• Journal of Embryo Transfer
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• v.10 no.3
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• pp.229-236
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• 1995

The experiments reported here take advantage of the large number of in vitro matured and in vitro fertilized(IVM /IVF) bovine oocytes which can be produced, permitting the design of controlled experiments to establish a simple defined medium for the study of early embryo requirements. A total of 1,386 IVM /IVF oocytes were used to compare a simple defined medium(KSOM) with more complex culture conditions used successfully for culture of bovine embryos but do not permit study of specific requirements. All experiments were extensively replicated factorials. In Experiment 1, KSOM was superior to Menezo B$_2$ medium in producing morulae plus blastocysts from IVM /IVF oocytes(33 vs 20%, P<0.()5). The yield of morulae plus blastocysts with KSOM was 22% and with BRLC added was 30%. In Experiment 2, (a 2x2 factorial of KSOM with or without BRLC and 0, 1 ng /ml of platelet derived growth factor, PDGF) more morulae plus blastocysts (40%) were produced in KSOM-BRLC co-culture containing 1 ng /ml PDGF than in the control KSOM(12%). In Experiment 3, there was no dose response when 0, 1 and 5 ng /ml of PDGF were added. The results with simple defined KSOM medium are sufficiently promising to indicate that specific requirements of the embryo may be examined in future studies with KSOM as a base.

• Journal of Life Science
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• v.6 no.3
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• pp.204-212
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• 1996

The aim of this study was to evaluate the effects of phosphate, aimno acid, and BSA on in vitro development of mammalian embryos. In vitro-matured and -fertilized(IVM/IVF) bovine embryos were cultured in simple, chemically defined, protein-free medium(mTLP-PVA0. When the phosphate concentration of mTLP-PVA supplemented with 19 amino acid were adjusted to 0.0, 0.10, 0.35, 1.05 and 2.10mM by the concentration of sodium phoshpate, there were no significant different in development ability of IVM/IVF bovine embryos cultured in the medium containing from 0.00 to 1.05mM phosphate until 48 hours post-insemination, However, proportion of embryos developing to $$8-cell and morula at 96 and 144 hours post- insemination, respectively, was significantly increased in the medium with o.35 mM phosphate(p<0.05). There was significant difference between O.10(18%)-0.35(24%)mM phosphate and 1.05(13%)-2.10(1%)mM phosphate in supporting development to blastocyst(p<0.05). When IVM/IVF bovine embryos were cultured in the medium supplemented with 19 amino acids, significant different was observed in the proporton of embryos reaching $$8-cell(49-50%), morula(38-40%) and blastocyst (29-32%) stages at 96, 144, and 192 hours post-insemination, respectively(p<0.05). Glutamine alone had no benefit on embryo development. When BSA was added to mTLP-PVA with 0.35mM phosphate, glutamine and 19 amino acids at 8, 48, 120 hours post-insemination, BSA significantly enhanced the development ability ofb embryos reaching $$2-cell (74-77%), $$8-cell (49-53%), morula(43-47%), and blastocyst(38-42%) stages at 48, 96, 144, and 192 hours post-insemination, respectively, regardless of the time of BSA addition.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.311-317
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• 2000

This study was carried out to compare the temperature and time of heat shock, and the effect of heat shock on development of embryos after in vitro maturation and fertilization in bovine oocytes. The results obtained were as follows. 1. The optimum temperature and time of heat shock were 41$^{\circ}C$ and 30sec on in vitro development of embryos from 4~8 cell to blastocyst. 2. The rates of cleavage on zygotes produced on in vitro were significantly increased by heat shock after IVM than before IVM(P<0.05). 3. When the oocytes were treated heat shock after IVM and 5 days cultured, developmental rates to blastocyst were increased than other experimental treatments.

• 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.2
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• pp.113-117
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• 2004

The present study was carried out to examine the effects of $O_2$ concentrations and culture media (North Carolina State University (NCSU)-23, porcine zygote medium(PZM)-3 or PZM-4) on in vitro development of porcine IVM/IVF embryos. Porcine oocyte-cumulus complexes were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.9 mM), $\beta$-mercaptoethanol (25 $\mu\textrm{g}$/$m\ell$), epidermal growth factor (10 ng/$m\ell$) and hormonal supplements (PMSG and hCG: 10 IU/$m\ell$) for 20∼22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20∼22 h. After culture, cumulus-free oocyte were coincubated with liquid boar spermatozoa for 5∼6h. Putative zygotes were transferred to NCSU-23, PZM-3 and PZM-4 medium under the condition of 5% $O_2$ or 20% $O_2$ concentrations. At 48 h, no mean differences were found in cleavage rates. However, the rates of blastocyst formation at day 7 after in vitro fertilization were significantly higher in PZM-3 medium under the condition of 5% $O_2$ concentration than other treatments (19.9$\pm$2.4 vs. 11.1$\pm$2.0 to 16.0$\pm$2.5%, P<0.05). The total cell numbers of blastocysts were significantly higher in 5% $O_2$ than in 20% O2 (P<0.05). However, no differences was found among the culture media within each $O_2$ concentrations. In conclusion, the use of PZM-3 medium in 5% $O_2$ concentration was effective on in vitro development of porcine IVM/IVF embryos.

• 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.59-60
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• 1996

• 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.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.8-17
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• 2002

There have been intensive attempts to establish reliable methods far in vitro production (IVP) methods for of porcine embryos. Although a great deal of progress has been made, our current IVP systems still need to be improved. In this review, we focused on studies about in vitro maturation and fertilization (IVM-IVF) of porcine oocytes and their in vitro culture (IVC), especially on an excellent piglets production system using modified IVP system producing porcine blastocysts with high Quality.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.128-128
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• 2003