Culture Measure For Bovine Homogeneity And Goat-Bovine Heterogeneity Cloned Embryos In Vitro

This study is conducted to explore an effective cultivated method for supporting the embryo development and to make these technologies going out of laboratory to be used in the fields of embryonic engineering. We studied the effect of different culture medium and the different order of supple macromolecules to mSOF on the culture development of the bovine and goat-bovine cloned embryo. The test and results is as following:1. Bovine ear Fetal Fibroblasts and Goat ear Fetal Fibroblasts were obtained either by the basic culture media DMEM or Goat Fetal tissues were digested by a mixture of 0.2% collagenase II and 0.25% trypsin at the ratio of 2/3 and Bovine Fetal tissues were digested by a mixture of 2.5 g/L trypsin +1 mmol/L EDTA. The cells could be stored both at-196℃for a long time and at -70℃for a short time under the condition that 10% DMSO was added into freezing media.2. The cattle ear fetal fibroblasts and the goat ear fetal fibroblasts are transplanted into the enucleated cattle oocytes separately by using oocyte intraplasmic nuclear injection method to develop bovine-bovine cloned embryos and goat-bovine cloned embryos. The embryos are cultivated in modified charles rosenkrans 2 amino acid medium (mCR2aa) and modified synthetic oviduct fluid medium (mSOF) separately. Result show: cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, cultivated in mSOF solution are all higher than those cultivated in mCR2aa (p<0.05).3. BSA(8mg/ml) or FBS(10%) can be added to mSOF according to the different periods of cultivation. The supplements and orders, added during the first three days and after three days are as follow: BSA and BSA, BSA and FBS, FBS and BSA, FBS and FBS. Result: the cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, adding BSA and FBS into mSOF, are in turn 79.8±7.1%, 49.7±3.5%, 21.5±1.8%, and 115.2±4.3 in bovine-bovine cloned embryo, and 40.1±6.3%,29.2±2.0%,13.4±2.1% and 100.1±3.0 in goat-bovine cloned embryo, which are evidently higher than other cultivation groups(p<0.05).