• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.37-41
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• 2005

The aim of this study was to improve efficiency and quality of the production of Korean Native Cow embryos. We examined effects of ovarian morphology and maturation vessel on the development and cell number of blastocysts. The development rates to the 2-cell embryos from oocytes collected from the ovaries of different morphological statues were similar ranging between 70.3 and 84.1%. The development rate to the 8 cell- and blastocyst-stage embryos was the highest in the group without both corpus luteum (CL) and follicle. The inner cell mass (ICM), trophectoderm (TE) and total cell number (TCN) were significantly higher in the groups of follicular cyst and regressive CL than other treatment groups, and the same pattern was observed in the ICM/TCN ratio. The development rate to the 2-cell stage was significantly higher in 0.5-㎖ straw group than 0.25-㎖ straw group. However, the development rates to the blastocyst stage were similar between the dish and the straw group. There were no differences in the number of ICM and TE cells, TCN and ICM/TCN ratio of blastocysts from oocytes matured in the different vessels.

• Journal of Embryo Transfer
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• v.22 no.3
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• pp.173-178
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• 2007

돼지 정자의 성 선별에는 일반적으로 유속 세포 분석기를 이용한다. 유속 세포 분석은 DNA량의 차이에 기초하여 정자를 분리하는 기술로써 X 정자와 Y 정자를 90% 정확도로 분리할 수 있다. 그러나 이러한 유속 세포분석 기술은 정자의 손상을 야기해 정자의 기능과 수정능에 영향을 미치므로, 본 연구에서는 특정한 핵산 서열을 탐지할 수 있는 Chromogenic in situ hybridization(CISH)을 그와 비교하여 평가하였다. 유속 세포 분석을 수행하기 위해 정자를 SYBR 14와 PI로 염색하였고, histogram, dotplot, density, contour를 측정하였다. Y 염색체 특이적인 primer를 이용한 PCR로 유속 세포 분석의 정확도를 검사하였다. HRP/DAB 시스템에 기초한 CISH 분석에는 X 또는 Y 염색체에 상보적으로 결합하는 probe가 사용되었다. CISH 분석은 기존의 방법들보다 빠르고 쉬우며 비용이 적게 든다는 장점이 있다. 또한, CISH는 보다 정화한 정자의 선별을 가능하게 하는 것으로 나타났다. 본 연구에 따르면 CISH가 기존의 선별 방법들을 평가하는 기술로서만이 아니라 특정한 성별을 가진 포유동물의 생산에도 사용될 수 있을 것이다.

• Journal of Embryo Transfer
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• v.8 no.2
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• pp.151-157
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• 1993

The present study was undertaken to determine the optimal condition for parthenogenetic activation of rabbit oocytes by electric stimulation in vitro in an attempt to develop nuclear transplantation techniques for cloning mammalian embryos and animals. Freshly ovulated oocytes were collected from superovulated rabbits from 13 to 26 hrs. after hCG injection. The cumulus-free oocytes were activated parthenogetically by repeated stimuli of square direct electric pulses in O.3M mannitol solution. After applying electric stimulations of different voltages, pulse durations and pulse times, all of the oocytes were cultured in TCM-199 with 10% FCS for 96 hours in a 5% $CO_2$ incubator, and their developmental potential in vitro was examined. The higher activation rate (68.9%) was achieved at the voltage of 2.0kv/cm, the pulse duration of 60 $\mu$sec and three pulse times and the activation rate of 100% was achieved at the pulse duration of 100 and 200 $\mu$sec, the voltage of 1.5kv/cm and three pulse times. Although the higher rates of activation of oocytes were achieved at 100 and 200 $\mu$sec, none of them developed to blastocyst in vitro. The oocytes collected 18~20 hours post hCG injection showed the highest rate of activation and development to blastocyst in vitro than the oocytes collected 13~15 or 25~26 hours post hCG injection. Therefore, it can be suggested that the application of electric stimulation of 2.0kv/cm, 60 $\mu$sec and three pulse times to the oocytes collected at 18~20 hours post hCG injection would be more beneficial for the parthenogenetic activation of oocytes in rabbits.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.255-260
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• 1994

원시생식세포(primordial germ cell; PGC)는 성성숙 이후에 기능을 갖는 생식세포의 근원이 되는 세포로서, 다능성을 갖고 있는 것으로 알려져 있다. 그러므로 chimera 및 유전자 변환동물 생산을 위해 널리 사용되어 온 배아주(embrynic stem; ES)세포를 대신할 다른 세포계라고 생각되어져 많은 연구가 진행되고 있다. 본 실험은 체외배양을 통하여 원시생식세포의 증식과 확립을 위해 배양조건을 구명하고, 또한 성장인자의 효과를 검증하기 위하여 실시되었다. 원시생식세포는 12.5일째의 ICR 생쥐태아의 원시생식선 융기조직으로부터 추출하였으며, DMEM + 20% FCS + nucleosides + antibiotics로 조성된 sDMEM 배양액을 사용하여 mitomycin C로 전처리한 되먹임세포단층(feeder layer)위에서 체외배양하였다. bFGF 및 LIF를 20, 40ng/ml농도로 각각 또는 함께 첨가하여 성장인자의 효과를 검토하였다. 원시생식세포는 성에 따라 유의적인 colony 형성율을 보였고(♂:1.9 colonies / genital ridge, ♀:1.3 colonies / genital ridge), bFGF 및 LIF의 첨가 및 첨가농도에 따라서도 유의성 있는 결과를 보였다(0.3~1.9 colonies / genital riege). 그러나 3회 이상 계대배양을 할 경우, 원시생식세포의 colony를 4% prarformaldehyde로 20분간 고정한 후, tris-maleate buffer(pH 9.0)로 10분간 3회 세정하였다. Fast Red로 염색을 실시한 결과, 대부분의 colony가 염색반응을 보여 다능성을 갖는 원시생식세포의 colony임이 입증되었다. 그러나 대부분의 colony가 3회 이상의 계대배양시 생종율이 급격히 떨어지는 것을 감안하면, 또 다른 미지의 성장인자나 보다 적절한 배양조건이 요구된다고 생각된다.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.109-115
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• 2002

The present study was conducted for the production of transgenic cloned cows those secrete human lactoferricin into milk by somatic cell nuclear transfer (NT). To estimate detrimental effects of gene transfection on transgenic cloned embryo production, development rates of NT embryos were compared between transfected and non-transfected cumulus and ear fibroblast cells. An expression plasmid for human lactofericin (pbeta-LFC) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human lactoferricin target gene into a pcDNA3 plasmid. Two bovine somatic cell lines (cumulus cell and ear fibroblast) were established and transfected with the expression plasmid using a liposomal transfection reagent, Fugene6 as a carrier. Cumulus cell and ear fibroblast were transfected at the passage of 2 to 4, trypsinized and GFP-expressing cells were randomly selected and used for somatic cell NT. Developmental competences (rates of fusion, cleavage, and blastocyst formation) in bovine transgenic somatic cell NT embryos reconstructed with non-transfectecd cells were significantly higher than those from transfected cells in cumulus cell and ear fibroblast (P＜0.05). This study indicated that transfection of done. cell has detrimental effect on embryo development in bovine transgenic NT.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.101-108
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• 2002

Human Prourokinase (proUK) offers potential as a novel agent with improved fibrin specificity and, as such, may offer advantages as an attractive alternative to urokinase that is associated with clinical benefits in patients with acute peripheral arterial occlusion. For production of transgenic cow as human proUK bioreacotor, we conducted this study to establish efficient production system for bovine transgenic embryos by somatic cell nuclear transfer (NT) using human prourokinase gene transfected donor cell. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human prourokinase target gene into a pcDNA3 plasmid. Cumulus cells were used as donor cell and transfected with the expression plasmid using the Fugene 6 as a carrier. To increase the efficiency for the production of transgenic NT, development rates were compared between non-transfected and transfected cell in experiment 1, and in experiment 2, development rates were compared according to level of GFP expression in donor cells. In experiment 1, development rates of non-transgenic NT embryos were significantly higher than transgenic NT embryos (43.3 vs. 28.4％). In experiment 2, there were no significant differences in fusion rates (85.4 vs. 78.9％) and cleavage rates (78.7 vs. 84.4％) between low and high expressed cells. However, development rates to blastocyst were higher in low expressed cells (17.0 vs. 33.3％), and GFP expression rates in blastocyst were higher in high expressed cells (75.0 vs. 43.3％), significantly.

• Journal of Embryo Transfer
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• v.10 no.1
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• pp.33-44
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• 1995

Genetic changes from improving female's reproductive rate through in vitro fertilization of large number of oocytes were studied. The breeding scheme employed was multiple ovulation and embryo transfer of juveniles and adults. Both balanced and unbalanced matings were examined for the four closed progeny population sizes, 10$^3$, 10$^4$, 105, 106. In balanced matings, all selected sires and dams were mated to each other(cross-classified mating) while unbalanced matings allowed selected dams and sires mated partially, eg. unbalanced matings allowed averages of .5 and .25 progeny per each mating. Various numbers of selected sires and dams were also examined in both balanced and unbalanced matings. In all mating schemes, selection of males and females was restricted to he one from each fullsib family to reduce the rate of inbreeding. The model calculations were deterministic and accounted for the effects of selection and inbreeding on loss of the genetic variation in succeeding generations. Balanced rectangular mating schemes, where more donors were selected than sires, resulted in larger selection responses than balanced square mating schemes, where equal numbers of sires and donors were selected, and unbalanced rectangular mating. The first round selection responses from the balanced rectangular matings of juvenile MOET, eg. number of progeny per mating equals 2 with 10 sires selected, were 1.192, 1.406, 1.580 and 1.735 times larger than the first round selection responses from the balanced square mating schemes for the given four progeny population sizes, 10$^3$, 10$^4$, 105 and 106, respectively. Similar results were obtained in adult MOET breeding schemes. However, balanced square matings gave greater selection responses than the unbalanced rectangular matings.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.171-179
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• 1997

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplant embryos. The oocytes collected from slaughterhouse ovaries were matured for 24 h and then enucleated and cultured to allow cytoplasmic maturation and gain activation competence. And then the donor embryos were treated for 12 h with 10 $\pi$g /ml nocodazole and 7.5 $\pi$g /ml cytochalasin B to synchronize the cell cycle stage at 26 h after the onset of culture. The blastomeres were transferred into the perivitelline space of the enucleated nocytes and blastomeres and oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. The age of the recipient(30 vs 40 h) had no significant effect on the fusion rates(82.4 vs 82.1%) and the developmental rates to morula /blastocyst(9.8 vs 11.0%). Effect of Nocodazole treatment on the donor cell cyle synchronization to improve the developmental rates of bovine nuclear transplant embryos was significantly higher than control group(21.4 vs 10.1%, p<0.05). Significant differences were in the percentage of fusion rates(72.9,77.1vs 61.9%) in three types of fusion medium(PBS(+), mannitol and sucrose, p<0.01). The developmental rates of bovine nuclear transplant embryos appeared to be highest in mSOF medium under 5% 0$_2$ condition, but no significant differences were found when compared with TCM199-BOEC and mSOF under two different oxygen ratio(5 and 20%).

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.43-49
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• 1997

The objective of this study is to determine developmental pattern and cell allocation to the ICM and TE in haploid and diploid of embryos following parthenogenetic activation and in vitro fertilization. The incidence of development to blastocyst was lower in the combined treatments of ethanol stimulation and cytochalasin B as compared to the control. However, the combined ethanol stimulation and cytochalasin B treatment (diploid) accelerated development to the blastocyst as compared to the ethanol treatment alone (haploid). Significantly reduction in the average number of total cells and ICM was observed in the parthenotes alone as compared to fetilized embryos, but those of combined ethanol stimulation and cytochalasin B treatment embryos were significantly increased as compared to ethanol alone embryos. These results suggested that the ploidy affects preimplantation developmental patters and cell allocation to the ICM and TE in the porcine.

• Journal of Embryo Transfer
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• v.15 no.2
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• pp.121-128
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• 2000

This study was conducted to enhance the efficiency of embryo transfer and embryos produced in vitro according to select good quality blastocyst among in vivo or in vitro produced embryos. After middle blastocys(MB) and late blastocyst(LB) stages embryos that developed in vitro for 7∼8 days were treated hypertonic solution(380 mOsm or 600 mOsm) and the rate of development to hatched blastocyst(HB) was examined. The results obtained were summarized as follows: The proportion of embryos developed to HB was higher than that of control regardless of hypertonic solution treatment. However, it was related to culture time in hypertonic solution The propotrion of embryos developed to HB of morphologically recovered embryos in 380 nOsm solution ws higher than that of morphologically shrinked embryos. However, treatment time was not significantly different the rates of HB development. Especially, the proportion of embryos developed to HB of morphologically recovered embryos of the 30-min-treated group was significantly higher than that of control (p<0.05). The results suggest that hypertonic solution treatment should enhance the efficiency for criterion of transferable blastocyst quality.