• Journal of Embryo Transfer
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• v.9 no.2
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• pp.173-180
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• 1994

The present experirnents on cryopreservation were carried out to investigate effect of solution toxicity, equilibration time and cell stages on the post-thaw survival of mouse morulae and blastocyst embryos cryopreserved by vitrification in EFS solution. The mouse embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen and thawed rapidly. After the mouse morulae embryos were exposed to EFS solution for 2 and 5 ruin. at room temperature and then they were washed in 0.5 M sucrose solution and basal mediurn(D-PBS + 10% FCS), they were cultured to examined cryoprotectant toxicity induced injury during exposure, most of embryos developed to expanded blastocysts(100 and 90.0%). However, when the exposure time was extended to 10 and 20 min, these development rates dropped dramatically in 10 ruin. (75.0%) and 20 ruin. (4.5%), respectively. When the compacted morulae were vitrified in EFS solution after equilibration for 2 and 5 min, the embryos have developed to normal blastocyst following thawing, washing and culture processes was 89.3 and 89.6%. However, when the exposure time was expanded to 10 ruin, this survival rate dropped to 68.8%. When the blastocyst were vitrified in EFS solution after equilibration for 2, 5 and 10 minutes, the survival rate of embryos which developed to normal blastocyst following thawing and culture processing were 58.5, 46.7 and 22.4%, respectively. The optimal time of equilibration of mouse morula and blastocysts in EFS solution seemed o be 2 and 5 ruin.

• Korean Journal of Animal Reproduction
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• v.16 no.1
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• pp.47-53
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• 1992

This study was carried out to investage the effect of cryoprotectant concentration and equilibration time on volume change and in vitro development of intact and bisected mouse embryos by rapid freezing. When compacted morulae were rapidly frozen in 3.0 to 4.0 glycerol or DMSO with 0.25M sucrose solution, the superior(P<0.05) post-thaw survival rate was obtained at the glycerol concentration of 4.0M(89.4%) than 3.0M(71.4%) or 5.0M(42.4%), but at the DMSO concentration of 3.0M(84.5%) than 4.0M(51.1%) or 5.0M(0.0%). The optimal equilibraton time for rapid freezing of ZP-free or bisected morulae in 4.0M glycerol with 0.25M sucrose was found tobe 3 minutes. The minimal volume of compacted morulaewhich corresponded with 61 to 62% of pre-equilibrated embryo volume was obtained from equilibration for 3 minutes in both 3.0 and 4.0M glycerol solutions with 0.25M sucrose.

• Development and Reproduction
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• v.18 no.2
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• pp.117-125
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• 2014

Vitrification method is widely used in oocyte cryopreservation for IVF but the birth rates are lower than that of the fresh oocyte. One of the known main reasons is structural instability of meiotic spindle and chromosome systems of mature oocyte. To get the best way for keeping competence of matured oocytes, we studied the best conditions for vitrification focused on equilibration times. The mature oocytes were underwent vitrification with current popular method and analyzed the survival rates, microtubule stability and DNA integrity. The survival rates of recovered oocyte are almost same between groups and are more than 93%. The structural configuration of meiotic spindle was well kept in 10 min equilibration group and the stability rate was almost same with that of control. The chromosomal breakdown was observed in all experimental groups, but the chromosomal stability was higher in 10 min equilibration group than the other groups. The 10 min equilibration group showed best condition compared with the other groups. Based on these results, the equilibration time is one of the key factors in successful keeping for competence of mature oocyte. Although, more fine analysis about the effects of physical stress on oocyte during vitrification is needed to define the optimal condition, it is suggested that the optimal equilibration time to get competent oocyte in mouse is 10 min. Information acquired this study may provide insight into intracellular structural events occurring in human oocytes after vitrification and application for cryopreservation of human oocyte.

• Development and Reproduction
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• v.1 no.1
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• pp.29-36
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• 1997

Experiments were performed to study the effects of diluents, cryoprotectant, equilibration time, thawing temperature and addition of BSA and egg yolk. Among the various diluents, Alsever's solution was the best for sperm cryopreservation. A combination of Alsever's solution and 15% ethylene glycol showed the better results than others did. Sperm activity indection and survival rate gradually decreased with the equilibration time. The appropriate thawing temperature was 30 ${\pm}1^{\circ}$C. These results indicate that sperm cryopreservation methods can be developed in tiger puffer.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.261-268
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• 1998

The present study was to assess the effect of ultrarapid freezing on the development of 1-cell mouse zygote using cryoprotectants, DMSO (dimethyl sulfoxide) or PROH (1,2-propanediol). We investigated the effect of the type and concentration of cryoprotectant, and of the temperature and time of prefreezing equilibration on their capacity to develop to the blastocyst stage in vitro. The concenration, the equilibration temperature, and the exposure time seemed to serve as an important factor in ultrarapid freezing of 1-cell mouse zygotes. In addition to the exposure time and the concentration of cryoprotectant appeared to playa key role in the development of the embryo. In general, the development of the embryo was more effective at $3^{\circ}C$ than $23^{\circ}C$ and 4.5 M than 3 M for 3 to 5 minutes. At $23^{\circ}C$ the development of the embryo was stimulated by DMSO while at $3^{\circ}C$ it was stimulated by PROH. Thus it has been suggested that there exists a correlation between the concentration of cryoprotectants and exposure time in the development of the embryo. In conclusion, we found that for ultrarapid freezing of mouse 1-cell embryos in DMSO, or PROH-based solution, viability shown optimum depending on the cryoprotectant, the concentration of the cryoprotectant and on the temperature and the duration of equilibration.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.315-324
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• 1997

This study was undertaken in an effort to develop a cryopreservation system of immature and mature porcine oocytes. For this aim, the experiments were designed to examine the effect of cryoprotectants and equdibration time on the viability of frozen-thawed oocytes by using trypan blue(TB) and fluorescene diacetate(FDA) test. The viability of frozen immature oocytes evaluated by TB test was slightly higher than that of frozen mature oocytes. The viability(25.O%) after IVM of frozen-thawed immature oocytes greatly decreased that(42.9%) of oocytes just after thawing, but it was higher than frozen-thawed mature oocytes(15.8%). When immature oocytes were equilibrated for 10, 20 and 30 minutes before freezing the oocyte viability was 20.0, 31.3 and 42.9%, respectively. There was a tendency for long equilibration before oocyte freezing to be more effective for the immature oocytes and a short equilibration time for mature oocytes. Although there was no difference in viability index of frozen oocytes hetween the viability test methods, the index of TB test was slightly higher than that of FDA test. The viability(FDA test) of frozen-immature oocytes with 3 different crtoprotectants was 22.2% for propylene glycol(PG), 9.3% for polyehtylene glycol(PEG) and 65.6% for PG+PEG, in which PG+PEG was more protective against freezing effect.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.103-109
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• 1997

The studies on the carried out to investigate to determine the optimum thawing temperature and equilibration time and 1 step straw method of frozen 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 concentration 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 developmental rate on in vitro culture or FDA-test. The results are summarized as followes : 1. The equilibration time on in vitro developmental rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20 min.). 2. The temperature thawed at 3$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher in vitro developmental rate than did at 2$0^{\circ}C$ and 35$^{\circ}C$. 3. The thawing time on in vitro developmental rates of bovine embryos was attained after short period of time(1~5 min.) in the freezing mediuim higher than long period of time(10min.). 4. The in vitro developmental rates of bovine embryos after rapid frozen-thawing by 1 step straw method in the freezing medium added 1.5M, 2.0M glycerol, DMSO, propanediol and 0.25M, 0.50M, 0.75M, 1.00M sucrose were 12.5~19.4%, 10.0~15.6%, 9.1~13.8% and 6.7~12.9%, respectively.

• Korean Journal of Animal Reproduction
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• v.15 no.1
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• pp.49-57
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• 1991

This study was carried out to investigate the effect of equilibration time, precooling and straw loading method on the post-thaw survival rates of mouse embryos cryopreserved by vitrification method. The effect of the vitrification procedure on the damage of the embryos was also examined by the straining of nuclei using Hoechst 33342. The results obtained were summarized as follows; 1. The equilibration in Medium-1 for 10 minutes was considered to be the optimum equilibration time. Embryos equilibrated in Medium-1 for 10 minutes(81.0%) showed significantly(P<0.05) higher survival rates than those equilibrated for 5 minutes(40.0%) or 15 minutes(74.1%). 2. The survival rate of embryos cryopreserved using the double Medium-2 column(81.0%) was significantly(P<0.01) higher than that using the single Medium-2 column, whish was considered to be due to the double Medium-2 column method being more reliable for preventing contamination of diluent solution of 1M sucrose. 3. The survival rate of compacted morula stage embryos cryopreserved with the precooled and non-precooled Medium-2 was not significantly(P<0.05) different. 4. The number of blastomeres of embryos at late blastocyst stage after culture of mouse morulae for 24 to 28hours was significantly(P<0.05) decreased by freezing embryos using vitrification(53.3${\pm}$1.6 vs 41.4${\pm}$1.5), which was considered to be due to the damage of embryos during vitrification and the delay of embryo development by handling in vitro. 5. The vitrification procedure is considered to be a very simple and efficient method for cryopreservation of embryos at early developmental stage.

• Korean Journal of Soil Science and Fertilizer
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• v.24 no.2
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• pp.116-123
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• 1991

Two solution to soil ratios, 2 : 1 and 5 : 1 were tested to determine the appropriate ratio in the sorption measurement off. sulfoxide for Wahiawa soil samples, 0-20 cm, 40-60 cm and 100-120 cm. and Salinas soil samples, 0-15cm and 115-130cm. One ${\mu}$ mol/L f.sulfoxide was used as an initial equilibration concentration. Sorption of f.sulfoxide at 5 : 1 ratio showed appropriate mixing, while sorption at 2 : 1 ratio indicated insufficient mixing during the various batch equilibration times (4, 12, 24 and 48 hours). For most samples the degree of sorption was about 20-50%, which falls in the desired range (20-80%) at the 5 : 1 ratio. An exception was with the low-sorptive Wahiawa subsoil in which the ranges were below 20%. Thus the 5 : 1 ratio can be used for f.sulfoxide sorption measurement. Four equilibration times (4, 12, 24 and 48 hours) and four concentrations(0.1, 1.0, 5.0 and $10{\mu}mol/L$) were used to determine the appropriate equilibration time for Wahiawa and Salinas soils. Sorption increased over all equilibration times, indicating no complete equilibrium within 48 hours. Apparent equilibrium was reached in 4 hours, and sorption increased slowly until 24 hours and faster thereafter, except for the Wahiawa soil, 100-120 cm. The recommended equilibration time is 24 hours, since it may eliminate the insufficient sorption and yet avoid undesirable transformation.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.65-71
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• 1994

This study were carried out to investigate the effective concentration of cryoprotectant agents and sucrose by one-step straw method, and to determine the optimum thawing temperature and equilibration time of frozen bovine embryos. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3O$^{\circ}C$ water. Survival rate was defined by FDA test. The results are summarized as follows : 1. The survival rate of bovine embryos thawed after rapid freezing in the freezing medium containing a various kinds of cryoprotective agents added 0.25M and O.50M sucrose were 28.6% and 25.0%, 35.1% and 31.6%, 32.4% and 24.4%, 34.2% and 28.2%, 18.9% and 17.6%, 14.7.% and 21.6%, respectively. 2. The survival rate of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol, i.5M and 2.OM DMSO and 1.5M and 2.OM propanediol were 22.9~37.8%, 2O.7~31. 3%, 19.2~30.0% and 17.2~25.0%, respectively. 3. The temperature thawed at 2$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 3$0^{\circ}C$ and 35$^{\circ}C$. 4. The equilibration time on the survival rate of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (1O~20 min.).