• 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.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.113-119
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• 1999

This study was performed to improve the development of the in vitro fertilized bovine embryos by the condition of in vitro maturation. COCs were matured in TCM 199 supplemented with 0.1% PVA, 10ng/ml EGF, Hormones (5$\mu\textrm{g}$/ml FSH, 10 IU hCG, 1 $\mu\textrm{g}$/ml estradiol 17-$\beta$) or granulsa cell+Hormones atmosphere 39$^{\circ}C$, 5% CO2, 95% air for 24hrs. Matured oocytes were fertilized with frozen-thawed semen capacitated with 5mM caffein in BO medium for 20 hrs. IVF embryos were cultured in TCM 199 containing with hormones(same as matured medium), 10% FBS and co-culture with bovine oviduct epitherial cells. Maturation rates of COCs were showed 73.8%, 78.5%, 83.2% and 87.6% respectively, and were significant differences between PVA, EGF, and Hormones, GC+Hormones(p<0.05). The cleavage rates of IVF embryos were revealed 72.5%, 78.4%, 82.3% and 84.2% and showed same tendency as maturation rates(p<0.05). The blastocysts matured by above maturation condition and cultured for 7~10 days after fertilization had 34.4, 43.6, 52.3 and 59.3 cells had no differences among the treatments. These results suggest that high molecules as a substitutes of serum and growth factor may induce nuclear resumption of COCs but we need more study to produce transferable IVF blastocysts by use of that agents.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.67-74
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• 1997

This study was carried out to increase the viability of bovine frozen4hawed in vitro produced (IVP) embryos and pregnancy rate by direct transfer method. Cumulus-oocyte complexes were aspirated from excised Hanwoo ovaries and matured in TGM 199 for 20~22 hours at 38.5$^{\circ}C$ in 2% $CO_2$ in air. Matured oocytes were fertilized with capacitated sperm for 6 hours and then co-cultured with cumulus cells for 9 days. 63% of the oocytes cultured was deaved and 29% out of them developed into blastocysts. Good or excellent grade of blastocysts on D 7 or 8 were frozen with 1.8M ethylene glycol as a cryoprotectant for direct transfer. Frozen embryos were thawed at 2$0^{\circ}C$ water for 10 sec following 4~5 second in air. For the survival assay of frozen4hawed lVP blastocysts, they were cultured in TCM 199 supplemented with 100$\mu$M $\beta$-mercaptoethanol and 20% FCS for 72 hours. The percentage of embryos developed to re-expanded or hatched after 72 hours culture was 95. 5 and 77.3%, respectively. When frozen-thawed Ivp embryos were transferred to 43 synchronized recipients by direct transfer method, eighteen recipients (41.8%) was pregnant. The highest pregnant was in naturafly synchronized recipients (71.4%), but induced estrus by using PRID(29.2%) and PGF$_2$$\alpha$(20.0%) was showed lower pregnancy rate. The pregnancy rate was higher in day 7 blastocysts(56.0%) than day 8 blastocysts(22.2%). (Key words: in vitro produced, blastocyst, frozen-thawed, direct transfer)

• Korean Journal of Medicinal Crop Science
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• v.7 no.4
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• pp.296-302
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• 1999

This experiment was conducted to study the effect of $CO_2$(0, 2, 500, 5, 000, 10, 000ppm) enrichment by enabling ventilation on micropropagation of multi-shoot and on the saponin contents in vitro in Korean ginseng (Panax ginseng C. A. Meyer). Embryo was cultured in Murashige and Skoog medium added 3mg/ l of Indolbutyric acid, Benzyladenin and Gibberellic acid $(GA_3)$, respectively. $CO_2$, enrichment had little effects on the number of adventitious buds and shoots originated from adventitious buds. The ratio of differentiated shoots to adventitious buds were about 50% in $CO_2$, enrichment treatment. The shoots originated from adventitious bud showed more rapid growth and had larger leaf area than the shoots originated from the leaf primordia did. The number of shoot primordia was the highest in 2, 500ppm of $CO_2$ enrichment treatment. On the contrary, 10,000ppm of $CO_2$, enrichment made smaller the number of shoot primordia and ratio of shoots to shoot primordia. The range of shoots differentiated was from shoot primordia were 15. 4 to 23. 9. The rate of dry weight of cultured shoots showed lowest (7. 5%) in control and highest (8. 59%) in 2, 500ppm of $CO_2$, enrichment. Rate of in vitro flower in control was 7.6% and that in 2500ppm of $CO_2$ was about twice (15.7-16.3%) as much as in control. Flower number per a embryo cultured was about 1.2-1.3. In the multi-shoots with callus enriched by 2, 500ppm of $CO_2$, the contents of crude saponin and ginsenosides in multi-shoots alone were higher than in multi-shoots with callus. The characteristics of ginsenosides in multi-shoots were especially the higher content of ginsenoside Rd, Re, and $Rg_1$.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.60-61
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• 2001

To produce reconstituted rabbit embryos with fetal fibroblasts, the present study was evaluated the efficiencies of the activation conditions as assessments of subsequent development and chromosome in the embryos. New Zealand White rabbits were used throughout the study. Fetal fibroblasts collected from 22-d of fetuses were cultured in DMEM＋10% FBS in 5% CO₂ in air. The culture was maintained for 10 passages. In every passage half of cell suspension were kept in frozen. (omitted)

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.1
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• pp.33-38
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• 2004

This study was aimed at testing the gene expression of antioxidant enzymes and apoptosis genes for in vitro culture in porcine embryos produced by in vitro maturation/in vitro fertilization (IVM/IVF). Pocine preimplantation embryos obtainted from IVM/IVF can be successfully culture in vitro, but they are delayed or stop to develop at specific developmental stage. Many factors such as reactive oxygen species and apoptosis in an IVM/IVF system followed by in vitro culture influence the rate of production of viable blastocysts. Porcine embryos derived from IVM/IVF were cultured in the atmosphere of 5% $CO_2$ and 20% $O_2$ at $38.5^{\circ}C$ in NCSU23 medium. The patterns of gene expression for antioxidant enzymes and apoptosis genes during in vitro culture in pocine IVM/IVF embryos were examined by the modified semi-quantitative single cell reverse transcriptase-polymerase chain reaction (RT-PCR). Porcine embryos produced by in vitro procedures were expressed mRNAs for CuZn-SOD, GAPDH and GPX, whereas transcripts for Mn-SOD and catalase were not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell 16 cell and blastocyst, but p53 mRNA was not detected at any stages. The fas transcripts was only detected in blastocyst stage. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in vitro culture of porcine IVM/IVF embryos.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.63-69
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• 2005

This study were carried out to evaluate the possibility of nuclear progression of canine immature oocytes, of which was cultured in a reproductive tract, such as oviduct, ovarian bursa and uterus of estrus bitch for 4, 5 and 6 days following immediately collection. Cumulus intact oocytes(COC) fore collected from domestic dog following ovariohysterectomy at local veterinary clinics. In Exp. 1, COCs $of>110\;{\mu}m$ diameter were selected and cultured in vitro at $39^{\circ}C$, $5\%\;CO\_{2} $ in air atmosphere. The nuclear progression of canine oocytes checked at 24, 48 or 72 h after in vitro maturation. There was not increased the nuclear progression to the M II stage depending on culture periods at 24, 48 and 72h $(1.3\%,\;3.7\%\;and\;4.7\%)$. In Exp. 2, to evaluate of nuclear progression of immature oocytes, collected or in vitro cultured oocytes were transfer into a canine reproductive tract (oviduct, ovarian bursa and uterus). The recovery rates of canine oocytes from a reproductive tract after 4 days $(33.7\%)$ in vivo culture were significantly higher than those 5 $(17.7\%)$ 6 day $(3.4\%)$ (P<0.05). The survival rates of collected oocytes after 4 days $(60.0\%)$ were also significantly higher than those of 5 days $(30.2\%)$ and 6 days $(38.9\%)$ (P<0.05). The meiotic resumption rates of canine oocytes were not significantly difference among the culture periods at 4 days $(5.9\%)$, 5 days $(0.0\%)$ and 6 days $(0.0\%)$. These results show that the nuclear progression of canine immature oocytes from in in vivo culture was not affect the nuclear resumption of oocytes.

• The Korean Journal of Malacology
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• v.32 no.1
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• pp.37-43
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• 2016

This study was carried out to determine the optimal water temperature for the embryonic development and laboratory culture of larvae of an intertidal mud snail, Nassarius festivus. The embryos and hatched veliger larvae of N. festivus were incubated at six different temperatures (5, 10, 15, 20, 25 and $30^{\circ}C$). Developmental time for each stage decreased as water temperature increased. The elapsed time to develop to the veliger larva at 15, 20, 25 and $30^{\circ}C$ was 559, 155, 131 and 103 hrs, respectively. At 5 and $10^{\circ}C$, embryo developed to veliger larvae but failed to hatch out of the egg capsule. In contrast, all embryos successfully hatched in the temperature range from 15 to $30^{\circ}C$. The biological minimum temperature during the embryonic development of N. festivus was estimated to be $9.5{\pm}0.4^{\circ}C$. The cumulative water temperatures for blastula, gastrula and veliger stages were calculated as $111{\pm}84$, $486{\pm}185$, $1,164{\pm}72^{\circ}C$, respectively. Temperature also affected the larval survival. Five days after hatching, more than 84% of larvae survived at all experimental temperatures. However, survival began to decrease after 6 days. It was 0% at $30^{\circ}C$. Survival of larvae incubated for 8 days was higher at 15 and $20^{\circ}C$ than other experimental temperatures. We therefore suggest that the optimal range of temperature for embryonic development and larval survival of N. festivus is $15-20^{\circ}C$.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.127-132
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• 2014

This study was carried out to investigate the effects of tissue inhibitor of matalloproteinase-1 (TIMP-1), Activin A and Heparin binding epidermal growth factor (HB-EGF) on in vitro production of bovine embryos. In experiment 1, presumptive zygotes were cultured in the medium supplemented with TIMP-1 ($0.5{\mu}g/ml$), Activin A (100 ng/ml), or HB-EGF (100 ng/ml) at $39^{\circ}C$ in a humidified atmosphere of 5% (v/v) $CO_2$, 5% (v/v) $O_2$ and 90% (v/v) $N_2$. In experiment 2, TIMP-1 + HB-EGF or Activin A + HB-EGF combinations were supplemented in the culture medium. The developmental rate to blastocysts, hatching rate and total cell numbers of the blastocysts were evaluated in both experiments. The embryos cultured in medium without growth factor supplementation was used as control group. In experiment 1, the embryos cultured in medium supplemented with TIMP-1 and Activin A showed significantly higher developmental rate to blastocysts than those cultured with HB-EGF and control (36.9%, 34.1%, 21.2% and 23.1%, respectively) (P<0.0001). However, the hatching rate of blastocyst was significantly higher in embryos with HB-EGF than those with TIMP-1, Actvin A and Control groups (84.4%, 58.8%, 51.4% and 49.3%, respectively) (P<0.001). Total cell number per blastocyst was also significantly higher in embryos with HB-EGF group ($174.3{\pm}2.5$) than those with TIMP-1, Activin A (149.7 and 150.0, respectively) (P<0.05) and Control (119.0) (P<0.001). In experiment 2, embryos cultured with combined treatment of Activin A and HB-EGF resulted in significantly higher rates of blastocysts formation (48.0%), hatching rate (89.7%) and total cell number in blastocyst ($182.3{\pm}2.1$) than those with TIMP-1 and HB-EGF combination group (32.0%, P<0.001; 76.6%, P<0.05; $165.7{\pm}4.2$, P<0.001, respectively). Our data demonstrate that in vitro production of bovine embryos could be improved by combined supplementation of Activin A and HB-EGF in culture medium.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.11-20
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• 1997

The influence of cryopreservation of donor embryos on the in vitro developmental potential in the nuclear transplant rabbit embryos was evaluated. The embryos of 16-cell stage were collected and cryopreserved with EFS solution by vitrification method. The frozen embryos were thawed and synchronized to S and G$_1$ phase of 32-cell stage. The recipient/ cytoplasms were obtained by removing the first polar body and chromosome mass from the oocytes collected by non-disruptive microsurgery procedure. The separated S and G$_1$ phase blastomeres of 32-cell stage were injected into enucleated recipient cytoplasms by micromanipulation. After culture until 20 hrs post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation. The fused nuclear transplant embryos were co-cultured with rabbit oviduct epithelial cells. After in vitro culture for 120 hrs, the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. The electrofusion rate was significantly (P<0.05) reduced in the frozen nuclear donor,compared with fresh donor nuclei as 80.0 vs 62.8% in S phase and 81.7 vs 64.8% in G$_1$phase, respectivley. The in vitro developmental rate to blastocyst stage with the S and G$_1$phase of fresh embryos(26.3 and 61.1%, respectively) was found significantly (P<0.05) higher, compared to the S and G]phase of frozen embryos(11.9 and 34.6%, respectively). When frozen as well as fresh donor embryos were synchronized to G$_1$ phase, the in vitro developmental rate to blastocyst stage was significantly (P<0.05) higher, compared with S phase donor nuclei. The cell counts of nuclear transplant embryos developed to blastosyst stage were significantly (P<0.05) more in G$_1$ phase of fresh or frozen embryos (180.1 and 125.7 cells, respectively), compared with S phase nuclear donor (145.1 and 103.7 cells, respectively). From the above results it was concluded that the rabbit embryos cryo- preserved by vitrification might be available as nuclear donor, though the developmentalpotential and cell counts of nuclear transplant rabbit embryos were decreased significantly.