Protocol Optimization For Production Of Porcine Cloned Embryos By Somatic Cell Nuclear Transfer

Pigs are considered as ideal organ donor for human future xenotransplantation. Somatic cell nuclear transfer provides an opportunity for producing HAR and endoviruses free pigs. Up to date, there is no somatic cloned pig produced in mainland of our country. So the current research was designed to study main items of pig somatic nuclear transfer systematically such as somatic donors, recipient oocyte, culture of cloned embryos and embryo transfer. It consists of 5 independent experiments.Experiment 1. Nine fetal fibroblast cell lines were set up from 9 fetus of Chinese miniature experimental pigs at 40d of gestation and 1 adult ear skin fibroblast cell lines was set up from fetuses' mother by using explant-seeding method. Cell cycle stage was synchronized by either serum starvation or contact inhibition. No significant difference of population at G0/G 1 between them and no further increases of G0/G1 cell population after extension of synchronization beyond 2d. It is concluded that fetal fibroblasts responded to serum starvation and contact inhibiton at similar degree and that synchronization for 2d is enough.Experiment 2. Effects of basic maturation media, oxygen tension, insulin and LIF supplementation on nuclear maturation of prepubertal gilts oocytes and subsequent development after parthenogenetic activation were studied. Effect of different activation method on in vitro development of parthenogenetically activated oocytes was also studied. Results are: 1) the rate of nuclear maturation was significant higher after cultured in NCSU-23 than in TCM199; 2) when cultured in NCSU-23, significant higher maturation rate was observed under high oxygen tension (20% O₂) than low oxygen tension (7% O₂). When cultured in a novel maturation media-PZM, 20% O₂ was better than 7% O₂ also. Regardless of oxygen tension, NCSU-23 supported nuclear maturation better than PZM. 3) Improved maturation rate was obtained after supplementation of insulin, although no significant increase in rates of cleavage and blastocyst after parthenogenetic activation was observed. LIF supplementation did not improve maturation rate. The rate of blastocyst formation decreased drastically after culture in LIF-contaning media although no significant influence on cleavage rate and total cell number of blastocyst were observed. 4) No significant differences among 1.4kv/cm-100μ s-1DC, 2.0kv/cm-30μ s-1DC, 2.0kv/cm-60μs-1DC activation groups as for the rates of cleavage and blastocyst formation. However, 1.4kv/cm-100μs-1DC treatment was significant higher than 2.0kv/cm-30μs-1DC or 2.0kv/cm-60μs-1DC as for death rate of oocytes treated. 5) Chemical activation using Ionomycin and 6-DMAP was poorer than electrical activation using either 2.0kv/cm-30μs-1DC or 2.0kv/cm-60μs-1DC. These results indicated that 1) NCSU-23 is an ideal maturation media and 2.0kv/cm-30μs-1DC or 2.0kv/cm-60μs-1DC electrical activation parameters could effectively activate MII oocytes. 2) High oxygen tension is beneficial to oocytes maturation while preimplatation of PAEs are media dependent. 3) Insulin is beneficial while LIF is detrimental tonuclear maturation of oocytes. 4) A novel media-PZM could be used as maturation media but further studies to optimize it are neededExperiment 3. In the current experiment, in vitro development potential and blastocyst quality of IVFEs, PAEs and NTEs were studied. Preimplantation development competence was similar among IVFEs, PAEs and NTEs. However, with regard to blastocyst quality i.e. ratio of ICM to total cell number, IVFEs and NTEs were superior to PAEs. Effects of culture media including PZM-3 and NCSU-23 on in vitro development potential of PAEs and NTEs were studied. For PAEs, PZM-3 was significant better than NCSU-23. For NTEs, PZM-3 was detrimental to cleavage rate while it had no obvious influence on blastocyst formation. When PAEs cultured in NCSU-23+4 mg/ml BSA supplemented with 5μg/ml Insulin or 1000 IU/ml LIF, no significant improvements of Insulin or no detrimental influence of LIF was observed. Results show that 1) an ide...