• Proceedings of the KSAR Conference
• /
• 2002.06a
• /
• pp.9-9
• /
• 2002

This study was to evaluate the in vitro survival of vitrified-thawed bovine MII enucleated (MIIe) oocytes according to activation timing and minimun volume cooling (MVC) method and their in vitro development after somatic cell nuclear transfer (SONT). Bovine oocytes were recovered from slaughtered bovine ovary and matured in TCM-199 supplemented with 10% FBS. (omitted)

• Clinical and Experimental Reproductive Medicine
• /
• v.24 no.3
• /
• pp.347-353
• /
• 1997

This study was to test whether in vitro/in vivo survival of vitrified mouse blastocysts was influenced by culture conditions and ET method. Mouse blastocysts were obtained from in vitro fertilization and cultured for 4 days in M16 medium, and they were vitrified in EFS40 which contained 40% ethlyene glycol, 18% Ficoll and 0.5 mol sucrose in PBS. In experiment I, in vitro and in vivo survival rate of these embryos were evaluated in different culture condition after thawing. When thawed embryos were cultured in M16 medium as a control, m-CR1 medium contained 20 amino acids (2% BME amino acis and 1% MEM non-essential amino acids solution) and 4 mg/ml BSA and cumulus monolayer cell co-cultured condition in mCR1 medium (10% FBS), their in vitro survival at 24 hr after thawing was not affected by culture condition (75.6, 83.1, 82.4%). However, in vivo survival rates of implantation in m-CR1 medium (80.4%) were significantly higher than those of M16 medium (51.2%), co-culture (57.1%) condition, although there was no difference in live fetuses rates on day 15 gestation (39.0, 49.0, 38.1%). In experiment II, the in vivo development potential of embryos by ET methods was examined. When blastocysts were transferred to the day 2, 3 pseudopregnant recipient without culture soon after thawing, no pregnant recipient was obtained on the day 2 pseudopregnancy, and 50% of pregnancy rates and 15.4% of live fetus rates were obtained on the day 3 pseudopregnant recipients. These results were significantly lower than those of transferred group (day 3 pseudopregnant recipients) after culture for 16 hr post thawing (73.5, 57.1%) (p<0.05). In experiment III, to elevate usability of delayed embryos in vitro/in vivo survival of vitrified embryos (day 4 early, day 5 early and expanding blastocyst) were examined. in vivo survival rates (live fetus, total implantation) were higher in day 4 early blastocysts (33.3, 66.7%) than in day 5 expanding blastocysts (29.0, 38.7%), although the highest in vitro survival rates were obtained in the day 5 expanding brastocysts (78.3%). Therefore, these results suggest that the in vitro/in vivo survival rates of vitrified embryos could be improve by the culture condition and ET method and that the in vivo development rates of delayed embryos were decreased with longer culture duration in vitro. It means that more effective cryopreservation was obtained in day 4 early blastocysts than in day 5 expanding blastocysts.

• Clinical and Experimental Reproductive Medicine
• /
• v.17 no.2
• /
• pp.167-172
• /
• 1990

This study was carried out to clarify the effects of various kinds of cryoprotectants which were frequently used in freezing embryos of domestic animals on the survival of frozen-thawed mouse embryos. Mouse embryos were collected by hyperstimulation induction of ICR mouse. The samples were slowly cooled ($l^{\circ}C/min$) to temperatures between $-7^{\circ}C$ and $-30^{circ}C$ before direct transfer to liquid nitrogen ($-196^{\circ}C$) and thawed rapidly ($-500^{\circ}C$/min). As cryoprotectants, Glycerol, DMSO, Ethylene glycol and Propylene glycol were used and applied each 2 cell, 8 cell, morula in embryo stage. After normal mouse embryos developed to blastocyst by in vitro culture, we observed recovery rate and developing rate of embryos at thawing. The results obtained in these experiments were as follows : 1. The in vitro development rate from the frozen-thawed 2 cell embryos to the blastocyst were 67.7% in ethylene glycol, 65.7% in Propylene glycol, 55.2% in glycerol and 50.0% in DMSO respectively. 2. The in vitro development rate from the frozen-thawed 8 cell embryos to the blastocyst were 83.6% in DMSO, 75.7% in glycerol, 52.2% in propylene glycol respectively. 3. The in vitro development rate from the frozen-thawed morula to the blastocyst were 84.2% in glycerol, 80.0% in DMSO, 66.6% in propylene glycol and 55.2% in ethylene glycol respectively.

• Korean Journal of Animal Reproduction
• /
• v.20 no.1
• /
• pp.9-17
• /
• 1996

This study was conducted to investigate freezability of in vitro and in vitro matured rabbit oocytes, possibility of NT using frozen-thawed unfertilized oocytes, and NT efficiency by zona-slit micromanipulation. After freezing of in vitro matured oocytes, 33 to 49% of oocytes appeared normal morphology and 1.0M DMSO and 1.5M glycerol showed slightly high survival rate, but there was no difference in survival between two cryoprotectants. Freezability of in vitro matured oocytes was low in 1.5M glycerol and more sensitive to freezing. Efficiency of enucleation and fusion rate in method B was higher than that in method A and no difference in this efficiency was between 3 groups of oocytes in method B. Cleavage rate and developmental capacity to M＋B stage of fused embryos derived from frozen oocytes was greatly lower than that from fresh oocytes, respectively(39.1% : 79.5% ; 3.1% : 19.3%) and there was no difference in cleavage rate between DC voltages in two group oocytes. Additional incubation in cytochalasin B after electrical stimulation did not affect embryo development. In conclusion, it is suggested that enucleation and nucelar transfer by slitting of zona is more effective method in rabbit and that further study on optimum freezing conditions for in vitro matured oocytes is necessary to use as recipient oocytes.

• Journal of Embryo Transfer
• /
• v.8 no.1
• /
• pp.31-36
• /
• 1993

The post-thaw survival of mouse embryos of the various developmental stages was determined after cryopreservation by vitrification in a solution containing ethylene glycol, Ficoll and sucrose (EFS). All the embryos were equilibrated for 2 minutes just prior to freezing. The number of blastomeres during in vitro development was counted by nuclei higher rates of post-thaw survival were obtained from the embryos of 2-cell(92.2%), 8-cell(77.2%) or morula stage(90.0%) than those of blastocyst stage(62.7%). The number of blastomeres per embryo following in vitro culture for 24 hours was significantly(P<0.05) smaller as 66.0f22.3 in vitrified and thawed morulae than fresh morulae(91.7$\pm$12.2).

• Proceedings of the KSAR Conference
• /
• 2001.03a
• /
• pp.5-5
• /
• 2001

This study was to test whether Hanwoo in vitro matured oocytes can be successfully cryopreserved by a new vitrification procedure using MVC method. For the vitrification, oocytes were pretreated in 10% ethylene glycol (EG10) for 5-10 min, exposed in EG30 for 30 sec, each oocytes were individually put on the inner wall of 0.25 $m\ell$ straw, and then straws were directly plunged into L$N_2$. Thawing was taken by 4-step procedures [1.0 Msucrose (MS), 0.5 MS, 0.25 MS, and 0.125 MS] at 37$^{\circ}C$. In vitro developmental capacity (survival, cleavage ($\geq$2-cell) and blastocyst rates) in vitrified group was no significant difference compared to that in other treatment groups (exposed; 100.0, 74.4, 32.3% and control; 100.0, 78.3, 36.3%): high mean percentage of oocytes (91.2%) was survived, 69.4% of them were cleaved and 27.9% of cleaved embryos were developed to blastocyst. Especially, after transfer of in vitro developed embryos in vitrified group, four of six recipient animals were found to pregnant and three of them were ongoing pregnant by manual palpation at 250 days after transfer. However, among them, two healthy female calves (23 and 25kg) were born. This result demonstrates that MVC method is very appropriate freezing method for the Hanwoo in vitro matured oocytes and that ovum bank can be maintained efficiently by MVC cryopreservation method.

• Korean Journal of Veterinary Research
• /
• v.27 no.2
• /
• pp.331-334
• /
• 1987

The effects of ethylene glycol as a cryoprotective agent on the survival of frozen-thawed mouse embryos were examined. The effects of the stage of development of ethylene glycol were also examined. Eight-cell embryos, morulae, early blastocysts and mid-blastocysts were recovered from superovulated immature ddY mice. Ethylene glycol was added to the embryos in 5 equal increments 5 minutes apart, giving a final concentration of 1.5M. The embryos were cooled to ${-6^{\circ}C}$ at ${1^{\circ}C/min}$ and seeding was induced at ${-6^{\circ}C}$. After being held for a further 5minutes at the seeding temperature, the samples were cooled to ${-35^{\circ}C}$ at ${0.3^{\circ}C/min}$ and then transferred to liquid nitrogen. Rapid thawing was done by placing the straws in ${37^{\circ}C}$ water. The thawed embryos were diluted in PBS of same time and manners as adding procedures. Survival of 8-cell embryos and morulae were assessed as a normal development of the embryos to the blastocyst stage and expanding blastocyst after 54 hours and 48 hours of in vitro culture, respectively. While those of the early and mid-blastocysts were assessed to the expanding blastocyst stage after 24 hours of in vitro culture. The survival rates of 8-cell embryos, morulae, early blastocysts and mid-blastocysts were 73.8%, 74.3%, 87.8% and 77.4%, respectively. Significant difference on the survival rate among the four stages of development was not observed.

• Korean Journal of Animal Reproduction
• /
• v.21 no.3
• /
• pp.287-292
• /
• 1997

This study was carried out to investigate the effects of concentration and kinds of cryoprotectants, equilibraction time, thawing temperature and time, sucrose concentration on the survival rates of frozen bovine demi-embryos. The bovine demi-embryos following dehydration by cryoprotectants a various concentration of sucrose were freezed by cell freezer and thawed in 3$0^{\circ}C$ water bath. Survival and in vitro developmental rates was defined as development rates on in vitro culture or FDA-test. The results are summarized as follows : 1. The high survival rates of demi-embryos after frozen-thawing in freezing medium was attained 2.0M glycerol. The high survival rates of demi-embryos after frozen-thawing in freezing medium was obtained using single cryoprotectant(25.0~30.0%) than mixed cryoprotectants(16.7~19.0%). 2. The survival rates of demi-embryos after frozen-thawing in freezing medium added 1.5M, 2.0M glycerol＋0.25M sucrose(37.5~33.3%) were higher survival rates than those of sucrose concentration of 0.50, 0.75M(12.5~26.7%). 3. The equilibration time on the survival rates of demi-embryos was attained after short period of time(30.0~35.0%) in the freezing medium higher than long period of time(21.1%). 4. The thawing temperature on the survival rates of demi-embryos was attained at 3$0^{\circ}C$ of thawing temperature(26.7~40.0%) higher than $25^{\circ}C$ or 37$^{\circ}C$ of thawing temperature(13.3~20.0%). 5. The thawing time on the survival rates of demi-embryos was attained at 1~5 minutes of thawing time(26.7~33.3%) in the freezing medium higher than 10 minutes of thawing time(13.3~18.8%).

• Journal of Embryo Transfer
• /
• v.17 no.2
• /
• pp.117-121
• /
• 2002

This study was carried out to investigate the in vitro fertilization rate of canine immature oocytes cryopreserved by vitrification freezing. The vitrification solutions of EPS and EDS were consisted of 40％ ethylene glycol 18％ Ficoll and 0.3M sucrose, and 20％ ethylene glycol, 16.5％ DMSO and 0.5M sucrose in TCM-199 medium supplemented with 10％ FCS, respectively. The oocytes were exposed The developmental rate of in vitro cultured oocytes recovered from ovaries collected at different stages of the reproductive cycle were 3.8％, 10.7％, 46％, respectively. to EFS or EDS at $25^{\circ}C$ and loaded into straw fer 30 sec. The straws was slowly immersed into L$N_2$. Fertilization and survival rate was defined as development rate on in vitro culture or FDA-test. 1. The fertilization rate after vitrification freezing of immature oocytes at 1, 6, 12 and 24 hrs after collection from ovaries was very low(5.3％～31.4％) than the unfrozen oocyte(60.0％). And the fertilization rate after vitrification freezing of immature oocytes was very higher than that of mature oocytes. 2. The survival rate after vitrification freezing of immature oocytes at 1, 6, 12 and 24 hrs after collection from ovaries was 55.0％, 40.0％, 28.6％ and 17.1％, respectively. And the survival rate after vitrification freezing of immature oocytes was slightly higher than that of mature oocytes.

• Journal of Embryo Transfer
• /
• v.24 no.4
• /
• pp.253-257
• /
• 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 $37^{\circ}C$.