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Regular Article |
a Comparative Medicine Center, Research Animal Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211
b Cryobiology Research Institute, Wells Research Center, Indiana University Medical School, Indianapolis, Indiana 46202
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Freezing protocols developed for certain cell types should be reliable and repeatable. It is our thesis that an improved understanding of fundamental cryobiological properties of murine spermatozoa is required to establish optimal freezing protocols that could be employed for all strains of mice. Therefore, it is critical to assess multiple levels of biological function when designing and optimizing cryopreservation protocols. Cells are exposed to multiple nonphysiological steps during the cryopreservation process in which they experience osmotically driven volume excursions. During permeating cryoprotective agent (CPA) addition, cells initially shrink as water flows out of the cell, and subsequently, the cell swells as water and CPA enter. The reverse process occurs during CPA removal. Superimposed on this aspect of cryopreservation is the issue that different cell types and even the same cell type among different species have different tolerances in response to these osmotically driven changes in their cell volume [10]. Discrete osmotic tolerance limits (OTL) have been determined for mouse spermatozoa [11], and the results suggest that these cells have an exquisitely narrow tolerance to such changes. These limits are so narrow that, when using even moderate concentrations of permeating CPA (e.g., 0.5 M glycerol [Gly]), maintenance of as little as 50% of the original motility requires very slow addition and removal of the CPA [12]. However, using low concentrations of CPA leaves the cells vulnerable to solute damage during cooling and warming [13].
Previous investigations have suggested that various non-membrane-permeable components used in extender media have cryoprotective affects on spermatozoa during cryobiological processing, but their mechanism(s) of action are largely unknown. These non-membrane-permeable extender components include chicken egg yolk (EY) [14], sugars [5, 15], and skim milk (SM) [16]. Therefore, one of the first goals of this study was to test the hypothesis that these components protect mouse spermatozoa during cryopreservation by broadening the plasma membrane OTL, thus allowing the cells to better endure the osmotically driven volume excursions during cryopreservation.
Additionally, previous studies have shown changes in membrane permeability characteristics upon exposure to CPAs, including significant reduction of the hydraulic conductivity (Lp) and an increase in the associated activation energy (Ea) of Lp in human and boar spermatozoa [17, 18] as well as a significant reduction of the Lp in murine spermatozoa upon exposure to Percoll separation gradients. Therefore, the second objective of this study was to test the hypothesis that the extender components would have an effect on murine spermatozoa permeability characteristics, specifically the Lp, CPA membrane permeability coefficient (PCPA), the reflection coefficient (
), and the associated Ea for each of these parameters. We also aimed to use the information gained in experiments 1 and 2 to theoretically predict a nondamaging method for the addition and removal of adequate concentrations of permeating CPAs while maintaining the cell volume within the OTL and thus provide critical information for the development of an improved mouse spermatozoa cryopreservation procedure.
Noiles et al. [19] suggested that mouse spermatozoa display no indication of swelling when examined under the microscope in hyposmotic media; they have thus proposed a low-amplitude swelling model to describe the osmometric behavior of mouse spermatozoa. According to this model, mouse spermatozoa swell to a very small extent in response to the osmotic difference across their semipermeable membranes due to resistance to stretching. As the osmotic difference increases, a critical pressure difference is reached at which point the cell lyses. As a result of this swelling, two regions of the plasma membrane come into contact, resulting in membrane fusion. In mouse spermatozoa, due to their extreme length relative to other mammalian spermatozoa, as a result of lysis, the tail tends to fuse with the midpiece or with other parts of the tail, causing irreversible bends or loops to form. This membrane fusion causes an irreversible loss of motility [11]. Therefore, a third experiment was conducted to determine the effects of permeating CPA (ethylene glycol [EG] and dimethyl sulfoxide [DMSO]) as well as nonpermeating CPA (sucrose and raffinose), combined with tests of mixtures of the two. This experiment tested the hypothesis that the morphology of mouse spermatozoa is more sensitive to permeating CPAs than nonpermeating CPAs, looking at percent membrane fusion with varying mixtures of permeating and nonpermeating CPAs.
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MATERIALS AND METHODS |
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All chemicals were obtained from Sigma Chemical Company (St. Louis, MO), unless otherwise stated. The extender stock medium consisted of either 1) 3% SM and 0.19 M (
6%) sucrose dissolved in distilled water to yield an isotonic solution (final osmolality 300 mOsm/kg), or 2) commercially obtained TEST Egg Yolk Buffer (Irvine Scientific, Santa Anna, CA). The 3% SM (skim milk dehydrated, Bacto Difco; Becton Dickinson, Bedford, MA) was dissolved in culture grade milli-Q water and centrifuged for 15 min at 12 000 x g in a microcentrifuge (Eppendorf; Model 5415). The supernatant was removed and filtered through a 0.45-µm acrodisc (Gelman Sciences Inc., Ann Arbor, MI). The CPA solutions were prepared by mixing the stock solutions with either Gly or EG to yield 1 M Gly and 2 M EG (2 M EG was used due to its rapid permeation, i.e., the cell volumetric response associated with 1 M EG removal was too rapid to measure accurately. Milli-Q water was added to 10x PBS to make an isotonic working solution for Coulter counter experiments.
Spermatozoa Collection and Processing
Male outbred ICR and hybrid B6C3F1 mice (Harlan Sprague Dawley, Indianapolis, IN) between 8 and 20 wk of age were used for the experiments. Mice were killed by cervical dislocation. The cauda epididymides and vas deferens were excised, placed in a 35 x 10-mm Falcon dish containing 500 µl isosmotic NaCl, SM, EY (Pacific Andrology, Inc., DE, Montrose, CA), sucrose, or choline at 22°C, then stripped and minced to release spermatozoa. Samples were allowed to incubate for 5 min at 37°C and were transferred to a 15-ml conical tube yielding 1–2 ml of a spermatozoa suspension.
Osmotic Tolerance Limits of Spermatozoa in the Presence of Extender
Spermatozoa were exposed to control solutions of isosmotic (300 mOsm) NaCl as well as anisosmotic NaCl solutions (150, 225, 450, and 600 mOsm). Osmolalities were determined using a freezing-point depression osmometer (Model 3D2; Advanced Instruments, Norwood, MA) with an accuracy of ±5 mOsm. Ten microliters of spermatozoa suspension (in 0.5 ml NaCl, SM, EY, choline, or sucrose) were transferred, in a single step, into 150 µl of anisosmotic solution and mixed by gently tapping the tube [20]. Samples were then allowed to equilibrate for 5 min before subsequent manipulation. The samples were returned to near isosmotic conditions by the addition of 1500 µl of isosmotic NaCl, SM, EY, choline, or sucrose, and motility was assessed. A total of three replicates (n = 3) for each strain were used and all data were collected at 22°C. The results for each anisosmotic solution were normalized to the isosmotic value for each treatment, for each replicate, and for each strain. Data were analyzed by standard ANOVA [21] using the General Linear Models procedures of the Statistical Analysis Software Program (SAS Institute, Cary, NC).
Analysis of Sperm Motility and Plasma Membrane Integrity
Control sperm samples (collected in PBS) and CPA-treated sperm samples were analyzed using a Hamilton Thorn IVOS computerized semen analyzer and bright-line hemocytometer cell counting chamber. For each sample, sperm concentration (total number of cells per milliliter), motility, and progressive motility were determined. Motility was defined as the percentage of spermatozoa that showed any movement of the sperm head. Progressive motility was the percentage of spermatozoa that moved with linear velocity greater than 50 µm/sec and whose straightness, derived from the ratio of absolute straight-line velocity to average path velocity, was not less than 50%. Fresh spermatozoa (collected in CPA-containing media) were observed with a hemocytometer for percent fusion. Spermatozoa were then centrifuged (500 x g for 5 min) to increase the concentration of spermatozoa and to remove nonmotile cells. The supernatant was removed and the cells were resuspended and observed with the hemocytometer to count percent fusion. Sperm plasma membrane integrity was evaluated by using the carboxyfluorescein diacetate/propidium iodide dual fluorescence staining method using a Nikon microscope equipped with an epifluorescence system [22].
Permeability of Murine Spermatozoa in the Presence of Skim Milk Extender Using Electronic Particle Counter
An electronic particle counter (Coulter Counter ZM model; Coulter Counter Electronics, Hialeah, FL) with 50-µm high-resolution aperture tube was used to measure the volume changes of mouse spermatozoa, which had been preequilibrated with either EG or Gly prepared in SM extender following abrupt introduction into isotonic PBS solution. Only SM extender was used in this experiment because the lipid droplets in the EY buffer resulted in high electrical noise in the Coulter Counter system. The values of membrane permeability were determined using a curve-fitting calculation as previously described [17]. The Coulter Counter generates electric pulses (voltage) that are proportional to cell volume. These voltage signals were then interfaced to a microcomputer using a CSA-2S interface (Great Canadian Computer Company, AB, Canada). Cell volume changes were then determined by calibrating the signals obtained when the cells reached an equilibrium state. Approximately 100 µl of the sperm suspension was introduced into 10 ml PBS (1:100 dilution); therefore, the extracellular CPA concentration was assumed constant. In order to determine the relationship between temperature and the values of membrane permeability, the volume measurements were repeated at 22, 15, and 2°C and the corresponding cell volume changes were measured and analyzed. Temperatures below 22°C (15 and 2°C) were obtained through the use of a cooling water bath and water-jacketed beaker. Filtered media, vials, and the Coulter Counter aperture tube were all cooled to the desired temperature before the start of the experiment.
Estimation of Membrane Permeability Coefficients> for Lp, PCPA, and Activation Energies
A pair of coupled nonlinear equations introduced by Kedem and Katchalsky [23] was used to describe the change rates of cell volume and intracellular CPA concentration. The cell volume and amount of intracellular solute concentration as functions of time can be presented as
and
where V is cell volume, A is surface area, 
is the molal concentration of CPA inside the cell, and
The superscripts i and e refer to the intra- and extracellular cell compartments, s is solute, and n is salt, respectively. The terms Lp, PCPA, and are parameters for the hydraulic conductivity of water (in the presence of CPA), the permeability coefficient of the CPA, and the reflection coefficient, respectively. The temperature and universal gas constant are given by T and R, respectively. In this study, nonideal solution effects were ignored. For the impermeable solute (NaCl), the intracellular osmolality of NaCl is given by
where Vb is the osmotically inactive cell volume and Vs = 
s is the CPA volume (s is the partial molar volume of the CPA). The superscript (0) represents the initial values at t = 0. The values for Lp, PCPA, and were determined. A fixed value for Vb (61% isotonic cell volume [11]) was used in the analysis.
The Arrhenius relationship was used to determine the Ea of the parameters LpCPA and PCPA [24]. The permeability value (LpCPA or PCPA) at any temperature T can be plotted as ln[Pa(T)] vs. 1/T (Arrhenius plot), i.e.,
where Ea for the process is expressed in Kcal/mole. The slope of the plot is defined as
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Theory of Optimal CPA Addition and Dilution
Methods for optimal permeating CPA addition and removal are defined here as the processes that minimize the number of addition and dilution steps while maintaining cells within their respective OTL. Theoretical considerations for this procedure are previously described by Gilmore et al. [25]. Briefly, computer simulations were performed to predict optimal CPA additions and removal procedures from mouse spermatozoa. Under the given experimental conditions (initial intracellular concentration, upper and lower OTL of the cell, and temperature), the program optimizes the addition and dilution steps automatically and provides the appropriate diluent concentration.
Morphology of Murine Spermatozoa in the Presence of Permeating and Nonpermeating CPA
In this experiment, only ICR mice were used. Sperm samples were suspended directly in permeating or nonpermeating CPA solution, gently stirred for 30 sec, and then placed in an incubator for 15 min before observation. The cryoprotectant solutions were prepared using 3% SM as the buffer or extender in which all of the CPAs were tested. Sperm samples were centrifuged at 500 x g for 5 min in order to increase concentrations and were analyzed before and after centrifugation for evaluating percent fusion using a hemocytometer. The CPAs used in the study were permeating (0.3 M Gly, DMSO, or EG), nonpermeating (12% sucrose and 18% raffinose), or combinations of equal molar concentrations of permeating and nonpermeating CPA (e.g., DMSO + raffinose, EG + raffinose, DMSO + sucrose, EG + sucrose, Gly + sucrose, and Gly + raffinose). All sugars and polyols that were tested in this experiment were dissolved in a 3% SM solution at a final concentration of 0.3 M. All SM solutions containing CPAs had a final osmolality between 300 and 420 mOsm.
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RESULTS |
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Spermatozoa from ICR and B6C3F1 mice were extended in isosmotic NaCl, SM, EY, choline, or sucrose, and aliquots were randomly assigned to treatment solutions in concentrations ranging from 150 to 600 mOsm. Motility estimates were determined after cells were returned to isosmotic conditions. Both B6C3F1 and ICR sperm motilities were significantly higher (P < 0.05) when the cells were extended in SM or EY than those extended in NaCl (Fig. 1). ICR spermatozoa have significantly higher (P < 0.05) motility when extended in EY or SM than cells from B6C3F1 mice (Fig. 2). Results also indicate that spermatozoa from both ICR and B6C3F1 that were exposed to choline have significantly lower (P < 0.05) percent motilities than those extended in sucrose or NaCl (Fig. 3).
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Experiment 2: Permeability of Murine Spermatozoa in the Presence of Skim Milk Extender
The values of LpCPA, PCPA, and are shown in Tables 1 and 2 (ICR and B6C3F1 mice). At 22°C, spermatozoa from B6C3F1 mice had the highest LpCPA and PCPA in the presence of EG. In the presence of Gly, there was no significant difference (P > 0.05) in the values of LpCPA and PCPA between strains. The type of CPA used and temperature had significant effects (P < 0.05) on the LpCPA and value PCPA in both strains. For ICR mice, the Ea for Lp in Gly and in EG were determined as 9.65 (r2 = 0.90) and 8.98 (r2 = 0.99) Kcal/mol, respectively. The Ea for PCPA in Gly and EG were determined as 12.84 (r2 = 0.96) and 10.55 (r2 = 0.96) Kcal/mol, respectively (Fig. 4). For B6C3F1 mice, the Ea for Lp in Gly and EG were determined as 8.34 (r2 = 0.99) and 16.65 (r2 = 0.91) Kcal/mol, respectively. The Ea for PCPA in Gly and EG were determined as 12.42 (r2 = 0.99) and 18.69 (r2 = 0.99) Kcal/mol, respectively (Fig. 4).
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Addition and Removal of CPAs in Extender Media
Based on the information gained from experiments 1 and 2, theoretical models were employed to predict the response of ICR and B6C3F1 mouse spermatozoa to the addition and removal of Gly or EG in the presence of SM extender. The results suggest that, for ICR spermatozoa in the presence of SM extender, both Gly and EG can simply be added and removed using one step (Figs. 5 and 6). On the other hand, for B6C3F1 spermatozoa, Gly required a two-step addition while EG only required a single step (Fig. 5). For removal from B6C3F1 sperm, Gly required a three-step removal process while EG required a two-step removal (Fig. 6).
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Experiment 3: Morphology Effects of Murine Spermatozoa in the Presence of Permeating and Nonpermeating CPA
Centrifugation of ICR sperm in SD-PBS using 500 x g force for 5 min did not have a significant effect (P > 0.05) on membrane fusion rate (1.77% ± 0.27% [mean ± SEM] and 2.68% ± 0.40% before and after centrifugation, respectively). The percent of spermatozoa observed to be nonlinear varied with the composition of the CPA in which they were collected. Sperm samples that were collected in Gly (46.88% ± 1.39%), EG (20.90% ± 1.27%), and DMSO (36.23% ± 2.82%) showed more fusion than sperm collected in sucrose (2.31% ± 0.22%) and raffinose (2.74% ± 0.19%) after centrifugation (Fig. 7) (P < 0.05). Although the percentage of sperm with nonlinear morphology obtained from the permeating CPAs was significant, Gly, the most widely used CPA, showed the highest percent of membrane fusion (P < 0.05). Sucrose slightly (although not statistically significant) resulted in a lower fusion rate than raffinose (P > 0.05) (Fig. 7). The results depicted in Figure 8 show that, when combining equal molar concentrations of permeating and nonpermeating CPAs, significantly less fusion (P < 0.05) occurs. Figure 9 represents light and fluorescent microscopic images of head-to-tail fused sperm collected in 0.3 M EG.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Permeating CPAs are known to protect cells during freezing via colligative action by minimizing the concentration of electrolytes and other solutes during freezing [26]. This principle has been well accepted and applied in almost all mammalian cell cryopreservation methods. However, the approach recently taken by several investigators toward mouse sperm cryopreservation suggests that permeating CPAs are not required for success. In fact, some reports have indicated that the inclusion of permeating CPAs actually caused marked reduction in mouse sperm survival after freezing and thawing. This is a fundamentally critical issue because very few cell types can be successfully cryopreserved in the absence of permeating CPAs and the fact that, to date, most attempts to cryopreserve mouse sperm have met with very limited success. Therefore, in order to develop improved mouse sperm cryopreservation methods, it is essential to gain an understanding of why permeating CPAs adversely affect mouse sperm viability. This information can be used to develop methods to prevent the adverse effects and allow these compounds to be used to prevent solute damage during freezing. In this regard, there is a need for a systematic examination to reveal each of those compounds' concentration-dependent beneficial or detrimental effects during each step of the freezing procedure such as CPA addition and removal, cooling, freezing, and warming [27].
Because they are nonpermeating solutes, the addition of sugars to media causes a concentration-dependent increase in osmolality resulting in dehydration of cells within the solution. This dehydration causes a proportional increase in the intracellular solute concentration, which in turn facilitates intracellular vitrification during cooling [28]. In addition, sugars provide an osmotic buffer that prevents excessive cell volume excursion during cryopreservation procedures [29]. The beneficial effects of SM and EY as membrane stabilizers during cooling have long been known, although mechanisms of action by which they protect the cell are still largely unknown [30, 31]. Over the past several years, our group has conducted extensive studies that have examined the low survival and the variability in success rates among various genetic backgrounds [2]. In the current study, we examined the effects of permeating and nonpermeating compounds on permeability characteristics and morphologic changes with the goal of enabling the use of permeating CPAs in standard procedures.
Osmotic Tolerance of Murine Spermatozoa in the Presence of Skim Milk Extender
During the cryopreservation process, cells are exposed to multiple steps in which they experience extensive volume excursions, which can be damaging to cells [8]. It has been previously shown that mouse spermatozoa are particularly sensitive to osmotically driven changes in cell volume and there is a genetic basis for this sensitivity of mouse spermatozoa to osmotic shock [9, 7]. Therefore, by broadening the OTL for mouse sperm, one can minimize the damage associated with volume excursions and improve cryopreservation. Prior studies have investigated the ability of extenders to protect cells during the steps of cryopreservation. One hypothesis of this study was that this could be explained by the extender's ability to expand the OTL of the cells. Certain extender media have been shown to broaden the OTL of spermatozoa from other osmotically sensitive species such as the boar [16]. Components such as EY, SM, and sugars have been commonly used media components and, more recently, choline has been observed to protect unfertilized mouse oocytes during cryopreservation [32]. Data from the current study confirmed the hypothesis that SM and EY effectively broaden the OTL of spermatozoa from ICR and B6C3F1 mice. However, effects of choline on OTL of mouse spermatozoa were unexpectedly negative. In addition, results indicate that ICR mouse spermatozoa appear to be more tolerant to osmotic change than B6C3F1 spermatozoa. On the other hand, the presence of sucrose and choline do not appear to enhance the OTL of murine spermatozoa because motilities in the presence of choline and sucrose were significantly lower than the control (NaCl).
Permeability of Murine Spermatozoa in the Presence of Skim Milk Extender
The present study shows that mouse sperm membrane LpCPA decreases in the presence of SM relative to previously reported values [10]. Muldrew and McGann [33] suggested that low permeability of a cell to water may have beneficial consequences on the degree of osmotic pressure that cells experience. If this is the case, the broader OTL of mouse sperm in the presence of SM and EY extender in the current study supports this early hypothesis. Values of Ea with or without SM extender are listed in Table 3. For ICR spermatozoa, Ea remained fairly consistent with or without SM extender. For B6C3F1, relatively larger changes were found in Ea of Lp in the presence of Gly and EG. As indicated by the Arrhenius plots of B6C3F1 sperm cells without SM extender [10], values of Ea deviated from a normal Arrhenius relationship at 15 and 1.5°C. In other words, in both Gly and EG, Ea of Lp and PCPA had larger values at 1.5°C than those at 15°C. In that study, this was explained as a potential lipid-phase transition in the plasma membrane occurring at these temperatures. In the present study, however, values of Ea followed a normal Arrhenius relationship at 15 and 1.5°C, with r2 values close to one (Fig. 4). If the phase transition of lipid in the plasma membrane is the actual reason for deviation at 15 and 1.5°C, SM extender may suppress this phenomenon.
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Optimal Addition and Removal of CPAs> in Extender Media
It has been suggested that osmotic damage caused by permeating CPAs can be minimized by adding and removing them in a manner that maintains cells within their OTL. The importance of appropriate CPA addition and removal procedures becomes apparent because there have been large numbers of reports that suggest extreme sensitivity of mouse spermatozoa to osmotic changes and handling such as pipetting and centrifugation [9, 34]. In this study, it was demonstrated that SM extender has a positive effect on mouse sperm survival. Combining the permeability measurements determined following exposure to the SM extender, theoretical models were employed to predict the response of mouse spermatozoa to the addition and removal of 1 M Gly or EG. According to the theoretical data, for ICR spermatozoa in the presence of SM extender, both Gly and EG (final concentration of 1 M) may be safely added and removed in a single step. On the other hand, Katkov et al. [35] have found that addition of 0.8 M Gly in the absence of SM required a multistep addition approach for ICR mice. However, in this study, due to the lower OTL of B6C3F1 spermatozoa, 1 M Gly required a two-step addition while 1 M EG only required a single step. For removal from B6C3F1 sperm, Gly required a three-step removal process while EG required a two-step removal. This is in sharp contrast with a similar study [10] in which the same types of simulations were performed in the absence of extender media. In that study, for ICR spermatozoa, a two-step addition process was required for both addition and removal of either Gly or EG, and for B6C3F1 spermatozoa, a three-step process was needed for Gly and a four-step process was needed for EG. Removal of either CPA was predicted to require three-steps. This data confirms that expansion of the OTL by SM allows an easier and more effective approach to mouse sperm cryopreservation.
Morphology Effects of Murine Spermatozoa in the Presence of Permeating and Nonpermeating CPA
Standard mammalian sperm cryopreservation protocols have typically used permeating CPAs (e.g., Gly, EG). However, to date, permeating CPAs have not been effective with mouse spermatozoa and have even been reported to be detrimental in some cases [35]. In the present study, it has been shown that, when ICR sperm were placed into various sugars or glycols at 0.3 M concentration in 3% SM under the same conditions, the number of sperm that survived incubation without membrane fusion and subsequent lysis in permeating additives was reduced in comparison with the samples placed in nonpermeating sugars. In this study, when mouse sperm were placed in sucrose or raffinose, no significant effect was shown in the frequency of membrane fusion (2.31% and 2.74%, respectively, after centrifugation), whereas mouse sperm placed in Gly, EG, or DMSO showed 46.88%, 20.90%, and 36.23% fusion after centrifugation, respectively. This finding is consistent with an early study that compared postthaw motility of CB6F1 (hybrid) and C57BL/6J (inbred) mouse sperm after exposing them to the same sugars and permeating CPAs (Gly, DMSO) at comparable concentrations (400 mOsm), resulting in a significantly higher postthaw motility (about 60%) than nonpermeating CPAs (less than 43%) [36]. These results led us to further investigate if this detrimental membrane fusion effect could be suppressed by combining equal molar concentrations of nonpermeating and permeating CPAs. The Gly, EG, and DMSO additives, when combined with sucrose and raffinose, showed very low membrane fusion (5.61%, 5.47%, 4.15%, 5.39%, 3.67%, and 5.62%, respectively) after centrifugation. On the contrary, a very early study by Tada et al. [5] reported that mouse sperm frozen in 18% raffinose alone showed 40% postthaw motility, while sperm frozen in a combination of permeating and nonpermeating CPAs (18% raffinose + 1.75 M Gly) had a postthaw motility of 60%. At this point, it is clear that more research on the use of nonpermeating compounds or possible combinations for mouse sperm freezing is necessary. This will increase our understanding of freezing differences in effective mouse sperm preservation protocols.
In conclusion, the current study determined that EY and SM extender media broadens the OTL of mouse spermatozoa. In addition, SM extender alters the membrane permeability characteristics, including the associated temperature dependence of these characteristics. Based on these results, new CPA addition and removal procedures have been predicted that are more efficient and allow a greater concentration of permeating CPAs to be used with less damage, potentially improving cryopreservation outcomes. Ongoing work is directed toward testing this hypothesis.
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FOOTNOTES |
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2 Correspondence: John K. Critser, Comparative Medicine Center, College of Veterinary Medicine, University of Missouri, 1600 East Rollins Street, Room E-109, Columbia, MO 65211. FAX: 573 884 7521; critserj{at}missouri.edu
Received: 14 March 2002.
First decision: 25 April 2002.
Accepted: 24 May 2002.
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