Development of OPS vitrified pig blastocysts: effects of size of the collected blastocysts, cryoprotectant concentration used for vitrification and number of blastocysts transferred

Unhatched blastocysts from Large White hyperprolific gilts (n=103) were identified, measured and vitrified using the Open Pulled Straw (OPS) technique to evaluate the effects of the collected blastocyst size and cryoprotectant concentrations used for vitrification, and the number of embryos transferred per recipient. Vitrified/warmed blastocyst viability was estimated in vitro, as the percentage of embryos developing after 72h, and in vivo, on pregnancy Day 30. In the in vitro study, we compared the use of three cryoprotectant concentrations (16.5, 18, or 20% DMSO+16.5, 18, or 20% EG+0.4M sucrose). Survival rates differed significantly between the control (98.3%) and the three cryoprotectant concentrations (67, 62.3, and 57%, respectively). Blastocyst size at vitrification determined the further in vitro development of embryos (26% survival for blastocysts 126-144microm versus 100% for blastocysts >199microm). For the in vivo study, blastocysts were vitrified using cryoprotectant concentrations of 16.5 or 18% DMSO+EG and transferred surgically in groups of 20 or 30 per recipient (n=40). Recipients were slaughtered on pregnancy D30. No significant differences were detected in gestation rates (50-70%) and embryo survival rates (14.7-25%), although survival was higher (P=0.0003) when 20 blastocysts were transferred compared to 30 (24.7% versus 15.5%). Our findings indicate that best results, in terms of subsequent in vivo embryo survival, were achieved after transferring 20 embryos at the blastocyst or expanded blastocyst stage, previously vitrified using cryoprotectant concentrations of 16.5 or 18%.     

Immature bovine oocyte cryopreservation: comparison of different associations with ethylene glycol, glycerol and dimethylsulfoxide

Research on different cryoprotectants and their associations is important for successful vitrification, since greater cryoprotectant concentration of vitrification solution may be toxic to oocytes. The aim of the present research was to compare the efficiency of immature bovine oocyte vitrification in different associations of ethylene glycol (EG), glycerol and dimethylsulfoxide (Me(2)SO). In the first experiment, oocytes were exposed to the cryoprotectant for either 30 or 60s in final solutions of EG+DMSO1 (20% EG+20% Me(2)SO) or EG+DMSO2 (25% EG+25% Me(2)SO) or EG+GLY (25% EG+25% glycerol). In the second experiment, the oocytes were vitrified in open pulled straws (OPS) using 30s exposure of final solutions of EG+DMSO1 or EG+DMSO2 or EG+GLY. Maturation rates of 30s exposure groups were not different from the control, but 60s cryoprotectant exposure was toxic, decreasing maturation rates. The vitrification with EG+DMSO2 resulted in enhanced maturation rate (29.2%) as compared with EG+DMSO1 (11.7%) and EG+GLY (4.3%) treatments. These data demonstrate that concentration and type of cryoprotectant have important effects on the developmental competence of vitrified oocytes.     

Cryoprotectants and components induce plasmolytic responses in sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) suspension cells

Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove cellular water and prevent ice crystallization, ensuring survival after liquid nitrogen exposure. Vitrification solutions can be comprised of a combination of components that are either membrane permeable or membrane impermeable within the timeframe and conditions of cryoprotectant exposure. In this study, the osmotic responses of sweet potato [Ipomoea batatas (L.) Lam.] suspension cell cultures were observed after treatment with plant vitrification solution 2 [PVS2; 15% (v/v) dimethyl sulfoxide (DMSO), 15% (v/v) ethylene glycol, 30% (v/v) glycerol, 0.4 M sucrose], plant vitrification solution 3 (PVS3; 50% (v/v) glycerol, 50% (w/v) sucrose), and their components at 25 and 0°C, as well as cryoprotectant solution, PGD (10% (w/v) PEG 8000, 10% (w/v) glucose, 10% (v/v) DMSO) at 25°C. At either 25 or 0°C, sweet potato cells plasmolyzed after exposure to PVS2, PVS3, and PGD solutions as well as the PVS2 and PVS3 solution components. Cells deplasmolyzed when the plasma membrane was permeable to the solutes and when water re-entered to maintain the chemical potential. Sweet potato suspension cells deplasmolyzed in the presence of 15% (v/v) DMSO or 15% (v/v) ethylene glycol. Sweet potato plasma membranes were more permeable to DMSO and ethylene glycol at 25°C than at 0°C. Neither sucrose nor glycerol solutions showed evidence of deplasmolysis after 3 h, suggesting low to no membrane permeability of these components in the timeframes studied. Thus, vitrification solution PVS2 includes components that are more membrane permeable than PVS3, suggesting that the two vitrification solutions may have different cryoprotectant functions. PGD includes DMSO, a permeable component, and likely has a different mode of action due to its use in two-step cooling procedures.     

Effect of cryopreservant combinations on the motility and morphology of curimba (Prochilodus lineatus) sperm

This study investigated the application of intra- and extra-cellular cryoprotectant combinations on the quality of curimba Prochilodus lineatus semen subjected to cryopreservation. Semen treatments were tested with 8% DMSO or methanol as intracellular cryoprotectant, 5% egg yolk or lactose as extracellular cryoprotectant and 5% BTS. These cryoprotectant combinations are suitable for curimba but have not been tested at the lesser concentrations proposed or in combination with BTS. Semen samples collected from 19 curimbas were diluted into one of four cryoprotectant combinations: DMSO+yolk; DMSO+lactose; methanol+yolk; and methanol+lactose. After dilution, semen samples were cryopreserved in 0.5 mL straws for 10 days in a liquid nitrogen tank. Semen was thawed in a water bath at 60°C for 8s. We evaluated the quality of fresh, diluted (pre-freezing) and post-freezing semen according to sperm motility rate (%) and duration (s). Sperm morphology was also analyzed in thawed semen. Sperm motility rate decreased progressively after dilution and thawing. The motility rate in post-freezing semen was higher in the treatments using DMSO+lactose and methanol+yolk. Sperm motility duration in post-freezing sperm was greater in the treatments using methanol rather than DMSO as intracellular cryoprotectant, irrespective of the extracellular cryoprotectant used. Abnormality frequency in thawed sperm was less in semen treated with egg yolk than with lactose. Thus the use of methanol intracellular cryoprotectant is recommended along with yolk extracellular cryoprotectant in the cryopreservation process for curimba semen.     

Effect of an interaction between haematocrit and cryoprotectant concentration on freeze-thaw haemolysis of bovine erythrocytes.

The effect of the haematocrit and cryoprotectant concentration on freeze-thaw haemolysis of bovine red cells was studied. Two-milliliter samples of bovine blood with an haematocrit of either 20 or 60% were diluted with 2 ml of either 5, 4, 3, 2, or 1 m glycerol or DMSO in PBS or with PBS alone. The degree of haemolysis after freezing to ?79 °C and thawing was least in blood diluted with 4m cryoprotectant. At the lower concentrations of cryoprotectant, haemolysis was greater in blood with the higher haematocrit, but this effect decreased as the cryoprotectant concentration was increased and was negligible at the optimal concentration.     

Determination of residual dimethylsulfoxide in cryopreserved cardiovascular allografts

Dimethylsulfoxide (DMSO) is a solvent which protects the structure of allografts during the cryopreservation and thawing process. However, several toxic effects of DMSO in patients after transplantation of cryopreserved allografts have been described. The aim of this study is to determine the residual DMSO in the cardiovascular allografts after thawing and preparation of cryopreserved allografts for clinical application following guidelines of the European Pharmacopoeia for DMSO detection. Four types of EHB allografts (aortic valve-AV, pulmonary valve-PV, descending thoracic aorta-DA, and femoral artery-FA) are cryopreserved using as cryoprotecting solution a 10% of DMSO in medium 199. Sampling is carried out after thawing, after DMSO dilution and after delay of 30 min from final dilution (estimated delay until allograft implantation). After progressive thawing in sterile water bath at 37–42 °C (duration of about 20 min), DMSO dilution is carried out by adding consecutively 33, 66 and 200 mL of saline. Finally, tissues are transferred into 200 mL of a new physiologic solution. Allograft samples are analysed for determination of the residual DSMO concentration using a validated Gas Chromatography analysis. Femoral arteries showed the most important DMSO reduction after the estimated delay: 92.97% of decrease in the cryoprotectant final amount while a final reduction of 72.30, 72.04 and 76.29% in DMSO content for AV, PV and DA, was found, respectively. The residual DMSO in the allografts at the moment of implantation represents a final dose of 1.95, 1.06, 1.74 and 0.26 mg kg^?1 in AV, PV, DA and FA, respectively, for men, and 2.43, 1.33, 2.17 and 0.33 mg kg^?1 for same tissues for women (average weight of 75 kg in men, and 60 kg in women). These results are seriously below the maximum recommended dose of 1 g DMSO kg^?1 (Regan et al. in Transfusion 50:2670–2675, 2010) of weight of the patient guaranteeing the safety and quality of allografts.     

Cryopreservation of four valuable strains of microalgae, including viability and characteristics during 15?years of cryostorage

Recent developments in the technology of cryopreservation have permitted the long-term storage of many strains of microalgae with reliable rates of survival. However, many strains still cannot be recovered from storage in liquid nitrogen. Here, we investigated the effects of various cryoprotectants in achieving comparatively high survival rate around 50%. The strains tested included two freshwater algae, Chlorella vulgaris C-27 and M-207A7, and two marine algae, Nannochloropsis oculata ST-4 and Tetraselmis tetrathele T-501. Cells of these strains were suspended in various cryoprotective solutions and slowly cooled to ?40°C prior to immersion in liquid nitrogen. Little or no cryoprotection was seen with dimethyl sulfoxide (DMSO) alone or in combination with sorbitol or proline; with glycerol alone; or with ethylene glycol (EG) alone. However, survival rates of approximately 50% were observed using a cryoprotectant mixture of 5% DMSO, 5% EG, and 5% proline. Viability persisted during a storage period of 15?years. Similarly, chlorophyll content was not significantly changed during this 15-year interval. Thus, the present study demonstrates the advantage of cryopreservation using liquid nitrogen. We expect that this method will contribute to both basic and applied biology through the establishment of cryopreserved microalgal culture collections.     

Successful cryopreservation of mouse blastocysts using a new vitrification solution.

Mouse blastocysts were exposed to solutions containing four concentrations (10, 20, 30 and 40% v/v) of six permeating cryoprotectants (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide, 1,3-butanediol and 2,3-butanediol) in phosphate-buffered saline (PBS) with calf serum (CS) at room temperature (20-22 degrees C). Blastocysts were exposed to these solutions for various periods, diluted into PBS plus CS with or without 1 mol trehalose l-1 solution and their subsequent survival in vitro was examined. Two-way anova showed a significant interaction (P < 0.01) between cryoprotectant type, concentration of cryoprotectant and method of dilution. However, no significant interaction was observed between cryoprotectant type and duration of exposure. Results suggest that cryoprotectant-induced injury to nonfrozen blastocysts is variable and depends on the cryoprotectant used. On the basis of toxicity assays, ethylene glycol was the least harmful and was combined with dimethyl sulfoxide and 1,3-butanediol to produce a new vitrification solution. Mouse blastocysts were successfully cryopreserved using a vitrification solution (designated as VSv) consisting of 20% ethylene glycol, 20% dimethyl sulfoxide and 10% 1,3-butanediol (v/v). Embryos were equilibrated in two steps, first in an equilibration solution (designated as ESv: 10% ethylene glycol, 10% dimethyl sulfoxide and 5% 1,3-butanediol; v/v) and then to VSv or one-step in VSv at different exposure times at room temperature, and then vitrified by direct plunging into liquid nitrogen. High developmental rates were obtained in vitro when the embryos were exposed to ESv and VSv for 3 and 0.5 min, respectively (96.2%) or exposed to VSv for 0.5 min (95.4%). Prolonged exposure time proved detrimental to subsequent embryo development in vitro. When vitrified warmed embryos were transferred immediately to pseudopregnant recipients, the rate of development to normal fetuses did not significantly differ from that of the nonvitrified control (two-step, 54.2 and one-step, 45.0 versus 60.0%, P > 0.05). These results suggest that the simple vitrification solution described in this study is effective for the cryopreservation of mouse blastocysts.     

In-straw cryoprotectant dilution of IVP bovine blastocysts vitrified in hand-pulled glass micropipettes

The aim of this study was to determine the influence of two ethylene glycol-based vitrification solutions on in vitro and in vivo survival after in-straw cryoprotectant dilution of vitrified in vitro-produced bovine embryos. Day-7 expanded blastocysts were selected according to diameter (> or = 180 microm) and osmotic characteristics and randomly assigned to one of three groups (i) VSa: vitrification in 40% EG+17.1% SUC+0.1% PVA; (ii) VSb: vitrification in 20% EG+20% DMSO; (iii) control: non-vitrified embryos. Vitrification was performed in hand-pulled glass micropipettes (GMP) and cryoprotectant dilution in 0.25 ml straws after warming in a plastic tube. Embryo viability was assessed by re-expansion and hatching rates after 72 h of IVC and by pregnancy rates after direct transfer of vitrified embryos. No differences in re-expansion rates were observed between vitrified groups after 24 h in culture (VSa=84.5%; VSb=94.8%). However, fewer VSa embryos (55.2%, P<0.05) hatched after 72 h than the VSb (75.8%) and control embryos (80.0%). To evaluate in vivo viability, vitrified embryos (VSa=20; VSb=21) were warmed under field conditions and individually transferred to synchronous recipients. Pregnancy rates (day 60) were similar between groups (VSa=20%; VSb=19%). Greater hatching rates occurred after 72 h of IVC for EG+DMSO than EG+SUC+PVA vitrification solutions. However, using a GMP vitrification container and in-tube warming, both solutions provided similar pregnancy rates after the in-straw cryoprotectant dilution and direct embryo transfer.     

Potential antifouling strategies for marine finfish aquaculture: the effects of physical and chemical treatments on the settlement and survival of the hydroid Ectopleura larynx

The hydroid Ectopleura larynx is a common fouling organism on aquaculture nets. To contribute to the development of novel cleaning methods, laboratory and field studies determined the effects of heat (30, 40, 50 and 60°C for immersion times of 1 and 3 s) and acetic acid (0.2 and 2.0% for immersion times of 1, 3 and 10 s, 1 and 5 min) on the settlement of actinulae and the survival of juvenile and adult E. larynx. Laboratory studies showed that, regardless of immersion time, a temperature of 50°C was effective in preventing the settlement of actinulae and the survival of juveniles, while ≤12% of adult hydroids could survive. A temperature of 60°C killed all adult hydroids. For an acetic acid concentration of 0.2%, an immersion time of 1 min substantially reduced the settlement of actinulae and the survival of juvenile and adult hydroids, and none of the juvenile and adult hydroids survived after 5 min. For an acetic acid concentration of 2.0%, all immersion times were effective and reduced the mean settlement of actinulae and the survival of juvenile and adult hydroids to ≤10%. Field studies with fouled net panels exposed to selected heat or acetic acid treatments showed small reductions in mean wet weight and net aperture occlusion of the net panels 2 and 5 days after treatment. Visual inspections of the net panels showed that hydranths of the hydroids were shed, but the dead stolons of the hydroids remained on the treated net panels. Novel cleaning methods and devices may utilise these results to effectively kill E. larynx on aquaculture nets, while further studies are needed to determine the necessity of removing the dead hydroids before further biofouling accumulates on thenets.     

Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid frozen-thawed mature mouse oocytes

Experiments were conducted to develop a simple rapid-freezing protocol for mature mouse oocytes that would yield a high proportion of oocytes with developmental potential. The effects of concentration (3.5, 4.5 and 6.0 M dimethyl sulfoxide (DMSO) all with 0.5 M sucrose) and the duration of exposure (2.5 min vs 45 sec) of oocytes to the cryoprotectant and its extraction after thawing in 2, 3 or 4 steps of descending sucrose concentration were studied. The most effective of the rapid-freezing and thawing protocols (4.5 M DMSO; 45 sec exposure and 3-step thawing) was compared to slow freezing protocols using 1.5 M DMSO and 1.0 M 1,2 propanediol as cryoprotectants. The DMSO concentrations had an effect on survival, fertilization and embryo development using short (45 sec) but not long (2.5 min) exposure. The rate of morphological oocyte survival was significantly higher using 4.5 M DMSO than 3.5 or 6.0 M (92% vs 82 and 73%, respectively). The development of fertilized embryos to blastocysts was also significantly higher at 4.5 M than at 3.5 or 6.0 M (68% vs 42 and 53%, respectively). The extraction of cryoprotectant in 3 or 4 steps of descending sucrose concentration resulted in higher survival (P < 0.01) and fertilization than in 2 steps. The best survival, fertilization and development was achieved with the 3-step procedure. Optimal combinations of conditions were 4.5 M DMSO at 45 sec prefreeze exposure and 3-step extraction of the cryoprotectant. Oocytes frozen by conventional methods had a survival, fertilization and development to blastocyst rate significantly lower than those frozen under the optimal rapid conditions. Thus rapid freezing of mature mouse oocytes with 4.5 M DMSO + 0.5 M sucrose and short prefreeze exposure is effective and has the additional advantage of being less time-consuming than slow freezing methods.     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

Effect of cryoprotective agents on rat cutaneous nerves.

Desheathed rat cutaneous nerves were exposed to various concentrations of ethylene glycol (EG), glycerol and dimethyl sulfoxide (DMSO) at temperatures of 1, 24, and 38 °C for periods of time ranging from 5 to 60 min. Measurements of the percent recovery of the original action potential (AP) were determined after removal of the cryoprotective agent (CPA) under various conditions, i.e., temperature, time and sequence of rinsing. A comparison of the results obtained after the nerves were exposed directly to a 15% concentration of the three CPAs at 1 °C for a 15-min period showed that the percentage of recovery of the AP was 90, 69, and 36% of the original values when treated with DMSO, EG, or glycerol, respectively. In all three groups, the nerves were rinsed at 1 °C for 15 min. If the exposure to glycerol at 1 °C was carried out in a gradual stepwise manner, the recovery of the AP in 10 and 15% solutions ranged from 58 to 64%. If the temperatures of the exposure and rinse were increased to 24 and 38 °C, glycerol produced some toxicity within 10 min and after 25 min no recovery of AP was obtained. The results of a 10-min direct exposure to EG at 1 °C showed a moderate decrease in recovery of the AP as the concentration was increased from 10 to 15–20%. Increasing the exposure time to 15 and 30 min at 1 °C also contributed to further reduction in recovery. DMSO, however, in concentrations of 10, 15, and 20% produced only a slight decline of AP after a 5–15 min exposure at 1 °C. Recovery ranged from 96% after 10 min in a 10% solution to 88% after 15 min in a 20% solution. Toxicity became more apparent with DMSO when nerves were exposed to 30% concentrations for 5–10 min; the latter time resulted in a 49% recovery of the AP. Exposure of nerves to a CPA solution containing isotonic concentrations of electrolytes resulted in a 10–30% improvement in recovery when compared with specimens treated with lower levels of salt. The effect of raising the temperature of the rinse to 38 °C and increasing the wash time to 20 min was studied in a few selected experiments. After a direct 15-min exposure to a 15% solution of a CPA at 1 °C the recovery in the case of glycerol was significantly increased with such treatment whereas with EG and DMSO it remained unchanged. There was no evidence of thermal or cold shock in this work.     

三种不同冷冻保护剂对小鼠附睾冷冻效果的比较

目的探讨三种不同冷冻保护剂对C57BL/6J小鼠附睾冷冻的效果。方法性成熟并交配过的6~8周龄C57BL/6J雄鼠附睾,分别用3种不同冷冻保护剂二甲基亚砜(DMSO)、丙二醇(PROH)、R18S3进行玻璃化冷冻、复苏和精子采集,观察比较三个实验组的复苏精子形态、存活率以及生殖能力。结果三组冷冻复苏分离后的精子形态完整,具有镰刀头状结构;荧光染色后PROH组精子存活率为(88.17±3.43)%,明显高于DMSO组:(61.17±10.65)%和R18S3组(16.83±6.49)%(P0.05);经辅助体外受精后均具有受精能力。获得的胚胎移植后可得到正常子代小鼠(DMSO∶PROH∶R18S3=13∶8∶17)。结论 3种冷冻保护剂均适用于C57BL/6J小鼠附睾冷冻,但PROH冷冻效果较好。     

Cryopreservation of a water-bloom forming cyanobacterium, Microcystis aeruginosa f. aeruginosa

A method for the Cryopreservation of Microcystis aeruginosa f. aeruginosa is described. For the five strains tested, dimethyl sulfoxide (DMSO) (3% v/v) was the only effective cryoprotectant for freezing to, and thawing from -196°C and allowed the successful recovery (>50%) of all the strains. The viability of frozen material was independent of the period of storage in liquid nitrogen. The strain NIES-44 (National Institute for Environmental Studies) had a recovery level of greater than 90% at 3–10% (v/v) DMSO in both two step and rapid cooling methods. The other three strains, NIES-87, 88 and 89 had greater than 60% of viability after freeze/thawing in presence of both 3% and 5% DMSO concentrations. On the other hand, the strain NIES-90 showed approximately 50% of viability in only 3% DMSO solution after two step cooling to and thawing from -196°C. This strain was damaged by greater than 4% DMSO and by rapid cooling to -196°C. It was found that cold shock injury and the cytotoxicity of DMSO were different at a strain level.     

The effect of tannin concentration and egg unsticking time on the hatching success of tench Tinca tinca (L.) larvae

The influence of dose and time of eggs exposure in tannin solution on tench embryonic development, survival of embryos and percentage of larvae hatched were studied. The eggs obtained under controlled conditions from 11 tench females were fertilized with semen from 7 males and then rinsed for 1 h in Woynarovich solution (40 g urea, 30 g NaCl per 10 dm³ of water). Random samples (50 cm³) of the fertilised eggs were placed into three tannin solutions 0.05, 0.1 or 0.15% for 30, 60 or 90 s, then they were transferred to Weiss jars where incubation at 25°C took place. Tannin solution of 0.05% for 30–90 s or 0.1% for 30 s allowed effective unsticking of eggs and resulted in a very high per cent of hatched larvae. Extending the eggs rinsing time to 60 s in 0.10 and 0.15% solutions resulted in a high mortality of embryos during hatching because of hardened egg capsule.     

The effect of cooling rate and of dimethyl sulfoxide concentration on low temperature preservation of neonatal rat heart cells

The effect of five cooling rates, 1, 5, 10, 30, and 50 °C/min, and of four DMSO concentrations, 2.5, 5, 7.5, and 10%, on the survival of neonatal rat heart cells after freezing and thawing were studied. Growth area, contracting area and contraction frequency were used as viability parameters. Growth area and contracting area were measured in a number of fields in statistically adjusted locations of the culture dish on the second and on the fifth day of culturing.Without freezing, DMSO concentrations higher than 5% caused a considerable decrease of the growth area and of the contracting area. After freezing and thawing, biphasic survival curves were found with a narrow optimum at 2.5, 5, and 10% DMSO and a broad optimum at 7.5% DMSO. The survival based on the growth area and the survival based on the contracting area were about the same on the second day of culturing but differed on the fifth day. On the second day of culturing the highest survival was 73%, at a cooling rate of 5 °C/min and with 5% DMSO. On the fifth day of culturing the highest survival based on the growth area was 100%, at a cooling rate of 10 °C/min with 7.5% DMSO; the contracting area was the same as on the second day. The cooling rate of 5 °C/min was optimal at all DMSO concentrations tested. There was no correlation between the contracting area and the spontaneous contraction frequency after freezing and thawing when both were expressed as percentages of the control. The contraction frequency after freezing and thawing was independent of the cooling rate and was maximally 50% of the control value.     

Inhibiting ice recrystallization and optimization of cell viability after cryopreservation

The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220?mM solutions of disaccharides; however, the best cell viability was obtained when a 200?mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficiency of osmotic and chemical treatments to improve the permeation of the cryoprotectant dimethyl sulfoxide to Japanese whiting (Sillago japonica) embryos

Insufficient cryoprotectant permeation is one of the major obstacles for successful fish embryo cryopreservation. The purpose of this study was to test the effectiveness of osmotic and chemical treatments to enhance cryoprotectant uptake by fish embryos. Japanese whiting Sillago japonica embryos at the somites and tail elongation stages were treated with hyperosmotic sugar solutions (1 M trehalose and sucrose) for 2-6 min, or a permeating agent (2-6 mg/mL pronase) for 30-120 min, and then impregnated with 10-15% DMSO in artificial sea water or aqueous solutions containing inorganic salts (0.125-0.25 M MgCl₂ and CaCl₂). The viability of the embryos after the treatments was estimated from hatching rates and the internal DMSO concentration was measured by HPLC. Treatment with trehalose for 3 min prior to impregnation with DMSO enhanced the uptake of the cryoprotectant by 45% without significantly affecting embryo viability, whereas pronase had no noticeable effect on cryoprotectant permeation. Incorporation of DMSO into the embryos was enhanced by 143-170% in the presence of 0.25 M MgCl₂ and 0.125 M CaCl₂ compared to sea water. A combination of treatments with trehalose and MgCl₂ was even more effective in promoting DMSO permeation (191% compared to untreated embryos). Tail elongation embryos were less tolerant of the treatments, but had higher DMSO impregnation. In conclusion, the use of trehalose (as dehydrating agent) and MgCl₂/CaCl₂ (as a vehicle during impregnation) greatly promoted cryoprotectant uptake and may be a promising aid for the successful cryopreservation of fish embryos.