Instead of surgical embryo transfer (ET) in the pig, nonsurgical ET is a hopeful method to increase the efficiency of biotechnology applications such as cloning and transgenesis. In this study, we conducted surgical and nonsurgical ET methods after somatic cell nuclear transfer (SCNT) with MHC miniature pig cells to find out the best condition for production of cloned miniature pigs. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pigs. Fibroblast cells were cultured, passaged (3 to 8 passages), and used as donor cells for NT. After the enucleation and injection process, eggs were held in TCM-199. For fusion, 2 DC pulses of 1.2 kV cm-1 were applied for 30 μs. Both IVF and SCNT embryos were cultured in PZM-3 medium. After IVF, 84.9% (411/484) of embryos cleaved and 27.3% (132/484) of embryos reached the blastocyst stage. In the SCNT group, 80.8% (231/286) of eggs fused and 25.9% (60/286) of embryos developed to blastocysts. For surgical ET, approximately 200 SCNT embryos were transferred into oviducts of each synchronized recipient. For nonsurgical ET, embryos were cultured in PZM-3 for 6 days after SCNT and IVF, and then good quality blastocyst stage embryos were selected for ET. The pregnancy status of recipients at Day 30 was determined by ultrasound scanning. Using Day 30 of gestation as an endpoint, the nonsurgical ET method (47.3%, 9/19) had a similar pregnancy rate as the surgical ET method (56.5%, 13/23). Further study is needed to optimize the nonsurgical ET method especially for SCNT eggs. This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.
Porcine cloning by somatic cell nuclear transfer (SCNT) has limitations because of the high incidence of fetal failure after embryo transfer to recipient. The fetal failure may originate from disturbed embryo-uterine interactions in the early implantation period. In this study, we compared apoptotic-related gene expression and cytokines using real-time RT-PCR and protein chip array. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pig. After the enucleation and injection process, eggs were held in TCM-199. For fusion, two DC pulses of 1.2 kV cm-1 were applied for 30 μs. SCNT embryos were cultured under PZM-3 medium to the 1- to 4-cell stage. For embryo transfer, approximately 200 SCNT eggs were transferred into the oviduct of each synchronized recipients. We obtained the extraembryonic (embryo origin) and endometrium (maternal origin) tissues from 3 cloned fetus with that from normal fetus by natural mating at Day 30. Each experiment was repeated at least 3 times and the data were presented as mean ± SE. Values of P < 0.05 were considered statistically significant. Data from real-time RT-PCR are expressed as log (fold change) ± SE. Expression of Bax-α, p53, and caspase-3 mRNA was significantly higher in the SCNT group than in the control group (P < 0.05). Five cytokines were analyzed: Interleukin (IL)4, IL8, tumor necrosis factor-α, and epidermal growth factor were lower in the control group than in the cloned group (P < 0.05). However, the level of MCP1 was higher in the SCNT group (P < 0.05). Numbers of trophoblast cells in the cloned group were also lower than in the control group (P < 0.05). These findings indicate that failure of endometrium and extraembryonic tissues in cloned pregnancies may originate from abnormal embryo-maternal communication that develops during the implantation period. This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.
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