The Research On Porcine Somatic Nuclear Transfer

The major object of this paper is to research some fators on porcine somatic cloning effiency as well as to try to obtain the somatic cloning gilts. Some factors influencing the efficiency of porcine somatic cloning have been discussed. Our work may be useful to further research on xnotransplation. The results obtained were as follows: (1) The effects of different hormone requirements in different stages of porcine oocytes, different basic mature medium and with or without serum in mature medium on porcine oocyte maturation in vitro are studied. The experiments included,①Porcine oocytes were cultured in TCM199+10% fetal bovine serum(FBS)+10 IU/mL pregnant mare serum gonadotrophin(PMSG)+10 IU/mLhuman chorion gonadotrophin(hCG)+2.5 IU/mLfollicle stimulate hormone (FSH) for 44 h in vitro, revomal of hormone supplements from mature at 22 h after culture, culture for 22 h in the medium without hormone supplement subsequent supplement hormone for another 22 h and culture for 44h in medium with hormone supplement; ②porcine oocytes cultured for 44h in three different basic medium(TCM199,TCM199,modified TCM199)with the ways of hormone supplement in experiment ①; ③porcine oocytes cultured in modified TCM199 of experiment②with or without serum.The results show, ②three different stages'hormone supplement is not significant difference in the rate of maturation; ②the rate of maturation of the group of mM199 (54.01%) is higher than that of the group of TCM199 (46,16%) and (47,14%),but it has no significant difference; ③the rate of maturation of the group of free-serum(67.10%) is higher than that of the group with serum(52.22%) and it has significant difference. Therefore, the group of mM199+10 IU/mLMSG+10 IU/mLhCG+2.5 IU/mLFSH is adapted to culture porcine oocytes in vitro for 44h ,and the rate of maturation is to 67.10%. (2) The effects of ionomycin, eclectrical field strength, numbers of electrical pulse and electrical stimulation in combination with chemical stimulation on parthenogenetic activation of porcine oocytes were studied.The results are as follows: ①The activation rate with 10μM ionomycin dealing 5 min(62.97%,17/27) was not significantly different from that of dealing 5min(62.50%,15/24),10 min (65.22%,15/23) . ②The activation rate with three times electrical stimulation of 120 v/mm for 60μs (66.67%,30/45)was significantly higher than that of electrical stimulation of 60 v/mm(40.98%,17/42), 80 v/mm(44.11%,15/34), 100 v/mm (46.19%,18/39),but was not significantly different from that of 140 v/mm (63.89%,23/36),160 v/mm(64.10%,25/39). ③The activation rate with two times electrical stimulation of 120v/mm for 60μs (67.40%,31/46) was not significantly different from that of one time electrical stimulation (62.80%,27/43),three electrical stimulation(68.30%,28/41). ④The activation rate with three times electrical stimulation of 120 v/mm for 60 μs in combination with 10 μM ionomycin dealing 5 min(84.84%,29/33)was significantly higher than that of two times electrical stimulation of 120v/mm for 60μs alone(67.64%,23/34).The results of this experiment showed that two times electrical stimulation of 120v/mm for 60μs in combination with 10 μM ionomycin dealing 5min can effiently activate porcine oocytes. (3) Porcine fetal fibroblasts was isolated by trypsinization and explant culture respectively. Porcine cumulus cells was obtained and cultured easily and quickly after pocine oocytes maturation culture. These porcine fetal fibroblasts cells was cryopreserved in liquid nitrogen. After thawed, the cells could survived well(the survive rate 83.5%). After 3-6 days culture,it would be confluent.It suggests that the fetal fibroblasts and cumulus cells develop well and porcine fetal fibroblasts cells survived well after thawed,so they are a suitable donor cells. (4) The matured oocytes were enucleated at 43~44 h, donor cells were transferred into the enucleated oocytes by intracytoplasmic nuclear injection (ICNI), and the reconstituted embryos were activated by electrical pulse (120 v/mm, duration 60 μs, 2 times, interval 3 s) combinated with 10 μM Inomycin(5min) and 2 mM 6-DMAP(2h). When the porcine cumulus cells and porcine fetal fibroblast cells used as donor cells, the cleavage rate of reconstituted embryos was 23.29% and 24.14%, respectively, there was no significant difference (P>0.05). When the serum hungry cultured and the normal cultured porcine fetal fibroblasts used as nuclear transfer donor cells, the cleavage rate of reconstituted embryos was 21.82% and 22.95%, respectively, there was no significant difference (P>0.05). When the reconstituted embryos were activated in different time (0h,1-3h,4-6h) respectively, the cleavage rate was(8.51%,25.64%,26.83%) respectively , there was significant difference (P<0.05).