• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.69-76
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• 1994

The survival and pregnancy rates were compared between non-frozen embryos and cryopreserved embryos at either pronucleate or 2-4 cell stages using the freezing and thawing techniques being identical in both groups were compared with fresh embryos. 496 embryos were frozen with 1, 2-propanediol and sucrose and 117 2-4 cell stages embryos had been thawed and 79.6 and 66.0% of them respectively were survival. Clinical pregnancy rate was 19.2% for embryos frozen at the pronucleate stage and 19.0% for embryos frozen at the 2-4 cell stages while the pregnancy rate of non-frozen embryos was 21.3%. There were no significant difference in the survival and pregnancy rates of embryos frozen at pronucleate and 2-4 cell stages. The current cumulative pregnancy rate per retrieval in all cycles with frozen zygotes is 35.4 %, consid~ erably higher than observed in single transfers of embryos without cryopreservation(21.3%); predicted pregnancy rate after transfer of all frozen embryos is 43.3 %. It is concluded that firstly, the survival and pregnancy rate of cryopreserved embryos at pronucleate or 2-4 cell stages are very similar to those from their fresh embryos and non-frozen embryos and secondly, cryopreservation substantially enhances pregnancy attainment from in vitro fertilization.

• Development and Reproduction
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• v.17 no.3
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• pp.269-274
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• 2013

This study was performed to evaluate the effects of fibroblast co-culture on in vitro maturation (IVM) of prepubertal mouse preantral follicles. The intact preantral follicles were obtained from the ovaries of 12-14 day old mice and these were cultured individually in ${\alpha}$-minimal essential medium (${\alpha}$-MEM) supplemented with 5% fetal bovine serum (FBS), $100mIU/m{\ell}$ recombinant follicle stimulating hormone (rFSH), 1% insulin-transferrin-selenium, $100{\mu}g/ml$ penicillin and $50{\mu}g/m{\ell}$ streptomycin as base medium for 12 days. A total of 200 follicles were cultured in base medium co-cultured with mouse embryonic fibroblast (MEF) (MEF group) (n=100) or only base medium as control group (n=100). Survival rate of follicles on day 12 of culture were significantly higher in the MEF group of 90.0%, compared with 77.0% of the control group (p=0.021). Follicle diameters on day 6 and 8 of the culture period were significantly larger in the MEF group than those in the control group (p=0.021, p=0.007, respectively). Estradiol levels in culture media on day 4, 6, 8, 10 and 12 of the culture period were significantly higher in the MEF group (p=0.043, p=0.021, p=0.006, p<0.001 and p=0.008, retrospectively). Our data suggest that MEF cell co-culture on IVM of mouse preantral follicle increases survival rate and promotes follicular growth and steroid production.

• Journal of Embryo Transfer
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• v.26 no.4
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• pp.223-227
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• 2011

The objective of this study was to compare of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used collagenase. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (${\alpha}$-MEM) + 5% FBS + Insulin-Transferrin-Selenium (ITS) + 100 mIU/ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase II rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral folliclesin a culture of mouse preantral follicle.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.101-111
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• 2001

The present study was conducted to develop an in vitro culture system that would support bovine follicle growth from preantral to antral stage, oocyte maturation, fertilization, and embryonic development. Bovine preantral follicles (150$\pm$1.2 ${\mu}{\textrm}{m}$) surrounded by theca cell were isolated ezymatically and mechanically from ovarian cortical slides in Leibovitz L-l5 medium containing 1 mg/$m\ell$ collagenase and 0.2 mg/$m\ell$ DNase I and cultured for 25 days in the presence of different concentrations of bovine FSH and LH in $\alpha$MEM medium. The survival and growth rates of follicles cultured in the presence of FSH (10~150 ng/$m\ell$) were significantly higher than those of control group (P ＜ 0.001), but no significant differences were observed in survival and growth rates of follicles between the LH treatment groups (1~125 ng/$m\ell$) and the control. The survival (40%) and growth (244 $\pm$ 0.5 $\mu\textrm{g}$) of follicles cultured with FSH (90 ng/$m\ell$) and LH (25 ng/$m\ell$) were higher than those of control (25%, 160 $\pm$1.0 $\mu\textrm{g}$). Finally, 50% percent of healthy antral follicles were obtained, and almost 60% of them has complete meiotic division with 1st polar body (18.1%) and 10.0% have developed to the cleaved embryo and blastocyst stage. These results suggest that bovine preantral follicle with intact theca cell can grow to the antral stage using these culture conditions, and that oocytes from in vitro-matured bovine preantral follicle may acquire meiotic competence and can undergo fertilization and development.

• Toxicological Research
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• v.24 no.2
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• pp.151-159
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• 2008

Rhus verniciflua Stokes(RVS), one of traditional medicinal plants in Asia, was found to have pharmacological activities such as antioxidative and antiapoptotic effects, raising the possibility for the development of a novel class of anti-cancer drugs. Thus, potential genotoxic effects of RVS in three short-term mutagenicity assays were investigated, which included the Ames assay, in vitro Chromosomal aberration test, and the in vivo Micronucleus assay. In Ames test, the addition of RVS water extracts at doses from 313 up to 5000 mg/plate induced an increase more than 2-fold over vehicle control in the number of revertant colonies in TA98 and TA1537 strains for detecting the frame-shift mutagens. The similar increase in reversion frequency was observed after the addition of RVS ethanol extracts. To assess clastogenic effect, in vitro chromosomal aberration test and in vivo micronucleus assay were performed using Chinese hamster lung cells and male ICR mice, respectively. Both water and ethanol extracts from RVS induced significant increases in the number of metaphases with structural aberrations mostly at concentrations showing the cell survival less than 60% as assessed by in vitro CA test. Also, there was a weak but statistically significant increase in number of micronucleated polychromatic erythrocytes(MNPCEs) in mice treated with water extract at 2000 mg/kg while ethanol extracts of RVS at doses of up to 2000 mg/kg did not induce any statistically significant changes in the incidence of MNPCEs. Therefore, our results lead to conclusion that RVS acts as a genotoxic material based on the available in vitro and in vivo results.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.51-57
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• 2014

The objectives of this study were to investigate the effects of trehalose as a cryoprotectant for porcine freeze-dried spermatozoa, to find the optimal freeze-drying time and storage periods of freeze-dried spermatozoa, and to find out pronuclear formation rates, cleaved rates, and embryo development through intracytoplasmic injection of freeze-dried spermatozoa on porcine oocytes. The survival rates of spermatozoa after freeze-drying with trehalose treatment were significantly higher than those of them without trehalose treatment (p<0.05). The highest survival rates were found at 75 mM trehalose treatment. The longer storage periods after freeze-drying seemed to have a lower survival rates. Development in culture of pig by ICSI with trehalose treatment were significantly higher than those of them without trehalose treatment (p<0.05). Shorter freeze-drying time of spermatozoa was resulted in the highest cleaved rates and embryo development.

• Journal of Animal Science and Technology
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• v.49 no.2
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• pp.287-294
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• 2007

The present study was to conducted the sexing efficiency and accuracy of bovine embryo by LAMP (Loop-mediated isothermal amplification) method, the development of the biopsied embryos into re- reformation and the freezability of these blastocysts by slow-freezing and vitrification. In vivo embryos were superovaluted with gonadotropin(Antorin R-10) for 4 days combined with progestrone releasing intravaginal(CIDR) insertion in Hanwoo donors, and in vitro embryos were used blastocyst embryos at Day 7 or Day 8 after post-insemination in vitro. The biopsy of bovine embryo was carried out in a 80μl drop with Ca2+-Mg2+ free D-PBS and the viability of biopsied embryos were evaluated in IVMD medium at over 12 h culture time in 5% CO2 incubator.For embryo sexing, about five or seven blastomeres were isolated from in vitro and in vivo embryos at blastocysts with microblade. and were then subjected to LAMP. The survivability of biopsied embryos were no difference in the development rate to re-formation of blastocysts between in vivo and in vitro embryos(100% and 90% respectively). The rates of sexed embryos were compared according to two groups, the female rate was lower than that the male in the in vivo and in vitro embryos(46% vs, 54% and 40% vs, 60%, respectively). However, there were no difference in the overall sexing ratio between the two groups. The survivability of frozen-thawed sexed embryos were lower in the in vitro than in vivo embryos in the slow-freezing(Group 1) and vitrification method(Group 2), (41.7% vs. 58.8% and 57.1% vs, 77.8. respectively).

• Korean Journal of Oriental Medicine
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• v.4 no.1 s.4
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• pp.129-138
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• 1998

Angiogenesis, the formation of new blood vessels, is considered to be involved in many pathological symptoms such as diabetic retinopathy, arthritis, inflammation and solid tumour. In particular, it is thought that angiogenesis is critical for development and growth of solid tumour. Recent study shows that there is a highly significant association of microvessel density with overall survival and relapse-free survival in patients with breast tumour In this study, the inhibition effect of angiogenesis of traditional herbs used for the treatment of cancer was examined. It was found out that the extract of Agaricus blazei by boiling water is a possible inhibitor of angiogenesis. It inhibited normal developmental angiogenesis In the chorioallantoic membrane of chick embryos and also inhibited capillary-like tube formation by endotherial cells on matrigel in vitro. These results suggest that Agaricus blazei can be a potent angiogenesis inhibitor.

• Journal of Plant Biotechnology
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• v.8 no.1
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• pp.43-50
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• 2006

The goal of this research was to develop an efficient cryopreservation protocol for germplasms of yam (Diosorea batatas), that were cultivated in Korea. Comparative studies with four other cryogenic techniques and subsequent experiments for shoot regrowth were conducted. in vitro-grown shoot-apices of the D. batatas were successfully cryopreserved by encapsulation-dehydration. The maximum survival of shoot-apices could be achieved when the precultured (with 0.3 M of sucrose for one day) and encapsulated (with a 3%(w/v) Na-alginate solution) apices were dehydrated for $3.5{\sim}4\;h$ prior to direct immersion in LN (liquid nitrogen). The frequency of regrowth rate of cryopreserved apices was not decreased during 3-month storage period. The thawing method markedly affected survival of the cryopreserved apices, and thawing at $40^{\circ}C$ for 3 min produced the best results. When cryopreserved apices were post-cultured on the post-culture medium (MS), supplemented with $0.2mgl^{-1}$ of BA ($N_6$-benzyladenine) and $0.2mgl^{-1}$ of kinetin, they showed direct shooting without callusing.

• Korean Journal of Animal Reproduction
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• v.16 no.2
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• pp.117-123
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• 1992

This Study was carried out ot investigate the effects of concentration and equilibration time of cryoprotective aagents on survival rate of slowly and ultrarapidly frozen porcine embryos. The porcine embryos following dehydration by cryoprotective agents and 0.25M sucrose were slowly freezed(from 2$0^{\circ}C$ to -7$^{\circ}C$/-1$^{\circ}C$/min., from -7$^{\circ}C$ to -35$^{\circ}C$/-0.2$^{\circ}C$/min., from -35$^{\circ}C$ to -38$^{\circ}C$/-0.3$^{\circ}C$/min.) by Cell Freezer and directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water bath. Survival rate was defined as development rate to the morula and blastocyst stage after in vitro culture or by FDA test. The results are summarized as follows : 1. The survival rates of porcine embryos after slow frozen-thawing in the freezing medium of 0.25M sucrose added 2.0M glycerol, 3.0M DMSO, 2.0M propanediol or 2.0M glycerol＋2.0M propanediol was 80.6, 84.7, 75.0 or 78.8%, respectively. 2. The survival rates of porcine embryos after slow frozen-thawing in the freezing medium of 0.50M sucrose added 2.0M glycerol, 3.0M DMSO, 2.0M propanediol or 2.0M glycerol＋2.0M propanediol was 80.9, 82.4, 73.1 or 77.1%, respectively. 3. The survival rates of porcine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucroese added 2.0M glycerol, 3.0M DMSO, 2.0M propanediol or 2.0M glycerol＋2.0M propanediol was 65.3, 68.6, 63.2 or 59.9%, respectively. 4. The survival rates of porcine embryos after ultrapid frozen-thawing in the freezing medium of 0.50M sucrose added 2.0M glycerol, 3.0M DMSO, 2.0 propanediol or 2.0M glycerol＋2.0M propanediol was 67.5, 62.9, 56.9, or 62.8%, respectively. 5. The higher survival rate of porcine embryos was attained at the short period ofequilibration time(5min.) in the freezing medium added 0.25M sucrose and 3.0 DMSO compared to those of 10 or 20min. equilibration time in the same condition.