• Clinical and Experimental Reproductive Medicine
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• v.20 no.1
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• pp.31-36
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• 1993

The effects of freezing and 1,2-propanediol on early and late pronucleate stage mouse ova were investigated in terms of survival after thawing and development in vitro. The samples were divided into two groups according to different age in pronucleate ova: ova in(1) early pronuclear stage with two distant pronuclei at 18h after hCG injection, and (2) late pronuclear stage with adjacent pronuclei at 30h. Zygotes in the late pronuclear stage have been proven to be more resistant to 1,2-propanediol, showing a significantly higher developmental rate than zygotes in early stage (80.3 versus 66.3%, <0.05), but survival rate was similar in the two groups (91.0 versus 93.5%). After freezing and thawing, survival and developmental rates were decreased in both groups when compared to the control group (54.3 versus 92.3%, 47.7 versus 73.3%. respectively). And developmental rate in the late pronuclear stage zygotes showed significantly higher than in early (55.4 versus 40.0%) after thawing. In conclusion, early pronucleate mouse ova have a lower developmental capacity in vitro and a lower survival rate after freezing and thawing than late ova. These findings suggest that the timing of freezing could be important for survival and further development in vitro in cryopreservation of human pronucleate ova.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.73-73
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• 2003

The present study examined the possibility of long term storage, by cryopreservation in liquid nitrogen, of the sperm of Filefish (Thamnaconus septentrionalis), and the changes in motility, survival rate and ultrastructure of the sperm after freezing and thawing. The sperm was collected by stripping and stored on ice until experiments. For selection of the immobilizing solution, diluted artificial seawater (ASW) of 20, 30 and 40% were tested. The sperm motility was significantly inhibited in 30% ASW, and restored entirely after 100% ASW was added again. Two cryoprotectants, dimethyl sulfoxide ($Me_2$SO) and glycerol, were added to 30% ASW to formulate the extenders at the concentrations between 5 to 20% by volume for freezing. The sperm was diluted at the ratio of 1 :6 with the extenders, inserted into 0.5ml plastic straws and frozen at a freezing rate of $50^{\circ}C$/min to $-100^{\circ}C$ after equilibration for 10 min at room temperature, followed by plunging into liquid nitrogen. The straws were thawed in a $30^{\circ}C$ water bath for 15 sec. The highest post-thawed sperm motility and survival rate were obtained with 5% glycerol Afterward, the effect of different freezing rates was examined using 5% glycerol as a cryoprotectant, and the rate of $20^{circ}C/min to $-80^{\circ}C$ showed the best result Some ultrastructural changes of sperm, such as the detachment of plasmatic and nuclear membranes, destruction of mitochondria, were observed after cryopreservation. Morphological normality of the sperm in 5% glycerol frozen at the ratio of 1$0^{\circ}C$/min to $-80^{\circ}C$ was better than that of others.

• Journal of Animal Science and Technology
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• v.48 no.6
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• pp.797-804
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• 2006

The present study was undertaken to investigate the post-thawed survivability of bovine embryo depending on different dose of ethylene glycol and sucrose. Ovaries were collected at local slaughterhouse and the cumulus-oocyte-complexes aspirated from ovaries were in vitro matured, fertilized and cultured at 39°C in an atmosphere of 5% CO2 incubator. For conventional slow-freezing, d 7 or 8 expanded blastocysts were collected. Embryos were equilibrated in 1.5 M and 1.8 M ethylene glycol(EG) with 0.1 M and 0.3 M sucrose in Dulbecco's phosphate-buffered saline(D-PBS) supplemented with 0.5% bovine serum albumin. Embryos were then loaded individually into 0.25ml-straw and placed directly into cooling chamber of programmable freezer precooled to 󰠏7°C, after 2 min, the straw was seeded, maintained at 󰠏7°C for 8 min, and then cooled to 󰠏35°C at 0.3°C/min, plunged and stored in liquid nitrogen for at least 3 days. For thawing, the straw containing embryos were warmed in air for 10 sec and exposed to 37°C water for 20 sec. Straws were then removed from 37°C water. Rates of blastocyst survive and hatching were evaluated at 24 to 72 h post-warming. No difference of the survivability was shown between 1.5 M and 1.8 M EG (71 and 70%, respectively). Addition of 0.1 M sucrose to 1.5 M and 1.8 M ethylene glycol in the freezing solution did not differ significantly embryo survival (74 and 77%, respectively), whereas survival rates was higher(89%) in freezing solution contained 0.3M sucrose to 1.8M EG compared with 0.3M sucrose to 1.5M EG group(71%). However, there was no difference in the overall total cell number between the two groups (122±1.8 vs 131±1.4, respectively). In conclusion, the results suggest that 0.3 M sucrose in 1.8 M EG may be optimal condition for freezing and thawing methods with in vitro produced embryos and may be applied to on-farm conditions for embryo transfer.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.1
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• pp.9-17
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• 2004

Objective: The aim of this study were to compare the effects of EG and PROH on cryopreservation of mouse and human embryos, and to find the optimal protocol for embryo freezing. Methods: Human embryos derived from fertilized eggs showing 3 pronuclei (PN) and mouse embryos were divided into two groups respectively: dehydrated with 1.5 M EG + 0.2 M sucrose or 1.5 M PROH + 0.2 M sucrose using the slow freezing method. Moreover mouse embryos were controlled the exposure time of cryoprotectant during dehydration or rehydration steps. Results: The survival rates of human embryos were 79.2% (84/106) in EG group and 77.9% (88/113) in PROH group. In mouse embryos, the survival and development rates up to blastocyst were 70.6% (245/347), 44.1% (123/279) in EG group and 62.1% (198/319), 45.1% (123/279) in PROH group, respectively. However, in EG group, partially damaged embryos after thawing were decreased compared to PROH group. In combination group, when the exposure time during dehydration and rehydration were reduced, the survival and embryonic developments were increased slightly, but not significant. Conclusion: Cryopreservation of mouse and human embryos at cleavage stage by using EG or PROH exhibited no statistical difference in the survival rate and/or developmental rate to blastocyst. However, the use of EG for cryopreservation of embryos might reduce the exposure time of the cryoprotectant because of a high permeation of EG and result in lessen its toxic effects.

• Korean Journal of Agricultural and Forest Meteorology
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• v.16 no.1
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• pp.51-58
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• 2014

Freezing hardiness of winter bud and branch of several pear (Pyrus pyrifolia) cultivars according to degree and duration of low temperatures was investigated by sprouting, electrolyte leaching rate and triphenyltetrazolium chloride (TTC). Sprouting rate as infected by degree and duration of low temperature were different between cultivars. The lower temperature, the longer duration, sprouting rate was decreased. Electrolyte leaching rate was showed above 30% at below $-30^{\circ}C$ treatment regardless of cultivars and duration. The lower temperature and the longer duration, Electrolyte leaching rate was increased. Electrolyte leaching rates of Manpungbae, Niitaka and Chuwhangbae at $-30^{\circ}C$ for 9 hours treatment which were observed high sprouting rate, were lower than those of other varieties. Absorbance rates by TTC test at $-21^{\circ}C$ treatment were 66.0 to 96.5% for 6 hours, 49.4 to 91.9% for 9 hours, and 37.3 to 89.4% for 12 hours. Freezing hardiness of pear cultivars at ecodormancy was different according to degree and duration of low temperature treatments.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4669-4675
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• 2012

Objective: Nude mice with orthotopic transplantation of human ovarian epithelial cancer were used to investigate screening criteria for paraneoplastic normal ovarian tissue and the security of the freezing and thawing for ovarian tissue transplantation. Methods: Expression of CK-7, CA125, P53, survivin, MMP-2/TIMP-2 in paraneoplastic normal ovarian tissues were detected by RT-PCR as well as immunohistochemistry. The tissues of the groups with all negative indicators of RT-PCR, all negative indicators of immunohistochemistry, negative expression of CK-7, CA125 and survivin, positive expression of CK-7, CA125 and survivin, cancer tissues and normal ovarian tissues of nude mice were used for freezing and thawing transplantation, to analyze overt and occult carcinogenesis rates after transplantation. Results: When all indicators or the main indicators, CK-7, CA125 and survivin, were negative, tumorigenesis did not occur after transplantation. In addition the occult carcinogenesis rate was lower than in the group with positive expression of CK-7, CA125 and survivin (P<0.01). After subcutaneous and orthotopic transplantation of ovarian tissues, rates did not change (P>0.05). There was no statistical significance among rates after transplantation of ovarian tissues which were obtained under different severity conditions (P>0.05). Conclusion: Negative expression of CK-7, CA125 and survivin can be treated as screening criteria for security of ovarian tissues for transplantation. Immunohistochemical methods can be used as the primary detection approach. Both subcutaneous and orthotopic transplantation are safe. The initial severity does not affect the carcinogenesis rate after tissue transplantation. Freezing and thawing ovarian tissue transplantation in nude mice with human epithelial ovarian carcinoma is feasible and safe.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.195-199
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• 2011

The objective of this study was to evaluate the efficiency of the conventional slow freezing and vitrification methods for cryopreserving in vivo and in vitro-produced bovine embryos. Morphology of post-thawed embryos was evaluated and normal embryos were used for successive culture for 72 h. In experiment I, In embryo viability, There was no significant differences in blastocyst re-expansion rates were found between in vivo and in vitro embryos(89.6% vs. 81.5%). whereas hatched-BL and total cell number rates was significantly higher (p<0.05) for in vivo-derived embryos (76.9%, 136${\pm}$3.6 vs. 43.4%, 107${\pm}$3.8). In experiment II, There was no significant differences in blastocyst re-expansion and Expansion-BL rates were found between in slow freezing and vitrification methods (91.3% vs. 85.7% and 71.4% vs. 75.0%, respectively). in conclusion, These results suggested that the field application for bovine embryo transfer is in part supported by improvements of technologies in embryo conventional slow freezing and vitrification cryopreservation.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.225-230
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• 2000

This study was designed to investigate effect of cryoprotectant kinds and cell stages on the viability of bovine embryos cryopreserved by vitrification. The oocytes were collected from ovarian follicles of Korean native cows. The follicular oocytes were cultured in TCM-199 medium containing hormone and 10%(v/v) FCS for 24~48hrs in a incubator with 5% $CO_2$, in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 7~10 hrs with spermatozoa capacitated by preincubation. The vitrification solutions of EFS and EDS were consisted of 40%(v/v) ethylene glycol, 18%(v/v) Ficoll and 0.3M sucrose, and 20%(v/v) ethylene glycol, 16.5%(v/v) DMSO and 0.5M sucrose in TCM-199 medium supplemented with 10% FCS, respectively. The embryos were exposed to EFS or EDS at $25^{\circ}C$ and loaded into OPP straw for 30 sec. The plug end of each straws was heat-sealed and straws was slowly immersed into liquid nitrogen(L$N_2$). The results obtained were summarized as follows : 1 . The rates of cleavage and hatching of embryos frozen with vitrification, rapid and slow freezing methods were 67.5%, 27.5% and 42.5%, 20.0% and 52.5%, 25.0%, respectively And rates of cleavage and hatching of embryos frozen with vitrification method were significantly(p<0.05) higher than those in other methods, and the rates were lower than those in control group(82.5% and 37.5%). 2. The rates of cleavage and hatching of embryos were significantly(p<0.05) different between EFS(47.5% and 22.5%) and EPS(52.5% and 27.5%), and the rates were lower than those in control group(82.5% and 37.5%). 3. After vitrification freezing of bovine embryos at zygote, 2 cell, 8 cell, morulae and blastocyst stage, the rate of cleavage and hatching were 25.0% and 15.0%, 32.5% and 20.0%, 37.5% and 20.0%, 52.5%, 27.5%, 47.5% and 25.0%, respectively. And developmental rates to the expended blastocyst stage of embryos frozen at zygote stage was significantly(p<0.05) lower rather than those in 2, 8-cell and morulae stage.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.199-205
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• 2009

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 ${\mu}g/ml$ porcine FSH, 0.5 ${\mu}g/ml$ equine LH, 1.0 ${\mu}g/ml$ 17 $\beta$-estradiol ($E_2$) and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ${\mu}l$) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.3
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• pp.241-248
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• 2003

Objective: The purpose of this study was to evaluate the survival rate of vitrified blastocyst according to the freezing vessels, equilibration time in cryoprotectant and artificial dehydration method. Methods: Human blastocysts were vitrified after loading onto the plastic straw, open-pulled straw (OPS), electron microscopy grid (EM grid) for 1.5 min or 3 min. They also were directly plunged into LN2 within 30sec. For artificial shrinkage of blastocysts, 36 gauge fine needle was pushed at the cellular junction of the trophectoderm into the blstocoele cavity until it shrank without damage of inner cell mass. Results: The survival rate of vitrified blastocysts on plastic straw, OPS, EM grid as freezing vessels were 26.7, 13.0 and 60.5%, respectively. The survival rate of EM grid was significantly higher than that of plastic straw and OPS (p<0.05). For 1.5 min equilibrium, the survival rates of early blastocyst (EB), middle blastocyst (MB) and late blastocyst (LB) were 64.4, 81.0, and 20.0% respectively. For 3 min equilibrium, the survival rates of EB, MB, and LB were 69.9, 50.0 and 57.5% respectively. The survival rates of EB and MB were significantly higher than that of LB in 1.5 min equilibrium group (p<0.05), however, the significance was not observed in 3 min equilibrium groups. In cytoplasmic shrinkage before vitrification, the survival rates of EB, MB and LB were 92.9, 100 and 75.9% respectively. The survival rate of MB was significantly higher than that of LB (p<0.05). The survival rates of vitrified blastocysts by artificial dehydration and slow-frozen blastocysts were not significantly different as 88.9 and 66.7%, respectively. Conclusion: This study showed that the vitrification of human blastocysts using EM grid and artificial dehydration is an effective method. Therefore, these methods would be an useful techniques for blastocyst cryopreservation.