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The Effects of Cummulus Oocyte Complex Quality on the Embryo Production Via OPU Procesures
Dongkyo Kim,Namtae Kim,Sung Woo Kim,Chan-lan Kim,Min Su Kim,Ik Soo Jeon 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10
OPU (Ovum pick up) has been an important technique to increase genetic resources for early establishment of genetically important herd and in vitro embryo production system. This study compared relationship between OPU-derived COCs quality and embryo development. The recovery procedures of COCs were performed twice a week using ultrasound guided OPU tools. The 18 gauge disposable needle was connected into 50 mL conical tube by teflon tubing, The grade of COCs was evaluated by microscopic examination. Grade A had 3 or more layers of cumulus cell and compact cytoplasm. Grade B had 1~3 layers of cumulus cell and compact cytoplasm. Grade C had 1 layers cumulus cell and compact cytoplasm. Grade D was denuded oocyte and poor cytoplasm. The collected COCs were fertilized in vitro and developed for 7~8 day in mSOF media. The cleavage rate of grade A/B dominant pick-up group (above 60% of A/B grade rate from total recovered COCs) was 48.9% and that of poor quality was 51.9%. The rate of blastocyst rate in grade A/B dominant OPU procedures after IVF was 28.9%, but poor quality group (below 60% of A/B grade COCs) was 14.8%. The developed blastocysts were transferred into recipient or cryopreserved for further research. When total 11 blastocysts were transferred into surrogate cows with the pregnancy rate (4/11) of 36.3% and 4 calves are expected.
Seasonal effect of Hanwoo oocyte recovery & embryo development by OPU technology
Dongkyo Kim,Namtae Kim,Sungwoo Kim,Chan-lan Kim,Ik Soo Jeon,YeoungGyu Ko,Minsu Kim 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11
The ovum pick up(OPU) technique can be used to produce embryos after in vitro culture of ovarian oocytes, can be used for early securement for effective herd early proliferation and excellent Hanwoo genetic resources, It is attracting attention as a very important technique for establishing technology. In addition to in vitro culture techniques, the number of oocytes retrieved depends on the environment and timing of the OPU. This study was conducted to compare and examine seasonal effect to the differences in the number of recovered oocytes, recovery rate and embryo development rate using Korean cattle kept in animal genetic resource research center by OPU technique. The grade of COCs was evaluated by microscopic examination. Grade A had 3 or more layers of cumulus cell and compact cytoplasm. Grade B had 1~3 layers of cumulus cell and compact cytoplasm. Grade C had 1 layers cumulus cell and compact cytoplasm. Grade D was denuded oocyte and poor cytoplasm. The recovery rate was 47.8±3.4% in summer (June to August) and 51.6±3.8% in autumn (September to October). The number of oocytes was 5.7±0.6 in summer and 5.2±0.7 in autumn. Oocyte grade A and B was 46.2%±6.3% in summer and 51.1±5.0% in autumn. The cleavage rate was 46.1±7.1% in summer and 65.0±11.3% in autumn. Blastocyst development rate was 19.9±9.4% in summer and 29.0±8.7% in autumn. There was no difference the recovery rate of oocytes and the number of embryos between summer and autumn. Cleavage rate and blastocyst rate of autumn was higher than summer. we will investigate to study the appropriate method for the production of Hanwoo embryos and the systematic comparison.
Kim, Min-Su,Min, Kwan-Sik,Seong, Hwan-Hoo,Kim, Chan-Lan,Kim, Dongkyo,Imakawa, Kazuhiko,Kim, Sung Woo The Korean Society of Embryo Transfer 2016 한국동물생명공학회지 Vol.31 No.4
Multiple interferon tau (IFNT) genes exist in bovine. An antiluteolytic substance secreted by the bovine conceptus and primarily responsible for maternal recognition of pregnancy is bovine trophoblast protein 1 (bIFNT1), a new type I interferon tau (IFNT) genes. The objectives of this research were to investigate whether multiple, distinct gene encode bIFNT1 and other type I bIFNT gene in the bovine genome and to examine expression of bIFNT1 and other bIFNTc1 mRNAs during conceptus development. These transcrips could be regulated through caudal-related homeobox-2 (CDX2) and ETS2 and/or AP1 (JUN) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. The presence of mRNAs encoded by bIFNT1 and type I bIFNTc1 genes were examined quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis of total cellular RNA (tcRNA) extracted from on day 17, 20 and 22 bovine conceptuses. The expression level of bIFNT1 was higher on day 17 transcripts were gradually weakly detectable on day 20 and 22. However, the other bIFNTc1 gene examined transcripts was highly expressed on day 20 and transcripts were weakly detectable on day 17 and 22 bovine conceptuses. Furthermore, human choriocarcinoma JEG3 was co-transfected with an -1kb-bIFNT1/c1-Luc constructs and several transcription factor expression plasmids. Compared to each -1kb-bIFNT1/c1-Luc increased when this constructs were co-transfected with, ETS2, AP1(JUN), CREBBP and/or CDX2. Also, bIFNTc1 gene was had very effect on activity by alone ETS2, and AP1 (JUN) expression factors in choriocarcinoma JEG3 cell. However, bIFNT1 gene expression of the upstream region was not identified. We demonstrated that the activities of bIFN genes are regulated by differential, tissue-specific and developmental competence during pregnancy.
Min-Su Kim,Kwan-Sik Min,Hwan-Hoo Seong,Chan-Lan Kim,Dongkyo Kim,Kazuhiko Imakawa,Sung Woo Kim 한국수정란이식학회 2016 한국동물생명공학회지 Vol.31 No.4
Multiple interferon tau (IFNT) genes exist in bovine. An antiluteolytic substance secreted by the bovine conceptus and primarily responsible for maternal recognition of pregnancy is bovine trophoblast protein 1 (bIFNT1), a new type I interferon tau (IFNT) genes. The objectives of this research were to investigate whether multiple, distinct gene encode bIFNT1 and other type I bIFNT gene in the bovine genome and to examine expression of bIFNT1 and other bIFNTc1 mRNAs during conceptus development. These transcrips could be regulated through caudalrelatedhomeobox-2 (CDX2) and ETS2 and/or AP1 (JUN) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. The presence of mRNAs encoded by bIFNT1 and type I bIFNTc1 genes were examined quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis of total cellular RNA (tcRNA) extracted from on day 17, 20 and 22 bovine conceptuses. The expression level of bIFNT1 was higher on day 17 transcripts were gradually weakly detectable on day 20 and 22. However, the other bIFNTc1 gene examined transcripts was highly expressed on day 20 and transcripts were weakly detectable on day 17 and 22 bovine conceptuses. Furthermore, human choriocarcinoma JEG3 was co-transfected with an -1kb-bIFNT1/c1-Luc constructs and several transcription factor expression plasmids. Compared to each -1kb-bIFNT1/c1-Luc increased when this constructs were co-transfected with, ETS2, AP1(JUN), CREBBP and/or CDX2. Also, bIFNTc1 gene was had very effect on activity by alone ETS2, and AP1 (JUN) expression factors in choriocarcinoma JEG3 cell. However, bIFNT1 gene expression of the upstream region was not identified. We demonstrated that the activities of bIFN genes are regulated by differential, tissue-specific and developmental competence during pregnancy.
Sung Woo Kim,Min Su Kim,Chan-Lan Kim,Dongkyo Kim,Hwan-Hoo Seong 한국수정란이식학회 2016 한국수정란이식학회 학술대회 Vol.2016 No.10
During the oocyte maturation, antioxidants may be beneficial for futher developmental competence against reactive oxygen species (ROS) because the media for oocytes lack boiomolecules that serve as scavengers. In this study, N-acetyl-L-cysteine (NAC), N-acetyl-L-cysteine amide (NACA), glutathione (GSH) and cysteamime were compared to determine the effects of protection for ROS from GV to MII stage when supplemented during in vitro maturation (IVM) and in vitro culture (IVC) of bovine oocytes. NAC is one of well known ROS scavanger and NACA is modified form of NAC to help permeation into cytosolic area of oocytes. Significant improvement on the development undergoing blastocysts (32%, vs 18%, 22%) were found when cysteamine (0.1mM) was added to the maturation medium than NAC (0.3 mM), NACA (0.2mM) or GSH (0.5 mM) as compared to control medium with antioxydents. However, the addition of NAC(18%) or NACA(21%) to media did not improve the proportion of oocytes undergoing development to morula and blastocysts than control (24%) and GSH (26%). Our study showed that medium supplementation with cysteame during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like NAC, NACA and GSH that caused no improvement.
Sung Woo Kim,Min Su Kim,Chan-Lan Kim,Dongkyo Kim,Hwan-Hoo Seong 한국수정란이식학회 2016 한국수정란이식학회 학술대회 Vol.2016 No.10
The periods of elevated temperature and high humidity has been longer since last ten years and cause problems in program of artificial insemination or at the efficiency of in vitro production of transferable embryos. The aims of this study were to evaluate the relationship between time of heat shock (0, 1, 2 and 4), during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. The develpmentat rate and percetage of apoptotic cells were evaluated on matured oocyte and day 8. 41℃ Heat treatment after IVM culture significantly decreased the developmental capacity of IVF embryos. Also the number of apoptotic cell in COCs, morula and blatostcysts was started to increase at 2 hr heat treatment but did not affect on the rate of maturation. These results indicate that heat treatment for 2 to 4 hr at 41℃ have negative effects on maturation rate of COCs and lower the developmental competence of heat shocked oocyte derived embryos.