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ABSTRACT: The temporal progressive increase of interferon tau (IFNτ) secretion from the bovine trophoblast is a major embryonic signal of establishing pregnancy. Here, we cultured and isolated bovine trophoblast cells (BTs) from IVM/IVF oocytes and in vitro produced blastocysts, used them, for the first time, as donor cells for nuclear transfer and compared them with adult fibroblasts (AFs) as donor cells. BTs were reprogrammed in enucleated oocytes to blastocysts with similar efficiency to AFs (14.5% and 15.6% respectively, P≤0.05). The levels of IFNτ, CDX2 and OCT4 expression in IVF-, BT- and AF-derived blastocysts were analyzed using reverse transcription polymerase chain reaction and reverse transcription quantitative polymerase chain reaction (RT-PCR and RT-qPCR). IVF-produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid, expanded and hatching blastocysts. RT-PCR and RT-qPCR studies showed that IFNτ expression was higher in BT-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and BT-derived blastocysts showed a progressive increase in IFNτ expression as blastocyst development advanced when it compared with AF-derived blastocysts. OCT4 was inversely related with IFNτ expression, while CDX2 was found to be directly related with IFNτ temporal expression. Persistence of high expression of IFNτ and CDX2 was found to be higher in BT-derived embryos than in IVF- or AF-derived embryos. In conclusion, using BTs expressing IFNτ as donor cells for bovine NT could be a useful tool for understanding the IFNτ genetics and epigenetics.
Journal of Reproduction and Development 04/2012; 58(4):425-31. · 1.46 Impact Factor
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ABSTRACT: Summary It is increasingly evident that conditional gene expression in pigs is necessary to make transgenic models. In this study, we investigated conditional expression in porcine fetal fibroblasts using Cre-loxP recombination, a system that has had limited application in large animals to date. Transformed fibroblasts were reprogrammed in enucleated oocytes to support further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with a plasmid that contained a red fluorescent protein marker (pCALNL-DsRed) and a floxed neomycin-resistance gene. Cells were selected with 750 μg/ml neomycin for 2 weeks following transfection but did not express DsRed after visualization under a fluorescence microscope. Expression was achieved only after transient transfection with plasmid DNA that expressed the Cre recombinase enzyme. The cells that expressed DsRed were used for somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%) were seen to have cleaved. Six blastocysts developed after SCNT and expressed DsRed. Deletion of the floxed neomycin-resistance gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in cloned blastocysts. This study demonstrated that Cre-loxP recombination can be conducted successfully in miniature pig fibroblasts and that the sequentially transformed cells can develop to the pre-implantation embryo stage via SCNT.
Zygote 01/2012; 20(4):423-5. · 1.17 Impact Factor
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Transgenic Research 11/2011; 20:1176-1177. · 2.75 Impact Factor
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ABSTRACT: The aim of this study was to investigate the influence of key parameters (donor parity, milk production, post-parturient day, season and milk recording data) associated with efficiency of embryo recovery (ER) in Holstein cattle. Elite Holstein cows and heifers were selected for ER, while Holstein heifers were used as recipients. The numbers of transferable embryos (TEs) produced were not significantly different when analyzed in terms of donor parity, milk production, postparturient day and season. However, the numbers of TEs were significantly increased when the milk protein (%; P)/fat (%; F) ratio was over 0.95 and/or the milk urea nitrogen (MUN) was between 12 and 18 dl/ml. The results from ET showed no differences in pregnancy rates among Holstein heifers receiving other types, developmental stage codes and quality grades of embryos. The mean interval from ER to artificial insemination was 60.6 days. Moreover, 19 offspring that had milk recording data showed a similar milk yield performance to that of the donor cows. In conclusion, this study showed that in Holstein cows, embryos were recovered and transferred and resulted in production of viable calves. Furthermore, P/F ratio and MUN could be candidate indicators for selection of high-efficiency donor cows.
Journal of Veterinary Medical Science 09/2011; 74(2):167-74. · 0.85 Impact Factor
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ABSTRACT: Because cloned dogs are genetically identical, variations among these animals can be a useful tool to elucidate mechanisms underlying phenotypic differences. To estimate the influence of genetic factors on phenotypic variation, changes in concentration patterns of growth hormone (GH), insulin like growth factor-1 (IGF-1), and IGF binding protein 3 (IGFBP-3) were compared among cloned and age-matched control dogs. In addition, the concentrations of GH and IGF-1 following administration of growth hormone releasing hormone (GHRH) and somatostatin (SRIF) were measured in both groups. In comparing hormone profiles, the control dogs had larger standard deviations from the means for GH, IGF-1, and IGFBP-3 than the clones. Also, the mean concentration of IGFBP-3 in clones was significantly lower than in the controls between 7 to 12 months of age, whereas the IGFBP-3 changes in clones and controls followed the same pattern. GHRH induced increased serum growth hormone concentration both in clones and controls. However, the concentration of IGF-1 was lower in clones than in controls, and larger standard variations were noted in the control group. In conclusion, the measured traits were more homogeneous in cloned animals than in controls, so cloned animals could be valuable for assessing effects of genotype and environment interactions.
Cellular reprogramming. 07/2011; 13(5):451-7.
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Hyunil Kim,
Hye Kwon Kim,
Jung Ho Jung,
Yoo Jung Choi,
Jiho Kim,
Chang Gyu Um,
Su Bin Hyun,
Sungho Shin, Byeongchun Lee,
Goo Jang,
Bo Kyu Kang,
Hyoung Joon Moon,
Dae Sub Song
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ABSTRACT: There have been many efforts to develop efficient vaccines for the control of porcine reproductive and respiratory syndrome virus (PRRSV). Although inactivated PRRSV vaccines are preferred for their safety, they are weak at inducing humoral immune responses and controlling field PRRSV infection, especially when heterologous viruses are involved.
In all groups, the sample to positive (S/P) ratio of IDEXX ELISA and the virus neutralization (VN) titer remained negative until challenge. While viremia did not reduce in the vaccinated groups, the IDEXX-ELISA-specific immunoglobulin G increased more rapidly and to significantly greater levels 7 days after the challenge in all the vaccinated groups compared to the non-vaccinated groups (p < 0.05). VN titer was significantly different in the 106 PFU/mL PRRSV vaccine-inoculated and binary ethylenimine (BEI)-inactivated groups 22 days after challenge (p < 0.05). Consequently, the inactivated vaccines tested in this study provided weak memory responses with sequential challenge without any obvious active immune responses in the vaccinated pigs.
The inactivated vaccine failed to show the humoral immunity, but it showed different immune response after the challenge compared to mock group. Although the 106 PFU/mL-vaccinated and BEI-inactivated groups showed significantly greater VN titers 22 days after challenge, all the groups were already negative for viremia.
Virology Journal 06/2011; 8:323. · 2.34 Impact Factor
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ABSTRACT: As shown by the birth of the first cloned dog 'Snuppy', a protocol to produce viable cloned dogs has been reported. In order to evaluate optimum fusion conditions for improving dog cloning efficiency, in vivo matured oocytes were reconstructed with adult somatic cells from a female Pekingese using different fusion conditions. Fusion with needle vs chamber methods, and with low vs high pulse strength was compared by evaluating fusion rate and in vivo development of canine cloned embryos. The fusion rates in the high voltage groups were significantly higher than in the low voltage groups regardless of fusion method (83.5 vs 66.1% for the needle fusion method, 67.4 vs 37.9% for the fusion chamber method). After embryo transfer, one each pregnancy was detected after using the needle fusion method with high and low voltage and in the chamber fusion method with high voltage, whereas no pregnancy was detected using the chamber method with low voltage. However, only the pregnancy from the needle fusion method with high voltage was maintained to term and one healthy puppy was delivered. The results of the present study demonstrated that two DC pulses of 3.8 to 4.0 kV/cm for 15 μsec using the needle fusion method were the most effective method for the production of cloned dogs under the conditions of this experiment.
Theriogenology 03/2011; 75(4):777-82. · 1.96 Impact Factor
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ABSTRACT: We have previously shown that the in vitro embryonic development and the yield of viable calves were increased by using a two-step chemically defined medium for post-fertilization culture of bovine embryos. In this study, we explored the embryonic development and the temporal behavioral interaction of the genes involved in IFNτ gene expression and how they behave in an orchestrated manner to increase the developmental competence of IVF produced embryos by culturing in the chemically defined medium. Behavior of genes included ETS2, CDX2, GATA2, GATA3, OCT4 and NANOG was analyzed in early bovine IVF produced embryos, (from compact morulae to the blastocyst hatching stages), by semi- and relative quantitative PCR and compared between two in vitro culture (IVC) systems, two-step chemically defined medium and modified synthetic oviductal fluid (mSOF) containing 8 mg/mL, BSA. Early embryonic development was found to be better in two-step chemically defined culture system than that of mSOF as indicated by the increment of blastocyst yield, 33.1% in two-step culture system vs 18.8% in mSOF medium, and the blastocyst hatching, 52.3% in two-step culture system vs 33.5% in mSOF medium. Relative quantitative gene expression showed harmonic behavior in the two-step culture system rather than the culture in mSOF, IFNτ showed even increase throughout the embryonic development in the two-step culture medium while it decreased with blastocyst hatching in mSOF culture condition. Temporal dominance of OCT4 over all the transcription factors was found in regulation of IFNτ expression (the major factor of expression regulation but in inverse manner). However, ETS2, CDX2, GATA2 and GATA3 are potent IFNτ stimulator in cumulative manner but in case of OCT4 decrement. CDX2 directly related with IFNτ, but still under OCT4 dominance and also regulated by the subservient of OCT4 which is NANOG. In conclusion, this study confirmed our previous results about the usefulness of using the two-step chemically defined culture medium for increasing the developmental competence of IVF produced embryos and elucidated the dominance of OCT4 over the other genes implicated in regulation of IFNτ expression.
Theriogenology 01/2011; 75(6):995-1004. · 1.96 Impact Factor
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ABSTRACT: A recent emerging technology, somatic cell nuclear transfer (SCNT), has been considered for conserving threatened or endangered species. Sapsaree is a native breed in Korea and has been designated as a Natural Monument. The aim of this study was to produce a Sapsaree by SCNT for breed conservation. Donor fibroblasts from a 9-year-old male Sapsaree were placed into the perivitelline spaces of enucleated in vivo matured oocytes and fused electrically. A total of 309 cloned embryos were transferred into the oviducts of 15 naturally synchronized recipients. Two recipients were diagnosed as pregnant, and each delivered one cloned puppy, both of which weighed 530 g. Overall, this study demonstrated that an endangered canine breed can be conserved by SCNT.
Journal of Veterinary Medical Science 09/2009; 71(9):1217-20. · 0.85 Impact Factor
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ABSTRACT: The present study was conducted to establish an efficient production system for bovine transgenic somatic cell nuclear transfer (SCNT) embryos, the effect of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos were examined with their expression rates of a marker gene. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected with the expression plasmid using a liposomal transfection reagent, Fugene6, as a carrier. In Experiment 1, three types of bovine cells were transfected at passages 2 to 4, and then trypsinized and GFP-expressing cells were randomly selected and used for SCNT. Developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In all cell types used, GFP expression rates of SCNT embryos gradually decreased with the progression of embryo development. In Experiment 2, the effect of passage number of cumulus cells in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed, but significantly higher GFP expression was shown in blastocysts reconstructed with cumulus cells at early passage. In Experiment 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 microm) or small cell (<30 microm)] at passages 2 to 4 were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells as well as fetal cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.
Journal of Veterinary Medical Science 12/2004; 66(12):1567-73. · 0.85 Impact Factor
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ABSTRACT: The objective of this study was to determine if the transfection of human prourokinase (ProU) gene and passage number of transfected ear fibroblasts affected in vitro development of bovine transgenic nuclear transfer (NT) embryos. An expression plasmid for human ProU was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker and human ProU gene into a pcDNA3 plasmid and transfected into bovine ear fibroblasts using a lipid mediated method. Abattoir derived oocytes were enucleated at 18-20 hr post maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the couplets were cultured in modified synthetic oviductal fluid (mSOF) medium for 168 hr. In Experiment 1, significantly lower rate in blastocysts formation (10.3%) was observed in transfected donor cells at early passage than that in nontransfected counterparts (22.1%, P<0.05). In Experiment 2, development to blastocysts and GFP expression in blastocysts were not significantly different between early (3-7) and late (8-12) passage donor cells (10.3 vs. 11.3% and 54.5 vs. 41.7%, respectively). This study indicates that in vitro development of bovine transgenic NT embryos is negatively influenced by transfection of human ProU gene into donor fibroblasts. However, passage number of transfected ear fibroblasts does not affect in vitro development of bovine transgenic NT embryos.
Journal of Veterinary Medical Science 03/2004; 66(3):257-61. · 0.85 Impact Factor
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Sanghwan Hyun,
Gabsang Lee,
Daeyoung Kim,
Hyesoo Kim,
Sohyun Lee,
Donghyun Nam,
Yeonwoo Jeong,
Sue Kim,
Soocheong Yeom,
Sungkeun Kang,
Jaeyong Han, Byeongchun Lee,
Woosuk Hwang
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ABSTRACT: A system for somatic cell nuclear transfer (SCNT) was developed and led to the successful production of GFP-transfected piglets. In experiment 1, two groups of SCNT couplets reconstructed with porcine fetal fibroblasts (PFF) and enucleated sow (S) or gilt oocytes (G): 1). received a simultaneous electrical fusion/activation (S-EFA or G-EFA groups), or 2). were electrically fused followed by activation with ionomycin (S-EFIA or G-EFIA groups), or 3). were subjected to electrical fusion and subsequent activation by ionomycin, followed by 6-dimethylaminopurine treatment (S-EFIAD or G-EFIAD groups). The frequency of blastocyst formation was significantly higher in S-EFA (26%) compared with that observed in the other experimental groups (P < 0.05), but not with S-EFIA (23%). Sow oocytes yielded significantly higher cleavage frequencies (68%-69%) and total cell numbers of blastocysts when compared with gilt oocytes, regardless of fusion/activation methods (P < 0.05). However, the ratio of inner cell mass (ICM)/total cells in G-EFA and S-EFA was significantly lower than in the other groups (P < 0.05). In experiment 2, SCNT couplets reconstructed with PFF cultured in the presence or absence of serum and enucleated sow oocytes were subjected to EFA. There were no effects of serum starvation on cell-cycle synchronization, developmental competence, total cell numbers, and ratio of ICM/total cells. In experiment 3, SCNT couplets reconstructed with PFF transfected with an enhanced green fluorescence protein (EGFP) gene using FuGENE-6 and enucleated sow oocytes were subjected to EFA and cultured for 7 days. Expression frequencies of GFP gene during development were 100%, 78%, 72%, 71%, and 70% in fused, two-cell, four to eight cells, morulae, and blastocysts, respectively. In experiment 4, SCNT embryos derived from different recipient cytoplasts (sows or gilts) and donor karyoplasts (PFF or GFP-transfected) were subjected to EFA and transferred to the oviducts of surrogates. The pregnancy rates in SCNT embryos derived from sow oocytes (66%-69%) were higher than those with gilt oocytes (23%-27%) regardless of donor cell types. One live offspring from GFP-SCNT embryos and two from PFF-SCNT embryos were delivered. Microsatellite analysis confirmed that the clones were genetically identical to the donor cells and polymerase chain reaction (PCR) from genomic DNA of cloned piglets and subsequent southern blot analysis confirmed the integration of EGFP gene into chromosomes.
Biology of Reproduction 09/2003; 69(3):1060-8. · 4.01 Impact Factor
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ABSTRACT: To evaluate the embryotrophic role of three hexoses (glucose, fructose, and galactose), bovine embryos derived from somatic cell nuclear transfer (SCNT) or in vitro-fertilization (IVF) were cultured in a modified synthetic oviductal fluid (mSOF), which contained either glucose (1.5 or 5.6 mM), fructose (1.5 or 5.6 mM), or galactose (1.5 or 5.6 mM). Compared to 1.5 mM glucose, use of 1.5 mM fructose significantly enhanced blastocyst formation in both SCNT (23 vs. 33%) and IVF embryos (26 vs. 34%), while 5.6 mM fructose did not improve blastocyst formation. Using 1.5 mM galactose did not improve blastocyst formation in SCNT embryos (22 vs. 23%), whereas it significantly inhibited blastocyst formation in IVF embryos (26 vs. 0%). In both SCNT and IVF embryos, 5.6 mM glucose or galactose significantly inhibited embryo development. In a second experiment, in glucose-free mSOF, fructose at concentrations of 0.75, 1.5, 3.0, or 5.6 mM was able to support to morula (32-42 vs. 12%) and blastocyst formation (30-38 vs. 12%) compared to 0 mM fructose. In Experiment 3, addition of fructose (1.5, 3.0, or 5.6 mM) to mSOF containing 1.5 mM glucose did not further promote blastocyst formation in SCNT embryos compared with replacement with 1.5 mM fructose only. Replacement of glucose with 1.5 mM fructose significantly increased total blastomeres (143 vs. 123 cells) and trophectodermal (TE) cells (116 vs. 94 cells) and decreased inner cell mass (ICM) to TE cell ratio (0.24 vs. 0.31) in blastocysts, compared to 1.5 mM glucose. The combined addition of 1.5 mM fructose and glucose significantly increased ICM cell number (36.7 cells) and ICM/TE ratio (0.46). In conclusion, fructose might be a more efficient energy substrate than glucose for producing large number of transferable blastocysts derived from SCNT.
Molecular Reproduction and Development 07/2003; 65(2):167-74. · 2.53 Impact Factor
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ABSTRACT: Intergeneric embryos were constructed by nuclear transfer using Mountain Bongo antelope somatic cells fused with enucleated bovine oocytes and their subsequent development in vitro was investigated. After two to six passages, starved or non-starved skin fibroblast cells were used as donor nuclei. In vitro matured bovine oocytes were enucleated by squeezing the first polar body and surrounding cytoplasm through a slit in the zona pellucida. After injection of a somatic cell into the perivitelline space, couplets were fused electrically and activated chemically, then subjected to different embryo culture treatments. Serum starvation had no effect on the frequency of cleavage to two cells or on development to the blastocyst stage in either sequential hamster embryo culture medium (HECM)-6/TCM-199 + serum or HECM-9/TC-199 + serum, or modified synthetic oviduct fluid (mSOF) culture medium. When couplets from non-starved donor nuclei were cultured, the frequency of cleavage (66 +/- 8% vs. 44 +/- 5%), development to >/=9 cells (46 +/- 6% vs. 24 +/- 4%), and formation of blastocysts (24 +/- 5% vs. 11 +/- 2%) were all significantly higher (p < 0.05) in the HECM-6 medium than in mSOF medium. In conclusion, bovine oocytes can support blastocyst development after intergeneric fusion with bongo fibroblasts. This technique could potentially be used as an alternative to using scarce bongo oocytes in attempts to propagate these endangered animals.
Cloning and Stem Cells 01/2003; 5(1):25-33. · 2.66 Impact Factor
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ABSTRACT: To evaluate whether oocytes excluded from somatic cell nuclear transfer (SCNT) could be utilized for embryo production by parthenogenetic activation (PA), porcine oocytes with poor morphology after maturation culture were excluded from SCNT and subsequently used for PA with different stimuli. In the first set of experiment, either electric pulse of different strengths (1.75, 2.0 or 2.25 kV/cm for 30 microsec each) or chemicals with different treatment durations [7% ethanol for 5 min followed by exposure to 6-dimethylaminopurine (6-DMAP) for 0, 2, 3 or 4 hr] was employed. Development to the 8-cell and morula stages was significantly (P<0.05) improved by electric stimulation of 2.0 kV/cm, while blastocyst formation was enhanced by chemical treatment of ethanol and 6-DMAP for 4 hr. Subsequently, oocytes were parthenogenetically activated by one of four stimuli; 1) optimal electric (2.0 kV/cm for 30 microsec), 2) optimal chemical (ethanol followed by 6-DMAP for 4 hr), 3) electric then chemical and 4) vice versa. On the other hand, oocytes with normal morphology were subjected to the same experimental treatments for the control. Regardless of oocyte type, a combination of electric and chemical stimulations did not further stimulate preimplantation development, compared with electric activation only. However, combinational treatment greatly increased the cell number of blastocysts in SCNT-excluded oocytes (21.9 to 22.9 vs. 16.9 cells/blastocyst), while such effect was not found in normal oocytes (22.2 to 23.3 cells/blastocyst). In conclusion, porcine oocytes excluded from SCNT still have a potential to develop blastocysts after PA and this might contribute to increasing the efficiency of SCNT for various purposes. A combined activation by electricity and chemical yielded the best rate of preimplantation development with increasing the quality of blastocyst.
Journal of Veterinary Medical Science 01/2003; 65(1):51-6. · 0.85 Impact Factor
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ABSTRACT: The objective of this study was to evaluate whether changes in NaCl concentration in a fertilization medium could improve normal fertilization and preimplantation development of bovine oocytes. In vitro matured bovine oocytes were inseminated with frozen-thawed semen for 18 hr in a Tyrode's medium with albumin, lactate and pyruvate (TALP), to which 114 (TALP-114), 96 (TALP-96) or 78 (TALP-78) mM NaCl was added. Presumptive zygotes were cultured for 192 hr in a modified TALP containing 90 mM NaCl, 1.5 mM glucose, 0.3% (w/v) BSA, minimal essential medium (MEM) essential and nonessential amino acids, and insulin-transferrin-selenium complex. Lower polyspermy rate was obtained by the insemination in TALP-96 (7.8 +/- 2.3%) than by the insemination in TALP-114 (25.6 +/- 1.4%), without decrease in male pronucleus (MPN) formation. Fertilization in TALP-78 also yielded decreased polyspermic fertilization (3.8 +/- 1.5%), but significant decrease in MPN formation was found (63.1 +/- 3.1%). In preimplantation development, more blastocysts developed from oocytes inseminated in TALP-96 (24.1 +/- 1.7%) than from oocytes inseminated in TALP-114 (16.8 +/- 1.4%). TALP-78, however, did not improve preimplantation development beyond the 8-cell stage compared with TALP-114. Mean cell number of blastocyst was higher when oocytes were fertilized in TALP-96 (137.0 +/- 4.5) than in TALP-114 (123.1 +/- 5.1) and in TALP-78 (102.3 +/- 4.5). These results demonstrate that insemination of bovine oocytes in a TALP with decreased NaCl concentration (96 mM) improves blastocyst formation and embryo viability. Decrease in NaCl concentration below 96 mM, however, may delay or inhibit MPN formation, and inhibits subsequent development in vitro.
Journal of Veterinary Medical Science 09/2002; 64(8):667-71. · 0.85 Impact Factor
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Jongki Cho,
Seungeun Park,
Hyungmin Chung,
Hosup Shim, Byeongchun Lee,
Kihyeong Rhee,
Sungkeun Kang,
Jaeyong Han,
Changkyu Lee,
Eunsong Lee,
Woosuk Hwang,
Jeongmook Lim
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ABSTRACT: This study was conducted to evaluate how exogenous amino acids could affect preimplantation development of ICR mouse embryos. Two-cell embryos collected from naturally mated mice were cultured in amino acid-, glucose- and phosphate-free preimplantation (P)-1 medium. In Experiments 1, 19 amino acids (aa; 1% and 0.5% of MEM essential and nonessential amino acid solutions, respectively) were added to P-1 medium supplemented with either fatty acid-free bovine serum albumin (BSA; 3 mg/mL) or human follicular fluid (hFF; 10%). Regardless of BSA or hFF addition, embryo development to the morula (84 to 86% vs. 97 to 100%) and the blastocyst (54% vs. 93 to 94%) stages was significantly (P<0.05) enhanced by the addition of aa compared with no addition. In Experiment 2, the cell number of blastomeres and inner cell mass (ICM) cells in blastocysts and the ratio of ICM cell to trophectodermal cell (TE) were evaluated after aa addition. In both BSA- and hFF-containing P-1 medium, a significant increase in total blastomere number were found after aa addition (47 to 52 vs. 62 to 63 cells) compared with no addition. However, the ICM/TE ratio was not significantly affected by aa supplementation in both media, while ICM cell number was greatly increased after aa addition in hFF-containing medium (12 vs. 17 cells). When blastocysts were further cultured up to 162 hr post-hCG injection, development to the hatched blastocyst stage was significantly promoted by aa addition (0% vs. 11 to 20%) in both BSA- and hFF-containing media. In conclusion, aa significantly promote the preimplantation development to the hatched blastocyst stage and such effect mainly exerted on supporting blastomere proliferation.
Journal of Veterinary Medical Science 09/2002; 64(9):797-801. · 0.85 Impact Factor
