• 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.

• 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.

• Korean Journal of Animal Reproduction
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• v.11 no.2
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• pp.122-126
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• 1987

This study was carried out to investigate the effects of the glycerol equilibration methods and embryo grades before freezing on the survival rate after thawing in mouse embryos. The results obtained from this study were as follows: 1. The number of embryos of grade I(Excellent), II(Good) and III(Fair) before freezing in this study was 97(27.4%), 160(45.2%) and 97(27.4%), respectively. 2. The average survival rate of frozen-thawed embryos in 3 and 5 steps glycerol equilibration was 66.7% and 64.1%, and the rate of transfererable embryos after culture was 68.9% and 69.0%, respectively. 3. Out of embryo grade I and II before freezing, the transferable rate after thawing was 75.2% and 48.1%, respectively, and grade I embryos before freezing was higher transferable rate than that of grade II.

• 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.

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.143-152
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• 1992

The present experiments were focussed to modify a short slow-cooling protocol used for freezing of early stage embryo(Testart et al., 1986) and also to apply the modified method for the cryopreservation of hamster oocytes with Zona or without. The protocol was modified by changing the 4-step equilibration into 1-step and the 1-step thawing into 2-step. The oocytes were added in 1.5M PROH and 0.1M Sucrose, seeded at $-7^{\circ}C$, slow cooled($0.3^{\circ}C$/min) to $-30^{\circ}C$ before plunging to $-196^{\circ}C$. The oocytes were thawed at $23-25^{\circ}C$ air(20sec/150sec) and/or 33-35 water(10sec). The survival of the frozen-thawed oocytes was determined by morphologic criteria and their fertilizing ability was also estimated by Sperm Penetration Assay(SPA) system(Chang et al, 1990) using fertile men semen sample. One-step equilibration showed slightly higher survival rate(83.9% vs. 71.0%) and fertilization rate(83.9% vs. 71.0%) compared with four-step(p>0.05). And two-step thawing(air & water exposing) of oocytes frozen after 1-step equilibration showed significantly higher survival rate(96.3%) than one-step thawing at air(85.2%) or water(65.0%) only(p<0.05). Therefore, by the modified method(l-step equilibration & 2-step thawing), Zona-intact(ZI) and Zona-free(ZF) oocytes were frozen and thawed. ZI-oocytes showed significantly higher survival rate(95.4%, 308/323 vs. 67.6%, 240/355) than ZF-oocytes(P<0.01). But the survival of ZF-oocytes was as high as ZI-oocytes in fourteen of twenty-four replicates. ZI-oocytes was also significantly higher fertilization rate($92.4{\pm}8.9%$ vs. $63.7{\pm}18.5%$) and higher mean number of penetrated sperm($6.2{\pm}4.2$ vs. $3.9{\\pm}3.3$) than ZF-oocytes, but not higher than control(fresh oocytes;$99.3{\pm}2.4%$, $8.4{\pm}4.2$)(P<0.001). Conclusively, this modified method will contribute to freeze effectively the hamster oocytes for simplifing of the logical consideration of performing SPA and also to freeze the human and other animal oocytes.

• The Korean Journal of Physiology
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• v.4 no.1
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• pp.13-17
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• 1970

The changes in the red cell volume and the plasma chloride level were measured when the blood $CO_2$ content was altered by equilibration with the atmospheric air or pure $CO_2$ for 20 minutes. The red cell volume was expressed in terms of hematocrit and mean corpuscular volume (M.C.V.). The results obtained were as follows. 1) On equilibration with the atmospheric air, the MCV and the plasma chloride level were $91.6{\pm}1.26\;c.{\mu}$ and $110.7{\pm}6.28mEq/L.$ respectively. 2) On equilibration with pure $CO_{2}$, the MCV and the plasma chloride level were $109.6{\pm}2.0\;c.{\mu}$ and $90.7{\pm}5.17\;mEq/L.$ respectively. 3) When the blood was subjected to equilibration with the atmospheric for air 20 minutes after equilibration with pure $CO_{2}$ for the same period of time the MCV and the plasma chloride level were $89.9{\pm}6.34\;c.{\mu}$ and $100.3{\pm}5.50\;mEq/L.$ respectively. From the above results it can be concluded that an increase of the blood $CO_2$ content in the experimental condition causes definitely a decrease of the plasma chloride level and a concomitant increase of the red cell volume, and that a decrease of the blood content $CO_2$ in the experimental condition causes definitely an increase of the plasma chloride level and a concomitant decrease of the red cell volme. Apparantly there exists a parallel relationship between the extent of the decrease of the plasma chloride level and that of the increase of the red cell volume when the blood $CO_2$ content increased in the experimental condition. When the blood $CO_2$ content decreased, the extent of the decrease of the red cell volume exceeds that of the increase of the plasma chloride level.