• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.73-73
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• 2001

This study was to examine whether the vitrified, one-step diluted and direct transferred Hanwoo IVM/IVF/IVC blastocysts can be successfully survived in vivo and they were succeeded into the live birth. For vitrification, blastocysts were serially exposed in glycerol (G) or/and ethylene glycol (EG) mixtures [10% (v/v) G for 5 min, 10% G plus 20% EG (v/v) for 5 min, and 25% G plus 25% EG (v/v) for 30 sect] which is diluted in 10% FBS added D-PBS. Thawing of straw was carried out in air for 10 sec and then in water bath of $25^{\circ}C$ for 20 sec. One-step dilution within the straw was done in water bath of $25^{\circ}C$ for 1 min. Vitrified and one-step diluted embryos were directly transferred into 36 (natural or hormone induced synchronized) recipient cows in 6 areas of Kyungsang Buk-Do. Pregnancies were confirmed at first when recipient cows did not return to the subsequent estrus cycle, and later by manual palpation per rectum on day 45, 90 and then living calves were derived into parturition. Overall pregnancy was 33.3%(12/36), However, higher pregnancy was obtained when the recipients exhibited estrus one day earlier than the age of transferred embryos (53.3 vs 25.0-27.3%), irrespective of synchronization methods. Also, parous recipients became pregnant higher than nulliparous heifers, And, there were not different in pregnancy rates by the aspect of corpus luteum (CL) quality of recipients (good, 29.4; fair, 37.5; poor, 33.3%). One hundred eight of frozen-thawed Hanwoo blastocysts were directly transferred into 36 recipient cows. In 12 of pregnant cows, 3 cows were aborted and 9 cows were calved [single, 66.7% (6/9): twin, 33.3% (3/9)]. Total embryo implantation rate was 11.1% (12/108). However, 9 Hanwoo calves were lived. Therefore, these results demonstrate that direct transfer technique of vitrified and one-step diluted bovine blastocysts can be applied easily and effectively with the higher pregnancy rate on field trial without the equipment and embryological skills.

• 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.18 no.1
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• pp.47-54
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• 1994

The objective of this study was to produce Korean native calves following transfer of in vitro matured, fertilized and cultured embryos into Holstein cows. The ovaries of Korean native cows or heifers were obtained from an abattoir and kept on 25 to 28$^{\circ}C$ and transported to laboratory within 2 hrs. The oocytes were matured in vitro (IVM) for 24 hrs in TCM-199 supplemented with 35$\mu\textrm{g}$/ml FSH, 10$\mu\textrm{g}$/ml LH, 1$\mu\textrm{g}$/ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% CO2 in air. They were fertilized in vitro (IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro (IVC) with bovine oviductal epithelial cells for 7 to 9 days. Late morulae and blastocysts produced in vitro were nonsurgically transferred to recipient cows by unilaterial. Recipients were monitored for estrus and for pegnancy by rectal plapation in 60 days after embryo transfer. One of them was pregnant to term and produced a female weighing 42.5kg at birth.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.459-465
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• 1997

The present study was undertaken to determine the effect of NT time on the rate of fusion a and suhseguent development In vitro and determine the optimal strength and duration of DC pulse for electrofusion of IVF donor embryo nuclei and IVM recipient oocytes. The recipient oocytes were enucleated 25 ~ 2Sh after IVM and further cultured for 18 ~ 20h prior to fusion for oocyte aging. IVF embryos as donor nuclei were C co cultured with BOEC for 16- to 32-cell stage development. The transfer time of donor bIas tomeres was 1~3h post-enucleation in early NT group and 1 ~ 18h post-enucleation in late NT group, respectively and fusion was performed 43~4Sh post-IVM. The fusion rate did not differ between the early NT and late NT group, but the rate of cleavage and 8- to 16-cell stage embryos in the late NT group was more higher than that in the early NT group. The fusion, cleavage and M+B development was high from O.7SkV /cm DC than from 1.0kV /cm DC voltage, resulting in 17.6% M+B from 0.75kV /cm DC voltage. No difference in fusion rate was among pulse durations, but 50 and 70 usec pulse duration showed slight high cleavage and M + B d development. The results indicate that the best NT time of IVF donor blastomeres into the enucleated oocytes was 42~44 post-IVM and the most suitable condition for electrofusion was a single 0.7SkV /cm DC voltage for SO~70$\mu$sec.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.161-166
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• 2004

This study was designed to evaluate the effects of nitric oxide scavenger (hemoglobin) and inhibitor (L-nitro-L-arginine methyl ester; L-NAME) with or without cumulus cell on the development of bovine IVM/IVF embryos. When CR/sub 1aa/ medium were supplemented with different dosage (lug/m, 5ug/m and 10ug/ml) of hemoglobin at 48hrs for in vitro culture, the proportion of embryos developing beyond morulae stage in 0, 1ug/ml and 5ug/ml with or without cumulus cell were 23.8%, 33.3 % and 26.8%, and 39.5%, 54.8% and 48.8%, respectively. There was a significantly difference the developmental rate of 1ug/ml hemoglobin intact cumulus cells to any other groups (P<0.05). On the other hand, when added to hemoglobin at 96 hrs, 1 ug/ml hemoglobin with cumulus cell group was significantly increased the percentage of developing into morulae and blastocysts to any other groups (P<0.05), and similar trend that of added at 48hrs. The overall means of the percentage of developing into morulae and blstocysts in 1ug/ml hemoglobin group was significantly increased than those of any other groups (P<0.05) and cumulus co-culture with hemoglobin was increased the in vitro developing rate of IVM/IVF embryos. In CR/sub 1aa/ medium treated with L-NAME 0, 10, 50 and 100mM, the developmental rate of morula plus blastocysts were 55.6%, 64.9%, 58.8% and 66.7%, respectively. The L-NAME did not affect the developmental rate and the cell numbers of blastocysts in all treated groups. These results indicate that hemoglobin and cumulus co-culture can increase the proportion of embryos that developed into morulae and blastocysts, but cell numbers of blastocysts were not affect in all groups.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.181-186
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• 2005

This experiment was conducted to investigate effects of the two different in vitro production systems, serumcontaining system (IVM, IVF and IVC; TCM199, TALP and CR1aa) and serum-free system (IVM, IVF and IVC; IVMD101, IVMD100 and IVMD101), on the development of in vitro fertilized or DNA-microiniected embryos. We also examined the effect of DNA dosage and its expression pattern in embryos. The DNA used for microinjection was a green fluorescence protein gene. The development rates to $\geq$ 2cell, 8cell and blastocyst stage were significantly higher in vitro fertilized embryos than those in DNA-microinjected embryos. The development rate to the 8-cell stage was significantly higher in serum-free system than in serum-containing system (p<0.05; $3.3\%\;vs.\;15.5\%\;and\;21.4\%$, respectively). The development rates to the blastocyst stage of in vitro fertilzed or DNA-microinjected embryos between two different culture system ($2.7\%\;vs.\;2.3\%\;and\;23.0\%\;vs.\;23.6\%$, respectively) were not different. The development rates of embryos injected 2 ng/uL DNA was higher. than those of embryos injected 4 or 8 ng/uL DNA. The GFP expression rate of 1-cell embryos was significantly higher than that of 2-cell and 4-cell embryos, whereas the rates were not different between 4-cell and blastocyst-stage embryos.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.67-75
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• 2000

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 $\mu\textrm{g}$/ml epidermal growth factor(EGF) at 39$^{\circ}C$ under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.7$\pm$5.1 and 96.6$\pm$4.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.4
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• pp.495-499
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• 2006

In our previous studies, we demonstrated that Vascular Endothelial Growth Factor (VEGF) enhances bovine oocyte maturation and early embryonic development in serum supplemented media. In this experiment, to determine the synergistic effect of VEGF with serum components on early embryonic development in vitro in cattle, 1 mg/ml polyvinyl-alcohol (PVA) was replaced with foetal bovine serum (FBS) in maturation and culture media. Bovine oocytes were matured in Synthetic Oviduct Fluid (SOF) supplemented with PVA, PVA+5 ng/ml of VEGF, FBS, or FBS+VEGF. Fertilized oocytes were cultured in the same conditions for 8 days. The development of embryos was examined at 48 h post- insemination and on days 6, 7 and 8. The results were analyzed using repeated measures two- factor ANOVA, in which the effects of VEGF and serum were assigned as two factors. The development rate to 4- to 8-cell embryos at 48 h was significantly higher in the PVA+VEGF group than in the PVA group (44.7% and 31.5%, respectively). However, the highest development rate to 4- to 8-cell embryos was obtained from the FBS+VEGF group (58.8%). On day 8, the blastocyst rates were higher in the PVA+VEGF (22.8%), FBS (32.1%, p<0.05) and FBS+VEGF (42.1%, p<0.05) groups than in the PVA group (17.1%). Two- factor ANOVA of the development rates indicates that VEGF had a significant effect, but had no synergistic effect with serum components on early embryonic development. The results of the present study demonstrate that VEGF improves the in vitro developmental competence of bovine oocytes and/or embryos independent of the effect of serum components.

• 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.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.128-128
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• 2003

The aims of this study are 1) to test oocytes and embryos collected from in-vivo and in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al. Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at 39$^{\circ}C$, and 10% fetal bovine serum was added to the culture medium thereafter. Embryos were treated with 7.5$\square$g/ml cytochalasin-B for 30 min, centrifuged at 13,000 ${\times}$ g for 13 min and then exposed sequentially to an ethylene glycol (EG) vitrification solution, aspirated into OPSs, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three donors after Al. Forty-six embryos (18, 9 and 19 embryos, respectively) were washed 3 times in mPBS+10%FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients received surgically 34(control), 188 and 184 embryos (derived from abattoir), respectively. Another three recipients were received nonsurgically 150, 100 and 150 embryos, respectively. All recipient sows exhibited delayed returns to estrus. To our knowledge, these results suggest that required an improved techniques, more vigorous embryos preparation and cleaner uterous condition(use gilt).