CryoLetters 31 (6), 438-444 (2010)
© CryoLetters, [email protected]
A SIMPLE OSMOTIC STRESS TEST TO PREDICT BOAR SPERM
CRYOSURVIVAL
Cesar Garzon-Perez1, Hector F. Flores2 and Alfredo Medrano1*
1
Departamento de Ciencias Pecuarias, Facultad de Estudios Superiores – Cuautitlan,
Universidad Nacional Autonoma de Mexico, Carretera Cuautitlan –Teoloyucan s/n.
Cuautitlan Izcalli, Estado de Mexico. 54714. Mexico.
2
Departamento de Genetica, Facultad de Medicina Veterinaria y Zootecnia, Universidad
Nacional Autonoma de Mexico, Ciudad Universitaria, Mexico, D. F.
*Corresponding author
email: [email protected]
Abstract
This work was carried out to test whether viability of pig spermatozoa subjected to an osmotic
test is correlated to sperm cryosurvival. Spermatozoa were cooled from 22°C to -5°C, aliquots
were exposed to a series of hyperosmotic solutions (300-2100 mOsm/kg) for 15 min,
immediately spermatozoa were re-warmed to 37ºC and isosmolarity was restored.
Spermatozoa were cooled from 22°C to -5°C and one aliquot was exposed to the osmotic test
while diluted spermatozoa were frozen-thawed. Plasma membrane-intact spermatozoa
decreased as osmolarity increased (P<0.0001), a further decreased (P<0.0001) was observed
when isotonicity was restored. Proportions of plasma membrane-intact and acrosome-intact
cells from the osmotic test were no different from those after freeze-thawing: 36% vs. 35%,
80% vs. 80%, respectively. A significant correlation was found between the proportion of
acrosome-intact cells after freeze-thawing and that from the osmotic test (r=0.81, P<0.01).
This test provides a useful and economical mean to predict in vitro boar sperm cryosurvival.
Keywords: semen cooling, chlortetracycline assay, sperm capacitation, lectins
INTRODUCTION
Among agricultural species pig possess the most sensitive spermatozoa to freezethawing protocols, this sensitivity is manifested as decreased fertility and prolificacy (11, 12),
for this reason artificial insemination using frozen-thawed spermatozoa is not a routine
practice in pig industry. Low fertility and prolificacy are the result of a number of factors
involved in the cryopreservation process that affect key cell structures impairing sperm
functionality (32). Cooling causes alterations to the sperm plasma membrane such as
premature capacitation (33) that may reduce sperm lifespan in the female reproductive tract;
chlortetracycline (CTC) assay allows monitoring of those capacitation-like changes in the
sperm plasma membrane (30, 13). The effect of freeze-thawing protocols on sperm
cryosurvival depends on inter-male variability, the so-called good and bad freezers, thus
spermatozoa from each group of males will be affected differently (19, 20). This is an ever
present factor in sperm cryopreservation, therefore a simple test based on simulation of the
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osmotic stress spermatozoa suffer during freeze-thawing may be useful to predict sperm
cryosurvival from different males and ejaculates in order to save time. Osmotic stress may
affect cellular responses in several ways such as structure and dynamic of plasma membrane,
mitochondrial function and signaling (22). Exposition of spermatozoa to hyperosmotic
solutions reduces cell viability in terms of plasma and acrosome membranes integrity; when
isosmolarity is restored that effect is more intense (2, 17). Cryomicroscopy studies showed
viability of ram and pig spermatozoa is maintained during freezing but it decreases after
thawing (9, 19); thus, that sort of osmotic tests may partially mimic sperm cryodamage. The
aim of this work was to test the hypothesis that viability of pig spermatozoa exposed to hyperand hypo-osmotic solutions, to simulate the osmotic stress that occurs during freeze-thawing,
is positively correlated to that after cryopreservation.
MATERIALS AND METHODS
Source and preparation of semen
Semen was collected by the gloved hand method, once a week, from 11 hybrid boars
and transported diluted 1:2 v/v in MR-A medium (Kubus, Spain) in insulated containers at
ambient temperature; semen arrived at its destination after 1-2 h. Semen was centrifuged for
10 min at 300 g., supernatant was removed by aspiration and spermatozoa were resuspended
in up to half of the final volume in standard BF5 freezing medium (27), without glycerol, at
room temperature. The criterion for selecting the semen samples to be used in these
experiments was that motility before freezing (after resuspension in BF5 freezing medium)
was at least 70%. Sperm suspensions were cooled over 3 h to 5ºC (approximately 0.1ºC min1
); BF5 medium with glycerol (final concentration 1% v/v) was added at 6.5ºC and diluted
spermatozoa were then packaged in 0.5 ml plastic straws; the final sperm concentration was
200 X 106 spermatozoa ml-1. Each ejaculate was processed and used separately.
Experimental design
In a preliminary experiment, diluted spermatozoa (n= 5 boars, 33 ejaculates) packaged
in plastic straws (aliquots) were cooled at three different target temperatures before freezing:
1) 5ºC, 2) 0ºC, and 3) -5ºC. When semen reached target temperatures, straws (5 per each
cooling treatment; 15 per ejaculate) were immediately frozen over nitrogen vapour, 4 cm
above the nitrogen level for 15 minutes at about -80ºC, plunged and stored in liquid nitrogen.
Temperature of sperm suspensions was monitored by means of a thermocouple located within
one straw. Cooling from 5ºC to 0ºC and to -5°C spent 40 and 50 min respectively, in addition
to the time spent (3 hrs) to reach 5ºC (Graph 1).
Figure 1. Cooling curve of boar
spermatozoa from 22ºC to -5ºC. At
this temperature (-5ºC) sperm
aliquots were subjected to osmotic
test and freezing.
Experiment 1. Diluted spermatozoa (n= 5 boars, 20 ejaculates) were cooled to -5ºC,
four aliquots were then taken and exposed to: 1) 300, 2) 900, 3) 1500 and 4) 2100 mOsm/kg
for 15 min; immediately, aliquots were rewarmed to 37ºC and exposed to suitable hyposmotic
solutions for 15 min, to restore isosmolarity.
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Experiment 2. Diluted spermatozoa (n= 6 boars, 30 ejaculates) packaged in plastic
straws (aliquots) were cooled to -5ºC, at this moment one additional aliquot was subjected to
the anisosmotic test; then, straws (n=5 per ejaculate) were immediately frozen as mentioned.
Thawing was carried out after 30 days of cryopreservation, plunging the straws in a
water bath at 39ºC for 30 seconds, thawed spermatozoa were then poured in dry plastic tubes
and one drop was diluted 1:10 (v/v) in BTS medium (27) for sperm assessment.
The anisosmotic test
Hyperosmotic solutions were prepared by adding glucose to BF5 freezing medium;
hyposmotic solutions were prepared by adding TES, TRIS and glucose to distilled water.
When diluted spermatozoa (in BF5, 200 X106 sperm/ml) reached -5°C, four aliquots were
taken and mixed (1:1 v/v) with the correspondent hyperosmotic solution to obtain target
osmolarity: 300 (control), 900, 1500 and 2100 mOsm/Kg for 15 min. Immediately, each
aliquot (spermatozoa in hyperosmotic solution) were mixed with its correspondent
hyposmotic solution (1:6 v/v) at 37°C for 15 min to reestablish isosmolarity. At the end of
each period (hyperosmolarity, restoration of isosmolarity), samples from each treatment were
taken to assess plasma and acrosome membranes integrity.
Sperm assessment
After collection and thawing, progressive motility, plasma membrane integrity,
acrosome membrane integrity and the capacitation status were assessed. Progressive motility
(percentage of cells showing forward movement) was estimated from semen diluted in BTS
1:10 (v/v), on a microscope stage at 37ºC, viewed at x100 magnification in an optical
microscope. Plasma membrane-intact spermatozoa were estimated by counting 200 cells per
sample from smears stained by SYBR14/PI fluorescent stain (7); acrosome-intact
spermatozoa were estimated by counting 200 cells per sample from smears stained by PSAFITC lectin (4) and fluorescent microscopy. Sperm capacitation status was assessed by
counting cells (200 per sample) showing any of the CTC assay patterns: F, B and AR (13)
under fluorescent light at x1000 magnification. The CTC staining assay was carried out as
described by Green & Watson (8); semen samples were previously filtered through a
Sephadex column (G-50 Sigma, St Louis MO, USA) to select a viable sperm subpopulation
so that CTC patterns were assessed on live cells only (25).
Statistical analysis
Data from motile, plasma membrane-intact, acrosome-intact and capacitated
spermatozoa was analyzed by ANOVA using the general linear model procedure from the
Statistica for Windows 5.5 software (StatSoft Inc., Tulsa OK, USA, 2000). Data expressed as
percentages were arcsine-transformed before ANOVA. Tukey’s multiple range tests was used
to compare means. Within-treatments and between-treatments (Experiment 1) were analyzed
by Friedman ANOVA and Wilcoxon Matched Pair Test, respectively. The Spearman´s rank
correlation coefficient was employed to measure the association between sperm survival after
(1) the osmotic test and (2) after cryopreservation.
RESULTS
Preliminary experiment
Slow cooling to -5ºC increased (P<0.05) the percentage of acrosome-intact
spermatozoa: 81 ± 1.4 vs. 78 ± 1.1 and 75 ± 1.4 at 0°C and +5°C respectively (mean ± SEM);
there were no differences regarding motile, plasma membrane-intact and capacitated
spermatozoa (Table 1).
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Table 1. Effect of three pre-freeze cooling temperatures on boar sperm cryosurvival
Plasma
AcrosomeCapacitated
Target
Motile sperm
membrane-intact
intact sperm
(B+AR patterns)
temperature
(%)
sperm (%)
(%)
sperm (%)
+5ºC
36 ± 1.7
56 ± 2.7
75 ± 1.4a
62 ± 1.8
0ºC
37 ± 1.4
56 ± 2.6
78 ± 1.1ab
64 ± 1.8
-5ºC
39 ± 1.5
60 ± 2.5
81 ± 1.4b
58 ± 1.9
Values are means ± SEM. Different letter in columns differ significantly (P<0.05).
Experiment 1
Proportion of plasma membrane-intact spermatozoa significantly decreased as
osmolarity increased (P<0.0001); a further decrease was observed when isotonicity was
restored (P<0.0001). In contrast, percentage of acrosome-intact spermatozoa did not change
as osmolarity increased but it decreased significantly when isosmolarity was restored
(P<0.002), see Graph 2. The biggest numeric change produced by the treatments was
observed at 2100 mOsm/Kg, thus this value was selected to be performed in parallel to freezethawing.
Experiment 2
Values of fresh and frozen-thawed spermatozoa are shown in Table 2, both groups of values
are normal for this species.
Figure 2. Plasma membrane-intact spermatozoa (% left panel), acrosome-intact
spermatozoa (% right panel) at different values of hyperosmolarity and at restoration of
isosmolarity. Bars represent SEM.
There was no difference between the percentage of plasma membrane-intact cells at
hyperosmotic condition from the osmotic test (36 ± 7.1%) and that after freeze-thawing (35 ±
8.6%), see Table 2. However, there was a small and non significant correlation value (r=0.20,
P>0.05). In contrast, there was a significant difference (P<0.05) between the proportion of
plasma membrane-intact cells after restoration of isosmolarity in the osmotic test (14 ± 6.8%)
and that after freeze-thawing (35 ± 8.6%).
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Table 2. Boar sperm variables during the osmotic test and after freeze-thawing
Motile
Plasma
AcrosomeCapacitated
Sperm
sperm (%)
membrane-intact
intact sperm (B+AR patterns)
condition
sperm (%)
(%)
sperm (%)
Fresh
74 ± 3.7a
91 ± 2.8a
89 ± 3.2a
16 ± 6.2a
Hyperosmolarity
na
36 ± 7.1b
80 ± 7.6b
na
Restoration of
na
14 ± 6.8c
69 ± 8.6c
na
isosmolarity
Frozen-thawed
45 ± 11.9b
35 ± 8.6b
80 ± 4.6b
73 ± 6.1b
Values are means ± SEM. Different letter in columns differ significantly (P<0.05). na= non
assessed
There was no difference between the proportions of acrosome-intact cells at
hyperosmotic condition from the osmotic test (80 ± 7.6%) and that after freeze-thawing (80 ±
4.6%), see Table 2. Correlation value was high and significant (r=0.81, P<0.01). In contrast,
there was a significant difference (P<0.05) between the proportions of acrosome-intact cells
after restoration of isosmolarity in the osmotic test (69 ± 8.6%) and that after freeze-thawing
(80 ± 4.6%).
DISCUSSION
Slow cooling of diluted spermatozoa close to their freezing point (around -5ºC)
improved sperm cryosurvival with respect to acrosome integrity, this confirm previous
observations on the use of slow cooling rates (3 to 6ºC min-1) from +5ºC to -5ºC in pig sperm
cryopreservation (1, 19, 20). Similar results were obtained in buck (18) and ram (28)
spermatozoa, cooled to -5 and -2°C before freezing, respectively. Slow cooling below 0ºC
may allow adequate packaging of plasma membrane phospholipids and proteins after lipid
phase transition takes place. Noiles et al. (24) identified a range of temperature (+4 to 0ºC) in
which a further lipid phase transition may occur in mouse spermatozoa. In addition, slow
cooling to -5ºC could allow spermatozoa to slowly pass through a stage in which cold shock is
still likely (31, 5).
Exposition of spermatozoa to hyper- and hypo-osmotic conditions was carried out to
simulate the osmotic stress that occurs during freeze-thawing; pig spermatozoa exposed to
hyperosmotic solutions showed a decrease in the proportion of both plasma membrane-intact
and acrosome-intact cells as osmolarity increase, this effect was far more intense when
isosmolarity was restored. This has been observed in ram (10, 6), man (6), horse (2, 26),
mouse (14) and rat spermatozoa (29). To explain post-hyperosmotic injury, phenomenon
investigated time ago (15, 21, 16), Muldrew (23) has proposed a model based on the
hypothesis that a group of intracellular proteins, salted during hypertonic exposure, may catch
dissolved ions in the cytoplasm; the process is reversed at rewarming when those ions come
back to the cytoplasm provoking cell swelling that may cause its breakage. Observations from
cryomicroscopy have revealed sperm plasma membrane integrity is maintained during
freezing but it decreases after thawing (9, 19). Regarding the effect of temperature on posthypertonic injury, incubation at 37°C caused more damage on viability and plasma membrane
integrity of pig spermatozoa than incubation at 4 and 16°C (3). In this work, spermatozoa
were maintained in hyperosmotic conditions at -5°C (near the freezing point of common
sperm extenders), since post-hyperosmotic injury is reduced by effect of temperature, slow
cooling of spermatozoa to subzero temperatures before freezing makes sense. Proportions of
plasma membrane-intact and acrosome-intact cells at hyperosmotic condition in the osmotic
test and those after freeze-thawing were similar suggesting these processes (osmotic stress
and cryopreservation) are, in some way, alike. In contrast, Medrano (17), using a similar test
442
of osmotic stress in boar spermatozoa, found a small and non significant correlation value for
those variables; it was argued, temperature of hyperosmotic condition (5°C) could be no
relevant. Taking in consideration that observation, this osmotic stress test was modified with
respect to temperature: -5 instead of +5°C, and time of exposition to hyper- and hypo-osmotic
solutions: 15 instead of 5 min. Thus, these modifications improved sensibility of this assay. In
conclusion, a simple test provides a feasible mean to predict in vitro boar sperm cryosurvival;
more research is needed to see whether results from the osmotic stress test may correlate to in
vivo fertility, i.e., artificial insemination using frozen-thawed spermatozoa.
Acknowledgements: C Garzon-Perez and HF Flores were supported by the Mexican
government (CONACYT). Project partially supported by Universidad Nacional Autonoma de
Mexico (PAPPIT IN207009). All experiments comply with the Institutional Subcommittee for
Care of Animals in Experimentation (SICUAE) from the National Autonomous University of
Mexico.
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Accepted for publication 8/7/2010
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