Papers by Duangjai Boonkusol
Reproduction, Fertility and Development, 2007
The purpose of the present study was to investigate the effects of two vitrification procedures o... more The purpose of the present study was to investigate the effects of two vitrification procedures on developmental capacity and ultrastructural changes of matured swamp buffalo oocytes. In vitro-matured oocytes were vitrified by using 35 and 40% ethylene glycol as vitrification solution for solid surface vitrification (SSV) and in-straw vitrification (ISV), respectively. Survival rate of vitrified-warmed oocytes, evaluated on the basis of ooplasm homogeneity, oolemma integrity and zona pellucida intactness, as well as parthenogenetic blastocyst rates of vitrified-warmed oocytes were significantly higher with SSV (89.3 and 13.6%, respectively) than ISV (81.8 and 5.5%, respectively). However, they were still significantly lower than that of control oocytes (100 and 34.2%, respectively). For examining the ultrastructural changes, fresh, VS-exposed (ISV and SSV), and vitrified-warmed (ISV and SSV) oocytes were processed for transmission electron microscopy. In VS-exposed oocytes, reduction of microvilli abundance and damage of mitochondrial membrane were found only in the ISV group. In vitrified-warmed oocytes, however, it was clear that both methods of vitrification induced profound ultrastructural modifications to microvilli, mitochondria, oolemma and cortical granules as well as to the size and position of vesicles. Damaged mitochondria were, however, more abundant in ISV vitrified oocytes than in SSV vitrified oocytes, which correlated with the developmental data, showing the superiority of the SSV method. The present study demonstrated the feasibility of vitrification of in vitro-matured swamp buffalo oocytes.
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Reproduction, Fertility and Development, 2007
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Reproduction, Fertility and Development, 2010
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Molecular Reproduction and Development, 2006
The analysis of differences in gene expression, responding to cryopreservation may explain some o... more The analysis of differences in gene expression, responding to cryopreservation may explain some of the observed differences in further development of the preimplantation stage embryos. The aim of this study was to create a link, for the first time, between morphological/developmental observations and gene activity changes following cryopreservation of embryos. Efficiency of two vitrification methods, solid surface and in-straw vitrifications for pronuclear-stage mouse zygotes and 8-cell stage mouse embryos was compared based on morphological survival, blastocyst formation, and changes in embryonic gene expression. Both stages of embryos were vitrified by SSV using 35% ethylene glycol (EG) for vitrification solution (VS) and in-straw vitrification using 40% EG for VS. No significant differences were found between immediate survival rates of embryos vitrified by SSV and in-straw vitrification in both stages. Blastocyst rates were significantly higher with SSV and not significantly different from that of control. These results showed that SSV was more efficient than in-straw vitrification. Treatment with cytochalasin-b did not improve cryosurvival during SSV. The quantification of selected gene transcripts from single embryo (6 embryos/treatment group) were carried out by quantitative real-time RT-PCR. It was performed by adding 1/8 of each embryo cDNA to the PCR mix containing the specific primers to amplify housekeeping gene (beta-actin), heat shock protein gene (Hsp70), genes related to oxidative stress (MnSOD and CuSOD), cold stress (CirpB, Rbm3), and cell-cycle arrest (Trp53). We found upregulation of all six stress-related genes at 3 hr post-warming in pronuclear stage embryos. Expression of these genes showed much higher level (2-33-fold) in in-straw vitrification than in in vitro control embryos. In SSV-treated embryos we could detect only slight changes (0.3-2-fold). At 10 hr post-warming, all genes were downregulated in embryos vitrified by in-straw method. In SSV-treated group expression of Hsp70 showed slight increase and Trp53 showed decrease. In contrast to pronuclear stage, there was no clear pattern of gene expression changes after vitrification in 8-cell stage embryos. Several genes were upregulated both at 3 and 10 hr post-warming. Moreover, we found upregulation of beta-actin gene which we expected to use as a reference gene in in-straw treated embryos in both 3 and 10 hr post-warming, while in pronuclear stage embryos and in SSV treatment there was no effect on beta-actin expression level. There was no difference in gene expression between blastocysts developed from fresh or vitrified embryos. In conclusion, the real-time RT-PCR method from single embryo opened new opportunities for the understranding of molecular events following cryopreservation. The upregulation of stress-related genes at 3 hr post-warming in pronuclear stage embryos might have been an early indicator of reduced viability following in-straw vitrification in good correlation with the developmental data to blastocyst stage.
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Acta Scientiae Veterinari
Although intensive research has been conducted on a worldwide scale, cryopreservation of embryos ... more Although intensive research has been conducted on a worldwide scale, cryopreservation of embryos in many species has not yet been possible with sufficient success rates. Embryo cryopreservation in farm animals is a valuable tool to maintain genetic diversity and for breeding purposes. In endangered species, banking of cryopreserved embryos would help in preservation efforts. Cryopreservation of human embryos, in combination with human ART methods, is of outmost importance in the treatment of infertility. This review is focused on the advances and problems of embryo cryopreservation in the most important laboratory and farm animal species
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Papers by Duangjai Boonkusol