• Korean Journal of Veterinary Research
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• v.27 no.2
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• pp.331-334
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• 1987

The effects of ethylene glycol as a cryoprotective agent on the survival of frozen-thawed mouse embryos were examined. The effects of the stage of development of ethylene glycol were also examined. Eight-cell embryos, morulae, early blastocysts and mid-blastocysts were recovered from superovulated immature ddY mice. Ethylene glycol was added to the embryos in 5 equal increments 5 minutes apart, giving a final concentration of 1.5M. The embryos were cooled to ${-6^{\circ}C}$ at ${1^{\circ}C/min}$ and seeding was induced at ${-6^{\circ}C}$. After being held for a further 5minutes at the seeding temperature, the samples were cooled to ${-35^{\circ}C}$ at ${0.3^{\circ}C/min}$ and then transferred to liquid nitrogen. Rapid thawing was done by placing the straws in ${37^{\circ}C}$ water. The thawed embryos were diluted in PBS of same time and manners as adding procedures. Survival of 8-cell embryos and morulae were assessed as a normal development of the embryos to the blastocyst stage and expanding blastocyst after 54 hours and 48 hours of in vitro culture, respectively. While those of the early and mid-blastocysts were assessed to the expanding blastocyst stage after 24 hours of in vitro culture. The survival rates of 8-cell embryos, morulae, early blastocysts and mid-blastocysts were 73.8%, 74.3%, 87.8% and 77.4%, respectively. Significant difference on the survival rate among the four stages of development was not observed.

• Biomedical Science Letters
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• v.23 no.1
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• pp.25-29
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• 2017

This study was to investigate the role of antioxidants on development in arsenite exposed porcine embryos. Oocytes were collected from porcine ovary, and then matured for 44 h. Maturated oocytes were incubated with sperm for 6 h, and fertilized oocytes with sperm (embryos) cultured for 48 h. After, embryos were culture with arsenite and/or antioxidants (melatonin, silymarin, curcumin and vitamin) for 120 h. Formation of pre-morulae, morulae and blastosysts rate was measured using microscope. In results, 10, 100 and 100 nM arsenite significantly decreased morulae and blastocysts formation compared to control in pigs (P<0.05). $10{\mu}M$ silymarin and $100{\mu}M$ vitamin E increased blastocyst formation compared to 10 nM arsenite exposed embryos, but there were no significantly among the treatment, and 1 nM melatonin and $5{\mu}M$ curcumin did not influence blastocysts formation in 10 nM arsenite exposed embryos. In summary, arsenite decreased embryo development, $10{\mu}M$ silymarin, $100{\mu}M$ vitamin E, 1 nM melatonin and $5{\mu}M$ curcumin had no positive effect to blastocyst formation in arsenite exposed porcine embryos. Therefore, we suggest that little arsenite may have negative effect to embryo development, and silymarin, vitamin E, melatonin and curcumin could not rescue embryo development from damage by arsenite in pigs.

• 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.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.163-170
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• 1996

The effect of several potential antioxidants and amino acids were examined as a means of increasing the development of in vitro matured and in vitro fertilized oocytes into morulae or blastocysts. Bovine embryos developed to the 2~8 cell stage after in vitro fertilization were cultured for 5 to 6 days at 39$^{\circ}C$ in CR1aa containing varing concentraton of the antioxidants and amino acid in a gas phase consisting of 5% CO2, high humidified air. At 5~6 days, embryo developments were reduced, and embryos were fixed and stained with Hochest 33342 DNA stain to facilitate counting of cells. In experiment 1, the proportion of embryos developed to morulae and blastocysts in CR1aa containing 1mM, 2.5mM taurine (22.6% and 20.4%) was slightly higher than those of 0, 5 and 10mM Taurine (5.7, 5.7 and 3.9%, P<0.05). In experiment 2, addition of glutathione did not improve blastocyst development (P>0.05). In experiment 3, concentations of superoxide dismutase(SOD) ranging from 300 to 1,000 U did not affect the propotion of embryos developing into blastocysts (P>0.05). In experiment 4, addition of 250 U catalase(38.5%) was slighty higher than those of 0, 500 and 1,000U. In experiment 5, the proportion of embryo developed beyond morula stage in CR1aa with taurine plus EDTA was slighty higher than other treatments(15.7, 26.0 and 29.2%), there were no significantly increases in cell number among treatments(P>0.05). These results are indicating that antioxidants and amino acids can increase the proportion of embryos that develop into morulae and blastocysts, but did not increas in cell number of blastocysts.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.275-284
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• 1995

In sexing early mammalian embryos, viability of biopsied embryos and accuracy of sexing are both important. We have been previously developed efficient methods for biopsy of mouse embryos and sex identification from a single blastomere by PCR. In this study, squeeze method used for biopsy of mouse embryos was applied to bovine embryos. Compact bovine morulae were obtained by flushing uteri on Day 6 after the onset of standing estrus. A small number of blastomeres could be isolated from bovine morulae by the biopsy method. All 13 biopsied morulae were survived and 10 embryos developed to normal blastocyst after 24 h of culture. Subsequently, sex of the bovien embryos was identified from a few blastomeres by PCR amplifying a Y-specific bovine DNA sequence. Among 13 embryos analyzed, 7 embryos were determined as males and 6 embryos as females. Thus, bovine embryos at morular stage could be successfully biopsied by the squeeze method and sex of the bovine embryos determined from biopsied material by PCR.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.73-78
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• 2001

This study was carried out to investigated developmental ability of IVM/IVF derived hanwoo embryos after DNA microinjection. Microinjected hanwoo embryos were cultured fur 7 days. The cleavage rates of DNA injected embrys(36.3%) was significantly lower than those of non-injected embryos(67.4%; p<0.05). The percentage of injected embryos reaching to the morulae and blastocyst was significantly lower than those of non-injected embryos(p<0.05). When injected embryo were cultured contaning L-ascorbic acid and $\alpha$-tocopherol for 168 hrs, the morulae and blastocyst rates were significantly higher than control(p<0.05). These results suggested that the addition of L-ascorbic acid and $\alpha$-tocopherol can enhanced development to the morulae and blastocyst of microinjected embryos and improved culture condition increased the transgenic hanwoo embryos.

• Journal of Embryo Transfer
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• v.9 no.2
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• pp.173-180
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• 1994

The present experirnents on cryopreservation were carried out to investigate effect of solution toxicity, equilibration time and cell stages on the post-thaw survival of mouse morulae and blastocyst embryos cryopreserved by vitrification in EFS solution. The mouse embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen and thawed rapidly. After the mouse morulae embryos were exposed to EFS solution for 2 and 5 ruin. at room temperature and then they were washed in 0.5 M sucrose solution and basal mediurn(D-PBS + 10% FCS), they were cultured to examined cryoprotectant toxicity induced injury during exposure, most of embryos developed to expanded blastocysts(100 and 90.0%). However, when the exposure time was extended to 10 and 20 min, these development rates dropped dramatically in 10 ruin. (75.0%) and 20 ruin. (4.5%), respectively. When the compacted morulae were vitrified in EFS solution after equilibration for 2 and 5 min, the embryos have developed to normal blastocyst following thawing, washing and culture processes was 89.3 and 89.6%. However, when the exposure time was expanded to 10 ruin, this survival rate dropped to 68.8%. When the blastocyst were vitrified in EFS solution after equilibration for 2, 5 and 10 minutes, the survival rate of embryos which developed to normal blastocyst following thawing and culture processing were 58.5, 46.7 and 22.4%, respectively. The optimal time of equilibration of mouse morula and blastocysts in EFS solution seemed o be 2 and 5 ruin.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.76-82
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• 1986

This experiment was conducted to determine the effects of trophoblastic vesicles (TV) and estradiol-17$\beta$ on the development in vitro of rabbit embryos. Thirty matured female rabbits were treated with PMSG followed by HCG injection and mating. Embryos were recovered with D-PBS (Dulbecco's Phosphate Buffered Saline) after superovulation, and normally developed to two-to four-cell embryos were used in the subsequent in vitro culture. Basal medium was Medium-199 su, pp.emented with 1.5% bovine serum albumin. Embryo on Day 5 after mating (Day 0) was cut into two or three pieces to remove the embryonic disc. Each piece of tissue was cultured for 24 hours at 37$^{\circ}C$ in 0.5 mlMedium-199 in 5% CO2. During culture, peices of trophoblastic tissue changed into spherical vesicles which were used for co-culture. These spheres were called trophoblstic vesicles. Two-to four-cell embryos were cultured for 4 days in Medium-199 in the absence or presence of trophoblastic vesicle, and two-to four-cell embryos cultured with varing concentration (0, 0.1, 1, 10ng/ml) of estradiol-17$\beta$ for 4 dyas. Culture vessels used were watch glass for coculture with trophoblastic vesicles and micortube for estradiol-17$\beta$ infusion. Compared with the Medium-199 alone as basal culture medium, more blastocysts (46.7% vs 15.1%; P<0.01) and morulae (84.4% vs 56.6%; P<0.05) were developed in the co-culture with trophoblastic vesicles. Estradiol-17$\beta$ infused in culture medium was not effective for embryo development to blastocysts (78.3% in control, 50.0% in 0.1ng/ml, 61.5% in 1ng/ml and 64.4% in 10ng/ml) and also to morulae (91.3% in control, 84.2% in 0.1ng/ml, 92.3% in 1ng/ml and 91.1% in 10ng/ml). Compared with the watch glass culture mehotd, more (P<0.01) blastocysts were developed in microtube culture (78.3% vs 56.6%) and more (P<0.01) morulae in microtube culture (91.3% vs 56.6%).

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.277-282
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• 1997

The purpose of this experiment was to determine the effects of thiol compounds, $\beta$-mercaptoethanol($\beta$-ME) and cystearrone with buffalo rat liver cell(BRLC) co-culture on the development and intracellular glutathione(GSH) concentrations of bovine embryos produced by in vitro inaturation(IVM) and in vitro fertilization(IVF). Bovine IVM /IVF embryos developed to 2~8 cell stage were co-cultured with BRLC in GRlaa with or without thiol compounds. The developmental rate beyond morulae stage in CRlaa containing 0, 10,25 and 50$\pi$M $\beta$-ME with BRLG were 63.0, 74.0, 72.3 and 77.1%, respectively. And the developmental rate with 0, 25, 50 and 75$\pi$M cystearnine with BRLC were 69.6, 77.6, 81.0 and 76.8%, respectively. The developmental rate beyond morulae stage of GRlaa containing thiol compound with BRLG group was higher than that of control group. The intracellular GSH concentrations of blastocysts cultured for 5 days in GRlaa containing 0 and 50$\pi$M $\beta$-ME or cysteamine with BRLG were 81.2 and 86.4, 83.2 and 84.2pM, respectively. The intracellular GSH concentrations of blastocysts in GRlaa containing thiol compounds with BRLG was slightly higher than that of control group The cell numbers of blastocysts were not difference in all experimental groups. These results indicate that thiol compounds with BRLG co-culture was increased the percentage of developed into morulae and blastocysts, and intracellular GSII concentrations of blastocysts embryos.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.63-69
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• 2001

This study was carried out to examine the effects of nitric oxide compounds (hemoglobin and L-NAME) on the development of porcine in vitro maturation (IVM) and in vitro fertilization (IVF) oocytes. Cumulus cell free embryos derived from porcine IVM/IVF oocytes were cultured in NCSU23 medium containing 1~5 $\mu\textrm{g}$/$m\ell$ hemoglobin added to 44 and 96hrs in culture times, and in NCSU23 medium containing 0, 10, 50 or 100mM L-NAME. The developmental rates beyond morulae stage in 0, 1 and 5 $\mu\textrm{g}$/$m\ell$ hemoglobin groups add to 44hrs in vitro culture times were 52.4%, 57.6% and 57.4%, respectively. The addition of hemoglobin groups made it slightly higher than the control group. The proportion of embryos developed to morulae and blastocysts in 1 $\mu\textrm{g}$/$m\ell$ hemoglobin add to 96hrs after in vitro culture (70.8%) was a little higher than those of 0 and 5 $\mu\textrm{g}$/$m\ell$ hemoglobin (66.2% and 62.8%). There was no significant difference in all groups (P〉0.05). The developmental rates beyond morulae stage in 0, 10, 50 and 100mM of L-NAME groups add to 96hrs after in vitro culture were 65.2%, 73.5%, 70.1% and 53.3%, respectively 10mM and 50mM L-NAME groups were significantly higher than in 0 and 100mM of L-NAME groups (P＜0.05). In conclusions, these results indicate that L-NAME (10mM, 50mM) can increase the proportion of embryos that develop into morulae and blastocysts but hemoglobin did not affect.