• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.693-700
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• 2007

The present study was conducted to examine the effect of different levels of essential and nonessential amino acid in NCSU-23 medium on the in vitro-produced porcine embryo as it develops from the zygote to the blastocyst stage. Four experiments were performed, each with a completely randomized design involving 5 to 8 replications of treatments. In order to know the effect of nonessential amino acids in NCSU-23 medium, 0, 5, 10 and $20{\mu}/ml$ MEM were supplemented there to, (Exp. 1) and the medium was supplemented with same level of essential amino acids (Exp. 2). The combined effect of nonessential (0, 5, 10 and $20{\mu}/ml$ MEM) and essential amino acids (0, 5, 10 and $10{\mu}/ml$ MEM) in NCSU-23 medium (Exp. 3), first 72 h with non-essential amino acids (at 0, 5, 10 and $20{\mu}/ml$ MEM), and last 4 d with essential amino acids with the same level as NEAA (Exp. 4) were examined. The embryo development was monitored and the quality of blastocysts was evaluated by counting the number of total cells and determining the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells. When Eagle's nonessential amino acids (MEM) added to NCSU-23 medium, it significantly increased the likelihood of development to the 2- to 4-cell stage and subsequent blastocyst development. Supplementation of different levels of essential amino acids in the NCSU-23 medium decreased cleavage rate, rate of morula and blastocyst development and the number of ICMs. In the case of the combined effect of essential and nonessential amino acids, better and significant results were found for blastocysts, hatching blastocysts and for ICM numbers which were also dose dependent. With respect to the biphasic effect of nonessential and essential amino acids, nonessential amino acids increased cleavage whereas essential amino acids increased the total cell number. Neither the nonessential nor the essential group of amino acids, on their own, affected blastocyst cell number or the differentiation of cells in the blastocyst. In conclusion, this study determined the role of nonessential and essential amino acids in the culture of the porcine embryo and showed that the embryo requires different levels of amino acids as it develops from the zygote to the blastocyst stage.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.3
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• pp.334-339
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• 2007

Reactive oxygen species (ROS) generate during electrical activation of oocytes which has detrimental effects on embryo survival when overwhelmed. The present study was designed to investigate the ability of L-ascorbic acid, a novel water soluble antioxidant, to reduce the ROS level in developing embryos and their subsequent effects on embryo development in vitro. The compact cumulus oocyte complexes (COCs) were cultured in tissue culture medium (TCM)-199 supplemented with 10 ng/ml epidermal growth factor, 4 IU/ml pregnant mare serum gonadotropin (PMSG), and human chorionic gonadotropin (hCG) and 10% (v/v) porcine follicular fluid (pFF) for 44 h. After maturation culture, the denuded oocytes were activated with a single DC pulse of 2.0 kV/cm in 0.3 M mannitol solution containing 0.5 mM of HEPES, 0.1 mM of $CaCl_2$ and 0.1 mM of $MgCl_2$ for $30{\mu}s$ using a BTX Electro-cell Manipulator. The activated oocytes were cultured in modified North Carolina State University-23 (mNSCU-23) medium for 168 h. The level of $H_2O_2$ in each embryo was measured by the dichlorohydrofluorescein diacetate (DCHFDA) method at 48 h after activation. The blastocyst formation rate was significantly higher when culture medium was supplemented with 50 and $100{\mu}M$ L-ascorbic acid (31.2 and 38.7%, respectively) compared to non-supplemented (16.1%) group. Accordingly, significantly more cells in blastocyst were found for 50 and $100{\mu}M$ L-ascorbic acid (50.0 and 56.4, respectively) compared to 0 and $200{\mu}M$ L-ascorbic acid (36.5 and 39.8, respectively). L-ascorbic acid reduces the $H_2O_2$ level in developing embryos in a dose-dependant manner. The $H_2O_2$ level (pixels/ embryos) was 191.5, 141.0, 124.0 and 163.3 for 0, 50, 100 and $200{\mu}M$ L-ascorbic acid, respectively. So, we recommend to supplement 50 or $100{\mu}M$ L-ascorbic acid in porcine in vitro culture medium.

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.21-27
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• 2009

This study was performed to elucidate the effects of addition of ${\beta}-lactoglobulin$ and bovine serum albumin (BSA) in vitro maturation (IVM) and in vitro culture (IVC) medium on porcine embryo production. The development rate to the 2 cell ($71.4{\sim}75.6%$) and blastocyst stages ($6.8{\sim}13.3%$) with different BSA concentrations in IVM medium were similar among treatment groups. Blastocyst hatching rate was significantly higher in the control group (0.0mg/ml) than in the group of 1.0mg/ml supplement (20.0% vs. 0.0%; p<0.05). The development rate to the 2 cell ($62.0{\sim}70.6%$) and blastocyst stages ($15.4{\sim}38.5%$) with different ${\beta}-lactoglobulin$ concentrations in IVM medium was similar among treatment groups. The development rate to the blastocyst was significantly higher in the group of 1.0mg/ml(15.3%) than in the group of 0.5mg/ml supplement (7.6%, p<0.05). The development rate to the 2 cell and blastocyst stages following the first addition of ${\beta}-lactoglobulin$ in IVM medium was significantly higher in the control group (77.0% and 18.9%) and was $0{\sim}44\;hr$(77.2% and 16.9%) greater than that observed in other treatment groups (p<0.05). The development rate to the 2 cell stage ($68.1{\sim}74.8%$) and blastocyst stages ($9.2{\sim}12.7%$) with different BSA concentrations in IVC medium was similar among treatment groups. However, blastocyst hatching rate was significantly higher in the group of 3.0mg/ml supplement (30.0%) than in the control group (0.0%; p<0.05). The development rate to the 2 cell stage ($72.9{\sim}78.0%$), blastocyst ($7.1{\sim}14.2%$) and hatching stages ($33.3{\sim}38.1%$) were not different. The development rate to the 2 cell stage ($63.6{\sim}72.5%$), blastocyst ($8.4{\sim}16.1%$) and hatching stages ($18.2{\sim}37.5%$) at the different culture periods were similar among treatment groups. This study suggested that if the addition level and periods of ${\beta}-lactoglobulin$ addition are adjusted, it is possible to replace BSA in the in vitro porcine embryo production.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.95-104
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• 2017

This study was conducted to establish the optimal chemical post-activation conditions in porcine embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) using 4 different chemical compositions (cytochalasin B (CB), cyclohexamide (CHX), demecolcine (DC), 6-dimethylaminopurine (DMAP). Porcine embryos were produced by PA and SCNT and then, cultured for post-activation with CB ($7.5{\mu}g/mL$), CB ($7.5{\mu}g/mL$) + CHX ($10{\mu}g/mL$), CB ($7.5{\mu}g/mL$) +DC ($0.4{\mu}g/mL$), and CB ($7.5{\mu}g/mL$) + DMAP (2 mM). In PA embryonic development, cleavage rates have been significantly higher in CB group (94.7%) and CB+DMAP group (94.1%) than that of CB+CHX and CB+DC group (88.1 and 84.3%, respectively). There have been no significant differences in blastocyst formation rates among the four groups. In cell number of blastocyst was shown in CB group (42.3%) significantly higher than CB+CHX and CB+DC group (40.6 and 40.6%, respectively). In SCNT embryonic development, CB+DMAP group (89.7%) significant differences were found on embryo cleavage rates when compared with other three groups. Blastocyst formation rates in CB+DMAP group (26.9%) were significantly higher when compared with CB, CB+CHX, and CB+DC groups (25.5, 20.2, and 22.1%, respectively). In blastocyst cell number, CB+DMAP group (41.4%) was found higher significant difference compared with other three groups. Additionally, we have investigated survivin expression in early development stages of porcine SCNT embryos for more confirmation. Our results establish that CB group and CB+DMAP group for 4 h during post-activation improves pre-implantation improvement of PA and SCNT embryos.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.120-121
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• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2006.10a
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• pp.78-78
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• 2006

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2006.10a
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• pp.26-27
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• 2006

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.90-90
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• 2004

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.108-108
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• 2004

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2001.05a
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• pp.27-28
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• 2001