![Best-fit water transport parameters of rat and mouse sperm determined by DSC and fit by FORTRAN (Formula Translation Computer Language) optimization as previously reported in Devireddy et al. [19].](https://www.researchgate.net/profile/Willem_Wolkers/publication/26300774/figure/tbl1/AS:601583830171662@1520440170997/Best-fit-water-transport-parameters-of-rat-and-mouse-sperm-determined-by-DSC-and-fit-by_Q320.jpg)
Content uploaded by Willem Wolkers
Author content
All content in this area was uploaded by Willem Wolkers
Content may be subject to copyright.
BIOLOGY OF REPRODUCTION 81, 700–706 (2009)
Published online before print 17 June 2009.
DOI 10.1095/biolreprod.109.076075
Cellular Biophysics During Freezing of Rat and Mouse Sperm Predicts
Post-thaw Motility
1
Mie Hagiwara,
5
Jeung Hwan Choi,
5
Ramachandra V. Devireddy,
9
Kenneth P. Roberts,
4,7,8
Willem F. Wolkers,
3,5
Antoine Makhlouf,
7
and John C. Bischof
2,5,6,7
Departments of Mechanical Engineering,
5
Biomedical Engineering,
6
Urologic Surgery,
7
and Integrative
Biology & Physiology,
8
University of Minnesota, Minneapolis, Minnesota
Department of Mechanical Engineering,
9
Louisiana State University, Baton Rouge, Louisiana
ABSTRACT
Though cryopreservation of mouse sperm yields good
survival and motility after thawing, cryopreservation of rat
sperm remains a challenge. This study was designed to evaluate
the biophysics (membrane permeability) of rat in comparison
to mouse to better understand the cooling rate response that
contributes to cryopreservation success or failure in these two
sperm types. In order to extract subzero membrane hydraulic
permeability in the presence of ice, a differential scanning
calorimeter (DSC) method was used. By analyzing rat and
mouse sperm frozen at 58C/min and 208C/min, heat release
signatures characteristic of each sperm type were obtained and
correlated to cellular dehydration. The dehydration response
was then fit to a model of cellular water transport (dehydra-
tion) by adjusting cell-specific biophysical (membrane hydrau-
lic permeability) parameters L
pg
and E
Lp
.A‘‘combined fit’’ (to
58C/min and 208C/min data) for rat sperm in Biggers-Whitten-
Whittingham media yielded L
pg
¼0.007 lm min
1
atm
1
and
E
Lp
¼17.8 kcal/mol, and in egg yolk cryopreservation media
yielded L
pg
¼0.005 lm min
1
atm
1
and E
Lp
¼14.3 kcal/mol.
These parameters, especially the activation energy, were found
to be lower than previously published parameters for mouse
sperm. In addition, the biophysical responses in mouse and rat
sperm were shown to depend on the constituents of the
cryopreservation media, in particular egg yolk and glycerol.
Using these parameters, optimal cooling rates for cryopreser-
vation were predicted for each sperm based on a criteria of
5%–15% normalized cell water at 308C during freezing in
cryopreservation media. These predicted rates range from
538C/min to 708C/min and from 288C/min to 368C/min in rat
and mouse, respectively. These predictions were validated by
comparison to experimentally determined cryopreservation
outcomes, in this case based on motility. Maximum motility
was obtained with freezing rates between 508C/min and 808C/
min for rat and at 208C/min with a sharp drop at 508C/min for
mouse. In summary, DSC experiments on mouse and rat sperm
yielded a difference in membrane permeability parameters in
the two sperm types that, when implemented in a biophysical
model of water transport, reasonably predict different optimal
cooling rate outcomes for each sperm after cryopreservation.
cryopreservation, DSC, motility, mouse sperm, rat sperm
INTRODUCTION
Mice and rats are the animal models used in about 97% of
biomedical research [1]. Although mice and rats are relatively
easy to house and breed, it is expensive to maintain and
transport transgenic or genetically modified strains [2].
Breeding can also lead to naturally occurring genetic mutations
[3]. Cryopreservation of sperm is one solution used in a variety
of species to facilitate long-term storage and transportation,
thereby reducing the cost of maintaining rodent strains [4, 5].
Cryopreservation of sperm is routinely used for some species.
For example, human sperm is cryopreserved for in vitro
fertilization and storage using glycerol and egg yolk media as
cryoprotective agents (CPAs) [4]. In the breeding of domestic
animals and cattle, where artificial insemination (AI) is
extensively used, cryopreservation in glycerol-based media
facilitates transport of semen [6, 7]. The maintenance of many
mouse sperm lines is achieved by cryopreservation, predom-
inantly using raffinose and skim milk as CPAs [2, 8, 9].
Despite successful sperm cryopreservation of some species, for
many others cryopreservation remains a challenge [10].
One important rodent model that remains difficult to
cryopreserve is the rat. Rats are a preferred model over mice
for many studies because of their larger size [11]. Therefore,
there continues to be a strong interest in developing a method
to cryopreserve rat sperm successfully. Only one group has
reported successful cryopreservation of rat sperm, subsequently
used for AI and yielding live offspring [12, 13]. This result has
not yet been repeated by other investigators or labs, and further
investigation of the ability to yield viable rat sperm after
cryopreservation is urgently needed [10]. This work examines
the cellular biophysics that occurs during rat sperm cryopres-
ervation and compares these results to those of a successfully
cryopreserved rodent species, the mouse. The goal of this study
is to determine if there are underlying differences in the
biophysical mechanisms of water transport (dehydration) and
injury that differentiate these two species’ sperm and to suggest
improved approaches for rat sperm cryopreservation.
Biophysical responses in cells during freezing have been
studied extensively, although challenges exist for sperm due to
their size and nonspherical shape. When cells are cooled in
suspension, ice nucleates first in the extracellular space, leading
to the biophysical responses of cellular dehydration and/or
1
Supported by the National Institutes of Health (NIH) grant R21
RR021698-01 and by the Institute for Engineering in Medicine at the
University of Minnesota.
2
Correspondence: John C. Bischof, Department of Mechanical Engi-
neering, University of Minnesota, 111 Church St. SE, Minneapolis, MN
55455. FAX: 612 625 4344; e-mail: bischof@umn.edu
3
Current address: Institut fu
¨r Mehrphasenprozesse, Leibniz Universita
¨t
Hannover, Hannover, Germany.
4
Current address: WWAMI Medical Education Program, Washington
State University-Spokane, Spokane, WA.
Received: 7 January 2009.
First decision: 14 March 2009.
Accepted: 28 May 2009.
Ó2009 by the Society for the Study of Reproduction, Inc.
eISSN: 1259-7268 http://www.biolreprod.org
ISSN: 0006-3363
700
Downloaded from www.biolreprod.org.
intracellular ice formation (IIF). Both extreme dehydration at
slow cooling rates and large, stable IIF at fast cooling rates can
destroy cells. However, optimal survival can be obtained at
intermediate cooling rates that minimize both of these injury
mechanisms, as has been demonstrated for a variety of cells
[14–16]. Optimal cooling rates that yield the highest post-thaw
survival can be predicted based on experimentally determined,
cell-specific parameters, including the membrane hydraulic
permeability, L
p
(which is dependent upon parameters E
Lp
and
L
pg
and is further described in Materials and Methods), and
heterogeneous ice nucleation parameters [17]. The relationship
of these parameters to the biological response of cells has been
explained previously [10, 18]. Experimental techniques to
determine cell-specific membrane hydraulic permeability
parameters (E
Lp
and L
pg
) during freezing traditionally rely
on optical cryomicroscopy, which works well for larger
spherical cells where two-dimensional cell areas can be
extrapolated to three-dimensional cell volumes. However, this
technique does not work for sperm, which are nonspherical and
too small. Thus, new approaches to measure sperm biophysical
responses during freezing are needed.
Several techniques are available to measure the biophysics
of sperm. However, only the differential scanning calorimeter
(DSC) technique allows measurement of subzero dehydration
behavior in the presence of ice and CPAs to be tested [19].
Previous work on sperm has shown that membrane hydraulic
permeability values from DSC (ice present) are much smaller
than permeability values obtained with suprazero techniques
(ice absent) [10, 19]. Recent work suggests the reduction in
membrane permeability in the presence of ice is due to extreme
lipid or membrane packing (i.e., creation of a gel phase), a
mechanism absent at suprazero temperatures [20]. The DSC
measures excess latent heat due to cellular dehydration in the
presence of ice in sperm and other cell types during a specific
cooling rate protocol [19, 21, 22]. This dehydration response
can be used to extract the membrane hydraulic permeability,
L
pg
, and the corresponding activation energy, E
Lp
, for rat and
mouse sperm. Using these parameters, water transport can be
simulated during freezing, and optimal cooling rates for
cryopreservation can be predicted. The predicted optimal
cooling rates can be separately verified for both rat and mouse
by assessing cryopreservation outcomes, in this case by
motility measurements. The present work uses DSC to reveal
differences in freezing biophysics between rat and mouse
sperm that help explain differences in cryopreservation
outcome between these two rodent sperm types.
MATERIALS AND METHODS
All procedures described within were reviewed and approved by the
University of Minnesota Institutional Animal Care and Use Committee and
were performed in accordance with the Guiding Principles for the Care and Use
of Laboratory Animals.
Sperm Collection and Handling
Rat. Caudal sperm of Hsd:SD:Hsd proven breeder rats (Harlan, India-
napolis, IN) were collected. This species was chosen because it was readily
available and inexpensive. A pair of cauda epididymides was excised from a
rat. Several small incisions were made to allow sperm to elute into 1 ml of
elution buffer for 5 min at 378C. Two elution buffers were used for rat sperm
DSC study: modified Biggers-Whitten-Whittingham (mBWW) [23] and a
cryopreservation media containing 23% (v/v) egg yolk, 8% (w/v) lactose
monohydrate, and antibiotics (described by Nakatsukasa [12] as media I). For
samples to be used in DSC measurements, the eluted sperm were centrifuged
for 5 min at 700 3g. The supernatant was removed, and the remaining sample
size was 0.2 ;0.5 ml. For freeze-thaw experiments and motility assessments,
rat sperm were cryopreserved according to the method of Nakatsukasa et al.
[12], the only method to date used to successfully cryopreserve rat sperm.
Briefly, rat sperm were eluted into Nakatsukasa media I but did not undergo a
centrifugation step. Following elution in Nakatsukasa media I, the sperm were
cooled and maintained at 158C for 30 min and then at 58C for 30 min.
Following the second cooling step, an equal volume of Nakatsukasa media II,
containing media I þ1.4% (v/v) Equex Stem (Nova Chemical Sales, Inc.,
Scituate, MA), was added. This final cryoprotective medium is referred to as
Nakatsukasa media III (521 mOsm total, 224 mM lactose) [12]. Rat sperm
motilities were assessed before and after freeze-thaw (see Cryopreservation
Outcome (Motility after Freeze-Thaw)).
Mouse. Caudal sperm of Hsd:ICR (CD-1) retired breeder mice (Harlan)
were collected by excision from two mice. For DSC study, Dulbecco
phosphate-buffered saline solution (D-PBS [pH 7.2]; Life Technologies, Grand
Island, NY) was used as elution buffer. For samples to be used in DSC
measurements, the eluted sperm were centrifuged for 5 min at 300 3g. The
result was compared to previously published data using D-PBS with 15% egg
yolk as well as to the low CPA media of D-PBS containing 15% egg yolk,
0.135 M glycerol, and 0.13 M raffinose, described in Devireddy [19]. For
freeze-thaw experiments and motility assessments, mouse sperm were eluted
into the 15% egg yolk in D-PBS elution buffer, and CPA was added stepwise to
a final concentration of 0.135 M glycerol and 0.13 M raffinose, described in
Devireddy [19]. The samples did not undergo a centrifugation step for freeze-
thaw experiments and motility assessments. Mouse sperm motilities were
assessed before and after freeze-thaw (see Cryopreservation Outcome (Motility
after Freeze-Thaw)).
DSC Studies
A DSC dynamic cooling program was used to measure water transport out
of rat and mouse sperm cells as previously described [19]. Briefly, sperm
samples were placed in standard aluminum sample pans (Perkin Elmer Life and
Analytical Sciences, Inc., Waltham, MA) with ,0.1 mg powdered Pseudo-
monas syringae (Snomax, York International, CO). The samples were
nucleated by cooling to 58C and then rewarmed to a temperature slightly
below the melting point (T
m
) so that small amounts of ice crystals remain. The
samples were then exposed to 58C/min or 208C/min cooling rates until a
temperature of 308C was reached. Subsequently, the cells were lysed by
performing a rapid freeze to 1508C, then the cooling step from ;T
m
to 308C
was repeated with the now-lysed cells. Figure 1 shows the heat-release
thermogram for the initial and final cooling steps. The difference in total area
under the curve Dq
total
¼q
initial
q
final
, represented by the shaded area, is a
measure of the water transport out of the cells [24] and is used to calculate the
volumetric change as shown in Equation 1:
VðTÞVb
VoVb¼DqðTÞ
Dqtotal
;ð1Þ
where V(T) is the cell volume at temperature T,V
b
is the osmotically inactive
cell volume, V
o
is the isotonic cell volume, and Dq(T) is the accumulated partial
area of Dq
total
evaluated at temperature T. For example, in Figure 1, Dq(T)/
Dq
total
would be 0 at T¼0.538C and 1 at T¼128C. It should be noted that
Equation 1 is based on an assumption that the cell exists in an isotonic medium
prior to freezing, so V
o
needs to be replaced by V
i
, the actual initial volume
prior to freezing, if it is placed in a nonisotonic medium.
The measured difference in heat release for rat sperm in mBWW ranged
from 5.9 to 13.1 mJ/mg, which is similar to previous work (9–11 mJ/mg for
mouse sperm [19]). This is within the range expected, given the cell
concentration of the sample (;100 million/ml) and the estimated volume of
osmotically active water in the sperm.
Prediction of Cellular Biophysics: A Model for Water
Transport During Freezing
A previously developed mathematical model [25–28] was used to simulate
the water transport of sperm cells during freezing as follows:
dV
dT ¼LpAc
BðDpÞð2Þ
where, Vis the sperm cell volume (lm
3
) at temperature T(K), A
c
is the
effective membrane surface area for water transport (lm
2
), which is assumed to
be constant during the freezing process, Dp is the difference in osmotic pressure
between the intracellular and extracellular compartment, Bis cooling rate (K/
min), and L
p
is the membrane hydraulic permeability to water defined by Levin
[27] as
Lp¼Lpgexp ELp
R
1
T1
TR
;ð3Þ
CRYOPRESERVATION OF RAT SPERM 701
Downloaded from www.biolreprod.org.
where T
R
is the reference temperature (273.15 K), L
pg
is the membrane hydraulic
permeability at T
R
(lm min
1
atm
1
), E
Lp
is the activation energy for water
transport (kcal/mol), and Ris the universal gas constant (8.314 J mol
1
K
1
).
In this study, the sperm cells are modeled as long cylinders. The sperm cells
are modeled using a length of 188.7 lm and a diameter of 1.42 lm for rat, and a
length of 122 lm and a diameter 0.92 lm for mouse. These geometric
parameters were taken from Devireddy et al. [19] and Cummins and Woodall
[29]. The osmotically inactive cell volume, V
b
, was assumed to be the same as
for mouse sperm (0.61V
o
), as reported by Willoughby et al. [30]. The
membrane permeability parameters (L
pg
and E
Lp
) were determined by selecting
values that would bring about a best fit of the volumetric change based on
Equation 2, with the volumetric change measured using the DSC.
The best-fit parameters were then used for simulation of rat and mouse
sperm biophysical responses, with adjustment of initial osmolality due to media
(rat in media III) and ice-seeding temperature (38C). Optimal rates of cooling
were defined as those that leave between 5%–15% of the initially active water
trapped inside the cells at 308C. This condition is based on the premise that
optimal survival will be obtained by minimizing dehydration or solution effects
injury during slow cooling (such that at least 5% of water remains) and by
minimizing IIF during fast cooling (so that less than 15% of the water changes
to ice within the cell). This approach has been presented in numerous studies
[14, 15, 18, 19].
Cryopreservation Outcome (Motility After Freeze-Thaw)
Motility was measured in rat and mouse sperm as an indirect measure of
sperm viability prefreeze and post-thaw. We have shown that motility is
comparable to dye exclusion assays for measurement of mouse sperm viability
[19]. Our own experience and the data in the literature suggest that dye
exclusion assays underestimate viability in rat sperm [12, 31]. Control rat sperm
motility (progressive) measured using computer-assisted semen analysis
(CASA) was 63% 610% in mBWW. However, progressive motility was
reduced to zero in freezing medium, though a high percentage of sperm
remained nonprogressively motile (twitching) when assessed visually using a
microscope, indicating that many sperm were still viable. Twitching is often
used as a measure of sperm viability in the clinical setting when selecting a
human sperm for ICSI [32, 33]. Upon dilution of the rat sperm out of the
freezing medium and into mBWW, progressive motility returned to 18% 6
8%, indicating that manipulating sperm into and out of freezing medium injures
the cells, as measured by progressive motility. A freeze-thaw step after
introduction into the freezing medium reduced the progressive motility to
essentially 0% upon dilution into mBWW. Therefore, it was clear that both
freeze-thaw and handling contributed to the loss of progressive motility in rat
sperm. As a result, we elected to measure nonprogressive sperm motility
directly after freeze-thaw by direct observation using a light microscope while
the rat sperm were still within the freezing medium. This technique appeared to
be the best possible approach for yielding new information on the survival of
rat sperm directly after freeze-thaw.
Both rat and mouse sperm samples of 150 ll, prepared as described below,
were frozen on the Linkam cryostage (Linkam BCS196 Cryobiology System,
Surrey, U.K.) in a circular quartz crucible. Samples were frozen at various
cooling rates ranging from 28C/min to 1308C/min, with an end temperature of
808C. The samples were then rewarmed to room temperature at 1308C/min,
and motility was assessed as noted below.
Rat. Rat sperm samples in media III (or mBWW negative control) were
nucleated with a chilled needle at 38C and then frozen to an end temperature
of 808C at various cooling rates. As discussed above, nonprogressive motility
was determined as a measure of rat sperm viability. Briefly, a light microscope
(Olympus BH2 light microscope, Tokyo, Japan) at 203was used both before
and after freezing to visually assess nonprogressive motility. A microslide
(#HTR 1099; VitroCom Inc., Mountain Lakes, NJ) containing a 0.1 32.0 mm
cannula was placed directly into the control or frozen-thawed sperm solution.
After the sample was loaded into the cannula by capillary action, manual
assessments of motility were performed at 203. Three hundred sperm per slide
were counted. For rat sperm samples in media III, motility was assessed before
(control) and after freezing, both with 1:10 dilution in media I at 378C. For
negative control sperm samples in mBWW, motility was also assessed before
and after freezing, but the 1:10 dilution was performed using mBWW only. The
post-thaw motility was normalized to the prefreeze motility. No motility was
found in the negative control of rat sperm frozen in mBWW.
Mouse. Mouse sperm (150 lm) in low-CPA media (or D-PBS as negative
control) were nucleated with a chilled needle at 88C and then warmed to 28C
before freezing on the Linkam cryostage to 808C at various cooling rates. This
nucleation temperature was necessary to overcome the higher concentration of
CPA in mouse cryopreservation media compared to rat media, and was further
chosen to match Devireddy et al. [19], in which the nucleation temperatures
varied between 7.48C and 8.58C for freeze-thaw study. The samples were
cooled to an end temperature of 808C at various cooling rates. The samples
were then rewarmed to room temperature at 1308C/min, and motility was
assessed. The initial (control) motility of the samples was assessed after 1:10
dilution with 1% bovine serum albumin/Hepes-buffered saline solution (Life
Technologies), as described in [19]. Post-thaw mouse sperm motility was
similarly assessed and normalized to the prefreeze motility using the same
dilution conditions. Factory-preprogrammed CASA settings for mouse were
used to assess motility. Freeze-thaw treated samples (10 ll) were transferred to
glass slides (20 lm in depth). The CASA software was set to report the average
of 10 readings of different fields per sample. The control was kept at room
temperature. Progressive motility of the frozen-thawed sperm was normalized
with respect to the unfrozen control. No motility was found after freeze-thaw
for the negative control of mouse sperm frozen in D-PBS.
RESULTS
DSC Studies on Water Transport During Freezing
Figure 2A shows the rat sperm water-transport data from
DSC experiments at 58C/min and 208C/min in media I. For
comparison, Figure 2B shows the DSC water-transport data for
mouse sperm in low CPA, published previously [19]. It is
noted that the osmolality of the suspending solutions for rat and
mouse sperm were different, thereby leading to different initial
normalized volumes. The water-transport data for all cases
investigated were fit to Equation 2, and the results are
summarized in Table 1. The combined best-fit membrane per-
meability parameters for rat sperm were determined in mBWW
as L
pg
¼0.007 lm min
1
atm
1
and E
Lp
¼17.8 kcal/mol (R
2
¼
0.95) and in media I as L
pg
¼0.005 lm min
1
atm
1
and E
Lp
¼
14.3 kcal/mol (R
2
¼0.95). The combined best-fit membrane
permeability parameters for mouse sperm were determined in
D-PBS as L
pg
¼0.009 lm min
1
atm
1
and E
Lp
¼21.8 kcal/
mol (R
2
¼0.97). Previously reported values in D-PBS and 15%
egg yolk and in low-CPA media for mouse sperm are also
shown in Table 1 for comparison [19]. The data show that rat
sperm in mBWW and egg yolk media have lower L
pg
and E
Lp
than does mouse sperm.
FIG. 1. The heat flow versus temperature curves for DSC measurement.
The lower and upper curves correspond to the heat release measured for
the live osmotically active and lysed osmotically inactive cells,
respectively. The difference between the initial and final heat flows (Dq)
is the measure of osmotically active water during sperm dehydration in the
system.
702 HAGIWARA ET AL.
Downloaded from www.biolreprod.org.
Modeling: Prediction of Cell Dehydration During Freezing
To simulate water transport of sperm under a variety of
cooling rates, experimentally obtained values of L
pg
and E
Lp
(Table 1), dimensional parameters (see Prediction of Cellular
Biophysics: A Model for Water Transport During Freezing in
Materials and Methods), and the osmolality of the solution
(media III for rat and low CPA for mouse) were taken as input
parameters for the water transport Equations 2 and 3. Figure 3
depicts the predicted volumetric response of sperm cells at
various cooling rates as a function of subzero temperatures.
The predicted optimal cooling rates (CR
opt
) for rat sperm in
media III were found to be 538C/min to 708C/min (shown
as the shaded region). For comparison, previously published
membrane permeability parameters and initial conditions were
used to predict the optimal cooling rate for mouse in low CPA
to be 288C/min to 368C/min [18]. Clearly, the optimal rates are
lower for mouse than for rat sperm in their respective
cryopreservation media.
Motility/Outcome
Results of rat and mouse sperm post-thaw motility with egg
yolk-containing media were normalized to prefreeze controls
and are shown in Figure 4. The optimal cooling range from
Figure 3 is also shown as the shaded area for comparison.
Motility was taken as a measure of cell viability, and cell
membrane integrity was then assumed intact if the cell
maintained motility. As both plots show, survival versus
cooling rate shows the expected inverted U-shaped curve,
indicating that the highest motility occurs between the slow
cooling rate, which yields excessive dehydration, and the fast
cooling rate, which favors large, stable IIF. Figure 4A shows
the normalized motility of post-thaw rat sperm, indicating the
peak motility at cooling rates between 508C/min and 808C/min.
Figure 4B shows that mouse sperm achieved the optimal
survival following cryopreservation at 208C/min, with progres-
sive motility of approximately 30% that drops sharply by 508C/
min. Additionally, negative control freeze-thaw experiments
were also performed for both rat and sperm with elution media
that did not contain egg yolk, and no progressive motility or
twitching was observed after freeze-thaw.
DISCUSSION
In the current study, differential scanning calorimetry was
used as the biophysical measurement technique to study the
freezing response of rat and mouse sperm and to yield insight
into their differential cryopreservation responses. Other
biophysical measurement techniques exist, including time to
lysis, Coulter counter, and electron spin resonance/electron
paramagnetic resonance approaches [30, 34–39]. All of these
measurements are at suprazero temperatures, or in the absence
of extracellular ice. Importantly, the DSC technique in sperm
has generated membrane permeability parameters that are
significantly less than those from suprazero studies mentioned
above and recently reviewed [10]. This reduction in membrane
hydraulic permeability is due to an increase in activation
energy and a reduction in the reference permeability compared
to suprazero techniques [19, 22]. We recently published a study
describing a correlative technique (Fourier transform infrared
spectroscopy—FTIR) that shows that the cell membrane
TABLE 1. Best-fit water transport parameters of rat and mouse sperm determined by DSC and fit by FORTRAN (Formula Translation Computer Language)
optimization as previously reported in Devireddy et al. [19].
Experimental system Cooling rate L
pg
(lm min
1
atm
1
)E
Lp
(kcal/mol) R
2
value
Rat sperm (mBWW) 58C/min 0.008 26.4 0.98
208C/min 0.01 20.2 0.98
Combined best fit 0.007 17.8 0.95
Rat sperm (Nakatsukasa media I) 58C/min 0.005 18.2 0.97
208C/min 0.005 11.5 0.99
Combined best fit 0.005 14.3 0.97
Mouse sperm (D-PBS) 58C/min 0.01 56.5 0.99
208C/min 0.009 20.2 0.99
Combined best fit 0.009 21.8 0.97
Mouse sperm (D-PBS þ15%egg yolk)* 58C/min 0.01 50.6 0.98
208C/min 0.01 20.5 0.96
Combined best fit 0.01 22.5 0.94
Mouse sperm* (low CPA) 58C/min 0.008 34.3 0.99
208C/min 0.009 26.9 0.99
Combined best fit 0.01 29.2 0.98
* Data from Devireddy et al. [19].
FIG. 2. Volumetric response of rat (A) and
mouse (B) sperm cells as a function of
subzero temperatures obtained using the
DSC technique. Rat sperm were cooled in
egg yolk media I and mouse sperm in low-
CPA raffinose glycerol media. The filled and
unfilled symbols show 208C/min and 58C/
min data, respectively. Values are given as
mean 6SD.
CRYOPRESERVATION OF RAT SPERM 703
Downloaded from www.biolreprod.org.
changes dramatically in the presence of extracellular ice. In
fact, there is an intense lipid phase change as the membrane
enters the gel phase during freezing-induced cellular dehydra-
tion that we refer to as ‘‘membrane packing’’ [40]. We further
show that the activation energy for lipid (i.e., membrane)
packing during freezing in three cell types is comparable to the
activation energy for membrane hydraulic permeability during
freezing in these same cell types obtained by cryomicroscopy.
This not only suggests that the mechanism for a reduction in
the permeability (with ice) is membrane packing (i.e., less
space for water movement through membrane), but also
indicates the necessity of using a subzero technique with ice
present in order to accurately measure water transport. Indeed,
it is likely that membrane packing in the presence of ice
explains the controversy over why dehydration predictions
based on suprazero membrane permeability parameters (with
no membrane packing) do not match experimentally deter-
mined optimal rates for cryopreservation (i.e., optimal rates of
cooling with ice present) [10].
At higher cooling rates, one postmortem approach in the
presence of ice and CPA uses electron microscopy (EM)
techniques to search for ice crystals within the cytoplasm of
frozen sperm [41]. The author claims, by EM evaluation of a
number of sperm, that no IIF occurs even though dehydration
is minimal and that rapidly cooled sperm experience an
osmotic shock that injures the membrane upon thawing. This is
an interesting, if controversial, result that still does not
challenge the well-known inverted U curve survival behavior
of sperm with cooling rate. In short, injury to sperm is linked to
the dehydration response (or lack of it), which currently can
only be measured with DSC in the presence of ice and CPA
(egg yolk, raffinose, and/or glycerol) [19, 21, 22].
Clear differences were found between the rat and mouse
sperm membrane biophysical parameters obtained from these
DSC experiments. In subsequent modeling, these parameters
were used to predict different optimal cooling rates for
cryopreservation of these sperm. To validate these predictions,
motility after freezing was used as a measure of cryopreser-
vation outcome. The results confirm that different rates of
cooling are needed for these two sperm types. Specifically,
predicted optimal rates for mouse sperm are significantly less
(288C/min to 368C/min) than for rat (538C/min to 708C/min) in
their respective cryopreservation media.
DSC results show clear differences in freezing responses at
the cellular level between rat and mouse sperm (Fig. 3) and the
effect of the suspending media on this freezing response (Table
1). Changes in freezing response are captured in the differences
between the ‘‘combined fit’’ biophysical parameters (L
pg
and
E
Lp
) that are obtained by fitting the DSC measurements as
shown here and in previous studies [19]. Using this approach in
mouse sperm, we have tested the effect of egg yolk on
biophysical measurements. No significant difference was found
between mouse sperm samples frozen in D-PBS and in D-PBS
with 15% egg yolk. However, previous work with mouse
sperm in low-CPA media, which includes 15% egg yolk, 1%
glycerol, and 6% raffinose, showed an increase in parameters.
This is in contrast to rat sperm, in which significantly higher
values of L
pg
and E
Lp
were found for rat sperm frozen in
mBWW vs. media I (23% egg yolk and 8% lactose). It should
be noted that though the ‘‘combined fit’’ parameters show the
trends as noted, specific fits at 58C/min and 208C/min do not
necessarily hold this trend. Clearly, these differences in the
biophysical parameters in rat vs. mouse with different media
require further measurement and understanding. The ‘‘com-
bined fit’’ parameters represent our current best understanding
of how these sperm behave in these media during freezing.
The difference in rat and mouse sperm biophysical response
may be due to differences in membrane composition and
FIG. 3. Predicted response to cooling for
rat (A) and mouse (B) sperm. Predictions are
based on rat sperm in egg yolk media III and
mouse sperm in low-CPA raffinose glycerol
media. The model-simulated dynamic
cooling response using the combined-fit L
pg
and E
Lp
values were tested for various
cooling rates. The nondimensional volume
is plotted along the y-axis and the subzero
temperatures are shown along the x-axis.
The optimal cooling rate range is shown as
the shaded area.
FIG. 4. Recovered motility of (A) rat and
(B) mouse sperm after nucleation and
freezing at various rates to 808C and
thawing at 1308C/min. Rat sperm were
frozen in egg yolk media III and mouse
sperm in low-CPA raffinose glycerol media.
Values are given as means 6SD. The
optimal cooling range from Figure 3 is also
shown as the shaded area for comparison.
704 HAGIWARA ET AL.
Downloaded from www.biolreprod.org.
membrane media interactions. It is suggested that the head-
group saturation level of the lipid acyl chains and the
membrane cholesterol content change membrane fluidity and
thus permeability to water. According to Hall et al. [42] and
Rejraji et al. [43], the phospholipid fraction of rat sperm
membranes is composed of 19.8% phosphatidylcholine (PC)
and 28.8% phosphatidylethanolamine (PE), whereas mouse
sperm membranes contain 41.4% PC and 18.8% PE. In
addition, mouse sperm contain relatively high percentages of
polyunsaturated membranous fatty acids compared to rat
sperm, and the cholesterol/phospholipid ratio is higher in rat
(0.46) than in mouse (0.29) sperm [42, 43]. It is, therefore, not
surprising that FTIR studies of several cell types suggest that
cell membranes undergo cell-dependent phase changes and
lipid alterations during dehydration, and that these events
correlate with changes in biophysical response [20, 40].
One approach to predict and explain differences in freezing
responses in various cells is through measurement and
prediction of biophysical (dehydration) responses. In this study
we have measured and used the ‘‘combined fit’’ biophysical
parameters in rat and mouse sperm to predict optimal freezing
rates for cryopreservation. These predictions were found to
compare closely to measured cryopreservation outcomes (in
this case motility). The predicted optimal cooling rate in rat
sperm based on dehydration is 538C/min to 708C/min, which
compares well to the experimentally obtained maximum
motility results of 508C/min to 808C/min. Similarly, in mouse
sperm, optimal cooling rates range from 288C/min to 368C/min,
and the maximum motility is measured at 208C/min, with an
abrupt drop at 508C/min. It should be noted that DSC and
motility experiments were performed under somewhat different
cytocrit and nucleation conditions, which may explain the
small differences between optimal cooling rate predictions
(from DSC measurements) and cryopreservation outcome
(from motility).
Although mouse motility is routinely assessed after freeze-
thaw in the literature, we are not aware of a routine recovery of
motility after freeze-thaw for rat sperm in the literature other
than that reported in [12, 13, 31], and thus our data is among
the first for rat sperm and the only work to tie motility to
biophysical responses. Our studies were not designed to
produce optimal results for cryopreservation in either species
but rather to study biophysics in a way that rat and mouse
sperm can be compared during freezing.
In summary, DSC was used to measure biophysical
parameters governing dehydration of rat sperm in media both
containing and not containing egg yolk during freezing, and
these parameters were compared with behavior in mouse sperm
in several media with and without egg yolk and glycerol. Using
these biophysical parameters, optimal rates of cryopreservation
in both sperm types were predicted and found to match well
with optimal cooling rates measured by cryopreservation
outcomes (i.e., motility after freeze-thaw). Further studies are
needed to reveal the underlying mechanisms (especially the
importance of membrane hydration and phase change) that
determine the biophysical response of a cell during freezing
and how this response is affected by the media. Future work
will also need to extend motility results to assessment of
fertilizing ability of post-thaw rat sperm under optimal and
suboptimal conditions.
ACKNOWLEDGMENTS
We would like to thank Kathy Bowlin and Laura Piehl for their
assistance in preparing the materials and obtaining experimental data. We
also would like to acknowledge Raghava Alapati for performing the
corroborative DSC experiments at LSU.
REFERENCES
1. Crawford RL. Information resources on the care and welfare of rodents.
AWIC Resource Series [serial online] No. 37. January 9, 2007. Available
at: http://www.nal.usda.gov/awic/pubs/Rodents/rodents.htm. Accessed Ju-
ly 29, 2009
2. Landel CP. Archiving mouse strains by cryopreservation. Lab Anim (NY)
2005; 34:50–57.
3. Buehr M, Hjorth JP, Hansen AK, Sandoe P. Genetically modified
laboratory animals—what welfare problems do they face? J Appl Anim
Welf Sci 2003; 6:319–338.
4. Audrins P, Holden CA, McLachlan RI, Kovacs GT. Semen storage for
special purposes at Monash IVF from 1977 to 1997. Fertil Steril 1999; 72:
179–181.
5. Woelders H, Matthijs A, Zuidberg CA, Chaveiro AE. Cryopreservation of
boar semen: equilibrium freezing in the cryomicroscope and in straws.
Theriogenology 2005; 63:383–395.
6. Pinkert CA, Dyer TJ, Kooyman DL, Kiehm DJ. Characterization of
transgenic livestock production. Domest Anim Endocrinol 1990; 7:1–18.
7. Pursel VG, Johnson LA. Freezing of boar spermatozoa: fertilizing capacity
with concentrated seman and a new thawing procedure. J Anim Sci 1975;
40:99–102.
8. Nakagata N. Cryopreservation of embryos and gametes in mice [in
Japanese]. Jikken Dobutsu 1994; 43:11–18.
9. Nakagata N. Cryopreservation of mouse spermatozoa. Mamm Genome
2000; 11:572–576.
10. Mazur P, Leibo SP, Seidel GE Jr. Cryopreservation of the germplasm of
animals used in biological and medical research: importance, impact,
status, and future directions. Biol Reprod 2008; 78:2–12.
11. Abbott A. Laboratory animals: the renaissance rat. Nature 2004; 428:464–
466.
12. Nakatsukasa E, Inomata T, Ikeda T, Shino M, Kashiwazaki N. Generation
of live rat offspring by intrauterine insemination with epididymal
spermatozoa cryopreserved at 196 degrees C. Reproduction 2001; 122:
463–467.
13. Nakatsukasa E, Kashiwazaki N, Takizawa A, Shino M, Kitada K,
Serikawa T, Hakamata Y, Kobayashi E, Takahashi R, Ueda M, Nakashima
T, Nakagata N. Cryopreservation of spermatozoa from closed colonies,
and inbred, spontaneous mutant, and transgenic strains of rat. Comp Med
2003; 56:511–513.
14. Mazur P. The role of intracellular freezing in the death of cells cooled at
supraoptimal rates. Cryobiology 1977; 14:251–272.
15. Mazur P. Cryobiology: the freezing of biological systems. Science 1970;
168:939–949.
16. Mazur P. Manifestations of injury in yeast cells exposed to subzero
temperatures, II: changes in specific gravity and in the concentration and
quantity of cell solids. J Bacteriol 1961; 82:673–684.
17. Toner M, Cravalho EG, Stachecki J, Fitzgerald T, Tompkins RG,
Yarmush ML, Armant DR. Nonequilibrium freezing of one-cell mouse
embryos. Membrane integrity and developmental potential. Biophys J
1993; 64:1908–1921.
18. Mazur P. Freezing of living cells: mechanisms and implications. Am J
Physiol 1984; 247:C125–C142.
19. Devireddy RV, Swanlund DJ, Roberts KP, Bischof JC. Subzero water
permeability parameters of mouse spermatozoa in the presence of
extracellular ice and cryoprotective agents. Biol Reprod 1999; 61:764–775.
20. Balasubramanian SK, Wolkers WF, Bischof JC. Membrane hydration
correlates to cellular biophysics during freezing in mammalian cells.
Biochimica et Biophysica Acta (BBA). Biomembranes 2009; 1788:945–
953.
21. Devireddy RV, Swanlund DJ, Olin T, Vincente W, Troedsson MH,
Bischof JC, Roberts KP. Cryopreservation of equine sperm: optimal
cooling rates in the presence and absence of cryoprotective agents
determined using differential scanning calorimetry. Biol Reprod 2002; 66:
222–231.
22. Devireddy RV, Swanlund DJ, Roberts KP, Pryor JL, Bischof JC. The
effect of extracellular ice and cryoprotective agents on the water
permeability parameters of human sperm plasma membrane during
freezing. Hum Reprod 2000; 15:1125–1135.
23. Biggers JP, Whitten WK, Whittingham DG. The Culture of Mouse
Embryos In Vitro. San Francisco, CA: Freeman; 1971.
24. Devireddy RV, Raha D, Bischof JC. Measurement of water transport
during freezing in cell suspensions using a differential scanning
calorimeter. Cryobiology 1998; 36:124–155.
25. Karlsson JO, Cravalho EG, Borel Rinkes IH, Tompkins RG, Yarmush
CRYOPRESERVATION OF RAT SPERM 705
Downloaded from www.biolreprod.org.
ML, Toner M. Nucleation and growth of ice crystals inside cultured
hepatocytes during freezing in the presence of dimethyl sulfoxide. Biophys
J 1993; 65:2524–2536.
26. Kedem O, Katchalsky A. Thermodynamic analysis of the permeability of
biological membranes to non-electrolytes. Biochim Biophys Acta 1958;
27:229–246.
27. Levin RL, Cravalho EG, Huggins CE. A membrane model describing the
effect of temperature on the water conductivity of erythrocyte membranes
at subzero temperatures. Cryobiology 1976; 13:415–429.
28. Mazur P. Kinetics of water loss from cells at subzero temperatures and the
likelihood of intracellular freezing. J Gen Physiol 1963; 47:347–369.
29. Cummins JM, Woodall PF. On mammalian sperm dimensions. J Reprod
Fertil 1985; 75:153–175.
30. Willoughby CE, Mazur P, Peter AT, Critser JK. Osmotic tolerance limits
and properties of murine spermatozoa. Biol Reprod 1996; 55:715–727.
31. Yamashiro H, Han YJ, Sugawara A, Tomioka I, Hoshino Y, Sato E.
Freezability of rat epididymal sperm induced by raffinose in modified
Krebs-Ringer bicarbonate (mKRB) based extender solution. Cryobiology
2007; 55:285–294.
32. Stalf T, Mehnert C, Hajimohammad A, Manolopoulos K, Shen Y,
Schuppe HC, Diemer T, Schill WB, Weidner W, Tinneberg HR. Influence
of motility and vitality in intracytoplasmic sperm injection with ejaculated
and testicular sperm. Andrologia 2005; 37:125–130.
33. Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan R, Nuhoglu A.
In-vitro culture of spermatozoa induces motility and increases implanta-
tion and pregnancy rates after testicular sperm extraction and intracyto-
plasmic sperm injection. Hum Reprod 1999; 14:2808–2811.
34. Noiles EE, Mazur P, Watson PF, Kleinhans FW, Critser JK. Determination
of water permeability coefficient for human spermatozoa and its activation
energy. Biol Reprod 1993; 48:99–109.
35. Curry MR, Redding BJ, Watson PF. Determination of water permeability
coefficient and its activation energy for rabbit spermatozoa. Cryobiology
1995; 32:175–181.
36. Watson PF. Recent developments and concepts in the cryopreservation of
spermatozoa and the assessment of their post-thawing function. Reprod
Fertil Dev 1995; 7:871–891.
37. Noiles EE, Bailey JL, Storey BT. The temperature dependence in the
hydraulic conductivity, Lp, of the mouse sperm plasma membrane shows a
discontinuity between 4 and 0 8C. Cryobiology 1995; 32:220–238.
38. Gilmore JA, McGann LE, Liu J, Gao DY, Peter AT, Kleinhans FW,
Critser JK. Effect of cryoprotectant solutes on water permeability of
human spermatozoa. Biol Reprod 1995; 53:985–995.
39. Kleinhans FW, Travis VS, Du J, Villines PM, Colvin KE, Critser JK.
Measurement of human sperm intracellular water volume by electron spin
resonance. J Androl 1992; 13:498–506.
40. Wolkers WF, Balasubramanian SK, Ongstad EL, Zec HC, Bischof JC.
Effects of freezing on membranes and proteins in LNCaP prostate tumor
cells. Biochim Biophys Acta 2007; 1768:728–736.
41. Morris GJ. Rapidly cooled human sperm: no evidence of intracellular ice
formation. Hum Reprod 2006; 21:2075–2083.
42. Hall JC, Hadley J, Doman T. Correlation between changes in rat sperm
membrane lipids, protein, and the membrane physical state during
epididymal maturation. J Androl 1991; 12:76–87.
43. Rejraji H, Sion B, Prensier G, Carreras M, Motta C, Frenoux JM, Vericel
E, Grizard G, Vernet P, Drevet JR. Lipid remodeling of murine
epididymosomes and spermatozoa during epididymal maturation. Biol
Reprod 2006; 74:1104–1113.
706 HAGIWARA ET AL.
Downloaded from www.biolreprod.org.
