Bovine oocyte vitrification: effect of ethylene glycol concentrations and meiotic stages

Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0–10, 10–14, and 18–24 h of IVM, respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 h of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-1 or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However, after vitrification in VS-2, blastocyst rates were less at 18 h than 0 h. Both cleavage rates and blastocyst rates were significantly less in all vitrification groups when compared to control group and only control oocytes hatched. In conclusion, both EG concentration and stage of meiotic maturation affect the developmental potential of oocytes after vitrification.     

Bacterial metabolism of ethylene glycol

Metabolism of ethylene glycol as the sole source of carbon by a species of Flavobacterium was affected by the dissolved oxygen tension of the growth medium. Under strongly aerobic conditions the diol was exclusively metabolised to glycollate by an initial oxidase, subsequently metabolised to acetyl-CoA with no net change in ATP, and then oxidised to CO₂, by the tricarboxylic acid cycle yielding large amounts of reduced nicotinamide nucleotides which were used to generate a net gain in ATP by oxidative phospsorylation. Under miccroaerophilic conditions, some ethylene glycol after initial metabolism to acetyl-CoA by the oxidase-initiated pathway, was subsequently catabolised to acetyl phosphate and then acetate, yielding a net gain in ATP by substrate-level phosphorylation: additionally some diol was catabolised by an inducible diol dehydratase to acetaldehyde and subsequently reduced to ethanol as a terminal metabolite.     

Glass transition behavior of the vitrification solutions containing propanediol, dimethyl sulfoxide and polyvinyl alcohol

Knowledge of the glass transition behavior of vitrification solutions is important for research and planning of the cryopreservation of biological materials by vitrification. This brief communication shows the analysis for the glass transition and glass stability of the multi-component vitrification solutions containing propanediol (PE), dimethyl sulfoxide (Me₂SO) and polyvinyl alcohol (PVA) by using differential scanning calorimetry (DSC) during the cooling and subsequent warming between 25 and −150 °C. The glass formation of the solutions was enhanced by introduction of PVA. Partial glass formed during cooling and the fractions of free water in the partial glass matrix increased with the increasing of PVA concentration, which caused slight decline of glass transition temperature, T_g. Exothermic peaks of devitrification were delayed and broadened, which may result from the inhibition of ice nucleation or recrystallization of PVA.     

Effect of common cryoprotectants on critical warming rates and ice formation in aqueous solutions

Ice formation on warming is of comparable or greater importance to ice formation on cooling in determining survival of cryopreserved samples. Critical warming rates required for ice-free warming of vitrified aqueous solutions of glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol 200 and sucrose have been measured for warming rates of order 10–10⁴ K/s. Critical warming rates are typically one to three orders of magnitude larger than critical cooling rates. Warming rates vary strongly with cooling rates, perhaps due to the presence of small ice fractions in nominally vitrified samples. Critical warming and cooling rate data spanning orders of magnitude in rates provide rigorous tests of ice nucleation and growth models and their assumed input parameters. Current models with current best estimates for input parameters provide a reasonable account of critical warming rates for glycerol solutions at high concentrations/low rates, but overestimate both critical warming and cooling rates by orders of magnitude at lower concentrations and larger rates. In vitrification protocols, minimizing concentrations of potentially damaging cryoprotectants while minimizing ice formation will require ultrafast warming rates, as well as fast cooling rates to minimize the required warming rates.     

Purification of aqueous ethylene glycol

Reagent-grade ethylene glycol has been shown to contain substantial amounts of aldehydes, peroxides, iron, and uv-absorbing hydrocarbons. These impurities can be removed by reduction with sodium borohydride, dilution with H2O, passing through a train of four columns, and filtering through a 0.45-micron filter. The product is stable for at least several months and perhaps much longer; storage under nitrogen in acid-washed dark bottles is preferable. Ten liters of 25% (v/v) aqueous ethylene glycol can easily be purified in about 1 week using equipment commonly available in a biochemical laboratory. This purification is also applicable to aqueous glycerol.     

Validated gas chromatographic–mass spectrometric assay for determination of the antifreezes ethylene glycol and diethylene glycol in human plasma after microwave-assisted pivalylation

A gas chromatographic–mass spectrometric assay is described for identification and quantification of the antifreezes ethylene glycol (EG) and diethylene glycol (DEG) in plasma for early diagnosis of a glycol intoxication. After addition of 1,3-propanediol as internal standard, the plasma sample was deproteinized by acetone and an aliquot of the supernatant was evaporated followed by microwave-assisted pivalylation. After gas chromatographic separation, the glycols were first identified by comparison of the full mass spectra with reference spectra and then quantified. The quantification has been validated according to the criteria established by the Journal of Chromatography B. The assay was found to be selective. The calibration curves for EG and DEG were linear from 0.1 g/l to 1.0 g/l. The limit of detection for EG and DEG was 0.01 g/l and the limit of quantification for both was 0.1 g/l. The absolute recoveries were 50 and 65% for the low quality control samples and 51 and 73% for the high quality control samples of EG and DEG, respectively. Intra- and inter-day accuracy and precision were inside the required limits. The glycols in frozen plasma samples were stable for more than 6 months. The method was successfully applied to several authentic plasma samples from patients intoxicated with glycols. It has also been suitable for analysis of EG and DEG in urine.     

Dynamics of water in supercooled aqueous solutions of glucose and poly(ethylene glycol)s as studied by dielectric spectroscopy

The dielectric behaviour of aqueous solutions of glucose, poly(ethylene glycol)s (PEGs) 200 and 600, and poly(vinyl pyrrolidone) (PVP) has been examined at different concentrations in the frequency range of 10(6)-10(-3) Hz by dielectric spectroscopy and by using differential scanning calorimetry down to 77 K from room temperature. The shape of the relaxation spectra and the temperature dependence of the relaxation rates have been critically examined along with temperature dependence of dielectric strength. In addition to the so-called primary (alpha-) relaxation process, which is responsible for the glass-transition event at T(g), another relaxation process of comparable magnitude has been found to bifurcate from the main relaxation process on the water-rich side, which continues to the sub-T(g) region, exhibiting relaxation at low frequencies. The sub-T(g) process dominates the dielectric measurements in aqueous solutions of higher PEGs, and the main relaxation process is seen as a weak process. The sub-T(g) process was not observed when water was replaced by methanol in the binary mixtures. These observations suggest that the sub-T(g) process in the aqueous mixtures is due to the reorientational motion of the 'confined' water molecules. The corresponding dielectric strength shows a noticeable change at T(g), indicating a hindered rotation of water molecules in the glassy phase. The nature of this confined water appears to be anomalous compared to most other supercooled confined liquids.     

Statistical prediction of the vitrifiability and glass stability of multi-component cryoprotective agent solutions

Long-term biologic storage of articular cartilage has proven elusive due to cellular degradation over time or acute damage during attempts at cryopreservation. Vitrification is one option that may result in successful cryopreservation but difficulty with cryoprotective agent (CPA) toxicity at high concentrations of a single cryoprotectant has hindered development of successful protocols. This study was designed to determine the vitrifiability and glass stability of solutions containing combinations of commonly used CPAs and to document CPA interactions that occur. One hundred and sixty-four multi-CPA combination solutions of 6-9 M were evaluated for vitrifiability and glass stability using direct visualization after immersion in liquid nitrogen for 30 min and upon warming. Binary and ordinal logistic regression analysis was used to statistically analyze each CPA for its ability to vitrify and its effect on glass stability in multi-component CPA solutions. Propylene glycol had the greatest incremental contribution to vitrification while formamide had the least contribution. A threshold was established whereby the ability of a solution to vitrify could be determined by calculation. Glass stability was not as clearly defined due to variability in the results; however, contributions of interactions between CPAs to the glass stability of solutions were determined. This study provided values that predict if a solution will vitrify. Furthermore, the glass stability of solutions containing multiple CPAs do not behave as linear additions of binary solutions and interactions between CPAs have a significant effect on the glass stability of these solutions. These variables should be considered when designing vitrification solutions.     

Metabolic engineering of Corynebacterium glutamicum for the de novo production of ethylene glycol from glucose

Development of sustainable biological process for the production of bulk chemicals from renewable feedstock is an important goal of white biotechnology. Ethylene glycol (EG) is a large-volume commodity chemical with an annual production of over 20 million tons, and it is currently produced exclusively by petrochemical route. Herein, we report a novel biosynthetic route to produce EG from glucose by the extension of serine synthesis pathway of Corynebacterium glutamicum. The EG synthesis is achieved by the reduction of glycoaldehyde derived from serine. The transformation of serine to glycoaldehyde is catalyzed either by the sequential enzymatic deamination and decarboxylation or by the enzymatic decarboxylation and oxidation. We screened the corresponding enzymes and optimized the production strain by combinatorial optimization and metabolic engineering. The best engineered C. glutamicum strain is able to accumulate 3.5 g/L of EG with the yield of 0.25 mol/mol glucose in batch cultivation. This study lays the basis for developing an efficient biological process for EG production.     

6-Shogaol attenuated ethylene glycol and aluminium chloride induced urolithiasis and renal injuries in rodents

The 6-shogaol, is a flavanone type flavonoid that is abundant in citrus fruit and has a wide range of pharmacological effects. The present study attempted to evaluate the antiurolithic effect of 6-shogaol on ethylene glycol (EG) and ammonium chloride (AC)-induced experimental urolithiasis in rats. The efficacy of 6-shogaol 50 mg/kg and 100 mg/kg was studied in EG 0.75% (V/V) and AC 1% (W/V) experimentally induced urolithiasis in rats for 21 days. The weight difference, urine volume, the levels of calcium, phosphate, magnesium, oxalate and uric acid in urine was observed. The blood urea nitrogen, creatinine, uric acid in serum and levels of malondialdehyde (MDA) and glutathione (GSH) were also measured. Histopathological analyses in kidneys were also performed. The rats weights were higher in the 6-shogaol groups than the urolithiasis group. EG caused a significant increase in serum creatinine (p < 0.05), BUN (P < 0.001), and uric acid (p < 0.01) while treatment with Cystone (750 mg/kg), and 6-shogaol (50 and 100 mg/kg) showed the significant reduction in increased serum levels of creatinine (p < 0.001), uric acid (p < 0.01) and BUN (p < 0.001). Administration of EG and AC showed statistically significant (p < 0.001) elevated levels of MDA and reduction in GSH levels. Treatment of Cystone (750 mg/kg), and 6-shogaol (50 and 100 mg/kg) significantly (p < 0.001) reduced MDA levels and an increase GSH levels as compared to EG and AC-treated group. The histological findings further attested antiurolithiatic properties of 6-shogaol. The present study attributed clinical shreds of evidence first time that claiming the significant antiurolithic effect of 6-shogaol and could be a cost-effective candidate for the prevention and treatment of urolithiasis.     

Anaerobic ethylene glycol degradation by microorganisms in poplar and willow rhizospheres

Although aerobic degradation of ethylene glycol is well documented, only anaerobic biodegradation via methanogenesis or fermentation has been clearly shown. Enhanced ethylene glycol degradation has been demonstrated by microorganisms in the rhizosphere of shallow-rooted plants such as alfalfa and grasses where conditions may be aerobic, but has not been demonstrated in the deeper rhizosphere of poplar or willow trees where conditions are more likely to be anaerobic. This study evaluated ethylene glycol degradation under nitrate-, and sulphate-reducing conditions by microorganisms from the rhizosphere of poplar and willow trees planted in the path of a groundwater plume containing up to 1.9 mol l⁻¹ (120 g l⁻¹) ethylene glycol and, the effect of fertilizer addition when nitrate or sulphate was provided as a terminal electron acceptor (TEA). Microorganisms in these rhizosphere soils degraded ethylene glycol using nitrate or sulphate as TEAs at close to the theoretical stoichiometric amounts required for mineralization. Although the added nitrate or sulphate was primarily used as TEA, TEAs naturally present in the soil or CO₂ produced from ethylene glycol degradation were also used, demonstrating multiple TEA usage. Anaerobic degradation produced acetaldehyde, less acetic acid, and more ethanol than under aerobic conditions. Although aerobic degradation rates were faster, close to 100% disappearance was eventually achieved anaerobically. Degradation rates under nitrate-reducing conditions were enhanced upon fertilizer addition to achieve rates similar to aerobic degradation with up to 19.3 mmol (1.20 g) of ethylene glycol degradation l⁻¹ day⁻¹ in poplar soils. This is the first study to demonstrate that microorganisms in the rhizosphere of deep rooted trees like willow and poplar can anaerobically degrade ethylene glycol. Since anaerobic biodegradation may significantly contribute to the phytoremediation of ethylene glycol in the deeper subsurface, the need for “pump and treat” or an aerobic treatment would be eliminated, hence reducing the cost of treatment.     

Simple method for cryopreservation of an anaerobic rumen fungus using ethylene glycol and rumen fluid

Abstract The cryopreservation of an anaerobic rumen fungus, Piromyces communis OTS1, was examined at −84 °C using dimethyl sulfoxide, propylene glycol or ethylene glycol as cryoprotectants. Ethylene glycol was the most effective agent, combining high survival and low toxicity, followed by dimethyl sulfoxide and propylene glycol. Cell-free rumen fluid in the cryopreservation medium decreased the toxicity of the cryoprotectant agents and also had a protective action per se. A survival of 80% after 1 year storage was obtained when samples with an initial zoospore density of 5 × 10⁴ zoospores/ml were equilibrated for 15 min in medium containing 0.64 M ethylene glycol and 5% cell-free rumen fluid, then frozen with dry ice and stored at −84 °C.     

Efficient utilization of pentoses for bioproduction of the renewable two-carbon compounds ethylene glycol and glycolate

The development of lignocellulose as a sustainable resource for the production of fuels and chemicals will rely on technology capable of converting the raw materials into useful compounds; some such transformations can be achieved by biological processes employing engineered microorganisms. Towards the goal of valorizing the hemicellulose fraction of lignocellulose, we designed and validated a set of pathways that enable efficient utilization of pentoses for the biosynthesis of notable two-carbon products. These pathways were incorporated into Escherichia coli, and engineered strains produced ethylene glycol from various pentoses, including simultaneously from D-xylose and L-arabinose; one strain achieved the greatest reported titer of ethylene glycol, 40 g/L, from D-xylose at a yield of 0.35 g/g. The strategy was then extended to another compound, glycolate. Using D-xylose as the substrate, an engineered strain produced 40 g/L glycolate at a yield of 0.63 g/g, which is the greatest reported yield to date.     

Preferential hydration and solubility of proteins in aqueous solutions of polyethylene glycol

This paper is focused on the local composition around a protein molecule in aqueous mixtures containing polyethylene glycol (PEG) and the solubility of proteins in water + PEG mixed solvents. Experimental data from literature regarding the preferential binding parameter were used to calculate the excesses (or deficits) of water and PEG in the vicinity of β-lactoglobulin, bovine serum albumin, lysozyme, chymotrypsinogen and ribonuclease A. It was concluded that the protein molecule is preferentially hydrated in all cases (for all proteins and PEGs investigated). The excesses of water and deficits of PEG in the vicinity of a protein molecule could be explained by a steric exclusion mechanism, i.e. the large difference in the sizes of water and PEG molecules.

The solubility of different proteins in water + PEG mixed solvent was expressed in terms of the preferential binding parameter. The slope of the logarithm of protein (lysozyme, β-lactoglobulin and bovine serum albumin) solubility versus the PEG concentration could be predicted on the basis of experimental data regarding the preferential binding parameter. For all the cases considered (various proteins, various PEGs molecular weights and various pHs), our theory predicted that PEG acts as a salting-out agent, conclusion in full agreement with experimental observations. The predicted slopes were compared with experimental values and while in some cases good agreement was found, in other cases the agreement was less satisfactory. Because the established equation is a rigorous thermodynamic one, the disagreement might occur because the experimental results used for the solubility and/or the preferential binding parameter do not correspond to thermodynamic equilibrium.     

Peculiarities of state diagrams of aqueous solutions of cryoprotective agents

The phase transitions in aqueous solutions of glycerol and PEO-1500 within the temperature range of +30 to −150 °C have been studied using the methods of thermoplastic analysis and volumetric scanning tensodilatometry. We present the revealed phenomenon of cluster cyrystallization of these solutions as well as principles of describing this phenomenon using state diagrams, containing the intervals of concentration corresponding to the existence of amorphous and cryocolloid fractions. We note that for the cryocolloid fraction, a low temperature association of molecules of cryoprotective agents leads the formation of ice nanocrystals either close to or directly inside the aggregations. These fractions exist in cooled cryoprotective solutions near the vitrification temperatures of the liquid phase and may contribute to the initiation of damaging events in cryopreserved biological systems. Our data may be helpful in explaining the peculiarities observed during crystallization of cryoprotective solutions and may further contribute to a broader understanding of the principles of protection and protocol optimization of biological materials at temperatures approaching vitrification.     

On the assessment of the stability of vitrified cryo-media by differential scanning calorimetry: A new tool for biobanks to derive standard operating procedures for storage,access and transport

When a vitrified sample is heated over the glass transition temperature it may start to devitrify endangering the sample. The ability to estimate the stability of the vitrified state can help in the development of new vitrification media as well as handling procedures. By employing differential scanning calorimetry, we can measure the ice crystallization rate in a vitrified sample and thus study the devitrification kinetics. Using this technique, we have studied samples comprised of PBS with cryoprotective additives (CPA) as dimethylsulfoxide (Me₂SO), ethylene glycol (EG) and mixtures thereof, regarding the dependence of the devitrification kinetics on the CPA concentration. We found that already small concentration changes lead to significant changes in the devitrification times. Changing the CPA concentration by 4 wt% changed the devitrification time with a factor of 342 and 271 for Me₂SO and EG, respectively. Concentration changes in EG/Me₂SO mixtures was found to have a smaller impact on the devitrification kinetics compared to the pure CPA samples. Our data suggest that these significant increases in the devitrification times are primarily due to a relation between nucleation rates and the CPA concentration. Finally, we investigated an established vitrification medium used to preserve human embryonic stem cells. This medium was found to have the poorest glass stability in this study and reflects the tradeoff between stability and biocompatibility. The present work finally provides a tool to evaluate handling and storage procedures when employing vitrification as a cryopreservation method and underlines the importance of these.     

The dielectric properties of aqueous solutions of poly(ethylene glycol) and their influence on membrane structure

The dielectric constant of water is reduced drastically on addition of poly(ethylene glycol). The behaviour is not described by a linear mixture equation. The decreased dielectric constant can lead to the general perturbation of the membrane structure which is necessary in such a manner that a strong aggregation of membranes would lead to their fusion. The changed cation permeability in the presence of poly(ethylene glycol) can explained as the effect of the lowered dielectric constant on the transfer energy.     

Pregnancy and conception rate after two intravaginal inseminations with dog semen frozen either with 5% glycerol or 5% ethylene glycol

The primary goal of this study was to compare the effects of 5% ethylene glycol (EG) and 5% glycerol (G) on fertility of frozen–thawed dog semen following intravaginal insemination. The sperm-rich fraction of the ejaculate of three male dogs was collected, pooled and divided into two aliquots, and then frozen with a Tris–glucose–egg yolk–citric acid extender containing either 5% G or 5% EG. A total of 10 bitches were inseminated twice, five with G-frozen–thawed semen and five with EG-frozen–thawed semen; intravaginal inseminations were performed the 4th and the 5th day after the estimated LH peak; four straws, thawed in a 37 °C water bath for 1 min and diluted in a Tris buffer, were used for insemination (200 × 10⁶ spermatozoa); the insemination dose was introduced in the cranial vagina of the bitch using a sterile plastic catheter. Ovariohysterectomy was performed in all bitches between days 29 and 31 after the calculated LH surge, and pregnancy status, and the number of conceptuses and corpora lutea were recorded. All bitches were pregnant. Neither the number of conceptuses, nor the ratio of conceptuses to corpora lutea (conception rate) was significantly different between groups. In this first screening, with a limited number of bitches, EG-frozen semen did not show a higher fertility than G-frozen semen when used for two intravaginal inseminations. Irrespective of the semen used, conception rate was 0.50.     

两种冷冻保护剂对小鼠2-细胞胚冷冻效果的比较

乙二醇（ETG）和1,2-丙二醇（PROH）具有高细胞渗透性和低毒性特点,常被用于人及多种哺乳动物早期胚胎冷冻保存。为了比较ETG和PROH对小鼠2-细胞胚的冷冻保护效果,本试验分别采用这两种冷冻保护剂,对小鼠2-细胞胚进行冷冻保存,并采用冻后体外培养和囊胚移植进行冷冻效果检测。结果表明,PROH组胚胎解冻后胚胎存活率与ETG组无显著差异,但PROH组4-细胞胚发育率和囊胚发育率显著高于ETG组（82．7％vs．64．6％,61．2％vs．29．1％,P〈0．01）。囊胚移植结果表明,2-细胞胚胎冻存后能够发育为正常的后代,PROH组和ETG组的囊胚移植后妊娠产仔率无统计学差异（P〉0．05）,但均显著低于对照组（P〈0．05）。为了分析两组胚胎冻存后损伤情况,埘解冻后的胚胎细胞微丝进行检测,结果显示ETG组微丝受损的胚胎数高于PROH组。本研究结果证明采用PROH作为冷冻保护剂冷冻保存小鼠2-细胞胚的冻存效果优于ETG[动物学报54（6）：1098—1105,2008]。