One-step Dilution Research Articles

Comparison of the Efficacy of Conventional Slow Freezing and Rapid Cryopreservation Methods for Bovine Embryos

Day 7 bovine morulae and early blastocysts were randomly assigned to one of four cryopreservation methods: (i) a modified conventional controlled slow freezing and stepwise dilution after thawing; and three methods which enable direct transfer of the embryo into the recipient upon thawing: (ii) conventional controlled slow freezing and a modification of a one-step procedure, (iii) vitrification with 6.5 M glycerol plus 6% BSA (w/v), and (iv) vitrification with 25% glycerol (v/v) and 25% propanediol (v/v). In a comparative in vitro study, the percentage of grade 1 and 2 embryos developing into expanded blastocysts in culture for cryopreservation methods 1-4 were, respectively, 53% (29/55), 33% (20/61), 44% (26/59), and 51% (17/33). Method 2 yielded a significantly lower survival rate than methods 1 ( P < 0.1) and 4 ( P < 0.05) and was excluded from a subsequent test of in vivo development. Pregnancy rates (Day 60) after transfer of embryos cryopreserved by methods 1, 3, and 4 were, respectively, 59% (20/34), 43% (17/40), and 24% (5/21). Method 4 yielded a significantly lower pregnancy rate than method 1 ( P < 0.05). Method 3, however, did not yield a statistically different pregnancy rate ( P > 0.1) when compared to method 1. Method 3 has considerable promise in providing a successful method for the cryopreservation of bovine embryos that (i) reduces the time required for equilibration and cooling, (ii) provides for simple and rapid one-step dilution of cryoprotectant after thawing, and (iii) enables more embryos to be thawed and transferred per unit time.

Pregnancy rates in a comparative field trial of vitrification and one-step dilution or conventional slow freezing and three-step dilution of bovine embryos are similar

Pregnancy rates in a comparative field trial of vitrification and one-step dilution or conventional slow freezing and three-step dilution of bovine embryos are similar

Pregnancy rate and survival in culture of in vitro fertilized bovine embryos frozen in various cryoprotectants and thawed using a one-step system

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours (38.5°C, 5% CO 2) in modified TCM-199 medium supplemented with 5% superovulated cow serum (SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3 M methyl cellosolve (MC), 1.1 M diethylene glycol (DEG), 1.8 M ethylene glycol (EG), 1.6 M propylene glycol (PG) and 1.1 M 1, 3-butylene glycol (BG) solutions. They were then loaded into 0.25-ml straws, placed into an alcohol bath freezer at 0°C, cooled from 0°C to −6°C at −1°C/minute, seeded, held for 10 minutes, and cooled again at −0.3°C or −0.5°C/minute to −30°C. Straws were then plunged and stored in liquid nitrogen. After thawing in 30°C water, the embryos were rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred nonsurgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows: EG (50.0%), MC (53.6%), DEG (56.9%), PG (58.0%) and BG (11.5%). The survival rate of embryos cooled at −0.3°C vs −0.5°C/minute was not significantly different (P>0.05), however, blastocysts hatched most often (P<0.01) in vitro when cooled at a rate of −0.3°C/minute (64.6%, 31 48 ) than at −0.5°C/minute (22.6%, 12 53 ). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows: MC (48%, 10 21 ); DEG (30%, 3 10 ); EG (74%, 20 27 ); and PG (40%, 4 10 ). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF bovine embryos.

Slow and ultrarapid freezing of fully grown germinal vesicle-stage mouse oocytes: optimization of survival rate outweighed by defective blastocyst formation.

The cryopreservation of mature metaphase II-stage mouse oocytes is associated with decreased fertilizability, spindle damage, and increased polyploidy. Therefore, we investigated the outcome of cryopreservation of immature germinal vesicle-stage mouse oocytes. Oocytes were punctured from Graafian follicles in primed F1 hybrid mice and were then released into maturation medium containing the meiotic inhibitor dibutyryl cyclic AMP. Both slow and ultrarapid freezing protocols with dimethyl sulfoxide, 1,2-proponediol, or a mixture of both agents were tested. We recorded morphological survival rates, in vitro maturation rates, and two-cell and blastocyst formation rates. Each group of frozen oocytes was compared with both unfrozen germinal vesicle-stage oocytes and metaphase II-stage oocytes. An optimal cryosurvival rate of 78% was reached after ultrarapid freezing with 3 M dimethyl sulfoxide followed by one-step dilution, but a decreased rate of two-cell formation was observed. Freezing with a combination of dimethyl sulfoxide and 1,2-propanediol did not improve this fertilization-decreasing effect. Very low cryosurvival rates after freezing with 1,2-propanediol indicated its inappropriateness for ultrarapid freezing of immature oocytes. The rates of in vitro maturation were equivalent for frozen-thawed and freshly collected germinal vesicle-stage oocytes, independent of the freezing protocol used. We report, nevertheless, as a general characteristic for both slow and ultrarapid freezing of fully grown germinal vesicle-stage oocytes, that the in vitro development up to the blastocyst stage is inhibited despite full nuclear maturation. We report that cryopreservation of immature germinal vesicle-stage oocytes is invariably associated with a low developmental capacity after fertilization. The rate of in vitro nuclear maturation did not equate with developmental competence. This in turn suggests the importance of cytoplasmic maturation for embryonic development.

Ambient temperature gas purifier suitable for the trace analysis of carbon monoxide and hydrogen and the preparation of low-level carbon monoxide calibration standards in the field

A novel gas purifier based upon Sofnocat 682, a catalyst containing platinum and palladium on a hydrophobic tin oxide support, is described for the quantitative removal at ambient temperatures of ppm (v/v) and sub-ppm (v/v) levels of carbon monoxide and hydrogen from air and nitrogen gas cylinders. This method provides a simple means of generating either a laboratory or field source of “zero grade” gas for both the trace analysis of carbon monoxide and hydrogen and the preparation of working calibration gas standards of carbon monoxide by using a simple one-step dilution of a higher concentration certified gas standard.

In search of an optimal dilution algorithm for feedforward networks

In a recent paper the authors presented a dilution algorithm that yields a storage capacity per synapse alpha eff larger than 2. In this letter, two new algorithms with even higher alpha eff values are introduced. They study a one-step dilution algorithm, where the perceptron is used to select a fraction of the couplings to be removed. For the remaining bonds the perceptron of optimal stability is relearned. They further compare simulation data from an iterative version of the one-step dilution algorithm with phase-space volume calculations.

Viability of vitrified mouse embryos using various cryoprotectant mixtures

Mouse morulae and blastocysts were cryopreserved by vitrification using six types of solutions. Each solution was composed of two types of cryoprotectants, glycerol (GL) + ethylene glycol (EG), GL + propylene glycol (PG), GL + dimethyl sulfoxide (DMSO), EG + PG, EG + DMSO, and PG + DMSO at an each cryoprotectant concentration of 25% v/v. Embryos were exposed to each type of vitrification solutions, which had been diluted 50% in PBS, for 5 minutes at room temperature, then for another 5 minutes at 4 °C. The embryos were loaded into straws containing vitrification solution at 4°C and plunged into liquid nitrogen within 30 seconds. After warming in water at 0°C and following one-step dilution of the cryoprotectant in 0.5 M sucrose+PBS, the embryos were cultured in vitro. The survival rates of morulae were 51, 16, 78, 44, 79 and 50%, respectively, for the six solutions. The survival rates of the morulae using GL + DMSO and EG + DMSO were significantly higher than those of the other solutions (P<0.01). The survival rates of blastocysts were 72, 29, 55, 46, 79 and 46%, respectively, for the six solutions. The survival rates using GL + EG and EG + DMSO were significantly higher than those of the other solutions (P<0.05). A high in vitro survival rate was obtained when both morulae and blastocysts were vitrified using EG + DMSO. Therefore, embryos vitrified in EG + DMSO were transferred to recipient mice, and their development into live fetuses was investigated. The in vivo development rates of morulae and blastocysts were 34 and 49%, respectively. These values were not significantly different from those of fresh control embryos.

Glycerol-sucroseで凍結したウシ体外受精由来胚のOne step希釈による移植成績

Glycerol-sucroseで凍結したウシ体外受精由来胚のOne step希釈による移植成績

The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method

The survival of whole and bisected rabbit morulae cryopreserved by the vitrification method was investigated. The embryos were loaded in a column of vitrification solution (VS, a mixture of 25% glycerol and 25% 1, 2-propanediol in PBS+16% calf serum), which was located between two columns of 1 M sucrose solution in a plastic straw. The embryos were frozen by being plunged into liquid nitrogen and thawed in a water bath at 20 degrees C. Two methods of loading embryos into straws were used: the single and double column vitrification solution methods. The embryonic survival rates between these two methods were compared. Seventy-one (86.6%) out of 82 morulae vitrified in double column straws developed into the blastocyst stage in vitro. Eleven (18.3%) live fetuses were obtained after the transfer of 60 frozen-thawed morulae to four recipients. By contrast, the survival rate (36.5%, 27 74 ) of embryos vitrified in the single column straws was significantly lower (P<0.05). The vitrification solution of the single column straws became opaque, indicating ice-crystal formation, upon thawing in 5 of 11 straws, which was assumed to have damaged the embryos. More than 80% (29 36 ) of the bisected morulae frozen and thawed in the double column straws developed to the blastocyst stage in vitro when cryoprotectant was diluted stepwise with 1 M and 0.25 M sucrose solution. When the cryoprotectant was removed by one-step dilution with 1 M sucrose solution, swelling in blastomeres was observed and the development rate of the recovered embryos decreased (45.8%, 11 24 ). These results indicate that the vitrification method employed in our experiment is not only efficient for the cryopreservation of rabbit morulae, but it can also be used for the preservation of bisected rabbit morulae, which had not been successful using previous methods.

Quick freezing of mouse embryos using ethylene glycol with lactose or sucrose

Mouse compacted morulae were quickly frozen in liquid nitrogen (LN 2) vapor at approximately −170°C after 5 min equilibration in 2 M or 3 M ethylene glycol or glycerol in the presence of either 0.25 M lactose or sucrose to examine their post-thaw viability. After thawing in a water bath at 37°C and a one-step dilution of the cryoprotectant, the embryos were cultured for 48 h. Significantly higher in vitro survival rates were obtained with 3 M ethylene glycol than with 2 M ethylene glycol or 2 or 3 M glycerol irrespective of the sugar used ( P < 0.001). However, 2 M ethylene glycol with lactose gave a significantly higher survival rate than in combination with sucrose ( P < 0.05). On the contrary, 3 M glycerol with sucrose gave a significantly higher survival rate than when combined with lactose ( P < 0.001). The transfer of frozen-thawed embryos cryopreserved with 3 M ethylene glycol with 0.25 M lactose or 0.25 M sucrose resulted in the development of normal fetuses at rates of 51.7% and 47.7%, respectively, as compared to 73.8% for fresh embryos. This study suggests that a higher concentration of ethylene glycol in combination with lactose or sucrose is an effective cryoprotective agent during quick freezing.

Effect of sucrose concentration used for one-step dilution upon in vitro and in vivo survival of bovine embryos refrigerated in glycerol and 1, 2-propanediol

Bovine embryos collected on Day 7 of pregnancy were suspended in 1.4 M glycerol or 1.6 M 1, 2-propanediol solution. Embryos were loaded into 0.25-ml plastic straws in phosphate buffered saline (PBS) containing 0, 0.2, 0.4 or 0.8 M sucrose. The straws were placed directly into a cooling chamber and cooled from 0°C to −6°C at 1°C/min and then seeded at −6°C and cooled at a rate of 0.3°C/min to −30°C. The straws were then plunged and stored in liquid nitrogen. Embryos frozen in each cryoprotectant were thawed by placing the straws into a 30°C water bath. The embryos were cultured in small dishes in Ham's F-10 medium supplemented with 10% fetal calf serum (FCS). When the embryos were frozen in 0.0, 0.2, 0.4 or 0.8 M sucrose, respectively, in Ham's F-10 the survival rates after culture were 0, 21, 82 or 88%, respectively, in 1.4 M glycerol and 88, 89, 84 or 72%, respectively, in 1.6 M 1, 2-propanediol. Embryos suspended in both cryoprotectants were cooled with 0.2 M sucrose in PBS, and the nonsurgical transfers were performed without removing the cryoprotectant. A pregnancy rate of 61% was obtained when embryos were suspended in 1.6 M 1, 2-propanediol and cooled with 0.2 M sucrose in PBS. However embryos suspended in 1.4 M glycerol and subjected to the same cooling regimen had a lower (P<0.01) developmental rate in vivo following nonsurgical transfer of embryos without the removal of cryoprotectant. These results indicate that 1, 2-propanediol can be used successfully to freeze bovine embryos and that its dilution from bovine embryos can occur in the absence of or at low levels of sucrose.

Cryopreservation of murine embryos with trehalose and glycerol

Several concentrations of trehalose (0.0, 0.04, 0.1, 0.25 M) in combination with three concentrations of glycerol (1.0, 1.5, 2.0 M) were evaluated for the Cryopreservation of murine embryos. Embryos were transferred through increasing concentrations of glycerol in Dulbecco's phosphate-buffered saline with 10% fetal calf serum (PBS + PCS) to reach the final glycerol concentrations. They were then randomly assigned to one of the concentrations of trehalose. A total of 506 morulae were packaged individually in 0.25-ml plastic straws and cooled from ambient temperature at 1.0 °C/min in a programmable methanol freezer. Embryos were seeded at −7 °C and then cooled to −25 °C at 0.3 °C/min before being plunged into liquid nitrogen. After thawing and a one-step dilution of glycerol, embryos were cultured for 48 hr and viability was determined by blastocoel formation. Highest viability (70.0%) after 48 hr in culture was obtained for embryos frozen in 1.5 M glycerol plus 0.10 M trehalose as compared to 31% viability for embryos frozen with glycerol alone. These observations suggest that trehalose can be used in combination with glycerol as a cryoprotectant and that a high rate of viability can be achieved after a one-step dilution of the cryoprotectants.

The effects of 1,2-propanediol as a cryoprotectant on the freezing of mouse oocytes

Cryopreservation of mammalian eggs has been successfully accomplished using 1,2-propanediol (PG). Effects of holding times of 0 and 30 min at −40° C and storage times of 1 d and 1 mo at −196° C were investigated in combination with various concentrations of PG (1.0, 1.5, and 2.0M) to determine the survival and fertilizability of mouse oocytes rapidly frozen and thawed in straws. A rapid one-step dilution using 0.5 M sucrose solution inside the straws was used following the thawing of oocytes. A significant effect of PG concentration was found between 1.0 M and 1.5 or 2.0 M (P<0.01), but no significance was discovered between 1.5 M and 2.0 M (P>0.05) on subsequent survival and fertilizability of frozen and thawed mouse oocytes. With 2.0 M PG, the best survival rate (58.3%) and fertilizability rate (19.0%) were obtained by holding at −40° C for 30 min and by storage at −196° C for 1 d. Thirty minutes of holding at −40° C reduced oocyte damage during the procedure but not significantly (P>0.05). In addition, there was no significant difference in the various storage periods (P>0.05). This study demonstrated that mammalian oocytes can be cryopreserved in the presence of 1,2-propanediol by utilizing a rapid freezing and thawing procedure.

Direct Determination of Benzalkonium Chloride in Ophthalmic Systems by Reversed-Phase High-Performance Liquid Chromatography

High-performance liquid chromatography has been used to quantitate benzalkonium chloride (alkylbenzyldimethylammonium chioride) in complex ophthalmic formulations at or below concentration levels of 50ppm. The method involves a one-step dilution for sample preparation and direct injection; therefore, recovery and/or conversion problems are nonexistent. The assay is quick, specific, reproducible, and simple. This new approach makes routine determinations far simpler than previous methods and is especially useful for product stability studies and quality control procedures.

Oriented reconstitution of red cell membrane proteins and assessment of their transmembrane disposition by immunoquenching of fluorescence

The two major membrane glycoproteins of human red cells, glycophorin and band 3, the anion exchange protein, were isolated from cells exofacially labeled with fluorescein and reconstituted into vesicles with defined transmembrane disposition. Uniform orientation of polypeptides was accomplished by two procedures: (i) Vesicles with single protein units were obtained by a one-step dilution of a protein/detergent suspension with a vast excess of phospholipid. Vesicles with uniform orientation of protein were selected by affinity chromatography on derivatized Sepharoses (organomercurial, wheat germ agglutinin, aminoethyl or diethylaminoethyl). (ii) Vesicles with multiple protein units with uniform orientation were generated by vectorial immobilization of detergent solubilized proteins on the above affinity matrices and in situ formation of proteoliposomes by detergent substitution for phospholipid. The proteoliposomes were released from the column by addition of excess free ligand. The orientation of band 3 and glycophorin in the reconstituted vesicles was first assessed by immunofluorescence quenching, using anti-fluorescein antibodies, to quantitatively quench fluorescein residues exposed on the outer surface of vesicles. Further assessment was achieved by chromatographing the vesicles through various affinity and ionic matrices. Vesicle populations of higher than 90% homogeneity in protein orientation (right-side-out or inside-out) were obtained with both procedures. The above methods provide a convenient experimental tool for the oriented reconstitution of proteins and the evaluation of their transmembrane disposition.

Poliovirus aggregates and their survival in water

Inactivation of aggregated poliovirus by bromine is characterized by a continuously decreasing reaction rate. Poliovirus released from infected cells in these experiments by alternate freezing and thawing in water without electrolytes has always been aggregated. The aggregates persist even on 7,000-fold dilution in ion-free water. Virus similarly released into phosphate-buffered saline solution may be well dispersed, but it aggregates when sedimented into a salt-free sucrose gradient or when it is diluted as little as 10-fold in water. Large one-step dilutions of dispersed virus in water remain dispersed. Aggregated virus was not dispersed by one-step dilution (7,000-fold) in distilled or untreated lake water but was dispersed if phosphate-buffered saline or clarified secondary sewage plant effluent was used as diluent. Dispersed virus aggregates at all dilutions in alum-treated, finished water from the city filter plant. This may be the result of complex formation with insoluble material rather than virion-virion aggregation. A simple procedure is described for rendering a very dilute suspension of mixed virion aggregates into a three-part spectrum of sizes.