The effect of cooling rate and of dimethyl sulfoxide concentration on the ultrastructure of neonatal rat heart cells after freezing and thawing

The ultrastructure of neonatal rat heart cells in suspension and in tissue culture after freezing at optimal, suboptimal, and supraoptimal cooling rates with 2.5, 5, 7.5, and 10% DMSO was investigated. The effect of DMSO treatment only on the structure of the cells was also studied. A comparison was made with the survival in culture.Without freezing, increasing DMSO concentrations caused an increase of morphological damage, correlating with a decrease of the survival in culture. With 2.5% DMSO there was no difference with untreated cells. At higher DMSO concentrations, the ultrastructural damage increased from spaces between cell membrane and cytoplasm at 5% DMSO to interrupted cell membranes, swollen or destroyed mitochondria, and nuclei with clumped chromatin at 10% DMSO.After freezing at optimal or nonoptimal cooling rates with 5 or 7.5% DMSO, the ultrastructure correlated well with the survival. After freezing with 2.5 or 10% DMSO at optimal or nonoptimal cooling rates, differences in survival were found, which were not reflected in the ultrastructure of the cell. After 8 days of culturing, cells which were frozen at all the different cooling rates and DMSO concentrations appeared to have a normal structure.     

Morphogenesis of the hydrogenosome: An ultrastructural study

Summary— The morphogenesis of hydrogenosomes in several trichomonad species (Tritrichomonas foetus, Trichomonas vaginalis, Tritrichomonas suis, Trichomonas gallinae, Tritrichomonas augusta and Monocercomonas sp) was investigated by transmission electron microscopy of thin sections and freeze-fracture replicas of whole cells or the isolated organelle. Close proximity, and even continuity, between endoplasmic reticulum and hydrogenosomes was observed. Structures were seen connecting hydrogenosomes to each other and to cytoplasmic structures. Morphological evidence is presented showing that in all the trichomonads here studied, hydrogenosomes, like mitochondria, may divide by two distinct processes: segmentation and partition. In the segmentation process, the hydrogenosome grows, becoming enlongated with the appearance of a constriction in the central portion. Microfibrillar structures appear to help the furrowing process, ending with a total fission of the organelle. In the partition process, the division begins by an invagination of the inner hydrogenosome membrane, forming a transversal septum, separating the organelle matrix into two compartments. We suggest that myelin-like structures seen either in close contact with or in the vicinity of the hydrogenosomes may be a source of membrane lipids for hydrogenosome growth.     

Hydrogenosome autophagy: an ultrastructural and cytochemical study.

The process of autophagy was studied in Tritrichomonas foetus under serum deprivation, drug treatment (hydroxyurea, zinc sulfate), and also in normal conditions using routine electron microscopy, freeze-fracture, freeze-substitution, and enzyme cytochemistry. We also used gold particles conjugated with bovine albumin to better characterize the participation of lysosomes in the process of hydrogenosome degradation. Apparently normal hydrogenosomes and also giant, abnormal hydrogenosomes presenting internal membranes were seen in the autophagic process. The first event observed was the rough endoplasmic reticulum surrounding and enclosing the hydrogenosome, forming an isolation membrane. The hydrogenosomes were first sequestered from the remaining cytoplasm and then degraded within lysosomes. The autophagic vacuoles were limited by double or multiple concentric membranes and many contained recognizable hydrogenosomes, probably in the preliminary steps of degradation. Lysosomes seemed to fuse with autophagic vacuoles forming a degradative structure bound by a single membrane and containing hydrogenosomes in various stages of degeneration. Hydrogenosomes appeared partially degraded, forming hydrogenosomal remnants. It was observed that there is a removal of hydrogenosomes in normal cells and in cases of cell toxicity.     

Cell death in trichomonads: new insights

Tritrichomonas foetus is an amitochondriate parasite that possesses hydrogenosomes, unusual anerobic energy-producing organelles. In these organisms the “mitochondrial cell death machinery” is supposed to be absent, and the mechanisms that lead to cell demise remain to be elucidated. The presence of a cell death program in trichomonads has already been reported, suggesting the existence of a caspase-like execution pathway in such organisms. Here we demonstrate the alterations provoked by the fungicide griseofulvin and raise the possibility that other cell death pathways may exist in T. foetus. Dramatic changes in trichomonads morphology are presented after griseofulvin treatment, such as intense plasma membrane and nuclear envelope blebbing, nucleus fragmentation, and an abnormal number of oversized vacuoles. One important finding was the exposition of phosphatidylserine (PS) in the outer leaflet of the plasma membrane in cells after drug treatment, and also the presence of a high amount of misshapen flagella and tubulin precipitates as vacuolar contents, suggesting an autophagic process of abnormal cellular elements. Interestingly, immunoreactivity for activated caspase-3 was not detected during griseofulvin treatment, a finding distinct from the observed when this cell was treated with H₂O₂. The possibility of the existence of different pathways to cell death in trichomonads is discussed.     

Membrane permeabilization of phosphatidylcholine liposomes induced by cryopreservation and vitrification solutions

Membranes are the primary site of freezing injury during cryopreservation or vitrification of cells. Addition of cryoprotective agents (CPAs) can reduce freezing damage, but can also disturb membrane integrity causing leakage of intracellular constituents. The aim of this study was to investigate lipid-CPA interactions in a liposome model system to obtain insights in mechanisms of cellular protection and toxicity during cryopreservation or vitrification processing. Various CPAs were studied including dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), dimethyl formamide (DMF), and propylene glycol (PG). Protection against leakage of phosphatidylcholine liposomes encapsulated with carboxyfluorescein (CF) was studied upon CPA addition as well as after freezing-and-thawing. Molecular interactions between CPAs and phospholipid acyl chains and headgroups as well as membrane phase behavior were studied using Fourier transform infrared spectroscopy. A clear difference was observed between the effects of DMSO on PC-liposomes compared to the other CPAs tested, both for measurements on CF-retention and membrane phase behavior. All CPAs were found to inhibit membrane leakiness during freezing. However, exposure to high CPA concentrations already caused leakage before freezing, increasing in the order DMSO, EG, DMF/PG, and GLY. With DMSO, liposomes were able to withstand up to 6 M concentrations compared to only 1 M for GLY. Cholesterol addition to PC-liposomes increased membrane stability towards leakiness. DMSO was found to dehydrate the phospholipid headgroups while raising the membrane phase transition temperature, whereas the other CPAs caused an increase in the hydration level of the lipid headgroups while decreasing the membrane phase transition temperature.     

The ability of cryopreservatives to prevent motility loss and freeze-thaw damage to the acrosome of chicken spermatozoa

Proteolytic membrane digestion and motility were used to determine the effects of cryopreservatives and freezing on acrosomal damage and survival of chicken spermatozoa. None of the cryopreservatives, glycerol, DMSO, or ethylene glycol caused a decrease in proteolytic membrane digestion by the chicken spermatozoon before freezing. After rapid freezing, high levels (16 and 24%) of glycerol prevented significant freeze-thaw reductions in proteolytic digestion. High levels of ethylene glycol (16 and 24%) and DMSO (12 and 14%) significantly reduced the ability of frozen spermatozoa to completely digest the protein membrane. Motility after freezing was highest (63%) in 16% glycerol.Glycerol at high levels appeared to be the best cryopreservative under these conditions.     

Improving sperm cryopreservation with antifreeze proteins: effect on gilthead seabream (Sparus aurata) plasma membrane lipids

Changes in the plasma membrane lipid composition have been related to a decrease in sperm quality during cryopreservation. Antifreeze proteins (AFPs) have been tested in different species because of their ability to depress the freezing point and their potential interaction with membranes, but controversial effects were reported. In the present study we analyzed separately the lipid composition of two sperm membrane domains, head plasma membrane (HM) and flagellar membrane (FM), after cryopreservation with an extender containing 5% dimethyl sulfoxide (DMSO) either alone or with AFPI or AFPIII (1 μg/ml). We used sperm from a teleost, Sparus aurata, because the lack of acrosome avoids changes of lipid profiles due to capacitation process or acrosomal losses during freezing/thawing. Comparing with the control (cryopreservation with 5% DMSO alone), the addition of AFPIII increased the velocity, linearity of movement, and percentage of viable cells. In addition, freezing with DMSO alone increased the phosphatidyl-serine content as well as the saturated fatty acids and decreased the unsaturated ones (mainly polyunsaturated) both in HM and FM. These changes in the lipid components were highly avoided with the addition of AFPIII. HM had a higher amount of saturated fatty acids than FM and was more affected by cryopreservation without AFPs. The percentage of viable cells was positively correlated with the amount of unsaturated fatty acids in the HM, whereas the motility parameters were positively correlated with both FM and HM amount of unsaturated fatty acids. AFPs, especially AFPIII, seem to have interacted with unsaturated fatty acids, stabilizing the plasma membrane organization during cryopreservation and contributing to improve sperm quality after thawing.     

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.     

Effect of external cryoprotectants as membrane stabilizers on cryopreserved rainbow trout sperm

The process of freezing and thawing induces certain cellular damage in rainbow trout (Oncorhynchus mykiss) spermatozoa. We have previously demonstrated that after freezing and thawing decreased fertility in rainbow trout (Oncorhynchus mykiss) spermatozoa, is related to sublethal damage to the plasma membrane. External cryoprotectants are known to stabilize the sperm cell membrane against such damage. In the current study, we used a basic freezing extender containing #6 Erdahl and Graham and 7% DMSO and added egg yolk, BSA, and a soybean-protein complex (DanPro S760) singly and in various combinations. To assess the effect of these cryoprotectants we evaluated the percentage of cells with progressive motility, permeability of cells to propidium iodide (viability) after exposure for 30 sec, 2, 5, 10 and 15 min. to hypo- and isoosmotic solutions of 10 and 300 mOsm, and the in vitro fertility rate. Fertility trials were performed using 1.87 x 10(7) spermatozoa/egg. Some of the tested stabilizers increased motility, increased viability, or reduced cell fragility after freezing and thawing. Nevertheless these quality improvements demonstrated by the "in vitro" tests do not always correlate with high fertility. The best membrane protection in terms of resistance to hypoosmotic shock was achieved when BSA and egg yolk were added to the extender. The highest fertility rates were obtained with DanPro S760 alone or in combination with BSA; the use of BSA with egg yolk did not improve this parameter. Our results demonstrated that some external cryoprotectants effectively increased membrane resistance during freezing and thawing, but some of the tested mixtures interfered with fertilization. Soybean protein concentrate provided good protection and increased fertility rates in cryopreserved trout spermatozoa.     

Mitochondrial type iron-sulfur cluster assembly in the amitochondriate eukaryotes Trichomonas vaginalis and Giardia intestinalis, as indicated by the phylogeny of IscS

Pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS) is an essential enzyme in the assembly of FeS clusters in bacteria as well as in the mitochondria of eukaryotes. Although FeS proteins are particularly important for the energy metabolism of amitochondrial anaerobic eukaryotes, there is no information about FeS cluster formation in these organisms. We identified and sequenced two IscS homologs of Trichomonas vaginalis (TviscS-1 and TviscS-2) and one of Giardia intestinalis (GiiscS). TviscS-1, TviscS-2, and GiiscS possess the typical conserved regions implicated in cysteine desulfurase activity. N-termini of TviscS-1 and TviscS-2 possess eight amino acid extensions, which resemble the N-terminal presequences that target proteins to hydrogenosomes in trichomonads. No presequence was evident in GiiscS from Giardia, an organism that apparently lacks hydrogenosmes or mitochondria. Phylogenetic analysis showed a close relationship among all eukaryotic IscS genes including those of amitochondriates. IscS of proteobacteria formed a sister group to the eukaryotic clade, suggesting that isc-related genes were present in the proteobacterial endosymbiotic ancestor of mitochondria and hydrogenosomes. NifS genes of nitrogen-fixing bacteria, which are IscS homologs required for specific formation of FeS clusters in nitrogenase, formed a more distant group. The phylogeny indicates the presence of a common mechanism for FeS cluster formation in mitochondriates as well as in amitochondriate eukaryotes. Furthermore, the analyses support a common origin of Trichomonas hydrogenosomes and mitochondria, as well as secondary loss of mitochondrion/hydrogenosome-like organelles in Giardia.     

Ultrastructure-function correlative studies for cardiac cryopreservation. V. Absence of a correlation between electrolyte toxicity and cryoinjury in the slowly frozen,cryoprotected rat heart

Hearts were frozen to ?17 °C in the initial presence of 2.1 m DMSO. Attempts were made to prevent or minimize the consequences of an osmotic shock based on Lovelock's classical hypothesis of freezing injury. Substitution of mannitol or potassium for NaCl before freezing did not improve the results, nor did perfusion of thawed hearts with hyperosmotic perfusate. It was found that freezing and thawing resulted in a significant attenuation of coronary flow and that, as a result of this, DMSO was apparently retained within the heart after thawing. DMSO was also difficult to remove at 30 °C in the absence of prior freezing and caused a significant drop in coronary flow upon institution of DMSO washout with balanced salt solution. The blanching of freezing and thawing was also seen, in milder form, in nonfrozen hearts. For both frozen-thawed and nonfrozen hearts, the blanching was associated with DMSO washout with balanced salt solution. Flow was improved by perfusion with hyperosmotic perfusate in both nonfrozen and in frozen-thawed hearts, but the improvement was largely temporary. Evidence from earlier studies indicates that electrolyte concentrations during freezing cannot be correlated with cardiac cryoinjury, in support of the present findings. It is suggested instead that cryoprotectant toxicity may be the chief agent of injury under the conditions studied.     

Fine-structure changes in Toxoplasma gondii trophozoites after deep-freezing with dimethyl sulphoxide (author's transl)]

The changes observed in trophozoites of Toxoplasma gondii after deep-freeze preservation were examined by electron microscopy. Toxoplasmas (strain BK) from peritoneal exudate of infected NMRI mice were supended in Ringer's solution, deep-frozen in liquid nitrogen with 5% dimethylsulphoxide (DMSO), and compared after thawing with control samples with and without the addition of DMSO. Slight structural changes such as widening of endoplasmic reticulum, formation of fissures in the cytoplasm, and loosening of chromatin were only observed in some of the free toxoplasmas of the DMSO control. Among the deep-frozen parasites, about 1/5 of the free stages showed no or only slight morphological changes. In contrast to this, almost all intracellular forms found in macrophages showed lesions. The most remarkable change was a partial destruction of the inner cell membrane complex. The outflow of ribosome-containing protoplasm with ballon-like swelling of the outer elementary membrane was observed as a consequence of this frequent lesion. The outflow of protoplasm induced a drastic decrease in the electronic density of the whole cytoplasm. Other characteristic degenerative signs were vacuolation of cytoplasm up to formation of great optically empty spaces, widening of the perinuclear space, swelling of mitochondria, disintegration of rhoptria, micronemata, and Golgi zone, coarse-plaque loosening, and displacement of electron-dense areas of the nucleus up to disintegration with maintenance of the karyoplasm. In some almost completely disintegrated trophozoites, enlarged mitochondria with remarkable electronic density were observed. Apart from the cell membrane, the conoid was the longest-persisting organelle. The alterations observed after deep-freezing permit the conclusion that the free cells, which were only slightly impaired or not at all, remained infective.     

Effect of glycerol and dimethyl sulfoxide on cryopreservation of rhesus monkey (Macaca mulatta) sperm

Glycerol and dimethyl sulfoxide (DMSO) are widely used as penetrating cryoprotectants in the freezing of sperm, and various concentrations are applied in different species and laboratories. The present study aimed to examine the effect of these two cryoprotectants at different concentrations (2%, 5%, 10%, and 15% glycerol or DMSO) on rhesus monkey sperm cryopreservation. The results showed that the highest recovery of post-thaw sperm motility, and plasma membrane and acrosome integrity was achieved when the sperm was frozen with 5% glycerol. Spermatozoa cryopreserved with 15% DMSO showed the lowest post-thaw sperm motility, and spermatozoa cryopreserved with 15% glycerol and 15% DMSO showed the lowest plasma membrane integrity among the eight groups. The results achieved with 5% glycerol were significantly better for all parameters than those obtained with 5% DMSO. The functional cryosurvival of sperm frozen with 5% glycerol was further assessed by in vitro fertilization (IVF). Overall, 85.7% of the oocytes were successfully fertilized, and 51.4% and 5.7% of the resulting zygotes developed into morulae and blastocysts, respectively. The results indicate that the type and concentration of the penetrating cryoprotectant used can greatly affect the survival of rhesus monkey sperm after it is frozen and thawed. The suitable glycerol level for rhesus monkey sperm freezing is 5%, and DMSO is not suitable for rhesus monkey sperm cryopreservation.     

CRYOPROTECTANT-INDUCED REDISTRIBUTION OF INTRAMEMBRANOUS PARTICLES IN MOUSE LYMPHOCYTES

This study demonstrates, by freeze fracture, clustering of intramembranous particles caused by cryoprotectant treatment of intact unfixed mouse lymphoid cells. Both T and B cells react in a similar fashion, while similar clustering of particles is not observed in some other cell types. The intramembranous particles can be aggregated by incubating unfixed cells in glycerol or dimethylsulfoxide (DMSO) before freezing. The aggregation phenomenon is dependent on the length of time of exposure and the concentration of the cryoprotectants. Further, the cells remain viable and the cryoprotectant-induced clustering is completely reversible. Prefixation of glycerol-treated cells with glutaraldehyde prevents the formation of these particle clusters, and unfixed nonglycerinated cells show no clusters. Thin sections of cells exposed to glycerol or DMSO without previous fixation exhibit bizarre membrane alterations and numerous other degenerative changes. These observations stress the importance of prefixation of lymphoid cells before exposure to glycerol or DMSO, as well as indicate that the membrane characteristics of certain cell types may be probed by glycerol treatment of unfixed cells.     

Cryopreservation of platelets using trehalose: The role of membrane phase behavior during freezing

In blood banks, platelets are stored at 20–24°C, which limits the maximum time they can be stored. Platelets are chilling sensitive, and they activate when stored at temperatures below 20°C. Cryopreservation could serve as an alternative method for long term storage of platelet concentrates. Recovery rates using dimethyl sulfoxide (DMSO) as cryoprotective agent, however, are low, and removal of DMSO is required before transfusion. In this study, we have explored the use of trehalose for cryopreservation of human platelets while using different cooling rates. Recovery of membrane intact cells and the percentage of nonactivated platelets were used as a measure for survival. In all cases, survival was optimal at intermediate cooling rates of 20°C min^?1. Cryopreservation using DMSO resulted in high percentages of activated platelets; namely 54% of the recovered 94%. When using trehalose, 98% of the platelets had intact membranes after freezing and thawing, whereas 76% were not activated. Using Fourier transform infrared spectroscopy, subzero membrane phase behavior of platelets has been studied in the presence of trehalose and DMSO. Furthermore, membrane hydraulic permeability parameters were derived from these data to predict the cell volume response during cooling. Both trehalose and DMSO decrease the activation energy for subzero water transport across cellular membranes. Platelets display a distinct lyotropic membrane phase transition during freezing, irrespective of the presence of cryoprotective agents. We suggest that concomitant uptake of trehalose during freezing could explain the increased survival of platelets cryopreserved with trehalose. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Isolation and low temperature preservation of adult rat heart cells

Adult rat heart cells were isolated by perfusion of the coronary system of the heart with a 0.05% collagenase solution.In one method (A), cells were finally isolated by shaking the heart fragments in a collagenase solution, after which the cells were washed and suspended in a Ca- and Mg-free buffered salt solution. The effect of different DMSO concentrations, 5, 10, 15, 20, 25, and 30% and the effect of the addition and dilution rate of DMSO on the number of trypan blue-excluding, intact, and contracting cells were studied. The highest DMSO concentration which was tolerated by the isolated adult heart cells was 15%. Variation of addition rate and the dilution rate of DMSO had no effect. After freezing at external cooling rates of 1, 5, 10, 30, and 50 °C/min to ?100 °C, and then rapidly to ?196 °C, in the presence of 5, 10, or 15% DMSO, reanimation of these cells was not achieved.In another method (B), heart fragments, after collagenase perfusion of the heart, were first treated with 5, 10, or 15% DMSO, after which the cells were isolated. If these cells were frozen at 1 °C/min with 10% DMSO, 15% of the cells, expressed as a percentage of the control, remained morphologically intact and 38% of the cells were contracting after thawing. Significantly higher survival percentages of 30 and 61%, respectively, were obtained if the heart fragments were left intact during freezing.     

Polypeptides of hydrogenosome-enriched fractions from rumen ciliate protozoa and trichomonads: Immunological studies

Abstract The evolution of hydrogenosomes, energy-generating organelles of rumen ciliate protozoa and the flagellate trichomonads has been the subject of much speculation. Polypeptides of the hydrogenosome-enriched fractions from the rumen ciliates, Dasytricha ruminantium, Isostricha spp., Polyplastron multivesiculatum and Eudiplodinium maggii were separated by SDS-PAGE and compared to analogous polypeptide preparations from Tritrichomonas foetus . Immunoblotting with antisera specific to the hydrogenosomes of T. foetus identified common immunoreactive polypeptides present at estimated molecular masses of 28, 35, 38, 44, 48, 58, 100 and 120 kDa. That at 120 kDa corresponds to a single subunit of the purified pyruvate: ferredoxin oxidoreductase from the hydrogenosome of Trichomonas vaginalis .     

Mechanisms of intracellular ice formation.

The phenomenon of intracellular freezing in cells was investigated by designing experiments with cultured mouse fibroblasts on a cryomicroscope to critically assess the current hypotheses describing the genesis of intracellular ice: (a) intracellular freezing is a result of critical undercooling; (b) the cytoplasm is nucleated through aqueous pores in the plasma membrane; and (c) intracellular freezing is a result of membrane damage caused by electrical transients at the ice interface. The experimental data did not support any of these theories, but was consistent with the hypothesis that the plasma membrane is damaged at a critical gradient in osmotic pressure across the membrane, and intracellular freezing occurs as a result of this damage. An implication of this hypothesis is that mathematical models can be used to design protocols to avoid damaging gradients in osmotic pressure, allowing new approaches to the preservation of cells, tissues, and organs by rapid cooling.     

Effect of cytochalasin B and demecolcine on freeze-thaw survival of murine embryos in vitro

The effects of cytochalasin B (CB), a microfilament inhibitor, and demecolcine (DC), a microtubule inhibitor, on freeze-thaw survival and culture survival of early cleavage stage mouse embryos, was evaluated. In the first experiment, eight-cell mouse embryos were frozen in dimethylsulfoxide (DMSO) or DMSO + 0.1 microgram/ml DC + 7.5 micrograms/ml CB. In the second experiment, eight-cell embryos were dehydrated and cultured in the presence of either DMSO, DMSO + DC, DMSO + CB, or DMSO + DC + CB for 45 min prior to rehydration and culture to stimulate the osmotic and chemical changes encountered during the dehydration and rehydration procedures, but without the consequences of freezing and thawing. In the third experiment, additional eight-cell embryos were frozen in either DMSO, DMSO + DC, DMSO + CB, or DMSO + DC + CB. The survival of embryos frozen in DMSO (75%) was significantly higher (P less than 0.01) than that of embryos frozen in DMSO + DC + CB (55%). No differences (P = 0.55) were observed after a 48-hr culture period in the development of embryos dehydrated, cultured, and rehydrated but not frozen. Embryos frozen in the presence of both DC and CB had a lower (P = 0.06) survival rate (55%) than that of embryos frozen in the presence of DMSO, DC, or CB (approximately 70%). These results suggest that both microfilaments and microtubules have a role in maintaining the structural integrity of the plasma membrane during the freeze-thaw process and that the loss of either loss does not seem to be detrimental to survival, but the loss of both results in lower survival.