• Korean Journal of Poultry Science
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• v.28 no.2
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• pp.125-133
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• 2001

The goal of this paper was to examine the qualify zygote-acquiring method for in-vitro culture and the in-vitro culture method of the acquired zygote from a technological perspective. We have reported the results on the introduction of foreign DNAs using the described culturing method. After performing in-vitro and surrogate eggshell culture on a zygote acquired from the abdomen of a hen, 25.8% hatchability was acquired. After microinjecting foreign DNAs into the acquired zygote and performing in-vitro and surrogate eggshell culture using the same method, 13.1∼11.7% hatchability was acquired. Having compared the developments of the control subjects and the experimental subjects, the viability of the experimental subjects on the 4∼5th day of culturing was much lower compared to that of the control subjects. This is a result that shows that the microinjection process of foreign DNAs might have a negative effect on the existence of the embryo; therefore, various technical attempts should be made to minimize such negative effects. Having microinjected foreign DNAs into the zygote of a hen to produce transgenic chickens, 3 transgenic founders were Produced and 70 G1 progeny were produced as a result of the progeny test that had been performed to the present.

• Journal of Plant Biotechnology
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• v.3 no.1
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• pp.19-25
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• 2001

Induction of somatic embryogenesis from Brazilian eggplant variety F-100 was studied in response to four auxin types. NAA, at the optimal concentration of 54 $\mu\textrm{m}$, was the only one that resulted in the induction of somatic embryos in either leaf and cotyledon explant and, at murk lower intensity and frequency, in hypocotyl and epicotyl explants. The optimal temperatures for embryo induction were 28 and 35$^{\circ}C$ for cotyledon and leaf explants. Incubation at 22$^{\circ}C$ caused a significant reduction both in the frequency and intensity of induction. This system was used to study the effects of position and orientation of the tissue on the culture medium as well as of antibiotics and explant co-cultivation with Agrobacterium on the efficiency of somatic embryo induction. The intensity of embryo induction was greater in the midsections of cotyledons relative to apical and basal regions, when the abaxial surface was in contact with the culture medium. The presence of antibiotics resulted in approximately 40-60% reduction of embryo induction relative to control explants, which originated 335$\pm$26.6 embryos. Co-cultivation with Agrobacterium before treatment with antibiotics caused a more drastic reduction (80-99%). Ampicilin treatment after cocultivalion with Agrobacterium caused the least inhibitory effect, allowing the production of 60 embryos/explant.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.9
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• pp.1152-1158
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• 2010

In differentiating human embryonic stem (d-hES) cells there are a number of types of cells which may secrete various nutrients and helpful materials for pre-implantation embryonic development. This study examined whether the d-hES could function as a feeder cell in vitro to support mouse embryonic development. By RT-PCR analysis, the d-hES cells revealed high expression of three germ-layered differentiation markers while having markedly reduced expression of stem cell markers. Also, in d-hES cells, LIF expression in embryo implantation-related material was confirmed at a similar level to undifferentiated ES cells. When mouse 2PN embryos were cultured in control M16 medium, co-culture control CR1aa medium or co-cultured with d-hES cells, their blastocyst development rate at embryonic day 4 (83.9%) were significantly better in the d-hES cell group than in the CR1aa group (66.0%), while not better than in the M16 group (90.7%)(p<0.05). However, at embryonic days 5 and 6, embryo hatching and hatched-out rates of the dhES cell group (53.6 and 48.2%, respectively) were superior to those of the M16 group (40.7 and 40.7%, respectively). At embryonic day 4, blastocysts of the d-hES cell group were transferred into pseudo-pregnant recipients, and pregnancy rate (75.0%) was very high compared to the other groups (M16, 57.1%; CR1aa, 37.5%). In addition, embryo implantation (55.9%) and live fetus rate (38.2%) of the d-hES cell group were also better than those of the other groups (M16, 36.7 and 18.3%, respectively; CR1aa, 23.2 and 8.7%, respectively). These results demonstrated that d-hES cells can be used as a feeder cell for enhancing in vitro and in vivo developmental potential of mouse pre-implantation embryos.

• Journal of Embryo Transfer
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• v.4 no.1
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• pp.28-34
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• 1989

These experiments were carried out to determine the effect of cell stage in embryo bisection on the sub-Sequent in vitro and in vivo development in mouse. The embryos of ICR mouse were microsurgicaily bisected at 2-cell, 4-cell, 8-cell, morula and blastocyst stage using a microsurgical blade attached a micromanipulator. These demi-embryos without zona pellucida were cultured up to blastocyst stage and transferred to pseudopregnant mice, and the development of these demi-embryos was compared with the results of intact embryos of the corresponding cell stage. The successful rate of mouse embryo bisection at 4-cell stage (59.0%) was significantly (p <0.05) lower than those at 8-cell (75.6%), 2ce11 (80.7%) or morula stage (84.8%), and highest at blastocyst stage (95.7%). When the bisected embryos without any damage from microsurgery were cultured in vitro up to blastocyst,the in vitro de'velopment of demi-embroys bisected at morula to blastocyst was 91.6 to 95.3%, which was similar to the culture result of intact embryos of corresponding stage. However, the in vitro development of demi-em-bryos bisected at 2- to 8-cell stage was signiflcantiy (p <0.05) lower.The post-transfer implantation rate of demi-embryos developed in vitro to eu-blastocyst were 19.6 and 25.4% in demi-embryos bisected at morula and blastocyst stage,respectively and not significantly (P <0.05)different from the result of intact embryos of the same stage. However, the implantation rates of demi-embryos bisected at 2- or 8-cell stage were significantly (P <0.05) lower than the result from the intact embryos of the corresponding stage.

• Korean Journal of Plant Tissue Culture
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• v.24 no.5
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• pp.291-294
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• 1997

The experiments were carried out to determine the optimum conditions for the direct embrogenesis from the cotyledon derived zygotic embryo culture of Paeonia lactiflora Pall. Different peony tissues derived from zygotic embryos were cultured on MS medium with and without 2,4-D. Somatic embryos were formed from the cotyledons cultured on the medium without 2,4-D. The somatic embryogenesis from cotyledons was promoted in the growth regulator-free MS medium containing 1.65~3.3 g/L $NH_4NO_3$ and 30~40 g/L sucrose. The maximum frequency (80.0%) of somatic embryo formation was obtained from the cotyledons excised from zygotic embryos that cultured on MS medium containing 3.3 g/L $NH_4NO_3$. Epicotyl and roots were elongated from a somatic embryo by adding 0.3 mg/L GA$_3$ in the medium or the cold treatment at 4$^{\circ}C$ more than three weeks at 4$^{\circ}C$.

• The Korean Journal of Zoology
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• v.31 no.3
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• pp.207-217
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• 1988

A myotrophic protein that seemed to he eseentiai for the hision of chick embryonic myoblasts in culture was isolated from chick embryo extrad and was found to be identical or at least similar to the iron-transporting protein, transferrin. Embryo extract seemed to contain, in addition to this myotrophic protein, a heat stable protein that inhibits the fusion of myoblasts. Iron seemed to he necessary for myoblasts to fuse and it was supposed that the role of the myotrophic protein m myoblast fusion is to supply iron to the cell. The numher of the myotrophic protein receptors on myoblast surface membrane decreased immediately after the start of myoblast fusion, supposedly due to the decreased need of iron after the fusion once commenced. It was estimated that endocytosis of myotrophic protein took about 10 minutes and one recycling about 2 hours. The accumulation of iron in myoblasts continued linearly with cultre time and endocytosis of the myotrophic protein occured at a constant rate.

• Korean Journal of Veterinary Research
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• v.29 no.1
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• pp.61-68
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• 1989

This study was carried out to determine suitable selection factors for recipients and embryos which could improve pregnancy rates following bovine embryo transfer. The experiment included 52 surgical transfers from February, 1985 through June, 1986 performed on Kyungpook Breeding Center in southern Korea. The pregnancy rate was highest when recipients were in estrus within 6 hours before the donor to 12 hours after the donor (78.3% versus 50% for recipients in estrus earlier or later). Pregnancy rates were acceptable following culture under field conditions for up to 17 hours. More recipients over 15 months of age (76.1%) remained pregnant than those under 15 months (66.7%). Embryos transferred during the months from February to July resulted in higher pregnancy rates than those transferred during the remaining 6 months (77.3% versus 57.1%). Transferrable embryos were classified A (best) to C (worst); those graded A or B resulted in significantly higher pregnancy rates than those graded C (81.8% and 73.3% versus 25.0%, p<.05). Pregnancy rates among recipients of the Korean native breed tended to be higher than among Holstein recipients (100% versus 71.1%). Similarly, when the embryo was transferred to the right uterine horn, pregnancy rates tended to be higher than when it was transferred to the left (81.3% versus 65%). Pregnancy rates did not differ according to the stage of development of the embryo; they were for morulae, tight morulae, blastocysts, and advanced blastocysts, respectively: 75.0%, 66.7%, 75.0%, and 77.4%.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.1
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• pp.77-84
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• 2003

Objective : The aim of this study was to evaluate the influence of three different media on preimplatation embryo development and the expression of Bcl-2, Mcl-1, Bax, and Bok in mouse. Materials and Methods: Two-cell embryos were retrieved from ICR female mice (4 weeks old) at 48 hr after hCG injection and cultured in Ham's F-10, HTF, and G1.2 media. The developmental rate of 2-cell embryos was evaluated from 24 hr to 72 hr after culture. RT-PCR was performed for the detection of Bcl-2, Mcl-1, Bax, and Bok gene expression. Results: The rates of morula and blastocyst in HTF and G1.2 media (88%, 98.1%) were significantly higher than those in Ham's F-10 media (39.6%) at 48 hr. Likewise, the rates of hatching and hatched blastocyst in HTF and G1.2 media (21.9%, 52.9%) were higher than those in Ham's F-10 media (3.5%) at 72 hr. Bcl-2 and Bax mRNAs were highly detected in embryos cultured in Ham's F-10 when compared in embryos cultured in HTF and G1.2. In contrast, the expression of Mcl-1 and Bok was not significantly different. Conclusion: These results show that HTF and G1.2 culture media increase the rate of blastocyst formation and stimulate Bcl-2 and Bax gene expression in mouse preimplantation embryos.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.161-169
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• 1997

This study was conducted to compare the insemination time of bovine oocytes and determine the effects of glucose(1.5 mM) on the development of bovine embryos at early cleavage stage. Oocytes were matured for 24 h, followed by exposure to sperm and cultured in modified Tyrode's media drops or with bovine oviduct epithelial cell monolayer prepared in TCM199(BOECM). Insemination time and culture system were varied in each experiment. In experiment 1, to investigate the developmental capacity of bovine embryos after different time of exposure to sperm, bovine ova and sperm were co-incubated for 18, 30 or 54 h, respectively. The development to blastocysts of 30 and 54 h insemination groups were significantly higher(P<0.05) than 18 h group, and in case of blastocysts of cleaved embryos, 30 h group were significantly higher(P<0.05) than other groups. In experiment 2, we investigated the effect of glucose on early bovine embryos. After 18 h insemination, in vitro fertilized oocytes were separated following 3 groups ; G+0, C+24 and C+48. Oocytes of G+0 group were cultured in glucose added Tyrode's medium after fertilization, oocytes in C+24 and C+48 groups were cultured in glucose free Tyrode's medium after fertilization. After 24 h culture, G+24 group was moved to glucose added medium. All oocytes of 3 groups were moved to BOECM after 48 h culture. The rates of cleavage and development to blastocysts in G+0 group were significantly lower than other groups. In experiment 3, we determined the effects of glucose exposure from 8 to 20 h after insemination on the cleavage and development of oocytes. The oocytes in glucose added group had high capacity of cleavage and further development. This study shows that in bovine oocytes, the optimal exposure to sperm is 30 h and glucose exposure to bovine one-cell embryos is detrimental to their first cleavage and further development in vitro but there has no evidence of detrimental effect of glucose(1.5 mM) exposure to bovine embryos over the two-cell stage in vitro.

• Journal of Plant Biotechnology
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• v.7 no.1
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• pp.75-80
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• 2005

Eleutherococcus senticosus (also called Acanthopanax senticosus), belonging to Araliaceae family, has been used as an important medicinal woody plant. Mature seeds of Eleutherococcus senticosus have rudimentary (extremely immature) zygotic embryos and require a long-term stratification for about 18 months to induce germination. Here, through the methods of endosperm removal and other exogenous treatments, we investigated the factors for inducing rudimentary embryos by in vitro culture, Rudimentary zygotic embryos in seeds were at globular to heart-shaped stage at about $250{\mu}m$ in length just after harvest of fruits. When the seeds without testa were cultured on 1/2 MS (Murashige and Skoog 1962) medium, they did not germinate regardless of medium and sucrose concentrations but the removal of endosperm tissue markedly stimulated the growth of rudimentary zygotic embryos. The embryo reached ear-lier maturation, once when the endosperm surrounding the rudimentary embryos was removed. Rudimentary zygotic embryos developed cotyledons within 3 weeks of culture after endosperm emoval. However, post-mature zygotic embryos failed to germinate though they were morphologically normal, indicating another dormancy of embryos. $GA_3\;(2.0\;\cal{mg/L})$ and/or charcoal ($0.2\%$) treatment rapidly enhanced the germination of zygotic embryos. These results suggest that E. senticosus seeds have double dormancy; i. e. morphological rudimentary dormancy influenced by surrounding endosperm and physiological dormancy after post-maturation of zygotic embryos. Based on the above findings, we established the rapid germination of rudimentary zygotic embryos by in vitro culture of excised seeds with endosperm removal and $GA_3$ treatment.