• Journal of Embryo Transfer
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• v.11 no.3
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• pp.301-307
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• 1996

This study was carried out to produce twin calves by embryo transfer in Hanwoo and investigate the pregnancy and twin rate by recipient's conditions. All recipients were bred at estrus by artificial insemination with Hanwoo semen and then transfered an additional embryo produced in vivo or in vitro to tbe uterine horn contralateral to the corpus luteum on Day 7. The results obtained were as follows ; 1. The pregnancy rate was higher in young recipients of 3 years (68.8%) than in old ones of 10 years and greater(36.4%). And for CL size pregnancy rate was 57.9, 45.4 and 60.1% in large, medium and small size of CL of recipients, respectively. 2. 447recipients were transferred an additional embryos at 7th day after Al and average pregnancy rate was 57.5% and twin production rate was 22.2%. 3. Average pregnancy and twin production rate by direct transfer methods of frozenthawed IVF embryos was 56.0 and 16.7%. 4. The ratio of male to female twin in a total of 55 twin pairs was 54.6%, and average gestation lengths of male to female and female to female twin were 280.6$\pm$5.4 and 279.715.4 days, respectively. Average birth weight of twins was beavior in male and male twin(23.2i5.8kg) than in male and female twin(20.5$\pm$2.6kg).

• Journal of Embryo Transfer
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• v.24 no.4
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• pp.281-287
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• 2009

As a simple and economical method for in vitro produced embryos, we have used BSA instead of serum for the production and embryo transfer of Hanwoo in vitro fertilized (IVF) embryos and obtained the following results: 1) When using serum (FBS; fetal bovine serum) or BSA-containing culture media as the initial culture media for immature oocytes, it is regarded as inappropriate to add only BSA to the culture solutions from maturation of the immature oocytes to development stage culture, but serum still needs be added though there is no significant difference in the concentration, with a change from 5% to 10%. 2) The results of culturing IVF embryos after development (4 cell stage) in the Medium199 solutions containing BSA instead of serum (FBS) showed that 0.3% BSA concentration is not optimal and 0.5% or higher BSA concentration has no significant difference among 0.5%, 0.7%, 1% and 2% (p > 0.05). 3) The post-freezing survival ratio after development in 5% FBS-Medium199 showed that 1% BSA concentration of the culture solution is the most suitable in the BSA concentrations of 0.3% (51%), 0.5% (67%), 0.7% (69%), 1% (77%) and 2% (75%). 4) The pregnancy rates of the transplanted fresh(not frozen) blastocyst had no significant concentration dependency (p > 0.5), and the average pregnancy rate was 63.8%. 14% of overweight calves were found among the calves given birth to by the transfer of IVF blastocysts cultured in the serum-added culture solution, but none was found in the experimental groups in which BSA was added instead of serum.

• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.389-396
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• 1997

Arrest in embryo development during in vitro culture has been reported in various mammals. Although some cause of the arrest have been suggested, little is known of the way that can overcome the arrest using in vitro culture system. The antioxidant, $\beta$-mercaptoethanol($\beta$-ME), has been shown to play an important role in embryo development. This study was designed to examine the effect of $\beta$-ME on the developing boving embryos produced in vitro by IVM and IVF. To select a, pp.opriate concentration of $\beta$-ME during whole culture period (7 days), various concentrations (10, 50 and 100$\mu$M) of $\beta$-ME were added to the CZB medium and their effects was significantly higher in 100$\mu$M of $\beta$-ME. The effects on development of embryos cultured with and without somatic cells to blastocyst stage were greater in FCS treatment (56.6 and 29.3%) than in BSA treatment(25.5 and 12.8%). We also evaluated the effects of $\beta$-ME addition on the blastocyst formation when embryos at different stages were exposed to 100$\mu$M $\beta$-ME. $\beta$-ME promoted increased development of embryo to blastocyst stage and the effect was greater in 6-cell to morula embryos than in embryos fewer than 4-cells at the initiation of treatment. The results suggested that $\beta$-ME can improve bovine embryo development by overcoming the arrest in early development.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.307-313
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• 1997

Experiments were conducted to assess the effect of quality and viability of bovine blastocysts derived from in-vitro culture(IVC) of in vitro matured and fertilized(IVM-IVF) oocytes during their transport 2 hours. Follicular oocytes were collected form ovaries obtained at a slaughterhouse and were cultured for 24 hours in TCM-199. The IVM oocytes were fertilized in vitro with caudal epididymis spermatozoa. Fertilized oocytes were cultured for 7 to 9 days, and embryos that developed to the blastocyst stage were used for the experiment. The blastocysts, packed in straws with storage medium that consisted TCM-199 with HEPES equilibratd in air and supplemented with 10% FCS were transported at 39~(2.0 h). The quality of blastocysts was assessed and ranked as A(excel-lent), B(Good), fair or poor after transportation. The percentages of A and B grade blastocysts after transport duration for < 1 hours(97.7%) were similar to the result from transport duration for 1~2 hours (92.9%) and 2~3 hours(89.6%), but significantly(P<0.05) higher than transpot duration for 3~4 hours(76.3%). The percentages of A and B grade blastocysts after transport duration for two hours from developed blastocyst at 7day(100%) and 8day(85.0%) were higher 9day(96.6%) and >9day (40.0%). And early to expanded blastocyst produced in vitro were transferred to recipient cow by additional embryos at 7 and 8th day after AI. Three of them were pregnant to term and produced four twin calves, and two calves was premature birth. The gestation lengths of male to female and female to female twin were 282 and 281 days, respectively. And birth weight of twin calves were male to female(22.Skg) and female to female twin(20.3Okg), respectively.

• Korean Journal of Veterinary Research
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• v.33 no.4
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• pp.757-762
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• 1993

The ovaries of Korean Native cows or heifers were obtained from an abattoir and kept on 20 to $25^{\circ}C$ and transported to laboratory within 2 hrs. The follicular oocystes were collected from 2~6mm follicles in diameter and classified into 3 grades by the morphology of cumulus cells attached. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with $23{\mu}g/ml$ FSH, $10{\mu}g/ml$ LH, $1{\mu}g/ml$ estradio-17 ${\beta}$ and granulosa cells at $39^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by incubation for 12 hrs. of epididymal spermatozoa pretreated with heparin, and then the zygotes were co-cultured in vitro(IVC) with oviductal epithelial cells for 7 to 9 days. Assessment of maturation revealed that 93.0%(147/158) of grade I oocytes had expanded of cumulus cells, which was higher(p<0.05) than the 79.4%(85/107) of grade II oocytes. Compared to epididymal sperm(32.9%), the insemination with frozen and thawed sperm resulted in slightly lower(20.5%), but not significant, development to morulae and blastocysts from grade I oocytes. Co-culture of bovine IVF embryos with oviductal epithelial cells improved the development to transferable embryos significantly(38.1%), compared to co-culture with granulosa cells(20.0%). When VF bovine embryos were vitrified at blastocyst, the post-thaw survival rate was obtained higher resulf for 1 min. equilibration time(82.6%) or 2 min.(73.9%) than 3 min.(18.2%) in EFS solution.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.2
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• pp.178-188
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• 2013

The glycosaminoglycans (GAGs) present in the female reproductive tract promote sperm capacitation. When bovine sperm were exposed to 10 ${\mu}g/ml$ of one of four GAGs (Chondroitin sulfate, CS; Dermatan sulfate, DS; Hyaluronic acid, HA; Heparin, HP) for 5 h, the total motility (TM), straight-line velocity (VSL), and curvilinear velocity (VCL) were higher in the HP- or HA-treated sperm, relative to control and CS- or DS-treated sperm. HP and HA treatments increased the levels of capacitated and acrosome-reacted sperm over time, compared to other treatment groups (p<0.05). In addition, sperm exposed to HP or HA for 1 h before IVF exhibited significantly improved fertilizing ability, as assessed by 2 pronucleus (PN) formation and cleavage rates at d 2. Exposure to these GAGs also enhanced in vitro embryo development rates and embryo quality, and increased the ICM and total blastocyst cell numbers at d 8 after IVF (p<0.05). A real-time PCR analysis showed that the expression levels of pluripotency (Oct 4), cell growth (Glut 5), and anti-apoptosis (Bax inhibitor) genes were significantly higher in embryos derived from HA- or HP-treated sperm than in control or other treatment groups, while pro-apoptotic gene expression (caspase-3) was significantly lower in all GAG treatment groups (p<0.05). These results demonstrated that exposure of bovine sperm to HP or HA positively correlates with in vitro fertilizing ability, in vitro embryo developmental potential, and embryonic gene expression.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.181-186
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• 2005

This experiment was conducted to investigate effects of the two different in vitro production systems, serumcontaining system (IVM, IVF and IVC; TCM199, TALP and CR1aa) and serum-free system (IVM, IVF and IVC; IVMD101, IVMD100 and IVMD101), on the development of in vitro fertilized or DNA-microiniected embryos. We also examined the effect of DNA dosage and its expression pattern in embryos. The DNA used for microinjection was a green fluorescence protein gene. The development rates to $\geq$ 2cell, 8cell and blastocyst stage were significantly higher in vitro fertilized embryos than those in DNA-microinjected embryos. The development rate to the 8-cell stage was significantly higher in serum-free system than in serum-containing system (p<0.05; $3.3\%\;vs.\;15.5\%\;and\;21.4\%$, respectively). The development rates to the blastocyst stage of in vitro fertilzed or DNA-microinjected embryos between two different culture system ($2.7\%\;vs.\;2.3\%\;and\;23.0\%\;vs.\;23.6\%$, respectively) were not different. The development rates of embryos injected 2 ng/uL DNA was higher. than those of embryos injected 4 or 8 ng/uL DNA. The GFP expression rate of 1-cell embryos was significantly higher than that of 2-cell and 4-cell embryos, whereas the rates were not different between 4-cell and blastocyst-stage embryos.

• Journal of Embryo Transfer
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• v.25 no.3
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• pp.189-193
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• 2010

We investigated the cleavage rate and blastocyst yield for each culture condition to enhance tolerance of cryo-preservation of bovine IVF embryo with relatively lower cryo-tolerance compared to in vivo embryo. The cleavage rate and blastocysts yield for CR1aa, IVMD, IVD, CR1aa+10% FBS were 73.2, 69.3, 72.8, 68.5% and 44.1, 30.8, 33.3, 48.0%, respectively. The values did not differ among each treatments without serum. For embryo vitrification, In vivo and In vitro blastocysts were exposed to VS1(10% glycerin, 0.1 M glucose, 0.1 M sucrose, PEG 1%) for 5 min, and VS2 (10% glycerin, 10% EG, 0.2 M glucose, 0.2 M sucrose, PEG 2%) for 5 min and then VS3 (10% glycerin, 30% EG, 0.3 M glucose, 0.3 M sucrose, PEG 3%) for 1 min. The exposed embryos were then loaded into the 0.25 ml plastic straws and then plunged into liquid nitrogen. The straws were held for period of 1 to 2 weeks before thawing. In embryo viability, no differences in blastocyst re-expansion rates were found between in vivo and in vitro embryos. whereas expansion-BL rates was significantly higher for in vivo-derived embryos (72.7%) when compared to in vitro-derived embryos (51.4%), respectively (P<0.05). In conclusion, our results indicate that combined use of CRIaa culture medium with vitrification might enhance tolerance of cryopreservation for bovine IVF embryo production.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.199-205
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• 2013

The present study was conducted to compare on embryo survival rates by blastomere isolation methods, and establish the optimal PCR procedure for perform the sexing of bovine blastocysts produced by IVF. IVF embryos used in the study was used the Bisected or Sliced methods for blastomere isolation, and the survival rates of blastocyst with rapid way of sexing PCR was assessed. In the present study for survival rates in blastocyst was the total cleavage rate was 75% and a blastocyst development among cleaved embryos was 40%. Survival rate of embryos treated with intact, bisected or sliced method was 100, 63.3 or 81.3%, respectively. Therefore, survival rate of embryos treated with sliced method was higher compared to that of embryos treated with bisected method. The sexing rate of female or male was not significantly different between S4BFBR primer and BSY + BSP primer (1.75 : 1 vs. 1.43 : 1), respectively. Because of the PCR amplification using the S4BFBR primer was simpler method than multiplex PCR amplification method. Furthermore, the accuracy of sexing rate and reduction of PCR work time between 2-step and 3-step of PCR methods was 98.0% / 1.5 hr and 97.0% / 3.5 hr, respectively. Based on these results, it can be suggested that the sliced and PCR methods we developed was very effective method to reduce time consuming and procedure of PCR amplification for sexing with the increase of survival rate on the blastocyst.

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