• Journal of Embryo Transfer
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• v.30 no.3
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• pp.161-170
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• 2015

These studies were conducted to establish the practical Hanwoo (Korean native cattle) improvement system through the combining of embryo transfer technology and confirming individual Hanwoo oocyte culture system and to investigate that correlation of Hanwoo carcass classification (intramuscular marbling) and oocyte donor for blastocyst production in vitro. In case of Hanwoo, the carcass meat quality grades were divided to grade $1^{{+}{+}}$, $1^{+}$, 1, 2, and 3 depends on fat distribution of longest muscle cross-sectional surface. As results, the numbers of follicular oocytes collected from individual fundamentally-registered Hanwoo yielded $1^{{+}{+}}$, $1^{+}$, 1, 2 and 3 meat quality were 9.5, 9.4, 8.5, 8.8 and 8.8 per ovary, respectively. The numbers of retrieval oocyte from follicles were significantly higher in the cattle yield $1^{{+}{+}}$, $1^{+}$ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). The rates of blastocyst formation were 18.2, 21.3, 29.4, 30.9, and 31.5% in the cattle yield $1^{{+}{+}}$, $1^{+}$, 1, 2 and 3 meat quality of after in vitro maturation, respectively. It was significantly lower in the cattle yield $^{{+}{+}}$ and $1^{+}$ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). In order to evaluate embryos quality, TUNNEL assay was conducted for each meat quality grade using blastocyst stage embryo on day 8. The results showed that apoptosis cell number was higher tendency in the cattle yield $1^{{+}{+}}$and $1^{+}$ meat quality (81 and 79, respectively) than in the cattle yield 1, 2 and 3 meat quality (51, 48 and 50, respectively) but there was no statistical significance in each group. After embryo transfer, the conception rate of recipient was 53.5 (23 out of 43), 52.1 (38 out of 73) and 58.0% (58 out of 100) in the meat quality of 1, $1^{+}$ and $1^{{+}{+}}$, respectively. These results showed that the conception rate was significantly higher in the cattle yield 1 meat quality than in the cattle yield $1^{{+}{+}}$, $1^{+}$, 2, and 3 meat quality (p<0.05). In summary, these results indicate that the application of confirming Hanwoo individual oocyte culture system and embryo transfer technology can make good use of the genetic resources conservation and improvement of Hanwoo. Relevance of the meat quality grade and reproductive ability of carcasses of Hanwoo will be considered to be one of the effective means for the associated research with obesity and reproduction.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.151-159
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• 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
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• v.7 no.2
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• pp.125-131
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• 1992

The nudear changes of bovine oocytes during 24 hrs. of culture for mejotic maturation were examined. Bovine oocytes were collected from small(<2 mm), medium(2~6 mm) and large(>6mm) follicles and classified into three grades by their morphological characteristics. A total of 242 oocytes collected were obtained:from 184 small, 157 medium and 1 large follicles, respectively and were classified into 95 grade I, 155 grade H and 92 grade III oocytes. All the bovine oocytes collected and graded were washed with a basal medium and incubated in groups of 10 for 24 hrs in 5% $CO_2$ and 39$^{\circ}C$. The basal medium used was composed of TCM-199 supplemented with sodium bicarbonate, sodium pyruvate, streptomycin, penicillin G and 10% FCS. The oocytes were cultured in drops of 50,$\mu$l basal medium supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH and 1$\mu$g /ml estradiol-17$\beta$. The oocytes were fixed and examined on their chromosomal status by 1% acetorcein staining in the interval of 3 hrs. Most of the grade I oocytes developed to germinal vesicule stage at 0 to 3 hrs., germinal vesicle breakdown at 6 hrs., metaphase I at 9 to 15 hrs., anaphase I and telophase I at 18 hrs., and metaphase II and the first polar body at 24 hrs. after culture for meiotic maturation. However, it was found that compared to grade I oocytes, grade H and W oocytes reached earlier to germinal vesicle breakdown and most of them developed earlier to M II stage at 21 hrs. after culture.

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

• Clinical and Experimental Reproductive Medicine
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• v.22 no.3
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• pp.279-285
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• 1995

Despite the frequent incidence of embryo fragmentation in early human embryos, the reason of the embryo fragmentation has not been known yet. This study was conducted to investigate the histological difference(s) between fragmented (FR) and non-fragmented (NFR) human embryos focusing on comparison of mitochondrial distribution and protein synthesis. Multi-pronuclei zygotes (MPZ) such as three or more pronuclei containing in human in vitro fertilization and embryo transfer (IVF-ET) program were used for this study. MPZ were cultured in TCM-199 supplemented with 10% of human fetal cord serum (hFCS) in 5% $CO_2$ incubator at $37^{\circ}C$ for 24 hours. The cleaved embryos to 2-4 cells after 24 hours were grouped by their grade of fragmentation. Embryos were stained with Rhodamine123 (Rh123) and fluorescence was evaluated under the fluorescence microscope through PB 450-490 filter (Leitz). Regarding to protein synthesis during early human embryogenesis, there is no significant difference in the amount of synthetic proteins between FR and NFR embryos. Distribution of cytoplasmic organelles in embryos was evaluated by transmission electron microscope (TEM). The cytoplasmic distribution of mitochondria was different between FR and NFR embryos. The mitochondrial distribution was even in NFR, whereas severely aggregated in FR. It is not able to clarify in the present study whether this uneven mitochondrial distribution in FR embryo is the reason for embryo fragmentation or is the result from fragmentation. Physiological disparity related to the mitochondrial distribution may be one of the reasons for embryo fragmentation. Further studies should be addressed to investigate the physiological differences between FR and NFR embryos.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.257-264
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• 2014

Recently, several approaches have been used to measure the oxygen consumption rates of individual embryos, but relationship between oxygen consumption and pregnancy rates of Hanwoo following embryo transfer has not yet been reported. In this study, we investigated the correlation between oxygen consumption rate and pregnancy rates of Hanwoo embryo using a SECM. In addition to, the expression of apoptosis-related genes was determined using real-time PCR by extracting RNA according to the oxygen consumption of in vivo embryo. First, we found that the oxygen consumption significantly increased in blastocyst-stage embryos (blastocyst) compared to early blastocyst stage embryos, indicating that oxygen consumption reflects the embryo quality (Grade I). The oxygen consumption or GI blastocysts were significantly higher than those of GII blastocysts ($10.2{\times}10^{14}/mol\;s^{-1}$ versus $6.4{\times}10^{14}/mol\;s^{-1}$, p<0.05). Pregnant rate in recipient cow was 0, 60 and 80% in the transplantation of embryo with the oxygen consumption of below 10.0, 10.0~12.0 and over $12.0{\times}10^{14}/mol\;s^{-1}$, respectively. Apoptosis regulatory genes, Hsp-70.1 were significantly increased in over-10.0 group than below 10.0 group but in Caspase-3, Bax and P53 gene, there was no significant difference. In conclusion, These results suggest that measurement of oxygen consumption maybe help increase the pregnant rate of Hanwoo embryos.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.167-175
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• 2008

This study was performed to investigate the result that in-vivo or in-vitro embryos of Hanwoo cows were transferred to Holstein cows. Seventeen Hanwoo cows were used as donors for production of in-vivo embryos and fresh hanwoo in-vivo embryos were transferred to 1,150 Holsteins. And 2 embryos were transferred to 188 Holstein recipients to produce twin calves. Diagnosis on pregnancy was performed by rectal palpation at $60\sim90$ days after transfer. The pregnancy rate of Holstein recipients was 55.8% after transfer with Hanwoo in-vivo embryos and 38.2% after transfer with Hanwoo in-vitro embryos. The delivery rate of pregnant Holstein recipients was 88.4% after transfer with Hanwoo in-vivo embryos and 75.6% after transfer with Hanwoo in-vitro embryos. The rate of delivery of Holstein recipients transferred with two Hanwoo embryos was 36.2% and the rate of twin production was 25.9%. The rate of twin production by embryo transfer with in-vivo embryos was 30.4%, whereas the fate with in-vitro embryos was 15.6%. The pregnancy rate according to the grade of corpus luteum of Holstein recipients transferred with Hanwoo in-vitro embryos was 41.5 and 36.0% for A and B grade, respectively. The pregnancy rate according to the transfer in site in the uterine lumen of recipients was 40.9 and 32.7% for anterior and middle site, respectively. The pregnancy rate according to day of embryo transfer after estrus of recipients was 45.5, 38.8 and 39.7% for day 6, day 7 and day 8, respectively. There was difference of pregnancy rate according embryo transfer technician ($30.5\sim45.8%$) individual dairy farm ($21.1\sim51.0%$). These results are supposed to indicate that the rate of pregnancy after transfer with Hanwoo embryos to Holstein recipients was similar to that within the same breed, and consequently that this method would be beneficial to enhance the productivity in Hanwoo reproduction.

• Clinical and Experimental Reproductive Medicine
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• v.11 no.2
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• pp.59-67
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• 1984

In vitro fertilization have been performed to know whether the frozen semen has fertilizing ability and can be used clinically. The results of cultured and developed embryos obtained are as follows: 1. The semen was frozen in three media for the good viability. The viability was more than 50% and the motility was also moderate (grade III), 2. As the 33 oocytes were collected from 45 follicles, the oocyte recovery rate was 73.3%. Among them, mature and immature ova were 5% each, and premature ova were 69.7%, When the first polar body was appeared, above ova were inseminated after adequate incubation with activated sperms. 3. The main components of three freezing medium containing egg yolk, glycerol and pyruvate respectively were the best for sperm viability, and Ham's F-10 medium was used for the fertilization and culture of eggs. 4. The results of in vitro fertilization of 33 ova, showed the second polar body developed in 12%, polyspermia in 24%, 1-cell embryo in 21% and 2-cell embryo in 9%. One mature ova developed to blastocyst via 16-cell to 32-cell embryo. The fertilization rate was 66%. 5. Above mentioned results represent that the frozen semen has fertilizing ability and can be used practically in the clinic.

• Journal of Embryo Transfer
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• v.3 no.1
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• pp.38-42
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• 1988

These studies were conducted to investigate the effect of repreated superovulation on embryo production, the effect of the frozen-thawed embryos transferred on the developmental stage and grade, and donor-recipient synchrony on pregnancy rate in Korean native cattle. The results obtained in these studies were as follows: 1. Repeated superovulations in Korean Native Catile were not affected on the number of corpus luteum (CL), embryos recovered and embryos cleaved (range: 4.8 $\pm$ 4.21 to 9.5 $\pm$ 6.50, 1.8 $\pm$ 2.53 to 8.2 $\pm$ 8.04 and 1.6 $\pm$ 2.32 to 4.0 $\pm$ 4.59, respectively). 2. Blastocyst embryos (38.5%) showed higher pregnancy rate than morula (31.6%). 3. The pregnancyrates of cattle transferred with good and fair embryos were 33.3% and 40.4%, respectively. 4. The pregnancy rate when the donors exhibited estrus 12 hours earlier than the recipients (62.5%) was higher than when the donors and recipients exhibited estrus at the same time (33.3%) or when the donors exhibited estrus 12 hours later than the recipients (20.0%).

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.305-310
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• 2019

Embryos produced with serum show the alterations in their ultrastructure, impaired compaction, abnormal blastulation, aberrant mRNA expression profiles and large calf syndrome with greater incidences of stillbirths and deaths after birth. The aim of the present study was to describe in vitro embryo production by analyzing embryo production, fetal production and pregnancy rate in free-serum medium. The OPU-IVP data used in this study from 2016. Approximately, sixteen cows (Hanwoo), which belonged to the Institute of Gyeongsang National University, were used. Two experimental group is used in this study. Serum groups were conducted in March to July and free-serum group was conducted in September to December. The recovered cumulus-oocyte complexes were morphologically classified to four grades based on the compaction of cumulus cells layers and homogeneity of the cytoplasm. The number of oocyte was significantly greater in serum groups than that in free-serum groups (29.61 ± 0.63 vs. 15.6 ± 0.62; p < 0.05). Between serum and free-serum groups indicate that average of 1st and 2nd grade oocytes were no difference (2.38 ± 1.67 vs. 2.38 ± 1.48; p > 0.05), but number of 3rd and 4th grade oocytes were greater in serum groups than that in free-serum groups (7.31 ± 7.64 vs. 5.60 ± 6.29; p < 0.05). Embryo cleaved competence was higher in rate in free-serum groups than that in serum groups (62.1% vs. 58.3; p < 0.05). However, blastocyst developmental rate was no difference between serum and free-serum groups (33.1% vs. 43.5%; p < 0.05). 986 recipients were used for embryo transfer. Pregnancy rate was indicated that between serum and free-serum group was no difference (54.6% vs. 56.3%; p < 0.05). In conclusion, we developed the free-serum system for production of in vitro bovine embryos in order to meet the developmental and qualitative requirements for large scale commercial use.