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NARUSE, Kenji,QUAN, Yan-Shi,CHOI, Su-Min,PARK, Chang-Sik,JIN, Dong-Il 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10
This study was carried out to investigate the effects of minimum essential medium (MEM) vitamins during in vitro maturation (IVM)/in vitro culture (IVC) of porcine nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. Porcine cumulus-oocyte complexes (COCs) were divided into five groups, matured for 44 h in maturation medium with various concentrations of MEM vitamins (0, 0.05, 0.1, 0.2 and 0.4%), and observed for maturation rate. Also, COCs were matured in NU5U-23 media without MEM vitamins for 44 h and cultured in PZM-3 media with various concentrations of MEM vitamins (0, 0.05, 0.4 and 1.0%) for 6 days following nuclear transfer. Factorial (IVM/IVC) experiments were also performed in NCSU-23 medium with or without 0.05% MEM vitamins and PZM-3 medium with or without 0.4% MEM vitamins. They were then tested by examining in vitro development of the porcine reconstructed embryos. The maturation rates of the COCs treated with the MEM vitamins did not differ significantly among the MEM vitamin-treated groups. Addition of vitamins to culture medium did not affect development of porcine reconstructed embryos in vitro. However, addition of low concentrations of MEM vitamins only to maturation medium increased (P<0.05) the proportion of NT embryos developing into blastocysts compared with the control group. Addition of MEM vitamins to IVC medium did not enhance the developmental rate compared with the control group. Thus, addition of MEM vitamins to IVM medium could improve subsequent blastocyst development of porcine NT embryos.
Naruse, Kenji,Kim, Hong Rye,Shin, Young Min,Chang, Suk Min,Lee, Hye Ran,Tarte, Vaishali,Quan, Yan Shi,Kim, Beak Chul,Park, Tae Young,Choi, Su Min,Park, Chang Sik,Jin, Dong Il 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10
Electrical treatment has been widely used for porcine oocytes activation However, developmental rates following electrical activation of porcine oocytes is relatively inefficient compared to other domestic animals. To investigate the effects of porcine oocytes on combined activation by both chemical and electrical treatment, in-vitro matured oocytes were activated by combined cycloheximide and electrical pulses treatment. Cumulus-free oocytes were exposed with NCSU-23 medium containing cycloheximide (10 μg/ml) for 0, 5, 10, 20, 30 min and then activated by electrical pulse treatment and cultured in PZM-3 for 8 days. Also effects of exposure to 6.25 μM calcium ionophore for 2 min for cumulus-free oocytes were tested. The percentage of blastocyst formation in 10 min exposure to 10 μg/ml cycloheximide and electrical pulse treatment was significantly increased (P<O.05) than in the control group. And exposure to 6.25 μM calcium ionophore for 2 min with 10 μg/ml cycloheximide for 10min and electrical pulse treatment significantly increased (P<O.05) the percentage of blastocyst developmental rates than the control group. In conclusion, activation by combined cycloheximide and electrical stimulation treatment promoted the subsequent development of porcine oocytes and improved the subsequence blastocyst development.
Naruse, Kenji,Quan, Yan Shi,Park, Tae Young,Kim, Beak Chul,Park, Chang Sik,Jin, Dong Il 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10
Abnormal fusion is thought to be related to the incorrect reprogramming of nucleus following transfer into the oocytes. For this reason, the purpose of current experiments was to investigate the development of porcine nuclear transfer embryos reconstructed by using treated donor cells with streptolysin O (SLO). SLO-treated group (200 ng/ml, 50 min) significantly increased (P<0.05) the percentage of fusion rates and blastocyst developmental rates compared with control. Exposure to SLO-treated donor cells with the matured cytoplasm for 40 min significantly increased (P<0.05) the rates of fusion and cleavage and blastocysts formation of nuclear transfer (NT) embryos. In conclusion, treatment to fetal fibroblast cells with Streptolysin O (SLO) and exposed to the matured cytoplasm could lead to improvements of the nuclear transfer methods.
Naruse Kenji,Kim Hong-Rye,Shin Young-Min,Chang Suk-Min,Lee Hye-Ran,Tarte Vaishali,Quan Yan-Shi,Kim Beak-Chul,Park Tae-Young,Choi Su-Min,Park Chang-Sik,Jin Dong-Il 한국동물생명공학회(구 한국동물번식학회) 2006 Reproductive & developmental biology Vol.30 No.1
Electrical treatment has been widely used for porcine oocytes activation. However, developmental rates following electrical activation of porcine oocytes is relatively inefficient compared to other domestic animals. To investigate the effects of porcine oocytes on combined activation by both chemical and electrical treatment, in-vitro matured oocytes were activated by combined cycloheximide and electrical pulses treatment. Cumulus-free oocytes were exposed with NCSU-23 medium containing cycloheximide (10μgml) for 0, 5, 10, 20, 30 min and then activated by electrical pulse treatment and cultured in PZM-3 for 8 days. Also effects of exposure to 6.25μM calcium ionophore for 2 min for cumulus-free oocytes were tested. The percentage of blastocyst formation in 10 min exposure to 10μgml cycloheximide and electrical pulse treatment was significantly increased (P<0.05) than in the control group. And exposure to 6.25μM calcium ionophore for 2 min with 10μgml cycloheximide for 10min and electrical pulse treatment significantly increased (P<0.05) the percentage of blastocyst developmental rates than the control group. In conclusion, activation by combined cycloheximide and electrical stimulation treatment promoted the subsequent development of porcine oocytes and improved the subsequence blastocyst development
Naruse, K.,Quan, Y.S.,Kim, B.C.,Lee, J.H.,Park, C.S.,Jin, D.I. 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10
To investigate the effects of cycloheximide exposure before electrical activation of in vitro-matured porcine oocytes on the subsequent development of parthenogenetic embryos, cumulus-free mature oocytes were exposed to NCSU-23 medium containing cycloheximide (10μg/mL) for 0, 5, 10, 20, 30 and 60 min, activated by electrical pulse treatment (1.5 kV/cm, 100μs) and then cultured in PZM-3 for 7 days. To evaluate the effects of cycloheximide on the activation of nuclear transfer embryos, reconstructed embryos were electrically activated by two DC pulses (1.2 kV/cm, 30μs) before or after exposure to cycloheximide. The reconstructed embryos were allocated into four groups: electrical pulse treatment alone (Ele); exposure to cycloheximide for 10 min followed by electrical activation (CHX + Ele); electrical activation followed by exposure to cycloheximide for 6 h (Ele + CHX); exposure to cycloheximide for 10 min, followed by electrical activation and a further exposure to cycloheximide for 6 h (CHX + Ele + CHX). The activated reconstructed embryos were cultured in PZM-3 for 6 days. Oocytes treated with 10 min exposure to cycloheximide followed by electrical activation had a significantly higher percentage of blastocyst formation compared to control oocytes and oocytes exposed for≥30 min. In the reconstructed embryos, the blastocyst development rates of embryos exposed to cycloheximide (CHX + Ele, Ele + CHX and CHX + Ele + CHX) were significantly higher than those of the control group (Ele). Among the cycloheximide-treated groups, the CHX + Ele group had increased development rate and total blastocyst cell number, though these values were not significantly different from those observed in the other cycloheximide-treated groups. To evaluate the quality of NT embryos treated with cycloheximide, apoptosis in blastocysts was analyzed by TUNEL assay. The 10 min exposure to cycloheximide prior to electrical activation significantly reduced cell death compared with longer exposure to cycloheximide after electrical fusion. In conclusion, brief exposure to cycloheximide prior to electrical activation may increase the subsequent blastocyst development rates in porcine parthenogenetic and reconstructed embryos.