• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.92-92
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• 2003

Accurate analysis of nuclear status is needed when biopsied-blastomeres are used for embryo sexing. In this study, the nuclear status of blastomeres derived from 8- to 16-cell stage IVF bovine embryos was analyzed to evaluate the representative of single blastomere for embryo sexing. When 55 embryos were analyzed by PCR following biopsy, the coincident rate of sex determination between biopsied-single blastomere and matched blastocyst by PCR was 80 %. Karyotyping of biastomeres in 8- 16-cell stage bovine embryos was conducted to assess chromosome status of IVF embryos. To establish karyotyping of blastomeres, concentrations of vinblastine sulfate and duration of exposure time for metaphase plate induction with 8- to 16-cell stage bovine embryos were tested. The most effective condition for induction of metaphase plate (>45%) was 1.0 ug/ml vinblastine sulfate treatment for 15 h. In 22 embryos under the condition, only 8 embryos out of ten that had a normal diploid chromosome complement showed a sex-chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four out of the other 11 embryos having a mixoploid chromosomal complement contained haploid blastomere with wrong sex chromosome (18.2%). These results suggested that morphologically normal bovine embryos derived from IVF had considerable proportion of mixoploid and sex-chromosomal mosaicism which could be the cause of discrepancies of the sex between biopsied-single blastomere and matched blastocyst by PCR analysis.

• Asian-Australasian Journal of Animal Sciences
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• 제14권1호
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• pp.7-12
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• 2001

Factors Affecting the Survival of Frozen Thawed Bovine In Vitro Produced Blastocysts. The effect of some factors on the post-thaw survival of a total of 240 in vitro produced bovine blastocysts was investigated using logistic regression analysis. The explanatory variables tested were: type of culture medium before freezing (TCM 199 supplemented with BSA, BSAITS (BSA+insulin+transferrin+selenium), ECS (estrous cow serum) with or without BOEC (bovine oviductal epithelial cells), age of the blastocyst (Day 7, Day 8+9), morphological appearance before freezing (distinct=Q1 or indistinct=Q2 inner cell mass) and type of cryoprotectant (glycerol, 1.0 M or ethylene glycol, 1.6 M). The survival after thawing based on the post-thaw quality and the development after co-culture with BOEC for 24 and 48 hours. Day 7 blastocysts had an almost three times better chance of survival than Day 8+9 blastocysts. Q1, Day 8+9 blastocysts had higher odds to survive after 48 hours in culture than Q2 blastocysts (p<0.05). Blastocysts produced in BSAITS medium had the best chances of survival; however, the odds were not always significant. Blastocysts frozen in glycerol had a better post-thaw quality rating than those frozen in ethylene glycol; however, the difference in post-thaw development at culture was not significant. The relationship between post-thaw quality and post-thaw development at culture was significant (p<0.05). The developmental stage and/or age of the embryo and culture medium where development up to blastocyst takes place affect the post-thaw survival of the bovine embryos.

• 한국가축번식학회지
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• 제20권2호
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• pp.171-177
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• 1996

This study was designed to evaluate the efficacy of antioxidants and amino acid with buffalo rat liver cell(BRLC), bovine oviductal epithelial cell(BOEC) and STOC monolayers in supporting the development of in vitro matured(IVM) and in vitro fertilized(IVF) bovine oocytes. Bovine embryos developed to the 2~8 cell stage after in vitro fertilization were cultured for 5 to 6 days at 39$^{\circ}C$ in CR1aa containing antioxidants and amino acids with various somatic cells. Embryo development was examined and cell numbers of blastocysts were counted by fluorescence staining method. In experiment 1, the proportion of embryos that reached the blastocyst stage in control, catalase(250U), SOD(600U), glutathione(100$\mu$M) and taurine(2.5mM) with BRLC were 11.4, 8, 0, 16.7 and 43.4 respectively. Taurine(2.5mM) with BRLC group was significantly the highest among treatments(P<0.05). In experiment 2, in vitro development rate into blastocyst in control, catalase(250U), SOD(600U), glutathione(100$\mu$M) and taurine(2.5mM) with BOEC were 15.8, 23.5, 22.8, 28.6 and 56.9 respectively. In experiment 3, embryonic development in all treatments as control, catalase(250U), SOD(600U), glutathione(100$\mu$M) and taurine(2.5mM) added to CR1aa with STO cells were 23.5, 24.5, 17.0, 28.8 and 50.0 blastocysts. These results show that antioxidants and amino acids with somatic cells can provide a significant benefit for coculture of early bovine embryos derived from IVM and IVF.

• Asian-Australasian Journal of Animal Sciences
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• 제22권4호
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• pp.500-506
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• 2009

A modified Tris-buffered medium (mTBM) has been widely used as an insemination medium for porcine in vitro fertilization (IVF). We examined whether mTBM could be used for bovine IVF. Bovine cumulus-oocyte complexes (COCs) were cultured in a serum-free medium containing 30 ng/ml EGF for 22 h. After culture, COCs were inseminated with spermatozoa for 12 h in mTBM containing 5 mM caffeine and 10 g/ml heparin. The penetration of oocytes increased significantly (p<0.05) as the sperm concentration increased from 0.1 (30%) to 1-10 $(87-100%){\times}10^6$ cells/ml. This was significantly different from values obtained at 1 (87%) and 10 $(100%){\times}10^6$ cells/ml. However, when COCs were inseminated with spermatozoa from different bulls, the proportions (62-100%) of oocytes penetrated varied according to the bull. The proportion (18%) of oocytes penetrated was significantly (p<0.05) lower in a fertilization medium without caffeine and heparin but increased with the addition of caffeine and/or heparin to the medium, and the proportion (93-96%) of oocytes penetrated increased significantly (p<0.05) when the medium was supplemented with heparin and caffeine. In this medium, sperm penetration was first observed at 3 h after insemination. Irrespective of the presence of glucose in the fertilization medium, the proportion (93-97%) of oocytes penetrated and the proportion (83-84%) of embryos at the ${\geq}2$-cell stage cultured in a chemically defined medium were not significantly different. However, the proportion of embryos developing to the blastocyst stage was significantly (p<0.05) higher in the presence (11%) of glucose in the fertilization medium than in its absence (2%). In conclusion, the present study demonstrated that bovine oocytes penetrated in vitro in mTBM can develop to the blastocyst stage and mTBM may be used for the in vitro production of bovine embryos.

• 한국가축번식학회지
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• 제17권2호
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• pp.121-125
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• 1993

Early development of bovine oocytes fertilized in vitro in the medium with caffeine and heparin was examined in different culture systems. When the oocytes were transferred into culture medium 8 h after insemination, 12%(7/60) of penetrated oocytes cleaved to 4-cell stage 24 h after insemination. The proportions of oocytes cleaved to 80to 16-cell stage 48 h after insemination had also a to be higher in oocytes transferred into culture medium 8 h (29%) than 16 h(10%) or 24 h(4%) after insemination. 52% of the 4-cell embryos developed to morula and blastocyst stages when they were co-cultured with oviductal epithelia, whereas only 5% of embryos cultured without the epithelial cells(P<0.001). In another experiment, embryos were co-cultured with ampulla, isthmus or utero-tubal junction of oviducts. There are no significant differences in the proportions of embryos developed to morula and blastocyst stage.

• 한국수정란이식학회지
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• 제16권1호
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• pp.41-46
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• 2001

This study was carried out to evaluate the effect of culture methods on development of embryos with each developmental stage after heat shock in bovine oocytes. The results obtained were as fellows. 1. The culture method after heat shock on development of embryos was better drop-culture than co-culture. 2. The medium without amino acids were not effect of heat sock on development of embryos but it was in need of amino acid during formation of blastocyst.

• 한국가축번식학회지
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• 제22권2호
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• pp.163-169
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• 1998

본 실험은 체외 생산된 소 배반포기배에 대한 laser drilling 처리가 배의 부화율에 미치는 영향을 조사하고자 실시하였다. 그 결과는 다음과 같다. 소 수정란의 체외 발달율을 조사하였던 바, 82.3%의 난할율( 2-세포기)과 체외수정 후 배양 7일째에 32.6%의 배반포 발달율을 나타내었다. 이렇게 생산된 배반포기배에 laser drilling 효과를 조사하였던 바, 처리 후 24시간째의 부화진행율(90.0%)은 대조군(44.4%)보다 유의하게 높게 나타났다(p<0.0001). 또한, 처리 후 48시간째의 부화율(68.0%)도 대조군(33.3%)보다 유의하게 높게 나타났다. 이러한 결과는 laser drilling이 체외 생산된 소 배반포기배의 부화진행율과 부화율을 유의하게 증가시킬 수 있다는 것을 알 수 있었다(p<0.001).

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.134-134
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• 2003

Bovine embryos produced by in vitro maturation, feretilization and development was examined for presevation and transfer. The fertilization medium used BO medium with 5 mM/$m\ell$ caffeine and 10$\mu\textrm{g}$/$m\ell$ heparin and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to 1$\times$$10^{6}$ cells/$m\ell$ motile sperm during fertilization in vitro. At 8~10 hrs after insemination, the oocytes were transferred into CR1aa medium and cultured for 7 days. Embryos were preserved by vitrification method for transfer. When the embryos of early, blastocyst and expanded blastocyst stages were frozen-thawed, the proportions of embryos with normal morphology 83.6, 88.1 and 85.2%. (중략)

• 한국수정란이식학회지
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• 제9권3호
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• pp.221-227
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• 1994

This study was conducted to examine the condition of in vitro culture system and the viability after embryo transfer of in vitro matured-in vitro fertilized (IVM-IVF) bovine embryos. The in vitro development to the blastocyst stage was enhanced by supplying bovine serum albumin(BSA) to co-culture medium with bovine oviduct epithelial tissue(BOET) compared with that in medium supplemented with fetal bovine serum(FBS) (41.2% vs. 26. 3%, P<0.05). After transfer of IVM-IVF blastocysts into the uterine horn of recipient females (Aberdeen Angus), one was pregnant to term and produced a head of male Korean native calf. These results confirm that the in vitro development of IVM-IVF bovine embryos is affected with different protein source in co-culture with BOET, and IVM-IVF embryos can develop to term after in vitro culture and embryo transfer.

• 한국가축번식학회지
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• 제22권2호
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• pp.153-161
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• 1998

This study was conducted to examine the viability of nuclear transfer bovine embryos following embryo transfer. Donor embryos were treated with nocodazole to arrest their cell-cycle-stage at mitotic(M) phase. After releasing from nocodazole blastomeres were separated and transferred into the enucleated oocytes(BC), or cultured in medium with aphidicolin. Freshly cleaved blastomeres within 1.5h after cleavage(AC) and non-cleaved ones up to 3h after releasing from nocodazole(NC) were transferred into the enucleated oocytes. Blastocysts derived from nuclear transfer were transferred to Day 7~8 recipient cows. Some blastocysts were vitrified and thawed before embryo transfer. Developmental rates to the blastocyst stage were higher in AC(18.1%, P<0.05) than BC(8.6%) and NC(5.1%). Blastocyst development slightly enhanced with aphidicolin(1~2$\mu\textrm{g}$/ml) treatment(16.9~22.6%) compared to non treated control(11.1%). Survival rate fo vitrified nuclear transfer embryos after thawing was 75%(24/32). Twnety-three vitrified nuclear transfer embryos and 3 fresh ones were transferred to 23 recipients, 6 heads were pregnant and 1 male calf(24 kg) was born from a recipient cow recevied one vitrifiedthawed nuclear transfer embryo at 277 days after embryo transfer. This result suggests that the nuclear transfer embryos can developed to term after vitrification andembryo transfer.