Stage-specific effects of the osmolarity of a culture medium on the development of parthenogenetic diploids in the pig

Nguyen, Van Thuan; Kure-bayashi, S.; Harayama, Hiroshi; Nagai, Takashi; Miyake, Masakazu

doi:10.1016/s0093-691x(02)01085-3

Cited by 35 publications

(31 citation statements)

References 24 publications

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“…Although failure of porcine parthenogenetic haploids to develop to the blastocyst stage has also been reported, the development of diploids in these reports was also very low [7,11]. Because the developmental ability of diploids has become remarkably high due to improvements in culture system for porcine embryos [6,13,20], we here investigated and elucidated the cause of the failure of the development in haploids.…”

Section: Discussionmentioning

confidence: 92%

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The Development of Porcine Parthenogenetic Diploid Oocytes with Homogeneous Genomic Components In Vitro.

Thuan

Harayama

Miyake

2002

Journal of Reproduction and Development

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Abstract. The aim of this study was to determine the influence of the homogeneity of genomic components and ploidy on the in vitro development of porcine parthenogenetic oocytes to the blastocyst stage. In vitro matured oocytes were subjected to a single pulse of electro-stimulation (El-St; 100 µsec, 1,500 V/cm) for activation. First, the activated oocytes were cultured for 6, 16, 18, 20 and 22 h after El-St, and examined for the timing of the first cleavage of parthenogenetic haploids. Next, the effects of the timing and duration of cytochalasin B (CB) treatment on inhibition of the first cleavage of haploids were examined in order to produce homogenous parthenogenetic diploids. Then the developmental ability to the blastocyst stage was compared among activated oocytes with genomic components of haploid (without CB treatment), homogeneous diploid (nn-diploid, CB treatment for 6 h from 20 h after El-St), and heterogeneous diploid (nn'-diploid, CB treatment for 4 h immediately after El-St). Most haploids were at the prophase to telophase of the first cleavage between 16 and 22 h after El-St. When the haploids were treated with 5.0 µg/ml CB for 6 h from 20 h after El-St, their first cleavage was efficiently suppressed, and most of them (84%) became diploids. The frequency of parthenogenetic development to the blastocyst stage was significantly lower in haploids (5%) than in nn-diploids (48%, P<0.01) or nn'-diploids (57%, P<0.01). These results shows that ploidy of activated oocytes, but not the homology of genomic components, affects the development of porcine parthenogenetic oocytes to the blastocyst stage. Key words: Porcine parthenogenesis, Ploidy, In vitro development (J. Reprod. Dev. 48: [157][158][159][160][161][162][163][164][165][166] 2002) he genotype of the parthenogenetic embryo var ies d e pe nd ing on the ex per ime ntal conditions and on the postovulatory age of the oocyte. Matured oocytes have two genomic sets of chromosomes, one which is delivered to the egg and the other which is shared by the second polar body. Each chromosome consists of a mixture of paternal and maternal genomic components by the process of crossing-over during the meiotic prophase I. Thus the two genomic sets are not exactly identical. Therefore, the possible genotypes arising from parthenogenetic activation are uniform haploids with the extrusion of the second polar body (n), mosaic haploids through an immediately equal cell-division but not extrusion of the second polar body (n+n'), and heterogeneous diploids without the extrusion of the second polar body (nn') according to the theoretical classification by Kaufman [1]. It has been reported, however, that porcine oocytes matured less than 48 h followed by artificial activation actually yielded activated eggs of uniform haploid type (about 90%) or of heterogeneous diploid type with two haploid (n + n') nuclei or a single diploid (nn') nucleus (a few percentage each), but no mosaic-haploid activated oocytes [2,3].

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Section: Discussionmentioning

confidence: 92%

“…The osmolarity of the culture medium was kept at 309 mOsmol (mWM309) for the first 48 or 72 h by addition of 26.2 mM NaCl to mWM and then 256 mOsmol (mWM256) until 192 h after El-St [20].…”

Section: Culture Media For Developmentmentioning

confidence: 99%

The Development of Porcine Parthenogenetic Diploid Oocytes with Homogeneous Genomic Components In Vitro.

Thuan

Harayama

Miyake

2002

Journal of Reproduction and Development

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“…Based on the percentage blastocyst, both parthenogenetic (Nguyen et al 2003) and IVF embryos can be cultured in media with higher osmolalities (parthenogenetic embryo: 280-320mOsM; IVF embryo: 330mOsM) for the first 2-3 days after electric activation and IVF, and then in the media with lower osmolalities (parthenogenetic embryo: 220-270mOsM; IVF embryo: 250 mOsM). These results are consistent with the higher osmolality of D3OF and the lower osmolality of D5UF.…”

Section: Discussionmentioning

confidence: 99%

Concentration and composition of free amino acids and osmolalities of porcine oviductal and uterine fluid and their effects on development of porcine IVF embryos

Whitworth

Lai

et al. 2007

Molecular Reproduction Devel

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The concentration of free amino acids and the osmolalities in porcine oviductal (OF) and uterine fluids (UF) on Day 3 (D3) and Day 5 (D5) were measured by HPLC and Vapor Pressure Osmometer, respectively. Based on these measurements we designed new media based on PZM3 by modifying the amino acid composition and osmolality. The effectiveness of the modified PZM3 on the development of porcine IVF embryos was then investigated. A total of 24 free amino acids were measured, including 20 protein and 4 non-protein amino acids (β Alanine, Taurine, Ornithine and Citrulline). There was no significant difference in the total concentration of amino acids among D3OF (13.06±3.63 mmol/L), D3UF (10.54±5.16 mmol/L) or D5UF (10.23±6.69 mmol/L). But the total concentration of amino acids in D5OF (5.89±1.47 mmol/L) was significantly lower than the three fluids above. Some individual amino acids varied significantly depending on where they were collected from and the day. The blastocyst rates of porcine IVF embryos were not improved when embryos were cultured in PZM3 with amino acids at D3OF (PZM3-D3OF, 20.3±7.9%) or D5UF (PZM3-D5UF, 14.3±10.7%) concentrations or in PZM3-D3OF for the first 48 (20.5±15.1), 72 (25.6 ±10.4) and 96 (18.7±10.0) hours and then transferred into PZM3-D5UF compared with PZM3 with Sigma amino acid solution (PZM3-SAA) (30.8±9.1%). However, when IVF embryos were cultured in PZM3-D5UF, the average nuclear number per blastocyst (57.6±8.3) was increased compared to PZM3-SAA (40.5±3.5). The osmolalities in D3OF, D3UF, D5OF and D5UF were 318±8, 320±32, 321 and 293±8 mOsM, respectively. When the IVF embryos were cultured in PZM3-SAA and PZM3-D3OF at a variety of osmolalities (150-360mOsM), higher blastocyst rates were obtained at 270-300 mOsM in the PZM3-SAA group (24.6%-33.9%) and 270-290 mOsM in PZM3-D3OF group (22.4%-24.2%). The blastocyst rate gradually decreased when the osmolality was increased or decreased in both groups. When the embryos were cultured in PZM3-SAA at 330 mOsM for the first 72 hours and then transferred to 250 mOsM (33.3±3.4%), the blastocyst rate was higher than original PZM3 (21.2±2.2%)(288 mOsM).

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“…Embryos with normal 1-cell (Day 1) or 2-to 8-cell stage (Day 2) morphology were selected for transfer and were surgically transferred into the oviducts of the recipient approximately 48 or 72 h after hCG injection. In vitro culture of embryos was carried out using NCSU23 (290 mOsm [19]) modified by the addition of 0.17 mM sodium pyruvate and 2.73 mM sodium lactate instead of D-glucose [20] and supplemented with 4 mg/ml BSA and 0.5 mg/ml hyaluronic acid [21]. A group of 20-25 embryos were cultured in 30-µl droplets of medium under paraffin oil in a plastic Petri dish maintained in a humidified atmosphere of 5% CO 2 , 5% O 2 and 90% N 2 at 38.5 C.…”

Section: Transfer Of Parthenogenetic Oocytesmentioning

confidence: 99%

“…In vitro culture was carried out for one day for evaluation of the recovered embryos. In vitro culture of the recovered embryos was carried out using NCSU23 (256 mOsm [19]) supplemented with 4 mg/ml BSA and 0.5 mg/ml hyaluronic acid [21]. After culture, development and survival of the embryos were evaluated under an inverted microscope (magnification: 100-200 ×).…”

Section: Recovery and Evaluation Of Embryosmentioning

confidence: 99%

Association Between Embryonic Loss and Damage to the Zona Pellucida by Invasive Micromanipulation During Oviductal Transfer of Early-Stage Embryos in Pigs

Ueno

Kurome

Tomii

et al. 2007

Journal of Reproduction and Development

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Abstract. The aim of this study was to examine the impact of zona pellucida damage, which might arise during somatic cell nuclear transfer (SCNT), on the development and survival of transferred embryos. The zonae pellucidae of in vitro matured oocytes were either punctured with 8-to 10-µm square-ended nuclear injection pipettes and piezo pulses or slit with 35-to 40-µm enucleation pipettes. Intact oocytes were used as controls. These oocytes were electroactivated to induce parthenogenesis and transferred to the oviducts of estrus-synchronized recipient gilts. After 5 to 7 days, the recipient uteri were flushed to collect embryos, and embryonic development (morula-blastocyst stage embryos/collected embryos) and survival (viable embryos/collected embryos) were determined. In total, 221 zona-punctured, 129 zona-slitted and 57 intact embryos were transplanted into four, two and two gilts, respectively. The efficiency of embryo recovery was similar in all groups (64.3 to 79.1%). However, the zona-penetrated and incised embryos exhibited unstable development and survival compared with the controls; development and survival of the control embryos were 94.7 and 87.7%, whereas those of the zona-punctured embryos were 69.0 and 47.9% (P<0.01) and those of the zona-slit embryos were 64.7 and 50.0% (P<0.01). Cells with large foci that appeared to be macrophage giant cells were observed at the surface or inside the degenerated zona-damaged embryos. These results indicate that the recipient's immune response to damage to the zona pellucida may impair embryonic development after transplantation to the oviduct. This may be one of the factors causing the reduced efficiency of live progeny production by SCNT.

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Stage-specific effects of the osmolarity of a culture medium on the development of parthenogenetic diploids in the pig

Cited by 35 publications

References 24 publications

The Development of Porcine Parthenogenetic Diploid Oocytes with Homogeneous Genomic Components In Vitro.

The Development of Porcine Parthenogenetic Diploid Oocytes with Homogeneous Genomic Components In Vitro.

Concentration and composition of free amino acids and osmolalities of porcine oviductal and uterine fluid and their effects on development of porcine IVF embryos

Association Between Embryonic Loss and Damage to the Zona Pellucida by Invasive Micromanipulation During Oviductal Transfer of Early-Stage Embryos in Pigs

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