• Korean Journal of Animal Reproduction
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• v.18 no.2
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• pp.105-111
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• 1994

The effects of human or mouse leukemia inhibitory factor(hLIF or mLIF) were examined as a means of increasing the development of in vitro matured(IVM) and in vitro fertilized (IVF) oocytes into morulae or blastocysts. Cell numbers of blastocysts were also counted using Hochest dye staining. Two-to 8-cell embryos derived from bovine IVM/IVF oocytes were cultured 5 to 6 days in CRI aa with or without mLIF or hLIF. All culture media were contained 3mg/ml bovine serum albumin. In experiment 1, the proportion of embryos developed to morulae and blastocysts in CRI aa containing 5,000U/ml mLIF(37.8%) was slightly higher than those of CRIaa containing 1,000U/ml mLIF(34.6%) and 0 U/ml mLIF(27.4%; P>0.05). In experiment 2, 0, 1,000 and 5,000U/ml of hLIF added to CR1aa media yielded 27.6%, 43.0% and 35.5% morulae and blastocysts, respectively(p>0.05). These were no significant increases in cell number among treatments(p>0.05). These results were indicating that mLIF or hLF can increase the proportion of embryos that develop into morulae and blastocysts without and increase in the cell number.

• Korean Journal of Animal Reproduction
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• v.17 no.3
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• pp.193-199
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• 1993

Bovine follicular oocytes were matured in two different conditions, TCM 199＋10% FBS with or without hormones (0.01 unit/ml ovine follicle stimulating hormone, 0.01 unit/ml ovine luteinizing hormone and 1$\mu\textrm{g}$/ml $\beta$-estradiol). There was no significant difference in maturation and fertilization rates of the oocytes between two groups. The result indicates that hormonal treatment does not have beneficial effect on in vitro maturation and fertilization of follicular oocytes. IVF-derived cone-cell bovine embryos were injected with foreign DNA (CChcLf) by microinjection method and then co-cultrued with bovine oviductal epithelial cells. Developmental rate of microinjected embryos to blastocyst stage (21%) was similar to that of non-injected embryos(29%). This result represents that microinjected bovine embryos produced in vitro have a potential of development to normal blastocysts.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.78-78
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• 2002

The role of heat shock proteins in shielding organism from environmental stress is illustrated by the large-scale synthesis of these protein by the organism studied to date. However, recent evidence also suggests an important role for heat shock protein in fertilization and early development of mammalian embryos. Effects of elevated in vitro temperature on in vitro produced bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (HSP70) by control and frozen-thawed after in vitro fertilization (IVF) or nuclear transfer (NT). The objective of this study was to assess the developmental potential in vitro produced embryos with using of the various containers and examined expression and localization of heat shock protein 70 after it's frozen -thawed. For the vitrification, in vitro produced embryos at 2 cell, 8 cell and blastocysts stage after IVF and NT were exposed the ethylene glycol 5.5 M freezing solution (EG 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min, and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid, cryo-loop. However, survival rates by straw were relatively lower than other containers. Only, nuclear transferred embryos survived by using cryo-loop. After IVF or NT, in vitro matured bovine embryos 2 cell, 8 cell and blastocysts subjected to control and thawed conditions were analysed by semiquantitive reverse transcription polymerase chain reaction methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNA were higher thawed embryos than control embryos. Immunocytochemistry used to localization the hsp70 protein in embryos. Two, 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed frozen-thawed. However, under control condition, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform in distribution.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.121-129
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• 1999

The objective of this study was to produce calves by transfer of embryos derived from slaughter house(SHD) and ultrasound-guided ovum pick-up (OPU). At 60 hrs after injection of 400 mg FSH dissolved in 25% polyvinylpyrrolidone(PVP) by single dose, ultrasound-guided follicular oocyte aspiration was ferformed. Day-7 and day-8 blastocysts produced by in vitro maturation (IVM), fertilization (IVF) and culture(IVC) of the oocytes derived from SHD and OPU were nonsurgically transferred into recipients. The results obtained were as follows. The cleavage rate and the development rate to blastocysts were not significantly (P<0.05) different between the oocytes obtained by SHD (72.9% vs. 34.1%) and OPU (75.9% vs. 38.4%). The oocyte recovery rate from the number of follicles by ultrasound-guided aspiration were not significantly (P<0.05) different between Holstein (61.7%) and Hanwoo (60.1%), but the rate of oocytes useful for IVF was significantly (P<0.05) higher in Hanwoo (69.3%) than Holstein (59.6%). The cleavage rate and the development rate to blastocysts was not significantly (P<0.05) different between Holstein (74.9% vs. 39.2%) and recipients on day 8 of estrus cycle resulted in 13 pregnancies (34.2%). One of them was sacrificed during gestation period due to mastitis and another was aborted spontaneous. The resulting 14 calves were morphologically normal at birth. Seventy eight fresh OPU-IVF embryos were transferred into 21 recipients on day 8 of estrus cycles, resulting in pregnancy of 12 recipients (41.4%). Two of them were sacrificed during gestation period due to mastitis and the other two were aborted. Nevertheless, the 11 OPU-calves have been born normally.

• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.335-343
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• 1997

The effect of thiol compounds on development and intracellular glutathione(GSH) concentrations of bovine embryos produced by in vitro maturation and in vitro fertilization(IVM/IVF) was examined in CRlaa medium with or without $\beta$-mercaptoethanol(0, 10, 25 and 50$\mu$MME) and cysteamine(0, 25, 50 and 75 $\mu$M). Numbers of cells comprising blastocysts were also counted using double fluorescence stain and the total glutathione levels(oxidized and reduced form) of morula and blastocyst embryos were than measured by an enzymatic method. Following routine IVM/IVF procedures oocytes and zygotes were cultured for 40 to 44h in CRlaa medium. Then 2 to 8-cell embyos had cumulus cell removed and were allotted randomly to the experimental medium. In Experiment 1, the proportion of embryos developing to and beyond morulae stages in 0, 10, 25 and 50 $\mu$M $\beta$-ME was 42.9%, 50.0%, 53.7% and 65.6%, respectively. Fifty $\mu$M $\beta$-ME group was significantly higher than those of any other groups (P<0.05). In Experiment 2, the percentages of embryos developed beyond morulae stages in 0, 25, 50 and 75 $\mu$M cysteamine was 42.9%, 40.4%, 60.0% and 59.2%, respectively. Fifty and 75$\mu$M cysteamine groups were significantly higher than in 0 and 25 $\mu$M cysteamine groups, but all of culture medium containing cysteamine(52.6%) was not significantly difference in control group(42.9%). In Experiment 3, the intracellular GSH concentrations of morulae and blastocyst embryos in 0 and 50 $\mu$M $\beta$-ME was 42.4 pM and 44.9 pM, 49.5 pM and 67.8 pM, respectively. Morulae embryos were not difference, but blastocyst embryos were significantly difference between treatments(P<0.05). In Experiment 4, the intracellular GSH concentrations of morulae in CRlaa with or without cysteamine were 39.8 pM and 45.6 pM, and blastocysts were 59.3 pM and 66.8 pM, respectively. Cell numbers of blastocysts were similar to in all experimental groups. These experiments indicate that thiol compounds can increase the proportion of embryos that developing to and beyond morulae stage and the intracellular GSH concentrations.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.311-320
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• 2002

The objective of this study was to assess the developmental potential in vitro produced embryos frozen-thawed with the various containers, and also examined expression and localization of heat shock protein 70 at these embryos. For the vitrification, 2-cell, 8-cell and blastocyst stage embryos produced by in vitro fertilization (IVF) and nuclear transfer (NT) were exposed the ethylene glycol 5.5 M freezing solution (EC 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop, and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min. and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid and cryo-loop. However, survival rates by straw were relatively lower than other containers. The use of cryo-loop resulted in only survival of nuclear transferred embryos (43.7%). Also, there embryos after IVF or NT were analysed by semi-quantitive reverse transcription-polymerase chain reaction (RT- PCR) methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNh were higher thawed embryos than control embryos. Immunocytochemistry used to localize the hsp 70 protein in embryos. Two and 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some frozen-thawed embryos. However, in the control, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform In distribution. Therefore, this result suggests that the exploiting Hsp 70 in the early embryos may be role for protection of stress condition for increase viability of embryos within IVF, NT and there frozen-thawed embryos.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.307-313
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• 1996

This study was conducted to investigate the effect of S-phase synthronized nuclear transfer on the development of nuclear transplant bovine embryos. A blastomere derived from the 16~32 cell stage bovine embryos was transferred into an enucleated metaphase II(MII) oocytes or activated S-phase eggs. From the MII-phase and S-phase nuclear transfer, 6.3%(4/63) and 13.8%(9/65) of nuclear transplant embryos developed to the blastocyst stage, respectively. In the S-phase nuclear transfer, maximal proportion of embryos developed to the blastocyst stage(16.6%) was obtained after the recipient cell was activated 8 h prior to receving a donor nucleus. MII-phase nuclear transplant embryos showed the PCC state of their nuclear at 1.5~2 h after fusion, whereas, S-phase nuclear transplant embryos did not undergo PCC. The result of this study suggests that if blastomeres of unknown cell-cycle-stage are used, S-phase nuclear transplantation through the activation of enucleated oocytes prior to fusion enhances development of nuclear transplant embryos. This result also suggests that the interval time from oocyte activation to cell fusion may affect the development of nuclear transplant embryos.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.235-239
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• 2005

This study was conducted to test whether the transgenic cattle pass the transgene to their progeny through germ cells, and whether the transgene is expressed in the mammary gland of ransgenic cows. Two male ransgenic calves were born from IVF-derived embryos injected with bovine $\beta-casein/human$ lactoferrin fusion gene and then grew up to be reproducible. Semen was collected from a transgenic bull after 18 mon of age and then frozen. Bovine oocytes matured in vitro were fertilized with spermatozoa of the transgenic bull and cultured in $50\;{\mu}L$ drops of CRlaa medium supplemented with 3 mg/mL BSA. After 48 h of culture, cleaved embryos were determined for the presence of transgenes by DNA polymerase chain reaction (PCR). Proportion of transgene positives among bovine embryos fertilized with sperm of the transgenic bull was $20.9\%$ (28/134). One of transgenic bulls did not produce transgenic sperm. Out of 34 calves produced from recipient heifers inseminated with semen of the other bull, 3 $(8.8\%)$ were transgenic animals (2 females and 1 male). Thus, one transgenic bull showed a low transmission frequency below Mendelian levels in both the IVF-derived embryos and his progeny. It was demonstrated by Southern blot that copy numbers of the transgene in the transgenic progeny enhanced about 1.8 times as compared to those of the founder bull The results demonstrate that the transgenic bull carrying human lactoferrin gene could pass his transgene to the progeny through germ cells, although he is a germ-line mosaic.

• 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.

• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.345-353
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• 1997

Antioxidants and antioxidants with somatic cell co-culture, bovine oviduct epithelial cells(BOEC) and buffalo rat liver cells(BRLC), were studied as a mean of increasing the development and intracellular glutathione(GSH) concnetrations of bovine embryos derived from in vitro matured(IVM) and in vitro fertilized(IVF) oocytes. Cell numbers and intracellular GSH concentrations of blastocysts were also counted. The developmental rate beyond morula stages in CRlaa containing taurine(2.5mM), superoxide dismutase(SOD, 600U) and catalase(250U) were 1%, 75.0%, 64.8% and 62.3%, respectively. The developmental rate in antioxidant groups was significantly higher than in control(P<0.05). The intracellular GSH concentrations of blastocysts cultured in 0, 2.5mM taurine, 600U SOD and 250U catalase were 33.8pM, 39.3pM, 42.3pM and 54.8pM, respectively. This result indicated that the developmental rates and intracellular GSH concentrations of catalase group was significantly higher than any other groups(P<0.05). The developmental capacity in CRlaa plus various antioxidants co-cultured with BOEC were 55.3%(control), 74.1%(2.5mM taurine), 66.7%(600U SOD) and 70.7%(250U catalase) and in CRlaa plus various antioxidants co-cultured with BRLC in control, 2.5mM taurine, 600U SOD and 250U catalase were 63.8%, 75.5%, 71.0% and 73.5%, respectveily, the intracellular GSH concentrations of blastocyst embryos co-cultured with BOEC and BRLC in CRlaa with 0.25mM taurine, 600U SOD and 250U catalase were 73.4pM and 64.4pM, 79.9pM and 67.5pM, 82.3pM and 71.7pM, and 83.0pM and 80.0pM, respectively. Cell numbers of blastocysts were not difference in all experimental groups. These studies indicate that andtioxidants and antioxidant with somatic cell co-culture can increase the proportion of embryo that developed into morula and blastocysts, and the intracellular GSH concentrations of blastocyst embryos.