• Journal of Embryo Transfer
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• v.11 no.2
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• pp.125-135
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• 1996

The present study was carried out to investigate the effects of cryoprotectants, equilibration step, freezing rate, culture condition following in vitro fertilization, and age and development stage of embryo by freezing with conventional slow freezing and vitrification on survival of frozen-thawed Korean native cattle(KNC) blastocysts produced in vitro. The KNC blastocysts produced in vitro were equilibrated in 1.8M ethylene glycol or 1.4M glycerol and cooled from -6$^{\circ}C$ to -35$^{\circ}C$ at -0.3$^{\circ}C$ or -O.6$^{\circ}C$ /minute. When equilibrated in 1.8M ethylene glycol, survival rate of fiozen4hawed blastocysts was sarne in both -0. 3$^{\circ}C$ /min and -0.6$^{\circ}C$ /min cooling rate(71.4%). With the equilibration in 1.4M glycerol, survival rate was higher in -0.3$^{\circ}C$ /min(63.6%) than in -0.6$^{\circ}C$ /min cooling rate(53.8%). For vitrification of the KNC blastocysts produced in vitro, they were equilibrated in 2-step or 3-step exposure to vitrification solution(25% ethylene glycol + 25% glycerol). Survival rate was sirilar in both 2-step(45.0%) and 3-step exposure(47.4%). According to culture condition following in vitro fertilization, higher survival rate was obtained for blastocysts co-cultured with bovine oviductal epithelial cell(BOEC, 77.3%) than for those cultured with epidermal growth factor(EGF, 65.7%) or for those co-cultured with BOEG + EGF (54.8%). According to embryo age and development stage, higher survival rate was obtained for 7-day ernbryos(70.0%) than 8-day(56.8%) or 9-day(20.0%) for blastocyst stage and obtained for 8-day embryos(74.3%) than 7-day(62.5%) or 9-day(42.9%) for exponded blastocyst. In surnmary, higher survival rate of frozen4hawed KNC blastocysts produced in vitro were obtained by using ethylene glycol for cryoprotectant and -0.3$^{\circ}C$ /min for cooling rate. And higher survival rate were obtained with co-culture with BOEC for culture condition following in vitro fertilization and with 7-day blastocyst or 8-day expanded blasto cyst for embryo age and development stage.

• Korean Journal of Animal Reproduction
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• v.19 no.4
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• pp.265-270
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• 1996

This study was carried out to investigate on the survival rates and in vitro developmental rates of bisected bovine embryos by micromanipulator and micropipette. Bisected embryos were cultured for 1∼5 days in 20% FCS＋TCM-199 medium. Survival rate and in vitro developmental rate were defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The survival rates of intact or free zona pellucida of bisected embryos were 30.3 and 25.0%, respectively. And the survival rates of bisected embryos by micromanipulator and micropipette were 33.3 and 26.7%, respectively. The survival rate of bisected embryos was significantly lower than that of non-bisection embryos(65.0%). 2. The survival rates of bisection embryos in cultured for 12, 24, 48, 72 hrs with 20% FCS＋TCM-199 medium were 40.0, 30.0, 23.3 and 13.3%, respectively. 3. The in vitro developmental rates of intact of free zona pellucida of bisected embryos by micromainipulator and micropipettes were 33.3 and 26.7%, respectively. The survival rate of bisection embryos was significantly lower than that of non-bisection embryos(45.0%).

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2006.10a
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• pp.40-41
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• 2006

• Korean Journal of Animal Reproduction
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• v.19 no.2
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• pp.129-134
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• 1995

This study was carried out to investigate on the survival rate and in vitro developmental rate of bisected porcine embryos and immature oocytes by manipulator and micropipette. Bisected embryos and immature oocytes cultured for 1∼5 days in TCM 199 medium with 20% FCS. Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The survival rate of bisected porcine embryos and oocytes were 26.1%, 22.7%, respectively. The survival rate of bisected embryos and oocytes was significantly lower than that of non-bisection embryos(62.5%). 2. The survival rate of bisected porcine embryos in cultured for 12, 24, 48, 72 hrs with 20% FCS＋TCM-199 medium were 26.9%, 19.2%, 19.2% and 11.5%, respectively. 3. The in-vitro developmental rate with and without zona pellucida of bisected porcine embryos by micromanipulator were 30.8%, 25.0%, respectively.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.25-29
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• 1994

This study was carried out to investigate the in-vitro fertilization rate survival rate of rapidly frozen porcine immature embryos. The porcine embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water bath. Survival rate was defined as development rate on vitro culture or FDA test. The results are summarized as follows : 1. The in vitro fertilization rate of all frozen immature oocytes(6.7~26.7%) was very low, 35.0% of unfrozen oocytes and the rate of immature oocytes was very higher than that of mature oocytes. 2. The survival rate of all frozen immature oocytes(10.3~25.0%, 13.3~30.0%) was very low, 45% of unfrozen oocytes and the rate of immature oocytes was slightly higher than that of mature oocytes.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.115-119
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• 2009

In the present study, effects of concentration of cryoprotectant solutions on the nuclear maturation of vitrifiedthawed porcine oocytes were examined. Oocytes were cultured in TCM-199 medium supplemented with 5% FBS at $38^{\circ}$C in 5% $C0_2$ and air. The percentage of monospermy in the toxicity group and vitrification group (22.0 ${\pm}$ 3.0% and 31.5 ${\pm}$ 3.5%) was decreased compared with that of the control group (44.0 ${\pm}$ 4.0%). The percentage of in vitro development to blastocyst in the toxicity group and vitrification group (12.0 ${\pm}$ 2.5% and 14.8 ${\pm}$ 2.8%) was decreased compared with that of the control group (28.0 ${\pm}$ 3.0%, p<0.05). The survival and in vitro developmental rate of oocytes vitrification-thawed with EDS and EDT + TCM-199 medium supplemented with 0.1% PVA were 46.3 ${\pm}$ 3.0%, 54.5 ${\pm}$ 3.8% and 14.8 ${\pm}$ 2.5%, 16.4 ${\pm}$ 2.7%, respectively. This results were lower than the control group (28.0 ${\pm}$ 3.5%). The in vitro developmental rate of embryos vitrified with EDS and EDT supplemented PVA did not have a significant difference. The survival and in vitro developmental rate of vitrified-thawed morula and blastocyst embryos were 44.2 ${\pm}$ 3.5%, 17.3 ${\pm}$ 3.0% and 48.1 ${\pm}$ 4.2%, 18.5 ${\pm}$ 3.5%, respectively. Vitrified morulae and blastcyst embryos had a lower survival and developmental rates than their control counterparts.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.1
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• pp.7-12
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• 2001

Factors Affecting the Survival of Frozen Thawed Bovine In Vitro Produced Blastocysts. The effect of some factors on the post-thaw survival of a total of 240 in vitro produced bovine blastocysts was investigated using logistic regression analysis. The explanatory variables tested were: type of culture medium before freezing (TCM 199 supplemented with BSA, BSAITS (BSA+insulin+transferrin+selenium), ECS (estrous cow serum) with or without BOEC (bovine oviductal epithelial cells), age of the blastocyst (Day 7, Day 8+9), morphological appearance before freezing (distinct=Q1 or indistinct=Q2 inner cell mass) and type of cryoprotectant (glycerol, 1.0 M or ethylene glycol, 1.6 M). The survival after thawing based on the post-thaw quality and the development after co-culture with BOEC for 24 and 48 hours. Day 7 blastocysts had an almost three times better chance of survival than Day 8+9 blastocysts. Q1, Day 8+9 blastocysts had higher odds to survive after 48 hours in culture than Q2 blastocysts (p<0.05). Blastocysts produced in BSAITS medium had the best chances of survival; however, the odds were not always significant. Blastocysts frozen in glycerol had a better post-thaw quality rating than those frozen in ethylene glycol; however, the difference in post-thaw development at culture was not significant. The relationship between post-thaw quality and post-thaw development at culture was significant (p<0.05). The developmental stage and/or age of the embryo and culture medium where development up to blastocyst takes place affect the post-thaw survival of the bovine embryos.

• Korean Journal of Animal Reproduction
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• v.15 no.2
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• pp.141-147
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• 1991

This stduy was carried out in order to investigate the effects of cryoprotective concentration and equilibration time on survival rate of ultrarapidly frozen in vitro fertilized bovine embryos. In vitro fertilized bovine embryos, following dehydration by cryoprotective agents and sucorese were directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water. Survival rate was defined by development rate to the morula and blaqstocyst stage after in vitro culture of by FDA test. The results are summarized as follows : 1. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucroese added 2.0M, 2.5M, 3.0M, 3.5M, 4.0M glycerol were 75.0%, 72.0%, 67.6%, 44.8% and 18.3% respectively. 2. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0M, 2.5M, 3.0M, 3.5M, 4.0M DMSO were 64.0%, 66.7%, 70.8%, 52.7% and 18.6, respectively. 3. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0M, 2.5M, 3.0M, 3.5M, 4.0M propanediol were 68.4%, 64.9%, 63.2%, 62.2% and 34.7%, respectively. 4. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing medium of 2.50M glycerol added 0.1M, 0.25M, 0.5M, 0.75M, sucrose were 60.5%, 72.2%, 70.1% and 54.9%, respectively. The survival rate of in vitro fertilized embryos after ultrarapid frozen-thawing in the freezing medium of 2.5M glycerol added 0.25M sucrose were higher than concentration of 0.10M, 0.50M and 0.75M sucrose. 5. The equilibration time on the survival rate of in vitro fertilized bovine embryos was attained after short period of time(2.5~5min.) in the freezing medium added 0.25M sucrose and 3.0M DMSO higher than long period time(1~20min.).

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.173-178
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• 1998

The objective of this study was to evaluate the embryonic development ability and the appearance of blastocysts of bovine in vitro fertilized oocytes cultured in different culture media, and also to evaluate survival rate after thawing of frozen embryos by using 1.5 or 1.8M ethylene glycol(EG) with sucrose or trehalose. Fertilized oocytes were divided into three groups; i ) monolayer of cumulus /granulosa cell prepared by TGM 199+5% calf serum(TGM199), ii)GRlaa+5% CS, iii)SOF+5% CS, and they were cultured after insemination for 9 days, at 39˚C, under 5% $CO_2$ in air, but SOF+5% CS was cultured at 39˚C, under 5% 02, 5% GO2, 99% N2. Blastocysts derived from GRlaa + 5% CS on day 7~8 after insemination were frozen by using 1.5M EG or 1.8M EG with/without 0.2M sucrose or O.1M trehalose. The development rate of blastocysts on day 7 after insemination in SOF+5% CS was significant higher than in TCM199 or CR1aa(P<0.05). The appearance rate of blastocysts on day 7-8 after insemination was higher than in TCM199, when fertilized oocytes were cultured in GRlas or SOF. The survival rate of frozen blastocysts after thawing tended to increase, when blastocysts were frozen by using 1.8M EG with 0.2M sucrose or O.1M trehalose. These results indicated that SOF or CRlaa media with amino acids was superior to TCM199 with monolayer in terms of blastocyst development in culturing of in vitro fertilized bovine nocytes, and sucrose or trehalose was supposed to prevent embryos from the freezing shock.

• Korean Journal of Animal Reproduction
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• v.19 no.2
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• pp.135-140
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• 1995

The study was conducted to investigate on the survival rate of bisected porcine embryos co-cultured in 10% FCS(v/v)＋TCM-199 media containing hormones, oviductal epithelial cells and cumulus cells 0 to 72 hrs after bisection. In vitro survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The survival rate of bisected porcine embryos cultrued in 10% FCS＋TCM-199 media contaning PMSG, hCG, PMSG＋hCG, hCG＋$\beta$-estradiol 0 to 20 hrs and 20 to 40 hrs were 37.6% and 37.5%, 28.6% and 28.6%, 35.7% and 28.8%, 30.8% and 23.1%, 38.5% and 30.7%, respectively. The survival rate of bisected embryos co-cultured in TCM-199 media containing hormones, oviductal epithelial cells and cumulus cells was significantly higher than that of non co-culture. 2. The survival rate of bisected porcine embryos co-cultured 10% FCS＋TCM-199 media containing oviductal epithelial cells 4 to 5 hrs and 20 to 24 hrs were 42.9% and 38.5%, respectively. 3. The survival rate of bisected porcine embryos co-cultured in 10% FCS＋TCM-199 media containing cumulus cells 4 to 5 hrs and 20 to 24 hrs were 40.0% and 35.7%, respectively.