• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.349-357
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• 1998

This study was to investigate whether the viability of Hanwoo IVM/IVF/IVC blastocysts was maintained after vitrification and thawing. In vitro produced Hanwoo blastocysts were vitrified by two-step method: equilibrated in EG20 for 3 min, and then exposed in EFS40 ［40% ethylene glycol (EG), 18% ficoll and 10.26% sucrose in mDPBS containing 10% FBS ］and vitrified in L$N_2$for 30 - 45 sec. After thawing, in vitro survival was assessed as the re-expanded and hatched rates at 24 hand 48 h, respectively. The results obtained in these experiments were summarized as follows: From the 12 replicates, 52.5% of Hanwoo blastocysts were produced in vitro at day 7 after IVF. When the effects of freezing solution to the embryo survival were examined, there is no significant toxicity in exposure (100.0, 73.8%) compared to that af control group (100.0, 87.0%). However, when embryos were vitrified, high survival (86.2, 55.4%) was obtained although it was significantly lower than those of exposure and control group (p＜0.05). When the in vitro survival of vitrified embryos according to developmental stage and culture day were examined, it showed that more advanced embryo stage exhibited a significantly higher survival rate irrespective of culture day (p＜0.05). Also, even in the same development stage, the in vitro survival of day 7 embryos (re-expanded: 75.0~87.5%, hatched: 21.4~66.7%) was higher than those of day 8 embryos(re-expanded: 58.6~78.3%, hatched: 10.3~52.2%). Therefore, these results suggested that in vitro produced Hanwoo blastocysts can be successfully cryopreserved by simple two-step vitrification method using EFS40 freezing solution, particularly at the expanded and early hatching blastocyst stage regardless of embryo culture duration (day 7 or day 8 after IVF).

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.169-175
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• 2013

Recently, the transgenic animal production technique is very important for the production of bio-parmaceutical as animal bio-reactor system. However, the absence of survival evaluation in vitro produced transgenic embryos has been a problem of the low productivity of transgenic animal because of absent of pre-estimate of pregnancy after transgenic embryos transferred into recipient. Therefore, this study is conducted to improve efficiency of transgenic cattle production by improving the non-surgical embryo transfer (ET) method. Transgenic bovine embryos were produced by injection of feline immunodeficiency virus enhanced green fluorescent protein (FIV-EGFP) lentiviral vector into perivitelline space of in vitro matured MII stage oocytes, and then in vitro fertilization (IVF) was occured. Normal IVF and EGFP expressing blastocysts were transferred into recipients. Results indicated that 2 expanded blastocysts (34.7%) transferred group showed significantly (P<0.05) higher pregnancy rate than 1 expanded blastocyst (26.8%) transferred group. In case of parity of recipient, ET to heifer (34.9%) showed significantly (P<0.05) higher pregnancy rate than ET to multiparous recipient (21.2%). However, there are no significant differences of pregnancy rate between natural induced estrus and artificial induced estrus groups. Significantly (P<0.05) higher pregnancy rate was obtained from recipient group which have normal corpus luteum with crown group (34.8%) than normal corpus luteum without crown (13.6%). Additionally, treatment of $100{\mu}g$ Gn-RH injection to recipient group (38.6%) 1 day before ET significantly (P<0.05) increase pregnancy rate than non- Gn-RH injection to recipient group (38.6%). We also transferred 2 EGFP expressing expanded blastocysts to each 19 recipients, 7 recipients were pregnant and finally 5 EGFP transgenic cattle were produced under described ET condition. Therefore, our result suggested that transfer of 2 good-quality expanded blastocysts to $100{\mu}g$ of Gn-RH injected recipient which have normal corpus luteum with crown is feasible to produce transgenic cattle.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.131-138
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• 2001

This experiment was designed to evaluate effects of nonspecific immunostimulator(NIS) Barodon-FX(equation omitted), anionic alkali mineral complex and far-infrared radiation solution on in vivo-produced mouse and in vitro-produced bovine embryos to blastocyst development. Proportion of mouse embryos developing into blastocyst was not greater in BSA- and Barodon-added medium than in BSA-control, but there was signifcantly different(P ＜ 0.05) in hatching and hatched blastocyst development between 0.25% Barodon-and PVP-contained medium(54.7%) than PVP-control(32.5%). BOEC and GC resulted in higher proliferation rate(24∼40% and 17∼22%, respectively) in 0.25∼0.5% Barodon-added medium than in controls, but proliferation of GC and CC greatly decreased in 1∼2% Barodon-added medium. Effect of Barodon on cell proliferation greatly varied among somatic cells. Proportion of early bovine embryos developing into morula and blastocyst was significantly greater(P ＜ 0.05) in 0.5% Barodon-added medium(50% and 63.6%) than in control(31.6% and 27.4%) under co-culture with BOEC and GC, but developmental rate was not different between other Barodon treatments and control. These data indicate that effect of Barodon on cell proliferation significantly varied between somatic cells and that addition of 0.5% Barodon in BOEC-coculture system may further improve blastocyst development in early bovine embryos.

• Journal of the Korean Society of Tobacco Science
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• v.13 no.2
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• pp.52-58
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• 1991

Interspecific cross between Nicotiana trigonophylla and N. tabacum cv. BY4 is highly sterile because of abnormal ovule and embryo development. In vitro culture of excised N. trigonophylla ovules after polination by N. tabacum allows significant numbers of hybrid embryos to develop into mature plants. Total yield of seedlings and number of normal seedlings were produced following in vitro culture of individual fertilized ovules of N. trigonophylla X N. tabacum at four days post-pollination on B5 medium containing 6% sucrose. Hybrids were uniform in morphology and peroxidase isozyme composition and the majority were cytologically stable: flower characteristics were generally intermediate between those of the parents. All hybrids evaluated were self-sterile.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.231-238
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• 1999

The objective of this study was to examine the effect of developmental stage and embryo age of in vitro produced bovine blastocysts after vitrification and thawing. In vitro cultured day 8 blastocysts after IVF were equilibrated 20% ethylene glycol (EG) for 3 min. and were vitrified using EFS40, which is consisted of 40% EG, 18% ficoll, 0.3M sucrose and 10% FBS added in mDPBS for 30 sec. before being plunged into $LN_2$. Also, survival in vitro was assessed by re-expansion and hatching or hatched at 24 hand 48 h postwarming, respectively. The results obtained in these experiments were summarized as follows; 1) When the embryos were cultured for 8 day after IVF, 41.0% of the cleaved embryos developed to the blastocysts (early; 7.6%, expanded; 22.9%, hatching; 4.6% and hatched; 5.9%). 2) When the embryos were exposed or vitrified to the freezing solution, the re-expansion of vitrified embryos (73.3%) was significantly lower than that of control and exposed embryos (100, 97.0%) (p<0.05). But the formation rate of hatching or hatched blastocysts of vitrified embryos (66.7, 46.7%) at 48h after thawing was similar to that of exposed embryos (66.7, 39.4%) but not control (100, 100%) (p<0.01). However, in the total cell numbers of those developed hatched blastocysts, there were not significantly different among the treatment groups. 3) When the embryo survival rates by different developmental stage were examined, the re-expansion was not different among the groups $(64.5{\sim}75.6%)$. After warming 48 h, the hatching and hatched formation of early blastocysts (25.8, 9.7%) was significantly lower than those of expanded (69.7, 39.4%) and hatching blastocysts (53.3, 43.3%) (p<0.05). 4) In addition, when the expanded blastocysts at day 7, 8 and 9 were vitrified, the re-expansion of day 8 and 9 embryos was significantly lower than that of day 7 (day 7; 93.9%, day 8; 75.8% and day 9; 87.5%) (p<0.05). However, the rates of development to hatched blastocysts were no difference among the groups (day 7; 36.4%, day 8; 36.4% and day 9; 31.3%). These results suggested that in vitro produced expanded or hatching blastocysts can be efficiently cryopreserved by the two-step vitrification method using EFS40.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.109-109
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• 2003

Even though success in birth of live offspring from nuclear transfer(NT) using somatic cells in many species, detailed information on processes or mechanisms of development are not well known. Cytoplasm of bovine oocyte has been known to support the development of nuclear transferred embryos using nuclear donor cells from different species. Therefore, interspecies NT might be used to find answers of some questions in basic aspect of nuclear transfer In this study, we examined the developmental potential of reconstructed embryos when bovine oocyte as a cytoplasm recipient and mouse embryonic fibroblast as a nuclear donor were used. The nuclear transfer units were aliocated in Group 1 (murine block media and normal media) and Group 2. (bovine block media and normal media). NT units were not blocked at 2-cell stage regardless of types of medium. On mouse media, poor development of interspecies NT units was observed compared to bovine media. However, as NT units cultured in bovine normal medium, embryos developed over 8-cell stage. Further studies performed to increase the developmental rate in condition of antioxidant treatment. Despite low development, bovine-murine interspecies nuclear transferred embryos could develop to blastocysts and they showed that blastocyts rate of antioxidant group was superior to those of non-antioxidant group. Next, we investigated gene expression pattern which is carried out for zygotic activation. The Xist gene is expressed in female mouse embryo after zygotic activation of 4-cell stage. But interspecies nuclear transferred embryos do not express Xist gene at 4-cell stage. As a result, it is suggested that the bovine cytoplasm controls the early preimplantation development in interspecies NT However, the development of later stages might require genomic control from transferred donor nucleus. Therefore, even though the involvement of several other factors such as mitochondrial incompatibility, effective development of embryos produced by interspecies NT requires proper genomic activation of donor nucleus after overcoming the cytoplasmic control of recipient oocytes.

• Proceedings of the Korean Society of Veterinary Clinics Conference
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• 2007.10a
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• pp.609-609
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• 2007

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.80-80
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• 2002

This study was carried out that to investigate the effects of amino acids supplemented with culture medium on development of porcine embryos cultured in vitro. Cumulus oocyte complexes (COCs) were cultured in the maturation medium containing hormones (0.5$\mu\textrm{g}$/$m\ell$ LH, 0.5$\mu\textrm{g}$/$m\ell$ FSH and 1$\mu\textrm{g}$/$m\ell$ estradiol-17${\beta}$) for 20-22 h at 39$^{\circ}C$ in an atmosphere of 5% CO$_2$in air. Subsequently, COCs were cultured in hormone-free maturation medium for 20-22 h. After maturation for 40-44h, oocytes were removed cumulus cells by pipetting and cultured with epididymal sperm for 5 h in the mTBM. Embryos obtained were divided in 4 groups (1) cultured in NCSU 23 containing 0.4% BSA to blastocyst stage(Control), (2) essential amino acids (EA), (3) non-essential amino acids (NA), (4) mixture of essential and non essential amino acid (EA+NA). All treated groups(2-4) were used a glucose free NCSU 23 medium supplemented with pyruvate (0.33 mM), lactate (4.5 mM) to morula stage. From morula to blastocyst stage embryos of all treated groups were cultured in NCSU 23 containing 0.4% BSA. The rates of cleaved oocytes at 48 h after IVF were from 82% to 88% in the groups of control, EA, NA and EA+NA, respectively. The in vitro developmental rates into blastocysts in the groups of EA and EA+NA were significantly (P<0.05) higher than those of group of control (35.1, 35.4 vs. 19.4%, respectively), however, no significant (P<0.05) between control and NA. In conclusion, supplemented with essential amino acid or mixture of essential and non essential amino acid in the culture medium at morula stage increased the rate of development to blastocyst on in vitro produced porcine embryos.

• 대한생식의학회:학술대회논문집
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• 1996.11a
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• pp.60.1-60.1
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• 1996

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.69-76
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• 2000

This study was carried out to improve of effective culture system on development of IVM/IVF/IVC bovine embryos. The cumulus-oocyte-complexes (COCs) collected from Korean cattle ovaries harvested at a local abattoir were matured in 50 ${mu}ell$ of TCM199 supplemented with 10% fetal bovine serum (FBS) and hormones (35 $\mu\textrm{g}$/$m\ell$ FSH, 10 $\mu\textrm{g}$/$m\ell$ LH, 1$\mu\textrm{g}$/$m\ell$ estradiol 17 $\beta$ under paraffin oil at 39$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. At 24 hrs after culture, matured oocytes were fertilized in vitro for 22~24 hrs with motile semen in which obtained by centrifugation of a frozen thawed semen on Percoll-density gradients (45% vs. 90%) at 500 g for 20 min. The presumptive zygotes were divided into three experimental groups. Single egg (Group 1), 25 (Group 2) or 50 eggs (Group 3) were cultured on cumulus cell in 50 ${mu}ell$ TCM199 supplement with 10% FBS for 6~9 days after fertilization. In vitro developmental rates into the blastocysts in the groups 2 and 3 were significantly (P＜0.05) higher than those of group 1 (37,27 vs. 6%, respectively). Cell number of blastocysts obtained in groups 2 and 3 at day 8 were significantly (P${mu}ell$) resulted in higher developmental competence and cell number of bovine blastocysts produced in vitro than those the culture of single embryos with cumulus cells.