Mouse Zona-Free Embryo Cultrue In Vivo

Based on Kun-ming strain mouse, this study firstly screened a more suitable culture medium from four culture mediums and perfected the in vivo culture way of zona-free embryos. Then through comparing the influence of the well of the well (WOW)method and agarose embed culture method on zona-free embryos'development in vivo, we select a more suitable culture method.. The purpose of the study is to set up an in vivo culture system for zona-free embryos which fits to the environment of our laboratory and complete the basic work which ultimately reduces techniques difficulties and increases the nuclear transfer efficiency. The main results were followed:1. Four different common mediums were used to culture mouse preimplantation embryos in vitro. The result showed that embryo development rate from 2-cell to 4-cell ranged from 30.5%～43.5% and the difference was not remarkable. The blastocysts rate in KSOM group was 40.9%, higher than other groups (27.9%～30.0%).2. To take KSOM and KSOMFBS as culture medium, the embryos separately co-culture with mouse oviduct epithelial cells (MOEC) or cumulus cells (CC). The changes of embryo numbers from 2-cell to 4-cell and blastocysts numbers were observed. The results suggest that co-culture has obvious effect for embryos to overcome in vitro development block. Co-culture with MOEC has better effect than CC. To Add FBS to KSOM culture medium had obvious effect to mouse earlier period embryos.3. Culturing zona-free embryos in vivo by the well of the well (WOW) culture method or agarose embed culture method, the blastocysts developmental rate, recovery rate and cell numbers of blastocyst were counted. The blastocysts developmental rate of zona-free embryos by WOW culture method (29.6%) has no obvious difference than agarose embed culture method(34.7%), but the recovery rate and cell numbers of blastocyst of the former(80.3%,36) were remarkable lower than the latter (90.5%,46).The result indicated that the agarose embed culture was the better method for the development of zona-free embryos in vivo.4. Zona-free embryos, embedded by agarose, directly cultured or co-cultured with MOEC. The 2-cell embryo rate of direct culture group (86.4%) has no obvious difference in co-culture group (88.7%). But 4-cell embryo rate and the blastocysts developmental ratio of co-culture group (46.9%, 44%) were remarkable higher than direct culture group (37.2%,...