The Optimization Of In Vitro Culture In The Pig

Intracytoplasmic sperm injection (ICSI) describes a procedure in which a single sperm is injected directly into the cytoplasm of an oocyte to facilitate fertilization. Clinically, this assisted fertilization approach is presently used for treating male infertility inmen with an extremely low number of and/or immotile spermatozoa.The application of ICSI in pigs has not been as simple as originally thought. It has been reported that fertilization rate by ICSI is low in pigs, e.g., about 20% on average. Although the causes of the low success rate in porcine ICSI are uncertain, incomplete oocyte activation and failure of male pronucleus formation have been suspected as the two pivotal factors for causing the low developmental capacity of ICSI-fertilized porcine embryos.It has been reported that U0126, as mitogen-activated protein kinase kinase (MEK-1 and-2) special inhibitors, could induce activation of oocytes by inhibiting the activation of mitogen-activated protein kinases, increase the rate of male pronucleus formation. Transgenic cloned pigs provide an outstanding animal model for human disease research and have great potential as human xenotransplantation. Although several transgenic pigs has been obtained in a few countries, the efficiency of cloned piglets production has been very low, with less than 1% of transferred embryos surviving to term. In vitro culture system of the porcine oocytes/embryos is the most basic and important procedure in somatic cell nuclear transfer technique. To optimize the culture condition be of great significance in increase cloned efficiency.This thesis included two studies. The first part was about ICSI. The effect of different activation regimes on pronuclear formation and developmental competence of in vitro matured porcine oocytes fertilized by intracytoplasmic sperm injection (ICSI) was examined. The injected oocytes were exposure to NCSU-23 for 0 h,2h,3 h, or 4 h after electrical pulse activation followed by U0126 activation, The results showed that ICSI oocytes activated with the delay of additional U0126 for 4 h after exposure to an electrical pulse achieved the highest fertilization rate. Blastocyst rate after 3 h intervals between electrical pulse and U0126 was significantly higher than that in the 2 h interval group (19.26% vs 13.64%, P<0.05). There was no difference in blastocyst rate between 3-h and 4-h interval groups (P>0.05).The second part was about the SCNT,the objective of this study was to determine the effect of optimized conditions on the efficiency of SCNT.Experimentl:The objective of this study was to investigate the effects of different chemical activators on development of somatic cell nucleus transfer (SCNT) embryos in pigs.Our results demonstrate that postactivation treatment with CHX and DMAP, there was no significant difference in cleavage (P>0.05). The blastocyst formation and the mean number of cells in the oocytes treated with CHX showed significant difference compared with the DMAP-treated groups. The mean rate of blastocyst formation in the groups treated with CHX was higher (P<0.05) than that treated with DMAP. The mean number of cells in the oocytes treated with CHX showed significant lower than the DMAP-treated groups.Experiment2:The objective of this study was to investigate the effects of thawed cells or the fresh cells on fusion rate and the development of somatic cell nucleus transfer (SCNT) embryos in pigs. The rates of blastocyst formation showed no significant difference between the two groups. The fusion rate and the cleavage rate showed significant difference.The use of the thawed cells was significant higher than the fresh cells.Experiment3:The objective of this study was to investigate the effects of injecting somatic cells or the transgenic cells on the development of somatic cell nucleus transfer (SCNT) embryos in pigs,there was no significant different in the fusion rate and the blastocyst rate between the two groups,the cleavage rate injected of somatic cells was higher than the group injected transgenic cells.Experiment 4:This study focused on the effect of melatonin on in vitro culture medium of porcine oocytes, supplemented with melatonin 10-7,10-9,10-11 or without melatonin.. In conclusion, no significant differences were observed in the blastocyst rate between the four groups.the highest cleavage rate was observed at 10-9 melatonin, which were significantly higher than others.The culture medium supplemented with 10-11 melatonin were significant differences between the groups supplemented with 10-7 and 10-9, no significant differences were observed between the groups supplemented with 10-7 and 10-9,The best results of this experiment was obtained when culture medium were supplemented with 10-9 melatonin.