groups. Group 1(n= 296) were activated with 5?M Calcium Ionophore for 5-7 minutes followed by treatment with 2.0 mM DMAP for 4 hr in mCR2aa medium while Group 2 (n= 228) matured oocytes were used for in vitro fertilization. Furthermore, inner cell mass (ICM) from hatched blastocysts of parthenogenetic activated embryos were used to produce ES cell-like cells while 2 cell embryos obtained from IVF were used for their production of tetraploid embryos. Chimeric embryos were produced by microinjection of parthenogenetic ESCs in to in-vitro fertilized tetraploid caprine embryos. The percentage of 2 cell, 4 cell, 8-16 cell, morula, hatched blastocyst and cleavage rate of parthenogenetic goat embryos were 20.03 ± 5.09%, 20.02 ± 6.30%, 24.41 ± 4.12%, 25.61 ± 7.63%, 9.92 ± 3.97% and 72.78 ± 13.39%, respectively while the cleavage rate following IVF was 37.08 ± 4.34%. Furthermore, 75.55 ± 12.37% embryonic stem cell colonies were formed from hatched blastocysts. The IVF two cell embryo used for tetraploid embryo production, resulting in 75.09 ± 7.86% fusion rate and 86.5 ± 13.02% cleavage rate. A total of eight microinjected chimeric embryo were transfer in two naturally synchronized goat. The result indicated that caprine ES-like cells can be incorporated into IVF tetraploid embryos by microinjection so as to produce chimeric embryos. However, in vivo development of chimeric embryos could not achieve.