• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.133-142
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• 2002

The objective of this study was to examine maturation, fertilization and developmental rate of the in vitro-grown mouse oocytes, and to compare these results with those of oocytes grown and matured in vivo. The preantral follicles isolated from 12-day-old mice were cultured on Transwell-COL membrane inserts. After in vitro growth and maturation, 72.5 % of oocytes grown in vitro produced polar body which can be comparable to in vivo growth (70.5 %). However, the mean oocyte diameter of the in vitro group (69.6$\pm$2.1$\mu$m) was smaller than that of the in vivo group (73.3$\pm$3.0$\mu$m). The fertilization rate was significantly lower (p<0.05) in the in vitro group (76.5%) than in the in vivo group (90.2%), however, there was no difference in the percentage of monospermic and polyspermic oocytes between two groups. The capacities of in vitro grown ova to cleave and develop to blastocyst were (57.8 and 14.4%, respectively) significantly lower (p<0.001) than those of the in vivo counterpart (84.4 and 56.6%, respectively). Moreover, the mean number of cells per blastocyst was significantly lower (p<0.05) in the in vitro group (39.0$\pm$10.8) than in the in vivo group (60.5$\pm$12.5). Live young were produced from transferred 2-cell embryos derived from in vitro-grown and matured oocytes. In conclusion, the results show that in vitro-grown oocytes did not achieve the developmental capacity of in vitro-grown oocytes.

• Toxicological Research
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• v.19 no.4
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• pp.267-275
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• 2003

The purpose of our study was to evaluate the toxicity of the thimerosal in embryos and neonates. Thimerosal (also known as mercurothiolate) is a mercury-containing compound used in trace amounts to prevent bacteria and other organisms from contaminating vaccines, especially in opened multi-dose vials. The toxicity of mercury is well known and those most at risk occurrs in unborn babies and newborn babies. Test methods included in vitro whole embryo culture (WEC) system and in vivo test of neonatal toxicity in Wistar rats. Ethylmercury and methylmercury were used as positive controls for the evaluating of toxic effects of mercury. In WEC assay, treated concentrations of thimerosal, ethylmercury and methylmercury were up to 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5 and 5 $\mu\textrm{g}$/$\textrm{m}{\ell}$, respectively. All compounds didn't show any morphological abnormalities, but showed retardation of growth and development in dose dependent manner (> 0.5 $\mu\textrm{g}$/$\textrm{m}{\ell}$). These data indicated that thimerosal showed developmental toxicity in vitro. In vivo neonatal toxicity, Wistar rats were administered subcutaneously with thimerosal, ethyl mercury, or methylmercury (5, 25, 50, 250, and 500 $\mu\textrm{g}$/kg) during from postnatal day (PND) 4 to 25. Significant effects of these compounds on relative organ weights and organ morphology were not observed in this experiment. However, accumulation of mercury was detected in the kidney and testis when treated with thimerosal, ethylmercury, or methylmercury. These results suggest that thimerosal may be a harmful compound to embryo and neonate, but used concentration of thimerosal in these experiments is much higher than that of clinical application. Further investigation is needed on the safety of vaccine components, i.e. a thimerosal using in vitro and in vivo tests in the future.

• Korean Journal of Poultry Science
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• v.28 no.2
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• pp.135-142
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• 2001

A novel sterategy has been established to determine the origin of the Primordial Germ Cells (PGCs) in avian embryos directly and the developmental fate of the PGCs for the application to Poultry biotechnology. Cells were removed from 1) the centre of area pellucida, 2) the outer of area pellucida and 3) the area opaca of the stage X blastoderm (Eyal-Giladi & Kochav, 1976). When the cells were removed from the centre of area pellucida, the mean number of circulating PGCs in blood was significantly decreased in the embryo at stage 15 (Hamburger & Hamilton, 1951) as compared to intact embryos. When the cells were replenished with donor cells, no reduction in the PGCs number was observed. The removal of cells at the outer of area pellucida or at the area opaca had no effect on the number of PGCs. In case, another set of the manipulated embryos were cultured ex vivo to the hatching and reared to the sexual maturity, the absence of germ cells and degeneration of seminiferous tubules was observed in resulting chickens derived from the blastoderm in which the cells were removed from the centre of the area pellucida. It was concluded that the avian Primordial Germ cells are originated at the center of area pellucida. Developmental ability of the cells to differentiate into somatic cells and germ cells in chimeras were analyzed. Somatic chimerism was detected as black feather attributed from donor cells. Molecular identification by use of female - specific DNA was performed. It was confirmed that the donor cells could be differentiated into chimeric body and erythrocytes. Donor cells retained the ability to differentiate into germline in chimeric gonads. More than 70% of the generated chimeras transmitted donor derived gametes to their offspring indicating that the cells at the center of area pellucida had the high ability to differentiate into germ cells. A molecular technique to identify germline chimerism has been developed by use of gene scan analysis. Strain specific DNA fragments were amplified by the method. It would be greatly contributed for the detection of germline chimerism. Mixed- sex chimeras which contained both male and female cells were produced to investigate the developmental fate of male and female cells in ovary and testes. The sex combinations of donor and recipient of the resulting chimeras were following 4 pairs; (1) chimeras (ZZ/ZZ) produced by a male donor (ZZ) and a male recipient (ZZ), (2) chimeras (ZW/ZW) produced by a female donor (ZW) and a female recipient (ZW), (3) chimeras (ZZ/ZW) Produce by a male donor (ZZ) and a female recipient (ZW), (4) chimeras (ZW/ZZ) produced by a female donor (ZW) and a male recipient (ZZ). It was found that genetically male avian germ cells could differentiate into functional ova and that genetically female germ cells can differentiate into functional spermatozoa in the gonad of the mixed- sex chimeras. An ability for introduction of exogenous DNA into the PGCs from stage X blastoderms were analyzed. Two reporter genes, SV-$\beta$gal and RSV-GFP, were introduced into the PGCs. Expression of bacterial/gal was improved by complexing DNA with liposome detectedcc in 75% of embryos at 3 days embryos. At the embryos incubated for 1 day, expression of the GFP was observed all the embryos. At day 3 of incubation, GFP was detected in about 70% of the manipulated embryos. In case of GFP, expression of the transgene was detected in 30 %e of the manipulated embryos. These results suggested that the cells is one of the most promising vectors for transgenesis. The established strategy should be very powerfull for application to poultry biotechnology.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.47-52
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• 2016

The objective of this study was to investigate the result of in vivo embryo collection and pregnancy rate after embryo transfer using sex-sorted sperm of Korean brindle cattle. Donor Korean brindle cattle superovulation treated by decreasing dose of FSH injection. Embryos were recovered on 7 days after the third artificial insemination. Control group semen straw used artificial insemination contained 20 million sperm. Sex-sorted semen straws contained 4 million sperm or 10 million sperm. As for the result of the recovery of the in vivo embryos derived from sex-sorted sperm, the number of transferable embryos was significantly highly recovered to be $6.20{\pm}2.28/donor$ from the control group and was significantly lowly recovered to be $1.57{\pm}1.72/donor$ from the group treated at a sperm concentration of $10{\times}10^6$ (p<0.05). The number of unfertilized embryo was $0.8{\pm}1.30/donor$ in control group which was significantly lower than the group treated at a sperm concentration of $4{\times}10^6$ (p<0.05). However, there was no significant difference in the number of undeveloped ova between control and treatment groups. Pregnancy rate after embryo transfer was shown to be 35.00% in control group and 12.50% in treatment group. The karyotype analysis of the calf derived from sex-sorted sperm resulted in a similar chromosomal distribution pattern (2n=60, XX) compared to those of common Korean native cattle.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.127-134
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• 2010

This study was to investigate the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos, and their pregnancy and delivery rate after embryo transfer into recipient. In experiment 1, to optimize the flavonoid concentration, parthenogenetic day 2 ($\geq$ 2-cell) embryos were cultured in 0 (control), 1, 10 and $20\;{\mu}M$ flavonoid for 6 days. In the results, in vitro development rate was the highest in $10\;{\mu}M$ flavonoid group (57.1%) among treatment groups (control, 49.5%; $1\;{\mu}M$, 54.2%; $20\;{\mu}M$, 37.5%), and numbers of total and ICM cells were significantly (p<0.05) higher in $10\;{\mu}M$ flavonoid group than other groups. We found that $10\;{\mu}M$ flavonoid treatment can significantly (p<0.05) decrease the apoptotic index and derive high expression of anti-oxidant, anti-apoptotic, cell growth and development marker genes such as Mn-SOD, Survivin, Bax inhibitor, Glut-5, In-tau, compared to control group. In experiment 2, to produce the cloned Jeju Black Cattle, beef quality index grade 1 bull somatic cells were transferred into enucleated bovine MII oocytes and reconstructed embryos were cultured in $10\;{\mu}M$ flavonoid added medium. When the in vitro produced day 7 or 8 SCNT blastocysts were transferred into a number of recipients, $10\;{\mu}M$ flavonoid treatment group presented higher pregnancy rate (10.2%, 6/59) than control group (5.9%, 2/34). Total three cloned Jeju Black calves were born. Also, two cloned calves in $10\;{\mu}M$ flavonoid group were born and both were all healthy at present, while the one cloned calf born in control group was dead one month after birth. In addition, when the result of short tandem repeat marker analysis of each cloned calf was investigated, microsatellite loci of 11 numbers matched genotype between donor cell and cloned calf tissue. These results demonstrated that the flavonoid addition in culture medium may have beneficial effects on in vitro and in vivo developmental capacity of SCNT embryos and pregnancy rate.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.37-42
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• 2008

This study was performed to improve the pregnancy rates of recipients following transfer of bovine embryos produced in vivo. Superovulation response didn't showe significant differences between each season (4.18 in spring; 4.36 in summer; 5.50 in fall; 4.38 in winter). Pregnancy rate was significantly different (p<0.05) between fresh (43.4%) and frozen embryos (17.2%). In administration of hCG to recipients, the pregnancy rate of fresh embryos (45.7%) was slightly higher than that of control (35.3%), but the pregnancy rates of frozen embryos in control group (25.0%) was higher than that of hCG group (16.0%). When synchrony of recipient and embryo was -2, -1, 0 and 1, the pregnancy rates were 20.0, 45.0, 30.3 and 26.3%, respectively. The pregnancy rates of recipients synchronized by naturally or $PGF_{2{\alpha}}$, CIDR/$PGF_{2{\alpha}}$ and E/P/CIDR/$PGF_{2{\alpha}}$/E treatments were 35.3, 48.0, 29.0 and 40.0%, respectively. Gestation lengths and birth weights of female and male calf were 288 and 290.5 days, 28.3 and 30.0 kg, respectively. The results were showed that the superovulation response was not affected by seasons, and also pregnancy rate didn't increase by administration of hCG, synchrony of embryo and recipients, synchrony methods. Further study and concern should be focused on improving the embryo freezing and pregnancy rate for commercial embryo transfer.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.4
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• pp.345-351
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• 2000

Objective: Hatching of the blastocyst from the zona pellucida (ZP) is a key event in mammalian implantation. In vivo, two factors have been identified as possible mediators of hatching: lysis of the ZP by substances elaborated either from the embryo or female reproductive tract and pressure exerted on the zona by expansion of the blastocyst. Two methods of zona manipulation were already in use to enhance the ability of embryos to hatch: mechanical PZD and chemical ZD by acidic Tyrode's solution. But several controversies of each method have been reported. The purpose of this study was to investigate the effect of pronase for mouse embryo hatching. Methods: Mouse embryos were obtained following ovulation induction of $F_1$ animals. Fresh and cryo-thawed morula embryos were exposed to 0.5, 1.0, 2.0, 5.0 ${\mu}g/ml$ pronase in Ham's F10 for 72 hrs. Main outcome measures were the rates of partial hatching and completely hatched blastocysts, and cell number of it. Results: In fresh and cryo-thawed group, the rates of completely hatched blastocyst were significantly higher in 5 ${\mu}g/ml$ pronase treatment group than control group. There was no difference in completely hatched blastocyst total cell number between pronase treatment group and control group. This suggest that pronase treatment did not harmful in mouse embryo development. In pronase treatment group, zona pellucida were thinner than control group. Conclusion: The addition of pronase to culture media may accelerate the hatching of embryo. So, enzymatic treatment of the zona may provide a valuable and effective assisted hatching technique for human in-vitro fertilization-embryo transfer.

• Journal of Plant Biology
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• v.29 no.4
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• pp.285-294
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• 1986

The present study was attempted to investigate the effects of polyamines such as putrescine, spermidine and spermine on protein content and DNase activity in vivo and in vitro in carrot embryos. It was also investigated whether polyamines could replace role of cations required for DNase activity in vitro. The results obtained are as follows. Putrescine, spermidine and spermine increased protein content, although response to spermine reached plateau at the concentration of 0.1 mM. DNase activity was inhibited by polyamines, the inhibition being concentration-dependent and the highest att he concentration of 10 mM. The inhibition of DNase activity was the most prominent with spermine. Similar inhibitory effect to polyamines which was concentration-dependent was found in DNase activity but no change was shown on time-course in vitro. Putrescine and spermidine enhanced the DNase activity at low Mg2+ and Mn2+ concentrations, suggesting that the role of Mg2+ and Mn2+ for DNase activity could be, in part, replaced by these polyamines. These results, therefore, suggest that plyamines can modulate DNase activity through binding to DNA rather than direct effect on DNase activity.

• Environmental Analysis Health and Toxicology
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• v.21 no.1 s.52
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• pp.81-86
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• 2006

Embryotoxicity tests were performed in Daphnia magna to assess aquatic ecotoxicity of bisphenol A, a well known industrial compound showing estrogen-like activity in vivo, and to examine their effectiveness in the toxicological assessment. The whole embryonic developmental period was classified into 6 stages and developmental abnormality was checked to evaluate the embryotoxicity. In the present study, bisphenol A showed the ability to interfere with embryonic development, suggesting its antiecdysteroidal activity. The rates of mortality, delayed development, deformity and immobility all showed good concentration-response relationship, demonstrating their possibility as useful toxicological indices in daphnid embryotoxicity tests that have been rarely performed so far. It seemed favorable to the test sensitivity that embryos are removed from maternal daphnids around 7 hr after deposition from the ovaries to the brood chamber. These results suggest that daphnid embryotoxicity tests can be one of useful tools available for the assessment of ecotoxicity of various chemicals in the aquatic environment.

• Clinical and Experimental Reproductive Medicine
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• v.12 no.2
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• pp.65-69
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• 1985

1) Rabbit follicular oocytes from preovulatory follicles were cultured for 12 hr in vitro and fertilized in vivo by transferring the oocytes to the first foster-mother. 2) Two youngs were bron from transferred embryos from the first foster-mother to the second foster-mother. This demonstrates that in vitro cultured follicular oocytes are normal and they can develop into normal young born when transferred to the foster-mother. 3) A simple chemically defined culture medium, salt sol. with glutamine (2mM), which was developed by Bae and Foote(1975) proves fully good enough for rabbit follicular oocyte culture. We call this B-F medium. 4) Twelve hours culture in vitro of the rabbit follicular oocyte may be a proper culture time for further development.