• Asian-Australasian Journal of Animal Sciences
• /
• v.12 no.1
• /
• pp.22-27
• /
• 1999

Bovine oocytes with compact and complete cumulus cells were cultured in 6 groups for up to 24h in TCM199 buffered with 25 mmol/1 HEPES and supplemented with 10% FCS, 1 mg/ml $17{\beta}$-estradiol, 20 IU/ml hCG. Half of the oocytes at each group cultured in the presence of $25{\mu}g/ml$ cycloheximide at different times during maturation (0, 6, 12, 18, 20, 22 h) were fixed at 24 h of maturation to examine the nuclear progression. The rests of them were inseminated with frozen-thawed spermatozoa in medium BO with 10 mg/ml BSA and 10 mg/ml heparin and fixed after additional 18-20 h culture to evaluate the sperm head transformation. When a protein synthesis inhibitor was added at the onset of the maturation, the oocytes were prevented to proceed GVBD. A few of the oocytes (16%) were able to be penetrated and sperm head decondensation was inhibited either. Addition of cycloheximide after 6-12 h of culture resulted in an increasing percentage of GVBCD (more than 80%), but the oocytes became arrested in M-I (69.2%). More than half of the oocytes was penetrated with a decondensing sperm head. Formation of male pronucleus was first obtained at 12 h of culture in the presence of cycloheximide. When cycloheximide was added from 18 h of culture onwards, nuclear progression to M-II was increasingly restored (80.4-85.5%). The proportion of male and female pronuclear formation increased from 17.9% to 46.2%. It is concluded that protein synthesis is necessary not only for GVBD and development from M-I to M-II, but also for sperm head decendensation and male pronuclear formation in bovine oocytes.

• Journal of Embryo Transfer
• /
• v.19 no.2
• /
• pp.121-126
• /
• 2004

본 연구는 개의 불임해결과 체외수정란을 생산할 목적으로 난소의 보존 및 난구세포의 부착 여부가 신선 및 동결 개 정자를 이용한 투명대 반응에 미치는 영향을 조사하였다. 1. 적출한 난소를 4$^{\circ}C$ 와 salt에 각각 48시간 보존 후 회수한 난구세포 부착 난자와 나화난자의 정자침입율은 각각 62.5%, 37.5% 및 42.5% 및 22.4%로서 난소를 적출 후 곧 바로 회수한 난구세포 부착 및 나화난자 내 정자침입율인 93.3%와 56.7%에 비해 현저히 낮게 나타났다. 2. 적출한 난소를 4$^{\circ}C$ 에 각각 4시간, 14시간 및 48시간 보존 후 회수한 난구세포부착 난자의 정자침입율은 각각 92.5%, 90.0%, 85.0%로서 신선 난소 난자의 정자침입율 93.3%에 비해 유사하거나 약간 낮게 나타났다. 3. 적출한 난소를 salt에 각각 4시간, 14시간 및 48시간 보존 후 회수한 난구세포부착 난자의 정자침입율은 각각 85.0%, 77.5%, 72.5%로서 신선난소로부터 회수한 난자의 정자침입율 93.3%에 비해 낮게 나타났다. 4. 적출한 난소를 4$^{\circ}C$ 와 salt에 각각 24시간 보존 후 회수한 난구세포부착 난자의 체외수정율과 체외발생율은 각각 50.0%, 22.5% 및 40.0%, 15.0%로서 신선난소로부터 회수한 난구세포부착 난자의 체외수정율과 체외발생율 72.5%와 32.5%에 비해 낮게 나타났다.

• Proceedings of the KSAR Conference
• /
• 2004.06a
• /
• pp.200-200
• /
• 2004

The purpose of this study was carried out to determine the possibility of in vitro maturation of tiger oocytes. Immature oocytes were recovered from a pairs of ovaries. A total of 78 oocytes were collected, of which forty threes were identified as compact cumulus cells and uniform cytoplasm. 43 COCs were in vitro matured at 39℃, 5% CO₂ in air atmosphere for 48 h in a IVM medium (TCM-199 supplement with 10% FBS, 0.6 mM cysteine, 0.2 mM pyruvic acid and 10 IU/㎖ HMG). (omitted)

• Journal of Embryo Transfer
• /
• v.29 no.2
• /
• pp.111-117
• /
• 2014

This study was conducted to optimize the efficiency of cloning and to produce cloned mice. The majority of cloned mammals derived by nuclear transfer (NT) die during gestation and have enlarged and dysfunctional placentas. In this study, the optimized conditions were established to produce clone mice. The parthenogenetic oocytes were activated after 6 h regardless of cytochalasin B (CB) concentration. CB treatment ($2{\mu}g/ml$) was found second polar body. Lower concentration of CB was decreased the activation rate, but the second polar body was the best highly increased during 6 h incubation. The small fragments were exhibited in the $5{\mu}g/ml$ treatment of CB, but it was not found in lower concentration groups (> $2.5{\mu}g/ml$). To examine effects of $SrCl_2$ on the adult cumulus cells, somatic cell NT oocytes were exposed during 0.5, 1 and 6 hrs. The second polar body was significantly greater in 0.5 h exposure group (6.6%) than 1, 6 hrs. Developmental rate from 2-cell to 4-cell was the lowest in 7.5 mM Strontium chloride ($SrCl_2$) groups (84.1% and 64.3%) than 5, 10 m $MSrCl_2$. The implantation rate was not significantly difference among 5, 7.5 and 10 m $MSrCl_2$ group. Three live fetuses were produced by SCNT. SCNT placentas were remarkably heavier than IVF group (8 fetuses) (0.34, 0.34, 0.33 vs 0.14 g) compared with the placenta weight of IVF and SCNT clones.

• Asian-Australasian Journal of Animal Sciences
• /
• v.14 no.6
• /
• pp.754-758
• /
• 2001

Bovine oocytes with compact and complete cumulus cells were cultured for up to 24h in TCM199 buffered with 25mmol/l hepes and supplemented with 10% FBS (fetal bovine serum), 1mg/ml $17{\beta}$-estradiol, 20IU/ml hCG(human chorionic gonadotropin). All of the oocytes were divided into at 6 groups depending upon incubation times (control, 0 hour, 6 hours, 12 hours, 16 hours, 18 hours). To all experimental media, $200{\mu}g/ml$ puromycin was added at different incubation times mentioned above. Following these culture times, in vitro insemination was conducted with frozen-thawed bovine spermatozoa in medium BO (Brackett and Oliphant medium for in vitro insemination) with $10{\mu}g/ml$ BSA(bovine serum albumin) and 10 mg/ml heparin added. After 22h culture, the oocytes were fixed with acetic alcohol solution and stained with orcein acetic solution to evaluate sperm nuclear progression. Addition of puromycin after 0, 6 and 12 h of culture resulted in near of oocyte maturation at the M1 stage. Contrariwise, puromycin addition after 12 h of culture led to restoration of nuclear progression to M2 stage. On the one hand, puromycin affected the synthesis of Cyclin B protein that may be involved in the oocyte maturation and sperm capacitation for in vitro fertilization. The present study suggests the participation of protein synthesis, cyclin B, in the oocyte development from M1 to M2 stages in vitro.

• Asian-Australasian Journal of Animal Sciences
• /
• v.8 no.2
• /
• pp.123-127
• /
• 1995

In vitro embryo production (IVP) is affected by various factors during in vitro maturation, fertilization, and development. In this experiment, the effect of ovary type, quality of follicular oocyte, medium used for fertilization, presence of hormone in medium, sperm concentration on in vitro maturation and fertilization were examined for effective IVP. In vitro maturation was carried out using TCM-199 supplemented with 15% FCS and hormones in 5% $CO_2$ incubator for 24h. In vitro fertilization was performed with frozen-thawed sperm in modified mTALP medium containing 0.3% BSA, $10{\mu}g/ml$ heparin, and 5mM/ml caffeine for 24h. The fertilized embryos were co-cultured on monolayer of cumulus cells in TCM-199. When oocytes were collected from functionally active and inactive ovaries, maturation rate was 76.9 and 7.7%, respectively. When oocytes were classified morphologically to good and poor grades, maturation rate was 75 and 58.8%, respectively. FSH + LH + $E_2$ (86.4%) showed higher maturation rate than control (53.0%) and FSH (73%). The fertilization rate was 28.2, 100 and 91.7% in $1.6{\times}10^5$, $5.0{\times}10^5$ and $10.0{\times}10^5$ sperm concentration per ml. When oocytes were fertilized in mTALP and BO media, fertilization and cleavage rates of oocytes in mTALP were higher (84.3 and 56.9%) than those (67.4 and 23.3%) in BO medium. In this experiment, in vitro maturation, fertilization and development of oocytes were affected by type of ovary, grade of oocyte, hormones, sperm concentration and fertilization medium.

• Korean Journal of Animal Reproduction
• /
• v.17 no.2
• /
• pp.75-83
• /
• 1993

Purine has been identified in the preparation of follicular fluid and shown an activity in maintaining oocyte meiotic arrest. Therefore this study was performed to examine the inhibitory effect of purine on germinal vesicle break down(GVBD) in the presence and absence of human fetal cord serum(HFCS) or human mature follicular fluid(HMFF), as a protein source, in vitro culture. Immature oocytes(GV stage) were collected from ovaries of 21∼28 days old ICR mice by puncturing the antral follicles with a fine needle, at 48 hrs after PMSG injection. Some of the oocytes were denuded by drawing the cumulus-enclosed(complex) oocytes in and out of a pasteur pipet. Complex oocytes and denuded oocytes were cultured 3 hrs. in T6 media containing 0.75mM adenosine or/and 4mM hypoxanthine, with HFCS or HMFF. Their GVBD rates were observed at every 1 hr. during the culture time. Both adenosine and hypoxanthine have shown a time-dependent inhibitory effect on GVBD in complex and denuded oocytes and the inhibitory effect was maximized in culture medium containing hypoxanthine and adenosine. HFCS and HMFF increased the GVBD rates in the presence of the purines, thus HFCS and HMFF may contain a factor that could reverse the inhibitory effect of purines. Also complex oocytes were more sensitive to not only the inhibitory effect of purines but the promoting action of HMFF on GVBD than denuded oocytes. Therefore it was reconfirmed that granulosa cells play an important part in meiotic arrest and resumption.

• Asian-Australasian Journal of Animal Sciences
• /
• v.22 no.8
• /
• pp.1117-1123
• /
• 2009

This study investigated the effects of IGF-I and EGF on the development of blastocysts or hatched blastocysts during the in vitro culture of embryos from immature porcine oocytes. After the in vitro maturation and fertilization of cumulus-oocyte complexes (COCs) and their culture in vitro in PZM3 medium, we examined the embryo development rate for 168 h. When different concentrations of IGF-I (0, 1, 10, 20 ng/ml) were supplemented to fertilized porcine embryos in vitro, there were no significant differences in cleavage rate, blastocyst development rate or blastocyst hatching rate among the treated groups. On the other hand, when different concentrations of EGF (0, 1, 10, 20 ng/ml) were supplemented to the in vitro culture medium, blastocyst development rate was highest in the group in which EGF was not supplemented and, specifically, it was higher than in the 20 ng/ml treatment group (p<0.05). When 10 ng/ml IGF-I and 1 ng/ml EGF were supplemented separately or simultaneously, there were no significant differences among the treated groups in blastocyst hatching rate and the number of cells in each condition. This study demonstrated that the addition of IGF-I and EGF into PZM3 medium did not enhance development of the blastocyst stage and total cell number in blastocysts.

• Korean Journal of Veterinary Research
• /
• v.57 no.2
• /
• pp.89-95
• /
• 2017

This study was conducted to determine the effects of biophoton treatment during in vitro maturation (IVM) and/or in vitro culture (IVC) on oocyte maturation and embryonic development in pigs. An apparatus capable of generating homogeneous biophoton energy emissions was placed in an incubator. Initially, immature pig oocytes were matured in the biophoton-equipped incubator in medium 199 supplemented with cysteine, epidermal growth factor, insulin, and gonadotrophic hormones for 22 h, after which they were matured in hormone-free medium for an additional 22 hr. Next, IVM oocytes were induced for parthenogenesis (PA) or provided as cytoplasts for somatic cell nuclear transfer (SCNT). Treatment of oocytes with biophoton energy during IVM did not improve cumulus cell expansion, nuclear maturation, intraoocyte glutathione content, or mitochondrial distribution of oocytes. However, biophoton-treated oocytes showed higher (p < 0.05) blastocyst formation after PA than that in untreated oocytes (50.7% vs. 42.7%). In an additional experiment, SCNT embryos produced from biophoton-treated oocytes showed a greater (p < 0.05) number of cells in blastocysts (52.6 vs. 43.9) than that in untreated oocytes. Taken together, our results demonstrate that biophoton treatment during IVM improves developmental competence of PA- and SCNT-derived embryos.

• Journal of Embryo Transfer
• /
• v.15 no.2
• /
• pp.129-136
• /
• 2000

This study was carried out to investigate the effect of biological factors on the in vitro production(IVP) of bovine oocytes for development of simple culture methods and medium. Oocytes from the slaughterhouse ovaries were matured and fertilized using general protocol and this study was examined if there were necessary to co-culture, media change, media type and embryo density. This results were as follows: 1. The development rate according to co-culture with cumulus cells and non co-culture as drop culture was not significantly different in cleavage (68.9 vs 71.7%), 8-cell stage (41.2 vs 44.1%) and blastocyst stage (12.2 vs 13.8%), respectively (p<0.05) 2. The blastocyst development rates in YS and CRIaa were higher than that in TCM199 (12.4, 10.4$ vs 3.7%), but the cleavage (69.0, 77.8 and 61.0%) and 8-cell stage (31.7, 37.0 and 35.7%) development accoring to YS, TCM199 and CRIaa ws not significantly different, respectively (p<0.05). 3. There was no significantly different in cleavage (62.6, 59.5 and 61.2%), 8-cell(34.7, 37.9 and 34.0%) and blastocyst (9.5, 11.6 and 12.8%) development among medium change time as control, Group I and Group II, respectively (p<0.05). 4. Blastocyst formation of 8-cell stage according to embryo density was not significantly different in 1, 10 and 25 embryos (27.3, 22.5 and 34.0%), respectively (p<0.05). These results indicated that simple culture system could reduce bovine IVP embryos as drop culture as non co-culture system, high density embryo (25 embryos/50 $\mu$1 drop). YS defined medium and no medium change for whole culture period, although other biological factors need to examine in order to produce efficient IVP bovine embryos.