Volatile transport in frozen aqueous solutions. I. Development of a mechanism

Frozen aqueous butanol solutions are equilibrated at constant subzero temperature over activated charcoal. A fraction of the butanol is lost within 24 hr, the remainder being retained for over 350 hr. The retained butanol is lost only with the simultaneous loss of water. Pure ice is demonstrated to be permeable to the transport of butanol. Based on experiments which remove, the free surface, the butanol loss that is independent of water loss is shown to originate from a surface layer postulated to form during freezing of the solution.Three types of butanol-ice interactions are postulated: (1) Butanol in the surface layer; (2) butanol entrapped in interdendritic spaces; (3) butanol present in pores and cracks after sorption from the vapor state.     

Semen preservation in Macaca fascicularis.

Semen was collected from adult male Macaca fascicularis using a rectal probe for electro-ejaculation. The effect on sperm motility of varying semen extender egg yolk concentration, pH, glycerol concentration, and equilibration times of sperm with glycerol was examined. No significant difference was observed between motilities at extender egg yolk concentrations of 10% to 40%. Progressive motility was significantly greater at pH 7.2 and 8.0 than at 5.8, 6.5, and 8.7 (p less than 0.05). Glycerol concentrations of 7% and 10% yielded optimum progressive motility after freezing. A 1-minute equilibration of semen in extender containing glycerol resulted in greater sperm motility after freezing than did equilibration for 25 or 45 minutes.     

Comparison of equilibration times when freezing epididymal sperm from African buffalo (Syncerus caffer) using Triladyl or AndroMed

Because of risks of disease transmission, it is not possible to move African buffalo (Syncerus caffer) within South Africa. Therefore, new ways must be found to enable exchange of genetic material and to increase genetic diversity. In this study epididymal sperm from 11 African buffaloes was exposed to 8 different pre-freezing equilibration times, using 2 different semen extenders. To test the influence of equilibration time and to find a practical way of freezing sperm in the field equilibration times between 2 and 9 h were compared. The extenders used were Triladyl and the totally defined extender AndroMed (both Minitüb, Tiefenbach, Germany). Post-thaw motility, longevity and acrosomal integrity were compared. Different equilibration times did not result in different post-thaw qualities. The use of Triladyl resulted almost always in higher post-thaw motilities and in better acrosomal integrity. Individual bulls had a significant influence on measured parameters. Results indicate that sperm flushed in the field can be stored in freezing medium for up to 9 h before being further processed and that Triladyl is superior to AndroMed when freezing epididymal African buffalo sperm. This knowledge is important to plan fieldwork, since working conditions are usually far from the ideal of a laboratory.     

Cryopreservation of oyster (Crassostrea gigas) embryos

Several critical variables associated with successful cryopreservation of oyster embryos (Crassostrea gigas) were examined. These were 1) embryo developmental stage, 2) kind and concentration of cryoprotectant, 3) equilibration time, and 4) freezing rate. The percentage of survival was scored as the number of recovered embryos that swam actively 12 h after thawing and had developed into veliger stage. The oyster embryos became increasingly susceptible to the cryoprotectants as the concentration was increased and the equilibration time was lengthened. The stage of development appears to be a critical factor for survival of oyster embryos, with trochophore stage embryos more resistant than morula and gastrula stages embryos to cryoprotectant exposure and having better surviving after freezing. The optimum cryoprotectant concentration for the trochophore embryos differed markedly from the morula stage. Cryopreservation of fertilized eggs (2 to 8 cells) was unsuccessful. Varying degrees of success were achieved using gastrula- and trochophore-stage embryos. Maximum survival was obtained when trochophore embryos incubated in 10% propylene glycerol-artificial sea water were cooled at -2.5 degrees C/min to -30 degrees C and were then directly placed into liquid nitrogen. The results showed a clear effect of the stage of development on survival.     

Effect of equilibration period on the viability of frozen-thawed mouse morulae after rapid freezing

Mouse morulae were frozen with 1.5-4.0 M glycerol + 0.25 M lactose solution by direct plunging into liquid nitrogen vapor 0.5-30 min after equilibration at room temperature. After thawing, embryos were cultured in vitro, and the highest survival rates were obtained after exposure for 3 min at 3.0 and 4.0 M and for 5 min at 1.5 and 2.0 M glycerol levels. Significant reductions in the survival rates (P less than 0.05) were observed when equilibration periods were extended for 3-5 min at 3.0 and 4.0 M and for 5-10 min at 1.5 and 2.0 M glycerol levels. These results clearly demonstrate that the equilibration time of embryos in glycerol-lactose mixture is one of the most important factors in the present rapid freezing conditions. To clarify the factors that lower embryo viability after prolonged equilibration, we performed further experiments on the effects of exposure to glycerol-lactose mixture on the developmental potential of embryos without freezing and on the volume changes of embryos during the exposure to glycerol solution with or without lactose. It was suggested that the detrimental effects of prolonged equilibration are due not only to the toxicity and osmotic injury of higher concentrations of cryoprotectant solution but also to the influx of water into embryonic cells caused by the hypotonic salt concentration of the extracellular (freezing) solution.     

Effect of N-acetyl-D-glucosamine, and glycerol concentration and equilibration time on acrosome morphology and motility of frozen-thawed boar sperm.

A series of experiments were conducted to determine the effect of N-acetyl-D-glucosamine, glycerol concentration and equilibration time for the freezing of boar spermatozoa in 5 ml maxi-straws. The optimum final glycerol concentration in the diluent with 0.05% N-acetyl-D-glucosamine in the first diluent was 2-3% and the optimum glycerol equilibration time was 2-3h. In conclusion, we recommend the first diluent containing 11% lactose hydrate, 20% egg yolk and 0.05% N-acetyl-D-glucosamine in 100ml distilled water, and the second diluent containing 11% lactose hydrate, 20% egg yolk, 4% glycerol and 1% orvus es paste for the diluents of boar sperm freezing. Also, we found out that 0.05% soluble N-acetyl-D-glucosamine was the optimum concentration in the first diluent and a concentration of 0.05% soluble N-acetyl-D-glucosamine significantly enhanced the cryopreservation of boar spermatozoa.     

Contributions of unfrozen fraction and of salt concentration to the survival of slowly frozen human erythrocytes: Influence of warming rate

The general belief is that slow freezing injury is either the result of exposure to high salt concentrations or the result of excessive cell shrinkage. Increased salt concentration arises as increasing amounts of pure ice precipitate out of solution during freezing and cause the liquid-filled channels in which the cells are sequestered to dwindle in size. Cell shrinkage is an osmotic response to the concentration of external solutes. The consensus has been that the injury is related to the composition of the solution in these channels and not to the amount of residual liquid.Ordinarily, salt concentration and the amount of liquid in the unfrozen channels are reciprocally related; but they can be separated within limits by varying the total concentration of solutes in the suspending medium while holding the mass ratio of additive to salt constant, and by then slowly freezing samples to various subzero temperatures, chosen to produce various molalities of salt, while holding the unfrozen fraction constant, or vice versa. We have recently reported (9) that when human red cells are frozen under these conditions and thawed rapidly, survival is more dependent on the unfrozen water fraction than it is on the salt concentration in that fraction. The present work compares these results with those obtained with slow thawing. While the general conclusion remains unaltered, slowly thawed cells were able to survive the freezing of a higher fraction of extracellular water than were rapidly thawed cells.Calculations were made of the changes in cell volume during the equilibration with glycerol and the subsequent freezing involved in these experiments. Cell size and cell solute concentration were found to be independent of the fraction of unfrozen extracellular water, but cell survival was strongly dependent on that fraction. If applicable to other than human red cells, this finding is likely to require major modifications in current views of slow-freezing injury and its prevention.     

Energy and water conservation in frozen vs. supercooled larvae of the goldenrod gall fly, Eurosta solidaginis (fitch) (Diptera: Tephritidae)

Insects that tolerate severe cold during winter may either supercool or tolerate ice forming within the tissues of the body. To compare the relative advantages of freezing and supercooling, we measured rates of CO(2) production and water loss in frozen and supercooled goldenrod gall fly larvae (Eurosta solidaginis). As an important first step, we measured the time required for ice content and metabolic rate to stabilize upon freezing. Ice content stabilized after only three hours of freezing at -5 degrees C, whereas CO(2) production required 12 hours to stabilize. Subsequent experiments found that freezing greatly reduced both water loss and metabolic rate. Comparisons of supercooled and frozen larvae at -5 degrees C indicated that CO(2) production fell 47% with freezing and water loss decreased 35%. As temperature decreased to -10 and -15 degrees C, CO(2) production fell exponentially and was no longer detectable at -20 degrees C with our measurement system. Our results demonstrate that freezing significantly reduces energy consumption during the winter and may therefore improve winter survival and spring fecundity. The advantages of freezing over supercooling would drive selection toward insect freeze tolerance and also toward higher supercooling points to increase the duration of freezing each winter.     

Slow relaxation of the sub-main transition in multilamellar phosphatidylcholine vesicles.

The influence of ionic strength and equilibration time on the appearance of the sub-main transition in fully hydrated multilamellar vesicles composed of phosphatidylcholines has been investigated by means of calorimetry and densitometry. The heat capacity measurements show that the transition enthalpy of the sub-main transition is affected by both salt concentration (KCl) and equilibration time. The small heat capacity peak appearing in vesicles made in pure water is significantly increased upon addition of salt. Furthermore, equilibration of the multilamellar vesicles at low temperatures for several weeks results in a pronounced enhancement of the transition enthalpy of the sub-main transition. Neither salt concentration nor equilibration time affected the transition temperature of the sub-main transition. In the densitometry measurements a small volume change is detectable for high salt concentrations. In order to gain further insight into the physical mechanisms involved in the sub-main transition, a Monte Carlo computer simulation study has been carried out using a microscopic model. The combined experimental and simulation results suggest that the sub-main transition involves an acyl chain disordering of phospholipids in lipid bilayer regions that are characterized by a locally decreased lateral pressure most likely caused by a curvature stress.     

不同甘油浓度与平衡时间对食蟹猴精液冷冻效果的影响

探讨了不同甘油浓度(3%、5%、7%、11%)和不同平衡时间(30、60、90、120min)对食蟹猴(Macaca fascicularis)精液冷冻效果的影响,以建立和优化食蟹猴精液冷冻的程序。参照TTE稀释液成分组成改良型TTE,冷冻前和解冻后均检测精子的活力、畸形率、质膜完整性、顶体完整率。结果显示,平衡时间为30min时精子的冷冻解冻后活力、复苏率均高于平衡时间90min和120min组,差异显著(P<0.05),比60min组稍好;甘油浓度为3%、5%组的精子冷冻解冻后活力及复苏率均高于甘油浓度11%组,差异显著(P<0.05),比7%组好;不同甘油浓度各组间以及不同平衡时间各组间畸形率、质膜完整性、顶体完整率差异不显著(P<0.05)。由此得出如下结论,在食蟹猴精液冷冻中,在改良TTE中加入3%~5%的甘油且平衡30min可以获得较好效果,精子冻后活率和复苏率达到45%和62%。     

The cryoprotectant used, its concentration, and the equilibration time are critical for the successful cryopreservation of rabbit sperm: Dimethylacetamide versus dimethylsulfoxide

This study was designed to identify a suitable freezing protocol for rabbit semen by comparing the effects of different concentrations and equilibration times of dimethylacetamide (DMA) and dimethylsulfoxide (DMSO) on the postthaw quality of the semen. After establishing the best protocols for each cryoprotectant, their efficacy was compared by examining the in vivo fertilizing capacity of the semen samples. Pooled semen samples diluted in freezing medium containing 4%, 6%, or 8% DMA or DMSO (all combined with 1% sucrose as a nonpermeating cryoprotectant) were loaded in straws and equilibrated for 5, 15, or 45 min before freezing in liquid nitrogen vapor. The variables assessed after thawing were sperm motility, viability, osmotic resistance, and acrosome and DNA integrity. Marked effects on these variables were shown by the cryoprotectant concentration and equilibration time, with best results obtained using DMA 6% or DMSO 8% and equilibration times of 45 min. These freezing protocols were selected to compare the two cryoprotectants in an insemination trial. Three groups of 114 rabbit does (28 nulliparous and 86 multiparous in each group) were inseminated with fresh semen or with semen frozen using the optimized DMA or DMSO protocols. Fertility rates and numbers of kids born were similar, respectively for the DMSO-frozen (79.8% and 7.7 ± 0.3 young per kindling) and fresh semen (81.6% and 8.6 ± 0.3) yet higher (P ≤ 0.05) than the rates returned using the DMA-frozen semen (47.4% and 6.7 ± 0.4). Moreover, the numbers of rabbits born alive when DMSO was used in the freezing protocol, despite being lower than those recorded using fresh semen, were higher than when DMA was used as the cryoprotectant (P < 0.05). The physiological status of the does (nulliparous or multiparous) had no influence on the fertility and prolificacy results. Our findings indicate that the cryosurvival of rabbit sperm frozen using DMSO or DMA as the cryoprotectant is highly influenced by the concentration of cryoprotectant used and the time the semen is exposed to the agent before freezing. According to our in vivo fertility and prolificacy data, DMSO emerged as more effective than DMA for the cryopreservation of rabbit sperm.     

Bedeutung einiger nichtflüchtiger carbonsäuren für die frostresistenz des halophyten Halimione portulacoides unter dem einfluß verschieden hoher kochsalzbelastung

Summary Plants of Halimione portulacoides (L.) Aellen were grown in natural temperature and light conditions but with different concentrations of NaCl in the nutrient solution. From August 1971 to April 1972 freezing tolerance, water content, succulence, accumulation of different sugars, citrate, malate, and chloride were simultaneously determined. If no NaCl was supplied the chloride content of the leaves decreased continuously within the period of investigation. During repeated and increasing addition of NaCl the chloride content of the leaves generally increased. However, there was a reversible decrease during the frost period, although no new leaves were formed and loss through leaves and dilution of the nutrient medium by precipitation was prevented (Fig. 1).In spite of being in minimum the chloride content was relatively high in winter. No regulation of the concentration by increase of succulence was observed. The concentration did, however, increase due to a diminished water uptake in the coldest period.Sugars, which are regarded as protective agents against the influence of freezing and salts, accumulated only slightly in the frost period. Predominantly sucrose, raffinose and stachyose were remarkable. Their concentration was not sufficient to compensate the salt burden and thus could not increase the freezing tolerance. The sugar content was even lowered when the salt content was higher. In contrast, citrate and to a lesser extent malate were intensively increased in the cold season (Fig. 3). Thus organic acid to chloride ratios of between 1:2 and 1:6 were established for Halimione, which expresses the effective protection of the membrane systems against freezing injury (Fig. 4), as has been shown in vitro for e.g. spinach chloroplasts by Santarius (1971). Accumulation of these acids was even enhanced by an increasing salt burden. Consequently accumulation of organic acids or their salts such as citrate and probably malate indicates an adaptation of halophytes, which enables them to survive freezing under salt stress on the sea shore and in cold desert regions during the winter.     

Mechanism of cryoprotection by extracellular polymeric solutes.

To elucidate the means by which polymer solutions protect cells from freezing injury, we cooled human monocytes to -80 degrees C or below in the presence of various polymers. Differential scanning calorimetric studies showed that those polymers which protect cells best have a limiting glass transition temperature (T'g) of approximately -20 degrees C; those with a T'g significantly higher or lower did not protect. Freeze-etch electron micrographs indicated that intracellular ice crystals had formed during this freezing procedure, but remained smaller than approximately 300 nm in the same proportion of cells as survived rapid thawing. We propose that cryoprotection of slowly frozen monocytes by polymers is a consequence of a T'g of -20 degrees C in the extracellular solution. In our hypothesis, the initial concentration and viscosity of protective polymer solutions reduce the extent and rate of cell water loss to extracellular ice and limit the injurious osmotic stress, which cells face during freezing at moderate rates to -20 degrees C. Below -20 degrees C, glass formation prevents further osmotic stress by isolating cells from extracellular ice crystals, virtually eliminating cell water loss at lower temperatures. On the other hand, the protective polymer solutions will allow some diffusion of water away from cells at temperatures above T'g. If conditions are correct, cells will concentrate the cytoplasm sufficiently during the initial cooling to T'g to avoid lethal intracellular freezing between T'g and the intracellular Tg, which has been depressed to low temperatures by that concentration. Thus, when polymers are used as cryoprotective agents, cell survival is contingent upon maintenance of osmotic stress within narrow limits.     

The role of freezing in setting the latitudinal limits of mangrove forests

Mangrove trees dominate coastal vegetation in tropical regions, but are completely replaced by herbaceous salt marshes at latitudes above 32 degrees N and 40 degrees S. Because water deficit can increase damage caused by freezing, we hypothesized that mangroves, which experience large deficits as a result of saline substrates, would suffer freeze-induced xylem failure. Vulnerability to freeze-induced xylem embolism was examined in the most poleward mangrove species in North America, in an area where freezing is rare but severe, and in Australia, in an area where freezing is frequent but mild. Percentage loss in hydraulic conductivity was measured following manipulations of xylem tension; xylem sap ion concentration was determined using X-ray microanalysis. Species with wider vessels suffered 60-100% loss of hydraulic conductivity after freezing and thawing under tension, while species with narrower vessels lost as little as 13-40% of conductivity. These results indicate that freeze-induced embolism may play a role in setting the latitudinal limits of distribution in mangroves, either through massive embolism following freezing, or through constraints on water transport as a result of vessel size.     

The influence of different freezing procedures and different cryoprotective agents on the immunological capacity of frozen-stored lymphocytes

The influence of a series of factors on the frozen storage of lymphocytes was investigated. The cells were frozen using different freezing programmes, using the cryoprotectants dimethyl sulphoxide and glycerol in different concentrations in the freezing medium, and with variations in the period of exposure of cells to cryoprotectants before freezing and after thawing. Cell viability was evaluated by quantitative measurements of the cell-mediated immune response after stimulation with phytohemagglutinin, pokeweed mitogen, concanavalin A, and allogenic cells in mixed lymphocyte cultures. The factors investigated were found to have an important effect on the immune response, so that careful investigation and exact specification of the freezing system are necessary before frozen cells are used in blast-transformation tests. The best freezing programme had a duration of approximately 40 min with a smooth progression through the temperature range where phase transition takes place. The optimum dimethyl sulphoxide concentration in this programme was 8–10%. Dimethyl sulphoxide had no toxic effect on the cells, and no equilibration period was necessary prior to freezing. An equilibration period of 15 min with 10% glycerol was even better than the optimum programme with dimethyl sulphoxide.     

Osmotic and cryoprotective effects of glycerol-sucrose solutions on day-3 mouse embryos

The relative volume of Day-3 mouse embryos changed as a linear function of the reciprocal of osmolality [corrected] of non-permeating solutes after 10 min exposure to sucrose and glycerol-sucrose solutions at 20 degrees C. The slope of the linear regression line was less in glycerol-sucrose than in sucrose solutions because glycerol permeation caused re-expansion. Before freezing by direct transfer to -180 degrees C the embryos were placed into glycerol-sucrose in 1-step (1-step equilibration) or first into glycerol and then into glycerol-sucrose (2-step equilibration). Using 2-step equilibration the post-thaw survival rate was substantially higher at 3.0 and 4.0 M-glycerol levels and less dependent on changes in the sucrose concentration within the range of 0.125 to 1.0 M than with 1-step equilibration. Under optimal conditions 90-95% of rapidly frozen embryos developed to blastocysts in vitro and 30% into live young in vivo. It is suggested that the cryoprotective role of glycerol is due to its ability to reduce osmotic pressure differences between the extra and intracellular spaces during rapid freezing of embryos.     

Assessment of freezing procedures for rat immature testicular tissue

Fertility preservation has been included in the management of childhood cancer treatment. Cryopreservation of immature testicular tissue is the only available solution for pre-pubertal boys. Different freezing protocols have been developed in several species but without a clearly identified procedure. We tried to evaluate several protocols for cryopreservation of rat immature testicular tissue. Twelve different freezing protocols using different (i) cryoprotectant (dimethylsulphoxide [DMSO] or 1,2-propanediol [PROH]), (ii) cryoprotectant concentration (1.5M or 3M), (iii) equilibration time (30 or 60 min), (iv) equilibration temperature (4 °C or room temperature), (v) size of testicular fragment (7.5mg or 15mg), (vi) package (straws or cryovials), were compared using cord morphological damage evaluation. A testicular tissue piece of 7.5mg cryopreserved in cryovial using 1.5M DMSO, an equilibration time of 30 min at 4 °C showed fewer morphological alterations than the other protocols tested. The selected freezing protocol was able to maintain rat immature testicular tissue architecture, functionality after testicular pieces organotypic culture, and could be proposed in a human application.     

Effect of cryoprotectants on certain seminal attributes and on the fertility of buck spermatozoa

Semen samples were obtained from 12 bucks (3 Beetal, 3 Black Bengal and 6 Beetal x Black Bengal) and 10 different extenders were constituted with varying concentrations of glycerol, DMSO, glycerol + DMSO and glycerol + lactose as the sperm cryoprotective agents. After the collection of semen samples, they were assessed for quality, diluted in different extenders after removal of seminal plasma, packaged in ministraws and frozen after equilibration (5'C) for 5 h. The samples were evaluated immediately after equilibration and again 24 h after freezing for progressive motility, percentage of live spermatozoa and acrosome, head and tail abnormalities. Both motility and the percentage of live spermatozoa were most affected by extenders containing only DMSO and these values improved in glycerol + DMSO extenders as the concentration of glycerol was increased while DMSO was decreased. However, these values were significantly higher in extenders containing glycerol + lactose as the cryoprotective agents, and were found to increase with increased concentration of lactose, being highest in TYGL (180). Acrosomal and tail abnormalities tended to increase between post equilibration and post thawing stage, and were higher in extenders containing the higher levels of DMSO. Significantly (P < 0.01) lower percentages of abnormalities were recorded in the glycerol + lactose extenders. The fertility results showed nonsignificant effect of extenders on the conception rate of does.     

Ion and water distribution in pig lenses incubated at 0 degree C to disable ion transport pumps.

This study was designed to test how extended exposure of lenses to sera with different ionic strengths influences the distribution of ions and water in the lens. Pig lenses were incubated in cold sera (0 degree C), which were adjusted to variable concentrations of NaCl, and their K+, Na+, Cl-, and water contents were measured. Incubation at 0 degree C inhibits active transport processes and thereby allows equilibration of the mobile ions and water. The hypothesis was that lens water content (volume) would follow the ion-induced protein changes predicted by a model derived from previous osmotic studies on proteins. As expected, exposure of the lens to cold caused a gain of sodium and a partial loss of potassium. However, the potassium concentration in the lens remained several fold higher than that in the bathing solution (about 41 vs. 1.8-4.6 mM/kg H2O), indicating that a portion of the potassium within the cold-exposed lens was not free to diffuse. That the water content of the lens showed a negative rather than a positive relationship with the concentration of NaCl within the lens was explained by the idea that an increase in NaCl within the lens (up to at least 250 mM/kg H2O) causes a decrease in the osmotically unresponsive water volume associated with lens proteins.     

Lipid-protein interactions in mitochondria. Changes in mitochondrial adenosine triphosphatase activity induced by n-butyl alcohol.

Butanol at a concentration of 0.35 m decreases the oligomycin sensitivity of the mitochondrial ATPase; at the same concentration of butanol the activation energy of enzyme is increased threefold. Butanol does not detach the ATPase from the membrane of either mitochondria or submitochondrial particles. The same effect is exerted by butanol on the sensitivity of the ATPase to DCCD, which is covalently bound to the ATPase complex in the oligomycin inhibition site. Diethyl ether also makes the ATPase oligomycin- and DCCD-insensitive; however, its effect on the activation energy of the enzyme is different from that of butanol, since ether does not increase the activation energy but lowers the temperature where a transition occurs in an Arrhenius plot of ATPase. The effect of both organic solvents on ATPase may be closely related to changes occurring in the lipid environment which might be transferred to the enzymic activity via a conformational change of the enzymic protein.