Embryonic Stem Cells Culture Of Arbas Cashmere Goat And Sheep

Arbas cashmere goat and sheep, as the two main livestock species of Inner Mongolia, their embryonic stem cell lines have never been established.The successful establishment of embryonic stem cell line could improve the breeding of the Arbas cashmere goat and sheep, and promote the development of contemporary animal husbandry. Our research studied on establishment of embryonic stem cells of Arbas cashmere goat and sheep. With mouse fetal fibroblasts as feeder layer, embryonic stem cells were cultured the in different media and passaged them in different methods. Finally embryonic stem cells were identified by a series of detection, such as morphological identification, alkaline phosphates staining, immunofluorescence staining, in vitro differention and PCR of pluripotency factors. The results of this study should provide experimental basis for the establishment of livestock embryonic stem cell lines. 1. Establishment of embryonic stem cells of Arbas cashmere goat culture system1) Preparatory experiments(1) The nanny goats in natural and synchronized estrus were superovulated to get multi number of in vivo embryos with high quality. The results showed that the embryos obtained by both methods were in good quality, but the number of ovulation points were significantly different (P<0.01).The number of ovulation points of the nanny goats in natural estrus was10.6±3.8, higher than that of nanny goats in synchronized estrus (6.1±3.1). The results indicated that nanny goat in natural estrus was more suitable for superovulation to obtain in vivo embryos.(2)From homology analysis of4pluripotency genes, we found that the homology between domestic animal and human beings were closer than that between domestic animal and mouse. So we chosed media of human embryonic stem cells for Arbas Cashmere goat embryonic stem cells culture, the medium I:DMEMF12+10%fetal bovine serum+0.1mM β-mercaptoethanol+0.1mM non-essential amino acids+0.1mM L-Glutamine+20ng/ml LIF+10ng/ml bFGF+100IU/ml penicillin+0.05mg/ml streptomycin; and the medium Ⅱ: DMEMF12+BSA+N2+B27+0.1Mmβ-mercaptoethanol+0.1mM non-essential amino acids+0.1mM L-Glutamine+100IU/ml penicillin+0.05mg/ml streptomycin+20ng/ml LIF+10ng/ml bFGF.(3) By ear vein injection, the serum of rabbit anti Aerbasi Cashmere goat was prepared in our lab. The titer assays of different dilution ratio of serum were carried. The results showed that a1:5dilution of rabbit anti Arbas cashmere goat serum could reacted sufficiently with guinea pig serum complement, so the dilution of1:5is the optimal titer of the antiserum.2)The establishment of cell culture system (1) Mouse fetal fibroblasts were used as feeder layer, with culture medium I and II, the inner cell masses were obtained by embryo adherent directly. Mechanical massaging and trypsin digestion were used to passage Arbas cashmere goat embryo stem cells,90%fetal calf serum+10%of DMSO as cryoprotectant solution was used for cryopreservation. After being thawed and cultured, the Arbas cashmere goat embryonic stem cells was detected for identification. The results showed that the two kind of culture media both could be used to culture Arbas cashmere goat embryonic stem cells. Embryonic stem cells cultured in medium II grew faster than that in medium I, therefore the medium II was more suitable to culture Arbas cashmere goat embryonic stem cells. Mechanical passaging method was more suitable for the passage of Arbas Cashmere goat embryonic stem.After cryopreservation and thawing, Arbas Cashmere goat embryonic stem cells cultured in the two kind of media were still keeping the pluripotency of embryonic stem cells. It is suggested that the normal method of cryopreservation and thawing could be taken in preservation of Arbas cashmere goat embryonic stem cells.(2) Immune surgery method using the serum of rabbit anti Arbas cashmere goat was used to obtain the inner cell masses of Arbas cashmere goat blastocysts, and mouse fetal fibroblasts were used as feeder layer, we cultivated embryonic stem cells with the medium Ⅱ and mechanical passaging to find out if immune surgery could obtain inner cell mass. The results showed that inner cell mass of Arbas cashmere goat in high purity could be obtained by the method of immune surgery, and pluripotency of embryonic stem cells could be detected. The conclusion was that the method of immune surgery could be usesd in the culture of Arbas cashmere goat embryonic stem cells. 2. Establishment of culture system of ovine embryonic stem cells1) Preparatory experimentsWith the method of one-step, two-step and the frozen semen law of the Boer goat, sheep freezing semen were prepared. The motility and survival rates of the sperm obtained by one-step method after thawing were higher than that obtained by the other two methods. The frozen semen with high quality could be obtained by one-step method, and high hatch ability of ovine blastocysts could be obtained by IVF.2)The establishment of cell culture system(1) The inner cell mass was obtained by embryo adherent directly, then cultured on the feeder layer of mouse fetal fibroblasts with basic medium of SESCCDMEM F12+10%Fetal bovine serum+0.1mM p-mercaptoethanol+0.1mM non-essential amimo acids+20ng/ml LIF+10ng/ml bFGF+100IU/ml penicillin+0.05mg/ml streptomycin, passaged with mechanical passaging method. The culture system of ovine embryonic stem cells was optimizated for establishment. The results showed that the basic embryonic stem cell culture method could be taken to culture ovine embryonic stem cells, and the embryonic stem cells could be carried out for further research. (2) The inner cell mass was obtained from embryo adherent directly, then cultured on the feeder layer of mouse fetal fibroblasts with basic medium I and II, passaged with mechanical passaging method. The efffection of the two kind media on embryonic stem cells culture was observered. The results showed that the ovine embryonic stem cells could be cultured in both kind of media, but they grew better in medium...