• Journal of Embryo Transfer
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• v.9 no.1
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• pp.43-48
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• 1994

This study was carried out to produce monozygotic twin calves by transfer of bisected embryos. Four Korean native cattle donors were superovulated with FSH and flushed to collect embryos on day 6 or 7 of the estrus cycle. Morula and early blastocyst embryos showed 1 or 2 grade were bisected with microblade and each set of demi-embryos without zona pellucida were transferred nonsurgically to 10 recipients respectively. The results obtained were as follows; 1. Twenty four demi-embryos (92.3%) were separated from 13 original embryos and among them 20 demi-embryos (83.3%) had normal appearance without severe damage. 2. Four sets of fresh demi-embryos were transferred to 4 recipients and one recipient was twin pregnant 3. Six sets of frozen-thawed demi-embryos were transferred to 6 recipients. Two recipients were pregnant, one of them twin.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.1
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• pp.15-21
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• 1999

The aim of the present study was to determine the effect of paternal sex chromosome on early development of buffalo embryos fertilized and cultured in vitro. Embryos were produced in vitro from abattoir derived buffalo oocytes. The cleaved embryos were cocultured with buffalo oviductal epithelial cells and evaluated on day 7 under the phase contrast microscope to classify development. The embryos which reached the morula/blastocyst stage were fast developing, the embryos which were at 16-32 cell stage were medium developing and the embryos below 16 cell stage were slow developing. The embryos which showed some fragmentation in the blastomeres or degenerated blastomeres, were degenerating. Sex of emberyos (n=159) was determined using PCR for amplification of a male specific BRY. 1 (301 bp) and a buffalo specific satellite DNA (216 bp) fragments. The results thus obtained show that 1) X and Y chromosome bearing sperms fertilize oocytes to give almost equal numbers of cleaved XX and XY embryos, 2) male embryos develop faster than female embryos to reach advanced stage and 3) degeneration of buffalo embryos is not linked with the paternal sex chromosome. We suggest that faster development of males is due to differential processing of X and Y chromosome within the zygote for its activation and / or differential expression of genes on paternal sex chromosome sex chromosome during development of buffalo embryos fertilized and cultured in vitro which may be attributed to a combination of genetic and environmental factors.

• 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.

• Korean Journal of Veterinary Research
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• v.29 no.3
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• pp.343-359
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• 1989

To study the effects of ibaraki virus on preimplantation mouse embryos collected from prepubertal ICR and BALB/cByJ mice (30~40days old) by superovulation, zona pellucidaintact(ZPI) or free(ZPF) embryos(n=774) of 4- to 8-cell and morulae were exposed to $10^{5.8}$ $TCID_{50}$ of the virus up to 96 hours. The embryos were examined morphologically by observing the degeneration and hatching rates, and virologically and immunologically by determining the presence of infection with the virus, in addition, the effect of washing the embryos to remove virus possibly attached to was also investigated. The ZPI 4- to 8-cell embryos and morulae exposed to the virus showed considerably higher degeneration rate than those not exposed, for 96, and for 72 to 96 hours, respectively(p<0.01). The ZPF 4- to 8-cell embryos and morulae exposed to the virus showed considerably higher degeneration rates than those not exposed, throughout the whole culture hours in vitro (p<0.01). The ZPI 4- to 8-cell embryos and morulae not exposed to the virus showed considerably higher rates of hatched blastocyst than those exposed (p<0.01). The virus infection rates of the ZPF 4- to 8-cell embryos and morulae were significantly higher than those of the ZPI embryos according to cell culture system. The viral antigen was detected exclusively on the zona pellucida of ZPI embryos, while the antigen was evenly distributed in the blastomeres of ZPF embryos by the immunofluorescent assay. In the ZPI embryos exposed to ibaraki virus, the virus was detected in the two times-washing groups, but not in the ten times-washing groups. The results indicated that zona pellucida of murine embryos would provide an effective protection and that ten times-washing of the ZPI embryos previously exposed to the virus was effective to remove virus from the embryos.

• Korean Journal of Animal Reproduction
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• v.15 no.3
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• pp.179-187
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• 1991

This experiment was carried out to develop a new technique of identifying XX of XY-bearing bisected embryos prior to implantation by immunological method. H-Y antiserum prepared in inbred Wastar female rats by repeated immunization with spleen cells from males of the same strain. The reactivity of H-Y antibody was confirmed by culturing mouse embryos in the medium containing H-Y antiserum and complement obtained from the guinea pig. The optimal condition for the activity of H-Y antibody was also investigated by culturing embryos under the concentraton or affected H-Y antibody was also investigated by culturing embryos under the concentration or affected H-Y antibody and culture rate. However, production of live young or sex rates of male and female from embryos transferred with psudopregnant. The biological test with the morula stage embryos showed that H-Y antibody was formed in all female rats immunized with spleen cell, but it was formed only in 80% female rats immunized with the antigen. When the bisected mouse embryos were cultured in vitro for 5~6 hours in morula stage, of 457 bisected embryos 81.4% of then were developed to the blastocyst stage. When the concentration rate of complement to H-Y antiserum varied from 1.0~5.0${mu}ell$, the lysis-rate of embryo was 19.5 to 67.3%. The concentration rate of complement did not influence the lysis-rate of embryos(P<0.05). The morphology embryos of bisected, zona-free and intact embryos showed the embryos lysis rate of 58.6, 42.7 and 48.5% respectively(P<0.05). Pregnancy rate were 50.0, 45.5 and 57.1% in psudopregnant recipient transferred with bisected, zona-free and intact blastocyst embryos. However, production of live youngs, sexual rate of male or female was 24(50.0:50.0), 22(45.5:55.5) and 36(58.3:41.7)mice, but affected and non affected half embryos with H-Y antiserum treatment was 23.1 and 26.7%. Also production of live youngs and sexual rate was 14(92.9:7.1) and 17(17.6:82.4)mice in affected and non affected half embryos in H-Y antiserum treatment(P<0.05).

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.229-237
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• 2015

To improve the developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos, this study compared the developmental rates to blastocyst stage in the SCNT embryos using donor fibroblasts treated with 5-azacytidine (5AC) and S-adenosylhomocysteine (SAH) at different concentrations. Their reprogramming efficiency level was investigated with level of telomerase activity. Donor fibroblasts isolated from adult ear skin of a cow were exposed to 5AC and SAH at different concentrations during 2 passages. After nuclear transfer into enucleated recipient oocytes, the cleavage and developmental rates were significantly (p<0.05) decreased in the SCNT embryos using 5AC-treated fibroblasts (5AC-SCNT embryos), compared with those of non-treated control (control-SCNT embryos) and SAH-treated fibroblasts (SAH-SCNT embryos). The developmental rates to blastocyst stage tended to be slightly increased in the SAH-SCNT embryos at each of the concentrations, and especially, the developmental rates in the SCNT embryos using 1.0 mM SAH-treated fibroblasts were significantly (p<0.05) higher than that of control SCNT embryos. The mean numbers of total and ICM cell in blastocysts were also significantly (p<0.05) decreased in the 5AC-SCNT embryos, compared with those of other SCNT blastocysts. Further, the level of telomerase activity was also significantly (p<0.05) decreased in the 5AC-SCNT embryos than those of control and SAH-SCNT embryos. Whereas, a significantly (p<0.05) up-regulated telomerase activity was observed in SAH-SCNT embryos, compare with that of control-SCNT embryos. In conclusion, SCNT embryos using hypomethylated donor cells with SAH, not 5AC, may improve the developmental potential and reprogramming efficiency.

• Journal of Veterinary Clinics
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• v.13 no.2
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• pp.149-152
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• 1996

This study was carried out to evaluate the efficiency of production of cloned embryos by nuclear translatation (NT) when using 4-cell to compact morula stage embryos as nuclear donor. In micromanitulation and electrofusion of blastomeres from 4-cell to morula stage embryos, the successful injection rate was higher with late stage blastomeres, on the contrary the fusion rate was lower. The in vitro developmental rate of NT embryos was not significantly different between cell-stages of donor blastomeres. Although the overall rate of production of cloned embryos with 4-cell. 8-cell, early and late morula stage embryos was 14.0, 18.0, 15.3 and 14.1%, respectively, the mean number of blastocysts produced with a donor embryo was the most (4.51) with the compact morulae. Therefore, it can be suggested that the embryos at thelate stage is more beneficial for the mulciple production of cloned embryos, If the late stage blastomeres have maintained their totipotency to produce intact offspring.

• Journal of Plant Biology
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• v.37 no.3
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• pp.365-370
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• 1994

Excised cotyledon segments of ginseng zygotic embryos at various developmental stages were cultured on MS basal medium from which somatic embryos were directly induced. The frequency of somatic embryo formation on the segments declined with the advancing zygotic embryo maturity. All of the cells in the cotyledons of immature zygotic embryos were smaller and more densely cytoplasmic than those in mature embryos. Histological examinations revealed that the poly-somatic embryos formed on immature embryos were of multi-cell originand derived from the epidermal and subepidermal cell layers. However, in the cotyledon of germinating zygotic embryos, only theepidermal cells were densely cytoplasmic and singularly competent to develop into somatic embryos resulting into single embryos at a frequency of 100%.

• The Korean Journal of Zoology
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• v.36 no.2
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• pp.277-284
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• 1993

A possible involvement of maturation promoting factor (nfPF) in the two-cell block phenomenon was studied by fusion experiments. Germinal vesicle (GlF) ooeyte was fused with a blastomore from late or blocked 2-cell mouse embryos. and germinal vesicle breakdoum (GVBD) of fused GV oocvtes in the presence of dbcAMP (100$\mu$g/ml) was scored as an index of MPF aniviD. GnD was induced approximately 30% by fusion of a blastomere derived from late 2-cell embryos, but not from blocked 2-cell embryos. The rate of GVBD was changed when GV oocyte was fused with a blastomere from late 2-cell embryos which were treated with u-amanitin, puromvcin or colcemid before and after hsion: Treatment of late 2-cell embryos with puromycin (50 Is/mll but not with u-amanitin (100 Is/ml) clearly inhibited GVBD, indicating that do novo protein synthesis maw be required for the appearance of MPF activity in late 2-cell embryos. Treatment of late 2-cell embryos w기h colcemid (0.1 Is/mll doubled GVBD, presumably due to the maintenance of metaphase or mitotic phase. SDS-PAGE and twoiimensional electrophoresis revealed that there was no difference in protein synthetic pattern in late and blocked 2-cell embryos, but three phosphoproteins with 27, 35 and 46 M)a, presumsblv M-phase components were phosphorylated in late 2-cell embryos but not in blocked 2-cell embryos. It seems then that MPF activity is closely related to phosphorylstion of M-phase components in late 2-cell embryos.

• Journal of Embryo Transfer
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• v.13 no.1
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• pp.53-60
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• 1998

This study was carried out to establish an effective system for embryo transfer techniques by analyzing several factors affecting in-vivo embryo transfer in Korean cattle. Embryos produced in-vivo were transferred into a total of 301 recipients. The results obtained in studies on the factors affecting pregnancy rate after embryo transfer by condition of embryos were as follow ; 1. The pregnancy rate of 301 recipients was 45.2% and higher with fresh embryos than with frozen embryos(63.5% : 21.4%, P<0.01). Embryos superovurated by FSH-P had slightly greater than by SUPER-OV in pragnancy rate, athough these were no difference between two treatments. 2. The pregnancy rates of transferred morulae and blastocysts showed no difference between fresh and frozen embryos(63.5% : 63~6% ; 20.0% : 25.8%). However, the pregnancy rates by quality of flesh and frozen embryos were significantly different(P<0~05). The pregnancy rates were outstandingly high in the grade A, B of fresh embryos(59.0~66.4%), and in the grade A of frozen embryos(43.6%). 3. The number of transferred embryos showed no difference in pregnancy rate, but when frozen embryos transferred, the pregnancy rate was slightly higher with two embryos than that with one embryo.