• Journal of Embryo Transfer
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• v.15 no.3
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• pp.219-230
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• 2000

This study was carried out to determine the factors on achieving good viability of embryos biopsied fur sexing, to investigate pregnancy rate following embryo transfer(ET) with sexed embryos, and to confirm the accuracy for the calves bort following ET with sexed embryos by polymerase chain reaction(PCR). To investigate viability of Hanwoo embryos after biopsy for sexing, fresh and frozen/thawed embryos were biopsied according to different developmental day of blastocysts, different stage of blastocysts, and different biopsy grade and the embryos themselves were incubated for 2 hours in TCM199 after microsection to be evaluated morphologically for recovery as blastocyst. The results obtained were as follows : 1. The rate of oocytes cleaved in vitro and the rate of blastocyst of the cleaved oocytes were 52.5% and 21.6%, respectively. The rate of blastocyst on day 8 was 11.2%, denoting the highest rate during whole culture period posterior to in vitro fertilization(IVF) 2. After biopsy for sexing, the viability rate of blastocyst on day 7, 8 and 9 was 75.0%, 88.4%, and 100.0%, respectively and the viability of early, mid, and expanded blastocyst after biopsy was 75.0%, 88.9%, and 91.1%, respectively The viability rate of fresh and frozen/thawed embryos was 89.9%, 71.4%, respectively. And the viability of expanded, hatching, and hatched blastocyst of frozen/thawed embryos was : 75.0%, 75.0%, and 50.0%, respectively. The viability of embryos according to biopsy grade of 10∼20%, 21∼30%, and 31∼40% was 85.7%, 91.5%, and 71.4%, respectively. 3. Pregnancy rate after transfer with biopsied embryo between flesh and frozen/thawed embryos was 22.6% and 20.0%, respectively. 4. In comparison between sex by PCR method and sex of calves born after embryo transfer, the accuracy of sex deterimination was 92.3% (12/13).

• Korean Journal of Animal Reproduction
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• v.13 no.3
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• pp.164-170
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• 1989

These experiments were carried out to develop the practical technique for the production of identical twins in cattle. Morula and blastocyst stage embryos collected from superovulated donors were bisected into halves by micromanipulation. The resulting demi-embryos were transferred to the uterine horn ipsilateral to the corpus luteum of synchronous recipients. The viability of demi-embryos after splitting was also evaluated by culturing demi-embryos with and without a zona-pellucida. The results obtained in these experiments were summarized as follows : 1. Of total 132 embryos collected by superovulation from 29 donors, 37 embryos were morular and 30 at blastocyst stages. 2. Total 111 demi-embryos were produced from 67 embryos by bisection and 98% of those were normal in morphology. 3. The viability of the demi-embryos cultured with zona-pellucida ranged from 70 to 76.5% and that of the demi-embryos without from 53.8 to 69.2%. 4. The viability of demi-embryos obtained from morula was 63.6% and that of demi-embryos from blastocyst was 73.3%, respectively. 5. 35 demi-embryos were transferred to 21 recipients, 7 of which were confirmed to be pregnant by rectal palpation at 55∼60 days after embryo transfer. One of them produced a calf and 6 are still on pregnancy.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.193-202
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• 1995

In order to improve the cryopreservatory techniques of livestock embryos, the quick freezing method which is directly plunged in liquid nitrogen via prefreezing procedure without freezing machine was carried out for mouse embryos treated with permeable and nonpermeable cryoprotectants. The viability of frozen-thawed embryos were evaluated by FDA vital dye test. The results obtained was summaried as follows: 1. A total of 720 embryos were recovered from frozen embryos for viability test. Evalution of the fluorescein diacetate(FDA) vital dye test with mice embryos were resulted of 2.3 total mean score - evaluted in orderly higher mean grade of P3 453 (63%), P2 133(18%), P1 51(7%) and P0 83(12%). 2. An all-round evalution of these combination, the highest viability was showed in 3M ethylene glycol + 0. 25M trehalose treated with the copper prefreezing. 3. Effects of permeable and nonpermeable cryoprotectants combination were evaluated by means FDA score. 3M ethylene glycol + 0.25M trehalose showed the highest survival rates of 2.8 mean FDA score. 4. Effects of permeable cryoprotectants were evaluated by mean FDA score but the results were not significantly different each other. 5. In evalution of the nonpermeable cryoprotectants, 0. 25M trehalose obtalned higher mean FDA score than of 0.25M sucrose and it was significantly different(P<0.05). 6. There was no significantly difference between copper and stainless-steel in prefreezing procedures.

• Korean Journal of Animal Reproduction
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• v.19 no.1
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• pp.49-54
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• 1995

This experiments were carried out to investigate the viabilities and the pregnancy rate of frozen-thawed IVF bovine embryos in various media, cryoprotectants and age of embryos produced. Hanwoo oocyte were collected in size of 2~7mm follicles, matured for 20~22hrs at 38.5$^{\circ}C$ in 5% CO2 incubator and then in vitro fertilized with Hanwoo semen. Blastocysts or more developed embryos at Day 7, 8 and 9 were frozen in 1.5 or 1.8M ethylene glycol. Viability of frozen thawed IVF embryos were identified the reformation of blastocoele after thawing and culture for 24~48 hours at 38.5$^{\circ}C$ in 5% CO2 incubator. Production rate of Hanwoo IVF embryos in TCM 199 and CR1aa ws 21.3%(39/183) and 28.1%(41/146), respectively. The viability of frozen thawed IVF embryos was higher rate in 1.8M ethylene glycol and Day 7 embryos than that in 1.5M and Day 8.53 cows out of 100 Hanwoo receipients transfered IVF embryos were pregnant and twin production rate was 26.3%.

• Korean Journal of Veterinary Research
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• v.29 no.2
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• pp.123-128
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• 1989

Demi-embryos were successfully produced by bisection of ICR mouse embryos at preimplantation stages. They were microsurgically bisected using a microsurgical blade attached to a micromanipulator after pretreatment with 0.5% pronase in PBS for two minutes or not. Embryos with softened zona pellucida were more easily bisected and less damaged than intact embryos. The highest success rate in bisection has been achieved by selecting blastocysts(94.1% in success rate with intact blastocysts and 100% in success rate with zona softened blastocysts). Demi-embryos without zona pellucida were cultured in D-PBS or M-16 medium at $37^{\circ}C$, 5% $CO_2$ in air for 72 hours for 2-cell stage embryos, 48 hours for 4-to 8-cell stage embryos, 24 hours for morula stage embryos and 6~12 hours for blastocyst stage embryos. For the in vitro culture of 2-cell stage embryos, $100{\mu}M$ 2Na-EDTA was added to the media. M-16 medium was better for the in vitro development of mouse embryos than PBS, and PBS is not considered to be suitable for long-term culture of embryos, especially at early stage of cleavage. In M-16 medium, developing rate of demi-embryos of which pair underwent development to form eublastocysts was 15.8% at 2-cell stage, 16.8% at 4-cell stage, 38% at 8-cell stage, 89.6% at morula stage and 94.4% at blastocyst stage, respectively. The more rapid and efficient production of demi-embryos and higher viability after bisection can be expected by softening zona pellucida with pronase and by selecting morulae or blastocysts rather than embryos at early stage of cleavage.

• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.235-240
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• 2007

This study was conducted to examine the viability of Korean native striped cattle (Bos namadicus Falconer, Chikso) clone embryos after embryo transfer. Chikso somatic cell nuclear transfer (SCNT) embryos were produced by fusion of ear skin cells derived from a female Chikso with enucleated oocytes matured in vitro for 18-24 hr. After in vitro culture of SCNT embryos for 7 to 8 days, fresh or vitrified blastocysts derived from SCNT were transferred into a uterine horn of recipient cows. Fifteen of total 43 recipients were pregnant at Day 50 and 4 recipients were maintained to term. Three IVF-derived calves and 1 clone Chikso calf were born. Pregnancy rate was higher when fresh embryos were transferred to recipients compared to vitrified embryos, but development to term was not different between both groups. The clone Chikso calf died at 5 days after birth due to the fullness of amniotic fluid in rumen and the infection of umbilical cord. The result of the present study shows that clone Chikso calf can produced from the embryo transfer of SCNT embryos, however, solution of abortion problem is necessary to improve the cloning efficiency.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.325-330
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• 1994

The study has been carried out in order to evaluate the effects of embryonic stage, and cryopreservation method on the rates of viability and development of the cryopreserved mouse early embryos. The results were as following:In the treatment steps of cryoprotectant, for the fertilized oocyte with pronucleus(PN), 2-step was better than the others. And for the other embryos, 4-step was better than 2- or 3-step. In respect to the embryonic stage, as the embryos developed from fertilized oocytes to 8-cell embryos, the rates of viability and development were increased higher. Therefore, 8-cell embryo was better stage than the others. In respect to the kind of cryoprotectants, PROH was better than DMSO for the fertilized oocyte, as a cryoprotectant. DMSO, for the 2-cell embryos and PROH and DMSO for the 4- and 8-cell embryos were suitable for cryopreservation.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.2
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• pp.129-135
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• 1990

This study was done to verify factors affecting viability after cryopreservation and pregnancy rate after frozen-thawed embryo transfer into uterus. Embryos were cryopreserved slow freezing and slow thawing and used DMSO as cryoprotectant. The results were to follows. 1. Viability of frozen-thawed embryos were 75.5% (94/105), which compared with viability of embryos according to cell stage, $2{\sim}5$ cell was 68.4% and $6{\sim}16$ cell 80.4% were significant differences (p<0.05). 2. No significant difference in duration of cryopreservation on effects affecting pregnancy rate was observed. 3. Number of embryo transfered into uterus was significant differences (p<0.05). 4. Four pregnancies resulted following replacement of 35 frozen-thawed.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.394-400
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• 1998

Cryopreservation of embryos by vitrification is a simple method to preserve bovine embryos for subsequent embryo transfer, but embryonic viability after vitrification has been inconsistent and low compared with conventional slow freezing. The aim of the present study is to examine the effect of serum or serum albumin in a vitrification solution and epidermal growth factor(EGF) or fibroblast growth factor(FGF) on in vitro viability of bovine blastocysts frozen by vitrification. Bovine blastocysts were produced by in vitro maturation, fertilization of follicular oocytes and culture of embryos in a synthetic oviduct fluid medium(SOFM) containing BSA and 19 essential and nonessential amino acids. Blastocysts with excellent or good morphology were selected at 7 or 8 days after culture and utilized for vitrification. In experiment 1, blastocysts were vitrified in a solution containing semi-fetal calf serum(SFCS) or BSA(5 or 10mg/ml) and then their subsequent viabilities were examined by culturing thawed embryos in a SOFM containing BSA and 19 amino acids. Effect of EGF or FGF added to a SOFM containing polyvinyl alcohol(PVA) on the viability of vitrified-thawed blastocysts was investigated in experiment 2. BSA added at 5 or 10mg/ml to a vitrification solution showed significantly higher(p < 0.05) developmental rate to expanded and hatching blastocysts than SFCS, but there was no significant difference in the developmental rate to hatched blastocysts after thawing. Supplementation of a culture medium with EGF and/or FGF significantly increased(p < 0.05) embryo development to expanded blastocysts compared with control but showed no beneficial effect on the development to hatching or hatched blastocysts. Coculture of thawed embryos with granulosa cells in a TCM 199 containing 10% fetal calf serum(FCS) showed the highest developmental rate to expanded, hatching and hatched blastocysts among the groups tested. In conclusion, supplementation of a vitrification solution with BSA at 5mg/ml and culture of thawed blastocysts in a medium containing EGF and/or FGF can improve in vitro viability of bovine blastocysts frozen by vitrification.

• Fisheries and Aquatic Sciences
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• v.21 no.9
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• pp.23.1-23.8
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• 2018

Background: Russian sturgeon (Acipenser gueldenstaedtii) is an emerging candidate species in the Korean aquaculture domain owing to its highly valued caviar. Although the embryonic development of this species was previously described, the complete image data on the morphological differentiation of developing embryos have not been yet fully available. Further, with the viewpoint of larval production in hatchery, the effects of temperature on embryonic viability and the temporal window of hatching event have not been extensively studied. Hence, the objective of this study was to provide a complete set of photographic image data on the embryogenesis and also to examine the effects of incubation temperatures on embryonic viability and hatching event in farm-bred Russian sturgeon. Results: Typical characteristics of embryonic development including uneven, holoblastic cleavages with unequal blastomeres, followed by the formation of germ layer, neurulation, and organogenesis until hatching, were documented. Under different temperature conditions (12, 16, or $20^{\circ}C$), viability of embryos incubated at $12^{\circ}C$ was significantly lower as relative to those of 16 and $20^{\circ}C$ incubated embryos. Hatchability of embryos was higher, and the timing of hatching event was more synchronized at $20^{\circ}C$ than at 12 and $16^{\circ}C$. Conclusion: Data from this study suggest that the incubation of Russian sturgeon embryos at $20^{\circ}C$ would be desirable in the hatchery practice with respect to the good hatchability of embryos and the synchronization of hatching events. Additionally, the updated image data for complete embryonic development could be a useful reference guide for not only developmental researches but also artificial propagation of Russian sturgeon in farms.