Vitrification Technique Research Articles

Cryopreservation of ovine embryos: Slow freezing and vitrification

Different methods for the cryopreservation of ovine embryos were evaluated in vitro (survival upon culture in vitro) and in vivo (pregnancy and lambing rates after transfer in field conditions). In the first 2 experiments, slow freezing conditions were evaluated. When glycerol and ethylene glycol were compared, no differences in the overall pregnancy rate were found (40.2 vs 51.3%), but better results were obtained with ethylene glycol than with glycerol in morulae (29.7 vs 59.4%, P<0.05). In the second experiment, 2 methods of removing ethylene glycol were compared: a I-step procedure using 0.5-M sucrose and a 3-step process for decreasing ethylene glycol concentration. There were no differences in the overall pregnancy rate (48.0 vs 48.0%) between the 2 methods. The last series of experiments were designed to compare 2 vitrification solutions: propylene glycol - glycerol (PG) and ethylene glycol - Ficoll 70 - sucrose (EFS). There were no differences between the 2 vitrification solutions, based on the overall pregnancy rate (28.1 vs 40.0%). The vitrification technique and specially with EFS solution has resulted in good pregnancy rates. The EFS solution was particularly efficacious with morulae (55.5% pregnancy). These results demonstrate that vitrification with EFS can be used successfully for the cryopreservation of ovine embryos.

96/06594 Disposal of weapon plutonium: Approaches and prospects

Preface. Session I: Safe Disposal of Weapons Plutonium. 1. Weapons Plutonium Management: The Present Situation, the Methods and the Prospects A.I. Karelin. 2. Retrievability and Safeguards Concerns Regarding Plutonium in Geological Repositories J. Swahn. 3. Multibarriers: Their Efficacy Against Intentional Intrusion into a Geological Repository to Retrieve Disposed Plutonium E.R. Merz. 4. Prospects for Using Plutonium as a Fuel in Russia V.N. Mikhailov, et al. 5. Plutonium Handling and Vitrification: Main Process Steps and their Cost Evaluation E. Trauwert, M. Demonie. Session 2: Product Forms. 6. Dissolution of Materials Containing Metal Plutonium and Oxidized Forms of Plutonium G.P. Nikitina, et al. 7. Crystalline Ceramics: Waste Forms for the Disposal of Weapons Plutonium R.C. Ewing, et al. 8. The Crystalline Host-Phases for Immobilization of Weapons Plutonium and Waste Actinides B.E. Burakov, et al. 9. On the Long-Term Storage of Weapons Plutonium in Form of Criticality-Free Ceramics Containing Lithium J.A. Trutnev, et al. 10. Incorporation of Pu and other Actinides in Borosilicate Glass and in Waste Ceramics Hj. Matzke, J. van Geel. 11. Feasibility of Rock-Like Fuel and Glass Waste Forms for Disposal of Weapons Plutonium H. Furuya, et al. 12. Possible Ways of the Weapon Plutonium Processing to Produce the Steady Glass Materials Suitable for Further Safe Disposal Y.I. Matyunin, et al. 13. Management of Operational Waste from MOX Fuel Plants F.W. Ledebrink. Session 3: Vitrification Technology. 14. SRS Vitrification Studies in Support of the U.S. Program for Disposition of Excess Plutonium G.G. Wicks, et al. 15. On the Feasibility of Vitrifying High-Level Liquid Waste Containing High Amounts of Plutonium M. Demonie, H. Cuyvers. 16. Vitrification of Weapons Plutonium: A Hypothetic Option or a Real Way to Solve the Problem A.S. Aloy, E.B. Anderson. 17. Vitrification of Plutonium for Disposal J.A.C. Marples. 18. French Appraisal of the Vitrification Option. Weapons Plutonium -- or how to get rid of it! A. Jouan. 19. Safety Aspects with Regard to Plutonium Vitrification Techniques L.W. Gray, T. Kan. Session 4: Disposal in Deep Boreholes. 20. Disposition of Radionuclides in Deep Geological Formations either in Containers or Vitrified Form T.A. Goupalo. 21. Disposition of Plutonium in Deep Boreholes W.H. Halsey, et al. Session 5: Long Term Safety Aspects. 22. The Process of the Equipment and Room Surface Pu- Decontamination and Means to Prevent their Contamination A.B. Pavlov, Ye.I. Yulikov. 23. Technical Basis for Pu Safeguard Practices, Handling and Disposal, PERLA and TAME Experiences H. Dworschak. 24. Long-Term Safety Aspects of Geological Disposal of Large Quantities of Plutonium B. Baltes. 25. MPC&A for Plutonium Disposition in the Russian Federation W.G. Sutcliffe. 26. Ecological Safety Aspects with Regard to Plutonium Disposal V.F. Menschikov. 6. Summary of Workshop Presentations. 7. NATO Workshop Summary evaluation. Index.

Cryoprotectant Toxicity in Penaeid Prawn Embryos

Cryopreservation of penaeid prawn embryos has definite applications in the aquaculture industry. It can be considered a viable alternative to solve the “seed” scarcity faced during the “lean” season. Presently, there is no protocol for the cryopreservation of prawn embryos. As a study of cryoprotectant toxicity is an essential prerequisite for the development of a cryopreservation protocol, this study focuses on the toxicity of seven well known permeating, low molecular weight cryoprotectants: glycerol, formamide, acetamide, methanol, propylene glycol, dimethyl sulfoxide (Me2SO), and ethylene glycol (EG). In the cryoprotectant toxicity experiments embryos were exposed to cryoprotectant solutions at 15°C for a period of 15 min, whereas the toxicity neutralization experiments were conducted at 28°C with an exposure time of 15 min. The widely used permeating cryoprotectant, glycerol, was toxic to morula stage embryos at concentrations >0.5M.This study shows that the toxicity tolerance ofPenaeus indicusembryos varies with the developmental stage, the later stages being more resistant. Morulae did not tolerate any cryoprotectant above 5M,whereas the nauplii had survival rates of 45% in 12.5Mmethanol and 78% in 6.4MEG. The toxicity neutralization experiments do not seem to indicate any specific toxicity-blocking mechanism in Me2SO toxicity reduction by EG. Based on the response to cryoprotectant toxicity, nauplius stage larvae of penaeid prawns can be considered a suitable stage for cryopreservation, both by the conventional slow cooling method and also the vitrification technique as it allows use of high levels of cryoprotectant.

Vitrification of fly ash by swirling-flow furnace

According to the amendment of the Waste Disposal and Public Cleansing Law of 1992, fly ash is regulated as “Specially controlled waste” and wide attention is now being paid to the melting and vitrification treatment of fly ash, which can reduce overall volume, detoxify and recover sources. Kobe Steel has demonstrated its operation using a swirling-flow furnace and has perfected a vitrification technique. The demonstration test has confirmed stable melting, high decomposition ratio of dioxins and the soundness of the slag. Kobe Steel has successfully developed a new technique for heightening the quality of slag and a new process for the heavy metals recovery from collected dust.

Viability of immature horse oocytes cryopreserved by vitrification

The objectives of this study were, a) to compare two different vitrification techniques, the solid surface vitrification (SSV) and direct vitrification (DV) method, b) to evaluate the effect of four cryoprotectant agents and their toxicity on the morphological appearance and ultrastructural of camel ovarian cortex, c) to examine the development of oocytes recovered from the vitrified ovarian cortex. Fragments of ovarian cortex were exposed to equilibration solution consisting of TCM- 199 with 10% fetal camel serum (FCS); 0.10 M sucrose and including one of the following cryoprotectants; 20% glycerol (GLY); 3.5 M ethylene glycol (EG); 3.5 M propanediol (PROH) or 3 M dimethylsulphoxide (DMSO). After vitrification of ovarian fragments, they were warmed and evaluated by histological and transmission electron microscope. The oocytes isolated from vitrified ovarian cortex were cultured in TCM-199 at 38.5 °C under 5% CO2 for 44 h. Maturation was indicated through cumulus expansion and calculated by oocytes reaching first telophase and second metaphase (TI + MII). The percentage of morphologically normal and viable follicles of SSV was significantly higher P < 0.05 than DV group (52.9 vs. 38.1, respectively). In conclusion, viability, histological and ultrastructural observations revealed that SSV method and ethylene glycol-based freezing solution were able to remain morphology better follicle and oocyte. Additionally, most organelles of oocytes are able to recover their normal morphology in camel ovarian cortex following cryopreservation and thawing processes, and oocytes isolated from vitrified ovarian cortex can exhibit maturation and reaches to (TI + MII).

Cryopreservation of kunming mouse oocytes using slow cooling, ultrarapid cooling, and vitrification protocols

Cryopreservation of unfertilized mouse ova and 2-cell embryos by a vitrification technique was examined. Survival was defined by development to the hatching blastocyst stage after in vitro fertilization. With 19 embryos at the 2-cell stage, the authors obtained 100% morphologic survival and 89% development to hatching blastocyst stage. To define the optimal conditions for vitrification of ova, the authors treated a total of 845 unfertilized ova. In experiments done at 0°C, the concentration of vitrification solution (VS1) and the length of exposure of ova to VS1 both had significant (P < 0.01) effects on survival. The mean survival rate for controls in ten experiments was 52%. VS1 100% or 90% in HEPES buffered saline and 10 minutes’ exposure yielded rates that did not differ significantly from controls. Significantly lower survival rates followed the use of 70 and 80% solution and exposure for 5, 15, 20, or 30 minutes. Thus, under these conditions, exposure of unfertilized mouse ova to VSl and cooling to 0°C did not interfere with in vitro fertilization and development of embryos. However, in five experiments in which a total of 101 ova were plunged into liquid nitrogen after treatment with VS1 under the optimal conditions, none could be fertilized in vitro.

Application of OPU for embryoproduction and breeding in cattle

The objective was to determine if immature bovine oocytes with cumulus cells at the germinal vesicle (GV) stage could be vitrified by aluminum sheets (AS; pieces of sheet-like aluminum foil). Cleavage rates in fertilized oocytes previously vitrified by the AS procedure were higher than those vitrified by a nylon-mesh holder (NM) procedure (89.3 ± 2.1% vs. 65.0 ± 3.7%). Cleaved embryos derived from the AS but not from the NM procedures developed to blastocysts. Furthermore, to investigate the effects of vitrifying GV oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes, the intracellular phospholipid membrane (IM) was stained with the lipophilic fluorescent dye, 3,3′-dioctadecyloxa-carbocyanine perchlorate. After vitrification by AS, the IM remained intact relative to that of oocytes vitrified by NM. During in vitro maturation, reorganization of the IM was also undamaged in oocytes vitrified by AS before oocyte maturation, and the IM within oocytes vitrified by the NM procedure was evidently impaired. Finally, vitrification (AS) was used for GV oocytes collected using the ovum pick-up method. A bull calf was born after in vitro production and subsequent embryo transfer. The vitrification techniques described herein should facilitate generation of viable in vitro production bovine blastocysts using oocytes recovered using the ovum pick-up method.

Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses.

Vitrification of mouse oocytes adversely affected the subsequent developmental potential of embryos and fetuses derived from the fertilization of such oocytes after thawing. Only 5% of oocytes vitrified formed viable fetuses on the 15th day of gestation as compared to 47% in the controls. The incidence of chromosomally aneuploid zygotes, derived from cryopreserved oocytes, was approximately threefold higher than the controls irrespective of whether the oocytes were cryopreserved by vitrification or DMSO slow-freezing. Malformed fetuses were obtained from oocytes that had been vitrified as well as those that had been exposed to vitrification solutions only, whereas no malformed fetuses were obtained in oocytes slow-frozen by DMSO or fresh controls--thus demonstrating that the exposure of oocytes to the vitrification chemicals was responsible for the fetal malformations. The data in this study suggest that the vitrification technique should be cautiously applied to human oocyte cryopreservation. Furthermore, the data also demonstrate that the exposure of female gametes to carcinogenic and/or teratogenic chemicals may result in malformed embryos when such oocytes are subsequently fertilized.

Structural Evolution During the Sol to Gel Transition of Silicon-Alkoxide Based Sols Observed by Cryogenic Transmission Electron Microscopy (CRYO-TEM)

ABSTRACTThe gelation reaction for tetraethoxysilane in both acid and base catalyzed environments was studied using cryogenic transmission electron microscopy. The evolution of the gel structure was observed in wet samples by the technique of fast-freeze vitrification, in which the three dimensional gel structure is preserved in vitreous solvent. The sample is then observed in the electron microscope using a cold stage. Samples were prepared and allowed to react until a specific point of gelation was reached, then were vitrified at that point. Results from this technique show a coarse network structure for base catalyzed gels. This network is composed of silica clusters ≈40Å in size. The acid catalyzed gels show an extremely fine texture (&lt;10Å) at end point of gelation. The structures at intermediate stages of gelation were also determined.

Cryopreservation of mouse 2-cell embryos and ova by vitrification: methodologie studies

Cryopreservation of unfertilized mouse ova and 2-cell embryos by a vitrification technique was examined. Survival was defined by development to the hatching blastocyst stage after in vitro fertilization. With 19 embryos at the 2-cell stage, the authors obtained 100% morphologic survival and 89% development to hatching blastocyst stage. To define the optimal conditions for vitrification of ova, the authors treated a total of 845 unfertilized ova. In experiments done at 0 degree C, the concentration of vitrification solution (VS1) and the length of exposure of ova to VS1 both had significant (P less than 0.01) effects on survival. The mean survival rate for controls in ten experiments was 52%. VS1 100% or 90% in HEPES buffered saline and 10 minutes' exposure yielded rates that did not differ significantly from controls. Significantly lower survival rates followed the use of 70 and 80% solution and exposure for 5, 15, 20, or 30 minutes. Thus, under these conditions, exposure of unfertilized mouse ova to VS1 and cooling to 0 degree C did not interfere with in vitro fertilization and development of embryos. However, in five experiments in which a total of 101 ova were plunged into liquid nitrogen after treatment with VS1 under the optimal conditions, none could be fertilized in vitro.

Experience with the cryopreservation of human embryos using the mouse as a model to establish successful techniques.

Mouse embryos at the one-, two-, and eight-cell stages have been used to optimize the conditions for cryopreservation of human oocytes and embryos. For storage in glass vials using 1.5 M dimethyl sulfoxide (DMSO) as a cryoprotectant and slow cooling (approximately 0.3 degrees C/min), phosphate-buffered medium was superior to Hepes-buffered medium. Termination of slow cooling at -80 degrees C before transfer to liquid nitrogen with subsequent slow thawing (approximately 8 degrees C/min) resulted in more embryos surviving than when cooling terminated at -40 degrees C and rapid thawing (approximately 500 degrees C/min) was employed. Dilution of DMSO upon thawing with medium containing 0.5 M sucrose gave higher embryo survival rates than a stepwise (0.25 M decrements) dilution. Using these techniques, three pregnancies were established upon the transfer of 11 frozen-thawed embryos to seven patients. Rates of embryo survival using the simpler cryopreservation technique of ice-free vitrification in 0.25-ml straws have been disappointing.