• 한국가축번식학회지
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• 제20권2호
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• pp.163-170
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• 1996

The effect of several potential antioxidants and amino acids were examined as a means of increasing the development of in vitro matured and in vitro fertilized oocytes into morulae or blastocysts. 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 varing concentraton of the antioxidants and amino acid in a gas phase consisting of 5% CO2, high humidified air. At 5~6 days, embryo developments were reduced, and embryos were fixed and stained with Hochest 33342 DNA stain to facilitate counting of cells. In experiment 1, the proportion of embryos developed to morulae and blastocysts in CR1aa containing 1mM, 2.5mM taurine (22.6% and 20.4%) was slightly higher than those of 0, 5 and 10mM Taurine (5.7, 5.7 and 3.9%, P<0.05). In experiment 2, addition of glutathione did not improve blastocyst development (P>0.05). In experiment 3, concentations of superoxide dismutase(SOD) ranging from 300 to 1,000 U did not affect the propotion of embryos developing into blastocysts (P>0.05). In experiment 4, addition of 250 U catalase(38.5%) was slighty higher than those of 0, 500 and 1,000U. In experiment 5, the proportion of embryo developed beyond morula stage in CR1aa with taurine plus EDTA was slighty higher than other treatments(15.7, 26.0 and 29.2%), there were no significantly increases in cell number among treatments(P>0.05). These results are indicating that antioxidants and amino acids can increase the proportion of embryos that develop into morulae and blastocysts, but did not increas in cell number of blastocysts.

• 한국수정란이식학회지
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• 제19권2호
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• pp.165-172
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• 2004

본 연구는 보다 안정된 돼지 체외수정란 생산시스템 확립을 목적으로 체외성숙배양액 mNCSU-37, mNCSU-23 및 TCM-199 가 각각 미성숙 난포란의 체외성숙, 체외수정, 체외발달에 미치는 영향을 구명하기 위하여 수행하였다. 도축돼지의 난소에서 채취한 COCs를 10% pFF가 포함된 각각의 성숙배양액에서 최종동도가 1${\times}10^5/m{\ell}$ 의 농도로 체외수정 시킨 다음, NCSU-23 에서 체외발달을 유도한 결과는 다음과 같다. 1. TCM-199에서 성숙시킨 난자가 mNCSU-37 이나 mNCSU-23에서 성숙시킨 것보다 난핵포 붕괴율과 핵 성숙율에서 다소 낮은 경향을 보였으나 유의적인 차이는 아니었다. 2. 정자 침투율은 성숙배양액간 유의적인 차이를 나타내지 않았으나 웅성정핵 형성율은 mNCSU-37 에서 성숙시킨 난자가 88.0%로 TCM-199 에서 성숙시킨 것의 71.1%보다 유의적으로 높았다.(p<0.05). 3. 난분할율은 mNCSU-37(52.3%)이나 mNCSU-23(53.7%)에서 성숙시킨 난자가 TCM-199(43.1%)에서 성숙시킨 난자보다 유의적으로 높았다(p<0.05). 배반포발달율도 mNCSU-37이나 mNCSU-23에서 성숙시킨 것이 TCM-199에서 성숙시킨 것보다 다소 높았지만 유의적인 차이는 아니었다. 이상의 결과로 보아 돼지 미성숙 난포란 유래의 체외수정란 생산을 위한 체외성숙배양액으로 TCM-199보다 mNCSU-37이나 mNCSU-23이 적합한 것으로 사료된다.

• 한국수정란이식학회지
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• 제11권3호
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• pp.249-258
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• 1996

To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28$^{\circ}C$ and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH, 1 $\mu$g /ml at 39$^{\circ}C$ under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5$\mu$m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P

• Asian-Australasian Journal of Animal Sciences
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• 제11권4호
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• pp.398-403
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• 1998

The objective of the present study was to examine the developmental rates, and the stage of development in relation to time since fertilization, of in vitro produced buffalo embryos. Buffalo cumulus-oocyte complexes obtained from slaughterhouse ovaries were matured and fertilized in vitro. The fertilized oocytes (n = 248) were then co-cultured with buffalo oviductal epithelial cells and evaluated for the developmental stages on Days 2, 4, 6, 7, 8, 9 and 10 post-insemination. The peak of 4-cell stage embryos was observed on Day 2 (63.7 %), whereas Day 4 was marked by peaks of 6-8-cell stage embryos (20.9%) and 16-cell stage embryos to early morulae (50%). On Days 6, 7, 8, 9, and 10 post-insemination, 49.5, 48.3, 38.3, 33.8 and 33.4% embryos were found to be at morula/compact morula stages, 8.8, 12.5, 25.4, 6.0 and 1.2% at early blastocyst/blastocyst stages, 0, 6.8, 7.2, 15.3 and 2.0% at expanded blastocyst stage and 0, 1.6, 4.8, 19.3 and 38.5% hatching/hatched blastocyst stages, respectively. The peaks of early blastocyst/blastocyst, expanded blastocyst and hatching/hatched blastocyst stages were observed on Days 8, 9 and 10, respectively. The percentages of oocytes which initially became arrested and subsequently degenerated were 3.6, 4.8, 10.4, 14.5, 21.3 and 24.5% on Days 4, 6, 7, 8, 9 and 10 post-insemination, respectively.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.257-257
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• 2004

The study was carried out to investigate the effects of morphology, reproductive cycle, incubation time and activation of oocytes on in vitro maturation of cat oocytes and development of IVM/IVF embryos. 1. When recovered from ovaries collected at different stages of the reproductive cycle (inactive, follicular and luteal stage), the developmental rates of oocytes to GV and MI stage were 72.5% and 27.5%, 57.5% and 7.5%, 62.5% and 17.5%, respectively. (omitted)

• 한국가축번식학회지
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• 제18권4호
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• pp.257-263
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• 1995

소 체외수정란의 체외배양 체계는 현재 완전히 확립되지는 않은 상태로서 8∼16 세포기 발육억제현상, 수정란의 파편하 현상, 성장지연 등 여러 가지 문제점들이 현 배양체계에서 나타난다. 그러나 hepler cell들과의 공배양에 의해 이러한 문제점들은 상당히 극복되며 또한 체외발생의 촉진 및 공배양된 수정란의 이식에 의해 임신율도 향상된다. 현재 소 체외수정란의 공배양에는 난관상피세포가 가장 널리 이용되나 이러한 시스템은 몇가지 문제점이 있다. 즉, primary culture를 확보하기 위하여 신선한 조직을 주기적으로 채취해야 하며 따라서 난관채취에 시간이 소요되고, 불편하며 또한 공배양에 이용되는 세포들이 균일하지 않은바 난관상피세포에 따라 배 발생 촉진작용에 변이를 나타내기도 한다. 그러나 확립된 cell line을 이용할 수 있다면 이러한 난관상피세포의 이용에서 나타나는 문제점들이 해결될 수 있다. Buffalo Rat Liver cell은 이러한 목적에 이용될 수 있는 cell line 중의 하나로서 이들은 여러 가지 성장인자(growth factor)를 분비하는 것으로 알려져 있다. 따라서 본고에서는 소 체외수정란의 공배양을 위한 BRL(Buffalo Rat Liver)cell의 이용성, 이용방법 및 이용에 있어서 고려하여야 할 요인들에 대하여 살펴보고자 한다.

• 한국가축번식학회지
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• 제21권4호
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• pp.389-396
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• 1997

Arrest in embryo development during in vitro culture has been reported in various mammals. Although some cause of the arrest have been suggested, little is known of the way that can overcome the arrest using in vitro culture system. The antioxidant, $\beta$-mercaptoethanol($\beta$-ME), has been shown to play an important role in embryo development. This study was designed to examine the effect of $\beta$-ME on the developing boving embryos produced in vitro by IVM and IVF. To select a, pp.opriate concentration of $\beta$-ME during whole culture period (7 days), various concentrations (10, 50 and 100$\mu$M) of $\beta$-ME were added to the CZB medium and their effects was significantly higher in 100$\mu$M of $\beta$-ME. The effects on development of embryos cultured with and without somatic cells to blastocyst stage were greater in FCS treatment (56.6 and 29.3%) than in BSA treatment(25.5 and 12.8%). We also evaluated the effects of $\beta$-ME addition on the blastocyst formation when embryos at different stages were exposed to 100$\mu$M $\beta$-ME. $\beta$-ME promoted increased development of embryo to blastocyst stage and the effect was greater in 6-cell to morula embryos than in embryos fewer than 4-cells at the initiation of treatment. The results suggested that $\beta$-ME can improve bovine embryo development by overcoming the arrest in early development.

• 한국가축번식학회지
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• 제20권2호
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• pp.111-117
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• 1996

본 연구는 혈청첨가 또는 무첨가에 따른 소 난포란의 체외성숙에 있어서 참가된 TGF-$\beta$1과 IGF-I이 그후의 수정 및 발생에 미치는 영향과, 이들 growth factor의 농도에 따른 8세포기 소 체외수정란의 발달에 미치는 영향을 조사하고자 실시하였다. 도축장에서 얻어진 난소로부터 채취된 난포란을 20% FBS가 첨가 또는 첨가되지 않은 TCM-199에 TGF-$\beta$1, IGF-I 또는 TGF-$\beta$1＋IGF-I을 각각 10ng/ml 첨가하여 38.5$^{\circ}C$에서 24시간 배양하여 체외성숙을 유기하였다. 성숙된 난자를 1$\times$106/ml 정자농도로 수정후 24시간에 glucoserk 첨가되지 않은 CZB 배양액으로 옮겨 48시간 배양한 다음, TCM-199＋20%FBS에서 96시간 추가배양하였다. 본 연구에서 혈청이 첨가된 난포란의 체외성숙배양액에 첨가된 growth factor들은 수정후의 배분할 및 배발생에 영향을 미치지 않았다. 혈청이 첨가되지 않은 경우에서는 TGF-$\beta$1의 첨가는 배분할 및 배발생율을 향상시켰다(P<0.05). 한편 TCM-199＋20%FBS에 5, 10ng/ml의 TGF-$\beta$1 및 5, 10, 50, 100ng/ml의 IGF-I을 각각 첨가후 8세포기 체외수정란을 배양한 결과, 10ng/ml TGF-$\beta$1의 첨가는 배반포기로의 발생율을 향상시켰다(P<0.05). 결론적으로, 혈청이 포함되지 않은 소 난포란의 체외성숙 배양액, 또는 수정란의 체외배양액에 10ng/ml TGF-1의 첨가는 배반포기로의 발생율을 향상시킨다.

• Asian-Australasian Journal of Animal Sciences
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• 제19권4호
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• pp.495-499
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• 2006

In our previous studies, we demonstrated that Vascular Endothelial Growth Factor (VEGF) enhances bovine oocyte maturation and early embryonic development in serum supplemented media. In this experiment, to determine the synergistic effect of VEGF with serum components on early embryonic development in vitro in cattle, 1 mg/ml polyvinyl-alcohol (PVA) was replaced with foetal bovine serum (FBS) in maturation and culture media. Bovine oocytes were matured in Synthetic Oviduct Fluid (SOF) supplemented with PVA, PVA+5 ng/ml of VEGF, FBS, or FBS+VEGF. Fertilized oocytes were cultured in the same conditions for 8 days. The development of embryos was examined at 48 h post- insemination and on days 6, 7 and 8. The results were analyzed using repeated measures two- factor ANOVA, in which the effects of VEGF and serum were assigned as two factors. The development rate to 4- to 8-cell embryos at 48 h was significantly higher in the PVA+VEGF group than in the PVA group (44.7% and 31.5%, respectively). However, the highest development rate to 4- to 8-cell embryos was obtained from the FBS+VEGF group (58.8%). On day 8, the blastocyst rates were higher in the PVA+VEGF (22.8%), FBS (32.1%, p<0.05) and FBS+VEGF (42.1%, p<0.05) groups than in the PVA group (17.1%). Two- factor ANOVA of the development rates indicates that VEGF had a significant effect, but had no synergistic effect with serum components on early embryonic development. The results of the present study demonstrate that VEGF improves the in vitro developmental competence of bovine oocytes and/or embryos independent of the effect of serum components.

• 한국수정란이식학회지
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• 제18권3호
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• pp.237-242
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• 2003

본 연구에서는 소에 있어서 수정란 이식을 다각적으로 분석하고 개선하기 위해 이식한 결과를 비교 검토하였고 그 결과는 다음과 같다. 1. 채란된 한우 난포란을 이용하여 생산된 체외수정란을 서로 다른 지역별로 이식한 바 B지역(경주)에서는 106의 수란우에 이식하여 51두(48.1％)가 수태하였고, A지역(김천) 수태율(33.8％)과 D 지역(경산) 수태율(35.3％)보다 유의하게 높았지만, C 지역(탑리)에서는 유의차가 존재하지 않았다. 따라서, 수정란이식에 있어서 이식 지역과 사육 환경, 시술자의 숙련도에 따라 수태율이 달라질 수 있다는 것을 알 수 있었다. 2. 수란우의 산차에 따른 수태율은, 미경산우 42.9％로서 경산우의 36.6％에 비해 다소 높은 수태율을 보였으나 유의적인 차이는 인정되지 않았다(P<0.05). 3. 이식 수정란의 발육단계는 중기 배반포 또는 후기 배반포로서 구분하여 수란우에 이식하였다. 중기 배반포가 45.5％, 후기 배반포 41.0％로서 중기 배반포가 후기 배반포보다 높은 수태율을 나타내었지만 유의한 차이는 인정되지 않았다. 배반포기가 생성된 날짜에 따른 수란우의 수태율은 7일째 생성된 배반포 이식시 수태율은 43.8％, 8일째 생성된 배반포 이식시 수태율은 32.9％, 9일째 생성된 배반포 이식시 수태율은 20.0％로서 7일째 배반포의 수태율 이 다른 두 처리군보다 수태율은 높았지만, 유의차는 인정되지 않았다.