• Reproductive and Developmental Biology
• /
• v.28 no.4
• /
• pp.253-256
• /
• 2004

Porcine fibroblasts were transferred into enucleated bovine oocytes for the interspecies nuclear transfer (NT). After NT, the embryos were cultured in three different culture systems. The media used for the experiment were CR1aa and NCSU23. The culture systems used for the experiment were: 1. Culture in CR1aa for 7 days (CR). 2. Culture in CR1aa for 2 days and subsequently in NCSU23 for 5 days (CR-NC). 3. Culture in NCSU23 for 7 days (NC). Bovine (intraspecies) NT group was used as a control. The oocytes in bovine NT group were treated the same as interspecies NT embryos except using bovine fibroblasts as nuclear donors. Regardless of their nuclear origin (interspecies vs bovine), the embryos in CR (68.4% vs 77.2%) and CR-NC (67.8% vs 70.5%) showed better developmental competence to the 2-cell stage (p<0.05) than those in NC (41.0% vs 10.0%). Bovine NT embryos in CR-NC did not develop over the 4-cell stage after the medium replacement, while interspecies NT embryos in CR-NC continued to develop and could reach over the 8-cell stage (12.2%). Blastocysts were only found in bovine NT group (17.4%), but no blastocyst was found in interspecies NT group. This study suggests that the development of interspecies NT embryos mostly depends on their recipient cytoplasm during the culture in vitro.

• Korean Journal of Animal Reproduction
• /
• v.14 no.3
• /
• pp.183-190
• /
• 1990

These studies were carried out ot investigate the effects of estrous cow serum(ECS), fetal calf serum(FCS), bovine follicular fluid(BFF) and matured cumulus cell(MCC) on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3-5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24~48 hrs. in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 18$^{\circ}C$20 hrs. with motile capacitated sperm in the TCF(Tyrode calcium-free) solution containing 200$\mu\textrm{g}$/ml of heparin. The results obtained in these experiments were summarized as follows : 1. The oocytes classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 61.4%, 12.1%, 19.2%, 4.2% and 3.0% of the total oocytes harvested, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 33.3%, respectively. 2. The maturation and fertilization rate of the follicular oocytes cultured in TCM-199 medium supplemented with 5%~20% ECS and FCS were 74.0%~80.6, 26.2%~30.0% and 71.7%~76.9%, 51.9%~58.0%, and those values were higher the supplement of ECS than FCS. 3. The maturation rate(68.0%~64.6%) and fertilization rate(59.6%~60.4%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 20~30% BFF were higher than those of follicular oocytes cultured TCM-199 medium supplemented with 10% FCS and 10% and 50% BFF. 4. The maturation rate(76.5%) and fertilization rate(61.7%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1$\times$106/ml cumulus cells were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1$\times$104~5/ml and 1$\times$108/ml cumulus cells.lus cells.

• 대한생식의학회:학술대회논문집
• /
• 1997.11a
• /
• pp.47.1-47.1
• /
• 1997

• 대한생식의학회:학술대회논문집
• /
• 1997.11a
• /
• pp.83.2-84
• /
• 1997

• Journal of Embryo Transfer
• /
• v.12 no.2
• /
• pp.189-194
• /
• 1997

The ovaries of Korean native cows or heifers were obtained from a slaughter house and kept on 28~3O˚C and transported to laboratory within 2 hrs. The follicular oocytes were collected follicles. The oocytes were matured in vitro for 24 hrs. In TCM-199 supplemented with 35 $\pi$g /ml FSH, 10 $\pi$g /ml LH, 1 $\pi$g /ml estradiol-17 and granulosa cells at 39˚C under 5% $CO_2$ in air. The caudal epididymis of Korean native bulls were obtained from a slaughter house and transported to laboratory within 30 minutes. Swim-up of collected spermatozoa and freezing sperm was layered under 2ml fertilization B. 0. medium in two tissue culture tubes and held at a 45˚C angle for 0~2 hrs. They wrer fertilized in vitro by freezing sperm treated with heparin for 24 hrs, and then the zygotes were co-cultured in vitro with bovine oviductal epithelial cells for 7 to 9 days. The follicular oocytes recovered were classified into 41.7% as grade I, 51.5% as grade II and 6.8% as graed III. The number of oocytes recovered per ovary was averaged 8.3 and they were classifed into 2.3 as grade I, 2.5 as grade II and 2.3 as grade III. The cleavage rate of matured oocytes was significantly(P

• Korean Journal of Animal Reproduction
• /
• v.27 no.3
• /
• pp.233-240
• /
• 2003

This study was conducted to examine the effects of oocyte maturation period, phytohemagglutinin-P (PHA-P) treatment and activation agent on the enucleation, fusion, activation or in vitro development of bovine nuclear transfer embryos. Bovine oocytes were enucleated at 16∼24 h of in vitro maturation (IVM). Adult ear skin cells treated or non-treated with PHA-P were transferred into enucleated oocytes. Reconstituted oocytes treated or non-treated with PHA-P were fused by a pulse of 1.5 kV/cm for 30 $\mu$sec. Fused oocytes were activated with a combination of calcium ionophore (A23187) and cycloheximide (CHXM) or dimethylaminopurine (DMAP), and cultured in vitro for 7∼9 days. Enucleation rate was significantly increased when oocytes were matured for 16∼18 h (70.2∼92.3%, P<0.05) compared to that of oocytes were matured for 20∼24 h (44.3∼53.4%). The location of metaphase-II plate was far off from the 1st polar body as maturation time was increased. PHA-P treatment of donor cells or reconstituted oocytes significantly improved fusion rate (P<0.05). Cleavage and blastocyst formation rates were significantly increased after activation with a combination of A23187 and DMAP (78.6% and 32.9%, respectively) compared to those of embryos activated with a combination of A23l87 and CHXM (48.5 and 15.2%, respectively). From the present result, it is suggested that high enucleation efficiency can obtained by using oocytes matured for 18 h. It also shows that PHA-P treatment can improve the fusion rate, and activation with a combination of A23187 and DMAP can enhance the embryo development.

• Journal of Embryo Transfer
• /
• v.16 no.3
• /
• pp.223-231
• /
• 2001

The success of nuclear transplantation with mammalian oocytes depends critically on the potential of oocytes activation, which mainly caused to prevent the re-accumulation of maturation promoting factor (MPF). This study was conducted to compare the effect of combined treatment of lonomycin with a Hl-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on activation of bovine oocytes. In vitro matured bovine oocytes with the first polar body (PB) and dense cytoplasm were assigned to 3 experimental groups. For activation treatment, oocytcs were exposed to 5 $\mu$M lonomycin for 5 min (Group 1), and followed by 1.9 mM dimethylaminopurine (DMAP) for 3 h (Group 2) or followed by 2 mM sodium pyrophosphate (SPP) for 3 h (Group 3). The activation effects in the three treatments and the control group (untreated) were judged by the extrusion of the second PB and formation of a pronucleus (PN). Differences among groups were analysed using one-way ANOVA after arc-sine transformation of proportional data. All three treatments led to high activation rates (90% to 95%), with significant difference from the control. However, the extrusion of the second PB and the rate of PN formation differed remarkably among treatments. In Group I and 3, about 95% of the oocytes had extruded the second polar body, but one PN had formed in a higher proportion of oocytes in Group 3 than in Group 1 (90% vs. 5%). In experiment 2, the rates of cleavage and development into blastocysts in Group 1 were significantly lower than those of Group 2 and 3 (8.7% and 0% vs. 50.5% and 11.6%, and 44.6% and 7.2%, respectively, P<0.05). In experiment 3, ~80% of parthenotes in Group 1 were developed with haploid chromosomal sets. However, when ionomycin was followed immediately by DMAP (Group 2). only 20% of parthenotes were haploid. In Group 3, combined treatment with ionomycin and SPP, the appearance of abnormal chromosomal tracts was significantly (P〈0.05) reduced and the proportion of haploid parthenotes was increased to 85% (17/20) than in Group 2. These results demonstrate that SPP acted as a cdc2 kinase inhibitor and formed the haploidy in oocyte activation. Thus, the present study suggests that cdc2 kinase inhibitor, such as sodium pyrophosphate, may have an effective role in oocyte activation for the production of cloned embryos/animals by nuclear transplantation.

• Journal of Embryo Transfer
• /
• v.32 no.3
• /
• pp.123-130
• /
• 2017

This study was conducted to investigate the effects of alpha-lipoic acid (aLA) as an antioxidant that decrease the reactive oxygen species (ROS) in bovine embryonic development. Slaughterhouse derived bovine immature oocytes were collected and 4 different concentrations (0, 5, 10 and 20 mM) of aLA was supplemented in bovine in vitro maturation (IVM) medium. After 20 hrs of IVM, maturation rates, levels of ROS and glutathione (GSH), and further embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF) was investigated according to aLA concentrations. Maturation rate was significantly higher in 10 mM group than other groups (80.5% vs. 62.9, 73.9, 64.2%; P<0.05). In the levels of ROS and GSH in matured oocytes as an indicator of oocyte quality, significantly better results were shown in 5 and 10 mM groups compared with other 2 groups. After IVM, significantly higher rates of blastocyst formation were shown in 10 mM groups in both of PA (27.9% vs. 18.8, 22.3, 14.2%; P<0.05) and IVF (32.6% vs. 23.9, 27.3, 16.2%; P<0.05) embryos. In addition, significantly more cell total cell number and higher inner cell mass ratio in 10 mM PA and IVP blastocysts showed developmental competence in 10 uM groups. Therefore, based on the entire data from this study, using $10{\mu}M$ of aLA confirmed to be the optimal concentration for bovine oocyte maturation and embryonic development.

• Asian-Australasian Journal of Animal Sciences
• /
• v.11 no.5
• /
• pp.498-502
• /
• 1998

The present study was designed, fIrst to develop the new methodology to measure the bioluminescence activity easily in live developing bovine embryos by photoncounting, and secondly to compare the expression efficiency of four luciferase reporter genes in bovine embryos at four- to 16-cell stages. In experiment 1, equimolar pSVlacZ and pSVEluc were microinjected into the pronucleus of fertilized bovine oocytes. At 2 days after micro injection, bioluminescence activity of these embryos was measured by photoncounting with a luminometer for 1 min, and lacZ gene expression in the same embryos was assayed by X-gal staining. All the luciferase-positive oocytes showed some bacterial ${\beta}$-galactosidase activity irrespective of the intensity. In experiment 2, four firefly luciferase genes (pTKEluc, pTK6WEluc, pSVEluc and pMiwluc) were introduced by micro injection, and the injected embryos were cultured for the following 2 days. Detection of the luciferase gene expression was done by photoncounting at 5 to 55 min. Over the measurement period, the luciferase activity was almost constant irrespective of the transgenes microinjected. The luciferase activity and expression efficiency at 2 days after microinjection were not significantly affected by the difference in the microinjected transgenes. The present results demonstrated that the bioluminescence activity in live developing bovine embryos could be measured quickly by photoncounting.

• Korean Journal of Animal Reproduction
• /
• v.15 no.2
• /
• pp.125-132
• /
• 1991

The studies on the carried out to investigate the effects of capacitation method of spermatozoa on the in vitro fertilization and cleavage rate of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible of diameter 3~5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and FCS for 24~48hrs in an incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18hrs with motile capacitated sperm by preincubation of mKRB, treatment of HIS(high strength ion), Ca-IA(Inophore A), BFF(bovine follicular fluids) and heparin. The results obtained in these experiments were summarized as follows : 1. The in vitro fertilizatin and cleavage rate offollicular oocytes fertilized with capacitated spermatozoas in BO solution by preincubation of mKRB, treatment of HIS, Ca-IA, BFF and heparin method were 53.1%, 33.9%, 50.8%, 48.1%, 58.8% and 28.1%, 17.7%, 26.2%, 22.8%, 32.8%, respectively. And the fertilization and cleavage rate of heparin method was of highest of all. 2. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solutin by both caffeine, BSA and heparin methods were 65.8%, 70.3% and 40.8%, 47.3%, respectively, and those rates were higher treatment of heparin＋BSA, heparin＋caffeine than treatment of heparin. 3. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoa in BO solution with heparin concentrations of 2, 5, 10, 20, 40$\mu\textrm{g}$/ml were 50.0%, 54.7%, 58.1%, 51.7% and 27.9%, 32.8%, 37.1%, 30.0%, respectively. And the fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution with 10$\mu\textrm{g}$/ml of heparin was the highest of all. 4. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution containing heparin with caffeine concentraton of 10, 20, 30, 40$\mu\textrm{g}$/ml were 71.4%, 74.3% and 70.6%, 70.0% and 45.7%, 47.3%, 44.1%, 41.4%, respectively. The fertilization and cleavage rate of spermatozoa fertilized in BO solution with caffeine and heparin together(70.3~74.3%) was higher than that of spermatozoa fertilized in BO solution with heparin(58.8%). 5. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution containing heparin with BSA concentration of 5, 10, 20, 30$\mu\textrm{g}$/ml were 63.6%, 62.9%, 66.7%, 60.3% and 44.1%, 43.5%, 48.5%, 42.7%, respectively. The fertilization and cleavage rate of spermatozoa fertilized in BO solution with BSA and heparin together(60.3~66.73%) was higher than that of spermatozoa fertilized in BO solution with heparin(58.8%).