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PubMed1976

Examination of some processing methods for freezing boar semen.

Five experiments were conducted to examine the effect of processing methods and diluents on survival and morphology of boar spermatozoa after freezing. Post-thawing survival of spermatozoa was better for Beltsville-F3 (BF3) than for tris-fructose-EDTA freezing diluent when the seminal plasma and glycerol were removed prior to freezing (method A). Both freezing diluents yielded similar viability results when the spermatozoa were frozen in the presence of siminal plasma and glycerol (method B). Viability of spermatozoa after thawing was better when glycerol concentration in the prefreezing diluent (method A) or in the freezing medium (method B) was 2-5 and 5-0 rather than 7-5%. Cooling of diluted semen to 5 degrees C beyond 4 h decreased the post-thawing survival of spermatozoa. The proportion of spermatozoa with undamaged acrosomes after processing and thawing by different methods was indistinguishable and relatively low. When the semen was frozen at cell concentrations ranging from 0-25 to 2-0 X 10(9)/ml, the viability of spermatozoa declined with increasing concentration following freezing in BF3, and S-1 diluents. Viability results were very similar for all cell concentrations examined when tris-fructose-EDTA diluent was used, indicating the possibility of freezing boar semen in a concentrated state.

Animals↗
PubMed1998

Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant.

Temperate plants develop a greater ability to withstand freezing in response to a period of low but nonfreezing temperatures through a complex, adaptive process of cold acclimation. Very little is known about the signaling processes by which plants perceive the low temperature stimulus and transduce it into the nucleus to activate genes needed for increased freezing tolerance. To help understand the signaling processes, we have isolated mutants of Arabidopsis that are constitutively freezing-tolerant in the absence of cold acclimation. Freezing tolerance of wild-type Arabidopsis was increased from -5.5 degreesC to -12.6 degreesC by cold acclimation whereas the freezing tolerance of 26 mutant lines ranged from -6.8 degreesC to -10.6 degreesC in the absence of acclimation. Plants with mutations at the eskimo1 (esk1) locus accumulated high levels of proline, a compatible osmolyte, but did not exhibit constitutively increased expression of several cold-regulated genes involved in freezing tolerance. RNA gel blot analysis suggested that proline accumulation in esk1 plants was mediated by regulation of transcript levels of genes involved in proline synthesis and degradation. The characterization of esk1 mutants and results from other mutants suggest that distinct signaling pathways activate different aspects of cold acclimation and that activation of one pathway can result in considerable freezing tolerance without activation of other pathways.

Acclimatization↗
PubMed2004

Freeze-dried irradiated bone brittleness improves compactness in an impaction bone grafting model.

BACKGROUND: Defatted bone chips with or without freeze-drying and irradiation have mechanical advantages as compared to fresh-frozen controls in in vitro models of impaction. These improved results have been ascribed to replacement of viscous bone marrow by saline and embrittlement of the freeze-dried bone by irradiation. MATERIAL AND METHODS: To determine which of these hypotheses is correct, we compared the development of stiffness and compactness of morselized bone graft that had been: 1) fat-reduced with saline, and 2) fresh-frozen, solvent-detergent defatted, 3) freeze-dried irradiated and 4) not irradiated. We used 12 osteoarthrotic femoral heads to prepare these four batches of morselized bone, and impacted 18 samples from each batch in a cylinder. The frozen bone grafts were tested after thawing at room temperature for 2 hours and the freeze-dried grafts were tested after 30 minutes of rehydration. We monitored the development of compactness and stiffness of the material during impaction. RESULTS: The stiffness of the freeze-dried irradiated bone was greater than that of the other three series after 10, 50 and 150 impactions. The freeze-dried bone chips that were not irradiated and the chips defatted with saline alone were less stiff than the fresh-frozen control after 150 impactions. INTERPRETATION: The brittleness of freeze-dried irradiated bone, caused by loss of the capacity to absorb energy in a plastic way, increases the compactness and stiffness of the morselized grafts. Washing bone with saline alone or treating bone with solvent-detergent but no irradiation had no similar mechanical advantage and the bone did not impact better than fresh-frozen undefatted bone in our model.

Aged↗
PubMed2002

Use of freeze-dried autologous fascia to augment the vocal fold: an experimental study in dogs.

This study assessed the practicality of using autologous freeze-dried fascia to augment the vocal fold. Freeze-dried autologous fascia was injected into the vocal fold and skin of dogs in order to monitor sequential histological changes. Fascia lata was harvested from six adult dogs. After freeze-drying, minced fascia suspended in hyaluronic acid was injected subcutaneously into the abdominal wall and directly into the vocal fold. The specimens were extracted 3 weeks after injection and studied histologically. Freeze-drying destroyed all cellular components but did not affect the collagen fibers, which are the major components of fascia. There was no evidence of degeneration, necrosis or infection. Fibroblastic infiltration was seen in the fascia injected into the vocal fold, but the fascia remained as an unencapsulated mass at the site of injection. This study demonstrates that freeze-drying does not compromise the collagen in fascia and that the injection of freeze-dried collagen is well tolerated. Freeze-dried fascia is a promising new augmentation material.

Animals↗
PubMed2006

Beneficial effects of freezing rate determined by indirect thermophysical calculation on cell viability in cryopreserved tissues.

Many types of mammalian cells, such as sperm, blood, embryos, etc., have been successfully cryopreserved for the last few decades, while no optimal method for the cryopreservation of mammalian tissues or organs has been established, showing a poor survival after thawing with a low recovery of function. In this study, the freezing rate was determined by indirect thermodynamic calculation, and its potential effect on the cryoprotection of human saphenous veins and tissue-engineered bones was investigated. The vein segments were frozen according to the calculated freezing rate, using rate-controlled freezing devices, with a freezing solution composed of 10% dimethylsulphoxide and 20% fetal bovine serum in RPMI 1640 media. The efficacy of indirect calculation was assessed by the cell viability measured using fluorescence double-staining methods. The results indicated that the freezing rate determined by indirect calculation significantly (P < 0.05) maintained the post-thaw cellular viability of the blood vessel, particularly in terms of the endothelial cells. However, it exerted relatively less protective effect on the osteoblastic cell-cultured scaffolds. These results suggest that freezing-induced injuries may occur in tissues, and the freezing rate determined by indirect thermophysical calculation can be used for the optimization of tissue cryopreservation by minimizing the injuries.

Animals↗
PubMedDate not supplied

Cold tolerance in hatchling painted turtles (Chrysemys picta): supercooling or tolerance for freezing?

We studied tolerance for cold in hatchling painted turtles (Chrysemys picta) from Lake Metigoshe, Bottineau County, North Dakota, to determine whether neonates in populations near the northern limit of distribution rely on a tolerance for freezing or on a capacity for supercooling to survive their first winter of life. We placed hatchlings individually into artificial hibernacula constructed in jars of damp, loamy sand and then cooled the jars to approximately -0.45 degrees C, which was below the equilibrium freezing point for water held by the sand but above that for body fluids of the neonatal turtles. A piece of ice next was placed on the surface of the sand in each jar to induce freezing of the soil water. After the soil water had frozen to an equilibrium, the temperature in the jars was lowered by 1 degrees C/d to minima averaging -2.5 degrees C, -4.5 degrees C, -6.5 degrees C, and -10.5 degrees C in different treatments. These temperatures were maintained for varying periods, so that animals in each treatment were exposed to temperatures below the equilibrium freezing point for their body fluids for a total of 11 d. Thirty of 32 hatchlings survived exposure to -2.5 degrees C; 24 of 32 survived at -4.5 degrees C; 14 of 32 withstood -6.5 degrees C; and 7 of 32 tolerated -10.5 degrees C. Freezing exotherms were detected in temperature profiles for turtles that succumbed but not in those for hatchlings that survived. Thus, the ability of hatchlings to withstand subzero temperatures for extended periods apparently requires that they avoid freezing. Although other workers contend that tolerance for freezing is the key to survival over winter by hatchling painted turtles from the region of Lake Metigoshe, our findings indicate that neonates rely primarily on their ability to remain unfrozen and supercooled.

Animals↗
PubMed2005

Survival, injury, and virulence of freeze-stressed plasmid-bearing virulent Yersinia enterocolitica in ground pork.

The effect of freezing at -20 degrees C on survival, injury, stability of the virulence plasmid, and expression of virulence-associated determinants of Yersinia enterocolitica (YEP(+)) in ground pork was assessed. The viable counts on nonselective and selective media after 12 weeks of freezing were similar to counts obtained before freezing, indicating that the YEP(+) strain survived the freezing process. There was no evidence of freeze-stress injury. Results of a PCR assay targeting plasmid-associated virF gene and virulence determinants indicate that YEP(+) subjected to freezing are potentially capable of causing food-borne illness and freezing is not a substitute for safe handling and proper cooking of pork.

Animals↗
PubMed1992

The influence of slow and ultra-rapid freezing on the organization of the meiotic spindle of the mouse oocyte.

We have investigated the effect of ultra-rapid versus slow freezing on the meiotic spindle of the mouse oocyte. A slow freezing protocol [1.5 M dimethyl sulphoxide (DMSO)] and an ultra-rapid protocol (3.5 M DMSO/0.5 M sucrose) have been compared. Oocytes were fixed at different time points as follows: after prefreeze equilibration, immediately after thawing and after 60 or 180 min of post-thaw recuperation. The spindle was visualized with a monoclonal anti-alpha-tubulin antibody followed by an immunogold-silver staining technique. The chromosomes were stained with 4',6-diamidino-2-phenylindole (DAPI). Spindle morphology was classified as follows: normal barrel-shaped, abnormal shaped, partial/multipolar or absent. After slow freezing with 1.5 M DMSO, these spindle morphologies were found respectively in 67%, 17%, 11% and 6% of the oocytes after 60 min of post-thaw recuperation and in 72%, 11%, 11% and 6% after 180 min. After ultra-rapid freezing with 3.5 M DMSO/0.5 M sucrose, we observed the same categories of spindle morphology in 84%, 13%, 3% and 0% of oocytes after 60 min of post-thaw recuperation and in 86%, 7%, 7% and 0% after 180 min. The present study demonstrates that the majority of spindles exhibit a normal morphology after both slow and ultra-rapid freezing. The ultra-rapid freezing protocol preserved spindle integrity to the highest extent. Nevertheless, the occurrence of abnormal spindles and chromosome dislocation indicate that the genetic risk of oocyte freezing has to be evaluated in further detail.

Animals↗
PubMed1998

Evidence for the cell wall involvement in temporal changes in freezing tolerance of Jerusalem artichoke (Helianthus tuberosus L.) tubers during cold acclimation.

We studied the mechanism of cold acclimation of Jerusalem artichoke (Helianthus tuberosus L.) tubers with special reference to the role of the cell wall. During the cold-acclimation process from September to January, the freezing tolerance of tubers increased from -2.8 degrees C to -8.4 degrees C (LT50). By contrast, the isolated protoplasts constitutively showed a consistent high level of freezing tolerance (LT50; below -25 degrees C) throughout the period. In tuber tissues, freezing injury was effectively protected by the external addition of isotonic solutions. Cryomicroscopic observations revealed that tissue cells mounted in isotonic solutions plasmolyzed upon freezing; tissue cells mounted in water collapsed with a tight attachment of plasma membrane to the cell wall. Upon freezing of intact tissues in water to temperatures below the critical range, the cytoplasm was irreversibly acidified as revealed by a fluorescence pH-ratiometry, suggesting that occurrence of detrimental cellular events leading to permanent cell injury. The freeze-induced acidification of cytoplasm was also effectively prevented by the external addition of isotonic solutions. These results suggest that the tight attachment of the plasma membrane to the cell wall during freezing may have a harmful effect on cells, in particular on the plasma membrane, possibly due to mechanical or some sort of chemical/ physico-chemical interaction with the cell wall.

Acclimatization↗
PubMed2003

Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa.

The nuclei of freeze-dried mouse spermatozoa are able to retain their chromosome integrity and developmental potential. To optimize the conditions of freeze-drying, we examined whether pH values of the freeze-drying solution affect the chromosome integrity and developmental potential of sperm nuclei. The sperm freeze-drying solution we used contained a high concentration (50 mM) of calcium-chelating EGTA. Sperm chromosomes were examined at the metaphase of the first mitosis after injection of freeze-dried spermatozoa into matured oocytes. The developmental potential of sperm nuclei was assessed by examining the development of fetuses in midgestation. The results showed that both sperm chromosomes and sperm developmental potential are maintained better when the freeze-drying solution was slightly alkaline (pH 8.0) rather than near neutral or acidic (pH 7.4-6.0). The data indicated that the chromosome integrity and developmental ability of mouse spermatozoa are affected by the pH value of freeze-drying solution.

Animals↗
PubMed2004

Freeze-dried sperm fertilization leads to full-term development in rabbits.

To date, the laboratory mouse is the only mammal in which freeze-dried spermatozoa have been shown to support full-term development after microinjection into oocytes. Because spermatozoa in mice, unlike in most other mammals, do not contribute centrosomes to zygotes, it is still unknown whether freeze-dried spermatozoa in other mammals are fertile. Rabbit sperm was selected as a model because of its similarity to human sperm (considering the centrosome inheritance pattern). Freeze- drying induces rabbit spermatozoa to undergo dramatic changes, such as immobilization, membrane breaking, and tail fragmentation. Even when considered to be "dead" in the conventional sense, rabbit spermatozoa freeze-dried and stored at ambient temperature for more than 2 yr still have capability comparable to that of fresh spermatozoa to support preimplantation development after injection into oocytes followed by activation. A rabbit kit derived from a freeze-dried spermatozoon was born after transferring 230 sperm-injected oocytes into eight recipients. The results suggest that freeze-drying could be applied to preserve the spermatozoa from most other species, including human. The present study also raises the question of whether rabbit sperm centrosomes survive freeze-drying or are not essential for embryonic development.

Animals↗
PubMed1992

Freeze-induced membrane damage in ram spermatozoa is manifested after thawing: observations with experimental cryomicroscopy.

Cryoinjury in ram sperm was investigated by direct observation, using cryomicroscopy, to validate model hypotheses of freezing injury in such a specialized cell. Fluorescein diacetate was used to determine when during the freeze-thaw cycle the sperm membrane became permeable. In noncryoprotected sperm plasma membrane, integrity was maintained throughout the cooling and freezing process, but fluorescein leakage occurred during rewarming. The temperature of post-thaw permeabilization varied in relation to the minimum temperature reached during freezing; cells cooled to -10 degrees C retained fluorescence into the post-thaw temperature range of 9-24 degrees C (mean +/- SEM; 13.25 +/- 0.91 degrees C), whereas cells cooled to -20 degrees C lost fluorescence shortly after thawing (mean +/- SEM; 2.62 +/- 0.91 degrees C). Sperm cooled to 5 degrees C, but not frozen, retained fluorescence during rewarming up to 20-30 degrees C. The inclusion of glycerol and egg yolk in the freezing medium significantly and independently increased the post-thaw permeabilization temperature. Maintenance of fluorescence was also correlated with ability to resume motility after thawing. Sperm reactivation experiments were undertaken to examine deleterious effects of freezing upon the flagellar microtubular assembly. No direct evidence for such effects was obtained. Instead, a highly significant correlation between minimum freezing temperature and post-thaw temperature of initial reactivation was detected.

Animals↗
PubMed2002

Identification and characterization of a novel freezing inducible gene, li16, in the wood frog Rana sylvatica.

The wood frog Rana sylvatica survives for weeks during winter hibernation with up to 65% body water frozen as ice. Natural freeze tolerance includes both seasonal and freeze-induced molecular adaptations that control ice formation, deal with long-term ischemia, regulate cell volume changes, and protect macromolecules. This report identifies and characterizes a novel freeze-inducible gene, li16, that codes for a protein of 115 amino acids. Northern blot analysis showed that li16 transcript levels rose quickly during freezing to reach levels 3.7-fold higher than control values after 24 h; immunoblotting showed a parallel 2.4-fold rise in Li16 protein. Regulatory influences on gene expression were assessed. Nuclear runoff assays confirmed that freezing initiated an increase in the rate of li16 transcription, and analysis of signal transduction pathways via in vitro incubation of liver slices implicated a cGMP-mediated pathway in li16 expression. Gene and protein expression in liver was also strongly stimulated by anoxia exposure, whereas the gene was less responsive to dehydration stress. The strong response of li16 to both freezing and anoxia, and the rapid down-regulation of the gene when oxygen was reintroduced, suggest that the Li16 protein may play a role in ischemia resistance during freezing.

Amphibian Proteins↗
PubMed2003

Freezing sensitivity in the sfr4 mutant of Arabidopsis is due to low sugar content and is manifested by loss of osmotic responsiveness.

Protoplasts were tested to determine whether the freezing sensitivity of the sfr4 (sensitive to freezing) mutant of Arabidopsis was due to the mutant's deficiency in soluble sugars after cold acclimation. When grown under nonacclimated conditions, sfr4 protoplasts possessed freezing tolerance similar to that of wild type, with the temperature at which 50% of protoplasts are injured (LT(50)) of -4.5 degrees C. In both wild-type and sfr4 protoplasts, expansion-induced lysis was the predominant lesion between -2 degrees C and -4 degrees C, but its incidence was low (approximately 10%); below -5 degrees C, loss of osmotic responsiveness (LOR) was the predominant lesion. After cold acclimation, the LT(50) was decreased to only -5.6 degrees C for sfr4 protoplasts, compared with -9.1 degrees C for wild-type protoplasts. Although expansion-induced lysis was precluded in both types of protoplasts, the sfr4 protoplasts remained susceptible to LOR. After incubation of seedlings in Suc solution in the dark at 2 degrees C, freezing tolerance and the incidence of freeze-induced lesions in sfr4 protoplasts were examined. The freezing tolerance of isolated protoplasts (LT(50) of -9 degrees C) and the incidence of LOR were now similar for wild type and sfr4. These results indicate that the freezing sensitivity of cold-acclimated sfr4 is due to its continued susceptibility to LOR (associated with lyotropic formation of the hexagonal II phase) and associated with the low sugar content of its cells.

Acclimatization↗
PubMed2004

Phylogenetic analyses in cornus substantiate ancestry of xylem supercooling freezing behavior and reveal lineage of desiccation related proteins.

The response of woody plant tissues to freezing temperature has evolved into two distinct behaviors: an avoidance strategy, in which intracellular water supercools, and a freeze-tolerance strategy, where cells tolerate the loss of water to extracellular ice. Although both strategies involve extracellular ice formation, supercooling cells are thought to resist freeze-induced dehydration. Dehydrin proteins, which accumulate during cold acclimation in numerous herbaceous and woody plants, have been speculated to provide, among other things, protection from desiccative extracellular ice formation. Here we use Cornus as a model system to provide the first phylogenetic characterization of xylem freezing behavior and dehydrin-like proteins. Our data suggest that both freezing behavior and the accumulation of dehydrin-like proteins in Cornus are lineage related; supercooling and nonaccumulation of dehydrin-like proteins are ancestral within the genus. The nonsupercooling strategy evolved within the blue- or white-fruited subgroup where representative species exhibit high levels of freeze tolerance. Within the blue- or white-fruited lineage, a single origin of dehydrin-like proteins was documented and displayed a trend for size increase in molecular mass. Phylogenetic analyses revealed that an early divergent group of red-fruited supercooling dogwoods lack a similar protein. Dehydrin-like proteins were limited to neither nonsupercooling species nor to those that possess extreme freeze tolerance.

Cornus↗
PubMed1996

Isolation of mutations affecting the development of freezing tolerance in Arabidopsis thaliana (L.) Heynh.

We screened for mutations deleterious to the freezing tolerance of Arabidopsis thaliana (L.) Heynh. ecotype Columbia. Tolerance was assayed by the vigor and regrowth of intact plants after cold acclimation and freezing. From a chemically mutagenized population, we obtained 13 lines of mutants with highly penetrant phenotypes. In 5 of these, freezing sensitivity was attributable to chilling injury sustained during cold acclimation, but in the remaining 8 lines, the absence of injury prior to freezing suggested that they were affected specifically in the development of freezing tolerance. In backcrosses, freezing sensitivity from each line segregated as a single nuclear mutation. Complementation tests indicated that the 8 lines contained mutations in 7 different genes. The mutants' freezing sensitivity was also detectable in the leakage of electrolytes from frozen leaves. However, 1 mutant line that displayed a strong phenotype at the whole-plant level showed a relatively weak phenotype by the electrolyte leakage assay.

Adaptation, Physiological↗
PubMed1999

Cold-induced freezing tolerance in Arabidopsis.

Changes in the physiology of plant leaves are correlated with enhanced freezing tolerance and include accumulation of compatible solutes, changes in membrane composition and behavior, and altered gene expression. Some of these changes are required for enhanced freezing tolerance, whereas others are merely consequences of low temperature. In this study we demonstrated that a combination of cold and light is required for enhanced freezing tolerance in Arabidopsis leaves, and this combination is associated with the accumulation of soluble sugars and proline. Sugar accumulation was evident within 2 h after a shift to low temperature, which preceded measured changes in freezing tolerance. In contrast, significant freezing tolerance was attained before the accumulation of proline or major changes in the percentage of dry weight were detected. Many mRNAs also rapidly accumulated in response to low temperature. All of the cold-induced mRNAs that we examined accumulated at low temperature even in the absence of light, when there was no enhancement of freezing tolerance. Thus, the accumulation of these mRNAs is insufficient for cold-induced freezing tolerance.

Acclimatization↗
PubMed1989

The effect of cooling rate, freeze-drying suspending fluid and culture age on the preservation of Campylobacter pylori.

The effects of freezing rate, suspending fluid and age of culture on the ability of four strains of Campylobacter pylori to survive and recover from freeze-drying were examined. Freeze-drying by standard procedures generally resulted in an overall loss in viability of between 3 and 7 log units. The exact cause of poor recovery by C. pylori was not established but strain differences were detected, with NCTC 11637 (type strain) surviving better than NCTC 11638 and NCTC 11639. Recovery of the poorest growing strain (NE 26695) was notably more erratic. The largest loss in viability occurred at the primary drying stage. Losses resulting from freezing and secondary drying were less marked and the rate of freezing had only a marginal effect on recovery. Nineteen different freeze-drying suspending fluids were investigated. Overall the best recovery results were obtained with 5% inositol-broth (or horse serum) plus 25% glucose, at pH 7.0, in which loss of viability was typically about 4 log units. Other factors, such as age of culture and number of viable bacteria in the before-dry suspension, did not have a significant effect on survival. We conclude from these results that C. pylori can survive freeze-drying, albeit in small numbers, but the degree of recovery is apparently largely strain dependent.

Campylobacter↗