• Journal of Embryo Transfer
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• v.15 no.3
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• pp.219-230
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• 2000

This study was carried out to determine the factors on achieving good viability of embryos biopsied fur sexing, to investigate pregnancy rate following embryo transfer(ET) with sexed embryos, and to confirm the accuracy for the calves bort following ET with sexed embryos by polymerase chain reaction(PCR). To investigate viability of Hanwoo embryos after biopsy for sexing, fresh and frozen/thawed embryos were biopsied according to different developmental day of blastocysts, different stage of blastocysts, and different biopsy grade and the embryos themselves were incubated for 2 hours in TCM199 after microsection to be evaluated morphologically for recovery as blastocyst. The results obtained were as follows : 1. The rate of oocytes cleaved in vitro and the rate of blastocyst of the cleaved oocytes were 52.5% and 21.6%, respectively. The rate of blastocyst on day 8 was 11.2%, denoting the highest rate during whole culture period posterior to in vitro fertilization(IVF) 2. After biopsy for sexing, the viability rate of blastocyst on day 7, 8 and 9 was 75.0%, 88.4%, and 100.0%, respectively and the viability of early, mid, and expanded blastocyst after biopsy was 75.0%, 88.9%, and 91.1%, respectively The viability rate of fresh and frozen/thawed embryos was 89.9%, 71.4%, respectively. And the viability of expanded, hatching, and hatched blastocyst of frozen/thawed embryos was : 75.0%, 75.0%, and 50.0%, respectively. The viability of embryos according to biopsy grade of 10∼20%, 21∼30%, and 31∼40% was 85.7%, 91.5%, and 71.4%, respectively. 3. Pregnancy rate after transfer with biopsied embryo between flesh and frozen/thawed embryos was 22.6% and 20.0%, respectively. 4. In comparison between sex by PCR method and sex of calves born after embryo transfer, the accuracy of sex deterimination was 92.3% (12/13).

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.249-258
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• 1996

To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28$^{\circ}C$ and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH, 1 $\mu$g /ml at 39$^{\circ}C$ under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5$\mu$m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P

• Journal of Animal Science and Technology
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• v.65 no.4
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• pp.779-791
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• 2023

This study aimed to assess the effects of embryonic developmental stage, quality grade, and fresh or frozen/thawed conditions on the pregnancy rate and sex ratio of live offspring in Hanwoo (Bos taurus coreanae) cows. The quality and developmental stage of in vivo-derived (IVD) transferred embryos were evaluated using the standard criteria of the International Embryo Technology Society. The recipient cows were synchronized using conventional (estradiol benzoate and progesterone) protocols before embryo transfer. Embryos were transferred to 297 cows, and pregnancy was monitored for 60-70 days after embryo transfer. The pregnancy rates of fresh and frozen/thawed embryos were 56.90% and 52.49%, respectively. Pregnancy rates varied according to embryo quality (56.18% for grade 1 vs. 36.67% for grade 2). Pregnancy rates also varied by developmental stage and cryopreservation (67.86% vs. 63.49% for stage 4-1, 64.00% vs. 54.72% for 5-1, and 50.00% vs. 47.83% for 6-1, in fresh embryos vs. frozen/thawed embryos, respectively). For stage 7-1, the pregnancy rates were 72.73% for fresh embryos and 20.00% for frozen/thawed embryos. In 66 fresh embryos, the sex ratio of live offspring was 5:5, whereas it was 4(female):6(male) for frozen/thawed embryos among the 95 frozen/thawed embryos. The miscarriage rate was approximately 3% higher for frozen/thawed embryos than for fresh embryos (18.1% for fresh vs. 21.1% for frozen). Seasonal fertility rates were 33.3% in spring, 55.67% in summer, 52.8% in autumn, 60.0% in winter. The following male-to-female ratios were observed in different seasons: 6.7:3.3 in spring, 4.0:6.0 in summer, 5.5:4.5 in autumn, and 3.3:6.7 in winter. The current data revealed no significant differences in pregnancy rates between fresh and frozen/thawed IVD embryos. However, there was a lower pregnancy rate with advanced-stage frozen/thawed embryos (stage 7-1). The current study provides comprehensive results for the better optimization of embryo transfer in Hanwoo cattle to obtain the desired fertility rate, pregnancy rate, and sex ratio of calves. These results provide important insights into the factors that influence the viability and success of IVD embryo transfer in Hanwoo cows and may have practical applications for improving breeding programs and reducing production costs.

• Journal of Embryo Transfer
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• v.13 no.1
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• pp.61-67
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• 1998

This study was carried out to establish an effective system for embryo transfer techniques by analyzing several factors affecting in-vivo embryo transfer in Korean cattle. Embryos produced in-vivo were transferred into a total of 301 recipients The results obtained in studies on the factors affacting pregnancy rate after embryo transfer by condition of recipients were as follows ; 1. The pregnancy rate by age and parity of recipients showed high in 5~8 and over 12 years old(72.7~73.9%), and 3rd~4th parity(82.1%) for fresh embryos(P<0.05). The pregnancy rate did not differ by age and parity of recipients in frozen embryos. The pregnancy rate of frozen embryos tended to be similar to that of fresh embryos(38.5% and 25.0~36.7%). 2. The number of observation for normal estrus cycles of recipients did not differ In pregnancy rate between one and 2 times in fresh embryos(64.9%, 69.8%). The pregnancy rate by transferred frozen embryos showed significantly higher after 2 times of observation(P<0.05, 16.3%, 37.5%). The pregnancy rate by days open did not differ between fresh and frozen embryos. But the pregnancy rate was slightly higher in 12 months and 6 months of days open for fresh and frozen embryos, respectively(70.1~71.1% and 24.5%, respectively). 3. The pregnancy rate of transferred fresh and frozen embryos into right and left side of uterine horn did not differ(62.1% : 65.9% 25.0% : 24.3%, respectively). The pregnancy rate by the grade of CL was not different in fresh embryos, but the pregnancy rate was significantly higher in the grade A than B for frozen embryos(P<0.01, 43.2%, 16.2%).

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.1-6
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• 1994

This experiment was investigated the effect of presence of granulosa cells from follicles of different size on bovine oocyte maturation, cleavage and development to late stage. The nuclear and cytoplasmic maturation of oocytes in the IVM-IVF system are critical for subsequent embryo development. Granulosa cells when the co-cultured with oocytes may interact with cumulus-oocytes complexes and influence the development competence of the oocytes. Granulosa cells from medium (2~6 mm) and large(>1O mm) size follicles were recovered by aspiration, washed 3 times by centrifugation at 500 x g for 5 min. and used for co-culture at a concentration of 2~3 x 106 cells/mi. The oocytes were matured in vitro (IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g/ml FSH, 10 $\mu$g/ml LH, 1 $\mu$g/ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro (IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro (I VC) with bovine oviductal epithelial cells for 7 to 9 days. The assessment of maturation revealed that Grade J oocytes showed significantly(P

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.67-73
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• 2004

본 연구는 우리나라 고유의 유전자원인 재래산양의 체내수정란 생산기술을 확립하고자 수행하였다. 흡입법(aspiration)과 세절법(slicing)에 의해 난소 한 개당 회수된 난자의 수는 3.9개와 4.1개를 나타내어 slicing방법이 aspiration방법보다는 많은 숫자의 난자를 회수하였으나 유의적인 차이는 나타내지 않았다. 회수된 난자의 등급별 분포는 aspiration방법에서 Grade I, Grade II, Grade III, Grade IV의 비율이 10.3%, 20.5%, 38.5%, 30.8%를 나타내었으며, slicing법에서는 9.8%, 22.0%, 39.0%, 29.3%를 나타내어 Grade III과 Grade IV의 비율이 70% 이상을 차지하였다. 회수된 난자를 체외성숙 시킨 결과 Grade I과 Grade II에서는 85% 이상이 metaphase H (MII)까지 도달하였으나, Grade III과 Grade IV는 40% 이하의 체외성숙율을 나타내었다. 체외수정용 배양액으로 BO를 사용하였을 경우 Grade I 및 II에서 84.4%의 난분할율을 나타내어 TALP를 사용하였을 때의 58.8%보다 높은 난할율을 보였다. 또한 배양액의 종류별 체외발달율에 있어서는 상실배 및 배반포기배로의 발달은 mSOF를 배양액으로 이용하였을 경우 15.0%의 발달율을 나타내었으며, 체외배양 시 항산화물질인 glutathione (GSH)을 첨가함으로서 26.8%의 상실배 및 배반포배로의 발달율을 나타내었다.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.255-261
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• 2000

The objective of this study was to improve the efficiency of bovine embryo transfer by transferring of Hanwoo embryos into Hanwoo or Holstein recipients. The cryopreserved or fresh in vitro produced(IVP) embryos were transferred into uterine horn contralaterally or ipsilaterally to the corpus luteum. The recipients were inseminated by artificially on the next day of estrus. The pregnancy was diagnosed by rectal palpation at 60∼90 days after transfer of the embryos. The pregnancy rate by transfer of one or two embryos was 78%(7/9) and 74%(31/42), respectively. The pregnancy rates according to the grade of corpus lutea of recipients was 75% (20/27) and 82.0%(18/22) at the grade of A and B, respectively. Ten(67.0%) of 15 Holstein recipients transferred with IVP Hanwoo embryos and 5(42.0%) of 12 Holstein recipients transferred with frozen IVP Hanwoo embryos were pregnant. The single and twin calving ratio in Hanwoos was 77.0%(10/13) and 23.0%(3.13) in the recipients transferred with IVP embryos and 64.0%(7/10) and 27.0%(3/10) in the recipients transferred with frozen IVP embryos, respectively. Twenty-four pregnant cows following transfer of IVP embryos, 21(88.0%) calved the normal calves, and 2(8.3%) aborted. When the frozen IVP embryos were transferred, 16 pregnant cows calved 14(88.0%) normal calves and 2(13.0%) aborted. In conclusion, when one or two IVP bovine embryos were transferred into recipients, the A and B grade of corpus luteum resulted in high pregnancy rates. For the production of twin calves, transfer of the IVP or frozen IVP embryos could be suitable.

• Journal of Embryo Transfer
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• v.5 no.2
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• pp.38-44
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• 1990

This study was carried out to produce superior dairy cattle by embryo transfer. Seven dairy cows were superovulated with divided injection of FSH 4Omg for 5 days started on day 9 to 14 of the estrus cycle and injection of PGF$_2$$\alpha$ 45mg on day 4 of FSH injection. Donor cows were flushed to collect embryos on day 7 or 8 of the estrus cycle. Fresh embryos collected were transferred to synchronized dairy recipients or frozen using glycerol 3 step method to he equilibrated. And 35 embryos which were frozen using glycerol 6 step method were imported from U.S.A. After glycerol dilution of frozen embryos was done by reverse density during freezing. frozen-thawed embryos were transferred to synchronized dairy or beef recipients. The results obtained were as follows; 1. Total of 24 embryos were collected from 7 donor cows flushed and transferable embryos were 18 (75.0%). 2. Among 24 embryos. morula, early blastocyst, blastocyst, expanded blastocyst and unfertilized ova were 3 (12.5%), 1 (4.2%), 10 (41.6%), 4 (16.7%) and 6 (25.0%), respectively. 3. Heat inducing rate after 1st and 2nd injections of PGF$_2$$\alpha$ in Holstein and beef cattle was 83.3% and 71.4% and 62.5% and 69.2%, respectively. 4. Among 56 recipients, 23 head were pregnant (41.1%). The pregnancy rate of fresh embryos was 50.0% (1/2 heads) and the pregnancy rate of frozen embryos which were frozen using glycerol 3 step and using glycerol 6 step imported from U.S.A. was 52.6%(l0/19 heads) and 34.3%(12/35 heads), respectively. 5. The pregnancy rate of blastocyst (60.0%) was higher than that of morula (39.0%), early blastocyst (25.0%) and expanded blastocyst (0%). 6. The pregnancy rate of grade I embryos (52.2%) was higher than that of grade 2 (34.6%) and grade 3 (28.6%). 7. The pregnancy rate according to synchrony of recipient with donor was higher in simultaneous recipient (55.0%) and +l2hrs' (53.8%) than -24hrs' (23.5%), -l2hrs' (20.0%) and +24hrs' (0%).

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.265-271
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• 2014

Implementation of smart embryo technologies in cattle e.g. ovum pick-up followed by in vitro embryo production (OPU-IVP). Seasonal variation is important factor for follicular growth, oocytes quality, quantity and developmental competence. Therefore the aim of present study was carried out to investigated whether the seasons (hot and cool) effect on follicular development, oocyte recovery and subsequent embryo development. Follicular oocytes were aspirated from Korean native cows (Hanwoo) by the ovum pick-up (OPU) method, which was performed 24 times during two different seasons, the hot (July to September) and cool (October to December), from OPU donors. The recovered oocytes were classified according to morphological categories and used for in vitro embryo production (IVEP). The mean number of total follicles was significantly higher (p<0.05) during the hot season ($18.32{\pm}2.26$) compared to cool season ($15.41{\pm}3.34$). Furthermore, seasons did not significantly effect on the number of oocytes recovered (hot season: 41.16% vs. cool season: 46.14%). However, the average number of Grade A oocytes was significantly greater during hot ($1.75{\pm}1.86$) season compared to the cool season ($1.00{\pm}1.46$), but there was no significant difference of other grades oocytes. The cleavage rate (hot: 66.67% vs. cool: 63.3%) and embryo development (hot: 58.95% vs. cool: 56.97%) did not differ significantly between the seasons. In conclusion, the results of present study suggest that the season (hot and cool) does not have effects on the oocyte recovery and embryo developmental competence of in vitro cultured embryos.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.1
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• pp.50-56
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• 2019

These studies were conducted to evaluate developmental competence of follicular oocyte collected from the ovaries of Hanwoo cows with the high offspring meat quality (1++ and 1+ grade). Cumulus oocyte complexes from individual cows were matured, fertilized and cultured using protocols of in-vitro maturation (IVM), invitro fertilization (IVF) and in-vitro culture (IVC). The rates of blastocyst development from Hanwoo cows with the offspring meat quality grades of 1++ and 1+ were 18.6 and 21.2%, respectively. The rates of blastocyst development were 26.3, 20.7, 20.7, 17.2 and 31.2% from Hanwoo cows with the meat quality grades of 1++, 1+, 1, 2 and 3, respectively. Fiftyseven transferable embryos were recovered from 11 Hanwoo donor cows (5.2/head) with the high offspring meat quality grades of 1++ and 1+ in vivo, and the pregnancy rate after embryo transfer was 61.1%. In conclusion, these results suggest that in vitro embryo production from the ovaries of cows with the high meat quality grades using individual culture system can be used an efficient method for livestock improvement. In addition, for the successful industrialization of embryo transfer, conception rate should be improved.