• Journal of Embryo Transfer
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• v.13 no.1
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• pp.11-19
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• 1998

This study was to determine the effect of ionomycin and 6-dimethylaminopurine (6-DMAP) and/or elcetrical stimulation on the oocyte activation and production of rabbit nuclear transplant embryos. The oocytes were collected from the oviduct of superovulated rabbits at 14 h post hCG injection and cultured in TCM-199 containing 10% FBS until 19 h post hCG injection. To determine the optimum concentration and exposure time of 6-DMAP, some oocytes were activated with 5 $\mu$M ionomycin for 5 min and then in 2.0 mM 6-DMAP for 0.5 to 3.0 h, or in 1.0 to 3.0 mM 6-DMAP for 2.0 h. Other control oocytes were stimulated electrically(3X, 1.25 kV/cm, 60 $\mu$sec) in 0.3 M mannitol solution supplemented with 100 $\mu$M CaCl$_2$ and MgCl$_2$. The nuclear donor embryos of 8-cell stage were synchronized to G$_1$ phase of 16-cell stage, and the recipient cytoplasms were obtained from removal of the first polar body and a portion of membrane bound cytoplasm of the oocytes collected at 15 h post hCG injection. A separated blastomere was injected into the perivitelline space of the enucleated oocytes. The oocytes injected with nucleus were cultured until 19 h post hCG and then electrofused and activated by electrical stimulation with or without ionomycin and 6-DMAP. These nuclear transplant embryos were cultured in TCM-199 containing 10% FBS in 39˚C, 5% CO2 incubator for 120 h. For the oncytes activated parthenogenetically with electrical stimulation with or with-out ionomycin and the various concentration of exposure time of 6-DMAP, the highest cleavage(92.3%) and development to blastocyst stage(41.0%) were resulted from the oocytes activated by ionomycin and 2.0 mM 6-DMAP for 2.0 h, which were found to be significantly(P<0.05) higher than the cleavage(45.2%) and developement to blastocyst stage(14.3%) from the oocytes activated with electrical stimulation. The significantly(P<0.05) more oocytes(71.4%) developed to 4 cell stage at 24 h post activation by ionomycin and 6-DMAP than those by electrical stimulation(18.9%). For the nuclear transplant embryos, the cleavage rate was similarly high in oocyte activation by electrical stimulation with(79.4%) or without ionomycin and 6-DMAP(70.5%). However, the embryo development to blastocyst stage was significantly(P<0.05) higher in oocyte activation by electrical stimulation with ionomycin and 6-DMAP(44.4%) than by electrical stimulation only(25.0%). The significantly(P<0.05) more nuclear transplant embryos(45.6%) developed to 4 cell stage at 18 h post activation by electrical stimulation with ionomycin and 6-DMAP than those by electrical stimulation only(10.6%). These results indicated that the supplemental oocyte activation by ionomycin and 6-DMAP with electrical stimulation enhanced and accelerated the preimplanted in vitro development of the rabbit nuclear transplant embryos.

• 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).

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.161-170
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• 2015

These studies were conducted to establish the practical Hanwoo (Korean native cattle) improvement system through the combining of embryo transfer technology and confirming individual Hanwoo oocyte culture system and to investigate that correlation of Hanwoo carcass classification (intramuscular marbling) and oocyte donor for blastocyst production in vitro. In case of Hanwoo, the carcass meat quality grades were divided to grade $1^{{+}{+}}$, $1^{+}$, 1, 2, and 3 depends on fat distribution of longest muscle cross-sectional surface. As results, the numbers of follicular oocytes collected from individual fundamentally-registered Hanwoo yielded $1^{{+}{+}}$, $1^{+}$, 1, 2 and 3 meat quality were 9.5, 9.4, 8.5, 8.8 and 8.8 per ovary, respectively. The numbers of retrieval oocyte from follicles were significantly higher in the cattle yield $1^{{+}{+}}$, $1^{+}$ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). The rates of blastocyst formation were 18.2, 21.3, 29.4, 30.9, and 31.5% in the cattle yield $1^{{+}{+}}$, $1^{+}$, 1, 2 and 3 meat quality of after in vitro maturation, respectively. It was significantly lower in the cattle yield $^{{+}{+}}$ and $1^{+}$ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). In order to evaluate embryos quality, TUNNEL assay was conducted for each meat quality grade using blastocyst stage embryo on day 8. The results showed that apoptosis cell number was higher tendency in the cattle yield $1^{{+}{+}}$and $1^{+}$ meat quality (81 and 79, respectively) than in the cattle yield 1, 2 and 3 meat quality (51, 48 and 50, respectively) but there was no statistical significance in each group. After embryo transfer, the conception rate of recipient was 53.5 (23 out of 43), 52.1 (38 out of 73) and 58.0% (58 out of 100) in the meat quality of 1, $1^{+}$ and $1^{{+}{+}}$, respectively. These results showed that the conception rate was significantly higher in the cattle yield 1 meat quality than in the cattle yield $1^{{+}{+}}$, $1^{+}$, 2, and 3 meat quality (p<0.05). In summary, these results indicate that the application of confirming Hanwoo individual oocyte culture system and embryo transfer technology can make good use of the genetic resources conservation and improvement of Hanwoo. Relevance of the meat quality grade and reproductive ability of carcasses of Hanwoo will be considered to be one of the effective means for the associated research with obesity and reproduction.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.124-124
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• 2003

The successful development of embryos cloned by nuclear transfer (NT)have been dependent on a wide range of known factors including cell cycle of donor and recipient ooplast, oocyte quality, NT procedure and oocyte activation. The present study compared the development of cloned porcine embryos following different activation treatments. Cumulus-oocyte complexes (COCs) were aspirated from 26 mm follicles of slaughterhouse ovaries and cultured for 22 h in NCSU #23 medium supplemented with 10% porcine follicular fluid, 0.57 mM cysteine, 0.5 g/mL LH, 0.5 g/mL FSH and 10 ng/mL EGF. The COCs were further cultured for an additional 22 h in the same medium at $39{\cird}C$ in an atmosphere of 5% $CO_2$ in air, without hormonal supplements. Primary cultures of fibroblasts isolated from a female fetus on day 40 of gestation were established in DMEM + 15% FCS. For nuclear donation, cells at the 5th-6th passage were cultured in DMEM +0.5% FCS for 5 days in order to arrest the cells in G0/Gl. After enucleation, oocytes were reconstructed by transfer of donor cells and fusion with three DC pulses (1.4 KV/cm, 30 sec) in 0.28 M mannitol containing 0.01 mM $CaCl_2$ and 0.01 mM $MgCl_2$. Eggs were then divided into three treatment groups, control (without further treatment, Group 1), eggs cultured in 10 g/ml cycloheximide (CHX) for 5 h (Group 2), and eggs cultured in 1.9 mM 6-dimethylaminopurine (6-DMAP) for 5 h (Group 3). The eggs were then cultured in sets of 30 in 60 I drops of NCSU#23 supplemented with 4mg/ml BSA (essentially fatty acid free) until day 7 at $39{\circ}C$ in a humidified atmosphere of 5% $CO_2$. On day 4 the culture were fed by adding 20 I NCSU #23 supplemented with 10% FBS. Development rates into blastocysts were significantly higher (P<0.05) in Group 3 embryos compared to Group 1 controls ($27.6 \mu 2.7% vs. 20.1 \mu 4.1%$, respectively), but rates did not differ in Group 2 compared to control ($23.8 \mu 5.7%$). Total cell number in Group 3 blastocysts was however significantly higher (P<0.05) than in Groups 1 and 2 ($44.6 \mu 2.4 vs. 19.9 \mu 1.9 and 21.9 \mu 2.1$, respectively). These results suggest that 6-DMAP is more efficient than cycloheximide in the activation of electrically fused NT oocytes during in vitro production of cloned porcine embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.1
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• pp.1-8
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• 2011

Large quantities of high-quality recipient oocytes with uniform cytoplasm are needed for research in the promising field of somatic cell nuclear transfer (SCNT) and embryonic stem cell research. In canines, however, it is difficult to obtain large quantities of oocytes because each donor produces a limited number of mature oocytes in vivo. Although in vitro maturation (IVM) is considered an alternative approach to oocyte production, this technique is still too rudimentary to be used for the production of highquality, uniform oocytes in large quantities. One technique for overcoming this difficulty is to use oocytes obtained from different species. This technique is known as interspecies SCNT (iSCNT). This review provides an overview of recent advances in canine - porcine interspecies SCNT.

• Journal of Embryo Transfer
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• v.10 no.2
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• pp.105-113
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• 1995

This study was conducted to examine the efficiency of enucleation and blastomere isolation from recipient oocytes and donor embryos, respectively and to determine the effect of oocyte age and electric voltage on the fusion rate and in vitro development of the fused oocytes in rabbit nuclear transplantation. Immature oocytes collected from ovarian follicles were matured in vivo for 12 h in TCM-199 containing FCS and hormones and in vivo matured oocytes were collected 17 to 18 h post-HCG. The fresh and frozen donor embryos of 8- to 16-cell stage were collected from the oviduct of superovulated does. The proportion of successfully enucleated oocytes was greatly lower in in vitro matured oocytes (42.3%) than that (62.7%) in in vivo matured oocytes The level of cytochalasin B for in vivo matured oocytes did not affect the efficiency of enuleation, but 7.5 $\mu$g /mL cytochalasin B for in vitro matured oocytes showed a high enucleation rate significantly. The isolation efficiency of a single blastomere nucleus did not differ between 8- and 16-cell stage embryos. The percentage of single blastomeres isolated from 16-cell stage fresh embryos after 0.5% pronase treatment was greatly higher at 16-min treatment (94.4%) than at 8-min(78. 1%) and the blastomeres(61.5%) isolated from frozen-thawed embryos after 16-min pronase were significantly fewer than those of fresh embryos. The age of recipient oocytes affected nuclear fusion rate. The reconstituted oocytes fused at 24-h age showed slightly higher fusion rate (77.8%) than those (65.0%)fused at 18-h age. The fusion rate of in vitro and in vivo matured oocytes inserted with fresh blastomere did not differ among electric voltages, but the cleavage rate and development to morula-blastocysts of in vitro matured oocytes was more higher under 0.6 kV/cm than under 0.8 to 1.2 kV/cm, while the cleavage rate and development of in vivo matured oocytes was higher under 0.8 to 1.0 kV/cm than under 1.2 kV/cm. The fusion and cleavage rate fol1owing insertion with frozen-thawed blastomere was not different between the in vitro and in vivo matured oocytes and was similar to those from fresh blastomere insertion.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.842-849
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• 2007

The purpose of the present study was to produce live offspring from in vitro fertilized goat embryos. Oocytes were collected from abattoir ovaries and kept in oocyte collection medium. Oocytes were washed 4-5 times with maturation medium containing medium-199 with 5 ${\mu}g/ml$ FSH, 100 ${\mu}g/ml$ LH, 1 ${\mu}g/ml$ estradiol-$17{\beta}$ 50 ${\mu}g/ml$ gentamycin, 10% inactivated estrus goat serum, and 3% BSA (fatty acid free). Oocytes were placed in 100 ${\mu}l$ drops of maturation medium containing granulosa cell monolayer and incubated in a 5% $CO_2$ incubator at $38.5^{\circ}C$ for 27 h. For capacitation of spermatozoa fresh semen was processed and mixed in 3 ml fertilization TALP medium containing 50 ${\mu}g/ml$ heparin and kept in the above incubator for 2 h. The capacitated spermatozoa were coincubated with matured oocytes for fertilization. Cleaved embryos were separated and cultured in embryo development medium with oviductal cells and 494 embryos were produced. Recipient goats were synchronized with two injections of 15 mg $PGF_{{2}{\alpha}}$/goat 10 days apart. Eighty early stage embryos were transferred into the uterotubal junction of 14 surrogate mothers using laparoscopy techniques. One recipient delivered twin kids, whereas another two recipients each.delivered a single kid The parentage of these kids was evaluated using highly polymorphic co-dominant microsatellites markers. From the present study, it was concluded that live goat kids can be produced from in vitro matured and fertilized goat embryos, to the best of our knowledge for the first time in India.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.99-106
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• 1999

핵 이식에 공여되는 수핵난자의 효과적인 동결보존을 위하여 한우 성숙난자를 1.0 M dimethylsulfoxide(DMSO) 또는 1.0 M glycerol이 함유된 동결보호제를 이용하여 처리하거나, 동결보호제 처리 후 완만동결법을 이용한 동결융핼르 시행하여 상기 실험처리로 야기되는 투명대 경화현상을 관찰하였다. 도축장 유래의 난소에서 미성숙난자를 채취한 후 10% 소 태아혈청을 함유한 TCM-199을 이용하여 22∼24 시간 동안 체외성숙배양을 이해하였다. 배양후 작출된 성숙난자를 각각의 동결보호제로 처리, 혹은 처리 후 동격융해한 후 protease를 이용하여 투명 대의 경화현상 발생의 빈도를 조사하였다. 또한 동격란을 동결정액을 이용한 체외수정에 공여한 후 정자 침입농도능을 조사하였다. 동결보호제로 처리한 난자에 있어서 보호제의 종류와 관계없이 투명대 경화현상이 유의적 (P<0.05) 으로 증가하였으나 이후의 동결융해 처리에 의한 추가적인 경화현상의 발생은 증가하지는 않았다. 또한 투명대 경화현상의 발생양상을 동결보호제 처리 후 10분 간격으로 측정한 결과 DMSO의 경우 처리후 10분, glycerol의 경우 처리 후 20분 후부터 유의적으로 차를 발견할 수 없었으며, 수정율 및 난자 1개당 침입한 정자의 수는 동결란에서 유의적으로 증가하지 않았다. 본 연구의 결과 동결난자의 투명대 경화현상은 동결보호제 처리과정에서 이미 일어나지만, 이러한 투명대 경화현상이 난자의 동결보존 후 수정능에는 현자한 영향을 미치지 않는다는 사실이 규명되었다.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.23-29
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• 1994

The long term goal of this research is to develop an efficient procedure for large scale production of genetically identical or cloned animals. To improve nuclear transpalntation efficiency in the rabbit, this study evaluated the age of nuclear recipient oocytes on the different steps of nuclear transplantation. The ovulated oocytes in different ages were collected from the superovulated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) supplemented with 10% fetal calf serum(FCS) from 13 to 15, 17 to 20 and 23 to 26 hours after hCG injection. The denuded oocytes were used as nuclear recipient cytoplasm following enucleation by micromanipulation. The blastomeres separated from the 8-cell embryos were used as nuclear donor. The enucleated oocytes receiving a blastomere in the perivitteline space were fused in the 0.28 M mannitol solution at 1.5 kV/cm, 60 sec for three times. The fused oocytes were co-cultured with the monolayered rabbit oviductal epithelial cells in TGM-199 solution with 10% FCS for 72 hours at 37$^{\circ}C$ in a 5% $CO_2$ incubator. The cultured nuclear transplant embryos and in vivo developed embryos collected at 72 hours after hCG injection were stained with Hoechst 33342 dye. Their cell numbers were counted under a fluorescent microscope. The results obtained were summarized as follows ; 1. The aged oocytes(20 hrs. post hCG) showed significantly(P<0.05) higher fusionrates(70 ~ 90%) than the recently ovulated oocytes(30.8%) 2. The aged oocytes which were electrically activated and fused at 20 hours developed to blastocyst at significantly(P<0.05) high rate, while none of the recently ovulated oocytes developed to blastocyst. 3. Even though the aged oocytes at 23~26 hours showed higher fusion rate(85.7%), not only they were inadequate to manipulate but also their developmental potential to blastocyst was highly impaired. 4. The developmental potential in vitro of nuclear transplant embryos was significantly retarded than in vivo deveolped embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.11
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• pp.1665-1672
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• 2008

Interspecies somatic cell nuclear transfer (iSCNT) is a useful method to preserve endangered species and to study the reprogramming event of a nuclear donor cell by the oocyte. Although several studies of iSCNT using murine cells and bovine oocytes have been reported, the development of murine-bovine iSCNT embryos beyond the 8-cell stage has not been successful. In this paper, we examined the developmental potential of embryos reconstructed with a murine embryonic fibroblast as the nuclear donor and a bovine oocyte as the cytoplasm recipient. The reconstructed embryos were cultured in CZB (murine medium) or CR1aa (bovine medium). In addition, for the development of a murine-bovine iSCNT blastocyst, the antioxidant ${\beta}$-mercaptoethanol (${\beta}ME$) was supplemented to CR1aa medium. Furthermore, to verify the mouse genome activation in murine-bovine iSCNT embryos, RT-PCR analysis of murine Xist was performed. The development of the murine-bovine iSCNT embryos cultured in CR1aa was significantly higher than that in CZB (p<0.05). With respect to the effect of BME on the development of the murine-bovine iSCNT blastocyst, addition of BME produced a significant increase in blastocyst development (p<0.05). Karyotype analysis confirmed that the reconstructed embryos were derived from murine cells (40XX). The Xist gene was gradually increased from the 8-cell stage to the blastocyst stage. This is the first report of blastocyst development of iSCNT embryos derived from murine somatic cells and bovine oocytes. These results demonstrate that bovine cytoplasm can support the development of later stages of a preimplantation embryo from murine-bovine iSCNT.