• Journal of Embryo Transfer
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• v.6 no.1
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• pp.13-24
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• 1991

The objective of this experiment was to study some possibilities to simplify freezing, thawing and transfer procedure of one-step straw method comparing with the conventional methods using bovine embryos. The previous work are also designed to investigate the thawing effect by development stage and its quality using the embryos. Results obtained were summarized as follows: 1. A total of 87 embryos from 14 donor cows were frozen-thawed and an average of frozen embryo/donor was 6.2. 2. The survival rates of morula stage(65.4%) were higher than those of blastocyst stage(57.l%) and vice versa in rate of morphological recovery (80% vs 95.4%). However. no significant difference was denoted between them. 3. In difference between the groups of good quality and poor quality. good quality was resulted in a significantly higher embryo survival rate(75%) and recovery rates(95%) than poor quality(P<0.0l). 4. In effects of non-permeable sugar dilution in added to l.0M glycerol. higher survival rates were orderd in sucrose. lactose, raffinose and xylose. But lactose-raffinose, sucrose-trehalose and xylose in added to 2.OM glycerol. 5. The highest survival rates were obtained by direct plunge into the liquid nitrogen with 3.OM concentration both of glycerol and trehalose. 6. The survival rates in vitro condition of one-step and direct plunge methods(75%-87.5%) were significantly higher than those of multiple steps (21.4-52.6%) in in vitro (P<0.0l). However, the results of single-step were critical in comparing to other steps of final pregnant conformation.

• Korean Journal of Animal Reproduction
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• v.9 no.1
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• pp.36-39
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• 1985

This experiment was carried out to investigate the morphology of bovine embryos thawed after deep freezing at -196$^{\circ}C$ and the development of frozen-thawed embryos after in vitro culture in Ham's F-10 medium with 10% NBCS. The results obtained were summarized as follows: 1. The propotion of embryos which a, pp.ared mophologically normal was averaged 77.5% (79/102). 2. The morphologically normal rate of frozen-thawed blastocyst (78.6%) was higher than that of morula (76.7%), but there was no significant difference. 3. Normal development was observed in 20 of 68 embryos cultured for 24-72hr in medium and overall survival rate was 29.4%. 4. Survival rate fo blastocyst (33.3%) was higher than that of morula (25.7%).

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.59-64
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• 1998

This study was designed to evaluate the influence of pronuclear age on the survival and post-thawing development after cryopreservation of mouse embryos. Freezing and thawing were performed in the different pronuclear stages of mouse embryos after IVF. Embryos were obtained from $F_1$ hybrid mice and classified into 4 groups according to the pronuclear stage (6hr, 9hr, 12hr and 15hr after insemination). Pronuclear ova were slowly cooled in a biological freezer using 1.5M 1,2-propanediol and 0.1M sucrose as cryoprotectant. Thawing was done at room temperature and 1,2-propanediol was removed by multi-step dilutions. Both frozen-thawed embryos and control fresh embryos were cultured in vitro in Ham's F-10 medium supplemented with 4mg/ml BSA. In control group, the development rate after 48hr was 99.3%, and the complete hatching rate after 144hr was 61.3%. In experimental groups, the survival rate after thawing was 95.4% in 6hr, 88.7% in 9hr, 75.2% in 12hr and 62.4% in 15hr after insemination, the development rate after 48hr was 61.1, 77.0, 67.0 and 79.6%, respectively, and the complete hatching rate after 144hr was 25.7, 43.7, 42.2 and 60.0%, respectively. The survival rate in 15hr was significantly lower (p<0.05) compared with other groups. In vitro development rates after 48hr were similar in all groups, but complement hatching rate was significantly lower (p<0.05) in 6hr group. In conclusion, cryopreservation of mouse pronuclear ova with 2 distinct pronuclei (9hr and 12hr groups) showed better results after thawing compared with early (6hr group) or late pronuclear ova just prior to cleavage (15hr group).

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.13-19
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• 2014

This study was carried out to study the survival rate of thawed Hanwoo embryos frozen by the slow-rate freezing or the cryotop vitrification method. Hanwoo cumulus-oocyte complexes were recovered from ovaries at a slaughter house, matured for 20~22 hours, fertilized with Hanwoo semen for 5~6 hours, and cultured for 7~9 days in $38.5^{\circ}C$, 5% $CO_2$ incubator. For freezing, Day 7~9 blastocysts were collected. Embryos for the slow-rate freezing were equilibrated in 1.8 M ethylene glycol (EG) with Dulbecco's phosphate-buffered saline (D-PBS). Programmable cell freezer was precooled down to $-7^{\circ}C$, and the straw was seeded during 8 minutes-holding time, and was cooled to $-35^{\circ}C$ at the cooling rate of $0.3^{\circ}C/min$, and then was plunged and stored in liquid nitrogen. Embryos for the cryotop vitrification were treated in TCM199 with 0.5 M sucrose, 16% EG, 16% dimethylsulfoxide (DMSO). Embryos were then loaded individually onto cryotop and plunged directly into liquid nitrogen. The survival rates of embryos frozen by these two freezing methods were evaluated at 12 to 24h post-thawing. The survival rates of frozen/thawed Hanwoo embryos by the cryotop vitrification method ($56.86{\pm}26.53%$) were slightly higher than those by the slow-rate freezing method ($55.07{\pm}26.43%$) with no significant difference. Using the cryotop vitrification and the slow-rate freezing of Hanwoo blastocysts on Day 7 following in-vitro fertilization (IVF) treatment, the survival rates of frozen/thawed Hanwoo embryos were $72.65{\pm}18.3%$ and $79.06{\pm}17.8%$, respectively. The survival rates by the cryotop vitrification were higher than those by the slow-rate freezing on both Day 8 and 9 with significantly higher survival rate on Day 9 (p<0.05). Using the cryotop vitrification and the slow-rate freezing of Hanwoo embryos to compare between three different blastocyst stages, the survival rates of the blastocyst stage embryos were $66.22{\pm}18.8%$ and $45.76{\pm}12.8%$, respectively with higher survival rate by the vitrification method (p<0.05). And the survival rate of expanded blastocysts was higher than those of early blastocysts and blastocysts in two freezing methods with significantly higher survival rate by the slow-rate freezing method (p<0.05).

• Journal of Embryo Transfer
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• v.31 no.4
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• pp.367-374
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• 2016

This study was conducted to evaluate the effect of transfer temperature of epididymis on survival rate of semen and development ability of B6D2F1 mice embryos. No significant differences were noted in the survival rate of semen ($59.0%{\pm}0.1$ vs. $47.6%{\pm}0.1$), in vitro fertilization rate ($90.7%{\pm}0.1$ vs. $90.7%{\pm}0.1$), developmental rate ($90.0%{\pm}0.1$ vs. $90.0%{\pm}0.1$), and blastocysts formation rate ($53.1%{\pm}0.2$ vs. $52.3%{\pm}0.2$) between groups. (NS; P>0.05). However, the zona hatched rate was significantly higher in the $4^{\circ}C$ group compared to those of the $37^{\circ}C$ group ($47.8%{\pm}0.1$ vs. $25.6%{\pm}0.2$; p<0.05). When it comes to cell numbers of blastocysts, the % ICM (/total cells) was significantly higher in the group of $4^{\circ}C$ compared to the $37^{\circ}C$ ($27.0%{\pm}0.1$ vs. $18.3%{\pm}0.1$; p<0.05). However there were no differences in total cell numbers ($72.7{\pm}31.6$ vs. $62.0{\pm}36.6$), ICM cell numbers ($17.0{\pm}7.8$ vs. $14.6{\pm}8.6$), TE cell numbers ($55.8{\pm}29.8$ vs. $64.0{\pm}24.4$), the ratio of ICM:TE ($1:4.2{\pm}4.1$ vs. $1:6.4{\pm}7.2$) between two groups (NS; P>0.05). Taken altogether, it is expected to achieve the best developmental ability of B6D2F1 mice embryos in the transfer temperature of epididymis. Also these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice. In future, therefore, it is expected that results herein might be applied for in vitro culture of human embryos.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.95-102
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• 1997

The studies on the carried out to investigate the effective concentration of cryoprotectant agents and sucrose by one-step straw method of bovine embryos. The follicular oocytes were cultured in TCM-199 medium containing 10 IU/ml PMSG(Sigma, USA), 10 IU/ml hCG(Sigma, USA), 1$\mu\textrm{g}$/ml $\beta$-estradiol(Sigma, USA) and 10% FCS for 24~48 hrs in incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation of heparin. The bovine embryos following dehydration by cryoprotective agents and various concentrations of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival and in vitro developmental rate was defined as devellpmental rate on in vitro culture or FDA-test. The results are smmarized as followes : 1. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was attained 2.0M glycerol, 2.0M DMSO, 1M or 2.0M propanediol. 2. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was obtained single cryoprotectant(6.7~17.4%) than mixed cryoprotectants(6.7~16.7%). 3. In vitro developmental rate of bovine embryos after rapid frozen-thawing in the freezing medium added 0.25M and 0.50M sucrose were higher cleavage rate than those of sucrose concentration of 0.75M and 1.00M. 4. The freezing methods on in vitro developemental rates of bovine embryos was attained slow freezing method(9.70~15.6%) higher than rapid freezing method(9.4~13.3%).

• Korean Journal of Veterinary Research
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• v.35 no.3
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• pp.635-641
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• 1995

The purpose of this study was to investigate the effects of developmental stage and equilibration time on survival of rabbit embryos following freezing by vitrification. Adult New Zealand White female rabbits were superovulated with PMSG and hCG. The 8-cell stage embryos were collected from 40 to 45 hours after hCG injection by flushing oviducts with Dulbecco's phosphated buffered saline and in vitro cultured in TCM-199 containing 10% fetal calf serum(FCS). Each embryos developed in vitro to 16-cell, compact morula and blastocyst was cryopreserved and cultured following thawing to examine their developmental potential to expanded blastocyst stage in vitro. The frozen-thawed-cultured embryos were stained with Hoechst 33342, and their nuclei were counted using a fluorescence microscope. On the toxicity test of EFS solution as cryopreservation, the survival rates of 8-cell stage embryos was decreased in reverse to increasing of exposure time over 5 minutes. The post-thaw survival rates of embryos on equilibration times was significantly(P<0.05) higher for 2 min. than for 5 or 10 minutes. From morula to blastocyst of rabbit embryos was more suitable than 8-cell stage for cryopreservation by vitrification. The higher post-thaw survival rate of embryos can be achieved by keeping the cryoprotectant at $4^{\circ}C$ than at $20^{\circ}C$. The mean number of nuclei per embryo following freezing by vitrification and in vitro culture to expanded blastocyst at compacted morula and blastcyst was not significantly differ from fresh blastocyst.

• Korean Journal of Plant Tissue Culture
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• v.28 no.2
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• pp.103-108
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• 2001

The in vitro plantlets of cassava (Manihot esculenta Crantz cv. MColl 22) could be regenerated from nodal explant cultures in a liquid MS basal medium containing 0.01 mg/L zeatin for 2 weeks. The plantlets of 1.5∼2.5 cm in shoot length were transplanted to a glass bottle containing fine sand and acclimated under non-sterile conditions after excising their intact roots by: 1) prune leaving roots base of 1∼1.5 cm; 2) complete removal of roots; and 3) cutting off the rooting zone. The majority of in vitro-formed intact roots continued growth after transferred to soil, and all of the damaged roots stopped further growth. The plantlets with excised roots began to develop new roots within 7∼10 days after being transferred to a glass bottle, and a few of the pruned roots developed lateral roots from the remaining portion. Pruning and removal of in vitro roots resulted in a high survival rate (>87%), and did not significantly affect ex vitro root regeneration and acclimation, but the plantlets in which the rooting zone had been cut-off showed 73% survival rate. Pruning or removal of in vitro roots before transfer of plantlets is recommended for useful method of commercial micropropagation because of easier handling and high survival rate of plantlets.

• Korean Journal of Plant Resources
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• v.19 no.6
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• pp.665-669
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• 2006

Somatic embryos do not survive at exposure to liquid nitrogen temperatures without cryoprotective treatments. A simplified technique which simultaneously induces and cryoprotects embryogenic calli using plant vitrification solution 2 (PVS2) followed by dehydration was developed for the cryopreservation of Soap berry genetic resources. Vitrification is a way of removing the moisture in vegetation through PVS2. The PVS2 vitrification solution consisted of 30% glycerol (w/v), 15% ethylene glycol (w/v), 15% Dimethylsulfoxide (w/v) in B5 medium containing 0.4M sucrose. Two tests were done. The one was to eliminate moisture at $0^{\circ}C$ and the other at $25^{\circ}C$. In both cases the best results came out at a vitrification time of $10{\sim}20$ minutes. It was also found that the survival rate was higher at $0^{\circ}C$ than at $25^{\circ}C$. In particular, the survival rate reached more than 80%. Water-damaged embryos turned brown and stoped growth, but energetic embryos took on a milky hue and show a very vigorous growth rate. Successful cryopreservation of somatic embryos of soapberry can be used to establish in vitro genebanks for long-term conservation of Soapberry genetic resources to complement field genebanks and other in vitro methods already being used.

• Journal of Embryo Transfer
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• v.24 no.4
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• pp.253-257
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• 2009

To enhance the embryo preservation technology and better application of embryo transfer technique to the field (dairy science or animal reproduction. etc.), we examined the viabilities of bovine embryos produced in vitro and in vivo after cryopreservation according to their developmental stage and thawing temperature. Bovine embryos from in vivo/vitro fertilization (Hanwoo) were examined at day 7, 8, and 9. Survival rates and total cell numbers of in vivo fertilized embryos were as follows: morulae 68.8% and $67\;{\pm}\;6.0$; blastocysts 80.5% and $120\;{\pm}\;10$; expanded blastocysts 77.4% and $138\;{\pm}\;9.7$, respectively. Rates of embryo development for blastocysts and expanded blastocysts after thawing were significantly higher than that of morula stage embryos (p<0.05). While survival rates of in vitro fertilized embryos according to developmental stage showed no significant difference among groups (morula 67.9%; blastocyst 74.3%; and expanded blastocyst 79.4%), total cell numbers were significantly lower than those of other groups (morula $64\;{\pm}\;5.9$; blastocyst $116\;{\pm}\;8.7$; and expanded blastocyst $135\;{\pm}\;9.1$) For the viability according to thawing temperature, survival rate was higher in $37^{\circ}C$.