Optimization Of The Protocols Of Sheep Embryoes Produced In Vitro

Since the birth of Dolly, techniques of somatic cell nuclear transfer(SCNT) have been developed for over a decade. However, the efficiency of development of cloned sheep offspring remains low and can be determined by each step during in vitro production of sheep embryos. Therefore, the objects of this research was to compare and optimize the protocols during ovine in vitro fertilization(IVF) and SCNT processes so as to further increase the efficiency of in vitro production of sheep embryos. The abattoir-derived ovaries, a day 30 fetus obtained from Small Tail Han sheep and the ear tissues obtained from Ao Han Finewool sheep were collected for materials. This research mainly focused on the modification and improvement of key steps including oocyte recovery, in vitro maturation(IVM), donor cell culture, SCNT and in vitro culture of embryos. Specific experiments and outcomes were as follows.Follicle Flushing, Slicing, Syringe Aspiration and Pump Aspiration were performed as oocyte recovery methods to compare their effects on quality and quantity of oocytes. FSH/LH produced from BIONICHE or Ningbo Hormone Factory and different serums (Estrous Sheep Serum or Fetal Bovine Serum) were supplemented to IVM media to compare their effects on maturation rates. After IVF, development of embryos cultured in mCR or mSOF system was examined. Results showed that the percentage of grade A and B oocytes recovered using Follicle Flushing was significantly(P< 0.05) higher than the other methods. Supplementation with FSH/LH(BIONICHE) and Estrous Sheep Serum(ESS) significantly(P< 0.05) increased maturation rate compared with other treatments. MSOF system provided significantly(P< 0.05) higher rates of blastocyst and hatched blastocyst than mCR.Establishment of ovine fetal and ear primary fibroblasts were conducted and cultured for 5 passages. The optimal cell culture media, growth curves and chromosome karyotype of these fibroblasts were investigated. Results showed that the growth speed, death rate during passages and shape of ovine ear fibroblast in DMEM/F12 were better than those in DMEM. The growth curves of ovine fetal and ear fibroblasts showed "S" shape and growth speed of fetal fibroblasts was higher. Karyotype analysis showed that proportions of normal karyotype in these two cell lines were very high.After in vitro maturation, Micro-electrode fusion, Zona-free and Micro-injection cloning were conducted to compare their efficiencies in construction of cloned embryos. Optimal concentration of Vitamin C was explored by examining its effects on the development of parthenogenetic oocytes and was subsequently applied to in vitro culture of cloned embryos. Results showed that fusion rate obtained form Micro-electrode fusion with 25 volts was significantly increased compared with Chamber Fusion(P< 0.05). Twenty five volts resulted in less dead oocytes than 30 volts in Micro-electrode fusion(P< 0.05). Zona-free cloning failed to increase cleavage and blastocyst rate of cloned embryos(P>0.05), but increased morula rate significantly(P<0.05). Vitamin C supplementation with the concentration of 50 μM significantly increased blastocyst rate of ovine parthenogenetic and SCNT embryos(P< 0.05).Together, Follicle Flushing method introduced here was more appropriate for ovine oocyte recovery. The maturation rate of ovine oocytes could be increased significantly with the supplementation of ESS and FSH/LH(BIONICHE) into IVM medium. DMEM/F12 was optimal for in vitro culture of ovine ear fibroblasts in contrast to DMEM. Both fetal and ear fibroblasts had good performance of growth and could be used as donor cells for SCNT. Micro-electrode fusion significantly increased the fusion rate during SCNT. There was no significant effect of Zona-free cloning on improving the blastocyst development of ovine cloned embryos. MSOF system supplemented with 50 μM Vitamin C was beneficial for obtaining more blastocysts produced in vitro.