The objective of this study was to examine the effect of in vitro maturation (IVM) medium, cytochalasin B (CB) treatment during intracytoplasmic sperm injection (ICSI), and electric activation on in vitro development ICSI-derived embryos in pigs. Immature pig oocytes were matured in vitro in medium 199 (M199) or porcine zygote medium (PZM)-3 that were supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 21～22 h. ICSI embryos were produced by injecting single sperm directly into the cytoplasm of IVM oocytes. The oocytes matured in PZM-3 with 61.6 mM NaCl (low-NaCl PZM-3) tended to decrease (0.05<P<0.1) nuclear maturation when compared with oocytes matured in M199 (76.9% vs. 83.8%) but no significant differences were found in embryo cleavage, blastocyst formation, and mean number of cells in blastocyst (73.8% vs. 74.6%, 11.1% vs. 12.1%, and 28.4 cells vs. 30.1 cells, respectively). The oocyte degeneration was not reduced by CB treatment during ICSI (11.9%) when compared with no treatment control (11.3%) while the treatment showed detrimental effects (P<0.05) on embryonic cleavage (40.0%) and blastocyst formation (1.8%) rates when compared with control (60.0% and 11.5%, respectively). For activation of ICSI oocytes, additional electric stimulus has no positive or negative effect on in vitro development of preimplantation stage ICSI porcine embryos. Our results demonstrate that CB treatment during ICSI inhibits embryonic development of ICSI oocytes and additional electric activation after ICSI has no effect in improving ICSI embryonic development in pigs. Further studies are needed to improve ICSI efficiency by investigating factors influencing embryonic development after ICSI in pigs.

본 연구에서는 체세포를 이용한 돼지 복제수정란의 생산효율을 높이는 최적의 방법을 구명코자 핵이식 수정란에 각기 다른 조건들의 전기적 자극에 의한 융합과 활성화를 유도하여 융합율, 분할율, 후기배로의 발달율 및 배반포기배의 할구수를 비교ㆍ조사하여 다음과 같은 결과를 얻었다. 핵이식 복제 수정란과 전기자극에 의한 단위발생란과의 체외배양후 분할율을 비교한 결과 두 처리간에 유의적인 차이를 나타내지 않았으나, 배양7일째 배반포기배로의 발달율에 있어서는 복제수정

The present study was undertaken to examine the critical effect of + concentration on electrostimulation and post-electrostimulation media for electric activation of in vitro matured oocytes of Korean Native Cattle. Oocytes collected from slaughterhouse ovaries were matured in TCM 199 containing FSH, estradiol-17 and FBS with granulosa cell monolayer for 24 hours and denuded with hyaluronidase. And then cumulus-free oocytes were submitted to a DC field of 1.0 kV/cm for 60 sec in electroporation media(0.28 M mannito' and PBS) with different + concentations (0.00, 0.05, 0.10 and 0.15 mM). Stimulated oocytes were stained and examined for pronuclear formation after incuhation in SOF for 12 hours. The rates of pronuclear formation in hovine oocytes electrically stimulated in 0.28 M mannitol with 0.05, 0.10 and 0.15 mM +(60.3, 82.2 and 75.0%) were significantly higher than without +(6.3%) at 12 hours after an electric pulse(p<0.005). The activation rates of Korean Native Cattle oocytes stimulated in PBS supplemented with 0.05, 0.10 and 0.15 mM +(71.0, 75.8 and 75.4%) were significantly higher than without +(23.5%) after post-stimulation incubation(p<0.005). After incubation of oocytes in SOF with and without + following electric stimulation in 0.28 M mannitol with 0.10 mM +, the rates of pronuclear formation of bovine oocytes in +-free SOF(85.7%) was significantly higher than in SOF with 1.71 mM +(62.5%, p<0.05). When oocytes were stimulated in two electrostimulation media supplemented with + and incubated in +-free SOF, there were no significant differences in the rates of pronuclear formation hetween 0.28 M mannitol and PBS. These results indicate that a single electric pulse could induce activation of Korea Native Cattle oocytes in 0.28 M mannitol and PBS supplemented with +. Furthermore, to improve the activation rates, it was hetter that stimulated oocytes were incubated in +-free SOF after electric stimulation than in SOF with +.