• Journal of Embryo Transfer
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• v.33 no.4
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• pp.343-348
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• 2018

Although the efforts to establish fish embryonic stem cells (ESCs) have been made for a long time, derivation of authentic ESCs that possess pluripotency is still difficult suggesting a need for the stepwise optimization of the methods to establish fish ESCs. Primary culture of the blastomeres from the embryos at blastula stage is a critical step for establishing continuous ESC lines. Here, we evaluated the effects of temperatures and basal media on primary culture of blastula embryo-derived blastomeres in marine medaka (Oryzias dancena). The blastomeres were isolated from the blastula embryos and cultured in various conditions designed by the combination of 4 temperatures including $28^{\circ}C$, $31^{\circ}C$, $34^{\circ}C$, and $37^{\circ}C$ and 2 basal media including Dulbecco's modified eagle's medium (DMEM) and Leibovitz's L-15 medium (L15). With the exception of a case cultured in L15 at $31^{\circ}C$, the rate of primary cell adherence reached 100% when the blastomeres were cultured over $31^{\circ}C$. The period for primary adherence was significantly shorter in the groups cultured in $34^{\circ}C$ and $37^{\circ}C$ than in the ones in $28^{\circ}C$ and $31^{\circ}C$. The proportion of subculture was significantly high in the group cultured in DMEM at $31^{\circ}C$ compared to the other groups. Collectively, we demonstrated that the culture in DMEM at $31^{\circ}C$ was effective to primary culture of the blastomeres derived from blastula embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.11
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• pp.1541-1546
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• 2011

This study was carried out to examine the influence of minimum essential medium (MEM) vitamins supplementation to in vitro maturation medium and in vitro culture medium on the development of porcine embryos. Porcine embryo development was investigated following cultivation in both in vitro maturation and culture medium with the supplementation of MEM vitamins (0, 0.1, 0.2 and 0.4%) using immature oocytes collected from the ovary of prepubertal gilts. Embryo development was observed and the total cell number in each blastocyst generated under the culture conditions was quantified following supplementation of the medium. The embryonic development rate of the blastocyst and hatched blastocyst was higher, but not significantly so, when 0.4% MEM vitamins were supplemented to the in vitro maturation medium of the porcine oocyte. Interestingly, the total number of cells in the blastocyst was significantly higher in the in vitro maturation MEM vitamins supplemented group compared to either the untreated group or the group which had MEM vitamins supplemented to both in vitro maturation and in vitro culture medium simultaneously (p<0.05). Therefore, the supplementation of 0.4% MEM vitamins to the in vitro mature medium has a beneficial effect on the embryonic development of in vitro produced blastocysts derived from the immature porcine oocytes.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.43-50
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• 1998

The effect of oxygen tension on embryonic development in co-culture was evaluated from the standpoint of the reduction of dissolved oxygen concentration by the oxygen consumption of feeder cells. Three co-culture systems using bovine oviductal epitherial cells (BOEC), African green monkey kidney cells (Vero cells) or buffalo rat liver cells (BRLC) have been compared in terms of development of bovine embryos derived from oocytes matured and fertilized in vitro. Among the co-cultured embryo, Vero cells su, pp.rted the highest developmental rate (29%) and the other two showed the similar rates. When the co-cultures were incubated in three different oxygen tension such as 5, 10, 20% oxygen atmosphere, embryos co-cultured with Vero cells at 10%-O2 resulted in the highest percentage of development. From the measurement of oxygen consumption of feeder cells, BRLC consumed 1.38　10-10 mg-O2/min/cell which was higher than 0.94　10-10 and 0.26　10-10mg-O2/min/cell for Vero cells and BOEC, respectively. Based on the oxygen consumption data, the phenomena of optimum oxygen tension required in embryo development in vitro has been analyzed, and we suggested that gas phase oxygen concentration, oxygen consumption rate of feeder cells and the number of feeder cells should be considered for the design of optimal co-culture system for effective fertilization of embryos in vitro.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2000.11a
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• pp.57-57
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• 2000

• Journal of Life Science
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• v.12 no.1
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• pp.19-21
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• 2002

The pathway of embryos formed anther culture in herbaceous peony was influenced by addition of 2,4-D. MS medium with 2,4-Dichlorophenoxy acetic acid (2,4-D) alone did not arise direct embryogenesis, but was proliferate callus. Embryos through calli were produced on medium containing 0.2 mg/1 zeatin or without growth regulators. Direct embryogenesis was obtained from MS basal medium. However, after the anthers were cultured on medium with 0.1 mg/1 2,4-D, 3 g/1 AC, 30 g/1 sucrose, 2 g/1 gelrite for 40 days. Its efficiency (32.3 %) was markedly improved when anthers cultured on medium without 2,4-D. Embryo morphology was also affected by the 2,4-D used in medium. The induction of normal embryos with two cotyledons was higher in the embryos formed through direct embryogenesis than those formed callus. The embryos formed from calli were mainly showed abnormal embryo with one, three, four cotyledons or hors and bowling pin type.

• Journal of Plant Biotechnology
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• v.34 no.3
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• pp.181-187
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• 2007

Orostachys japonicus A. Berger is a Perennial herbaceous plant which has been traditionally used as an anti-inflammatory agent to treat hepatitis and as an anticancer agent. The objective of this study was 1) to establish and proliferate in vitro plant of O. japonicus 2) to induce indirect somatic embryogenesis from O. japonicus. General calli and embryogenic calli in all ranges of 2,4-D and BA combination, were induced and were best at 22% (embryogenic cell) in 5.0 mg/L 2,4-D and 0.5 mg/L BA combination. Embryogenic cell line was maintained by subculture at 2 week intervals and transferred to solid and liquid medium for embryo formation. In solid medium culture, globular and heart shaped embryos were observed in MS medium containing 5.0 mg/L 2,4-D and 0.5 mg/L BA combination. The number of embryos was 6.5 per 0.5 g cell, and then the immature embryos transferred to MS basal medium for embryo development. In a suspension culture of embryogenic cells, globular and heart shaped embryos were emerged in MS medium supplemented with 3.0 mg/L 2,4-D and 0.3 mg/L BA combination after 10 days of incubation. The embryo formation rate was about 33% by suspension culture. The ratio of embryo germination was 60.9%, on the other side, the root formation rate was 74.3% in 1/2 MS continuously.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.299-304
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• 1998

Despite the rapid development of assisted reproductive technologies (ART) in recent years, implantation rates after replacement of embryos into the uterine cavity remains low. Several techniques such as culture conditions based on formulations of human tubal fluid and various ART techniques as GIFT, ZIFT, TET have been adopted in recent years to improve embryo viability in vitro and implantation rates. Also, coculture of human IVF-derived embryos have been used in an effort to increase the number of viable embryos following IVF and to improve synchrony between the developing embryo and the uterine environment. The aim of this study was to evaluate whether the use of co culture with autologous cumulus cells has a significant beneficial effect on the development of embryos in vitro and its relation to the pregnancy rates in 120 patients with previous failed IVF-ET from September, 1995 to January 1998. We obtained the results from which significant improvement in the quality of viable embryos were observed using a coculture system with autologous cumulus cells, but pregnancy rates in this group of patients did not differ from the rate in the standard IVF group during the same period. Our study shows that a simplified short-term coculture system with autologous cumulus cells may help rescue moderate quality embryos to cleave regularly.

• Journal of Plant Biotechnology
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• v.6 no.4
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• pp.213-219
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• 2004

Zygotic embryos were excised from immature and mature seeds of the Himalayan yew, Taxus wallichiana. The embryos germinated precociously when kept in darkness for 5 weeks and developed into full seedlings within 10-12 weeks. The highest rate of embryo germination ($81\%$) was obtained in modified Lloyd & McCown' s woody plant medium containing macro and micronutrients at half strength supplemented with $1\%$ activated charcoal, which supported both the best embryonic growth ($43\%$) and seedling development ($32\%$). However, the supplementation of basal media with kinetin, thidiazuron, 6-benzyl aminopurine or $GA_3$ had no effect on the germination of the embryos. The embryos derived from immature seeds germinated but the frequency of embryonic growth was better in mature seeds. Stratification of seeds effected precocious germination of embryos. Seeds kept at $4^{\circ}C$ for 1 week germinated earlier and at a higher frequency irrespective of the stage of seed maturity, while the germination rate declined with prolonged cold treatment for 1 month at that same temperature. Analysis of taxanes in germinating seedlings revealed that root tissues contained high levels of taxol, 10-deacetyl-baccatin ill and baccatin ill as compared to shoots. Thus embryo culture technique appears to overcome the lengthy dormancy requirement of T. wallichiana seeds.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.229-236
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• 1995

The experiments reported here take advantage of the large number of in vitro matured and in vitro fertilized(IVM /IVF) bovine oocytes which can be produced, permitting the design of controlled experiments to establish a simple defined medium for the study of early embryo requirements. A total of 1,386 IVM /IVF oocytes were used to compare a simple defined medium(KSOM) with more complex culture conditions used successfully for culture of bovine embryos but do not permit study of specific requirements. All experiments were extensively replicated factorials. In Experiment 1, KSOM was superior to Menezo B$_2$ medium in producing morulae plus blastocysts from IVM /IVF oocytes(33 vs 20%, P<0.()5). The yield of morulae plus blastocysts with KSOM was 22% and with BRLC added was 30%. In Experiment 2, (a 2x2 factorial of KSOM with or without BRLC and 0, 1 ng /ml of platelet derived growth factor, PDGF) more morulae plus blastocysts (40%) were produced in KSOM-BRLC co-culture containing 1 ng /ml PDGF than in the control KSOM(12%). In Experiment 3, there was no dose response when 0, 1 and 5 ng /ml of PDGF were added. The results with simple defined KSOM medium are sufficiently promising to indicate that specific requirements of the embryo may be examined in future studies with KSOM as a base.

• Journal of Embryo Transfer
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• v.13 no.1
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• pp.43-52
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• 1998

The embryogenesis stimulating activity(ESA) had been shown in co-culture of embryos with bovine oviduct epithelial cell(BOEC) and culture in BOEG-conditioned medium. The present study was undertaken to purify and quantify the embryotropic proteins and to determine the optimum concentration of the embryotropic protein for the proper development of embryos. In BOEC-conditioned medium, five major bands of proteins were detected(66, 53, 40, 32 and 24 kDa) by SDS-PAGE. From these proteins, 288pg of protein that had a 32kDa molecular weight was purified by gel filtration column and perfusion chromatography ion-exchange column. When purified protein was supplemented to the in vitro culture media at various concentrations in protein-free media, 2.5$\mu$g /ml supplement group showed significantly higher rates of embryo development into morula /blastocyst stages than other groups(p<0.05). In conclusion, we purified 32kDa protein from BOEC-conditioned medium and this protein showed optimum embryogenesis stimulating effect at 2.5$\mu$g /ml.