• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2006년도 The 6th Intermational Symposium on Developmental Biotechnology
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• pp.106-106
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• 2006

• 한국수정란이식학회지
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• 제24권2호
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• pp.89-95
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• 2009

Successful in vitro embryo production heavily relies on the normal maturation and fertilisation of oocytes. We examined the normal and abnormal fertilisation of zebu cattle oocytes matured in vitro. Immature cumulus oocyte complexes (COCs) from zebu cattle ovaries at slaughter were matured in vitro (IVM) for 24 h. The oocytes were either fixed, stained and examined for nuclear changes or fertilised in vitro (IVF) with Percoll-separated, heparintreated spermatozoa (1.0 ${\times}$ $10^6$/mL) of zebu (n = 7) and crossbred bulls (n = 7). After 18 h of sperm-COCs co-incubation at $39^{\circ}$C with 5% $CO_2$ in humidified air, the presumptive zygotes were fixed, stained and examined for pronuclei. The number of oocytes retrieved per ovary was 5.4 ${\pm}$ 0.7. The percentage of matured oocytes was 73.0. The difference in motility of spermatozoa before and after Percoll seperation was significant (p<0.001). The percentages of normal and abnormal fertilisation (polyspermia and oocytes with one pronucleus) varied significantly depending on individual bulls (p<0.05). A protocol for IVF of IVM oocytes in Bangladeshi zebu cattle is developed. A future study may elucidate the capacity of such IVM-IVF oocytes to develop to the blastocyst stage for transfer to surrogate mother.

• 한국수정란이식학회지
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• 제9권1호
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• pp.1-6
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• 1994

This experiment was investigated the effect of presence of granulosa cells from follicles of different size on bovine oocyte maturation, cleavage and development to late stage. The nuclear and cytoplasmic maturation of oocytes in the IVM-IVF system are critical for subsequent embryo development. Granulosa cells when the co-cultured with oocytes may interact with cumulus-oocytes complexes and influence the development competence of the oocytes. Granulosa cells from medium (2~6 mm) and large(>1O mm) size follicles were recovered by aspiration, washed 3 times by centrifugation at 500 x g for 5 min. and used for co-culture at a concentration of 2~3 x 106 cells/mi. The oocytes were matured in vitro (IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g/ml FSH, 10 $\mu$g/ml LH, 1 $\mu$g/ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro (IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro (I VC) with bovine oviductal epithelial cells for 7 to 9 days. The assessment of maturation revealed that Grade J oocytes showed significantly(P

• 한국가축번식학회지
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• 제24권3호
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• pp.299-309
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• 2000

본 연구는 한우 체외수정란의 정확한 성판별을 위한 PCR의 적정조건을 검토하였고, Y 염색체 특이적 DNA primer(BOV97M : 141bp)와 소 특이적 DNA primer(216bp)를 이용한 2단계 PCR 방법으로 체외수정란의 발육속도에 따른 성판별율과 성비를 검토하였다. PCR 기법을 이용한 한우 체외수정란의 성판별은 Y 염색체 특이적 DNA primer와 소 특이적 DNA primer로 정확히 성을 판별할 수 있었으며, 웅성 수정란의 경우 141bp와 216bp에서 band가 나타났고, 자성 수정란의 경우 216bp에서 band가 나타났다. 한우 체외수정란의 경우 성판별 정확도를 높이기 위하여 투명대의 제거가 필요하며, 수정란의 DNA 추출방법에 따른 Y 염색체 특이적 band의 출현율은 Proteinase K와 반복 동결융해방법이 각각 45.2 및 53.3%로 나타났다. DNA의 양에 따른 성판별율은 zona free인 경우는 2$\mu$1와 10$\mu$1에서 각각 97.2%와 92.2% 였으며, zona intact의 경우는 12$\mu$1와 13$\mu$1에서는 각각 91.5%와 93.6%로서 zona free 수정란의 경우 2$\mu$1, zona intact의 경우 13$\mu$1의 DNA 양으로 성판별이 가능하였다. 한편, PCR 기법에 의한 한우 체외수정란의 성판별율은 96.0%였으며, 정확히 성판별을 할수 없는 비율은 4.0%였다. 성판별 수정란의 자성과 웅성의 비율은 46.3%와 49.7%로서 성비는 1.1:1이였으며, 발육단계간 자웅성비는 커다란 차이가 인정되지 않았으며, 발육단계가 진행될수록 성판별 정확도가 증가하였다.

• 한국수정란이식학회지
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• 제31권3호
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• pp.153-159
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• 2016

(-)-Epicatechin gallate (ECG) is a polyphenol compound of green tea exhibiting biological activities, such as antioxidant and anticancer effects. To examine the effect of ECG on porcine oocytes during in vitro maturation (IVM), oocytes were treated with 0-, 5-, 15-, and $25{\mu}M$ ECG. After maturation, we investigated nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). After 42 hours of IVM, the $5{\mu}M$ group exhibited significantly increased (p< 0.05) nuclear maturation (89.8%) compared with the control group (86.1%). However, the $25{\mu}M$ group observed significantly decreased (p< 0.05) nuclear maturation (83.5%). In intracellular maturation assessment the 5-, 15-, and $25{\mu}M$ groups had significantly increased (p< 0.05) GSH levels and decreased ROS levels compared with the controls. The 5- and $15{\mu}M$ group showed significantly increased (p< 0.05) embryo formation rates and total cell number of blastocysts after PA (18% and 68.9, 15% and 85.1 vs. 12% and 59.5, respectively) compared with controls. Although the $25{\mu}M$ group observed significantly lower blastocyst formation rates after PA (27.6% vs. 23.2%) than control group, the $5{\mu}M$ group showed significantly increased blastocyst formation rates after PA (37.2% vs. 23.2%) compared to the control group. Furthermore, the $5{\mu}M$ group measured significantly increased blastocyst formation rates (20.7% vs. 8.6%) and total cell number after IVF ($88.3{\pm}1.5$ vs. $58.0{\pm}3.6$) compared to the control group. The treatment of $5{\mu}M$ ECG during IVM affectively improved the porcine embryonic developmental competence by regulating intracellular oxidative stress during IVM.

• 한국수정란이식학회지
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• 제11권2호
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• pp.151-158
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• 1996

This study was carried out to investigate the effect of equilibration time, sucrose concentration and age of embryo on survival and developmental rates of bovine IVF expanding blastocysts frozen-thawed by direct transfer method. The bovine oocytes were collected from 2~5mm follicles, matured for 20~24hrs in 5% $CO_2$incubator and then fertilized with frozen-thawed semen. Expanding blastocysts at day 7, 8, 9, 10 and 11 after IVF were frozen in 1.8M ethylene glycol(EG). Survival and hatching rates of frozen-thawed IVF embryos were examined. The results were as follow ; Survival and hatching rate of TVF expanding blastocysts after 10, 20, 3Omin exposure in 1.8M EG were 100,0,90.9, 47.1, 85.0, 75.0 and 62.5% respectively. Survival rates of IVF expanding blastocysts frozen with 1.8M EG and various concentration(0, 0.25, 0.5, 1M) of sucrose were 73.3, 25. 0, 16.7, 9.1% respectively. Survival and hatching rates of IVF expanding blastocysts frozen-thawed according to age of embryo(Day 7, 8, 9,10, 11) were 86.1, 84.8, 79.3, 61.4, 51.3, 74.2, 76.9, 71.7, 63.0 and 65.0% respectively. In conclusion, the age of the embryo(Day 7, 8) is very important for the successful freezing of IVF bovine embryos and 1.8M ethylene glycol not containing sucrose may be effective cryoprotectant for direct transfer method.

• Asian-Australasian Journal of Animal Sciences
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• 제14권9호
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• pp.1342-1351
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• 2001

Over the years, the embryo transfer industry has grown from the simple collection & transfer of embryos into an advanced field of embryo biotechnology. Currently a large demand exists for bovine oocytes and early embryos in both research and commercial settings. Bovine embryos can now be produced in-vitro. Primary oocytes collected from antral follicles of abattoir - obtained ovaries can be induced to undergo the maturation process. In-vitor maturation system, however must ensure that the resulting oocyte is capable of undergoing normal fertilization and yields a zygote competent of developing to term after embryo transfer. Sperm preparation for IVF has improved with the use of heparine. The use of co-culture system has proved beneficial in circumventing the developmental block in IVM/IVF bovine embryos.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.128-128
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• 2003

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2006년도 The 6th Intermational Symposium on Developmental Biotechnology
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• pp.46-46
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• 2006

• 한국가축번식학회지
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• 제24권3호
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• pp.311-317
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• 2000

본 연구는 체외에서 한우 난포란의 성숙과 수정후 배의 체외발생에 있어서 온도충격의 영향이 처리온도 및 처리시간에 미치는 효과를 검토하였다. 1. 체외에서 생산된 4~8 세포기 배의 배반포로의 배발달율에 있어서 온도충격의 적정온도는 41$^{\circ}C$, 처리시간은 30초였다. 2. 체외성숙 후의 온도충격이 체외성숙 전의 온도충격보다 수정란의 분할율을 유의하게 상승시켰다(p<0.05). 3. 체외성숙 후와 배양 5일째 배에 각각 온도처리를 하였을 경우 비처리군보다 배반포의 배발달율이 향상되었다.