• Journal of Embryo Transfer
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• v.14 no.1
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• pp.23-29
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• 1999

The objectives of the study were to establish sperm separation method and duration of insemination for bovine IVF. Oocytes from slaughterhouse ovaries were matured and fertilized using general protocol. After 18 or 42 h of insemination, six to ten embryos were placed into a 30${mu}ell$ drop of each medium, and the embryos were examined 7~10d post in semination without medium renewal. First, we compared Percoll gradient will swim-up technique for sperm separation. There was no difference in cleavage rates between them, but the development rates over morula stage of oocytes fertilized with sperm separated by Percoll gradient was significantly higher than that sperm selected by swim-up technique (p<0.05). Second, we evaluated development of bovine embryos derived from the IVF procedure with different durations(18 vs 42 h) of fertilization. There was also no difference in cleavage rates, but the development to blastocyst stage of oocytes exposed in cleavage rates, but the development to blastocyst stage of oocytes exposed to sperm for 42 h was significantly higher than that exposed for 18 h (p<0.05). In conclusion, Percoll gradient can be used for sperm selecton, improving of embryonic development. Also, 42h of IVF may improve the development of bovine embryos.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.11-11
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• 2001

In this study, we examined the developmental potential of reconstructed bovine embryos and the fate of donor mitochondria during their preimplantation development after nuclear transfer. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69%(56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed among blastomeres and could be identified up to the 8- to 15-cell stages. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer These results suggest that donor mitochondria disappear from recipient cytoplasm before 16-cell stage following nuclear transfer in reconstructed bovine embryos.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.93-97
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• 1999

The development potential of bovine somatic cells was evaluated using nuclear transfer. A single donor cell derived from fetus of HanWoo(Korean Native Cattle) was selected and deposited into perivitelline space of each enucleated oocyte before electrical fusion and activation. Nuclei of donor cells starved for 7 days (37%) tended to support the development of reconstitute embryo the blastocyst stage better than those of donor cells starved 3, 14 and 30 days. The cleavage rate was significantly lower(P<0.05) in reconstitute embryos derived from large size donor cells(51.2%), than those from small medium size donor cells(76.6 and 73.5, respectively). The developmental rate to blastocyst of reconstructed embryos from medium size donor cells was higher than those from small and medium size donor cells. This study demonstrates that an appropriate culture period for induction into quiescent stage and the size of donor cells effect on the efficiency of nuclear transfer using cultured bovine cells.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.279-288
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• 1996

The objective of this study was to determine the effect of EGF on the development of IVM/IVF bovine embryos and their ICM and TE cell number. In addition, we examined the combined effect of EGF and coculture to the bovine embryo development and the expression of EGF-R protein on bovine embryos by indirect immunofluorescence. The results obtained in these experiments were summarized as follows: When the IVM/IVF 4- to 8-cell embryos were treated at 0, 1, 10, 100 ng/ml of EGF, EGF treatment group showed improved development to blastocyst and increased pattern of ICM and TE cell number compared with control, although there is not significantly different. The stimulating effect of EGF (10 ng/ml) to the develop ment level of IVM/IVF bovine embryos significantly increased development rate to blastocyst after 8-cell stage (p<0.05), although there is no significant effect to the increase of ICM and TE cell numbers. Also, expression of EGF-R on the bovine embryonic stage by indirect immunofluor escence presents after 4-cell stage and the intensity of the EGF-R staining was variable with the development progression. On the other hand, embryos cultured in coculture group added either with or without EGF commonly indicated the significant difference in development rate to blastocyst and Total cell number compared with control. These results suggest that the a addition of EGF to the coculture may stimulate the coculture effect between IVM/IVF bovineembryos and cumulus cells. Therefore, EGF could promote preimplantation bovine embryo development by binding with expressed EGF~R after 4-cell stage, and stimulate the production of embryotrophic factors from the coculture environment. Also, the present study showed that there was no significant effect of EGF to the increase of ICM and TE cell number although the rate of blastocyst significantly increased when treated with EGF after 8-cell stage (p<0.05).

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.7-14
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• 1994

The present experiments on cryopreservation were designed to examine the effects of solution toxicity, equilibration time and cell stages on the post-thaw survival of bovine IVF embryos. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g /ml FSH, 10 $\mu$g /ml LH, 1 $\mu$g /ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ 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. The bovine IVF 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. 1. after the bovine blastocysts were exposed to EFS solution for 2 min. at room temperature and then they were washed in 0.5 M sucrose solution and TCM-199, they were cultured to examined cryoprotectant induced injury during exposure, Most of the embryos(95.0%) developed to reexpanded blastocoels. However, when the exposure time was extended to 5 and 10 min, these development rates dropped dramatically in 5 min. (69.5%) and 10 min. (47.4%), respectively, 2. When the bovine IVF embryos were vitrified in EFS solution after the equilibration for 1 and 2 min. exposure, The embryos to have reexpanded blastocoels following thawing, washing and culture processes were found to he 82.6 and 73.9%, respectively. However, when the exposure time was extended to 3 min, this survival rate dropped to 18.2%. The optimal time for equilibration of bovine IVF blastocysts in EFS solution seemed to he 1~2 min. 3. When the bovine IVF embryos were equilibrated for 1 min. the significantly (P<0. 05) higher post-thaw survival rates were obtained from the embryos of blastocyst stage(81.3%) than morulae stage(5. 1%). The optimal cell stage for viterification with EFS solution proven to he blastocyst stage in bovine IVF embryos. 4. The number of blastomeres of blastocyst stage was examined with nuclear staining with Hoechst 33342 during 7 to 9 days post-insemination. The cell counts of frozen bovine IVF embryos were found significantly(P$\geq$7.5 and those of the fresh embryos 76.6$\geq$7. 1, which were cultured in the sarne period and conditions as frozen embryos.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.263-267
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• 2008

The purpose of this study was to determine toxic effect of sucrose and trehalose prior to cryopreservation on nuclear maturation and embryonic development in immature bovine oocytes. All cryoprotectant was prepared in tissue culture medium 199-HEPES (TCM 199-HEPES) with 10% fetal bovine serum (FBS). Immature oocytes were exposed to 1.2M ethylene glycol (EG) and 0.1M sucrose or 1.2M EG and 0.1M trehalose for 3 min and then were exposed to 3.2 M EG and 0.25 M sucrose or 3.2 M EG and 0.25 M trehalose for 1 min. Oocytes treated with cryoprotectants were exposed to 0.25 M sucrose or 0.25 M trehalose for 5 min and then 0.1 M sucrose or 0.1 M trehalose for 5 min. Depending on type of sugar added to cryopreservation solution, oocytes were allocated to sucrose group and trehalose group, respectively. Oocytes exposed to TCM 199-HEPES with 10% FBS were considered as control. Oocytes were cultured in TCM 199 supplemented with 10% FBS, 5 ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone, and $1\;{\mu}g/ml$ estradiol for 24 h in $39^{\circ}C$, 5% $CO_2$. Nuclear maturation was assessed by staining oocytes with 1% aceto-orcein. Oocytes were fertilized in vitro and were cultured in TCM 199 supplemented with 10% FBS, 5 mM sodium pyruvate, and antibiotics in $39^{\circ}C$, 5% $CO_2$. The rates of cleavage and blastocyst, and cell number in blastocyst were assessed. Metaphase II rates were not different among experimental groups regardless of type of sugar. The cleavage rate of trehalose group (73.3%) was significantly higher (p<0.05) than those of sucrose group (62.8%) and control group (60.8%). The blastocyst rate was significantly higher in trehalose group (p<0.05). Mean cell number in blastocyst were not different among experimental groups, although cell number of blastocyst in trehalose group was significantly higher on day 7 (p<0.05). In conclusion, sucrose and trehalose were not toxic to immature bovine oocytes prior to cryopreservation. In particular, trehalose was more effective on embryonic development.

• 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.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.28-28
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• 2001

The objective of this study was to the effect on subsequent development of EGF present in defined medium during bovine 1)oocyte maturation or 2)embryo culture. The presence of EGF during IVM, irrespective of concentration(1, 10, 100ng/$m\ell$), stimulated cumulus expansion and significantly increased the proportion of oocytes attaining metaphaseII, the rate of cleavage, and develop to blastocyst. 1. In the group of EGF-added medium(1, 10, 100ng/$m\ell$), nuclear maturation rate for in vitro maturation was 91% to 92% but was not significantly higher than control group(87%). 2. For in vitro maturation, in the group of EGF-added medium(1, 10, 100ng/$m\ell$)the rate of cumulus cell expansion degree 2 ranged from 81% to 87%, which was significantly higher than the control group(medium with EGF not added). The rate of in vitro fertilization, developing to 2-to 4- cell stage, was 76% to 80%, which was also significantly higher(p<0.05)than control group(62%). 3. For in vitro maturation, in the group of EGF added in medium(1, 10, 100ng/$m\ell$)the development rate to blastocyst was 24.3% to 27%, which was significantly higher than control group(13.7%). The total cleavage rate in the group of EGF-added medium was 77% to 82%, which was higher than control group. 4. The development rate to blastocyst for 6 days of cultivation and the hatching blastocyst were 30.6% and 59.1%, respectively, in the group of 100ng/$m\ell$ of EGF, which were significantly higher(p<0.05)than control group(14.0% and 24%, respectively), The numbers of cells in blastocyst were 140.2 and 148, respectively, in 10ng/$m\ell$ and 100ng/$m\ell$ of EGF-added medium, which were higher than 108.5 in control group. 5. The development rate of in vitro fertilized embryos to blastocyst in 10ng/$m\ell$ of EGF-added medium co-cultured with somatic cell was 28%, which was significantly higher(p<0.05)than control group(11.8%). The numbers of cells in blastocyst were 141.6 for EGF-added medium and 145 for EGF+co-culture group, which were higher than control(101.6)and medium co-cultured with somatic cells(110.6). These results showed that in vitro maturation and fertilization, EGF was found a significant effect of increase of development rate to blastocyst and cell number.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.1
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• pp.23-28
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• 2003

The present study was undertaken to investigate the effects of PVP concentration and exposure temperature to vitrification solution on the post-thaw survival, in vitro maturation and development of immature bovine oocytes (germinal vesicle stage). The vitrification solution (VS) consisted of 40% ethylene glycol (EG)+0.5 M sucrose (S)+10% FBS. PVP was added to VS: 0%, 5% or 10%. The cumulus-oocyte complexes (COCs) were diluted in VS as one step, after 2 min the COCs were loaded in straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were plunged into $30^{\circ}C$ water bath for 10s. After thawing, the oocytes were diluted in 0.5 M (in DPBS with 10% FBS) sucrose solution for 5 min. The survival rate (FDA-test and trypan blue) of immature bovine oocytes was measured. The survival rate was higher in 5% PVP (91.5%) than in 0% (64.2%) or in 10% PVP (79.7%). The proportion of metaphase II formation was 69.35% in control (no vitrified COCs), 9.3% in 40% EG+0.5 M S+0% PVP and 21.05% in 40% EG+0.5 M S+5% PVP (p<0.05). The effect of room temperature ($25^{\circ}C$ for 10 min) and cold temperature ($4^{\circ}C$ for 10 min) on COCs were determined in this study. After IVF, the cleavage and blastocysts rate of oocytes exposed to room temperature and cold temperature in VS+5% PVP was significantly different (2 cell: 63.20% vs 37.97%, blastocysts: 18.40% vs 2.53%). The cleavage rates of frozen-thawed oocytes were 20.53% with PVP and 22.13% without PVP (p>0.05). Two out of 151 oocytes (1.32%) developed to blastocyst stage after frozen-thawed with 5% PVP (p>0.05). Development of oocytes after frozen-thawing to the 2 cell were not significantly affected with or without PVP following IVF. However, the vitrification of immature bovine oocytes with PVP maintained the ability to develop to the blastocyst stage after IVM-IVF and IVC, while no blastocysts were obtained from oocytes vitrified without PVP. These results suggested that PVP has a protective role for vitrification of immature bovine oocytes as far as survival is concerned, however, the protection was not sufficient enough to support blastocyst formation.

• Korean Journal of Animal Reproduction
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• v.19 no.3
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• pp.191-195
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• 1995

Bovine follicular oocytes were matured in vitro for 3D hr and exposed to Dulbecco's phosphate buffered saline containing 7% ethanol for 7 minutes for their activation, and incubated in TCM199 containing 10% fetal calf serum (FCS) and cytochalasin D (CD). When the oocytes were exposed to cytochalasin D for 0, 5, 10 and 15 hr, 5 hr showed highest developmental rate to blastocyst (16%), expanded blastocyst (13%) and hatched blastocyst (7%). Also when the oocytes were cultured for 7 hours in medium contai ning 0, 2.5, 5 and 7.5 ${\mu}\textrm{g}$/ml cytochalasin D, 2.5 ${\mu}\textrm{g}$/ml showed highest developmental rate to blastocyst (13%), expanded blastocyst (7%) and hatched blastocyst (4%). In conclusion, cytochalasin D played very important role for parthenogenetic development of follicular oocytes and 5 hr of exposure time to CD and 2.5 ${\mu}\textrm{g}$/ml CD in the medium was optimal for development of the follicular oocyte.