• Reproductive and Developmental Biology
• /
• v.36 no.2
• /
• pp.115-120
• /
• 2012

This study was conducted to examine the optimal concentration and treatment time of antioxidants for inhibition of the ROS generation in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine oocytes were activated parthenogenetically, during which oocytes were treated with various antioxidants to determine the optimal concentrations and kind of antioxidants. Determined antioxidants were applied to oocytes during in vitro maturation (IVM) and/or SCNT procedures. Finally, antioxidant-treated SCNT embryos were compared with in vitro fertilized (IVF) embryos. $H_2O_2$ levels were analyzed in embryos at 20 h of activation, fusion or insemination by staining of embryos in $10{\mu}M$ 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA) dye, followed by fluorescence microscopy. $H_2O_2$ levels of parthenogenetic embryos were significantly lower in $25{\mu}M$ ${\beta}$-mercaptoethanol (${\beta}$-ME), $50{\mu}M$ L-ascorbic acid (Vit. C), and $50{\mu}M$ L-glutathione (GSH) treatment groups than each control group ($24.0{\pm}1.5$ vs $39.0{\pm}1.1$, $29.7{\pm}1.0$ vs $37.0{\pm}1.2$, and $32.9{\pm}0.8$ vs $36.3{\pm}0.8$ pixels/embryo, p<0.05). There were no differences among above concentration of antioxidants in direct comparison ($33.6{\pm}0.9{\sim}35.2{\pm}1.1$ pixels/embryo). Thus, an antioxidant of $50{\mu}M$ Vit. C was selected for SCNT. $H_2O_2$ levels of bovine SCNT embryos were significantly lower in embryos treated with Vit. C during only SCNT procedure ($26.4{\pm}1.1$ pixels/embryo, p<0.05) than the treatment group during IVM ($29.9{\pm}1.1$ pixels/embryo) and non-treated control ($34.3{\pm}1.0$ pixels/embryo). Moreover, $H_2O_2$ level of SCNT embryos treated with Vit. C during SCNT procedure was similar to that of IVF embryos. These results suggest that the antioxidant treatment during SCNT procedures can reduce the ROS generation level of SCNT bovine embryos.

• Journal of Embryo Transfer
• /
• v.12 no.2
• /
• pp.151-159
• /
• 1997

In vitro fertilization(IVF) derived morula and blastocyst embryos were bisected by a simple method and cultured in vitro without zona pellucida And also bisected embryos were frozen-thawed and cultured in vitro) to evaluate the survival rate. The results obtained were as follows : The average number of grade I or II immature follicular oocytes recovered by slicing method per ovary was 11.9 from 142 ovaries. Following in vitro fertilization, the rates of cleavage and in vitro development to morula and blatocyst were 61.7 and 32.2% respectively. The successful bisection rate of IVE embryos was 67.51%, and the embryos of blastocyst stage were bisected successfully at significantly(P

• Journal of Embryo Transfer
• /
• v.10 no.1
• /
• pp.83-90
• /
• 1995

Production of calves after transfer of nuclear transplant embryos is the latest technology to be applied in commercial livestock breeding. The objective of this study was to establish an efficient procedure to produce offsprings from nuclear transplant embryos. The fusion rates (72.7% vs. 80.8%), cleavage rates (62.5% vs. 71.4%) and rates of development in vitro (12.0% vs. 15.2%) of nuclear transplant embryos were not significantly different between 30 and 40h maturation age of cytoplast. The in vivo and in vitro-derived embryos as nuclei donor were used in this system of bovine nuclear transplantation. Fusion rates of nuclear transplant embryos were not significantly different between in vivo and in vitro-derived embryos (73.0 and 79.2%, respectively). The percentage of embryos reaching the morulae or blastocysts were 21.8% for in vivo-derived embryos and 11.9% for in vitro-derived embryos (p<0.01). Pregnancy rates after embryo transfer of nuclear transplant embryos were not significantly different between in vivo and in vitro-derived embryos (45.9 and 40.5%, respectively). However, calving rates after embryo transfer of nuclear transplant embryos were significantly higher in the in vivo-derived embryos than in vitro (p<0.01). Further research for age of cytoplast and use of in vitro-derived embryos as nuclei donor is required in this system. In conclusion, these results clearly show that the use of in vitro-derived oocytes as recipient cytoplast can improve the nuclear transplant system for genetic progress in cattle.

• Journal of Embryo Transfer
• /
• v.14 no.2
• /
• pp.107-111
• /
• 1999

The present study was performed to evaluate the best electric fusion condition in nuclear transfer, Korean Native Cattle fibroblasts were used as nucleic donors. Oocytes from slaughterhouse were matured in vitro for 22 h and enucleated. Each individual cells were transferred into enucleated ocytes and reconstructed embryo were placed into the fusion chamber. In experiment 1, pulse were performed by altering pulse duration at 1. 75kv/cm, 1 time. When pulse duration is 30$mutextrm{s}$, fusion and development rates is higher than other conditions. In experiment 2, the effect of different pulse number were studied at the pulse duration 30$mutextrm{s}$ and the same pulse intensity. When pulse number was one, fusion rates were higher than other conditions. The fused embryos were moved to culture medium and assessed their development to blastocyst. These results showed that best fusion condition was 30$mutextrm{s}$ and one time. And the fibroblasts derived from Han Woo can be reprogrammed by nuclear transplantation and develop subsequently in vitro.

• Journal of Embryo Transfer
• /
• v.14 no.3
• /
• pp.171-176
• /
• 1999

This study was carried out to investigate the effect of co-culture system(bovine oviduct epithelial cells; BOEC) and defined culture system (modified TALP ; mTALP) on the conception of embryos transferred, and pregnancy and twin birth rates after transfer of fresh or frozen-thawed bovine blastocysts produced in vitro were also evaluated. Oocytes from the slaughterhouse ovaries were matured and fertilized using general protocol. The results obtained were as the following. The pregnancy rate after transfer was higher in co-culture group than in mTALP group, but was not signficantly different, and there is no difference between fresh embryo group and frozen-thawed embryo group in conception rate. The conception rate was not different whether 3∼4 blastocysts or 2 blatocysts transferred into a recipient, but the production rate of twin calves was significantly higher (p<0.05) when 3∼4 embryos transferred. The average birth weight of twin calves(24.38kg) was numerically, but not significantly lighter than that of single calves(26.68kg).

• Proceedings of the KSAR Conference
• /
• 2001.10a
• /
• pp.37-43
• /
• 2001

1. About fifty thousand of cattle embryos were transferred and 16000 ET-calves were born in 1999. Eighty percents of embryos were collected from Japanese Black beef donors and transferred to dairy Holstein heifers and cows. Since 1985, we have achieved in bovine in vitro fertilization using immature oocytes Collected from ovaries of slaughterhouse. Now over 8000 embryos fertilized by Japanese Black bull, as Kitaguni 7 -8 or Mitsufuku, famousbulls as high marbling score of progeny tests were sold to dairy farmers and transferred to their dairy cattle every year. 2. Embryo splitting for identical twins is demonstrated an useful tool to supply a bull for semen collection and a steer for beef performance test. According to the data of Dr.Hashiyada (2001), 296 pairs of split-half-embryos were transferred to recipients and 98 gave births of 112 calves (23 pairs of identical twins and 66 singletons). 3. A blastomere-nuclear-transferred cloned calf was born in 1990 by a joint research with Drs.Tsunoda, National Institute of Animal Industry (NIAI) and Ushijima, Chiba Prefectural Farm Animal Center. The fruits of this technology were applied to the production of a calf from a cell of long-term-cultured inner cell mass (1998, Itoh et al, ZEN-NOH Central Research Institute for Feed and Livestock) and a cloned calf from three-successive-cloning (1997, Tsunoda et al.). According to the survey of MAFF of Japan, over 500 calves were born until this year and a half of them were already brought to the market for beef. 4. After the report of "Dolly", in February 1997, the first somatic cell clone female calves were born in July 1998 as the fruits of the joint research organized by Dr. Tsunoda in Kinki University (Kato et al, 2000). The male calves were born in August and September 1998 by the collaboration with NIAI and Kagoshima Prefecture. Then 244 calves, four pigs and a kid of goat were now born in 36 institutes of Japan. 5. Somatic cell cloning in farm animal production will bring us an effective reproductive method of elite-dairy- cows, super-cows and excellent bulls. The effect of making copy farm animal is also related to the reservation of genetic resources and re-creation of a male bull from a castrated steer of excellent marbling beef. Cloning of genetically modified animals is most promising to making pig organs transplant to people and providing protein drugs in milk of pig, goat and cattle.

• Reproductive and Developmental Biology
• /
• v.34 no.3
• /
• pp.127-134
• /
• 2010

This study was to investigate the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos, and their pregnancy and delivery rate after embryo transfer into recipient. In experiment 1, to optimize the flavonoid concentration, parthenogenetic day 2 ($\geq$ 2-cell) embryos were cultured in 0 (control), 1, 10 and $20\;{\mu}M$ flavonoid for 6 days. In the results, in vitro development rate was the highest in $10\;{\mu}M$ flavonoid group (57.1%) among treatment groups (control, 49.5%; $1\;{\mu}M$, 54.2%; $20\;{\mu}M$, 37.5%), and numbers of total and ICM cells were significantly (p<0.05) higher in $10\;{\mu}M$ flavonoid group than other groups. We found that $10\;{\mu}M$ flavonoid treatment can significantly (p<0.05) decrease the apoptotic index and derive high expression of anti-oxidant, anti-apoptotic, cell growth and development marker genes such as Mn-SOD, Survivin, Bax inhibitor, Glut-5, In-tau, compared to control group. In experiment 2, to produce the cloned Jeju Black Cattle, beef quality index grade 1 bull somatic cells were transferred into enucleated bovine MII oocytes and reconstructed embryos were cultured in $10\;{\mu}M$ flavonoid added medium. When the in vitro produced day 7 or 8 SCNT blastocysts were transferred into a number of recipients, $10\;{\mu}M$ flavonoid treatment group presented higher pregnancy rate (10.2%, 6/59) than control group (5.9%, 2/34). Total three cloned Jeju Black calves were born. Also, two cloned calves in $10\;{\mu}M$ flavonoid group were born and both were all healthy at present, while the one cloned calf born in control group was dead one month after birth. In addition, when the result of short tandem repeat marker analysis of each cloned calf was investigated, microsatellite loci of 11 numbers matched genotype between donor cell and cloned calf tissue. These results demonstrated that the flavonoid addition in culture medium may have beneficial effects on in vitro and in vivo developmental capacity of SCNT embryos and pregnancy rate.

• Korean Journal of Veterinary Research
• /
• v.37 no.3
• /
• pp.647-659
• /
• 1997

To overcome the difficulties of collecting and culture of primary cell from genital tract on embryonic development, the present study was carried out to investigate the critical effect of cell lines, such as BRL and Vero cell and its conditioned medium on the development of early Korean native cattle embryos in vitro. Oocytes collected from slaughterhouse ovaries were matured in TCM199 containing FSH, estradiol-$17{\beta}$ and FBS with granulosa cell monolayer for 24 hours and then fertilized in vitro using frozen-thawed, heparin-treated spermatozoa in TALP for 30 hours. And then early embryos (1-2cell) were cultured in TCM199 containing 10% FBS with BOEC, Granulosa, BRL, Vera cell monolayers and conditioned medium for 2~3 days. Development to morulae and blastocysts were recorded, also examined the number of blastomeres presented a valuable parameter for the evaluation of embryonic development. The early cleavage rates of in vitro fertilized embryos co-cultured, there was no differences between primary cell and cell lines(p<0.05). The rate of development to the later stage, coculture of BRL cell was significantly higher than that of the primary cell(p<0.05). The rates of development to morula and blastocyst were significantly higher in vero cell than BRL, Granulosa, Oviduct epithelial cell conditioned medium. In the result of effect of serum on development of early bovine embryos, the use of media containing serum were significantly higher than the use of not containing one on development of early and later stage of embryos. The result of number of blastomeres in blastocysts, there is no differences between primary cell and cell lines. The blastocysts from coculture were higher than from conditioned medium in blastomere cells. In summary, these experments have proved that the culture system in TCM199 with BRL, Vero cell monolayers is effective on in vitro development of early bovine embryos, In addition, it is effective to development of bovine embryos that containing serum in conditioned medium, or in co-culture rather than in conditioned medium alone. The use of cell lines opponent to primary cells is effective in bovine embryo culture.

• Journal of Embryo Transfer
• /
• v.31 no.3
• /
• pp.191-197
• /
• 2016

Historically, Korea old cattle had been consisted with various lines of coat color brindle, black and white-brown breeds or more. The two rare lines of black and white coat color are maintained for animal resources and preserved critically. The present study was carried out to evaluate potential usage of cysteamine supplementation during in vitro matration (IVM) and in vitro culture/production of embryo (IVP) by transvaginal ultrasound-guided follicle aspiration (Ovum Pick-Up: OPU) for the establishment of cryo-banking system. Immature slaughterhouse-derived cumulus-oocyte complexes (SL-COCs) were matured in IVM medium supplemented with 0, 0.1, 0.3 or 0.9 mM cysteamine, and then cultured in mSOF-BAS for 8 days after in vitro fertilization. The treatment of 0.1 mM cysteamine on SL-COCs showed higher rate of blastocyst, so OPU-derived COCs from rare breeds were matured in TCM media supplemented with or without 0.1 mM cysteamine, FSH and 5% FBS. The embryos were evaluated their developmental stages on day 8. During IVM, cysteamine treatment significantly increased the embryo production rate of slaughterhouse-derived COCs (19.6% vs. 30.5%). The presence of cysteamine during IVM of OPU-derived COCs from rare Korean cattle breeds (albino white and black line) also increased embryo production rates than those from SL-COCs (27.4% vs. 41.9% and 36.4%). With these results, cysteamine treatment during IVM is one of key factors IVP of blastocysts to establish banking system of endangered rare Koarean cattle with OPU derived transferable blastocysts.

• Reproductive and Developmental Biology
• /
• v.33 no.4
• /
• pp.257-262
• /
• 2009

This study was to investigate pregnancy rate of IVM/IVF/IVC Korean cattle (registered in government) embryos according to transport time course. For the production of embryos, oocytes recovered from slaughtered excellent grade cow and highly motile frozen-thawed bull semen (purchased from LIMC, KPN#497) was used. In vitro produced embryos were cultured in CR1aa medium for 8 days and some of them were frozen. The rate of average cleavage (>2-cell) was 83.0% (308/371) and blastocyst rate at day 8 was 34.7% (107/308). Among in vitro produced blastocyst embryos at day 8, most healthy embryos were freshly transferred on production day and some frozen embryos were direct transferred on appropriate day. These embryos were produced in a laboratory, embryo transfer (ET) was planned in 10 areas of the remote island (Jeju) from the laboratory by airplane. Thus, we examined the pregnancy rate in recipient cow according to embryo of transport time course before ET. From embryo transferred 44 recipient cows, overall pregnancy was 40.9% (18/44), these 18 cows were all calved [single, 94% (17/18); twin, 6% (1/18)] and total embryo implantation rate was 26% (19/66). Comparing transport time in the base of 6 hr, pregnancy rate in ET group required less 4 hr (60%, 9/15) was significantly higher than that required more 6 hr (26.3%, 5/19). In direct ET of freezing embryos, the pregnancy rate was 40% (4/10). However, it was difficult to find the meaning of temperature, pH and corpus luteum quality of recipients on comparison of pregnancy rate. When the cell death level of embryos according to storage time in thermos (straw container) before ET was measured by TUNEL staining, apoptotic index was increased with storage time-dependent. These results demonstrated that long distance transfer of IVM/IVF/IVC embryos is possible and the time of embryo transport is very important for the pregnancy rate on field trial.