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These studies were designed to identify the effects of sperm treatments and individual Korean native bulls on in vitro acrosome reaction, invitro fertilization and in vitro embryonic development. Sperm samples used were frozen-thawed ejaculated sperm from 9 bulls and epididymal sperm from 6 bulls and the acrosomes of sperm were evaluated at various intervals after the treatment with caffeine, heparin or caffeine-heparin. The treated sperm were fertilized in vitro with zona-free hamster eggs for sperm penetration assay and with in vitro matured bovine follicular oocytes for in vitro embryonic development. 1. When the frozen-ejaculated sperm were treated with caffeine, the percentage of live and dead sperm with detached acrosome increased significantly from 1.5h after preincubation (P<0.05) and these increases were observed at the other sperm treatments. The acrosome reaction of live sperm by each sperm treatment was not significantly different among the individual bulls, but the total sperm with detached acrosome were significantly different among them (P<0.05). 2. The acrosome reaction of epididymal sperm increased significantly from 3h after sperm treatment (P<0.05), but there was no significant difference between the sperm treatments and the time was slightly slower than that of ejaculated sperm. When the frozen-thawed epididymal sperm were treated with caffeine, the percentage of live sperm with detached acrosome ranged from 8.8 to 22.8 and there was significant difference between the individual bulls (p<0.05). 3. When the frozen-thawed ejaculated sperm were preincubated 2h after caffeine treatment, 42.1 to 52.6% of them penetrated into zona-free hamster eggs and the bull of a higher acrosome reaction tended also to show a higher penetration rate, but there was not significantly different between the individual bulls. When the frozen-thawed epididymal sperm were preincubated 3h after caffeine treatment, 41.2 to 59.4% of them penetrated into zona-free hamster eggs, There was no significant difference between the individual bulls and their penetration rates were not similar to their acrosome reaction rates. 4. The embryonic development from bovine follicular oocytes which were fertilized in vitro with 2h-preincubated frozen-ejaculated sperm was not accorded with the acrosome reaction rates of sperm, while the cleavage to above 8-cell was different between the bulls although there was not significant. The embryonic development from bovine follicular oocytes which were fertilized in vitro with 3-h preincubated frozen-epididymal sperm was not related to the acrosome reaction of bulls, but their cleavages to above 8-cell tended to be higher than those with ejaculated sperm. It is concluded that the caffeine treatment was more effective to induce the acrosome reaction of sperm and the effective preincubation time was 2h for ejacuated sperm and 3h for epididymal sperm. The results also indicated that the in vitro fertilization and embryonic development is not accorded with the in vitro acrosome reaction of sperm but can be affected by the individual bulls.
This study examined the effects of growth factors in TCM199 on bovine 1-cell embryos development in vitro. After 6 day to 11 day in culture, 15.8%(19/120), 15.3%(20/130), 21.8%(35/160), 27.0%(56/207), 26.3%(53/201) and 30.7%(40/130) of the 1-cell embryos developed into expanding blastocysts in su, pp.ementing TCM199 with control, insulin, IGF-I, IGF-II, FGF and EGF, respectively. Hatching rate of 1-cell embryos in su, pp.ementing TCM199 with FGF, EGF and IGF-II were 21.4%(53/247), 20.3%(42/206) and 16.8%(41/243), respectively. The beneficial effect of growth factors on embryo development in vitro could be duplicated. These data indicate that the presence of FGF, EGF or IGF-II in the culture medium is beneficial for embryo development in vitro and accelerate cell differentiation.
생쥐 초기배의 배반포 발달율이 BSA첨가 배양액에 Barodon-FX(equation omitted) 첨가로 증가되지 않았으나 PVP 첨가 배양액에 0.25% 첨가에서는 부화배반포 발달율이 54.7%로 대조구(32.5%)보다 크게 향상 되었다(P <0.05). 1∼2% Barodon 첨가는 배발달을 월등히 저하시켰다. Barodon 첨가에 따른 체세포 증식율은 BOEC와 GC의 경우 0.25∼0.5%에서 대조구보다 각각 24∼40%와 17∼22%더 크게 증가되었다(P<0.05). 그러나 GC와 CC에서는 1%이상 첨가시 세포증식이 크게 억제되었다(P <0.05). Barodon의 효과는 체세포간에 큰 차이가 있었다. 소 초기배에서 상실배이상의 배발달율은 BOEC와 GC 공배양조건에서 Barodon 0.5% 첨가에서 대조구보다 크게 향상되었다(P<0.05). 그러나 다른 처리 수준에서는 배 발달율이 대조구와 차이가 없었다. 결론적으로 Barodon의 세포증식효과는 세포종류에 따라 차이가 많았으며 0.5% 수준의 첨가는 소 초기배의 배반포발달율을 향상시킬 수 있었다. This experiment was designed to evaluate effects of nonspecific immunostimulator(NIS) Barodon-FX(equation omitted), anionic alkali mineral complex and far-infrared radiation solution on in vivo-produced mouse and in vitro-produced bovine embryos to blastocyst development. Proportion of mouse embryos developing into blastocyst was not greater in BSA- and Barodon-added medium than in BSA-control, but there was signifcantly different(P < 0.05) in hatching and hatched blastocyst development between 0.25% Barodon-and PVP-contained medium(54.7%) than PVP-control(32.5%). BOEC and GC resulted in higher proliferation rate(24∼40% and 17∼22%, respectively) in 0.25∼0.5% Barodon-added medium than in controls, but proliferation of GC and CC greatly decreased in 1∼2% Barodon-added medium. Effect of Barodon on cell proliferation greatly varied among somatic cells. Proportion of early bovine embryos developing into morula and blastocyst was significantly greater(P < 0.05) in 0.5% Barodon-added medium(50% and 63.6%) than in control(31.6% and 27.4%) under co-culture with BOEC and GC, but developmental rate was not different between other Barodon treatments and control. These data indicate that effect of Barodon on cell proliferation significantly varied between somatic cells and that addition of 0.5% Barodon in BOEC-coculture system may further improve blastocyst development in early bovine embryos.