Effect of high osmotic media on blood viscosity and red blood cell deformability

Effects of high osmotic media on the shape and deformability of RBC were examined for determining increasing factors of blood viscosity. Dog blood and Urographin (a hypertonic contrast medium) were used; the plasma osmolality was changed by Urografin suspended in blood. The viscosity was measured for normal RBC and glutaraldehyde-treated RBC suspensions with a cell volume concentration. The RBC deformability was evaluated from the difference in viscosity between the two suspensions. It was shown that normal RBC suspension increased the viscosity with increase in osmolality at high shear rate; hardened RBC suspension decreased the viscosity with increase in osmolality. It was concluded that the RBC deformability decreased with increasing osmolality.     

Resistance of mammalian red blood cells of different size to hypertonic milieu

1. The resistance of different mammalian red blood cells (RBCs) to hyperosmotic environments was studied. RBCs of six mammalian species were exposed to 10 increasingly hyperosmotic NaCl solutions for 24 hr at 5 degrees C. 2. The osmolality at which the amount of liberated haemoglobin reached a preset level (e.g. 3-4% of the total haemoglobin) showed a linear correlation with negative slope with RBC volume. This indicates that small RBCs are more resistant to hyperosmotic milieu than large ones. 3. A similar relation can be found from literature data when maximal urinary tonicities are plotted as a function of RBC volume, i.e. animals with the ability to produce highly concentrated urine have small RBCs. 4. RBC volume and maximal urinary tonicity in mammals are therefore tightly linked. Future research will have to show whether this correlation is fortuitous or not and whether, as can be speculated, RBC size is directly or indirectly regulated by the kidney.     

A new red blood cell filtration device with improved time resolution and its application to the impaired RBC deformability in the diabetic ob/ob mouse

A new filtration device and blood handling technique for the assessment of RBC deformability in small blood samples is described and used to study RBC deformability in adult obese-hyperglycemic ob/ob-mice and normoglycemic controls. The new filtration device was designed to improve the time resolution during RBC incubation. Test and control RBC suspensions were directly filtered from two identical incubation chambers under a constant pressure of 1200 Pa. Nuclepore filters (3 microns) were mounted on top of several standard test tubes into which the filtrate was subsequently collected and weighed. Because the RBCs were resuspended to a very low (0.01%) hematocrit, the average number of RBCs passing each pore was less than 10. Therefore, any detectable difference must reflect the physical properties of RBCs, e.g. shape or viscoelasticity, whereas the role of white blood cells is negligible. When ob/ob-mouse RBCs were studied with the new technique they showed impaired filtrability as compared with control RBCs, both when incubated without glucose and with glucose present at the same concentration as that recorded in the RBC donating mouse.     

Initial studies on sperm cryopreservation of a live-bearing fish, the green swordtail Xiphophorus helleri

Swordtails and platyfish of the genus Xiphophorus are valuable models for biomedical research and are also commercially raised as ornamental fish valued by aquarists. While research use and commercial interest increases yearly in these fish, cryopreservation of sperm is unexplored in this genus. Xiphophorus are live-bearing fishes characterized by small body sizes, limited sperm volumes, and internal fertilization, an atypical reproductive mode for fish. These attributes make research involving cryopreservation of Xiphophorus germplasm challenging. To explore methods for sperm cryopreservation, this study evaluated the effect of different loading volumes of sperm suspension in 0.25-ml French straws, different dilution ratios of sperm to extender, an osmolality range of extender without cryoprotectant and with dimethyl sulfoxide (DMSO) as cryoprotectant, and short-term storage at room temperature and 4 degrees C after thawing. No significant difference in sperm motility due to straw loading volume was observed after thawing. Sperm motility was observed to decrease with increasing dilution. The osmolality of Hanks' balanced salt solution (HBSS) without cryoprotectant in which the highest sperm motility (67%) was observed was 320 +/- 3 mOsm/kg, which was also the osmolality of X. helleri blood plasma. When cryopreserved with 10% DMSO, however, the highest motilities within 10 min after thawing were observed with HBSS in the range of 240-300 mOsm/kg. Sperm suspended in HBSS at 320 mOsm/kg with a dilution factor of 100 maintained motility for 24h at room temperature, but persisted for 10 days when stored at 4 degrees C. These results provided the first evidence that cryopreservation may be applied to conservation of genetic resources in live-bearing fishes.     

Quantitative autoradiographic assessment of 55Fe-RBC distribution in rat brain

A simple in vivo technique of labeling erythrocytes (RBCs) with 55Fe was developed for quantitative autoradiography (QAR). This procedure involved injecting 5-6 ml of [55Fe]ferrous citrate solution (1 mCi/ml) intraperitoneally into donor rats. The number of labeled RBCs reached a maximum at around 7 days and declined very slowly thereafter. Labeled RBCs were harvested from donor rats and used for RBC volume measurement in awake rats. Brain radioactivity was assayed by QAR, which yielded spatial resolution of greater than 50 microns. Tight nearly irreversible binding of 55Fe to RBCs was found in vivo and in vitro. More than 99.5% of the 55Fe in the blood of donor rats was bound to RBCs. Because of this, labeled blood can be taken from donors and injected into recipients without further preparation. The tissue absorption of 55Fe emissions was the same in gray and white matter. Microvascular RBC volumes measured with 55Fe-labeled RBCs agreed with those assayed with 51Cr-labeled RBCs for many, but not all, brain areas. In conclusion, 55Fe-RBCs can be readily prepared by this technique and accurately quantitated in brain tissue by QAR.     

Loading red blood cells with trehalose: a step towards biostabilization

A method for freeze-drying red blood cells (RBCs) while maintaining a high degree of viability has important implications in blood transfusion and clinical medicine. The disaccharide trehalose, found in animals capable of surviving dehydration can aid in this process. As a first step toward RBC preservation, we present a method for loading RBCs with trehalose. The method is based on the thermal properties of the RBC plasma membranes and provides efficient uptake of the sugar at 37 degrees C in a time span of 7 h. The data show that RBCs can be loaded with trehalose from the extracellular medium through a combination of osmotic imbalance and the phospholipid phase transition, resulting in intracellular trehalose concentrations of about 40 mM. During the loading period, the levels of ATP and 2,3-DPG are maintained close to the levels of fresh RBCs. Increasing the membrane fluidity through the use of a benzyl alcohol results in a higher concentration of intracellular trehalose, suggesting the importance of the membrane physical state for the uptake of the sugar. Osmotic fragility data show that trehalose exerts osmotic protection on RBCs. Flow cytometry data demonstrate that incubation of RBCs in a hypertonic trehalose solution results in a fraction of cells with different complexity and that it can be removed by washing and resuspending the RBCs in an iso-osmotic medium. The data provide an important first step in long-term preservation of RBCs.     

Cryopreservation of red blood cells: Effect on rheologic properties and associated metabolic and nitric oxide related parameters

AimHigh glycerol cryopreservation of red blood cells (RBCs) reduces metabolic processes at ultralow temperatures but less is known regarding the effect of cryopreservation on RBC nitric oxide (NO) metabolism, haemorheological properties, structural behaviour and membrane fragility.MethodsBlood from ten healthy participants was sampled, glycerolized and stored at −80 °C (SB). Aliquots were thawed and further processed after 4, 8 and 12 weeks, respectively. At these time points, fresh blood (FB) was additionally sampled from each participant. FB/SB mixtures were prepared corresponding to transfusion of 1–3 blood bags. Additionally, mixtures were exposed to shear stress similar to that found in the circulation and deformability was measured to estimate possible behaviour of cryopreserved RBC in vivo.ResultsAgeing of RBC was reduced during cryopreservation. Markers for RBC metabolism (ATP, 2,3-DPG) were not altered but RBC sodium levels increased and potassium and calcium decreased, respectively. Mean cellular volume was higher and accordingly, mean cellular haemoglobin concentration was lower in SB. Deformability was altered during storage with less shear stress necessary to deform RBCs. Changes were also detectable in blood mixtures. Deformability remained unaltered in shear stress settings in FB and SB. RBC viscosity was reduced in SB. RBC-NOS content and phosphorylation sites as well as nitrite and RxNO levels seem not to be affected by the intervention.ConclusionCryopreservation maintains RBC metabolic function in vitro, but structure and function of cryopreserved RBC seems to be altered. Impact of these alterations in vivo seems to be less but needs further investigation.     

Effect of hydration on the water content of human erythrocytes.

An ideal, hydrated, nondilute pseudobinary salt-protein-water solution model of the RBC intracellular solution has been developed to describe the osmotic behavior of human erythrocytes during freezing and thawing. Because of the hydration of intracellular solutes (mostly cell proteins), our analytical results predict that at least 16.65% of the isotonic cell water content will be retained within RBCs placed in hypertonic solutions. These findings are consistent not only with the experimental measurements of the amount of isotonic cell water retained within RBCs subjected to nonisotonic extracellular solutions (20-32%) but also with the experimental evidence that all of the water within RBCs is solvent water. By modeling the RBC intracellular solution as a hydrated salt-protein-water solution, no anomalous osmotic behavior is apparent.     

Quantitative Imaging of Human Red Blood Cells Infected with Plasmodium falciparum

During its 48 h asexual reproduction cycle, the malaria parasite Plasmodium falciparum ingests and digests hemoglobin in excess of its metabolic requirements and causes major changes in the homeostasis of the host red blood cell (RBC). A numerical model suggested that this puzzling excess consumption of hemoglobin is necessary for the parasite to reduce the colloidosmotic pressure within the host RBC, thus preventing lysis before completion of its reproduction cycle. However, the validity of the colloidosmotic hypothesis appeared to be compromised by initial conflicts between model volume predictions and experimental observations. Here, we investigated volume and membrane area changes in infected RBCs (IRBCs) using fluorescence confocal microscopy on calcein-loaded RBCs. Substantial effort was devoted to developing and testing a new threshold-independent algorithm for the precise estimation of cell volumes and surface areas to overcome the shortfalls of traditional methods. We confirm that the volume of IRBCs remains almost constant during parasite maturation, suggesting that the reported increase in IRBCs' osmotic fragility results from a reduction in surface area and increased lytic propensity on volume expansion. These results support the general validity of the colloidosmotic hypothesis, settle the IRBC volume debate, and help to constrain the range of parameter values in the numerical model.     

Effect of blood admixture on in vitro survival of chilled and frozen-thawed canine spermatozoa

Hematospermia in the dog usually occurs secondary to benign prostatic hypertrophy or trauma of the penis or prepuce during semen collection. Regarding the difficulty of removing blood cells from a hematospermic sample, the present study was performed to determine whether blood contaminated ejaculates can still be chilled (4 degrees C) or frozen (-196 degrees C) without an additional decrease in sperm quality. In the first experiment, blood additions of up to 10% exerted no negative effects on the functional characteristics of canine spermatozoa cooled (4 degrees C) and stored for 4 days in an egg-yolk-Tris extender. In contrast, in experiment 2, blood admixtures of 4% or more clearly caused negative effects on cryopreserved (-196 degrees C) spermatozoa, mainly on the motility parameters, on the membrane integrity and on the acrosomal status of the spermatozoa. In experiment 3, we showed that these negative effects of blood admixture on cryopreserved spermatozoa were mainly associated with the red blood cells (RBCs) whereas the addition of plasma, serum or inactivated serum exerted little or no negative effect. Moreover, in experiment 4, we showed that 58.3+/-11.6% of the RBCs hemolysed after a freeze-thaw process. In experiment 5, a clear and negative effect of hemoglobin on cryopreserved canine spermatozoa was observed. We conclude that the presence of up to 10% blood is not detrimental for the storage of chilled canine spermatozoa and that the detrimental effects of blood on cryopreserved spermatozoa are at least partly attributable to the high amount of hemoglobin originating from the RBC hemolysis observed after freezing and thawing.     

Osmolality- and hematocrit-mediated flow behavior of RBC suspensions in 33 micrometer ID tubes

The effects of suspending medium osmolality (166 to 736 mosm/kg) on relative viscosity (eta r) and tube hematocrit (HT) measured in 33 microns diameter tubes were studied for 40, 47 and 57% feed hematocrit (HF) suspensions of human RBC in buffer. At all feed hematocrits, eta r increased sharply for the hypertonic media, but was essentially insensitive to hypotonicity. HT/HF was less affected by osmolality (13% change over the entire range of osmolality and feed hematocrit). Viscosities could not be calculated from the experimental HT values. However, eta r could be predicted from RBC number concentration and the tube diameter/RBC volume ratio via a semi-empirical model. RBC transport efficiency depended on both feed hematocrit and osmolality, and was maximal at or near isotonic conditions. Our results appear applicable to non-isotonic regions of the microcirculation, and to estimation of flow resistance for RBC with abnormal cellular mechanical properties.     

Blood volume in inbred strain BALB/c, CBA/J and C57BL/10 mice determined by means of 59Fe-labelled red cells and 59Fe bound to transferrin.

Circulating red blood cell (RBC) and plasma volume was determined in male inbred strain BALB/c, CBA/J and C57BL/10 mice by parallel use of the 59Fe-labelled RBC dilution and the dilution of 59Fe bound to transferrin. The whole blood volumes values derived from the venous haematocrit and plasma volume were about double the values calculated from the venous haematocrit and circulating RBC volume. Comparison of the two methods thus explains the marked differences in different studies of blood volume in mice and shows that correct values can be obtained only by parallel measurements of RBC and plasma volume by separate methods, or by correcting the venous haematocrit to whole body haematocrit. Combination of the labelled RBC method and the 59Fe-transferrin method showed the blood volume values in the above strains of mice to be 10.35 +/- 0.16, 7.32 +/- 0.10 and 7.94 +/- 0.15 ml/g b.w. respectively. The ratio of whole body to venous haematocrit in these strains was was 57.3 +/- 1.6%, 68.0 +/- 1.8% and 69.5 +/- 2.2%. Significant interstrain differences were demonstrated in RBC, plasma and blood volume and in the venous and whole body haematocrit and their ratio.     

Nitric oxide red blood cell membrane permeability at high and low oxygen tension.

Red blood cell (RBC) encapsulated hemoglobin in the blood scavenges nitric oxide (NO) much more slowly than cell-free hemoglobin would. Part of this reduced NO scavenging has been attributed to an intrinsic membrane barrier to diffusion of NO through the RBC membrane. Published values for the permeability of RBCs to NO vary over several orders of magnitude. Recently, the rate that RBCs scavenge NO has been shown to depend on the hematocrit (percentage volume of RBCs) and oxygen tension. The difference in rate constants was hypothesized to be due to oxygen modulation of the RBC membrane permeability, but also could have been due to the difference in bimolecular rate constants for the reaction of NO and oxygenated vs deoxygenated hemoglobin. Here, we model NO scavenging by RBCs under previously published experimental conditions. A finite-element based computer program model is constrained by published values for the reaction rates of NO with oxygenated and deoxygenated hemoglobin as well as RBC NO scavenging rates. We find that the permeability of RBCs to NO under oxygenated conditions is between 4400 and 5100 microm s(-1) while the permeability under deoxygenated conditions is greater than 64,000 microm s(-1). The permeability changes by a factor of 10 or more upon oxygenation of anoxic RBCs. These results may have important implications with respect to NO import or export in physiology.     

Quantitative Analysis of Mechanisms That Govern Red Blood Cell Age Structure and Dynamics during Anaemia

Mathematical modelling has proven an important tool in elucidating and quantifying mechanisms that govern the age structure and population dynamics of red blood cells (RBCs). Here we synthesise ideas from previous experimental data and the mathematical modelling literature with new data in order to test hypotheses and generate new predictions about these mechanisms. The result is a set of competing hypotheses about three intrinsic mechanisms: the feedback from circulating RBC concentration to production rate of immature RBCs (reticulocytes) in bone marrow, the release of reticulocytes from bone marrow into the circulation, and their subsequent ageing and clearance. In addition we examine two mechanisms specific to our experimental system: the effect of phenylhydrazine (PHZ) and blood sampling on RBC dynamics. We performed a set of experiments to quantify the dynamics of reticulocyte proportion, RBC concentration, and erythropoietin concentration in PHZ-induced anaemic mice. By quantifying experimental error we are able to fit and assess each hypothesis against our data and recover parameter estimates using Markov chain Monte Carlo based Bayesian inference. We find that, under normal conditions, about 3% of reticulocytes are released early from bone marrow and upon maturation all cells are released immediately. In the circulation, RBCs undergo random clearance but have a maximum lifespan of about 50 days. Under anaemic conditions reticulocyte production rate is linearly correlated with the difference between normal and anaemic RBC concentrations, and their release rate is exponentially correlated with the same. PHZ appears to age rather than kill RBCs, and younger RBCs are affected more than older RBCs. Blood sampling caused short aperiodic spikes in the proportion of reticulocytes which appear to have a different developmental pathway than normal reticulocytes. We also provide evidence of large diurnal oscillations in serum erythropoietin levels during anaemia.     

Osmotic parameters of red blood cells from umbilical cord blood

The transfusion of red blood cells from umbilical cord blood (cord RBCs) is gathering significant interest for the treatment of fetal and neonatal anemia, due to its high content of fetal hemoglobin as well as numerous other potential benefits to fetuses and neonates. However, in order to establish a stable supply of cord RBCs for clinical use, a cryopreservation method must be developed. This, in turn, requires knowledge of the osmotic parameters of cord RBCs. Thus, the objective of this study was to characterize the osmotic parameters of cord RBCs: osmotically inactive fraction (b), hydraulic conductivity (L_p), permeability to cryoprotectant glycerol (P_glycerol), and corresponding Arrhenius activation energies (E_a). For L_p and P_glycerol determination, RBCs were analyzed using a stopped-flow system to monitor osmotically-induced RBC volume changes via intrinsic RBC hemoglobin fluorescence. L_p and P_glycerol were characterized at 4 °C, 20 °C, and 35 °C using Jacobs and Stewart equations with the E_a calculated from the Arrhenius plot. Results indicate that cord RBCs have a larger osmotically inactive fraction compared to adult RBCs. Hydraulic conductivity and osmotic permeability to glycerol of cord RBCs differed compared to those of adult RBCs with the differences dependent on experimental conditions, such as temperature and osmolality. Compared to adult RBCs, cord RBCs had a higher E_a for L_p and a lower E_a for P_glycerol. This information regarding osmotic parameters will be used in future work to develop a protocol for cryopreserving cord RBCs.     

Assay of the Ca pump ATPase activity of intact red blood cells.

An assay for the Ca pump ATPase of intact human red blood cells (RBCs) was developed. The assay utilized a small volume (typically 10 microliters) of packed RBCs in 1 ml of a buffer of known composition. The assay was based on the exposure of intact RBCs to the ionophore, A23187, in the presence of Ca. Such exposure caused a rapid degradation of ATP in RBCs. This degradation process is modeled in a numerical simulation in a companion paper (Vincenzi, F. F. and Hinds, T. R. (1992) Biochim. Biophys. Acta 1105, 63-70). The loss of ATP followed pseudo-first-order kinetics, and the rate constants for ATP degradation was taken as a measure of the capacity of the Ca pump ATPase. A number of variables were examined to optimize the activity of the ATPase. These variables included the concentrations of Ca and A23187. Because A23187 can promote loss of cellular Mg, it was necessary to include MgCl2 in the incubation medium to optimize ATPase activity. Likewise, it was determined that inclusion of iodoacetic acid optimized the rate of ATP loss, presumably by preventing the resynthesis of ATP from ADP and inorganic phosphate. Cobalt inhibited the ionophore-dependent loss of ATP by apparent competition with Ca for binding to A23187. Results of many assays demonstrated substantial differences in the rate constant for ATP loss in RBCs from different individuals. RBCs were selected according to density. Density associated loss of Ca pump ATPase activity was observed both by the intact RBC assay, and by assay of Ca pump ATPase activity in saponin lysates of RBCs. The correlation coefficient between the two assays was 0.93. It is suggested that the rate constant for ATP loss in intact RBCs exposed to A23187 and Ca can be taken as a measure of the Ca pump ATPase activity. This may be useful when isolated membrane ATPase assays fail (e.g., dog RBCs). The intact cell assay can also be carried out on very small volumes of cells and may be of particular value when RBC volumes are limited.     

Synergistic effects of liposomes, trehalose, and hydroxyethyl starch for cryopreservation of human erythrocytes

Red blood cells (RBCs) can be cryopreserved using glycerol as a cryoprotective agent, but one of the main disadvantages is the time-consuming deglycerolization step. Novel cryopreservation strategies for RBCs using nontoxic cryoprotective agents are urgently needed. The effect of DMPC, DOPC, and DPPC liposomes on survival of RBCs cryopreserved with trehalose and HES has been evaluated. DMPC caused hemolysis before freezing and affected RBC deformability parameters. DMPC treated RBCs displayed a strong increase in trehalose uptake compared to control cells, whereas DOPC treated liposomes only displayed a slight increase in trehalose uptake. High intracellular trehalose contents were observed after cryopreservation. The recovery of cells incubated with trehalose and liposomes, frozen in HES ranged between 92.6 and 97.4% immediately after freezing. Recovery values of RBCs frozen in HES, however, decreased to 66.5% after 96 h at 4°C compared to 77.5% for DOPC treated RBCs. The recovery of RBCs incubated and frozen in trehalose medium was 77.8%. After 96 hours post-thaw storage recovery of these cells was 81.6%. DOPC and DPPC treated RBCs displayed higher recovery rates (up to 89.7%) after cryopreservation in trehalose compared to control RBCs. Highest survival rates were obtained using a combination of trehalose and HES: 97.8% directly after thawing and 81.8% 96-h post-thaw. DOPC liposomes, trehalose and HES protect RBCs during cryopreservation in a synergistic manner. The advantage is that the protective compounds do not need to be removed before transfusion.     

The Effects of Ethanol on the Morphological and Biochemical Properties of Individual Human Red Blood Cells

Here, we report the results of a study on the effects of ethanol exposure on human red blood cells (RBCs) using quantitative phase imaging techniques at the level of individual cells. Three-dimensional refractive index tomograms and dynamic membrane fluctuations of RBCs were measured using common-path diffraction optical tomography, from which morphological (volume, surface area, and sphericity); biochemical (hemoglobin (Hb) concentration and Hb content); and biomechanical (membrane fluctuation) parameters were retrieved at various concentrations of ethanol. RBCs exposed to the ethanol concentration of 0.1 and 0.3% v/v exhibited cell sphericities higher than those of normal cells. However, mean surface area and sphericity of RBCs in a lethal alcoholic condition (0.5% v/v) are not statistically different with those of healthy RBCs. Meanwhile, significant decreases of Hb content and concentration in RBC cytoplasm at the lethal condition were observed. Furthermore, dynamic fluctuation of RBC membranes increased significantly upon ethanol treatments, indicating ethanol-induced membrane fluidization.     

Vitamin C in mouse and human red blood cells: an HPLC assay

Although vitamin C (ascorbate) is present in whole blood, measurements in red blood cells (RBCs) are problematic because of interference, instability, limited sensitivity, and sample volume requirements. We describe a new technique using HPLC with coulometric electrochemical detection for ascorbate measurement in RBCs of humans, wild-type mice, and mice unable to synthesize ascorbate. Exogenously added ascorbate was fully recovered even when endogenous RBC ascorbate was below the detection threshold (25 nM). Twenty microliters of whole blood or 10 μl of packed RBCs was sufficient for assay. RBC ascorbate was stable for 24h from whole-blood samples at 4°C. Processed, stored samples were stable for >1 month at -80°C. Unlike other tissues, ascorbate concentrations in human and mouse RBCs were linear in relation to plasma concentrations (R=0.8 and 0.9, respectively). In healthy humans, RBC ascorbate concentrations were 9-57 μM, corresponding to ascorbate plasma concentrations of 15-90 μM. Mouse data were similar. In human blood stored as if for transfusion, initial RBC ascorbate concentrations varied approximately sevenfold and decreased 50% after 6 weeks of storage under clinical conditions. With this assay, it becomes possible for the first time to characterize ascorbate function in relation to endogenous concentrations in RBCs.     

The effect of osmotic stress on the cell volume, metaphase II spindle and developmental potential of in vitro matured porcine oocytes

Porcine animal models are used to advance our understanding of human physiology. Current research is also directed at methods to produce transgenic pigs. Cryobanking gametes and embryos can facilitate the preservation of valuable genotypes, yet cryopreserving oocytes from pigs has proven very challenging. The current study was designed to understand the effects of anisotonic solutions on in vitro matured porcine oocytes as a first step toward designing improved cryopreservation procedures. We hypothesized that the proportion of oocytes demonstrating a normal spindle apparatus and in vitro developmental potential would be proportional to the solution osmolality. Oocytes were incubated for 10 min at 38 degrees C in various hypo- or hypertonic solutions, and an isotonic control solution and then assessed for these two parameters. Our results support the hypothesis, with an increasing proportion of spindles showing a disrupted structure as the levels of anisotonic exposure diverge from isotonic. Only about half of the oocytes maintained developmental potential after exposure to anisotonic solutions compared to untreated controls. Oocyte volume displayed a linear response to anisotonic solutions as expected, with an estimated relative osmotically inactive cell volume of 0.178. The results from this study provide initial biophysical data to characterize porcine oocytes. The results from future experiments designed to determine the membrane permeability to various cryoprotectants will allow predictive modeling of optimal cryopreservation parameters and provide a basis for designing improved cryopreservation procedures.