• Reproductive and Developmental Biology
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• 제34권3호
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• pp.223-227
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• 2010

These study was carried out to investigate the effects of the recovery time, diameter of oocytes on in vitro fertilization or intracytoplasmic sperm injection (ICSI). The in vitro maturation rates to MII stage of oocytes recovered at the inactive, follicular and luteal stages matured for 72 h were $1.4{\pm}0.0%$, $43.4{\pm}3.2%$ and $10.8{\pm}2.7%$, respectively. The fertilization rates of in vitro cultured oocytes recovered from ovaries at the in active, follicular and luteal stages were $0.0{\pm}0.0%$, $15.7{\pm}3.4%$ and $7.6{\pm}3.5%$, respectively. The in vitro maturation rate of oocytes recovered from ovaries at the follicular stage of the reproductive cycle was significantly higher than those at the inactive and luteal stages (p<0.05). The penetration rate determined that the percentages of oocytes with diameters in the < $100\;{\mu}m$, 100 to $100\;{\mu}m$ and 110 to $120\;{\mu}m$ ranges were $17.5{\pm}4.7%$, $43.9{\pm}4.5%$, $21.3{\pm}3.4%$, respectively. The penetration rate of oocytes with diameters between 100 to $100\;{\mu}m$ was significantly higher than that of oocytes whose diameters were < $100\;{\mu}m$ and $110{\sim}120\;{\mu}m$ (p<0.05). The penetration rate of oocytes determined that the percentages of ovaries with diameters between 1 to 5 mm and 6 to 10 mm were $32.9{\pm}3.2%$ and $17.5{\pm}3.7%$, respectively. Thus, the diameters of the ovaries were significantly higher at 1 to 5 mm (p<0.05). A total of 264 oocytes were fixed and stained after co-incubation with sperm, of which 72 had identifiable nuclear material. After in vitro fertilization for 20 hrs, 27.3% of oocytes were penetrated by spermatozoas. Oocytes were fixed and stained after ICSI, of which 38 oocytes contained identifiable nuclear material. After in vitro fertilization and ICSI for 20 hrs, to 27.3% and 67.9% of oocytes were penetrated by spermatozoas. The in vitro fertilization rates by ICSI was significantly higher than that in vitro fertilization method (p<0.05).

• 한국가축번식학회지
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• 제15권2호
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• pp.125-132
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• 1991

The studies on the carried out to investigate the effects of capacitation method of spermatozoa on the in vitro fertilization and cleavage rate of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible of diameter 3~5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and FCS for 24~48hrs in an incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18hrs with motile capacitated sperm by preincubation of mKRB, treatment of HIS(high strength ion), Ca-IA(Inophore A), BFF(bovine follicular fluids) and heparin. The results obtained in these experiments were summarized as follows : 1. The in vitro fertilizatin and cleavage rate offollicular oocytes fertilized with capacitated spermatozoas in BO solution by preincubation of mKRB, treatment of HIS, Ca-IA, BFF and heparin method were 53.1%, 33.9%, 50.8%, 48.1%, 58.8% and 28.1%, 17.7%, 26.2%, 22.8%, 32.8%, respectively. And the fertilization and cleavage rate of heparin method was of highest of all. 2. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solutin by both caffeine, BSA and heparin methods were 65.8%, 70.3% and 40.8%, 47.3%, respectively, and those rates were higher treatment of heparin＋BSA, heparin＋caffeine than treatment of heparin. 3. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoa in BO solution with heparin concentrations of 2, 5, 10, 20, 40$\mu\textrm{g}$/ml were 50.0%, 54.7%, 58.1%, 51.7% and 27.9%, 32.8%, 37.1%, 30.0%, respectively. And the fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution with 10$\mu\textrm{g}$/ml of heparin was the highest of all. 4. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution containing heparin with caffeine concentraton of 10, 20, 30, 40$\mu\textrm{g}$/ml were 71.4%, 74.3% and 70.6%, 70.0% and 45.7%, 47.3%, 44.1%, 41.4%, respectively. The fertilization and cleavage rate of spermatozoa fertilized in BO solution with caffeine and heparin together(70.3~74.3%) was higher than that of spermatozoa fertilized in BO solution with heparin(58.8%). 5. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution containing heparin with BSA concentration of 5, 10, 20, 30$\mu\textrm{g}$/ml were 63.6%, 62.9%, 66.7%, 60.3% and 44.1%, 43.5%, 48.5%, 42.7%, respectively. The fertilization and cleavage rate of spermatozoa fertilized in BO solution with BSA and heparin together(60.3~66.73%) was higher than that of spermatozoa fertilized in BO solution with heparin(58.8%).

• 한국가축번식학회지
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• 제15권1호
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• pp.41-47
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• 1991

미성숙 흰쥐에 있어서 과잉배란 유기시 PMSG용량이 체외수정에 의한 수정율과 수정란의 배양에 미치는 영향을 조사하고, 체외수정 및 배양용기로서 plastic mini-straw의 이용효과와 체외수정란의 이식성적을 조사한 바 다음과 같은 결과를 얻었다. 미성숙 흰쥐(체중 65~80g)에게 PMSG를 4, 10, 16 혹은 40IU를 1회 근육주사한 후 72시간에 채란한 난자 중에서 난구세포괴를 가진 정상형태의 난자와 정소상체 미부에서 채취하여 예비배양한 정자로 체외수정시켰다. 체외수정율은 PMSG 용량이 증가될수록 감소하였는데, 즉 4IU에서는 70.8%였으나, 40IU에서는 45.0%로 유의적으로 (P<0.05)저하하였다. 그러나 다정자 수정발생율은 2.3~9.7%로서 PMSG 용량에 따른 유의적인 증가는 없었다. 이 결과는 과량의 PMSG의 투여로 배란된 난자 중에서 일부는 비록 난자가 형태학적으로는 난구세포괴를 가지는 정상적인 난자일지라도 체외수정율의 저하는 정상적인 배란시간보다 조기배란으로 난자의 노화로 인하여 수정에 적합하지 않음을 제시하고 있다. 그리고 plastic mini-straw를 고안하여 straw내에서 체외수정시킨 후 66~72시간까지 배양시험한 결과 2-16와 4-16세포기까지 발달된 배의 비율은 petri dish보다 다소 우수(P<0.05)하였으며, straw용기에서 체외수정된 2세포기의 52개의 배를 7마리의 위임신 흰쥐에게 이식시켰던 바 6개의 배를 이식 받은 한 마리의 수란쥐가 2마리의 새끼를 분만하였다.

• Clinical and Experimental Reproductive Medicine
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• 제29권2호
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• pp.77-82
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• 2002

Objective : This study is to evaluate the efficacy of intracytoplasmic sperm injection (ICSI) for previous fertilization failure with conventional in vitro fetrtilization (IVF), compared with ICSI for male factor. Method: The author analyzed the 3 years of clinical experience with ICSI retrospectively, between the conventional IVF failure group (IVF failure) and male factor group (male factor). Surgically retrieved epididymal or testicular spermatozoa for ICSI were excluded. The IVF failure group was 13 cycles of 6 patients and male factor group was 30 cycles of 15 patients. Results: The fertilization rates of the IVF failure group and male factor group were 63% and 66% respectively (p=0.635). The clinical pregnancy rates of the both group were 23.1% and 26.7% (p=0.804), and that of live birth rates were 15.4% and 13.3% (p=0.858). There were no significant difference between the two groups. Conclusion: The author concluded that ICSI can overcome previous fertilization failure, with the same fertilization and clinical pregnancy rates seen in patients with male factor.

• 한국가축번식학회지
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• 제14권4호
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• pp.245-252
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• 1990

This study was carried out to investigate the factors affecting fertilization in vitro of follicular oocytes with frozen-thawed spermatozoa in Korean Native Cattle. The bovine ovaries were obtained at a slaughter house and the follicular oocytes were recovered by aspirating the follicular fluid from the visible follicles of 3~6mm. The bovine oocytes were matured in vitro for 20~24 hours in TCM-199 containing FCS and hormones. The matured oocytes were fertilized in vitro using Percoll-separated frozen-thawed spermatozoa in BO solution. The effects of dilution and fertilization media, capacitating method, concentration of inseminated sperm and time after insemination of fertilization, were observed. The results obtained are summarized as follows : 1. The fertilization rate of frozen-thawed sperm inseminated in BO solution with caffeine and heparin together(56.4%) was higher than that of sperm inseminated in BO solution with either caffeine(10.5%) or heparin(8.9%) and without both caffeine and heparin(0%)(P<0.05). 2. The fertilization rate(56.3%) of frozen-thawed sperm inseminated in BO solution with both caffeine and heparin without preincubation was higher than that of sperm preincubated(2.9%)(P<0.05). 3. The fertilization with high concentration of frozen-thawed sperm(1.4~1.8$\times$107cells/ml) in BO solution containing caffeine and heparin resulted in higher fertilization rate, 76.7%, than the low concentration of sperm(0.8~1.0$\times$107cells/ml), 32.7%(P<0.01). 4. When the oocytes were inseminated with frozen-thawed sperm in BO solution containing caffeine and heparin without preincubation, fertilization rate increased by time and the rates were 5.9, 46.0 and 59.4% at 8, 16 and 24 hours, respectively.

• 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.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.29-29
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• 2001

This study was carried out to examine, by the reverse transcription chain reaction(RT-PCR)and Immunostain assays, epidermal growth factor mRNA expression in bovine ova during oocyte maturation in vitro(0-2lh)and after fertilization in vitro(6-144hr: zygotes to blastocysts). In this study, the transcripts of EGF was detected in oocytes using primers for EGF. Transcripts for EGF mRNA was not detected in oocytes through in vitro maturation. But EGF mRNA were present after fertilization up to the 2-cell stage and the blastocyst stage. The highest mRNA levels in 4-cell stage embryos were decreased at 8cell stage and then reincreased upto morulae and blastocysts. The results of this study showed EGF mRNA are present in embryo after fertilization and this factors are involved in the regulation of bovine embryo development.

• 한국수정란이식학회지
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• 제26권2호
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• pp.117-122
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• 2011

In this study, we examined the effectiveness of in vitro fertilization of porcine immature oocytes on the embryo development of blastocysts or hatched blastocysts and the number of cells according to the in vitro fertilization conditions. In the in vitro fertilization of in vitro matured porcine oocytes, there were no significant differences between treatment groups regarding fertilization rate, blastocyst rate, and embryo development of hatched blastocysts according to the storage periods of liquid sperm of 24, 48, and 72 hours. The embryo development rate of hatched blastocysts after the fertilization according to different spermatozoa concentrations ($0.4{\times}10^5$, $1.2{\times}10^5$, and $3.6{\times}10^5$ cells/ml) showed the highest rate in the group with a spermatozoa concentration of $1.2{\times}10^5$ cells/ml; in particular, this rate was significantly higher than that in the $0.4{\times}10^5$ cells/ml group (p<0.05). The total number of blastocysts cells as well as trophectoderms (TE) that developed in each treatment group were also significantly higher in the $1.2{\times}10^5$ cells/ml group than in any other groups (p<0.05). In contrast, the embryo development rate of blastocysts according to different co-incubation periods of sperm and oocyte (1, 3, and 6 hr) was high in the 6-hour group; in particular, the rate was significantly higher than that of the I-hour group (p<0.05). Furthermore, the total number of oocytes cells and TEs that developed was significantly higher in the 6-hour group than any other group (p<0.05). In this study, the most effective treatment conditions for porcine embryo development and high cell number were found to be as follows: a sperm storage period of less than 72 hours, a spermatozoa concentration of $1.2{\times}10^5$ cells/ml, and a 6-hour co-incubation period for sperm and ooocyte.

• 한국수정란이식학회지
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• 제33권3호
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• pp.169-175
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• 2018

It has been claimed that artificial insemination (AI) of cows with frozen-thawed semen treated with commercially produced kits, Wholemom (in favour of female gender) increases the birth chance of calves with desired sex ratio by approximately 85% without decrease of pregnancy rates. Hence, this study was conducted to investigate the efficacy of wholemom kits as combined with frozen-thawed bovine semen during in vitro fertilization on the in vitro fertilization and developmental efficiency and sex ratios such as some reproductive parameters in bovine. For this, 1,737 oocytes were in vitro fertilized and developed. Agglutination effects on bovine after treatment of Wholemom kit were observed by time passage and dose respectively. To determine sex of embryos, Bovine embryo Y-specific gene primers(ConEY) and Bovine specific universal primer(ConBV) were used as multiple PCR method. Fertilization rate of wholemom-treated group was significantly lower than its of control group[66.9% (1,156/1,737) in Wholemom-treated group; 75.0% (610/813) in control group]. However, developmental rate after fertilization of both wholemom-treated and control groups were not significantly different [26.1% (404/1,156) in Wholemom-treated group; 27.4% (224/610) in control group]. Sex ratio of in vitro fertilized embryo with frozen-thawed semen treated with wholemom kit was determined by multi PCR. Female ratio in wholemom-treated group [85.4% (173/201)] was significantly higher than its of control group [47.2% (66/141)]. In conclusion, wholemom treatments of semen used in the in vitro fertilization and development of bovine oocytes provided increase in female ratio with decrease of fertilization rate.

• 한국가축번식학회지
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• 제17권2호
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• pp.133-139
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• 1993

The studies were carried out to investigate the effects of co-culture with cumulus cell, oviduct epithelial cells and uterine endometrial cells on the in-vitro fertilization and cleavage rate of porcine follicular oocytes. The ovaries were obtained from slaughtered swine. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible follicles of diameter 3~5 mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24~48 hrs in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation. The results obtained in these experiments were summarized as follows : 1. The in-vitro maturation and fertilization rate of porcine oocytes co-cultured with cumulus cells in TCM-199 meidum were 64.6%~74.5% and 37.5%~55.3%, respectively. And in-vitro fertilization rate of cumulus-enclosed oocytes(51.5%) were significantly(p<0.05) higher than cumulus-denuded oocytes(21.7%). 2. The in-vitro maturation and fertilization rate of porcine oocytes co-cultured with 1$\times$104 cells/ml, 1$\times$106 cells/ml, 1$\times$108 cells/ml and 1$\times$1015 cells/ml oviduct epithelial cells in TCM-199 medium were 53.5% and 37.2%, 61.7% and 46.8%, 54.5% and 31.8%, 42.2% and 26.7%, respectively. 3. The in-vintro maturation and fertilization rate of porcine oocytes co-cultured with 1$\times$106/ml, 1$\times$108/ml, 1$\times$1015/ml uterine endometrial cells in TCM-199 medium were 54.3% and 39.1%, 58.3% and 43.8%, 55.5% and 33.3%, and 45.7% and 30.4%, respectively. 4. When the in-vitro fertilized oocytes were co-cultured with porcine cumulus cells, ovdiduct epithelial cells and uterine endometrial cells, the development rate to the blastocyst stage was 9.5%, 10.7% and 11.8%, respectively and the rates were higher than that of control, 2.1%(p<0.05).