• Journal of Animal Science and Technology
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• v.58 no.3
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• pp.13.1-13.7
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• 2016

Background: In the present study, various freezing containers were tested for mouse embryos of respective developmental stages; embryos were vitrified and then their survival rate and developmental rate were monitored. Mouse two cell, 8 cell, and blastula stage embryos underwent vitrification freezing-thawing and then their recovery rate, survival rate, development rate, and hatching rate were investigated. Methods: EM-grid, OPS, and cryo-loop were utilized for vitrification freezing-thawing of mouse embryos. Results: It was found that recovery rate and survival rate were higher in the group of cryo-loop compared to those of EM-grid (p < 0.05). Embryonic development rate, two cell embryos to blastocyst, as well as hatching rate were higher in the control group compared to the EM-grid group and OPS group (p < 0.05), yet no difference was noted between the control group and cryo-loop group. Development rate and hatching rate of eight cell morulae and blastocysts were all lower in the treatment groups than the control group whilst hatching rate of blastocysts was higher in the control group compared to the groups of EM-grid and OPS (p < 0.05); although the cryo-loop group was shown to be slightly higher than other groups, it was not statistically significant. Conclusions: In the study, we investigate effects of freezing containers on vitrified embryos of respective developmental stages; it was demonstrated that higher developmental rate was shown in more progressed (or developed) embryos with more blastomeres. There was however, no difference in embryonic development rate was shown amongst containers. Taken together, further additional studies are warranted with regards to 1) manipulation techniques of embryos for various vitrification freezing containers and 2) preventive measures against contamination via liquid nitrogen.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.3
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• pp.110-115
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• 2018

Objective: To compare our in-house method of embryo freezing with Cryotop vitrification in terms of immediate survival, subsequent cleavage and blastocyst formation, and cell numbers in blastocysts. Methods: Two-cell mouse embryos were randomly allocated into three groups: a non-frozen control group (group 1, n = 300), a group that underwent Cryotop vitrification (group 2, n = 300), and a group that underwent our in-house freezing method (group 3, n = 300). Results: There were no significant differences between groups 2 and 3 in the immediate survival rate (96.3% vs. 98.6%, respectively; p= 0.085), the further cleavage rate (91.7% vs. 95.0%, respectively; p= 0.099), or the blastocyst formation rate (80.7% vs. 78.6%, respectively; p= 0.437). The cell numbers in the blastocysts from groups 1, 2, and 3 were comparable ($88.99{\pm}10.44$, $88.29{\pm}14.79$, and $86.42{\pm}15.23$, respectively; p= 0.228). However, the percentage of good-quality blastocysts in the Cryotop vitrification group was significantly higher than in the group in which our in-house method was performed, but was lower than in the control group (58.0%, 37.0%, and 82.7%, respectively; p< 0.001). Conclusion: At present, our method is inferior to the commercial Cryotop vitrification system. However, with further improvements, it has the potential to be useful in routine practice, as it is easier to perform than the current vitrification system.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.203-209
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• 2011

Objective: This study was performed to compare the efficiency of slow freezing and vitrification based on survival, development to blastocysts, and cell numbers of blastocysts. Changes in embryonic gene expression in fresh and frozen-thawed embryos were also examined. Methods: Eight-cell stage embryos were collected from superovulated female BDF1 mice. The collected embryos were randomly divided into three groups. One group was maintained as fresh controls (n=42), one was frozen by slow freezing (n=43), and one was cooled by vitrification (n=43). After thawing or cooling, survival rates, development to blastocyst, and cell numbers and inner cell mass (ICM) cell numbers of blastocysts were compared with those of the control group. The expressions of eight genes ($Rbm3$, $Birc5$, $Sod1$, $Sod2$, $Cirbp$, $Caspase3$, $Trp53$, $Hsp70.1$) were examined by real time-quantitative polymerase chain reaction in the fresh and frozen-thawed embryos. Results: There were no significant differences in the slow freezing and vitrification groups' survival rate after thawing (88.4% vs. 88.4%), development to blastocyst (100% vs. 97.4%), cell numbers ($107.0{\pm}21.0$ vs. $115.0{\pm}19.7$), or ICM cell numbers of blastocysts ($11.3{\pm}5.2$ vs. $11.1{\pm}3.7$). Cell numbers of blastocysts were significantly ($p$ <0.05) lower in the frozen-thawed embryos than the fresh embryos. There were no significant differences in the slow freezing and the vitrification groups' expressions of the eight genes. The expressions of $CirbP$ and $Hsp70.1$ were higher in the frozen-thawed embryos than in the fresh embryos but there were no significant differences. Conclusion: These results suggest that there were no significant differences between embryos that underwent slow freezing and vitrification.

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.397-401
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• 2014

The boar sperm has more lipid droplets and specialty of seminal plasma compared with other species, causing difficulties of freezing sperm and decreases for the utilization of frozen semen into the artificial insemination. However, several studies reported significant results for the recovery of sperm motility and reproductive by addition of cryoprotectants and seminal plasma after thawing. This study was designed to investigate the effects of supplementation of trehalose or glycerol in the LEY (lactose and egg yolk in BTS) solution for the conventional freezing and vitrification process. Two boars aged 16 months were used to collect semen for 2 times in a week. The samples were allotted to 3 freezing solutions (LEY + glycerol 10.5% + OEP 1.5%, LEY + trehalose 1M + OEP 1.5%, and sucrose 1.5M + trehalose 1 M + OEP 1.5%) after centrifugation at 800 g for 10 minutes. Semen was equilibrated in freezing solutions for 10 minutes and injected into plastic straws with 2~3 air bubbles to minimize freezing damages. Vitrification was performed to locate sperm in 5 cm above $LN_2$ for 5 minutes, and the conventional freezing was conducted with an automatic freezer. Motility and survival rates were measured by CASA (Computer assisted sperm an alyzing system) and FITC (Fluorescein isothiocyanate), respectively after thawing semen at $50^{\circ}C$ for 12 seconds. The results were analyzed by ANOVA with STATVIEW statistical program. The vitrificatioin solution (LEY + 10.5% glycerol + 1.5% OEP) presented higher motility (20.9%) than other solutions while the solution (LEY + 1M trehalose + 1.5% OEP) showed the lowest (motility : 5.2%). However, survival rates of vitrified sperms detected by FITC showed 1~4% live sperms in almost of dead sperms at all vitrification solutions' groups, but survival rate of freezing solution of LEY + 1M trehalose + 1.5% OEP LEY and LEY + 10.5% glycerol + 1.5% OEP were showed 49%, and 79%, respectively. There were differences (P<0.05) survival rate of conventional freezing in LEY + 10.5% glycerol + 1.5% OEP and LEY + 1M trehalose + 1.5% OEP and the remaining showed no differences. The results suggested that vitrified boar semen was not enough to be utilized for the artificial insemination, but it showed possibility to utilize for ICSI and conventional freezing with glycerol would be useful method for artificial insemination in pig while we choose the outstanding semen against tolerance to freezing damages.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.1
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• pp.75-81
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• 2004

Objective: The purpose of this study was to evaluate the efficacy and effect of various cryopreservation method on the survival and the cytoskeletal stability of metaphase II mouse oocyte. Methods: Mouse ovulated oocytes were collected and cryopreserved by a modified slow-freezing method with 1.5 M 1, 2-propanediol (PrOH)+0.1 M sucrose or by vitrification using cryo loop and EM grid with 40% ethylene glycol+0.6 M sucrose. Four hours after thawing, intact oocytes were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-$\beta$-tubulin antibody to visualize spindle and propidium iodide (PI) to visualize chromosome. Spindle morphology was classified as follows: normal (barrel-shaped), slightly and absolute abnormal (multipolar or absent). Results: Survival rate of the frozen-thawed oocytes in vitrification group was significantly higher than that of slow-freezing group (62.7% vs. 24.4%, p<0.01). Vitrification with cryo loop showed significantly higher survival rate than that with EM grid (67.7% vs. 53.5%, p<0.05). On the other hand, proportion of normal spindle and chromosome configurations of the frozen-thawed oocytes between two vitrification group was not significantly different. Conclusion: For mouse ovulated oocytes, vitrification with cryo loop may be a preferable procedure compared to slow-freezing method. Further study should be needed to investigate developmental competency of frozen-thawed mouse oocytes.

• Korean Journal of Animal Reproduction
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• v.15 no.3
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• pp.173-177
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• 1991

In vitro survival of the mouse morulae frozen by vitrification method(Kasai et al., 1990) was investigated in the present study. The embryos were plunged into LN2 directly after exposure to the vitrification solutions(EFS, GFS and DFS). The results were obtained as follows. The viability of morulae after freezing and thawing was high in EFS(96.7∼100.0%) and GFS vitrification solution(93.3∼96.7%), and the lowest in DFS vitrification solution(0.00∼0.03%).

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.191-200
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• 2000

Objective: This study was carried out to compare the effects of the stepwise exposure treatments on the morphological normality, fertilization and blastocyst formation rate of the frozen-thawed mouse mature oocytes by vitrification or ultra-rapid freezing and to use as a fundamental data for the cryopreservation of human oocytes. Materials and Methods: The morphological normality and fertilization rates of the vitrified and ultra-rapid frozen mouse mature oocytes after three-stepwise exposure treatments (1step, 3step and 5step) were observed. After choosing the 3step exposure treatment groups, we observed the morphological normality and fertilization, blastocyst formation rate of the vitrified and ultra-rapid frozen mouse mature oocytes. Results: The morphological normality and fertilization rates of the vitrified mouse mature oocytes after three-stepwise exposure treatments (1step, 3step and 5step) were 75%, 85%, 88% and 58%, 61 %, 54% respectively. There were no significant differences among treatments(p>0.05). The morphological normality and fertilization rate of the control was 92% and 65%. There were no significant differences in fertilization rate among control and treatments (p>0.05). The morphological normality and fertilization rates of the ultra-rapid frozen mouse mature oocytes after three-stepwise exposure treatments (1step, 3step and 5step) were 83%, 83%, 84% and 75%, 63%, 56% respectively. There were no significant differences among treatments (p>0.05). The morphological normality and fertilization rate of the control was 95% and 67%. There were no significant differences among control and treatments (p>0.05). The morphological normality and fertilization rate of the vitrified or ultra-rapid frozen mouse mature oocytes after 3step exposure treatment were 69% and 75%, respectively. The blastocyst formation rate was 60% and 57%. The results did not differ significantly between vitrification and ultra-rapid freezing (p>0.05). Conclusion: As known in the above results, there were no significant differences in the fertilization and blastocyst formation rate of the frozen-thawed mouse mature oocytes by vitrification or ultra-rapid freezing among the control and treatments. It is suggested that vitrification and ultra-rapid freezing method were effective for the cryopreservation of mouse mature oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.67-74
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• 2000

Objective: This study was conducted to investigate the effect of vitrification on the implantation and the pregnancy of human blastocysts. Method: The transfer of the frozen-thawed blastocysts by the slow freezing or vitrification was performed between January 1998 and July 1999. The zygotes derives from IVF were cocultured with cumulus cells in YS medium containing 20% hFF for 5days. Two or three of the best balstocysts produced on day 5 were transferred into the uterus, and then supernumerary blastocysts were randomly divided into two groups. One was frozen by slow freezing and the other was frozen by vitrification method. The slow freezing procedure was performed in two steps (5% glycerol and 9% glycerol + 0.2 M sucrose for 10 min, respectively) using programmed freezer ($-2^{\circ}C$/min to $-7^{\circ}C$, manual seeding at $-7^{\circ}C$, $-0.3^{\circ}C$/min to $-38^{\circ}C$ and plunged into $LN_{2}$). The blastocysts frozen by slow freezing were thawed at $36^{\circ}C$ then removed glycerol in 7 steps. The vitrification procedure was performed in three steps (10% glycerol for 5 min, 10% glycerol + 20% ethylene glycol for 5 min, 25% glycerol + 25% ethylene glycol and directly $LN_{2}$ within 1 min). The blastocysts frozen by vitrification were thawed at $20^{\circ}C$ water then removed cryoprotectant in 3 steps. In each group, thawed blastocysts were cocultured with cumulus cells in YS medium containing 20% hFF for 18h and transferred into the uterus. The implantation rate was evaluated per transferred blastocysts and the pregnancy rate was evaluated per transfers. Results: The survival rate of vitrified group (74.5%) was higher than slow freezing group (68.0%), but not significant. When 98 thawed blastocysts of vitrification were transferred in 40 cycles, 19 pregnancies (clinical pregnancy rate; 47.5%) were established. One miscarriage occurred in the eighth week of pregnancy (ongoing pregnancy rate; 45.0%). 7 pregnancies were ongoing, 11 pregnancies went to term, and 16 healthy infants were born. The Implantation rate was 31.6%. These results were higher than those obtained by the slow freezing (clinical pregnancy rate; 40.3%, ongoing pregnancy rate; 32.5% and implantation rate; 25.3%), but not significant. Conclusion: Vitrification is a simple, quick and economical method when compared to slow freezing. It will be chosen as a good method of human embryo freezing in IVF-ET programs.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.141-149
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• 1997

This study was carried out to investigate the effect of vitrification and slow freezing methods on the post-thaw developmental rate of rabbit zygotes. After exposing rabbit zygotes in EFS solution for 0.5, 1, 2, 3 and S min at room temperature, they were washed with 0.5 M sucrose solution, D-PBS and TCM-199 and then cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) to examine whether the cryoprotectant induced injury during the various exposure periods. The embryo development rates to hatched blastocyst after exposing in EFS solution for 3 and 5 min(40.0 and 16.7%) were significantly lower than in 0.5, 1 and 2 min(63.0, 72.0 and 54.5%), respectively. The post-thaw development rates to hatched blastocyst were significantly(P<0.05) higher in in vivo morula with intact mucin coat(85.2%) and mucin seperated morula(77.8%) than those of in vitro morula(58.5%) and zygote(5.9%), hut no difference was shown between in vitro morulae and mucin separated morula. The cryoprotectant dilution procedures showed no effects on the post-thaw development rates to hatched blastocyst under the present culture conditions. The post-thaw development to hatched blastocyst in the rabbit zygotes was not significantly different between the slow freezing(12.8%) and vitrification(5.9%). These results indicated that the rabbit frozen zygotes could he successfully developed in vitro to hatched blastocysts, though their developmental rate was very low, compared with morula stage embryos, in either vitrification or slow freezing procedure under the present conditions.

• Journal of Embryo Transfer
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• v.8 no.1
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• pp.1-7
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• 1993

This study was carried out to investigate the survival rates of late mouse molulae frozen in the state of vitrification and then thawed after equilibrating them separately in EFS 40, GFS 40 and DFS 40 at 1$0^{\circ}C$. The results obtained are as follows : 1. Freezing in the state of vitrification and thawing late mouse molulae after equilibrating them at l0$0^{\circ}C$ in EFS 40 for 30 seconds, one minute and two minutes, we obtained survival rates of 76.7%, 96.7% and 100%, respectively. 2. Freezing and thawing them after equilibrating at 1$0^{\circ}C$ in GFS 40 for 30 seconds, one minute and two minutes, we obtained survival rates of 60%, 96.7% and 10%, respectively. These results are as similar as in the case of EFS 40. 3. Freezing and thawing them after equilibrating at l$0^{\circ}C$ in DFS 40 for 30 seconds and one minute, we obtained survival rates of 62.1% and 0%, respectively. These results represent lower survival rates than those obtained with EFS 40 and GFS 40. In conclusion, even equilibrating late mouse molulae in EFS 40 and GFS 40 at 1$0^{\circ}C$ for more than one minute gives a survival rate of more than 97%, while equilibrating them in DFS 40 at 1$0^{\circ}C$ for more than one minute results in a 0% survival rate, which means that DFS 40 has a strong toxicity.