Study On The Establishment Of The Ear Fibroblast Cell Line And IVM,IFV Of The Sheep

China is one of the counties that have the richest on resources of specific of the domestic animals .However,in recent years,because of indiscriminate introduction of exotic high-productive breeds,a large number of breeds of the domestic animals face to extinct. So it is imperative to conserve national domestic animal genetic resources .Taking Guide Black fur sheep,Shanxi Tong sheep and Nei Menggu Wu Zhu Mu Qin sheep ear margin as the study materials. we succeeded in establishing a fibroblast cell line by the method of explant adherence culture and cell cryoconservation technology. Observations on morphology,dynamic growth ,determination of viability , analysis of karyotype, test of microorganism were carried out. The newly established cell line not only preserved the gemlplasm resources of this important Guide Black fur sheep on the cell level ,but also provided valuable materials for the research of somatic cloning. The cell freezen amount is about 3×106 cell/ml, sample contents of each breed were between 40 and 50, cryopreservation ampules were 240-360 each breed. And at the same time studied the effects of EGF, IGF-I separation or combination on sheep oocytes maturation in vitro and cleavage, cysteamine and different medium on sheep embryos development. In order to provide the adequate quality and high performance embryo for Cryopreservation. The experiment results were as follows:ⅠThe Establishment of the ear Fibroblast Cell Line1 The cultured cells were typical fibroblast morpha,which growed as irregular triangle or shuttle,appearing as radioactive blaze in shape.Their growth curve were "S",which showed since the second day, the cells understood latent phase, logarithmic growth phase and stagnate phase.2 Cell viability before freezing was 96.2%, The fibroblasts were frozen in DMEM/F-12+20%FBS by adding 10% DMSO, After thawing Cell viability was 93.6% , The attachment rates of thawed cells after 24h was 85%. Adherent cell after revival growed as fast as cell before freezing. Meantime the fibroblasts became more purified.3 The fibroblasts identified with Microbial detection test were negative ,which demonstrated cultured cells were not contaminated by bacteria,yeasts,fungi,virus and mycoplasmas.4 The number of chromosome is 2n=54 ,Diploid (2n=54)account for 96-98% in cells chromosome. Results of chromosomal karyotypes indicated that cells are stable diploid cell line.ⅡThe effects of the EGF and IGF-1 on IVM,different culture medium on the embryo development in vitro of the sheep1 The maturation rate and the cleavage rate derived from oocytes matured in medium supplemented with 50ng/ml EGF is 71.2% and 45.5% Seperatately. It′s significantly highter compared with controd and 10ng/ml,20ng/ml,30ng/ml,40ng/ml groups (P<0.05) ,when EGF solution arrive to 100ng/ml,the maturation rate and the cleavage rate is 72.9% and 45.7%.2 The maturation rate and the cleavage rate derived from oocytes matured in medium supplemented with 40ng/ml IGF-1 is 70.7% and 58.5% Seperatately. It′s significantly highter compared with controd and 10ng/ml,20ng/ml, 60ng/ml,80ng/ml,100ng/ml groups (P<0.05) ,when IGF-1 solution arrive to 100ng/ml,the maturation rate and the cleavage rate is 38.8% 20.0% Seperatately It′s significantly lower than other group(P<0.05).3 The maturation rate and the cleavage rate derived from oocytes matured in medium supplemented with 50ng/mL EGF and 40ng/mL IGF-1 combation group was significantly higher compared with controd and 50ng/mLEGF and 40ng/mLIGF-1 groups(P<0.05).The maturation rate and the cleavage rate is 85.6%and 61.0% Seperatately.4 The blastocyst rate derived from matured in medium mSOFaa +10%FCS supplemented with 100umol/L cysteamine is 43.6%,Its significantly highter compared with controd, 50 umol/L and 200umol/L (P<0.05) ,There is no differences between 50 umol/L and 200 umol/L (P>0.05).5 The Cleavage rate in medium(mSOFaa+3mg/mL BSA) and medium (mSOFaa+10%ESS) is 61.2%,62.5% respectively(P﹥0.05) .the blastocyst rate is 13.5% and 30.0% respectively(P﹤0.05).It is illuminated that the effect of BSA is worse than the effect of ESS in sheep embryo development.