• Journal of Embryo Transfer
• /
• v.30 no.3
• /
• pp.195-200
• /
• 2015

The objective of this study was to evaluate the toxicities of permeable cryoprotectants and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Toxicities of permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), Glycerol, and 1,2-PROH were investigated using a murine embryo model. Female $F-{_1}$ mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to among DMSO, EG, Glycerol, and 1,2-PROH. Embryo development was evaluated up to the blastocyst stage. The total cell count of blastocysts that were treated with DMSO and Glycerol at the 2-cell stage was significantly lower than that were treated with EG ($81.1{\pm}15.1$), 1,2-PROH ($88.0{\pm}21.1$) or the control ($99.9{\pm}21.3$) (p<0.001). On comparison of four cryoprotectant treated groups, the DMSO and Glycerol treated group showed a decreased cell count compared with the EG and 1,2-PROH treated group (p<0.05). Both DMSO ($14.7{\pm}1.3$), EG ($12.1{\pm}1.1$), Glycerol ($15.2{\pm}1.8$), and 1,2-PROH ($11.5{\pm}1.3$) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control ($6.5{\pm}0.7$, p<0.0001). In addition, the DMSO or Glycerol treated group showed more apoptotic cells than the EG or 1,2-PROH treated group (p<0.001). The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to among DMSO, EG, Glycerol, and 1,2-PROH at room temperature. When comparing four permeable cryoprotective agents, EG and 1,2-PROH appeared to be less toxic than DMSO and Glycerol at least in a murine embryo model.

• Journal of Embryo Transfer
• /
• v.29 no.3
• /
• pp.241-248
• /
• 2014

This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Two to eight cell embryos were obtained from oviducts of mated $F_1$ hybrid female mice superovulated by PMSG and hCG. Two-step EG, DMSO and 4-step EG, DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow-cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in EG and DMSO was significantly higher than 4~8 cell (65.4% versus 61.2%, 81.1% versus 72.5%) (p<0.01, p<0.01), but the development rates of 4~8 cell embryos in EG and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4~8 cell embryos in EG were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in EG(77.0% versus 64.4%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The development rate from morular to hatching blastocyst, however, was sinificantly higher in EG than in DMSO during 48 hr (p<0.01). The survival rate of 4~8 cell embryo was 62.5% in EG and 73.3% in DMSO. The development rates of embryo in EG were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. The survival rate of embryo in 2 cell stage was higher than in 4~8 cell stage, and EG appears more effective cryoprotectant than DMSO because EG showed better development rates of embryos in 2 and 4~8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

• Journal of the Korean Society of International Agriculture
• /
• v.23 no.5
• /
• pp.546-551
• /
• 2011

Chrysanthemums, often called mums or chrysanths, belong to the genus Chrysanthemum, which includes about 30 species of perennial flowering plants in the family Asteraceae. We extracted DNA from Dendranthema grandiflorum ('Smileball') to construct a simple sequence repeat (SSR)-enriched library, using a modified biotin-streptavidin capture method. GS FLX (Genome Sequencer FLX System which provides the flexibility to perform the broad range of applications) sequencing (at the 1/8 run specification) resulted in 18.83 mega base pairs (Mbp) with an average read length of 280.06 bp. Sequence analyses of all SSR-containing clones revealed a predominance of di-nucleotide motifs (16,375, 61.5%) followed by tri-nucleotide motifs (6,616, 24.8%), tetra-nucleotide motifs (1,674, 6.3%), penta-nucleotide motifs (1,283, 4.8%), and hexa-nucleotide motifs (693, 2.6%). Among the di-nucleotide motifs, the AC/CA class was the most frequently identified (93.5% of all di-nucleotide types), followed by the GA/AG class (6.1%), the AT/TA class (0.4%), and the CG/GC class (0.03%). When we analyzed the distribution of different repeat motifs and their respective numbers of repeats, regardless of the motif class, of 100 SSR markers, we found a higher number of di-nucleotide motifs with 70 to 80 repeats; we also found two di-nucleotide motifs with 83 and 89 repeats, respectively, but their product lengths were within optimum size (297 and 300 bp). In future work, we will screen for polymorphisms of possible primer pairs. The results will provide a useful tool for assessing molecular diversity and investigating the population structure among and within Chrysanthemum species.

• Korean Journal of Animal Reproduction
• /
• v.27 no.3
• /
• pp.215-223
• /
• 2003

This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) was slowly cooled to room temperature (20∼23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min 800 ${\times}$ g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0${\times}$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin B$_{12}$ , 25 mM HEPES, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 ${mu}ell$ mTBM fertilization media with 1.0${\times}$10$^{6}$ sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 ${mu}ell$ NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend the coincubation time of 6 h in 500 ${mu}ell$ TBM fertilization medium with 1${\times}$10$^{6}$ sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.

• Current Research on Agriculture and Life Sciences
• /
• v.7
• /
• pp.83-97
• /
• 1989

This study was carried out to obtain basic information necessary for aggregation and in-vitro culture of mouse embryos by treating phytohemagglutinin-p (PHA-P). The 4-, 8-cell and morula embryos were obtained from female mice of albino BALE/C, CBA and C57BL strains, those were injected 5 i.u pregenant mare serum gonadotrophin and 5 i.u human chorionic gonadotrophin to superovulation. The zona pellucidia was removed by placing the embryos in Acidic Tyrode solution containing 1.0% protease or/and 5 ug/ml PHA-P. The pairs of zona free embryos were subjected to aggregation by glassneedle in BMOC-3 containing 5 ug/ml PHA-P. The aggregation embryos were cultured in Brinster's mouse ova culture-3(BMOC-3) medium under the gas phase of 5% $CO_2$ in air $37^{\circ}C$ for 13 to 50 hours. The results obtained in this study are summarised as follows : 1. When 4-, 8-cell and morula embryos were zona-freed in acidic Tyrode solution containing 1.0% protease or/and 5 ug/ml PHA-P, and cultured in vitro to blastocysts, the 4- and 8-cell embryos showed slightly less development rates than the morula one did, and solution of 5 ug/ml PHA-P brought some higher development rate than negative control. 2. As 2, 5 or 10 ug/ml PHA-P was added to the solution to aggregate 4-, 8-cell or morula embryos, 2 ug/ml solution represented slightly lower aggregation rate than the higher levels solutions, and 4- and 8-cell embryos showed higher rates than morula one did (P<.05). 3. In respect to the development rates of aggregated embryos to morula no significant difference was found among PHA-P levels and between 4-and 8-cell embryos. With respect to those of aggregated embryos to blastocysts the different levels of PHA-P showed similar results, however, the 4- and 8-cell embryos represented higher rates than the morula one did (P<.05). 4. The mean time necessary for development of aggregated 4-, 8-cell and morula embryos to blastocysts were 38.5-40, 26-27 and 19-20hrs. Respectively in solution for aggregation. 5. The aggregation rates of embryos were 34-94%, when treated protease or/and PHA-P. Supplementation of 5 ug/ml PHA-P to the solution for aggregation showed a trend demonstrating higher aggregation rate compared to negative control, although no significance was found. However, 4- and 8-cell embryos represented significantly higher aggregation rates than the morula one did (P<.05). 6. The development rates of 4- and 8-cell embryos to morula were 52.7-84.7 and 73.8-87.2%, respectively, showing no significant difference between two cell stages. However, the aggregation rates of embryos treated with solution containing PHA-P were higher than negative control (P<.05). 7. The development rates of 4- and 8-cell and morula embryos to blastocysts were 41.7-77.7 78.7-83.0 and 0-19.2%, respectively. The rates of 4-cell embryos treated with PHA-P were significant higher than the negative control (P<.05). The 8-cell and morula embryos also showed more rates when treated PHA-P.

• Korean Journal of Soil Science and Fertilizer
• /
• v.18 no.3
• /
• pp.265-275
• /
• 1985

Present studies were carried out to investigate the distribution and formation of organic tiers contained paddy soils in Honam area characteristics to give basic informations on the effective utilization, management and improvement of the soils. The results obtained were summarized as follows; 1. The extent of organic tiers contained paddy soils in Honam area were 6.538㏊ and the amount of peat deposits were presumed about 2.41 million M/T. 2. Out of the total extent of the organic tiers contained paddy soils, about 97.6% was distributed in Honam plains (water-sheds of Mangyeong-Dongjin river), while about 1.5% in the Naju plains (water-sheds of Yeongsan river), and 0.9% in the Wando and Yeocheon areas. 3. The period of peat formation was presumed to be about the early of Seung Moon period (B.C. 4,250), and the Gongdeog series and the Bongnam series were formed in the bog conditions close to the valley mouth of near rolling and hill with small steram channels, and the Gimje series was formed in the out-skirts plains of the Gongdeog and Bongnam soils. 4. In the casue of peat formation, it was presumed to be the Gimje series that accumulated the fibrous peat out of the autochthonous peat such as reeds and grasses etc, to be the Gongdeog and Bongnam series that accumulated the autochtonous peat and the xylem and fibrous peat out of first allochthonous peat. 5. In the Organic horizons of these soils, the range of muck and peat horizons were in 62-68cm and 68-137cm of soil profile in the Gongdeog series, 52-84cm and 84-113cm in the Bongnam series respectively, one of muck horizon was in 46-71cm in the Gimje series. 6. The marks of soil horizons of the soils were expressed that the lower soils than the horizon of muck and peat were formed Cg, Aag for the muck horizon, 0 for the peat horizon, 0 of peat horizon were distingushed with Oag and Oig according to Organic forms. 7. The depthe occurred the muck and peat horizons were positively correlated with the width of local in the Gongdeog series ($r=0.881^{**}$, $r=0.827^{**}$), but not in the Bongnam series and Gimje series.

• Korean Journal of Heritage: History & Science
• /
• v.39
• /
• pp.351-378
• /
• 2006

With the development of media, the methods for the documentation of intangible cultural heritage have been also developed and diversified. As well as the previous analogue ways of documentation, the have been recently applying new multi-media technologies focusing on digital pictures, sound sources, movies, etc. Among the new technologies, the documentation of intangible cultural heritage using the method of 'Motion Capture' has proved itself prominent especially in the fields that require three-dimensional documentation such as dances and performances. Motion Capture refers to the documentation technology which records the signals of the time varing positions derived from the sensors equipped on the surface of an object. It converts the signals from the sensors into digital data which can be plotted as points on the virtual coordinates of the computer and records the movement of the points during a certain period of time, as the object moves. It produces scientific data for the preservation of intangible cultural heritage, by displaying digital data which represents the virtual motion of a holder of an intangible cultural heritage. National Research Institute of Cultural Properties (NRICP) has been working on for the development of new documentation method for the Important Intangible Cultural Heritage designated by Korean government. This is to be done using 'motion capture' equipments which are also widely used for the computer graphics in movie or game industries. This project is designed to apply the motion capture technology for 3 years- from 2005 to 2007 - for 11 performances from 7 traditional dances of which body gestures have considerable values among the Important Intangible Cultural Heritage performances. This is to be supported by lottery funds. In 2005, the first year of the project, accumulated were data of single dances, such as Seungmu (monk's dance), Salpuri(a solo dance for spiritual cleansing dance), Taepyeongmu (dance of peace), which are relatively easy in terms of performing skills. In 2006, group dances, such as Jinju Geommu (Jinju sword dance), Seungjeonmu (dance for victory), Cheoyongmu (dance of Lord Cheoyong), etc., will be documented. In the last year of the project, 2007, education programme for comparative studies, analysis and transmission of intangible cultural heritage and three-dimensional contents for public service will be devised, based on the accumulated data, as well as the documentation of Hakyeonhwadae Habseolmu (crane dance combined with the lotus blossom dance). By describing the processes and results of motion capture documentation of Salpuri dance (Lee Mae-bang), Taepyeongmu (Kang seon-young) and Seungmu (Lee Mae-bang, Lee Ae-ju and Jung Jae-man) conducted in 2005, this report introduces a new approach for the documentation of intangible cultural heritage. During the first year of the project, two questions have been raised. First, how can we capture motions of a holder (dancer) without cutoffs during quite a long performance? After many times of tests, the motion capture system proved itself stable with continuous results. Second, how can we reproduce the accurate motion without the re-targeting process? The project re-created the most accurate motion of the dancer's gestures, applying the new technology to drew out the shape of the dancers's body digital data before the motion capture process for the first time in Korea. The accurate three-dimensional body models for four holders obtained by the body scanning enhanced the accuracy of the motion capture of the dance.