• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.281-287
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• 2013

In mammal, unfertilized oocytes remain in the oviduct or under in vitro culture, which is called "oocyte aging". This asynchrony negatively affects fertilization in pre- and post-implantation embryo development. Caffeine a phosphodiesterase inhibitor is known to rescue oocyte aging in several species. The objective of this study is to determine the cytoskeleton distribution in aged oocytes and the embryo developmental ability of aged oocytes in the present or absence of caffeine during maturation. Caffeine treatment increased the incidence of normal spindle assembly of aged oocytes (treatment, $67.57{\pm}4.11%$ aging, $44.61{\pm}6.4%$) and no significant differences compared to control group. Fluorescence values were compared using ROS (Reactive oxidation species) stain. Fluorescence values appear of control group intensity rate ($51.53.{\pm}3.80$), aging group ($68.10{\pm}5.54$) and treatment of caffeine ($45.04{\pm}2.98$). Aged oocytes that were derived from addition of caffeine to the IVM (in vitro maturation) medium had significantly increased 2-cell that developed to the blastocyst stage compared to the aging group. Blastocysts, derived from caffeine treatment group, significantly increased the total cell number compare aging ($90.44{\pm}10.18$ VS $67.88{\pm}7.72$). Apoptotic fragments of genomic DNA were measured in individual embryo using TUNEL assay. Blastocyst derived from caffeine treatment group decreased significantly the apoptotic index compared to blastocyst derived from aging group. In conclusion, we inferred that the caffeine treatment during oocyte aging can improve the developmental rate and quality in bovine embryos developing in vitro.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2020.08a
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• pp.19-19
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• 2020

The Cassava(Manihot esculenta Crantz) is a tropical root crop, originally from Amazonia, that provides the staple food of an estimated 800 million people worldwide. It belongs to the family Euphorbiaceae which also includes rubber (Hevea brasiliensis) and castor bean (Ricinus communis). Among tropical crops, rice, sugarcane, maize and cassava are the most important sources of calories for human consumption. Problems in the propagation of cassava are virus diseases and low rates of seed germination. So we tried to optimize protocols for mass production of somatic embryo amenable to large-scale vegetative propagation of Cassava. After in vitro eight-week culture of leaves of Cassava, the medium which contained the 2,4-D, BAP and IBA showed the highest callus induction rate, embryogenesis callus formation rate and somatic embryo formation in Cassava culture. In the medium with GA₃ and myo-inositol, shoots were most vigorously regenerated from somatic embryos of Cassava. Our experiments confirmed that in vitro growth and multiplication of plantlets could depend on its reaction to the different medium composition, and this micropropagation techniques could be a useful system for healthy and vigorous plant production.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.117-123
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• 1992

Development in vitro of 2-cell mouse embryos was examined after appropriate exposure to oviductal milieu to demonstrate biological activity present in the oviducts. ICR and ($C57Bl/6{\times}Balb/c$) $F_1$ hybrid mice were superovulated and mated for the recovery of early embryos. Embryos were recoverd at every 2h intervals from 32h post-hCG(hph) to 56 hph. The proportions of developmental stages were determined in the recovered embryos. Development in vitro of 2-cell embryos was more rapid in $F_1$ hybrid than in ICR, showing high proportions of 4-cell embryo and blastocyst at 120 hph. 100% of blastocyst development was obtained at 38hph in $F_1$ hybrid and at 50 hph in ICR when 2-cell embryos were cultured upto 120hph in vitro. Moreover, in vitro culture of oviducts containing 2-cell embryos in ICR mice for 12h from 34hph to 46hph increased developmental capacity of ICR mouse embryo in vitro. The results indicate that oviductal environment contains substances having mitogenic activity and overcoming early cell block in vitro. The mitogenic activity is effective in vitro as well as in vivo.

• Reproductive and Developmental Biology
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• v.38 no.3
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• pp.93-98
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• 2014

The objective of this study was to investigate the changes of oxidative stress and antioxidant enzyme during in vitro development with washing culture oil in porcine embryos. During the culture, the four types of culture oil such as paraffin oil with or without washing and mineral oil with or without washing were examined. The oil was washed with PZM-3 during 7 days and collected oil only. The embryos were stained with CellTracker$^{TM}$ Red, DCFDA and Hoechst 33342 to confirm the effects of the oil. As a results, Cleavage rates and total cell number were no difference among the four oil groups. However, ${\geq}16$ cell embryos were significantly different in fore type oil treatment and blastocyst rate was significantly higher washing paraffin treatment than in other group(p<0.05). Also, the expression of free radical were lower in washing paraffin oil than in other groups (p<0.05). On the other hand, the expression of glutathione were not significant different among paraffin oil with or without washing and mineral oil with or without washing, however washing paraffin oil and washing mineral groups were higher than other treatment groups. In conclusion, the washing oil was expected with positive effects on in vitro development in porcine embryos.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.27-32
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• 2016

This study was conducted to evaluate the effects of different volume ($100{\mu}l$ vs. 2 ml) of microdrop culture on B6D2F1 mice oogenesis. In the present study, B6D2F1/CrljOri $F_1$ mice were utilized in order to maximize oogenesis. Also we used TCM-199, Dulbecco's medified Eagle's medium (DMEM), embryo culture medium (Fertilization medium, Cleavage medium, Blastocyst medium), G series medium and One step medium. Blastulation rate was not different between groups ($58.4{\pm}2.9%$ vs. $61.2{\pm}4.8%$). Zona hatched rate ($38{\pm}15.4%$ vs. $27{\pm}3.4%$) and attached rate ($55{\pm}13.9%$ vs. $46{\pm}3.9%$) did not differ by the volume of culture media. Total cell numbers ($59.8{\pm}9.7$ vs. $70.3{\pm}8.7$), ICM cell numbers ($15.8{\pm}0.6$ vs. $16.8{\pm}1.5$), TE cell numbers ($44.0{\pm}9.7$ vs. $53.6{\pm}7.3$), % ICM ($26.4{\pm}2.9%$ vs. $23.8{\pm}3.3%$) and ICM:TE ratio ($1:2.8{\pm}0.4$ vs. $1:3.2{\pm}0.6$) were not different between groups (i.e., $100{\mu}l$ vs. 2 ml). These results show that the capacity of the culture medium did not effect the cell numbers of B6D2F1 mice blastocysts. In summary, these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.51-57
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• 2008

The present study investigated the efficient methods to produce in vitro Hanwoo embryos, and to improve the pregnancy rate. The developmental rate, total cell number and ICM ratio of in vitro embryos were compared amongst different culture media. Comparisons were also made on the status of recipients, pregnancy rate along with day of transfer after the estrus. Development of embryos into blastocyst stage in IVMD101 supplemented with 5% fetal bovine serum (FBS) group was significantly higher (34.2%) than that of TCM-199 supplemented with 5% FBS (26.8%) and IVMD101 without FBS (25.9%) (p<0.05). The development rate to blastocyst stage was significantly faster in IVMD101(5% FBS) than that of other groups ($0.2{\sim}2.3%$) (p<0.05). The average number of inner cell mass and trophectoderm were similar among treatment groups, which were $36.0{\sim}44.7$ and $83.3{\sim}106.7$. However, total cell number in IVMD101(5% FBS and 0% FBS) was significantly higher than that of TCM199(5% FBS). There were no differences in the pregnancy rate among treatment groups (32.0%, 33.9% and 28.6%, respectively). However, the pregnancy rate of Day 6 embryos cultured in IVMD101(5% FBS) was significantly (p<0.01) higher than IVMD101 without FBS and TCM-199 + 5% FBS (38.0% vs. 17.2% and 32.4%, respectively). No significant difference was observed for the pregnancy rate between heifer and cow transferred with Day 6 embryos cultured in IVMD 101(5% FBS) (42.7% and 39.3%, respectively). However, there was a significant difference of pregnancy rate (p<0.05) in heifer between one and two embryos transferred (31.4% and 41.9%). There was no difference of pregnancy rate among transfer days after estrus between heifer and cow, but the pregnancy rate of transfer to heifer with day 6 after estrus was significantly higher (p<0.05) than that of day 7 and 8 (22.2% vs. 49.0% and 38.7% respectively). Based on the above findings, there is a possibility to produce in vitro produced embryos cultured in IVMD101(5% FBS) showed higher blastocyst rate and the increased cell number. In terms of the pregnancy rate of in vitro produced embryos, the highest pregnancy rate was observed when two embryos were cultured in IVMD101(5% FBS) and transferred.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.185-193
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• 1999

To improve the efficiency of in vitro production of embryos with follicular oocytes in pig, the recovery rates, in vitro fertilization and development. The results obtained were as fellows: The number of oocytes recovered 37 ovary was 1,365 by aspiration, 1,884 by slicing and 3,830 aspiration post slicing, per ovary was averaged 103.5 aspiration post slicing than 30.7 by aspiration and 50.8 by slicing (P<0.05). The percentage of grade I and II oocytes recovered was 0.05∼0.2% and 1.7∼2.3% respectively(p<0.05). The fertilization rates of ejaculate and epididymis sperm was 83.0 and 83.1%. And cleavaged rate was 60.8 and 69.0% respectively(P<0.05). However, there were no significant differences between sperm sources. The clevage rates of fertilized oocyte was significantly(P<0.05) higher as B.O(92.8%) than TALP (90.1%) or mTBM (80.1%). And in vitro developed to blastocyst rates of mTBM media used for fertilization was significantly (P<0.05) higher as 12.4%, compared with the results using the media of TALP(1.6%) or B.O (0.0%). The embryos developed 2-cell stage after in vitro fertilization were co-cultured with or without POEC and BOEC in NCSU-23 and TCM-199 media. In vitro developed to blastocyst rates was NCSU-23 with POEC(2.3%) or BOEC(1.2%), but in vitro cultured in TCM-199 medium with POEC or BOEC was not developed to blastocyst. The percentage of embryos that developed to morula stage in 0, 50, 100, 200 and 250uM was 16.6, 22.0, 13.5, 19.0 and 22.0%, respectively.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.237-242
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• 1995

Porcine follicular oocytes matured in culture were inseminated with frozen-thawed spermatozoa. When the oocytes were inseminated in the medium with oviductal epithelial cell monolayer, the penetration rates higher in those with (4.1, 31.7, 45.1, 54.5 and 69.4%) than without cells (0, 17.1, 34.8, 45.2 and 58.9%) at 4, 8, 12, 16 and 20 h after insemination. The proportions of polyspermy in penetrated oocytes in medium with or without cells increased with time of examine. In another experiment, the penetration rate was higher without (57.6%) than with (19.6~24.1%) preincubation of spermatozoa for 1~4 h in medium. However, when the oocytes were inseminated with spermatozoa preincubated for 1~2 h, the penetration rates significantly higher (P<0.05) in those with (65.6 and 55.9% for 1 and 2 h) than without (24.1 and 20.6% for 1 and 2 h) oviductal epithelial cell monolayer. On the other hand, the proportions of polyspermy decreased with time of spermatozoa preincubation. These results indicate the significant advantages of the spermatozoa preincubation with oviductal epithelial cell monolayer for 1 and 2 h to maintain penetration potential during in vitro fertilization in the porcine.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.11-20
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• 1997

The influence of cryopreservation of donor embryos on the in vitro developmental potential in the nuclear transplant rabbit embryos was evaluated. The embryos of 16-cell stage were collected and cryopreserved with EFS solution by vitrification method. The frozen embryos were thawed and synchronized to S and G$_1$ phase of 32-cell stage. The recipient/ cytoplasms were obtained by removing the first polar body and chromosome mass from the oocytes collected by non-disruptive microsurgery procedure. The separated S and G$_1$ phase blastomeres of 32-cell stage were injected into enucleated recipient cytoplasms by micromanipulation. After culture until 20 hrs post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation. The fused nuclear transplant embryos were co-cultured with rabbit oviduct epithelial cells. After in vitro culture for 120 hrs, the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. The electrofusion rate was significantly (P<0.05) reduced in the frozen nuclear donor,compared with fresh donor nuclei as 80.0 vs 62.8% in S phase and 81.7 vs 64.8% in G$_1$phase, respectivley. The in vitro developmental rate to blastocyst stage with the S and G$_1$phase of fresh embryos(26.3 and 61.1%, respectively) was found significantly (P<0.05) higher, compared to the S and G]phase of frozen embryos(11.9 and 34.6%, respectively). When frozen as well as fresh donor embryos were synchronized to G$_1$ phase, the in vitro developmental rate to blastocyst stage was significantly (P<0.05) higher, compared with S phase donor nuclei. The cell counts of nuclear transplant embryos developed to blastosyst stage were significantly (P<0.05) more in G$_1$ phase of fresh or frozen embryos (180.1 and 125.7 cells, respectively), compared with S phase nuclear donor (145.1 and 103.7 cells, respectively). From the above results it was concluded that the rabbit embryos cryo- preserved by vitrification might be available as nuclear donor, though the developmentalpotential and cell counts of nuclear transplant rabbit embryos were decreased significantly.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.135-142
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• 2003

본 연구는 사람의 시험관아기 프로그램에서 수정 후 1일째의 전핵 등급이 체외 수정란의 발달에 미치는 영향을 조사하였다. 정상적인 시험관 아기 시술을 시행한 실례 환자를 대상으로 과배란을 유기하여 배란 직전의 난자를 채취하여 정자를 주입한 다음 체외 수정을 유도하였다. 수정 유도 18시간 후 전핵과 핵인의 형태에 따라 전핵의 등급을 1 및 2등급으로 나누어 각각 3일 동안 체외 배양을 실시하여, 체외 수정란의 형태에 따라 1, 2 및 3등급으로 분류하였든 바, 전핵의 등급에 따라 각각 1등급 체외수정란의 발달율은 1등급 전핵란에서는 83.5%로서 2등급 전핵란의 5.5%보다 유의적으로 (P<0.05) 높았다. 1등급 및 2등급 전핵란에서 수정 후 3일째에 5-세포기 이상의 단계로 발달하는 경우는 각각 85.1% 및 24.5%를 나타내었고, 평균 할구수는 7.4$\pm$2.1 및 4.1$\pm$3.5개를 나타내어 발달 능력에서 1등급 전핵란이 2등급 전핵란보다 유의적으로 (P<0.05) 높게 나타났다.