• 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 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%).

• Korean Journal of Animal Reproduction
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• v.8 no.1
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• pp.29-35
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• 1984

These experiments were carried out to obtain basic information necessary for in vitro culture of aggregated mouse embryos. Inbred ICR mice were used to obtain embryos. The zona pellucida was removed by placing the embryos in Whittingham's medium containing 0.5% protease for about 5-10minutes at 37$^{\circ}C$. Total 263 pairs of 2-, 4- and 8-cell zona free mouse embryos were subjected to aggregation by physical pressure and cultured in Whittingham's medium under the gas phase of 5% CO2 in air at 37$^{\circ}C$ for 24 to 60 hours. The results obtained in these experiments were summarized as follows: 1. Time needed for fusion of 2-, 4- and 8-cell embryos were 0-3, 0-3 and 0-3 hours, respectively and average time needed for in vitro development of 2-, 4- and 8-cell embryos after aggregation to morula and blastocyst were 42, 30 and 13.5 hours, and 51, 39 and 27 hours, respectively. 2. Of total 263 pairs of naked embryos, 227 were firmly aggregated together and the rats of aggregation in 2-, 4- and 8-cell embryos were 71.8, 88.3 and 97.0%, respectively. 3. The rates of aggregated pairs which obtained from 2-, 4- and 8-cell embryos developed to morula were 96.7, 95.6 and 96.9%, respectively, and embryos developed to blastocysts were 88.5, 89.7 and 90.8%, respectively. 4. Conspicuous differences in size of volume and inner cell masses between single and double blastocysts were observed. Although a single blastocolic cavity was formed in most double blastocysts, several formed two distinct cavities from the very beginning.

• Reproductive and Developmental Biology
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• v.34 no.2
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• pp.73-79
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• 2010

We investigated the microtubule dynamics, including the inheritance of donor centrosomes and the mitotic spindle assembly occurring during the first mitosis of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were fixed 15 min and 1 h after fusion in order to assess the inheritance pattern of the donor centrosome. The distribution and dynamic of the centrosome and microtubule during the first mitotic phase of SCNT embryos were also evaluated. The frequency of embryos evidencing $\gamma$-tubulin spots (centrosome) was 93.2% in the SCNT embryos 15 min after fusion. In the majority of the SCNT embryos (61.5%), however, no centrosome was observed 1 h after fusion. The frequency of the embryos with no or abnormal mitotic spindles 20 h after fusion was 19.6%. The $\gamma$-tubulin spots were detected near the nuclei of somatic cells regardless of cell cycle phase, whereas $\gamma$-tubulin spots in the SCNT embryos were observed only during the inter-anaphase transition. These results showed that the donor centrosome is inherited into the SCNT embryos, but failed to assemble the normal mitotic spindles during first mitotic phase in some SCNT embryos.

• Journal of Embryo Transfer
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• v.11 no.2
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• pp.95-101
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• 1996

The present study investigated the effect of treatment with nocodazole on the efficiency of cell cycle synchronization and development of mouse 4-cefl embryos. In addition, developmental ability of reconstituted embryos that received nuclei from 4-cell embryos synchronized with nocodazole at M phase was examined in vitro and in vivo. (1) When 4-cell blastorneres exposed to culture medium containing l$\mu$g /ml nocodazole for 2, 4, 6 and 8 hrs, 40% (40/10l), 75% (l19/159), 85% (87/102) and 97% (155/160) of nuclei were synchronized at M phase, respectively. (2) Treated with nocodazole for 4 hrs, the proportion of 4-cell embryos developed to blastocysts (98%, 60/61) was not significanUy different from that of the control embryos (98%, 196/201). However, the developmental ability of 4-cell embryos treated for 8 (87%, P<0.05)and 12 hrs (76%, P

• Korean Journal of Animal Reproduction
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• v.16 no.4
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• pp.301-310
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• 1993

This experiment was performed to develop simple practical methods for short term preservation of rabbit embryos. A total of 55 cross bred does were superovulated by intramuscular injection of PMSG and HCG. Embryos were recovered at 25~30 hrs, 60~65 hrs and 80~85 hrs after mating and selected by morphological examination. Four cell stage, morulae and blastocyst embryos were stored in PBS enrich with 1, 10, 20 and 40% heat-treated FCS at 4, 20, 30 and 37$^{\circ}C$, respectively. Embryos were examined morphologically at 24, 48 and 72 hrs following storage. The result obtained in this experiment were summarized as follows: The superovulation was induced by PMSG 200 IU and HCG 100 IU. The average number of ovulation points and embryos recovered by collection time were 19.0, 15.6(25~30 hr), 17.3, 13.5(60~65 hr) and 19.2, 14.4(80~85 hr), respectively. And recovery rates of embryos recovered at 25~30 hr, 60~65 hr and 80~85 hr after mating were 62.8%(4 cell), 84.7%(morulae) and 79.6%(blastocyst), respectively. On the other hand, the average number of ovulation points collected by the no, of operations for the repeated collection was 17.3(60~65 hr), 19.2(80~85 hr) in 1st and 9.4(60~65 hr), 10.6(80~85 hr) in 2nd surgery, respectively. There was a significant decrease(P<0.05) in the number of ovulation points the 2nd surgery as compared to the 1st surgery. All of the 4-cell stage embryos stored at 4$^{\circ}C$ for 48 hrs showed the same morphology throught the storage period, on the contrary, 4-cell stage embryos stored at 2$0^{\circ}C$ and 3$0^{\circ}C$ for 48 hrs showed degeneration embryos and stored at 37$^{\circ}C$ for 24 hrs showed degeneration embryos. Morulae and blastodcyst stored at 4, 20, 30 and 37$^{\circ}C$ for 24 hrs showed degeneration embryos. All of the blastocyst stored at 37$^{\circ}C$ for 72 hrs showed degeneration embryos.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.255-261
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• 2000

The objective of this study was to improve the efficiency of bovine embryo transfer by transferring of Hanwoo embryos into Hanwoo or Holstein recipients. The cryopreserved or fresh in vitro produced(IVP) embryos were transferred into uterine horn contralaterally or ipsilaterally to the corpus luteum. The recipients were inseminated by artificially on the next day of estrus. The pregnancy was diagnosed by rectal palpation at 60∼90 days after transfer of the embryos. The pregnancy rate by transfer of one or two embryos was 78%(7/9) and 74%(31/42), respectively. The pregnancy rates according to the grade of corpus lutea of recipients was 75% (20/27) and 82.0%(18/22) at the grade of A and B, respectively. Ten(67.0%) of 15 Holstein recipients transferred with IVP Hanwoo embryos and 5(42.0%) of 12 Holstein recipients transferred with frozen IVP Hanwoo embryos were pregnant. The single and twin calving ratio in Hanwoos was 77.0%(10/13) and 23.0%(3.13) in the recipients transferred with IVP embryos and 64.0%(7/10) and 27.0%(3/10) in the recipients transferred with frozen IVP embryos, respectively. Twenty-four pregnant cows following transfer of IVP embryos, 21(88.0%) calved the normal calves, and 2(8.3%) aborted. When the frozen IVP embryos were transferred, 16 pregnant cows calved 14(88.0%) normal calves and 2(13.0%) aborted. In conclusion, when one or two IVP bovine embryos were transferred into recipients, the A and B grade of corpus luteum resulted in high pregnancy rates. For the production of twin calves, transfer of the IVP or frozen IVP embryos could be suitable.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.3
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• pp.167-178
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• 2002

Objective : The present study was performed to investigate whether apoptosis occur in human embryos by annexin staining and detect the expression of Fas, Fas-ligand (FasL), Bax, and Bcl-2 in human fragmented embryos derived from IVF-ET by immunofluorescence and Western blot analysis. Materials and Methods: Using annexin staining, immunofluorescence and Western blot analysis on normal and fragmented embryos, we were able to detect apoptotsis and apoptotic gene products in fragmented embryos. Result: Phosphatidylserine (PS) translocation, the marker for apoptosis, were detected frequently in fragmented embryos. Bcl-2 and Bax protein were detected in both fragmented and non-fragmented embryos. When fragmented embryos compared to normal embryos, immunofluorescent intensity of Bcl-2 tended to be lower in fragmented embryos. Bax gene expression increased in the fragmented embryos compared to the normal embryos. This result supports a model in which the molar ratio of Bcl-2 to Bax determines whether apoptosis induced or inhibited in human embryo. Fas was highly expressed in human preimplantation embryos but not FasL. It suggests that embryo may undergo apoptosis by binding with FasL produced by follicular or immune cells. Conclusion: The over expression of Bax and Fas will trigger apoptosis to lead embryo fragmentation and change embryo to be nonviable.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.8
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• pp.1188-1191
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• 1999

Primordial germ cells (PGCs) of White Leghorn chicken embryos as a donor were transferred to Rhode Island Red chicken embryos as a recipient. At 48-50 h (stage 13-15) of incubation of fertilized eggs, donor PGCs, which were taken out from blood vessels of donor embryos, were injected into blood vessels of recipient embryos. Sex of the treated embryos was determined after the transfer of PGCs using remaining blood samples. In the present experiments, survival rate of the treated embryos was 33.3% for homo-sexual and 35.4% for hetero-sexual transfers of PGCs, respectively, when determined at 17 days of incubation. In this study, most of the treated embryos could not survive more than 18 days of incubation, though the reason for that was not clarified in the present work. The gonalds removed from embryos that died after 18 days of incubation and the organs from newly hatched chicks were examined for morphological and histological features. The gonads removed from the embryos with homo-sexual transfer of PGCs showed normal development in appearance. On the contrary, some (35.3%) of the embryos with hetero-sexual transfer of PGCs possessed abnormal gonads similar to ovotestis by histological observation. In cases where the gonads developed to be normal organs (64.7%) the sex of embryos was the same as recipient ones. The present results suggest that hetero-sexual transfer of the PGCs may bring about the possibility of development of the embryos bearing sexually different gametes, spermatogonia or oogonia.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.4
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• pp.483-491
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• 2009

In vitro produced porcine embryos have potential application in reproductive biotechnology. However, their development potential has been very low. This study evaluated the in vitro developmental ability and quality of cloned and parthenogenetic porcine embryos having 2-4 cells or 5-8 cells on Day 2 of in vitro culture. Analysis of results showed that 2 to 4 cell embryos had higher ability to form blastocysts than 5 to 8 cell embryos (p<0.05). Blastocysts produced from culture of 2 to 4 cell embryos also contained higher cell numbers and had lower BAX:BCLxL transcript ratio than those produced from 5 to 8 cell embryos (p<0.05), thereby suggesting 2 to 4 cell embryos have higher development potential. Further investigation revealed that 5 to 8 cell embryos had higher incidence (100${\pm}$0.0%) of blastomeric fragmentation than 2 to 4 cell embryos (15.2${\pm}$5.5% for parthenogenetic and 27.7${\pm}$7.1% for cloned embryos). This suggests that low development potential of 5 to 8 cell embryos was associated with blastomeric fragmentation. In conclusion, we have shown that morphological selection of embryos based on cell number on Day 2 of in vitro culture could offer a practical and valuable non-invasive means to select good quality porcine embryos.