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PubMed2004

Cryopreservation of Trichomonas vaginalis: a trial of using four different cryoprotectants.

To reach the maximum survival rate of Trichomonas vaginalis after cryopreservation, four substances: dimethylsulphoxide (DMSO), glycerol, egg yolk and horse serum were used as cryoprotectants. T. vaginalis trophozoites, in the logarithmic phase, cultured on TYM-S medium were used. Rapid and slow freezing schedules were followed. Examinations for viable organisms were done after a month duration in liquid nitrogen. Immediate and delayed thawing were tried. Viability scores depended on the presence of live organisms in the thawed parasite suspensions determined by the presence of motile trophozoites under microscopic examination, by dye uptake and by re-suspension of the organisms in fresh TYM-S and watching for parasite growth and multiplication. Rapid freezing protocol failed to cryopreserve T. vaginalis with any cryoprotectant. DMSO was superior to glycerol especially in 10% conc. by slow freezing. Viable organisms were more when the suspensions were immediately thawed without delay.

Animals↗
PubMed1989

[A comparative study of the cryoprotective properties of dimethylacetamide and dimethyl sulfoxide in preserving thrombocytes at moderately low temperatures].

Comparative characteristics of cryoprotective properties of dimethylacetamide and dimethyl sulfoxide (DMAC and DMSO, respectively), used for freezing and storage of platelets at -80 degrees C, have been presented. It is shown that with the studied final concentrations of the cryoprotectors (5% of DMSO, 2.5% of DMAC), solutions containing DMSO display higher cryoprotective properties. Positive effect of autoplasma in the mixture with the platelet-protecting solution has been manifest in the maintaining of the functional properties of platelets after defrosting.

Acetamides↗
PubMed1988

Cryopreservation of human spermatozoa. III. The effect of cryoprotectants on motility.

A series of experiments was conducted to examine potential toxic effects of cryoprotectants on motility of human spermatozoa. The data indicated that exposure of spermatozoa to cryoprotectant medium for as little as 15 minutes at room temperature caused a reduction in motility. This reduction in motility was caused by glycerol. Lowering glycerol concentrations from 7.5% to 5.0% improved sperm motility at 24 hours post-thaw. Sperm motility was not affected by either slow or abrupt cooling rates above -5 degrees C. Motility was greater in cryopreserved sperm at 24 hours post-thaw when glycerol was added at -5 degrees C rather than at room temperature. These data suggest that avoiding glycerol toxicity either by reducing the concentration used or by adding glycerol at a lower temperature, or both, may improve human sperm cryosurvival rates.

Cryoprotective Agents↗
PubMed1978

[Cryoprotective ability of glycerin and DMSO in the deep freezing of dog sperm].

The sperm-bearing fractions of 163 ejaculates collected from 6 Beagles aged two years were used to test the cryoprotective effect of DMSO and glycerol in deep freezing investigations. It could be shown that the cryoprotective capacity of glycerol was better than the one of DMSO. Glycerol should rather be added to the sperms at + 5 degrees C than at + 25 degrees C. The best preservation results were achieved by a combined addition of DMSO and glycerol under the following conditions: time of cooling down to +5 degrees C of the extended sperm fractions 50 minutes, final concentration of DMSO 1% (max.), final concentration of glycerol 8% (max.), glycerol equilibration time 30 minutes.

Animals↗
PubMed1981

[Problems relating to low temperature preservation of ram semen. 1. Effects of various cryoprotective agents on sperm].

Studies into the action of several cryoprotective agents in sodium-citrate-yolk diluent on ram semen provided preliminary information regarding both concentration applicable in deep-freeze preservation and toxicity of the agents involved. Included in the tests were glycerin, dimethylsulphoxide, ethylene glycol, propylene glycol, polyethylene glycol, polyvinylpyrrolidome, and dimethylacetamide. The effect of adding cryoprotective-containing diluent at 5 degrees C was more favourable, in its impact on ram sperma, than that observed, when diluent was added at 30 degrees C.

Acetamides↗
PubMedDate not supplied

Effect of cryoprotective additives and cryopreservation protocol on sperm membrane lipid peroxidation and recovery of motile human sperm.

Sperm membrane damage during cryopreservation reduces the recovery of motile sperm. The present study investigates changes in sperm motility and membrane lipid peroxidation (LPO) in response to two changes in the standard sperm cryopreservation/thawing methodology: 1) the addition of platelet-activating factor (PAF) and pentoxifylline (PTX) as cryoprotective additives, and 2) the alteration of sample thawing time. PAF (1 microM) and PTX (3 mM) were added to fresh sperm samples prior to cryopreservation. After 2 weeks the samples were thawed either quickly (5 minutes at 37 degrees C) or slowly (30 minutes at 4 degrees C) and evaluated for sperm motility and LPO. Thawing time influenced both post-thaw motility and LPO. Samples thawed quickly exhibited a 31% increase in motility recovery (35.2 +/- 4.3% in quick-thaw samples; 24.3 +/- 3.9% in slow-thaw samples) and a 23% lower LPO level (23.3 +/- 3.4% in quick-thaw samples; 30.09 +/- 4.4% in slow-thaw samples) compared to samples thawed slowly. Results also demonstrated that PAF (49 +/- 1.7%) or PTX (42.6 +/- 1.5%) enhance post-thaw motility in comparison to control (35.8 +/- 1.2%), whereas neither PAF nor PTX affect post-thaw LPO (19.1 +/- 2.2% in controls; 20.2 +/- 1.7% in PAF samples; 20.5 +/- 1.4% in PTX samples). These results support observations that there is a negative correlation between sperm motility and LPO in cryopreserved samples. The results also discount the hypothesis that LPO protection is a result of the cryoprotective action of PAF or PTX.

Cell Membrane↗
PubMedDate not supplied

[Effect of cryoprotectants on protein-synthesizing activity of cell-free system from rat liver].

The protein-synthetizing activity in postmitochondrial supernatant (S15) was studied as affected by ethylene glycol, PEG-400, PEG-4000, DMSO, ethanol, methanol, and glycerol. It is found that cryoprotectants inhibit 14C-leucine incorporation in the presence of physiological concentrations of Mg2+ (5 mM) in the cell-free system. All cryoprotectants, exept of glycerol, shift the maximum of 14C-leucine incorporation towards lower Mg2+ concentrations in the cell-free system.

Alcohols↗
PubMedDate not supplied

[Low temperature and cryoprotectant effect on oxygen uptake in rat kidney cortex homogenates].

Respiration and oxidative phosphorylation of the rat kidney cortex mitochondria are studied as affected by low temperatures and cryoprotectants. There is no change in respiration in state 2 during freezing and thawing of the rat kidney cortex slices both in case of using the succinate oxidation substrate and alpha-ketoglutarate. Respiration in state 3 is inhibited in the medium with alpha-ketoglutarate during freezing under protection of polyethylene oxide-100 (PEO-100) and glycerol, and does not significantly change in case of using dimethyl sulphoxide (DMSO). The effectiveness of cryoprotectants used in the experiments reduces in the following sequence: DMSO--glycerol--PEO-100. Preservation of the main mitochondria function, ATP synthesis, is shown to be possible for the kidney cortex slices exposed to freezing and thawing.

Adenosine Triphosphate↗
PubMed1990

Improved cryoprotection and freeze-substitution of embryonic quail retina: a TEM study on ultrastructural preservation.

Conditions for cryofixation and freeze-substitution crucial to the ultrastructural preservation of embryonic quail retina were improved. As freeze-substitution makes gentle dehydration and chemical fixation of tissue possible, the suitability of different cryoprotectants were tested in the preceding cryofixation. Additionally, different conditions for chemical prefixation were studied. In cryofixation, all of the "classic" cryoprotectants caused more or less severe tissue destruction. Only dimethylformamide (DMF) and--with certain reservations--dimethylsulfoxide (DMSO) yielded improved structure preservation. Perfusion fixation with a mixture of formaldehyde/glutaraldehyde (FA/GA) was superior to GA alone. In comparison to conventional fixation and dehydration methods, freeze-substitution yielded better ultrastructural preservation of the embryos with fewer artifacts.

Animals↗
PubMed1992

Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs.

Ice formation in the freeze-tolerant wood frog (Rana sylvatica) induces the production and distribution of the cryoprotectant, glucose. Concomitantly, organs undergo a beneficial dehydration which likely inhibits mechanical injury during freezing. Together, these physiological responses promote freezing survival when frogs are frozen under slow cooling regimes. Rapid cooling, however, is lethal. We tested the hypothesis that the injurious effects of rapid cooling stem from an inadequate distribution of glucose to tissues and an insufficient removal of water from tissues during freezing. Accordingly, we compared glucose and water contents of five organs (liver, heart, skeletal muscle, eye, brain) from wood frogs cooled slowly or rapidly during freezing to -2.5 degrees C. Glucose concentrations in organs from slowly cooled frogs were significantly elevated over unfrozen controls, but no significant increases occurred in rapidly cooled frogs. Organs from slowly cooled frogs contained significantly less water than did those from controls, whereas water contents from rapidly cooled frogs generally were unchanged. Rapid cooling therefore inhibited the production and distribution of cryoprotectant and organ dehydration during freezing. This inhibition may result from an accelerated, premature failure of the cardiovascular system.

Adaptation, Physiological↗
PubMed2000

Cold acclimation and cryoprotectants in a freeze-tolerant Antarctic nematode, Panagrolaimus davidi.

Panagrolaimus davidi is a freeze-tolerant Antarctic nematode which survives extensive intracellular freezing. This paper describes the development of culture techniques which provide clean samples, with a high degree of freeze tolerance and in sufficient quantities for the analysis of potential cryoprotectants. Cultures grown at 20 degrees C survived a short-term freezing stress but survival declined with the time spent frozen. Acclimation of cultures at 5 degrees C enhanced the long-term survival of freezing. Starvation, however, reduced the nematode's ability to survive short-term freezing. The principal cryoprotectants detected by gas chromatography were trehalose and glycerol. The levels of trehalose, but not those of glycerol, increased significantly after acclimation. Trehalose may stabilise membranes and protect them against the dehydrating effects of the osmotic stresses resulting from freeze concentration effects but other factors, such as recrystallisation inhibition, may be involved in long-term survival.

Acclimatization↗
PubMed1987

Use of sucrose during removal of cryoprotectants after thawing eight-cell mouse embryos.

Eight-cell mouse embryos were frozen in 0.5-ml plastic straws in modified Dulbecco's phosphate buffered saline (PBS) plus 5% steer serum plus either 1.32 M dimethyl sulfoxide (DMSO) or 1.32 M glycerol. Upon thawing, embryos were diluted 1:4 with 0.0, 0.2, 0.6, or 1.0 M sucrose solutions within the straws. Thawing was either in air at ambient temperature or in 8 degrees C or 38 degrees C water. After 48 h of culture, more embryos frozen in DMSO and thawed in 8 degrees C and 37 degrees C water developed to blastocysts (87 and 93%, respectively) than embryos thawed in air (75%; P<0.05). No significant differences in development were noted among the three thawing regimens when embryos were frozen with glycerol. There was no significant effect of concentration of sucrose during dilution on development of embryos postthaw. With glycerol as the cryoprotectant, damage to zonae pellucidae increased as thawing rates increased, whereas the opposite was observed with DMSO as the cryoprotectant (P<0.05).

Journal Article↗
PubMed1993

Dimethyl-acetamide as a cryoprotectant for rainbow trout spermatozoa.

Dimethyl-acetamide was evaluated as a cryoprotectant for rainbow trout (Oncorhynchus mykiss ) semen on the basis of motility, percentage of dead spermatozoa as determined by fluorometry, and fertility of frozen-thawed spermatozoa. Dimethyl-acetamide performed significantly better (P<0.05) than the conventional cryoprotectant, dimethyl sulfoxide, by all evaluation methods. An extender comprised of 0.137 M NaCl, 0.011 M KCl, 0.004 M Na(2)HPO(4) 7H(2)O, 7.5 g/l L-alpha-lecithin and 10 % DMA showed promise for cryopreserving rainbow trout spermatozoa. Future evaluation of new extenders should be carried out by several in vitro techniques and fertility measurements to present a complete assessment of an extender's ability to cryopreserve sperm cells.

Journal Article↗
PubMed1995

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.

Journal Article↗
PubMed2000

Cyclitols as cryoprotectants for spinach and chickpea thylakoids.

Thylakoid membranes isolated from either spinach or chickpea leaves were used as a model system for evaluating the capacity of cyclitols to act as cryoprotectants. The effect of freezing for 3 h at -18 degrees C on cyclic photophosphorylation and electron transport was measured. The cyclitols, ononitol, O-methyl-muco-inositol, pinitol, quebrachitol and quercitol at 50-150 mol m(-3) decreased membrane damage by freezing and thawing to a similar degree as the well known cryoprotectants sucrose and trehalose. On addition of the cryotoxic solute NaCl (100 mol m(-3)) to the test system these methylated cyclohexanhexols again provided a protection comparable to that of the two disaccharides. Quercitol (cyclohexanpentol) was not effective when added in lower concentrations (50-100 mol m(-3)) and in case of this cyclitol a ratio of membrane toxic to membrane compatible solute of 0.66 was apparently needed to prevent a loss of cyclic photophosphorylation. Little difference was observed in the results from spinach or chickpea thylakoids although these plants naturally accumulate different cyto-solutes (spinach: glycinebetaine; chickpea: pinitol).

Journal Article↗
PubMed2000

Time course for cryoprotectant synthesis in the freeze-tolerant chorus frog, Pseudacris triseriata.

Increases in liver glycogen phosphorylase activity, along with inhibition of glycogen synthetase and phosphofructokinase-1, are associated with elevated cryoprotectant (glucose) levels during freezing in some freeze-tolerant anurans. In contrast, freeze-tolerant chorus frogs, Pseudacris triseriata, accumulate glucose during freezing but exhibit no increase in phosphorylase activity following 24-h freezing bouts. In the present study, chorus frogs were frozen for 5- and 30-min and 2- and 24-h durations. After freezing, glucose, glycogen, and glycogen phosphorylase and synthetase activities were measured in leg muscle and liver to determine if enzyme activities varied over shorter freezing durations, along with glucose accumulation. Liver and muscle glucose levels rose significantly (5-12-fold) during freezing. Glycogen showed no significant temporal variation in liver, but in muscle, glycogen was significantly elevated after 24 h of freezing relative to 5 and 30 min-frozen treatments. Hepatic phosphorylase a and total phosphorylase activities, as well as the percent of the enzyme in the active form, showed no significant temporal variation following freezing. Muscle phosphorylase a activity and percent active form increased significantly after 24 h of freezing, suggesting some enhancement of enzyme function following freezing in muscle. However, the significance of this enhanced activity is uncertain because of the concurrent increase in muscle glycogen with freezing. Neither glucose 6-phosphate independent (I) nor total glycogen synthetase activities were reduced in liver or muscle during freezing. Thus, chorus frogs displayed typical cryoprotectant accumulation compared with other freeze-tolerant anurans, but freezing did not significantly alter activities of hepatic enzymes associated with glycogen metabolism.

Adaptation, Physiological↗
PubMed2000

An osmotin-like cryoprotective protein from the bittersweet nightshade Solanum dulcamara.

Cold acclimation in plants is a polygenic phenomenon involving increased expression of several genes. The gene products participate either directly or indirectly towards increasing cold tolerance. Evidence of proteins having a direct effect on cold tolerance is emerging but limited. With isolated protoplasts from warm-grown kale (Brassica oleracea) as a model system, we tested protein fractions from winter bittersweet nightshade, Solanum dulcamara, stems for the presence of proteins that have a cryoprotective effect. Purification of one such fraction resulted in isolation of a 25 kDa protein. N-terminal Edman degradation amino acid sequence analysis showed that it has high homology to osmotin and osmotin-like proteins. When added to warm-grown protoplasts, it increased the cryosurvival of frozen-thawed protoplasts by 24% over untreated or BSA-treated controls at -8 degrees C. A cDNA library which was made in November from stems and leaves of S. dulcamara was successfully screened for the corresponding cDNA clone. The deduced amino acid sequence indicated that the protein consists of 206 amino acid residues including a N-terminal signal sequence and a putative C-terminal propeptide. The mature protein, without the N-terminal signal sequence, was expressed in Escherichia coli. The partially purified protein in the supernatant fraction of the culture medium had cryoprotective activity.

Acclimatization↗
PubMed1992

The effects of cryosurgery and cryoprotectants on peripheral nerve function.

Cryosurgery has many advantages in the treatment of musculoskeletal tumors, but complications can occur, with injuries to the peripheral nerves. Cryoprotectants such as dimethylsulfoxide (DMSO) and glycerol enhance the in vitro survival of cultured Schwann cells, but they have not been studied in vivo. In vitro, 10 percent DMSO provided the maximal survival after freezing to -80 degrees C. The neurologic recovery of a peripheral nerve, following cryoinjury and using liquid nitrogen, was evaluated both with and without pretreatment of the nerve with 10 percent DMSO. There were three experimental groups: 1) one sham operation; 2) with freezing of the sciatic nerve; and 3) with freezing of the sciatic nerve after treatment with DMSO. Gait analyses (sciatic function index, SFI), muscle weights, and histologic evaluations were performed in order to follow the neurologic recovery over a ten-week period. The freezing-only group and the sham-operated control had an excellent functional recovery in terms of the SFI following surgery (-4.2 percent +/- 4.0 percent and -2.4 percent +/- 5.7 percent, respectively), in contrast to the freezing group with DMSO (-34.1 percent +/- 5.5 percent, p less than .01). The average tibialis anterior muscle weight, reported as the ratio of the operated to the unoperated limb (88.2 percent +/- 1.0 percent) in the freezing-only group, was greater than the average for the freezing-with-DMSO-treated group (70.6 percent +/- 3.9 percent, p less than .01). Cryoprotectants are routinely used to optimize the survival of cells in tissue culture. However, DMSO not only failed to protect peripheral nerves from cryoinjury, but it also inhibited functional recovery.

Animals↗