• Clinical and Experimental Reproductive Medicine
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• v.12 no.2
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• pp.65-69
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• 1985

1) Rabbit follicular oocytes from preovulatory follicles were cultured for 12 hr in vitro and fertilized in vivo by transferring the oocytes to the first foster-mother. 2) Two youngs were bron from transferred embryos from the first foster-mother to the second foster-mother. This demonstrates that in vitro cultured follicular oocytes are normal and they can develop into normal young born when transferred to the foster-mother. 3) A simple chemically defined culture medium, salt sol. with glutamine (2mM), which was developed by Bae and Foote(1975) proves fully good enough for rabbit follicular oocyte culture. We call this B-F medium. 4) Twelve hours culture in vitro of the rabbit follicular oocyte may be a proper culture time for further development.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.1-10
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• 1997

The present study was conducted to investigate the influence of embryo cell stage at 18h post-fusion and oocyte preactivation on sebsequent in vitro developmental potential in the nuclear transplant rabbit embryos. The embryos of 16-cell stage were collected and synchronized to G$_1$ phase of 32-cell stage. The recipient cytoplasms were obtained by removing the first polar body and chromosome rnass from the oocytes collected by non-dis-ruptive microsurgery procedure. The separated G$_1$ phase blastomeres of 32-cell stage were injected into non-preactivated recipient cytoplasms. Otherwise, the enucleated recipient cytoplasms were preactivated by electrical stimulation at 18h post-hCG injection and the separated G$_1$ phase blastomeres of 32-cell stage were injected. Mter culture until 20h post-hOG injection, the nuclear transplant oocytes were electrofused by electrical stimulation. The fused nuclear transplant embryos were classified into 3~4-cell, 2-cell and 1-cell stage at 18 hrs post-fusion and cultured until the embryos reached blastocyst stage. The developmental rate to blastocyst stage was significantly (P <0.05) higher in all the reconstituted embryos of 3~4-cell stage(58.0%) than in 2 and icell stage. The developmental rate to blastocyst stage in the embryos of 3~4-cell stage at 18 hrs post-fusion was significantly (P<0.05) higher in the reconstituted without oocyte preactivation(77.8%) than in the oocyte-preactivated embryos (33.3%). These results indicated that the higher rate of in the in vitro development to blastocyst stage might be obtained form the embryos which were reconstituted with nuclear donor of G$_1$ phase and non-preactivated oocyte, and developed more rapidly for 18 hrs post-fusion.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.5
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• pp.617-620
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• 2004

The present study compares development of rabbit embryos generated using different oocyte activation protocols and reconstructed with embryonic or cumulus cells as nuclear donor. In vivo matured oocytes were collected from New Zealand White rabbits at 16 h after ovulation treatment and were activated at18 h of post-ovulation treatment. The following schemes of oocytes activation were tested: 1) single electric pulse (EP, 3.2 kV/cm, 3${\times}$20 $\mu$s, 0.3 M mannitol)+5 min culture in the presence of 5 mM Ionomycin, 2) single electric pulse (EP, 3.2 kV/cm, (${\times}$20 $\mu$s, 0.3 M mannitol)+1 h culture in the presence of 2 mM 6-DMAP, and 3) three electric pulses 30 min apart. Cleavage rate, percentage of expanded and hatched blastocysts as well as total cell number of blastomeres of parthenogenetic embryos were significantly higher using either EP+6-DMAP or 3${\times}$EP schemes, comparing with EP+Ionomycin. Development rate up to hatched blastocyst stage of cloned rabbit embryos using the EP+6-DMAP for activation of nuclei were 19% for embryonic cell nuclei and 36% for cumulus cell nuclei. The best activation protocol optimalized in this study was the combined treatment "P+6-DMAP" which may be potentially used for nuclear transfer protocol.

• 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.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.249-253
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• 2008

Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for studying the interactions between an oocyte and somatic nucleus. The object of this study was to investigate the developmental competence of in vitro-matured porcine oocytes after transfer of the somatic cell nuclei of 2 different species (goat and rabbit). Porcine cumulus oocytes were obtained from the follicles of ovaries and matured in TCM-199. The reconstructed embryos were electrically fused with 2 DC pulses of 1.1kV/cm for $30{\mu}s$ 0.3M mannitol medium. The activated cloned embryos were cultured in porcine zygote medium-3 (PZM-3), mSOF or RDH medium for 7 days. The blastocyst formation rate of the embryos reconstructed from goat or rabbit fetal fibroblasts was significantly lower than that of the embryos reconstructed from porcine fetal fibroblast cells. However, a significantly higher number of embryos reconstructed from goat or rabbit fetal fibroblasts cultured in mSOF or RDH, respectively, developed to the morular stage than those cultured in PZM-3. These results suggest that goat and bovine fetal fibroblasts were less efficacious than porcine-porcine cloned embryos and that culture condition could be an important factor in iSCNT. The lower developmental potential of goat-porcine and porcine-bovine cloned embryos may be due to incompatibility between the porcine oocyte cytoplasm and goat and bovine somatic nuclei.

• The Korean Journal of Zoology
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• v.18 no.4
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• pp.181-196
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• 1975

Rabbit follicular oocytes were cultured in a basic medium containing 0.4% bovine serum albumin (BSA), carbohydrates and amino acids in various combinations. Osmolarity of the medium was maintained at 308 mOsm. The carbohydrates, pyruvate, lactate and glucose were all about equally beneficial, but not essential for rabbit oocyte maturation. Glutamine and proline, but not methionine or phenylalanine stimulated oocyte develoment. Glutamine stimulated more follicular oocytes to develop to prophase and metaphase II than did any of the three carbohydrates tested alone or in combination. Ammonia production after 24 hours of culture was highest in medium containing glutamine(15.2$\\mu$g/ml) but this was not inhibitory to maturation. Negligible amounts of ammonia were found with the other amino acids added. The optimum level of osmolarity for rabbit oocyte maturation appears to be ranged from 250$\\sim$310 mOsm with the maximum level of 270 mOsm. With 0, 0.08, 0.4, 2, 10 and 50 mM of glutamine in the medium, plus BSA but without carbohydrates, 30, 73, 70, 71, 59, 45% of the oocytes developed to prophase or metaphase II respectively. This indicates that no carbohydrate is required of the maturation of rabbit oocytes when 0.08$\\sim$2 mM of glutamine is included, which are the optimum range. Follicular oocytes could develop in the medium containing $^14 C$-glutamine and BSA but without carbohydrates or other organic compound. From the $^14 CO_2$ produced and TCA precipitable material isolated, it is suggested that glutamine probably is utilized by oocytes and cumulus cells as a source of energy as well as for protein synthesis.

• Korean Journal of Animal Reproduction
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• v.15 no.3
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• pp.221-224
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• 1991

This study was undertaken to evaluate the effect of rabbit peritioneal fluid(rPF) on in vitro maturtion of porcine follicular oocytes. From does 20h after hCG injection, rPF was aspirated aseptically at laparatomy, and then centrifuged, filtrated, and preincubated immediately for 12h. Porcine follicular oocytes isolated from ovaries of slaughtered animals were incubated in TCM-HEPES＋10% FCS, TCM-HEPES＋rPF(v/v, 50/50), or rPE only and examined the nuclear maturation after aceto-orcein or hochest staining. After identifying the optimal incubation time, this experiment was repeated for 5 times. Under the TCM-HEPES containing hormones and serum codition, the time range of porcine follicular oocyte maturation was 38 to 44 hours and the optimal time of maturation of follicular oocyte in vitro was 42 hour cultivation, respectively. The maturatin rates(89.4% and 92.7%) of porcine follicular oocytes cultured in the media with 50% rPF or only rPF were signifciantly higher thanthat (84.6%) of oocytes cultured with TCM-HEPES, respectively. These results suggest that the unknown components(s) of rPF promoted in vitro maturation of porcine follicular oocytes.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.183-191
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• 1995

This experiment was carried out to study the determination of survival of vitrified and thawed mammal follicular oocytes by FDA-test. Oocytes were divided into 3 groups according to attachment of cumulus cell. Group A oocytes were tightly surrounded by cumulus cell, group B oocytes were partially surrounded by cumulus cell, and group C oocytes were poorly surrounded by cumulus cell. Vitrification solution developed by our previous study (Kim et al, 1992) which consisted of permeable agent (20 % glycerol + 10 % ethylene glycol) and nonpermeable agent (30 % Ficoll + 10 % sucrose). Oocytes (7~10) loaded into 0.25 ml straw after 10 min equilibration were plunged into liquid nitrogen (- 196$^{\circ}C$) directly. The FDA-score of vitrified and thawed group A oocytes was higher in rat (4.2) than in rabbit (3.9), cow (3.8), mouse (3.4) and porcine (2.4), however that of cumulus cell was higher in rabbit (4.7) than in rat (4.1), cow (2.9), porcine (2.6) and mouse (1.4). The FDA-score of vitrified and thawed group B oocytes were 3.1 (cow), 2.9 (rabbit), 2.9 (mouse), 2.6 (rat) and 2.5 (porcine), respectively. However that of cumulus cell was higher in rabbit (3.7) than in porcine (2.6), rat (2.3), cow (1.7) and mouse (0.3). The FDA-score of vitrified and thawed group C oocytes was higher in mouse (4.1) than in cow (2.9), rabbit (2.6), rat (1.3) and porcine (1.1). As shown in the above results, The survival rates of oocytes were higher in group A than in group B and C except in mouse and cow. These results suggest that the survival of cumulus cell as well as follicular oocytes can be reliably judged by their fluorescence with FDA-test.

• Korean Journal of Animal Reproduction
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• v.17 no.1
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• pp.57-68
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• 1993

This stduy was carried out to find a reliable method for the production of in vitro fertilized embryos having more excellent development capacity and freezability in the rabbit and cattle. The greatest number of rabbit oocytes was recovered 6hrs after HCG injection(P<0.05). The maturation rate in vitro was slightly higher in the oocytes(6-h-oocytes) from 6h than those (8-h-oocytes)from 8 hrs after HCG injection and the beneficial effect of FSH during oocyte maturation was significantly great in the oocytes from large follicles. The cleavage rate into 2-to-6-cell stage was not differ between the 6-h-oocytes and 8h-oocytes, but the cleavage of these oocytes was greatly promoted by FSH addition to maturation medium and the cleavge of 8-h-oocytes matured without FSH was significantly low. The embryo development into 16-cell to morula was not promoted by the co-culture with rabbit oviduct epithelial cells. The freezability by embryo stages was ovidusly high at 4-cell and morula stage in 6-h-oocytes and the viability of 16-cell embryos from 8h-oocytes was similar to that of morula stage. The implantation sites after surgical tranfer of fresh rabbit embryos were not implanted. In bovine experiment, the in vitro development into 16-cell and morula after in vitro maturation and fertilization in the follicular oocytes was slightly improved by the co-culture with granulosa cells compared to that with oviduct epithelial cells and the frozen-thawed viability rate of these embryos ranged from 14 to 40%. The excellent fresh embryos were transferred nonsurgically to 6 recipients, but were not pregnant.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.718-724
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• 2007

Considerable attention has been focused on the cloning of mammalian embryos, as a consequence of poor development, in order to enhance the application of genetic engineering. Experiments were conducted to compare the developmental competence of parthenotes and reconstructed (NT) rabbit eggs with fetal fibroblasts (FFs) following various activation regimens. Oocytes and NT eggs were exposed to: electric stimulation (EST, Group 1) and EST followed by 6-dimethylaminopurine (DMAP, Group 2), cycloheximide (CHX, Group 3) or DMAP/CHX (Group 4). Pronuclear (PN) status, cleavage, blastocyst development and the ploidy were assessed. In parthenote groups 1, 2, 3 and 4, the PN formation differed significantly. And, the cleavage and blastocyst rates were 41.7 and 5%, 75.6 and 53.7%, 68 and 36%, 82.1 and 52.6%, respectively, among treatments. Polyploidy was observed in 17.2% of EST plus DMAP and 44.9% of EST plus DMAP/CHX groups. In SCNT groups (Group 1, 2, 3 and 4), the cleavage and blastocyst rates were 28.6 and 7.1%, 58.3 and 29.2%, 56.8 and 24.1%, 64.5 and 27.8%, respectively. The chromosomal composition differed significantly (p<0.05) among treatments. In Group 2 and 3, 53.8% and 81.8% of embryos revealed diploid chromosomal sets, respectively. However, in Group 4, 53.3% of embryos showed abnormal ploidy (mixoploid). Although DMAP or combination with DMAP/CHX resulted in higher in vitro development of rabbit SCNT embryos, higher incidence of chromosomal abnormality may induce problems related to fetal loss of at late stage of development.