Theriogenology 58 (2002) 1373±1384
The role of osmotic resistance on equine
spermatozoal function
Angela C. Pommer, Josep Rutllant, Stuart A. Meyers*
Sperm Biology Laboratory, Department of Anatomy, Physiology, and Cell Biology, School of Veterinary
Medicine, University of California, Davis, CA 95616, USA
Received 27 March 2001; accepted 15 March 2002
Abstract
Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality.
The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by
analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume
(MCV). Spermatozoa were incubated at 22 8C for 10 min in isosmolal TALP (300 mOsm/kg), or a
range of anisosmolal TALP solutions (75±900 mOsm/kg), for initial analysis, and then returned to
isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0:05) in
anisosmolal conditions compared to sperm motility in control medium. When cells were returned to
isosmolal conditions, only sperm previously incubated in 450 mOsm/kg TALP were able to recover
to control levels of motility. Sperm viability and MMP were lower (P < 0:05) when exposed to
hypotonic solutions in comparison to control solutions. Sperm suspensions that were returned to
isosmolal conditions from 75, 150, and 900 mOsm/kg had lower (P < 0:05) percentages of viable
sperm than control suspensions (300 mOsm/kg). MMP was lower (P < 0:05) in cells previously
incubated in 75 and 900 mOsm/kg when returned to isosmolal, as compared to control cells. MCV
differed (P < 0:05) from control cell volume in all anisosmolal solutions. Cells in all treatments were
able to recover initial volume when returned to isosmolal medium. Although most spermatozoa are
able to recover initial volume after osmotic stress, irreversible damage to cell membranes may render
some sperm incapable of fertilizing an oocyte following cryopreservation.
# 2002 Elsevier Science Inc. All rights reserved.
Keywords: Osmotic tolerance; Stallion; Sperm function; Motility; Mitochondrial membrane potential
1. Introduction
It has been estimated that only 24% of stallions produce ejaculates that are suitable for
cryopreservation, and fertility of frozen semen is approximately 40% that of fresh,
*
Corresponding author. Tel.: ‡1-530-752-9511; fax: ‡1-530-752-7690.
E-mail address: [email protected] (S.A. Meyers).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 1 0 3 9 - 7
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A.C. Pommer et al. / Theriogenology 58 (2002) 1373±1384
nonfrozen semen [1]. Cooled or frozen storage of equine spermatozoa has been reported
to result in marked reductions in sperm fertility for a majority of stallions in commercial
breeding programs [2]. Although the mechanisms responsible for decreased fertility of
cryopreserved spermatozoa are unknown, disruption of certain cellular processes has
been reported [3,4]. Cryopreservation requires exposure of spermatozoa to extreme
variations in temperature and osmolality. Cells undergoing freezing are initially exposed
to extracellular ice crystallization that results in hyperosmolal concentration of solutes in
the unfrozen aqueous channels between ice crystals [3,5]. The cell responds to this insult
by losing water and shrinking in volume so that the solute concentrations between
intracellular and extracellular compartments can equilibrate. Conversely, as cells are
exposed to a hypotonic extracellular environment, as is the case during thawing, cell
volume is increased by passive diffusion of water. It is not known whether this osmotic
stress results in sublethal, irreversible damage to cell membranes, causing decreased
fertilizing capacity.
Determining osmotic resistance for equine spermatozoa is critical to understanding the
biophysical capabilities of spermatozoa during the freeze±thaw cycle. However, there are
few published studies concerning osmotic resistance of equine spermatozoa [6,7]. Therefore, the goal of the present study was to determine the osmotic tolerance limits of equine
spermatozoa by analyzing motility, viability, mitochondrial membrane potential (MMP),
and mean cell volume (MCV) in anisosmolal Tyrode's medium containing albumin,
lactate, and pyruvate (TALP). Once the osmotic resistance of equine spermatozoa is
determined, this knowledge can be used to understand membrane behavior and to modify
current cryopreservation methods that could increase the fertility of frozen±thawed stallion
spermatozoa.
2. Materials and methods
2.1. Chemicals and reagents
Propidium iodide and JC-1 were purchased from Molecular Probes (Eugene, OR). All
other chemicals were obtained from Sigma Chemical Company (St. Louis, MO).
2.2. Animals and semen
Semen was obtained from three stallions individually housed at the Veterinary Medicine
Teaching Hospital and the Animal Science Horse Barn located at the University of CA,
Davis. Stallions were maintained on a diet of mixed grass hay and Omolene 200, with fresh
water ad libitum and daily exercise according to Institutional Animal Care and Use
Committee protocols at the University of California. Semen was collected using an
arti®cial vagina and a phantom mare. A nylon mesh ®lter was used to eliminate the
gel fraction, and the gel-free semen was immediately diluted 1:1 (v/v) into TALP
capacitation medium [8]. The diluted semen was transported to the laboratory within
5 min of collection. Upon arrival at the laboratory, the sperm concentration was determined
using a hemacytometer and diluted to 150 106 ml 1.
A.C. Pommer et al. / Theriogenology 58 (2002) 1373±1384
1375
2.3. Media
The media used were isosmolal (300 5 mOsm/kg) and anisosmolal TALP. Anisosmolal solutions were prepared by diluting isosmolal TALP (hyposmolal solutions, 75 and
150 mOsm/kg) and by diluting 10-strength TALP (hyperosmolal solutions, 450, 600, and
900 mOsm/kg) with HPLC-puri®ed water for experiments in which MCV was determined.
To achieve the ®nal osmolalities of 75, 150, 450, 600, and 900 mOsm/kg in the experiments
in which the sperm motility, MMP, and viability were assessed, an adjusted set of
anisosmolal solutions was prepared (20, 115, 490, 675, and 1050 mOsm/kg, respectively)
in order to avoid the dilution effect (1:5) of the sperm sample in the anisosmolal media.
Final osmolalities were always con®rmed using a vapor pressure osmometer (Model 5100
C; Wescor Inc., Logan, UT).
2.4. Sperm processing
2.4.1. Evaluation of sperm motility
For motility experiments, 50 ml of the initial sperm suspension (150 106 ml 1) was
placed in 200 ml isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP
solutions to get a ®nal concentration of 75, 150, 450, 600, and 900 mOsm/kg, and
incubated at 22 8C for 10 min (30 106 ml 1) prior to motility analysis. Sperm suspensions were then returned to near-isosmotic conditions by adding 1.25 ml of isosmolal
TALP (5 106 ml 1) as described by Gilmore et al. [9]. After 5 min incubation, motility
was analyzed again. At least 200 cells were evaluated using computer-assisted sperm
analysis (CASA) for all experiments in which motility was determined (CEROS, Version
10.9i, Hamilton Thorne Biosciences, Inc., Beverly, MA). The settings used were: frame
rate 60 Hz, frames acquired 30, minimum contrast 80, minimum cell size 3, threshold
straightness 80, medium VAP cut-off 25, low VAP cut-off 5, low VSL cut-off 11, nonmotile
head size 6, nonmotile head intensity 160, static size limits 1.0±2.9, static intensity limits
0.6±1.4, static elongation limits 0±80.
2.4.2. Evaluation of mitochondrial function
For MMP experiments, 200 ml of the sperm suspension (150 106 ml 1) was added to
800 ml of isosmolal or anisosmolal TALP for a ®nal sperm concentration of 30 106 ml 1,
and incubated at room temperature for 10 min. Then, each 1 ml sample was divided into
two 500 ml samples. To the ®rst set of tubes in each treatment, a ®nal concentration of 2 mM
JC-1 was added for 10 min and the samples were evaluated using a ¯ow cytometer
(FACSCalibur, Becton-Dickinson Immunocytometry Systems, San Jose, CA). Using the
appropriate gating parameters for equine spermatozoa determined during preliminary
experiments, the cell population (10,000 events) was divided into either high membrane
potential (red/green ¯uorescence) or low membrane potential (green ¯uorescence),
according to natural partitioning. In the second set of tubes, sperm were diluted with
1.5 ml isosmolal TALP (7:5 106 ml 1) for 10 min, and then incubated another 10 min
with 2 mM JC-1 before being analyzed on the ¯ow cytometer. This same protocol was used
for the viability studies, the only difference being that propidium iodide (5 mM) was used
instead of JC-1.
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2.5. Determination of sperm volume
A Coulter counter (Z2 model; Coulter Corp., Miami, FL) with a standard 50-mm aperture
tube was used. Sperm MCV was calibrated using spherical polystyrene latex beads of three
different diameters (3, 5, and 10 mm, CC Size Standard reference: 6602793, 6602794,
6602796, respectively; Coulter Corp.) as suggested by the manufacturer. The Coulter
counter was interfaced to a microcomputer and data were acquired by means of speci®c
software (AccucompTM, Coulter Corp.). For MCV analysis, 20 ml of the sperm suspension
(150 106 ml 1) was added to 20 ml of isosmolal or anisosmolal TALP (150 103 ml 1)
for 10 min. The MCV was then determined by evaluating 10,000 cells. For determination
of MCV after return to isomolality, in separate tubes, 20 ml of sperm were added to 2 ml of
isosmolal or anisosmolal TALP and incubated for 10 min. Then, 18 ml of isosmolal TALP
was added to each treatment tube and incubated an additional 10 min before MCV was
determined. Since the instruments used to determine MMP and MCV count a preselected
number of cells for analysis, dilution factors do not affect results.
In order to determine the inactive cell volume and estimate whether equine sperm
membranes behave as linear osmometers, sperm volumes recorded in isosmolal and
anisosmolal conditions were ®tted to the following Boyle van't Hoff equation:
V
Miso
Vb
Vb
1
ˆ
‡
Viso
M
Viso
Viso
where V is the cell volume at the osmolality M, Viso is the cell volume at isosmolality (Miso),
and Vb is the osmotically inactive cell volume.
2.6. Statistical analysis
Data were analyzed using one-way ANOVA with Minitab1 statistical software (Minitab,
Inc., State College, PA). ANOVA was used to evaluate treatment differences over a range of
anisosmolality treatments. Endpoints included sperm total motility, progressive motility,
viability, MMP, and MCV.
3. Results
3.1. Motility
Normalized total sperm motility was lower (P < 0:05) in anisosmolal conditions as
compared to controls (Fig. 1). When sperm were returned to isosmolal conditions, only sperm
previously incubated in 450 mOsm/kg TALP were able to recover to control levels of motility.
In control samples (300 mOsm/kg) a decrease in motility was observed following dilution into isosmolal TALP; however, signi®cant differences were not observed (P ˆ 0:27).
3.2. Viability and mitochondrial membrane potential
Spermatozoal viability (Fig. 2A) and MMP (Fig. 3A) were lower (P < 0:05) when cells
were exposed to hypotonic solutions as compared to control cells and cells exposed to
A.C. Pommer et al. / Theriogenology 58 (2002) 1373±1384
1377
Fig. 1. Sperm samples were incubated for 10 min in isosmolal or anisosmolal TALP and motility was analyzed
using a CASA system. Samples were then diluted with isosmolal TALP, incubated 5 min, and motility was
analyzed again. Letters (a±d) denotes signi®cant difference (P < 0:05) from control (300 mOsm/kg) within each
treatment group, respectively; n ˆ 5 experiments, three stallions.
hypertonic solutions. Sperm suspensions that were returned to isosmolal conditions from
75, 150, and 900 mOsm/kg had lower (P < 0:05) numbers of viable spermatozoa than
control cells (Fig. 2B). MMP was lower in a majority of cells previously incubated in 75
and 900 mOsm/kg when returned to isosmolal medium, as compared to control cells
(P < 0:05) (Fig. 3B).
3.3. Mean cell volume
The isosmotic MCV (mean S:E:M:) of equine spermatozoa was determined to be
24:4 0:6 mm3 at 22 8C. MCV was different than control volume in all anisosmolal
solutions (P < 0:05). Spermatozoa in all treatments were able to recover initial volume
when returned to isosmolal medium (P > 0:05) (Fig. 4). MCV swelled to 1.60 times initial
volume when exposed to 75 mOsm/kg TALP and decreased to 0.81 times initial volume
when incubated in 900 mOsm/kg TALP (Table 1), indicating an active osmoregulatory
volume range.
The osmotic response of equine spermatozoa was determined over the range 150±
900 mOsm at 22 8C. Data are presented as a Boyle van't Hoff plot (volume versus
1/osmolality) and depicts a linear response (r 2 ˆ 0:999) and a Vb of 70.7% (Fig. 5).
4. Discussion
An understanding of the osmotic tolerance limits and cellular processes involved is
essential to develop new methods for semen processing and storage that will be useful for a
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A.C. Pommer et al. / Theriogenology 58 (2002) 1373±1384
Fig. 2. Sperm samples were incubated for 10 min at room temperature in isosmolal or anisosmolal TALP. (A) A
®nal concentration of 5 mM propidium iodide was added to each treatment for 10 min, and the samples were read
using a ¯ow cytometer. (B) Treatments were diluted with 1.5 ml isosmolal TALP for 10 min, and then incubated
another 10 min with 5 mM propidium iodide before being analyzed on the ¯ow cytometer. Nonlabeled cells were
considered live. Letter (a) denotes signi®cance from control (300 mOsm/kg) viability (P < 0:05); n ˆ 4
experiments, three stallions.
large number of genetically valuable sires. The rate at which osmotic volume regulation,
and hence, cooling or warming, may take place is highly dependent upon cell hydraulic
conductivity (water permeability), Lp. In spermatozoa, Lp has been shown to be dependent
on species [10], temperature, cryoprotective agent (CPA), and ice crystal formation [9].
Thus, further progress in improving sperm survival would not be achieved simply by
modifying established cryopreservation diluents. A more fundamental understanding of
the biophysical and biochemical processes that accompany sperm freezing and thawing
will be essential to design new successful cryopreservation protocols [11].
4.1. Osmotic behavior of equine spermatozoa
The results of our study showed that the mean volume (average of multiple mean values)
of isotonic equine spermatozoa measured with a Coulter counter was 24.4 mm3. Equine
sperm cell volume has not been previously reported, therefore, we cannot compare our
A.C. Pommer et al. / Theriogenology 58 (2002) 1373±1384
1379
Fig. 3. Sperm samples were incubated for 10 min at room temperature in isosmolal or anisosmolal TALP. (A) A
®nal concentration of 2 mM JC-1 was added to each treatment for 10 min, and the samples were read using a
¯ow cytometer. (B) Treatments were diluted with 1.5 ml isosmolal TALP for 10 min, and then incubated another
10 min with 2 mM JC-1 before being analyzed on the ¯ow cytometer. Red labeled cells were considered to have
high MMP, while those labeled green were considered to have low MMP. Letter (a) denotes signi®cance from
control (300 mOsm/kg) values (P < 0:05); n ˆ 3 experiments, three stallions.
results to other studies. However, using measures of equine spermatozoa obtained by
automated sperm morphometric analysis and optical microscopy [12,13], the estimated
cell volume is approximately 37 mm3. As has been described in mouse [14] and human
[15±17] sperm, the estimates of cell volume obtained from electronic cell sizers are smaller
than estimates obtained from microscopic measurements. The origin of these discrepancies
is not clear. However, considering that the resolution limits of optical microscopy are close
to some of the diameters measured in the spermatozoa, an over-estimation of as little as
10% is not unreasonable, and could explain the difference between the two estimated
volumes for equine spermatozoa.
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Fig. 4. Sperm samples were incubated for 10 min at room temperature in isosmolal or anisosmolal buffer. MCV
was then determined using a Z2 Beckman Coulter Counter. In separate tubes, after the initial 10 min incubation,
samples were diluted with isosmolal buffer and incubated for 10 min before volume was determined. Letter (a)
denotes signi®cant difference from control (300 mOsm/kg) within each treatment group, respectively
(P < 0:05); n ˆ 3 experiments, three stallions.
In the present study, equine spermatozoa behaved as linear osmometers in the range of
150±900 mOsm/kg (r 2 ˆ 0:999) with 70.7% of the total cell volume (Vb) (both solids and
water) being osmotically inactive. When a cell behaves as an ideal osmometer, the volume
of osmotically available water contained in the cell, in our case almost 30% of total sperm
volume in isosmotic conditions, will be inversely related to the osmolality of nonpermeable
solutes in the external medium [18]. In addition, the observed linear osmotic behavior
Table 1
Normalized cell volume, motility, viability, and MMP of equine spermatozoa in anisosmotic TALP
Osmolality
(mOsm/kg)
Normalized
volume
Normalized
motility (%)
Normalized
viability (%)
Normalized
high MMP (%)
75
150
300
450
600
900
1.60
1.28
1.00
0.89
0.85
0.81
13.03
56.67
100.00
70.30
15.15
0.61
43.13
60.64
100.00
99.69
99.47
96.96
45.36
69.91
100.00
86.70
94.45
85.13
Sperm samples were incubated for 10 min at room temperature in isosmolal or anisosmolal TALP prior to all
analyses. MCV was then determined using a Z2 Beckman Coulter Counter and motility was analyzed using a
CASA system. For viability determination, a ®nal concentration of 5 mM propidium iodide was added to each
treatment for 10 min and the samples were read using a ¯ow cytometer. Nonlabeled cells were considered live.
For MMP determination, a ®nal concentration of 2 mM JC-1 was added to each treatment for 10 min and the
samples were read using a ¯ow cytometer. Red labeled cells were considered to have high MMP, while those
labeled green were considered to have low MMP.
A.C. Pommer et al. / Theriogenology 58 (2002) 1373±1384
1381
Fig. 5. Boyle van't Hoff plot (volume vs. 1/osmolality) of equine spermatozoa at 22 8C. Sperm samples were
exposed to different osmolalities: 900, 600, 450, 300, and 150 mOsm/kg TALP. The y-intercept indicates that Vb,
the osmotically inactive volume, is 70.7% of the isosmotic cell volume.
included hypoosmolal and hyperosmolal conditions (150±900 mOsm/kg), suggesting that
the values for exosmotic and endosmotic hydraulic ¯ows were similar in the tested
osmolality range.
A recent paper addressing optimal cooling rates for equine sperm [19] used estimated
sperm measurements (sperm isosmotic volume, osmotically inactive cell volume) based on
geometrical shape models since objective data was not previously available. The data
reported here will be of help in determining water and CPA permeabilities in equine
spermatozoa during freezing±thawing processes.
The effect of four permeable CPAs on equine sperm function has been recently reported
[7]. In that study, the use of glycerol resulted in the most marked decline in sperm function
(viability, motility, and MMP) while propylene glycol, dimethylsulfoxide and particularly
ethylene glycol caused the least osmotic damage. During CPA addition, cells shrink due to
the increased external hyperosmotic environment and the reverse occurs during CPA
removal. These volume excursions associated with CPA use may be the cause of cell
function disorders. It has been demonstrated in other species [9,20] that an optimal CPA is
one that has rapid permeability to the cell minimizing cell volume excursions, low
temperature dependence, and low toxicity to the cell. Estimates from regression analysis
of our data indicate that equine spermatozoa can tolerate approximately 20% swelling and
11% shrinkage of their isosmotic cell volume and still maintain more than 70% of control
motility. These limits will be used in future studies to determine the optimal CPA type and
concentration to cryopreserve equine sperm.
4.2. Osmotic tolerance of equine spermatozoa
Osmotic tolerance limits are critical to understanding the capabilities of equine
spermatozoa during freezing and thawing. Resistance to anisosmolality is essential to
prevent cell lysis and death. The capacity for spermatozoa to respond with cell volume
adjustment is determined by several factors including membrane phospholipid composi-
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A.C. Pommer et al. / Theriogenology 58 (2002) 1373±1384
tion, water permeability, lipid phase transition temperature, Na‡/K‡ ATPase activity, ion
channels, and cytoskeletal elements. Semen from individual stallions and boars can have
signi®cant differences in the ability to withstand the freeze±thaw cycle suggesting
differences in sperm membrane characteristics and biochemical composition between
species [4,17]. Within the range of osmotic conditions evaluated in this study, our data
demonstrate that equine spermatozoa are capable of signi®cant and reversible adaptation of
mean cellular volume (Fig. 4). MCV was maximal at 75 mOsm/kg, the lowest osmolality
tested, and sperm cells reached an increase in volume of 60% over that of isosmolality.
Noiles et al. [6] reported that stallion spermatozoa are capable of swelling to volumes of 6.1
times that of isosmotic volume. In that study, equine spermatozoa were evaluated for cell
volume changes using a wider range of hyposmotic conditions (3±215 mOsm/kg) than in
the present study. Although the number of ejaculates was low, that study also indicated a
highly responsive osmoregulatory function of equine spermatozoa. The trend between the
two studies was similar, but there was a large difference observed in cell volume excursions
while subjected to hyposmolal solutions (1.6 versus 6.1 times isosmotic sperm volume).
There are two possible explanations for this discrepancy. First, the osmotic ranges tested
were lower in the study by Noiles et al. (3 mOsm/kg), and second, those authors used an
indirect ¯ow cytometric technique to estimate sperm volume in response to anisosmolal
conditions.
The present study demonstrates that equine spermatozoa are highly susceptible to
osmotic stress since sperm motility decreased in anisosmolal solutions and spermatozoa
from most treatments demonstrated poor return to normal motility following this brief
osmotic stress period. Both motility and viability of equine spermatozoa decreased
signi®cantly in hyposmotic media demonstrating a behavior similar to boar spermatozoa
[4], which appear to be more sensitive to osmolality changes and to have a limited osmotic
tolerance compared to mouse [14] and human sperm [17,20]. A recent study [7] reported a
limited osmotic tolerance of equine spermatozoa when subjected to anisosmolal conditions, in agreement with our results.
It was interesting that MCV returned to control levels in light of the failure of sperm
motility to return to control levels. It is likely that the 10 min anisosmotic stress induced
sublethal damage to equine spermatozoa. Although the appearance of the cells was normal
using light microscopy, and the cell volume returned to normal, there were signi®cant
detrimental changes in sperm motility. It is clear that for equine spermatozoa, the swelling
process is more detrimental to sperm function than shrinking, and that the mechanisms that
affect motility seem to be different in these situations. Spermatozoa subjected to a
hyperosmolal environment appear to have intact membranes (viable cells) and are capable
of preserving their mitochondria, as demonstrated by a high MMP. However, when
spermatozoa were subjected to hypotonic solutions, MMP, and viability markedly
decreased. At 450 mOsm/kg cell volume was reduced to 89% of initial volume, and
retained 70% of initial motility. However, at 150 mOsm/kg volume increased 28% from
control and motility decreased to 57% of initial motility. Furthermore, when the cells were
returned to isosmolal conditions, spermatozoa in 900 mOsm/kg showed decreased MMP
and viability as cell volume increased. This suggests that the sperm's ability to maintain
electrolyte balance and membrane potential is hindered when cells swell. Thus, the
thawing process may be more detrimental to spermatozoa than the freezing process.
A.C. Pommer et al. / Theriogenology 58 (2002) 1373±1384
1383
Future studies should involve determining the optimal cryoprotectant agent to minimize
such ¯uctuations in cell volume.
In summary, this study has determined several important cryobiological characteristics
of equine spermatozoa that can be applied to improve current cryopreservation protocols.
The results showed that: (a) equine spermatozoa exhibit a linear osmotic response in the
range of 150±900 mOsm/kg; (b) equine spermatozoa have a cell volume of 24.4 mm3, with
70.7% being osmotically inactive; (c) although most spermatozoa are able to recover initial
volume after osmotic stress, they are not able to recover initial motility and MMP; and (d)
the membrane changes resulting from cellular swelling or shrinkage causes fundamental
irreversible damage to plasma membrane and cellular organelles that, in turn, cause
perturbations in cell function.
Acknowledgements
The authors gratefully acknowledge Dr. Mary Delany, Department of Animal Science,
College of Agriculture and Environmental Sciences, for technical assistance with the Z2
Coulter Counter and Abigail Spinner, California Regional Primate Research Center, for
technical assistance with ¯ow cytometry. This work was supported by a grant from the
USDA (no. 98-35203-6584).
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