• Journal of Plant Biotechnology
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• 제2권1호
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• pp.29-33
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• 2000

To develop an efficient propagation method for yew tree, zygotic embryos were cultured under various conditions. When dissected embryos were cultured on GA$_3$ containing media, the highest germination frequency was observed on WPM medium contaning 1.0 mg/L GA$_3$. For germination of the embryos, two different conditions were compared; culturing embryos with endosperm (Method I), and 2) culturing embryos only (Method II). Maximum germination was achieved in 0.5 mg/L GA$_3$ when embryos with endosperm were cultured on the media. Of the media tested, White and WPM medium were the most suitable on germination of embryos. The abnormality of yew embryos found was observed when it cultured on GA$_3$ or culture media. About 40% of the precociously germinated embryos could be developed into full seedlings. Seedlings contained taxol in high quantity (535 $\mu\textrm{g}$/g dry weight). In vitro techniques will be sewed as a useful tool for the development of transformed root cultures and biosynthesis studies.

• 한국가축번식학회지
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• 제19권4호
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• pp.259-264
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• 1996

This study was carried out to investigate on the survival rates and in vitro developmental rates of bisected bovine embryos were by manipulator and micropipette. Bisected embryos were co-cultured in 20% FCS(v/v)＋TCM-199 media containing hormones, oviductal epithelial cells and cumulus cells 0 to 72 hrs after bisection. Survival rate and in vitro fertilization rate were defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The in vitro developmental rate of biseciton embryos co-cultured in 20% FCS＋TCM-199 medium containing PMSG, hCG, PMSG＋hCG, hCG＋$\beta$-estradiol 0 to 20 hrs and 20 to 40 hrs were 36.7, 26.7, 33.3, 40.0, and 30.0, and 30.0, 33.3, 30.0, 26.7, and 26.7%, respectively. The survival rate of bisection embryos co-cultured in TCM-199 medium containing hormones was significantly higher than that of non co-culture(25.0%). 2. The in vitro developmental rates of bisection embryos co-cultured in 20% FCS＋TCM-199 medium containing oviductal epithelial cells 4 to 5 hrs and 20 to 24 hrs were 40.0 and 33.3%, respectively. The survival rate of bisection embryos co-cultured in TCM-199 medium containing oviductal epithelial cells was significantly higher than that of non co-culture(25.0%). 3. The in vitro developmental rates of bisection embryos co-cultured in 20% FCS＋TCM-199 medium containing cumulus cells 4 to 5 hrs and 20 to 24 hrs were 43.3 and 36.7%, respectively. The survival rate of bisection embryos co-cultured in TCM-199 medium containing cumulus cells was significantly higher than that of non co-culture (25.0%).

• 한국수정란이식학회지
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• 제7권2호
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• pp.81-88
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• 1992

Cryopreservation of mouse embryos biopsied at 4-cell stage was investigated by ultrarapid freezing. Four-cell embryos were obtained from ICR mice on 55h after hCG injection. Zona pellucida of the embryos were partially dissected with a cutting pipet, and then single blastomeres were biopsied from the embryos followed by incubation in $Ca^2$+ and $Mg^2$+-free M16 medium for 30min. Biopsied embryos cultured for lh or 15h were frozen by ultrarapid freezing method using 3M DMSO or 5M glycerol as a cryoprotectant, respectively. The developmental rate of biopsied embryos after ultrarapid freezing and thawing to blastocysts was 81 % in the group of biopsied embryos cultured for lb and 98% in the group of biopsied embryos cultured for 15h, respectively. When biopsied embryos after ultrarapid freezing and thawing were transferred to the uteri of pseudopregnant recipients, normal live young were born. These results suggest that this freezing method can efficiently cryopreserve biopsied mouse embryos.

• 한국가축번식학회지
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• 제20권2호
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• pp.179-190
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• 1996

Sexing and developing from splitted embryos which were fertilized in vitro implicate a possibility of production of the superior and sex controlled individuals. This study was carried out to investigate the production of transferable late blastocysts from in vitro fertilized embryos and to analyze sex by chromosome analysis from same embryos. In results, the ratio of cleavage and fertility of bovine follicular oocytes matured in vitro was 90% in co-cultured with granulosa cells. The competence of embryonic development from in vitro matured and fertilized bovine oocytes was 38% in co-cultured with bovine oviductal epithelial cells. To produce a lot of transferable embryos, therefore, the best conditon of culture system was co-cultured with granulosa cells for immature bovine oocytes and then co-cultured with bovine oviductal eptithelial cells for matured and fertilized oocytes. In chromosome analysis, 93% of in vitro fertilized embryos were very important aspect in chromosome preparation from bovine embryos such as duration of colcemid treatment, weakening of zona pellucida, methods of hypotonic treatment and fixation treatment.

• Clinical and Experimental Reproductive Medicine
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• 제19권2호
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• pp.125-131
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• 1992

The possible effect of human follicular fluid(hFF) on the growth and development of fertilized oocytes and embryos is important because the fallopian tubes are exposed to FF after follicular rupture and the processes of fertilization and embryo cleavage occur inside the fallopian tubes. Previously, it was suggested that human FF might adversely affect on the development of early mouse embryos. In order to investigate the effect of hFF on the development of embryos, early mouse embryos were cultured in media containing various protein sources as bovine serum albumin(BSA), fetal cord serum(FCS) and FF. And we evaluated the development of early mouse embryos in terms of the morphology, cleavage rate, and cell count of blastcysts. There were no significant differences in the morula and blstocyst formation rates of 2-cell mouse embryos cultured in the media containg three different protein sources and three different concentrations of FF. The blastocyst formation rate of 1-cell mouse embryo cultured in FF group was significantly higher than that cultured in BSA group(P<0.05). The morula and blastocyst formation rates of 2-cell mouse embryos of the group cultured in the media containing FF were comparable with those of other two groups, in addition, the cell count of blastocysts of FF group in the 2-cell embryo culture was higher than those of BSA group and HCS group(P<0.01), and this finding was also noted in 1-cell embryo culture. There was no difference in the morula and blastocyst formation rates of the 2-cell mouse embryos cultured in the media containing different concentrations of FF. These results suggest that mature human follicular fluid has no inhibitory activity on the development of early mouse embryos even in high concentration and may be a good protein source which is positively associated with the development of mouse embryos in vitro especially in 1 cell embryo culture.

• Reproductive and Developmental Biology
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• 제30권4호
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• pp.307-311
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• 2006

The purpose of this study was to determine effects of oxytocin and $interleukin-1{\alpha}$ on in vitro development of bovine embryo cultured with endometrial epithelial and stromal cells isolated from bovine uterus. The expressions of COX-2 mRNA in bovine endometrium were also studied. When embryos were cultured with epithelial cells, the rate of blastocysts was significantly (p<0.05) higher in embryos treated with oxytocin than that of control group. The rate of hatched blastocysts was also significantly (p<0.05) higher in embryos treated with oxytocin than those of two control groups. On the other hand, when the embryos were cultured with stromal cells, the rate of blastocysts were significantly (p<0.05) higher than those of groups treated with $IL-1{\alpha}$, oxytocin and control with stromal cells than that of control group without stromal cells. The rate of blastocysts hatched were also significantly (p<0.05) higher in group treated with $IL-1{\alpha}$ than those of control group without stromal cells and oxytocin group. In another experiment, COX-2 gene was expressed in embryo group treated with oxytocin during the co-culture of embryos with epithelial cells. In contrast, COX-2 mRNA was expressed in group treated with $IL-1{\alpha}$ when the embryos were cultured with stromal cell. This result shows that oxytocin and $IL-1{\alpha}$ were stimulate embryo development in vitro when embryos were cultured with epithelial and stromal cells, and can affect the development of bovine embryos in the uterus.

• 한국수정란이식학회지
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• 제20권2호
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• pp.113-121
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• 2005

This study was conducted to investigate the effects of different hexoses (glucose, mannose, galactose and fructose) on in vitro development of parthenogenetic embryos in the pigs. When the parthnogenetic embryos were cultured in medium with concentrations of 5mM glucose or 1mM galactose, the rates of embyos developed to morula and blastocyst stages were significantly higher than those in another culture conditions (P<0.05). However, high concentration of galactose inhibited development to morula and blastocyst stages. Addition of hexoses at early stage of porcine parthenogenetic embryos were effective for in vitro development. Especially, the embryos cultured in medium with glucose at early stage were effective for development to 2-cell $(72\%)$ and blastocyst $(19\%)$ stages compared with embryo cultured without glucose. From the present results, it is suggested that development of porcine parthenogenetic embryos can improve in medium with 5mM glucose. The concentration of 1mM galactose was also effective for development of porcine parthenogenetic embryos. It also show that parthenogenetic embryos cultured with glucose at early stage can improve in vitro development.

• Asian-Australasian Journal of Animal Sciences
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• 제12권1호
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• pp.15-21
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• 1999

The aim of the present study was to determine the effect of paternal sex chromosome on early development of buffalo embryos fertilized and cultured in vitro. Embryos were produced in vitro from abattoir derived buffalo oocytes. The cleaved embryos were cocultured with buffalo oviductal epithelial cells and evaluated on day 7 under the phase contrast microscope to classify development. The embryos which reached the morula/blastocyst stage were fast developing, the embryos which were at 16-32 cell stage were medium developing and the embryos below 16 cell stage were slow developing. The embryos which showed some fragmentation in the blastomeres or degenerated blastomeres, were degenerating. Sex of emberyos (n=159) was determined using PCR for amplification of a male specific BRY. 1 (301 bp) and a buffalo specific satellite DNA (216 bp) fragments. The results thus obtained show that 1) X and Y chromosome bearing sperms fertilize oocytes to give almost equal numbers of cleaved XX and XY embryos, 2) male embryos develop faster than female embryos to reach advanced stage and 3) degeneration of buffalo embryos is not linked with the paternal sex chromosome. We suggest that faster development of males is due to differential processing of X and Y chromosome within the zygote for its activation and / or differential expression of genes on paternal sex chromosome sex chromosome during development of buffalo embryos fertilized and cultured in vitro which may be attributed to a combination of genetic and environmental factors.

• Reproductive and Developmental Biology
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• 제28권1호
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• pp.13-20
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• 2004

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS, and primary fibroblast cultures were established in TCM-199 with 10% FBS. After maturation, expanded cumulus cells were removed by vigorous pipetting in the presence of 0.3% hyaluronidase. The matured oocytes were dipped in D-PBS plus 10% FBS＋7.5 $\mu\textrm{g}$/ml cytochalasin B and 0.05 M sucrose. The reconstructed oocytes were electrically fused with donor cells in 0.3 M mannitol fusion medium. After the electofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. On the other hand, the NT embryos with porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6∼8 day at $39^{\circ}C, 5% CO_2$ in air. In caprine-bovine NT embryos, the cleavage(2-cell) rate was 36.8% in confluence and 43.8% in serum starvation. The developmental rate of morula- and blastocyst-stage embryos was 0.0% in confluence and 18.8% in serum starvation. In caprine-porcine NT embryos, the cleavage(2-cell) rate was 76.7% in confluence and 66.7% in serum starvation. The developmental rate of morula and blastocyst stage embryos was 3.3% in confluence and 3.0% in serum starvation, and no significant difference was observed in synchronization treatment between donor cells. In caprine-bovine NT embryos, the cleavage(2-cell) rate of cultured donor cells was 30.8% and 17.6% in 5∼9 and 10∼14 passage(P＜0.05). The developmental rate of morula and blastocyst stage embryos were significantly higher(P＜0.05) in 5∼9 passage(23.1%) than in 10∼14 passage(0.0%) of cultured donor cells. In caprine-porcine NT embryos, the cleavage rate was significantly higher(P＜0.05) in 5∼9 passage(86.7%) than in 10∼14 passage(50.0%) of cultured donor cells. The developmental rate of morula and blastocyst stage embryos were 3.3 and 0.0% in 5∼9 and 10∼14와 passage of cultured donor cells. In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer, 33.9% in in vitro fertilization and 28.1% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than in vitro fertiltzation(26.9%) and parthenotes(37.4%).

• Asian-Australasian Journal of Animal Sciences
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• 제2권4호
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• pp.595-598
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• 1989

Bovine embryos/ova obtained from in-vitro fertilization were either co-cultured on a monolayer of bovine cumulus cells or cultured in medium alone. Embryos/ova co-cultured with cumulus cells developed to 8-cell (30.9%), morula (29.8%) and blastocyst stages (26.6%) after 3-4, 5-6, and 7-8 days of culture, respectively, while embryos/ova cultured in medium alone failed to develop beyond 8-cell (0-13.3%), morula (0-1.5%) and blastocyst stages (0%). The results of this study demonstrated the beneficial effect of cumulus cells on the development of bovine embryos.