• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.29-29
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• 2003

The objective of this study is to examine an effective cryopreservation method and various vitrification containers on the survival vates of embroys. For the vitrification, in vitro produced embryos at blastocyst stage were exposed to ethylene glycol 5.5 M freezing solution (EG5.5) for 20 sec, loaded on containers such as grid, straw and paper and then immediately plunged into - 196$^{\circ}C$ L$N_2$. The blastocysts were thawed serially in 0.5, 0.25 and 0.125%; P < 0.05). Therefore, this study suggests that bovine embryos can be easily, effectively and successfully cryopreserved by grid, straw, and paper in the presence of freezing solution. Furthermore, vitrification using paper may be used as a no M sucrose in CR1aa, each for 1 min, and cultured in CR1 an medium supplemented with 10% FBS. After thawing, there were not significant differences in recovery rates of EM grid, straw and paper as 84.6, 88.3, and 93.7%, respectively (Table 1). However, survival rates of EM grid (78.1%) and paper (77.1%) showed significantly higher than straw (52. 1w method for bovine embryos.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.163-169
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• 1999

This study was carried out to investigate the effect of co-culture system(bovine oviduct epithelial cells; BOEC) and defined culture system(modified TALP ; mTALP) on the development of IVM-IVF embryos, and survival of in vitro produced blastocysts after freezing and thawing. Occytes from the slaugheterhous ovaries were matured and fertilized using general protocol. The results obtained were as the following: 1. Survival rates of frozen-thawed blastocysts using 10% glycerol as cryoprotectant was higher in day 7 blastocysts than in Day 8 and 9 blastocysts from co-cultrue system, but survival rate of frozen-thawed blastocysts was higher in Day 10 blastocysts than in day 8 and 9 blastocysts from defined culture system. Regardless of their age, survival rate of frozen-thawed blastocysts was significantly higher (p<0.05) in co-culture system than in defined culture system. 2. The cell number of blastocysts was significanlty higher (p<0.05) in Day 7 blasotcysts than in Day 8 and 9 blastocysts from co-cultures, but the cell number of blsstocysts was significantly higher (p<0.05) in Day 10 blastocysts than in Day 8 and 9 blastocysts from defined culture system. Regardless of the culture system, blastocysts with higher cell number showed higher survival rates after freezing and thawing.

• Journal of the Korean earth science society
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• v.32 no.7
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• pp.761-769
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• 2011

Daily minimum temperature and freezing data of the Seoul weather station ($37^{\circ}$34'N, $126^{\circ}$57'E, Songwol-dong Jongno-gu Seoul, hereinafter Songwol) and freezing data of the Han River station ($37^{\circ}$30'N, $126^{\circ}$57'E, hereinafter Han River) were used to study the long-term variation of the freezing climate for Seoul, Korea, for the period of 100 years from 1907 to 2006. 'Freezing' of Songwol is defined that the water in outdoor fields is frozen, and 'freezing' of the Han River located 6 km away from Songwol is defined as the region 100 meters upstream of the second and fourth piers in the south end of the Han River Bridge is fully frozen. The mean first freezing date for Songwol was October 28, and one for Han River was December 28; these showed a late tendency, with the rate of 0.78 days $decade^{-1}$ and 3.47 days $decade^{-1}$, respectively. The mean annual freezing days was 159.06 days for Songwol and 50.33 days for Han River; each showed a $decade^{-1}$shorter tendency, with rates of 2.01 days $decade^{-1}$ and 5.24 days $decade^{-1}$, respectively. All the seven no-freezing years (1960, 1971, 1972, 1978, 1988, 1991, and 2006) for Han River came after 1950. The mean daily minimum temperatures of the first freezing dates for Songwol and Han River were $0.55^{\circ}C$ and $-12.22^{\circ}C$. The first freezing occurred after 6.43 days for Songwol and after 8.94 days for Han River with daily minimum temperature below $0^{\circ}C$. The annual minimum temperatures of Songwol and Han River exhibited positive correlations with the first freezing date and negative correlations with freezing days. The result shows that the freezing climate change is relevant to temperature change and is a part of overall climate change. By conducting additional studies with various methods and wider region, we will be able to monitor the freezing climate.

• Transactions of the Korean Society of Mechanical Engineers B
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• v.25 no.10
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• pp.1400-1407
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• 2001

The size distributions of electrospray droplets from the Taylor cone in cone-jet mode are directly measured by using a freezing method and a transmission electron microscope (TEM) image processing technique. These results are compared with the data obtained by an aerodynamic size spectrometer (TSI Aerosizer DSP). The use of glycerol seeded with NaI and a freezing method make it possible to sample droplets with their original sizes preserved. Since pictures of droplets are taken with TEM with very low vapor pressure of the solution, evaporation is suppressed by freezing. For liquid flow rates below 1 nl/sec, the measured droplet diameters by the TEM image processing technique and the aerosizer are in the range of 0.25 to 0.32 m add 0.3B to 0.40m, respectively. Comparing the TEM data with the aerosizer measurements, it has been revealed that the TEM image processing technique can afford more accurate values of droplet size distributions in the submicron range of 0.1 to 0.4m.

• Korean Journal of Food Science and Technology
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• v.43 no.4
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• pp.509-512
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• 2011

To study the effect of freezing rate on the quality of freeze-dried rice porridge, freeze-dried rice porridge products were prepared with rice porridge pre-frozen at three different temperatures of -20, -40, and -70$^{\circ}C$. The porridge properties such as microstructure, mechanical properties, textural properties, and rehydration rate were determined. Scanning electron microscopy images indicated that fewer air cells were obtained with a larger size of freeze-dried rice porridge frozen at -20$^{\circ}C$ compared with that frozen at -40 and -70$^{\circ}C$. In contrast, quick frozen products at -70$^{\circ}C$ had more dense texture with higher mechanical strength, whereas slow frozen products exhibited higher rehydration rates than those of quick frozen products. In conclusion, the proper choice of pre-freezing temperature plays a decisive role when preparing freeze-dried rice porridge with optimum quality and convenience.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.44 no.6
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• pp.665-670
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• 2011

The rotifer Brachionus plicatilis is one of the most important food organisms in aquaculture. The resting eggs produced by mictic female rotifers are easily stored and hatched, making them useful as the starter for the mass culture of rotifers in marine larval culture. This study examined the optimum preservation method for resting eggs to ensure a high hatching rate. To produce resting eggs, the marine rotifer B. plicatilis was cultured with Nannochloris oculata (KMMCC 16). The resting eggs were harvested and cryopreserved using 5% and 10% methanol (MeOH), dimethylsulfoxide (DMSO), and glycerol as cryoprotectant agents (CPAs). The cryopreservation comprised slow or rapid freezing and the resting eggs were stored for one month in liquid nitrogen ($-196^{\circ}C$). The resting eggs were also dried at different temperatures (30, 40, and $50^{\circ}C$) and for different times (1, 2, and 3 h). In general, the hatching rates of the resting eggs preserved with CPA were higher than those without CPA and the slow freezing method was better than the rapid freezing method. However, the optimum CPA concentration for the hatching rate of the resting eggs varied with the freezing method and kind of CPA, and the CPA also affected the viability of the resting eggs. Dried resting eggs had a high, rapid hatching rate over 80%. The moisture content of the resting eggs cryopreserved in liquid nitrogen affected the hatching rate. Drying at $30^{\circ}C$ for 1 hour resulted in a high hatching rate of the resting eggs. In conclusion, drying at $30^{\circ}C$ for 1 hour and preservation in liquid nitrogen with the slow freezing method, without CPA, is recommended for a high hatching rate (ca. 95%) of rotifer resting eggs.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.1
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• pp.55-63
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• 2001

Objectives: 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). Materials and Methods: Two to eight cell embryos were obtained from oviducts of mated $F_1$ hybrid female mice superovulated by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Two-step 1,2-propanediol (PROH), dimethylsulfoxide (DMSO) and 4-step PROH 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. Results: As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in PROH and DMSO was significantly higher than 4-8 cell (64.5% versus 62.1 %,79.7% versus 73.2%) (p<0.01, p<0.01), but the development rates of 4-8 cell embryos in PROH 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 PROH 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 PROH (74.4% versus 64.5%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The developmen1 rate from morular to hatching blastocyst, however, was significantly higher in PROH than in DMSO during 48 hr (p<0.01). The survival rate of 4-8 cell embryo was 62.1% in PROH and 73.2% in DMSO. The development rates of embryo in PROH 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. Conclusions: The survival rate of embryo in 2 cell stage was higher than in 4-8 cell stage, and PROH appears more effective cryoprotectant than DMSO because PROH 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.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.3
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• pp.191-198
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• 2001

Objective : This study was conducted to examine the effect of vitrification on the survival and in vitro development of mice 1-cell zygotes. Method: Effects of exposure to vitrification solution and vitrification, with different concentrations of the cryoprotectant solution, were examined. The 1-cell zygotes were also subjected to a slow freezing-thawing method to compare with vitrification method. Solution composed of ethylene glycol (6.0 M, 5.0 M, 4.0 M) and sucrose (1.0 M) were used as cryopropectant. The experiments employed the method loading the embryos on electron microscope grids. Results: I. The effects of exposure in vitrification solution. 1-cell zygotes were non-toxic at all concentrations of the vitrification solution showing the survival rate between 88.1% and 97.5%. Development into 2-cell was more successful in the higher concentrations of the vitrification solution. Therefore, higher concentrations of the vitirification solution do not seem to cause any problems in vitrification procedure. II. The effects of vitrification method. 1-cell zygotes showed the survival rate between 78.8% and 92.4%. The lowest and the highest survival rate was observed in the 6.0 M and 4.0 M vitrification solution, respectively. 2-cell development rates varied from 77.6% to 91.3%. Blastocyst development rate was shown highest in 5.0 M and the lowest in 4.0 M solution. Therefore, the highest 2-cell and blastocyst development rate was observed in 5.0 M solution. III. Comparison of vitrification and slow freezing-thawing method on 1-cell zygotes. This experiment showed that 1-cell zygotes had the highest survival and development rates in 5.0 M vitrification solution. Vitrified group of 1-cell zygotes, in the 5.0 M vitrification solution, were compared with the group processed in slow freezing-thawing method. The development rate into 2-cell and blastocyst as well as the survival rate were higher in the vitrified group than in the slowly freezed group. Conclusion: 1. The results demonstrate that the best cryoprotectant is a 5.0 M vitrification solution for 1-cell zygotes. 2. Vitrification method significantly increases the survival rate of the 1-cell zygote and its development into 2-cell and blastocyst. Equilibration and exposure time during the vitrification was remarkerbly short in this experiment. Total time, from the exposure to vitirification solution to storage in the liquid nitrogen, was taken only 90 seconds. In contrast, the slow freezing-thawing method have taken more than four hours. Taken together, we presume that the overall time used for the procedure contributes to the results as an important parameter. 3. The loading of 1-cell zygotes on the EM grid is technically more simple and takes less time than the straw or cryo vial method.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.271-281
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• 1996

The effect of superovulation (PMSG, FSH) on the ovarian response of matured cows were tested. The survival rates of bovine embryos and ovarian oocytes frozen by slow, rapid freezing and vitrification were investigated. A total of 15 heads of cow were devided into 3 groups by injection dose of GTH (PSMG, FSH). Each group was superrovulated with injections of 2, 500, 3, OOOJU PSMG and 40mg FSH followed by injection of 30mg PGF2a. Embryos were non-surgically recovered from superovulated cows 6~7days after estrus. The recovered embryos were frozen in 10% glycerol + 10% sucrose by slow and rapid freezing. Ovarian oncytes were frozen in 20% g]ycerol+l0% ethylene glycerol + 30% Ficol + 10% sucrose by vitrification and the survival of frozen embryos and ovarian oncytes were judged by FDA-test. The results are summarized as follows; 1. Estrus after the injection of 2500, 3000 I.U. PMSG and 4Omg FSH were 32.8, 35.0 and 43.4 and the duration of estrus were 18.6, 18.8 and 22.4 hours respectively. 2. The average sizes of the left ovaries were 5.4cm (2, 500 IU PMSG), 5.1cm (3, OOOIU PMSG) and 6.4cm (FSH), and the right were 6.2cm (2, 5001U PMSG), 5.7cm (3, OOOIU PMSG) and 7.&m (FSH) respectively. There were significant differences in the right overies among treatments (P<0.05). 3. The average number of ovarian follicles in the left ovaries were 4.8 (2, 500 IU PMSG), 5.2(3, 000 IU PMSG) and 7.8 (FSH) respectively. There were significant difference in the right ovaries among treatments (P<0.05). 4. In the average numbers of ovulation points in the left ovaries were 3.0 (2, 5001U PMSG), 3.2 (3, OOOIU PMSG) and 4.4 (FSH) respectively, and the right were 7.2 (2, 5001U PMSG), 7.8(3, 000IU PMSG) and 11.4 (FSH). There were significant differences in the right ovaries among treatments (P<0.05). 5. The numbers of the recovered embryos were 20 (2, 5001U PMSG), 19 (3, 000 IU PMSG) and 21 (FSH) respectively, and oncytes and degenerted oncytes were 6.5 and 11.0 Estrus periods of post parturation were 52.4days (2, 5001U PMSG), 69.8days (3, OOOIU PMSG) and 62.4days (FSH) respectively. 6. The FDA score of cow morulae frozen by slow freezing, sernirapid frezing and vitrified freezing was higher in slow (3.1) and vitrified freezing (3.0) than that in semirapid freezing (1.28). The FDA-scores of cow, pig and rabbit ovarian oocytes frozen in 20% glycerol + 10% ethylene glycol + 30% Ficoll + 10% sucrose by vitrification were higher in cows (3.3) than both in pigs (2.6) and rabbits (2.3).

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.76-76
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• 2001

The objective of this study was to optimize the vitrification method of in vitro produced bovine embryos. Thus, in vitro produced embryos at 8 cell, morula and blastocyst stages were vitrified on electron microscope grids (EM grids) or in open pulled straws (OPS) with EG5.5 (5.5 M ethylene glycol, 1.0 M sucrose and 10% FBS in m-DPBS medium) freezing solution and their survival rates after thawing were compared. The embryos on EM grids or in OPS were briefly exposed to EG5.5 freezing solution and plunged directly into liquid nitrogen within 30 to 35 sec. Post-thawed embryos were serially diluted in 0.5, 0.25 and 0.125 M sucrose in m-DPBS, each for 1 min, and then cultured in CRI aa medium supplemented with 10% FBS. Embryonic survival rate was assessed as re-expanded and hatched rates of those embryos after warming. The rates of re-expansion embryos did not significantly different between EM grid (8 cell: 42.10%, morula: 66.66% and blastocyst: 77.08%) and OPS (8 cell: 47.36%, morula: 61.90% and blastocyst: 83.33%) methods. In addition, the hatched rates in EM grid (8 cell: 31.57%, morula: 57.14% and blastocyst: 72.91%) were similar to those in OPS (8 cell. 34.21%, morula: 50.00% and Blastocyst: 77.08%). Interestingly, even at the same blastocyst stage, the in vitro survival of day 7 embryos (EM grid: 79.48 and OPS: 87.18%) was higher than those of day 8 embryos (EM grid: 72.10 and OPS: 82.06%). The total cell number of blastocyst developed in vitro after vitrification was examined with Hoechst 33342 staining to compare the embryo quality among different treatment groups. The total cell number of blastocyst was not significantly different between vitrified groups (EM grid: 162.4$\pm$8.0 and OPS: 158.4$\pm$7.1) and unvitrified control (168.0$\pm$5.6). These results indicate that both vitrification containers can provide the high rate of embryo survival. Moreover, the OPS container may not need a cap to protect the container from floating after immersion in L$N_2$. Therefore, this study suggest that bovine embryos can be cryopreserved easily, effectively and successfully by vitrification method using EM grid or OPS with EG5.5 freezing solution. In the future, the Pregnancy rate would be investigated after transfer of our vitrified embryos into the appropriated recipients.