• 한국가축번식학회지
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• 제14권1호
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• pp.17-22
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• 1990

The present study was carried to investigate the development potential of bovine follicular oocytes matured and fertilized in vitro. Bovine oocytes matured in vitro were fertilized, cultured, and transfered to the rabbit oviduct for in vitro culture to evaluate the development potential. The rates of in vitro maturation, fertilization and polyspermy were 96%(355/369), 90%(320/355) and 15%(17/320), respectively. The percentage of oocytes cleaved after culture for 48 hours, to 2 cell, 3~4 cell, 6~8 cell stage were 17%(60/356), 21%(75/356) and 19%(67/356), respectively and overall cleavage rate was 57%(202/356). The rate of recovered embryos after 5 days in rabbit oviduct was 56%(80/142), and 21%(17/80) of recovered embryos developed over morula stage.

• 한국수정란이식학회지
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• 제15권1호
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• pp.33-38
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• 2000

This study was undertaken in an effort to select the sire bull in Hanwoo through in vitro fertilization of proven bull semen. It was used for in vitro fertilization that of the 20 proven bull semen with follicular oocytes derived from slaughterhouse ovaries of Hanwoo. The stage of maturation on the time course of bovine cumulus-enclosed oocytes incubated for 24 hours was found the highest(96.4%) than hose of other maturationi time. In vitro fertilization rate of bovine oocytes with proven bull sperm showed from 61.5 to 88.9%. Polyspermy of in vitro fertilized oocytes according to proven bulls were the highest KP 491(61.5%) nothing but KP 486, KP 491 and KP 497.

• 한국수정란이식학회지
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• 제10권3호
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• pp.243-250
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• 1995

Frozen storage of the oocytes has been used in a few mammalian species including mouse, hamster, human and cattle. However, frozen4hawed oocvtes show different sperm penetration on the levels of the zona pellucida and the plasma memhrane when compared with fresh oocytes. To elucidate biological changes occurring during freezing and thawing, we examined the kinetics of sperm penetration into frozen-thawed hamster oocytes. Oocytes obtained from superovulated female golden hamsters were frozen-thawed in an autofreezer according to an established method. Fresh and frozen4hawed oocytes were fertilized in vitro with capacitated hamster spermatozoa after removing the zona pellucida. The oocytes were examined at 1, 2, 3 and 6 h postinsemination. Sperm penetration found to be 1 h delayed in frozen-thawed oocytes. Other parameters such as degree of polyspermy and decondensing sperm heads were not affected by freezing and thawing. The results suggest that freezing and thawing may cause changes in the egg membrane surface and subsequently which leads to delay in the sperm-egg fusion.

• Clinical and Experimental Reproductive Medicine
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• 제25권3호
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• pp.315-322
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• 1998

본 연구는 xanthine과 xanthine oxidase가 첨가된 배양액내에서 전배양된 돼지동결정액의 수정능력에 있어서 catalase의 영향을 검토하였다. 체외수정을 위한 기본 배양액내에서 0 또는 30분간 전배양한 정자는 catalase를 첨가 (40 및 15%)한 경우 보다는 무첨가 (66 및 38%)시 유의적으로 높은 정자침입율을 나타냈지만 (p<0.05), 배양액내에 xanthine을 첨가해 정자를 0, 30 및 60분간 전배양했을 때에는 catalase 무첨가 (33, 41 및 19%) 보다는 첨가시 (68, 70 및 49%) 유의적으로 높은 정자침입율이 인정되었다 (p<0.05). 그러나 xanthine oxidase를 첨가하여 정자의 전배양을 행하지 않은 경우는 catalase의 첨가 (13%) 보다는 무첨가 (51%)시 유의적으로 높은 정자침입율을 나타냈지만 (p<0.01), 전배양(30 및 60분)후에는 catalase의 존재유무와 정자전배양시간에 관계없이 매우 낮은 정자침입율 $(10\sim21%)$을 나타냈다. xanthine과 xanthine oxidase를 동시에 첨가하여 0, 30 및 60분간 정자를 전배양한 경우 catalase의 무첨가 (14, 4 및 8%)보다 첨가 (75, 55 및 52%)시 유의적으로 높은 정자침입율을 나타냈다 (p<0.001). 한편, 다정자침입율은 xanthine, xanthine+xanthine oxidase의 첨가시 정자전배양기간이 길어짐에 따라 감소하였으며, catalase의 첨가보다는 무첨가시 낮은 다정자침입율을 나타냈다. 본 연구의 결과로부터 xanthine과 xanthine oxidase를 동시에 첨가시 catalase의 존재는 정자의 전배양후에도 다정자침입을 억제하면서 수정능력의 유지를 위해 매우 효과적인 것으로 추측되었다.

• 한국가축번식학회지
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• 제24권3호
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• pp.255-262
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• 2000

본 연구는 돼지난자의 체외수정시 Asc와 Fe$^{2+}$의 첨가하에서 정자 전배양의 영향을 검토하기 위하여 수행되었다. 체외에서 성숙시킨 돼지난포난자를 0, 1, 2, 3, 4 및 5시간 전배양된 돼지동결-융해정액을 이용하여 수정한 결과, 정자침입율(37~51%)은 0.1mM Asc의 첨가하에서 정자의 전배양 기간사이에서 유의적인 차이는 인정되지 않았다. 또한 정자의 전배양 기간동안 1.0mM Fe$^{2+}$의 첨가시에도 정자침입율에는 커다란 차이를 나타내지 않았다. 그러나 정자 전배양시 Asc와 Fe$^{2+}$ 를 동시에 첨가했을 때 정자의 전배양기간이 길어짐에 따라 정자침입율이 증가하는 경향을 나타냈으며, 5시간 전배양시 이들 물질의 첨가시 무첨가에 비해 유의적으로 높은 정자침입율을 나타냈다 (P<0.05). 한편 Asc와 Fe$^{2+}$ 가 첨가되지 않은 배양액내에서 전배양된 정자를 이용하여 수정했을 때, 수정배양 액내에 Asc 또는 Asc+Fe$^{2+}$ 가 첨가된 경우 보다는 Fe$^{2+}$ 첨가시 유의적(P<0.05)으로 높은 정자침입율을 나타냈으며, 다정자침입율은 정자의 전배양기간이 길어짐에 따라 감소하는 경향을 나타냈지만 이들 물질이 첨가된 배양조건하에서는 그 차이가 인정되지 않았다. 본 연구의 결과로부터 정자의 전배양시 Asc 와 Fe$^{2+}$ 의 첨가는 정자침입에 효과적으로 작용했으며, 전배양된 정자를 이용한 체외수정시 Fe$^{2+}$의 첨가는 다정자침입을 억제하면서 수정능력이 계속 유지되는 것으로 나타났다.

• 한국수정란이식학회지
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• 제16권3호
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• pp.163-172
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• 2001

본 연구는 돼지 난포란의 체외성숙에 적합한 배양액을 조사하고, 아울러 산소농도가 돼지 난포란을 이용한 체외수정란의 생산에 미치는 영향을 구명하고자 실시하였다. 본 연구에서 얻어진 결과를 요약하면 다음과 같다. 1. NCSU-23과 TCM-199 배양액에 10% PFF를 첨가하여 5% 및 20% 산소조건하에서 체외성숙을 유기한 결과, 배양액 및 산소조건에 따른 난핵붕괴률 및 핵성숙률에는 유의적 (P>0.05)인 차이가 없었다. 2. NCSU-23이 TCM-199 배양액보다 다정자침입률 및 평균정자침입률에 있어서 유의적으로 낮은 결과를 나타냈고 (P>0.05), 5% 및 20% 산소농도에 따른 차이는 인정되지 않았다. 3. 체외수정을 실시한 후, 5% 및 20% 산소조건하에서 NCSU-23 배양액에서 배양하여 배발달에 미치는 영향을 조사한 결과, 배달달 2일째 배반포형성에 있어서는 NCSU-23 처리구가 TCM-199 처리구에 비해서 유의적 (P<0.05)으로 높은 결과를 나타냈다. 또한, 처리구별 배반포기의 총세포수를 조사한 결과 5% 및 20% 산소농도 사이에는 유의적인 차이가 인정되지 않았다. 따라서, 효과적으로 돼지 난포란을 이용하여 체외발달을 유기하기 위한 체외성숙조건은 20% 산소조건하의 NCSU-23 배양액이 적합한 것으로 사료된다.

• 한국가축번식학회지
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• 제23권3호
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• pp.263-270
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• 1999

본 연구는 체외성숙배 양액에 L-ascorbic acid 와 selenium을 첨가 배양, 돼지 미성숙 난포란의 체외성숙, 체외수정 및 체외배발달에 미치는 영향을 검토코자 수행하였다. 배양액내 L-ascorbic acid를 0. 62.5. 100 그리고 30 $\mu$Ml 첨가 40~44시간동안 배양한 성적은 난핵포 붕괴율이 각각 86.8%, 92.9%, 91.7%, 그리고 92.6%였으며 핵성숙율은 각각 44.7%. 57.1%, 52.8%, 그리고 53.7%로 첨가구에서 유의적으로 높았다 (p＜0.05). 체외성숙배양액내 L-ascorbic acid와 selenium을 첨가 배양 후 체외 수정 유기 결과, 웅성전핵 형성율은 유의적으로 높았고 (p＜0.05) 다정자 침입율은 유의적으로 낮게 나타났으며 (p＜0.05), 체외수정 후 난할율, 상실배와 배반포배 발달율도 첨가구에서 유의적으로 높게 나타났다 (p＜0.05). 이러한 결과는 체외성숙 배양액내 L-ascorbic acid와 selenium을 첨가 배양했을 때 돼지 미성숙난포란의 체외 성숙율, 웅성 전핵 형성율 그리고 돼지 체외수정란의 배발달율을 향상 시킬 수 있으리라 사료된다.

• 한국가축번식학회지
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• 제17권2호
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• pp.103-111
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• 1993

These experiments were undertaken to establish the optimal culture systems for in vitro maturation, fertilization and subsequently embryonic development of porcine immature follicular oocytes isolated from the ovary of slaughtered pigs. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles (3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated (39$^{\circ}C$, 5% CO2 in air) for 42hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were prepared forfertilizaing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${mu}ell$ of capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the culture media. The fertilization rates of in vitro matured follicular oocytes cultured in B. O., TCM-HEPES, m-KRB, and TALP-II media were 61.3%, 83.0%, 88.9% and 89.2%, respectively. In addition, the polyspermy rates were 60.7%, 66.5%, 53.8%, and 43.9%, respectively. These data indicated that the highest of fertilization and the lowest of polyspermy rate was shown in TALP-II medium. Spermatozoa capacitated by caffeine, heparin, and percoll density gradient treatment in the 4 different media, the fertilization rates were 33.0~57.2%, 39.9~90.2%, and 52.6~92.8%, respectively, showing the lowest rate in caffeine treatment. The development rate of follicular oocytes, fertilized with the spermatozoa capacitated by caffeine, heparin, and percoll gradient in the TALP-II medium, upto 2 to 4-cell stages were 32.6%, 74.5% and 70.9%, respectively. Finally, fertilization rates of follicular oocytes cultured with follicular fluid containing medium from 10 to 100% were 61.2~94.1% and the rates (90~94%) with 10~20% follicular fluids were significantly higher than those (85.3%) of cultured in the media without follicular fluid. In addition, the rates of pronucleus formation were also higher in follicular fluid treated group (73.1~83.0%) than those (64.7%) of oocytes cultured without follicular fluid. The highest fertilization and pronucleus formation rates was found in oocytes cultured with 10% follicular fluid. These results suggest that the addition of heparin or percoll density gradient method is better capacitation method. Furthermore, the addition of porcine follicular fluid to the fertilization medium may improve the fertilization rates and formation of pronucleus.

• 한국가축번식학회지
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• 제16권3호
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• pp.199-208
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• 1992

These studies were carried out to investigate optimal physological conditions for in vitro fertilization (IVF) of mouse ova. The unfertilized ova were obtained by superovulation from ICR mice of 4 to 6 weeks old. Tyrode's 280 solution was used as basal media, and pH and osmolality of basal media were adjusted with the supplementation of sodium bicarbonate and sodium chloride, respectively. The optimal pH, and osmolality of culture media and the optimum period of sperm preincubation were examined in fertilization in vitro of mouse ova and the subsequent culture in vitro of embryos. The pH range of media examined was designed from 6.5 to 7.5 with 0.2 interval and the range of osmolality from 250 to 370 mOsm with 20 interval, and the period of sperm preincubation examined was 30, 60, 120, and 180 minutes. The ova developed to 2-cell embryosafter 26hrs. of incubation with preincubated sperm were evaluated as in vitro fertilized ones. The results obtained were summarized as follows: 1. The percentage of in vitro fertilized ova was highest (64.7%) in media of pH 7.1 and lowest (38.0%) in pH 6.7. No significant difference in % fertilized ova was found from the media of pH 7.1 to 7.5. Compared with the result from pH 7.1 medium, the pollyspermy was increased signifciantly (p<0.05) in the media of pH over 7.5 and below 6.9;, and the % degenerated ova was significantly (p<0.05) increased in the media of pH below 6.9. 2. The percentage of in vitro fertilized ova was highest (69.4%) in media of osmolality 330 mOsm and lowest (47.9%) in osmolality 250 mOsm. No significant difference in % fertilized ova was found from the media of osmolality 310 to 350 mOsm. Compared with the result from osmolality 330 mOsm in medium, the polyspermy aws increased significantly(p<0.05) in the media of osmolality over 350 mosmol and blow 290 mOsm, and the % degenerated ova was significantly (P<0.05) increased in the media of osmolality below 290 mOsm. 3. The percentate of in vitro fertlilized ova was highest (62.7%) in media of period sperm preincubation 180 min. and lowest (40.4%) in sperm preincubation 30 minutes. No significant difference in % fertilized ova was found from the media of sperm preincubation 120 to 180 minutes. Compared with the result from sperm preincubation 180 minutes in medium, the polyspermy was low differ no significantly(P<0.05) in the media of period sperm preincubation, and the % degenerated ova was signifciantly(P<0.05) increased in the media of sperm presincubation below 60 minutes.

• 한국가축번식학회지
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• 제14권1호
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• pp.50-56
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• 1990

Bovine oocytes obtained from follicles(2~5mm) of ovaries after slaughter were cultured in TCM 199 medium with 10~20% heat-inactivated estrus cow serum(ECS) for 25~27 hr, at 39$^{\circ}C$ under 5% CO2 in air. At the end of culture period, some oocytes were stained with 1% acetoorcein and examined for the evidence of oocyte maturation. The remainder were used to assess the potential of in vitro fertilization(IVF) with frozen-thawed spermatozoa and subsequent development in media with or without bovine oviduct epithelial cell (BOEC) co-culture. The results obtained were summarized as follows ; 1. The maturation rate of oocyte in vitro in TCM 199 medium with 15% ECS group(76.3) was superior to 10% ECS group(68.3%) and 20% ECS group(64.5%). 2. The IVF rates of oocytes matured in vitro, and formation rate of male and female pronuclei were 63.6%(77/121) and 93.5%(72/77), respectively. The incidence of polyspermy was very low(2.4%). 3. Of 73 oocytes fertilized in vitro and cultured in TCM 199 medium with 10% fetal calf serum for 7 days, 41(56.3%) were cleaved over 2-cell and only 1(2.4%) was developed beyond the 16-cell stage. 4. Of 76 oocytes co-cultured with BOEC, 58(76.3%) were cleavaged and 23(39.7%) were developed to morula and blastocyst stage. The results of this study indicate that co-culture with BOEC deserved a positive effect on the IVF oocyte development through the 16-cell block.