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Steps involved in the procedures of embryo loading and in-straw dilution. A) The straw was loaded in the following manner. (a) It was preloaded with the TCM-199 solution containing 1 M sucrose (8.0 cm) followed by the vitrification solution (approximately 1.0 cm). (b) The vitrification solution (30 µl) containing embryos was loaded using a pipette. (c) The rest of the straw was filled with an air bubble (approximately 0.1 cm) and vitrification solution (0.2 cm), and then it was sealed with powder. B) The cryoprotectants were diluted in the following manner. (a) The straw was plunged vertically into a 20 C water bath for warming until the TCM-199 solution melted (approximately for 10 sec). (b) It was then inverted and maintained in a vertical position for 1 min to allow mixture of the cryoprotectant and sucrose solutions. (c) After cutting off the layer of powder, the straw was placed in a horizontal position for 5 min to dilute the cryoprotectants.
Source publication
In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrifie...
Contexts in source publication
Context 1
... IVF and SCNT embryos. The vitrification solutions consisted of a TCM-199 solution supplemented with 0.6 M sucrose and either 40% GLY, 30% GLY + 10% (v/v) EG, or 20% GLY + 20% EG. In each vitrification solution group, the embryos were first embryos per straw) was loaded into a 0.25-ml French plastic straw (I.M.V., L'Aigle, France) using a pipette (Fig. 1A). The end of the straw was sealed with powder. After the vitrification solutions and embryos were loaded into the straw, the straw was placed vertically to prevent mixing of the vitrification and sucrose solutions. Approximately half of the straw, including the vitrification solution containing the embryos, was rapidly immersed in ...
Context 2
... liquid nitrogen, the straws were warmed in a 20 C water bath. They were then inverted and maintained in a vertical position for 1 min to allow mixture of the cryoprotectant and sucrose solutions without shaking. Subsequently, the layer of powder was cut off and the straw was placed in a horizontal position for 5 min to dilute the cryoprotectants (Fig. 1B). After dilution, the contents of the straws were expelled into a culture dish. The embryos were immediately transferred into fresh culture medium and washed twice. After washing, the embryos were cultured for 72 h on a feeder layer of cumulus cells in the culture medium in which the IVF embryos were cultured for 9 ...
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Purpose
Through the use of the trans‐vaginal ultrasound‐guided pinpoint transfer method, the aim was to ensure the safety of the embryo being transferred in cases of difficult cervixes. It also was aimed to be able to stop embryo transfers in difficult cases and to be able to restart the process under anesthesia or cryopreservation.
Methods
A new...
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Uteri of mice (6-8 weeks old) were isolated and kept at 4 degrees C in vitro for 24 or 48 h in 0.154 mol/l NaCl or University of Wisconsin (UW) solution. Viability was evaluated by assessment of morphology and contractility in vi...
The feasibility of modifying bovine cryopreservation methods for use with baboon embryos was evaluated. Twenty-six baboon embryos at the eight-cell to blastocyst stage of development were transferred after being frozen using glycerol as cryoprotectant. Either a two-step fast of a controlled linear temperature depression was achieved to -40 degrees...
There are conflicting results on whether the rate of blastocyst development before freezing influences the outcome of frozen-thawed blastocyst transfers.
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The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and co...
Citations
... SCNT, activation, in vitro culture of embryos and TSA treatment SCNT was conducted according to the methods previously described by Taniguchi et al. (2007). In brief, the zona pellucida above the first polar body was cut with a glass needle and a small volume of cytoplasm was then squeezed out (the metaphase spindle and first polar body were visualized after incubating the oocytes in 3 μg/mL of Hoechst 33342 (Sigma-Aldrich)). ...
... To compare with naturally fertilized embryos, bovine IVF embryos were used (Taniguchi et al. 2007). The PN and two-cell stage embryos were collected at 20 h and 24 h post-insemination (PI), respectively, and the fourand eight-cell stage embryos were collected at 48 h PI for immunofluorescence analysis. ...
... When using vitrification technology in veterinary practice, a practical approach is needed for the warming of vitrified embryos so that embryos can be directly and easily transferred to the uterus. Thus far, there have been several attempts to replace successive dilution steps with one-step in-straw cryoprotectant dilution [3][4][5][6][7][8][9][10][11][12][13][14][15] However, in some of these procedures, in-straw embryo warming requires more than one dilution step and the proper handling of the carrier system, thus demanding more accuracy when these techniques are to be used in the field by embryo-transfer practitioners [7,9,10,12]. Using the VitTrans device designed by our group, IVP embryos are easily warmed/diluted instraw for their transfer to recipient females in field conditions [16]. ...
This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p < 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p < 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFNτ gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution.
... When using vitri cation technology in veterinary practice, a practical approach is needed for the warming of vitri ed embryos so that embryos can be directly and easily transferred to the uterus. So far, there have been several attempts to replace successive dilution steps with one-step in-straw cryoprotectant dilution [3][4][5][6][7][8][9][10][11][12][13][14]. However, in some of these procedures, in-straw embryo warming requires more than one dilution step and proper handling of the carrier system, demanding more accuracy when these techniques are to be used in the eld by embryo-transfer practitioners [7,9,10,12]. ...
Background
VitTrans is a device that enables the vitrification and warming/dilution of in vitro produced bovine embryos followed by their direct transfer to recipient females in field conditions. This study sought to improve the VitTrans method by comparing two equilibration times: short (SE: 3 min) and long (LE: 12 min). Outcome measures recorded in vitrified D7 and D8 expanded blastocysts were survival and hatching rates, differential cell counts, apoptosis rate and gene expression.
Results
While survival rates at 3 h and 24 h post-warming were reduced (P < 0.05) after vitrification, hatching rates of D7 embryos vitrified after SE were similar to those obtained in fresh non-vitrified blastocysts. Hatching rates of vitrified D8 blastocysts were lower (P < 0.05) than of fresh controls, regardless of treatment. Total cell counts, and inner cell mass and trophectoderm cell numbers were similar in hatched blastocysts derived from D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values, regardless of treatment. The rate of apoptotic cells was significantly higher in both treatment groups when compared to fresh controls, although apoptosis rates were lower using the SE than LE protocol. No differences emerged in expression of the genes BAX, AQP3, CX43 and IFNτ between blastocysts vitrified after SE or LE, whereas a significantly higher abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE compared to LE.
Conclusions
The VitTrans device combined with a shorter exposure to the equilibration medium improves vitrification/warming outcomes facilitating the direct transfer of vitrified embryos under field conditions.
... Also in buffaloes, maturation rates after vitrification of immature buffalo oocytes using mixture of 3 M EG + 3 M DMSO and 3.5 M EG + 3.5 M DMSO were 61.8% and 69.6%, respectively (Mahmoud & El-Sokary, 2013). Furthermore, the combination of EG and DMSO is assumed to reduce not only the toxicity of each cryoprotectant, but also the osmotic damage at warming, since EG is more likely to diffuse out of the cell rapidly, whereas DMSO is less permeable (Taniguchi et al., 2007). It was found that maturation rate in equine oocytes was better in the group which is surrounded with cumu- CPAs. ...
Camel fertility faces many problems that could be solved by assisted reproductive technologies (ART). The experiment designed to explore the effect of different cryoprotectant concentrations and combinations on viability and maturation rates of vitrified‐warmed camel oocytes. Ovaries collected directly after slaughtering from local abattoir and transported to laboratory in a thermo flask contain normal physiological saline. Oocytes aspirated from follicles (2 – 8 mm) in diameter and washed three times in TCM‐199 then examined under stereo‐ microscope for selection. Morphologically normal oocytes with an evenly granulated cytoplasm and a compact cumulus cell layer selected. Morphologically normal oocytes equilibrated in equilibration solution (ES) which is half concentration of vitrification one. After equilibration, oocytes were transported to vitrification solution using ethylene glycol (EG, 40%), Dimethyl‐sulphoxide (DMSO, 40%) and EG 40% + DMSO 40%. The obtained results revealed that addition of EG 40% + DMSO 40% resulted in the best quality of vitrified‐warmed oocytes which demonstrated by higher percent (90.16%) survival rate and (58.95%) maturation rate. While DMSO 40% resulted in 62.79% and 29.54%, respectively, EG 40% reported 86.11% and 53.47%, respectively. It could be concluded that vitrification of immature camel oocytes by using 40% EG + 40% DMSO are suitable methods to limit drawbacks of vitrification methods and further studies are needed to assess the ability of in vitro produced blastocyst to develop in vivo and establish pregnancy after embryo transfer.
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... Also in buffaloes, maturation rates after vitrification of immature buffalo oocytes using mixture of 3 M EG + 3 M DMSO and 3.5 M EG + 3.5 M DMSO were 61.8% and 69.6%, respectively (Mahmoud & El-Sokary, 2013). Furthermore, the combination of EG and DMSO is assumed to reduce not only the toxicity of each cryoprotectant, but also the osmotic damage at warming, since EG is more likely to diffuse out of the cell rapidly, whereas DMSO is less permeable (Taniguchi et al., 2007). It was found that maturation rate in equine oocytes was better in the group which is surrounded with cumu- CPAs. ...
Camel fertility facing many problems which could be solved by assisted reproductive technologies (ART). The experiment was designed to explore the effect of different cryoprotectant concentrations and combinations on viability and maturation rates of vitrified-warmed camel oocytes. Ovaries were collected directly after slaughtering from local abattoir. Then ovaries transported to laboratory in thermo flask contain normal physiological saline. Oocytes aspirated from follicles (2 – 8 mm) in diameter. Oocytes were washed three times in TCM-199 then examined under stereomicroscope for selection. Morphologically normal oocytes with an evenly granulated cytoplasm and a compact cumulus cell layer were selected. Morphologically normal oocytes equilibrated in equilibration solution (ES) (which is half concentration of vitrification one). After a period of equilibration, oocytes transported to vitrification solution using ethylene glycol (EG, 40 %), Dimethyl-sulphoxide (DMSO, 40 %) and EG 40 % + DMSO 40 %. The obtained results revealed that addition of EG 40 % + DMSO 40% resulted in the best quality of vitrified-warmed oocytes which demonstrated by higher percent (90.16 %) survival rate and (58.95 %) maturation rate. While DMSO 40 % resulted in (62.79% and 29.54%, respectively), EG 40 % reported (86.11% and 53.47 %, respectively). It could be concluded that vitrification of immature camel oocytes by using 40 % EG + 40 % DMSO suitable methods to limit drawbacks of vitrification methods and further studies are needed to assess the ability of in vitro produced blastocyst to develop in vivo and establish pregnancy after embryo transfer.
... However, the warming of embryos cryopreserved by vitrification requires the removal of the cryoprotectant (CP) agent via successive dilution steps. The trouble this causes when working under farm conditions has led to efforts to develop vitrification procedures that make direct ET less problematic [11,[22][23][24][25]. We have previously reported the use of the cryologic vitrification method (CVM) for vitrifying IVP bovine blastocysts in fibreplugs to be associated with promising survival and PRs [19,26]. ...
... Other authors have developed vitrification and warming methods that show potential to allow direct ET. These use French straws (0.25 mL) [24,68,[72][73][74], open-pulled straws [22,23], cryotops [11,70], modified straws [75,76] and fibreplugs [71] to load the embryos for vitrification, but use different strategies for warming and preparing the embryos for the procedure. Rodriguez Villamil et al. [71] used a similar approach than ours (solid surface vitrification and fibreplugs as a cryodevice), but differed in the warming method for instraw dilution. ...
... These results were similar to those reported by other authors (Saha et al. 1996;Campos-Chillon et al. 2006), which also suggested that using or not using sucrose during warming of vitrification solutions is irrelevant when vitrification solutions are composed with solutions containing EG as the only intracellular cryoprotectant. Furthermore, vitrified embryos diluted in-straw have higher survival rates when embryos were cryopreserved in simplified solutions with one permeable cryoprotectant (Pugh et al. 2000;Taniguchi et al. 2007). In vitro assessment of the in-straw dilution system demonstrated that it is a feasible method for direct transfer, reducing the chances of manipulation errors and variations during the warming process. ...
Contents
Three experiments were designed to test a solid‐surface vitrification system for bovine in vitro ‐produced embryos and to develop a simple method of in‐straw dilution after warming, which can be potentially used for direct transfer in the field. Experiment 1 evaluated embryo survival rates (i.e. re‐expansion and hatching) after vitrification and warming in three different solutions: VS 1 (20% ethylene glycol ( EG ) + 20% propanediol ( PROH ) + 0.25 m trehalose (Tr)), VS 2 (20% EG + 1M Tr) or VS 3 (30% EG + 0.75 m Tr). Re‐expansion and hatching rates were higher (p < 0.05) for embryos vitrified in VS 3 (72.2 ± 1.9 and 58.2 ± 0.8) than VS 1 (64.4 ± 0.9 and 37.2 ± 2.5) or VS 2 (68.5 ± 1.5 and 49.6 ± 1.0; p < 0.05). Experiment 2 was designed to compare two methods of vitrification: glass micropipettes or solid surface, using the VS 1 or VS 3 solutions. No significant differences were detected between the two methods; but re‐expansion and hatching rates were higher (p < 0.05) with VS 3 (73.5 ± 3.1 and 47.1 ± 2.1) than VS 1 (63.3 ± 3.3 and 39.7 ± 2.8). In experiment 3, embryos were vitrified by solid surface in VS 1 or VS 3 solutions and cryoprotectants were diluted in‐straw after warming in a TCM 199, 0.25 m sucrose solution or holding media. Survival rates of embryos vitrified in VS 3 did not differ between those exposed to 0.25 m sucrose (74.7 ± 1.3 and 57.2 ± 2.2) or holding (77.3 ± 1.4 and 58.0 ± 2.5) medium after warming; however, survival rates of embryos vitrified in VS 1 were higher (p < 0.05) in those exposed to 0.25 m sucrose (67.7 ± 2.3 and 47.0 ± 1.7) than holding medium (54.5 ± 1.0 and 27.7 ± 3.1). In conclusion, solid‐surface vitrification using simplified EG ‐based solutions and in‐straw dilution with holding media may be a practical alternative for cryopreservation and direct transfer of in vitro ‐produced bovine embryos.
... Bovine oocytes were matured according to procedures described by Taniguchi et al. (2007) with minor modifications. Cumulus-oocyte complexes (COCs) were cultured in tissue culture medium-199 (TCM-199; Invitrogen, Carlsbad, CA, USA) supplemented with 2.5 lg/mL of taurine (Sigma-Aldrich, St. Louis, MO, USA), 0.02 IU/mL of folliclestimulating hormone (FSH; Kawasaki Mitaka Seiyaku K.K., Kawasaki, Japan), 5% fetal bovine serum (FBS; Invitrogen), 20 lg/mL of epidermal growth hormone (EGF; Sigma-Aldrich), and 50 lg/mL of gentamicin (Sigma-Aldrich) for 22 h at 38.5°C in a humidified atmosphere containing 5% CO 2 . ...
... SCNT, activation, in vitro culture of embryos, and TSA treatment SCNT was conducted according to the methods previously described by Taniguchi et al. (2007). Briefly, the zona pellucida above the first polar body was cut with a glass needle and a small volume of cytoplasm was then squeezed out (the metaphase spindle and first polar body were visualized after incubating oocytes in 3 lg/mL of Hoechst 33342; Sigma-Aldrich). ...
... To compare with naturally fertilized embryos, in vitro-fertilized (IVF) bovine embryos were used. IVF was carried out according to the method described by Taniguchi et al. (2007). The two-cell stage embryos were collected at 24 h postinsemination (PI), and the four-and eight-cell stage embryos were collected at 48 h PI for fluorescent immunodetection of AcH3K9. ...
Abstract Abnormal epigenetic reprogramming, such as histone acetylation, might cause low efficiency of interspecies somatic cell nuclear transfer (iSCNT). This study was conducted to evaluate the effects of trichostatin A (TSA) on the developmental competence and histone acetylation of iSCNT embryos reconstructed from cat somatic cells and bovine cytoplasm. The iSCNT cat and parthenogenetic bovine embryos were treated with various concentrations of TSA (0, 25, 50, or 100 nM) for 24 h, respectively, following fusion and activation. Treatment with 50 nM TSA produced significantly higher rates of cleavage and blastocyst formation (84.3% and 4.6%, respectively) of iSCNT embryos than the rates of non-TSA-treated iSCNT embryos (63.8% and 0%, respectively). Similarly, the treatment of 50 nM TSA increased the blastocyst formation rate of parthenogenetic bovine embryos. The acetylation levels of histone H3 lysine 9 (H3K9) in the iSCNT embryos with the treatment of 50 nM TSA were similar to those of in vitro-fertilized embryos and significantly higher (p<0.05) than those of non-TSA-treated iSCNT embryos (control), irrespective of the embryonic development stage (two-cell, four-cell, and eight-cell stages). These results indicated that the treatment of 50 nM TSA postfusion was beneficial for development to the blastocyst stage of iSCNT cat embryos and correlated with the increasing levels of acetylation at H3K9.
... These results were similar to those reported by other authors (Saha et al. 1996;Campos-Chillon et al. 2006), which also suggested that using or not using sucrose during warming of vitrification solutions is irrelevant when vitrification solutions are composed with solutions containing EG as the only intracellular cryoprotectant. Furthermore, vitrified embryos diluted in-straw have higher survival rates when embryos were cryopreserved in simplified solutions with one permeable cryoprotectant (Pugh et al. 2000;Taniguchi et al. 2007). In vitro assessment of the in-straw dilution system demonstrated that it is a feasible method for direct transfer, reducing the chances of manipulation errors and variations during the warming process. ...
... However, the most dramatic improvement has probably been achieved by the use of newer vitrification containers that aimed to minimize the volume of vitrification solution surrounding the oocyte/embryo (0.1-1.0 L) and thereby increase the speed of cooling and warming (up to 20 000°C/min) by facilitating the rapid transfer of heat to and from liquid nitrogen (LN2). Such containers included modified plastic straws [14], electron microscope (EM) grid [15], open pulled straw (OPS) [16], cryoloop [17], cryotop [9] and their various derivatives (See [18 -19] for detailed review). Unfortunately, most of these containers can provide physical support to hold a limited number of embryos per container (ϳ10 -15 embryos per container). ...
... We next determined if commonly available absorbent papers could be used as a substitute for existing vitrification device, such as EM grid [15], OPS [16], cryoloop [17] and plastic straw [14] for holding the embryos during the vitrification process. Since absorbent papers can absorb the vitrification solution, we hypothesized that their use may minimize the volume of vitrification solution surrounding the blastocysts and hence may increase the cooling and warming process by facilitating rapid transfer of heat to and from LN2. ...
