Cryobiology 41, 28–39 (2001)
`doi:10.1006/cryo.2001.2300, available online at http://www.academicpress.com on
`
`Comparison of Permeating and Nonpermeating Cryoprotectants
`for Mouse Sperm Cryopreservation
`
`J. M. Sztein, K. Noble, J. S. Farley, and L. E. Mobraaten
`
`The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, U.S.A.
`
`Mouse sperm has proven to be more difficult to cryopreserve than sperm of other mammalian species. Pub-
`lished reports show that only three cryoprotectant agents (CPAs), alone or combined, have been studied: glyc-
`erol and dimethyl sulfoxide (DMSO), as permeating agents, and raffinose, as a nonpermeating agent. To date,
`the most consistent results for mouse sperm cryopreservation have been achieved by use of raffinose/skim milk
`as cryoprotectant with rapid cooling at 20°C per minute. In this study, we compared the cryoprotection provided
`by permeating (glycerol, formamide, propanediol, DMSO, adonitol) or nonpermeating (lactose, raffinose,
`sucrose, trehalose, D-mannitol) compounds for freezing mouse sperm. Different solutions were made using 3%
`skim milk solution as the buffer or extender in which all different cryoprotectant agents were dissolved at a con-
`centration of 0.3 M, with a final osmolality of approx. 400 mOsm. Sperm samples from CB6F1 (hybrid) and
`C57BL/6J (inbred) mice collected directly into each CPA were frozen/thawed under identical conditions. After
`thawing and CPA elimination (centrifugation) raffinose (59%), trehalose (61%), and sucrose (61%) sustained
`the best motility (P 5 ,0.1) of the nonpermeating agents, whereas the best of the permeating agents was
`DMSO (42%). Membrane integrity was analyzed and showed that the simple exposure (prefreeze) to sugars was
`less harmful than the exposure to glycols. Coincidentally, sperm frozen in trehalose (41%), raffinose (40.5%),
`and sucrose (37.5%) were the samples less injured among all different postthawed CPA tested. The in vitro
`fertilization results demonstrated that hybrid mouse spermatozoa frozen with sugars (lactose 80%, raffinose 80%,
`trehalose 79% of two-cell embryos production) were more fertile than those frozen with glycols (glycerol 11%).
`© 2001 Academic Press
`
`The reviews on sperm preservation by A. U.
`Smith (32) and J. K Sherman (31) trace the first
`attempts at conservation at low temperatures to
`as early as 1776. In 1938 Luyet and Hodapp
`(13) published the first report of sperm vitrified
`in liquid air (2192°C); for that experiment the
`authors used frog spermatozoa dehydrated by a
`2 M sucrose solution. Later, Luyet and Keane
`(14) reported that the same procedure was not
`successful for rat sperm. They also found that a
`single exposure of sperm to hypertonic solutions
`was enough to kill 40% of cells; however,
`nearly 100% of those that survived exposure to
`sucrose also survived the vitrification. In 1949
`Polge and collaborators (28) discovered that
`glycerol protects fowl and human spermatozoa
`
`Received October 20, 2000; accepted January 4, 2001.
`This research was supported by NIH Grants RR09781
`and RR01262 and as part of the NICHD/NCRR National
`Cooperative Program on Mouse Sperm Cryopreservation to
`L.E.M. through Cooperative Agreement RR15012.
`
`28
`
`0011-2240/01 $35.00
`Copyright © 2001 by Academic Press
`All rights of reproduction in any form reserved.
`
`exposed to low temperatures. Since then, sperm
`from a variety of mammals have been success-
`fully frozen as a result of the addition of this
`“cryoprotectant.”
`Successful mouse sperm cryopreservation
`was reported in 1990 (23, 40, 41, 44). From re-
`ports that have been published since, only three
`cryoprotectant agents (CPAs), alone or com-
`bined, have been studied. Two of them have
`properties
`that
`facilitate
`their permeation
`through the cell membrane: glycerol and di-
`methyl sulfoxide (DMSO). The other one is raf-
`finose, a trisaccharide classified as a nonperme-
`ating compound. Up to now, more consistent
`results with mouse sperm cryopreservation have
`been obtained by use of the raffinose/skim milk
`combination (1, 15, 19–23, 38, 39, 44). Glyc-
`erol, the commonly used sperm cryoprotectant,
`did not provide the expected protection for
`mouse sperm when used at the usual concentra-
`tion (5 to 10%). However, glycerol at low con-
`centration (6 1.7%) combined with egg yolk
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`MOUSE SPERM CRYOPRESERVATION
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`29
`
`and raffinose has been shown to protect mouse
`sperm during freezing (26, 34, 35).
`Other mouse sperm cryopreservation studies
`such as those investigating osmotic tolerance
`(3, 10, 21, 35, 48), collection temperature (38,
`43), or mechanical damage (11, 12, 40) have
`been done with solutions containing either glyc-
`erol or raffinose. Therefore, since the optimal
`working conditions were not similar among
`those studies, their results cannot be directly
`compared.
`The goal of this project was to demonstrate
`that cryoprotection for mouse sperm can be
`achieved without the use of a permeating cryo-
`protectant. With the freezing protocol elabo-
`rated for the 18% raffinose–3% skim milk (38)
`cryoprotectant solution as reference for the
`present study, we analyzed the degree of cryo-
`protection that different permeating and non-
`permeating compounds provided to mouse
`sperm.
`A hypothesis of the difference seen between
`the nonpermeating and the permeating com-
`pounds is that whereas they both bring about
`dehydration of the spermatozoon (necessary for
`successful cryopreservation), it may be that the
`mouse spermatozoa does not tolerate the intra-
`cellular presence of permeating cryoprotective
`compounds as well as spermatozoa from other
`species. Maybe this has something to do with
`the fact that mouse spermatozoa have a smaller
`fraction of water content than other mammalian
`sperm.
`
`MATERIALS AND METHODS
`Sperm Donors
`Inbred C57BL/6J male mice were obtained
`from the Animal Resources colonies at The Jack-
`son Laboratory and hybrid CB6F1/J (BALB/cBy
`3 C57BL/6By) mice from our own colony.
`Mice were maintained under routine husbandry
`procedures according to standards set forth in the
`Guide for the Care and Use of Laboratory Ani-
`mals (5). They include a light–dark cycle of 14 h
`light and 10 h dark. Males were housed singly or
`at maximum of two per cage at least 5 days be-
`fore the experiment. Food and water were pro-
`vided ad libitum.
`
`Cryoprotectant Solution
`The cryoprotectant solution was prepared using
`3% skim milk as the buffer or extender in which
`all different cryoprotectant agents were tested.
`The CPAs used in this study were classified as
`permeating (acetamide, glycerol, formamide,
`propanediol, dimethyl sulfoxide, adonitol, and
`perseitol) or as nonpermeating (raffinose, lactose,
`sucrose, trehalose, and mannitol) (tissue culture
`grade; Sigma).
`The 3% skim milk (skim milk dehydrated,
`Bacto Difco; Becton–Dickinson) was dissolved
`in culture-grade water (Millie Q Plus PF sys-
`tem) and centrifuged for 30 min at 16,000g in
`a microcentrifuge (Eppendorf; Model 5410).
`The supernatant was removed and filtered
`through a 0.22-mm pore. This clear solution
`was stored at 4°C in a refrigerator for no more
`than 2 weeks. All sugars and polyols tested in
`this study were dissolved in this solution at
`0.3 M final concentration. For complete disso-
`lution of the sugars, the solution was warmed
`(,60°C), centrifuged at 10,000g for 10 min,
`and filtered again. All skim milk solutions con-
`taining CPAs had a final osmolality between
`380 and 450 mOsm.
`To analyze whether the 3% skim milk, the
`lactose that the skim milk contained, or the 18%
`raffinose alone has cryoprotectant activity,
`sperm samples were frozen in 3% skim milk so-
`lution, in 0.4 M lactose (0.4 M to obtain the cor-
`rect osmolality), or in 0.3 M raffinose dissolved
`in phosphate-buffered saline (PBS).
`
`Sperm Collection
`Sperm were collected from 3- to 5-month-old
`CB6F1 and C57BL/6J males. After an animal
`was euthanized, both epididymides and vas
`deferentia were removed and placed into a
`35-mm sterile plastic dish (Falcon 1008; Becton–
`Dickinson) containing 1 ml of CPA. Each cauda
`epididymis with vas deferens from one animal
`was combined with that from another animal to
`diminish individual variation. The tissue was
`cut five to seven times with the edge of a 30-g
`injection needle directly in the CPA equilibrated
`at 37°C, and sperm were allowed to “swim out”
`for 10 min.
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`30
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`SZTEIN ET AL.
`
`Freezing and Thawing Procedures
`Sperm samples collected into different cryo-
`protectants were frozen and thawed using the
`same protocol (38). Briefly, vials (1.8-cc Nunc
`Cryotubes) with 100-ml sperm aliquots col-
`lected at 37°C were frozen by exposure to liquid
`(LN2) vapors for 10 min (approx. 220°C) per
`minute) before storage under LN2. Samples
`were rapidly thawed by transfer from LN2 into a
`37°C water bath until the ice melted. Subse-
`quently, the sample was centrifuged at 735g for
`4 min. The supernatant (cryoprotectant) was
`discarded and replaced either with 50 ml of
`human tubal fluid (HTF) (29) for in vitro fertil-
`ization (IVF) or with bovine serum albumin
`(BSA)–Hepes-buffered saline for fluorescence
`microscopy. The sperm sample was gently re-
`suspended by tapping the tube with fingertips
`and incubated for 10 min at 37°C to allow a
`minimal selection by “swim up”.
`
`Sperm Analysis
`Sperm samples—control (collected in HTF or
`Hepes–BSA), prefreeze (collected in CPA), and
`postthawed—were analyzed using a Hamilton
`Thorn IVOS computerized semen analyzer. Para-
`meters of concentration (total number of cells per
`milliliter), motility, and progressive motility were
`determined in each sample. 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 a linear velocity greater than 50 mm
`per second and whose straightness, derived from
`the ratio of absolute straight-line velocity to aver-
`age path velocity, was not less than 50%.
`
`Live/Dead Fluorescence
`The cell membrane integrity was measured
`by the fluorescent staining method for determin-
`ing sperm viability (Live/Dead Sperm Viability
`Kit; Molecular Probes). Live spermatozoa with
`intact cell membranes fluoresce bright green
`with the membrane-permeant nuclei acid stain
`SYBR 14, whereas dead cells or cells with dam-
`aged membranes fluoresce red with propidium
`iodide. Control samples were collected directly
`into Hepes–BSA buffer, as the staining protocol
`
`required. Prefreeze and frozen/thawed sperm
`were centrifuged 4 min at 750g; the supernatant
`was removed and replaced with Hepes–BSA
`buffer before being stained. After being stained,
`15 ml of sperm sample was placed onto a micro-
`scope slide and covered with a cover slip. A
`Nikon Diaphot microscope with an epifluores-
`cence system was used for observation.
`
`In Vitro Fertilization
`The 20- to 23-day-old CB6F1 hybrid females
`were superovulated by intraperitoneal injection
`of 2.5 IU of PMSG (Sigma), followed by 2.5 IU
`of HCG (Sigma) 48 h later. At 13 h later, the fe-
`males were euthanized and their oviducts were
`removed. The oocyte–cumulus complexes were
`isolated in a sterile culture dish containing 2 ml
`of HTF medium.
`Forty microliters of the thawed sperm sam-
`ple, with a minimum count of 1 million progres-
`sively motile sperm per milliliter, were added
`to a 250-ml drop of HTF covered with light
`mineral oil (Sigma; embryo tested). The oocyte–
`cumulus complexes collected from three to five
`females were placed into each fertilization drop
`containing sperm that had been incubated for at
`least 10 min. Dishes were placed into a sealed
`modular incubator chamber (Billups–Rothen-
`berg) gassed with 5% CO2, 5% O2 balanced in
`90% N2 and maintained in the incubator at 37°C
`for 5 h. After that time, eggs were washed to
`eliminate excess sperm and then cultured over-
`night in a 250-ml drop of HTF under the same
`conditions. The following morning the two-cell
`embryos were counted and either were trans-
`ferred to a 200-ml drop of KSOM/AA medium
`(8) for culture until the blastocyst stage or were
`surgically transferred at the two-cell stage into a
`surrogate mother for in vivo evaluation. For
`each strain, an IVF control was done using only
`10 ml of the 1-ml sample of fresh sperm col-
`lected in HTF. Fertility was considered the per-
`centage of two-cell embryos produced by IVF
`scored 24 h after insemination.
`
`Embryo Transfer
`Forty-five two-cell embryos from each group
`selected for in vivo evaluation were surgically
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`MOUSE SPERM CRYOPRESERVATION
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`31
`
`three
`infundibulum of
`the
`into
`transferred
`pseudopregnant CB6F1 females. In most cases a
`maximum of 15 embryos per recipient were
`transferred using both uterine horns. Embryos
`were allowed to develop to term and the number
`of pups born was recorded.
`
`Statistics
`The arcsine transformation values of motility
`and progressive motility percentages were eval-
`uated by repeated-measures ANOVA test. The
`analysis was corroborated by Dunnett’s test for
`comparison of a control group (raffinose) with
`all the experimental groups.
`Fertility, considered the percentage of two-
`cell-stage embryos scored 24 h after insemina-
`tion, was compared by paired Student’s t test
`and expressed as the mean 6 SE. Differences
`were considered significant when P , 0.01. All
`analyses were carried out using the GraphPad
`prism version 2.0 computer program (GraphPad
`Software).
`
`RESULTS
`Effect of CPA Composition on Sperm Motility
`The sperm motility rate before freezing var-
`ied with the composition of the CPA in which it
`was collected. For both strains, sperm freshly
`collected in any of the sugars (nonpermeating)
`showed greater motility (P , 0.01) than sperm
`collected in all of the permeating CPAs used in
`the study. Although the postthaw motility was
`reduced for all groups, spermatozoa had greater
`motility when frozen in sugars than when
`frozen in glycols. Sucrose, slightly (but not sta-
`tistically significantly) better than trehalose and
`raffinose, produced the highest motility (Figs.
`1a and 1b). Sperm did not survive freezing
`when protected by perseitol or mannitol; 4 M
`lactose and 3% skim milk alone had very low
`protective activity.
`The results for progressive motility (Figs. 2a
`and 2b) were similar to those for motility. The
`prefreeze values were higher in the sugar (P ,
`0.01) than in the glycol groups. Although the
`progressive motility rates obtained for all groups
`diminished abruptly upon postthaw evaluation,
`
`sugars retained the highest rates. Progressive
`motility was slightly higher in sucrose than in
`raffinose or trehalose (P , 0.05) for CB6F1
`hybrid sperm, but not for C57BL/6J sperm in
`which DMSO produced progressive motility
`levels statistically similar (P . 0.05) to those of
`raffinose and trehalose.
`
`Cryoprotectants Evaluated by Live/Dead
`Fluorescence
`Sperm collected directly into different CPA
`solutions showed no significant decrease in
`motility after 10-min exposure. The motility and
`integrity of freshly collected spermatozoa were
`similarly maintained in the sugars and in glyc-
`erol, but not in the rest of the glycols (P , 0.05).
`The postthaw evaluation showed that raffinose
`and trehalose provided the best protection for
`hybrid sperm. However, trehalose was less effec-
`tive than raffinose in protecting C57BL/6J
`sperm. In general, glycols were less effective
`than sugars in protecting the spermatozoa mem-
`brane integrity, and from that group DMSO was
`better than glycerol (P , 0.05) (Figs. 3a and 3b).
`
`In Vitro Fertilization
`The fertilization rates achieved with hybrid
`sperm frozen in lactose, raffinose, trehalose, and
`glycerol are presented in Fig. 4.
`The frozen/thawed hybrid sperm cryopro-
`tected with lactose (80.4% (626/776)), raffi-
`nose (80% (591/736)), or trehalose (79%
`(476/600)) retained significantly better fertility
`(P , 0.001) than the sperm frozen in glycerol
`(11% (27/244)). For C57BL/6J sperm, trehalose
`(9% (54/541)) supported fertilization best among
`the sugars (P , 0.01), and lactose (4% (26/623))
`and raffinose (2% (14/493)) were better than
`glycerol (0% (1/521)).
`
`Embryo Transfer
`When embryos derived from hybrid sperm
`frozen in raffinose or lactose in skim milk were
`transferred into pseudopregnant recipients, the
`percentage of offspring born was the same as
`that for the unfrozen control (56%);
`those
`derived from sperm frozen in sucrose and
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`32
`
`SZTEIN ET AL.
`
`FIG. 1. CB6F1(a) and C57BL/6J (b) prefreeze and postthaw motility of sperm exposed to permeating and
`nonpermeating cryoprotectants at the same molal solution. The count was performed by computer-assisted sperm
`analysis.
`
`trehalose resulted in 42 and 33% live born, re-
`spectively.
`
`DISCUSSION
`Rodent sperm has proven to be more difficult
`to cryopreserve than other mammalian sperm.
`Rat sperm cryopreservation has not yet been ac-
`complished and mouse sperm cryopreservation
`was reported only 10 years ago. The studies
`published then, by Takeshima et al. (42), Tada
`et al. (41), Okuyama et al. (24), and Yokoyama
`
`et al. (45), followed different approaches con-
`cerning the CPA selected for freezing mouse
`sperm: glycerol or DMSO combined with raffi-
`nose or raffinose alone.
`Difficulties in reproducing the original results
`(4, 26) inspired modifications (21, 26, 33) to
`protocols that make freezing generally more
`reliable but still not equally successful for all
`mouse strains.
`Classic cryobiology studies suggest the use
`of glycerol or any permeating additive to effi-
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`MOUSE SPERM CRYOPRESERVATION
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`33
`
`FIG. 1––Continued
`
`ciently protect cells from freezing injuries
`through colligative or “solution effects” (16).
`This principle has been empirically applied in
`mammalian semen preservation since
`the
`early 1950s using protocols containing be-
`tween 5 and 10% glycerol
`in the CPA. It
`seems that the choice of additives is a matter
`of trial and error, because the action of cry-
`oprotectants is still not totally understood (9).
`It is not clear whether successful freezing of
`mouse spermatozoa requires glycerol or any
`permeating CPA for intracellular protection (9,
`25, 30). Glycerol has been shown to adversely
`
`affect fertility of frozen rooster sperm as re-
`sult of membrane disruptions (6, 7). Mouse
`spermatozoa are affected also by this cryopro-
`tectant; poor fertility was reported for mouse
`sperm frozen in 5% glycerol and 18% raffi-
`nose despite good recovery of sperm motility
`(37). Nevertheless, glycerol was used in many
`mouse sperm cryobiology studies (2, 10, 17,
`27, 43) and it was used as a CPA component
`in the solution in which different sugars were
`compared for their activity (36). In the present
`study we show that when sperm samples were
`frozen under
`the same cooling conditions
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`34
`
`SZTEIN ET AL.
`
`FIG. 2. CB6F1 (a) and C57BL/6J (b) prefreeze and postthaw progressive motility of sperm exposed to per-
`meating and nonpermeating cryoprotectants at the same molal solution. The count was performed by computer-
`assisted sperm analysis.
`
`using various sugars or glycols at 0.3 M con-
`centration in skim milk, the number of mouse
`spermatozoa that survived cryopreservation in
`glycerol or other permeating additives was re-
`duced in comparison with the samples pre-
`served with nonpermeating sugars. Raffinose
`was not the only sugar capable of providing
`protection to mouse sperm. Lactose, trehalose,
`and sucrose protect as well, and postthaw fer-
`tility was maintained at similar levels by these
`sugars
`(80, 79,
`and 80%,
`respectively),
`whereas sperm frozen in glycerol
`retained
`only 10% fertility.
`The reproductive capacity of the sperm, of
`course, has to be maintained after cryopreser-
`vation (9, 25, 31, 46); therefore, the concept of
`
`viability of cryopreserved sperm should not be
`misinterpreted by associating fertility directly
`with motility or membrane integrity. Fertility
`is essential for frozen/thawed sperm to be used
`for artificial insemination or in vitro fertiliza-
`tion. It has been shown that to overcome the
`low fertility that mouse sperm exhibit after
`freezing and thawing it is necessary to manipu-
`late the oocyte zona pellucida by drilling (18)
`or nicking (22) to facilitate sperm penetration.
`However, the status of the spermatozoa is not
`important if the cryopreserved—even without
`cryoprotectant—sperm will be used for intra-
`cytoplasmatic sperm injection (47).
`Skim milk has been shown to be an effective
`buffer or extender for mouse sperm preserva-
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`MOUSE SPERM CRYOPRESERVATION
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`35
`
`FIG. 2––Continued
`
`tion. It has been shown (45) and corroborated
`here that skim milk does not have cryoprotec-
`tive activity per se, but the association of skim
`milk with some sugars resulted in greater sur-
`vival than use of PBS (only results for PBS
`with 4 M lactose shown here). For this study
`we dissolved the cryoprotectant additive in a
`crystal clear skim milk solution that was made
`by centrifugation of macromolecules in sus-
`pension. This final product was more stable
`than standard 3% skim milk, since there are no
`suspended nuclei to initiate raffinose precipita-
`tion.
`The 220°C per minute cooling rate obtained
`by exposure of the samples to LN2 vapors was
`empirically determined and successfully applied
`(15, 23, 33, 38, 44); later, this temperature was
`experimentally corroborated to be optimal for
`
`mouse sperm cryopreservation (3). This obser-
`vation seems to be true at least for cryoprotec-
`tants containing glycerol or raffinose at 0.3 M
`concentration.
`Strain differences in freezing sensibility have
`been described in many studies (9, 15, 22, 23,
`35, 40). We corroborated (39) that sperm from
`different mouse strains retained almost 50% of
`their original motility rate as was described
`generally for other mammals (46). However,
`sperm fertility diminishes considerably with
`substantial variability among strains. The com-
`parison in this study between a hybrid and an
`inbred mouse strain arrived at a similar conclu-
`sion. Nonpermeating CPAs protect sperm of
`both strains more efficiently than permeating
`CPAs, as demonstrated by postthawed motility;
`however, the fertility was not equally retained
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`36
`
`SZTEIN ET AL.
`
`FIG. 3. CB6F1 (a) and C57BL/6J (b) prefreeze and postthaw spermatozoa membrane integrity. Bars represent
`the average percentage of live sperm analyzed by fluorescence microscopy (green fluorescence).
`
`in both strains. For example, CB6F1 sperm
`maintained similar fertility rate (80%) when
`freshly collected or after being frozen/thawed in
`skim milk/raffinose, but fertility rate decreased
`to 10% when sperm were frozen in CPA con-
`taining glycerol. C57BL/6J sperm frozen in raf-
`finose/skim milk retained 10% fertility rate, but
`for sperm frozen in glycerol the fertility rate
`was 0%, as was previously reported (35).
`This study corroborates empirical findings on
`mouse sperm cryopreservation and counters the
`opinion “that classical [cryobiology] theory ar-
`gues that a non-permeating sugar like raffinose
`should not protect; yet it is the major cryopro-
`tectant in nearly all published data” (10). It is
`
`clear that more research on the use of nonper-
`meating compounds or possible combinations
`for mouse sperm freezing, which may increase
`our understanding of
`freezing differences
`among strains of the same species, is warranted.
`
`REFERENCES
`
`1. An, T. Z., Iwakiri, M., Edashige, K., Shakurai, T., and
`Kasai, M. Factors affecting
`the
`survival of
`frozen–thawed mouse spermatozoa. Cryobiology 40,
`237–249 (2000).
`2. Dewit, M., Marley, W. S., and Graham, J. K. Fertilizing
`potential of mouse spermatozoa cryopreserved in a
`medium containing whole eggs. Cryobiology 40,
`36–45 (2000).
`3. Devireddy, R. V., Swanlund, D. J., Roberts, K. P., and
`Bischof, J. C. Subzero water permeability parameters
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`MOUSE SPERM CRYOPRESERVATION
`
`37
`
`FIG. 3––Continued
`
`FIG. 4. Fertility of the CB6F1 frozen/thawed sperm in different cryoprotectants. Fertility is expressed as the
`percentage of two-cell embryos produced by in vitro fertilization.
`
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`38
`
`SZTEIN ET AL.
`
`of mouse spermatozoa in the presence of extracellu-
`lar ice and cryoprotective agents. Biol. Reprod. 63,
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