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Theriogenology 74 (2010) 940 –950

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The HSP90AA1 sperm content and the prediction of the

boar ejaculate freezability
I. Casasa,*, S. Sanchoa, J. Ballesterb, M. Briza, E. Pinarta, E. Bussalleua, M. Yestea,
A. Fàbregaa, J.E. Rodríguez-Gilc, S. Boneta
a
Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology
(INTEA), University of Girona, Campus Montilivi, s/n, 17071 Girona, Spain
b
Center for Animal Biotechnology and Gene Therapy (CBATEG), Building H, Autonomous University of Barcelona, 08193 Bellaterra, Spain
c
Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, 08193 Bellaterra, Spain
Received 12 March 2010; received in revised form 12 April 2010; accepted 18 April 2010

Abstract
In a previous study we reported that the immunolabelling of GLUT3, HSP90AA1, and Cu/ZnSOD proteins on boar sperm did not
show differences between good and poor freezability ejaculates, in terms of a qualitative analysis based on location and reactivity of
these proteins at 17 °C and at 240 min post-thaw. Since predicting the ejaculate freezability is considerably important in sperm
cryopreservation procedures, the objective of the present study was to quantify the expression of these three proteins in good and poor
freezability ejaculates. For this purpose, 10 ejaculates from 9 Piétrain boars were cryopreserved and their sperm quality assessed in the
three main steps of the freezing process (17 °C, 5 °C, and 240 min post-thaw). After this assessment, the 10 ejaculates were clustered
for freezability on the basis of their sperm progressive motility and membrane integrity at 240 min post-thaw. From the whole ejaculates,
only four good and four poor freezability ejaculates displaying the most divergent values were selected for a western blot assay using
sperm samples coming from the three mentioned freezing steps. Protein levels through densitometry were significantly different between
good and poor freezability ejaculates for Cu/ZnSOD at 240 min post-thaw (P ⱕ 0.01) and for HSP90AA1 at 17 °C and 5 °C (P ⱕ 0.05).
This last finding claims the introduction of tests based on molecular markers in spermatozoa to accurately predict the freezability of
ejaculates in order to promote the use of frozen semen on artificial insemination programmes.
© 2010 Elsevier Inc. All rights reserved.

Keywords: Cryopreservation; Freezability; Sperm; GLUT3; HSP90AA1; Cu/ZnSOD

1. Introduction ejaculates seems to be more dependent on individual

The boar spermatozoon is, compared with other
mammalian species, the most sensitive to low temper-
atures mainly due to the characteristics of its plasma-
lemma, which displays a low content of cholesterol and
saturated phospholipids [1,2]. The freezability of boar
* Corresponding author. Tel.: ⫹ 34 972 18 32 16; fax: ⫹ 34 972 41
81 50.
E-mail address: isabel.casas@udg.edu (I. Casas).
features rather than on the cryopreservation process
itself [3], and thus the capacity of a boar to produce
cold-shock resistant sperm has several implications on
its own ejaculate characteristics.
In the field of boar sperm cryopreservation there is
scarce literature conducting research to address differ-
ences between good and poor freezability ejaculates.
This omission is partly due to the focus on improve-
ments of the cryopreservation procedures and extenders
in frozen-thawed (FT) sperm rather than on increasing

0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2010.04.021
 I. Casas et al. / Theriogenology 74 (2010) 940 –950
current knowledge about the molecular basis of sperm
cold-shock, both in FT sperm and in refrigerated se-
men.
The particular features that lead some ejaculates
(known as good freezability ejaculates, GFEs) to resist
cryopreservation better than others (known as poor
freezability ejaculates, PFEs) remain unidentified. Pre-
vious works have linked this aspect with the genetic
origin of boars [4,5], which also explains why ejacu-
lates collected from the same boar tend to display the
same freezability [6 –9]. Notwithstanding, it has been
noticed that there could be changes in freezability
among collections for which the breeding conditions,
along with genetics, may affect their resistance to cold-
shock. Such heterogeneity in boar ejaculate freezability
conducts to the cryopreservation of a large amount of
sperm with poor recovery that can decrease, by two
times, the probabilities of pregnancy after insemination
[10]. Research in this field should make feasible a
previous selection for GFEs.
Until now, the highest reliability in the prediction of
the boar ejaculate freezability has been separately
achieved by analyzing the morphometry of the sperm
head in refrigerated semen [11,12] and by assessing the
indexes of sperm linearity (LIN) and straightness (STR)
at 5 °C during the cryopreservation protocol [13]. The
value of the first descriptor lies on the size of the sperm
head, since it is proved that sperm with large heads
appears to be more sensitive to osmotic changes during
freezing, as observed in bull [14], dog [15], and boar
[11,12]. Regression models based on sperm morpho-
metric characteristics in refrigerated semen account for
up to 36% of intact sperm membranes post-thaw [11].
On the other hand, the combination of LIN and STR
motility indexes at 5 °C, related to a higher hyperacti-
vation of PFEs at this temperature, can predict the
freezability of ejaculates with 70% of confidence [13].
As it can be observed, routine sperm quality param-
eters still do not guarantee 100% reliability in predic-
tion of cryopreservation success. Flores et al [16] sug-
gest that the predictive value could be increased by
assessing subpopulations in the ejaculate instead of ana-
lyzing it as a whole, and by developing new tests. The
introduction of novel tests for freezability prediction re-
quires that we look for indicators of the sperm function-
ality interfering, in turn, with sperm freezing resistance.
In a previous study, three sperm proteins were se-
lected in our laboratories to test their adequacy as
indicators of the ejaculate freezability: the solute carrier
family 2 (facilitated glucose transporter) member 3
(SLC2A3, alias GLUT3), the heat shock protein 90-
941
KDa alpha A1 (HSP90AA1), and the copper- and zinc-
containing superoxide dismutase (Cu/ZnSOD) [13].
The potential of the three proteins in predicting the boar
ejaculate freezability was qualitatively tested under flu-
orescence microscopy. However, this technique was
not able to solve differences between GFEs and PFEs.
The objective of the present study was to test the
potential ability of these three proteins for freezability
prediction by applying the technique of western blot,
using boar sperm samples from GFEs and PFEs in three
main steps of the cryopreservation process: in refriger-
ated semen (17 °C), after adding glycerol (5 °C), and at
240 min post-thaw.
2. Materials and methods
2.1. Animals and obtainment of samples
Nine mature (20 mo) and healthy boars (Sus scrofa
domestica) from the Piétrain breed were stalled in a
herd under supervision of Selecció Batallé SA (Girona,
Spain). They weighed around 250 Kg and were fed
under standard protocols while being provided with
water ad libitum. Boars were routinely submitted to a
rhythm of two semen collections per week. Ten sperm
rich fractions were obtained within two weeks using the
gloved-hand method and immediately diluted at the rate
2:1 (v:v) using a long-term commercial extender free
from BSA (Vitasem LD, Magapor SL, Zaragoza,
Spain). Each boar contributed one ejaculate to the
study, except one boar from which two ejaculates were
collected (ejaculates number 1 and 9). Other ejaculates
collected from these boars were used for commercial
purposes. The 10 sperm rich fractions were diluted in
the AI centre (Girona, Spain) to the final proportion 1:5
(v:v) in the long-term commercial extender (Vitasem
LD). They were thereafter transported to our laborato-
ries where they were maintained at 17 °C overnight.
Cryopreservation was carried out the next day, im-
mediately after having assessed the sperm quality to
ensure that minimal standards were fulfilled (see Sperm
quality assessment, section 2.2.). Ejaculates collected
on the same day were frozen together, performing up to
four different collections/cryopreservations during the
two weeks: ejaculates 1 and 2; ejaculates 3, 4, and 5;
ejaculates 6 and 7; ejaculates 8, 9, and 10. Later, it was
verified that at least one ejaculate was out of the PFEs
group in each of the four cryopreservations performed,
to discard any effect of the freezing protocol on freez-
ability results.
The experimental protocol was designed in accor-
dance with the guidelines established by the Animal
942 I. Casas et al. / Theriogenology 74 (2010) 940 –950
Welfare Directive of the Autonomous Government of
Catalonia (Spain).
2.2. Sperm quality assessment
The sperm quality assessment was performed in
three different steps during the cryopreservation pro-
cess: Step 1 (17 °C; refrigerated semen), Step 2 (at 5
°C), and Step 3 (240 min post-thaw; FT sperm). In Step
2, analyses were carried out 30 min after glycerol was
added. Eleven motility parameters and the sperm mem-
brane integrity were evaluated in these three steps.
Sperm morphology was evaluated in Step 1 and the
osmotic resistance of sperm in Steps 1 and 3. Minimal
values were verified to be accomplished on all param-
eters in Step 1 to continue with cryopreservation, ac-
cording to Casas et al [13].
Sperm motility parameters were examined at ⫻ 100
magnification under a phase contrast Olympus BX41
microscope (Olympus Europa GmbH, Hamburg, Ger-
many) through computer assisted analysis (Sperm Class
Analyzer, SCA 2002, Microptic SL, Barcelona, Spain).
Three replicates were assessed per ejaculate in a Makler
chamber (Sefi-medical Instruments, Haifa, Israel) after
sperm was heated at 37 °C for 20 min. For each repli-
cate different fields were captured up to 1500 sperma-
tozoa (spz) were counted. Eleven motility parameters
were assessed: total motility, circular trips, curvilinear
velocity (VCL), straight-linear velocity (VSL), average
velocity (VAP), linearity index (LIN ⫽ VSL/VCL),
straightness index (STR ⫽ VSL/VAP), oscillation in-
dex (WOB ⫽ VAP/VCL), amplitude of lateral head
displacement (ALH), beating frequency (BCF), and
progressive motility (sperm showing more than 45% of
STR).
Sperm membrane integrity was evaluated using the
LIVE/DEAD® Sperm viability kit (Molecular Probes,
Eugene, OR, USA). Each sample was diluted to 0.010 ⫻
109 spz mL⫺1 in Beltsville Thawing Solution (BTS;
[17]). Two replicates were assessed per ejaculate, the
first by flow cytometry (20000 events per sample; Cell
Lab Quanta SC®, Beckman Coulter, Fullerton, CA,
USA) and, the second at ⫻ 400 magnification under a
fluorescence microscope (200 spz per sample; Zeiss
Axio Imager.Z1, Carl Zeiss, Germany). A single mean
was retrieved from the two replicates of sperm mem-
brane integrity.
Sperm morphological assessment was performed
in Step 1 as quality control. The percentage of sperm
without anomalies (absence of abnormal head shape,
proximal droplets, tail folding, or tail coiling) and
the percentage of sperm with proximal droplets were
obtained at ⫻ 200 magnification under a phase con-
trast microscope (Olympus BX41) through SCA
2002 software. A minimum of 300 spz was analysed
per each ejaculate. Distal droplets were not consid-
ered.
The osmotic resistance of sperm was assessed in
Steps 1 and 3 using the osmotic resistance test (ORT;
[13,18,19]). Two samples of 0.5 mL from each ejacu-
late were assessed at ⫻ 400 magnification under a
phase contrast Olympus BX41 microscope. The per-
centage of non-reacted sperm was the mean obtained
from the number of non-reacted sperm in each of the
two solutions in the test (150 mOsm kg⫺1 and 300
mOsm kg⫺1). One hundred spz was counted per solu-
tion.
2.3. Sperm cryopreservation
The 10 ejaculates were cryopreserved using the
Westendorf method for porcine [20], modified by Car-
vajal et al [21] and adapted in our laboratory. After
being held overnight at 17 °C [22,23] an aliquot of each
ejaculate was taken for analysis (Step 1). Ejaculates
were centrifuged at 17 °C and 640 ⫻ g for 3 min and
the pellets obtained were diluted to 1.5 ⫻ 109 spz mL⫺1
into a freezing medium containing lactose and egg yolk
(LEY). After cooling to 5 °C for 150 min in a cooling
cabinet, sperm was diluted to 1 ⫻ 109 spz mL⫺1 in a
second freezing medium (LEYGO) containing LEY
supplemented with 6% (v:v) glycerol and 1.5% (v:v)
Orvus Es Paste (Equex STM, Nova Chemical Sales
Inc., Scituate, MA, USA). An aliquot from each sample
was diluted to 1:3 (v:v) in BTS and left for 30 min at
5 °C in the cooling cabinet before sperm quality assess-
ment (Step 2). The rest of sperm was packed into 0.5-mL
plastic straws using a semiautomatic filling system (SFS
133, Minitub Ibérica SL, Spain). The straws were trans-
ferred to a programmable freezer (Icecube 14S-B with
SY-LAB software, Minitub Ibérica SL, Spain) and cooled
for 5 min and 13 sec at the following rates: ⫺6 °C min⫺1
from 5 to ⫺5 °C (1 min 40 sec), ⫺39.82 °C min⫺1 from
⫺5 to ⫺80 °C (1 min 53 sec), held for 30 sec at ⫺80 °C,
and ⫺60 °C min⫺1 from ⫺80 to ⫺150 °C (1 min 10 sec).
The straws were finally plunged into liquid nitrogen tanks
(⫺196 °C) for preservation.
After the samples had been stored for at least 12 h in
liquid nitrogen, four straws per ejaculate were thawed
for 20 sec in water at 37 °C and then diluted to 1:3 (v:v)
in BTS at the same temperature. Sperm was left for 240
min under these conditions until post-thaw quality anal-
yses were carried out (Step 3).
 I. Casas et al. / Theriogenology 74 (2010) 940 –950
2.4. Western blot assay
Only eight ejaculates, belonging to extreme groups of
freezability (GFEs and PFEs), were selected for the west-
ern blot assay (see Results, section 3.1.).
Samples were taken at the three different steps dur-
ing the cryopreservation protocol. They were centri-
fuged twice at 600 ⫻ g for 10 min in PBS and pellets
were stored at ⫺80 °C until the beginning of the assay.
Pellets were then resuspended and sonicated in 300 ␮L
of ice-cold lysis buffer (pH 6.8) containing 0.01 mol
L⫺1 Tris-HCl, 0.015 mol L⫺1 EDTA, 0.15 mol L⫺1
potassium fluoride (KF), 0.6 mol L⫺1 saccharose, 0.014
mol L⫺1 ␤-mercaptoethanol and, a commercial mixture
of protease inhibitors (1:100; v:v) (Protease inhibitor
cocktail ref. P8340, Sigma-Aldrich, St Louis, MO,
USA). After 30 min in ice, the homogenized suspensions
were centrifuged at 4 °C at maximal speed (22600 ⫻ g) for
15 min and total protein amount in supernatants was
calculated through the Bradford method [24] using a
commercial kit (Bio-Rad Laboratories; Fremont, CA,
USA). The volumes of supernatants containing 2 ␮g of
protein were boiled for 5 min in sodium dodecyl sul-
phate (SDS) sample buffer (1:1; v:v) (0.25 mol L⫺1
Tris pH 6.8; 0.05 mol L⫺1 dithiothreitol (DTT); 10%
(wt/vol) SDS; 50% (v:v) glycerol; 0.5% (wt/vol) bro-
mophenol blue). Finally, samples were loaded into 1.5
mm gels containing 10% acrylamide (wt/vol) to per-
form SDS-PAGE [25].
After running the gels at constant voltage (180 V),
the proteins were transferred to nitrocellulose mem-
branes [26] (75 min at 100 V). Membranes were sub-
sequently submerged for 30 min in blocking solution
(Tris-buffered saline (TBS) with 3% (wt/vol) BSA and
0.1% (v:v) Tween20) to be incubated at 4 °C overnight
with the primary antibody. Incubations were performed
sequentially on each membrane and antibodies were
diluted to different concentrations in blocking solution:
1:500 (v:v) (rabbit polyclonal anti-GLUT3 ref. RB-
9096-P; Thermo Fisher Scientific, Fremont, CA, USA;
Mouse monoclonal anti-HSP90AA1 ref. SR-B830;
MBL, Nagoya, Japan) and 1:1000 (v:v) (rabbit poly-
clonal anti-Cu/ZnSOD ref. SOD-100; Stressgen, Ann
Arbor, MI, USA; Mouse monoclonal anti-alpha tubulin
ref. MA1-19401; Thermo Fisher Scientific, Fremont,
CA, USA).
The immunoreaction was tested for 1h using two
different horseradish peroxidase (HRP) conjugated
polyclonal antibodies at 1:5000 (v:v) in blocking solu-
tion: goat anti-rabbit immunoglobulins (for GLUT3
and Cu/ZnSOD; Dako, Denmark) and rabbit anti-
mouse immunoglobulins (for HSP90AA1 and alpha-
943
tubulin; Dako, Denmark). The reaction was developed
for 5 min with a chemiluminescent HRP substrate (Im-
mobilon Western detection system; Millipore Corpora-
tion, Billerica, MA, USA). The experiment was per-
formed at least for triplicate and each replica consisted
of nine protein patterns (three proteins per three cryo-
preservation steps).
The protein patterns were scanned and quantified
using a gel documentation system (Gel Doc XR; Bio-
Rad; Hercules, CA, USA) and Quantity One Version
4.6.2. software package (Bio-Rad Laboratories). Pro-
tein levels were expressed as “band volume”, which is
defined as the total signal intensity inside the boundary
of a band measured in pixel intensity units (density) ⫻
mm2, the lowest intensity in a pixel being 0 (white).
Band boundaries were defined onto amplified digital
images of the protein patterns, avoiding background
signal. Alpha-tubulin was used as an internal standard
to normalize the volume of protein bands, stripping and
reprobing membranes when necessary.
2.5. Statistics
Values obtained for sperm quality parameters and
band volumes were expressed as mean ⫾ SEM. The
statistical software SPSS v15.0 for Windows (SPSS
Inc., Chicago, IL) was used for analyses and the sig-
nificance level was set at P ⫽ 0.05 (bilateral). Data in
percentages (x) were previously transformed to arcsine
square-root (x/100).
Selection of GFEs/PFEs among the 10 ejaculates
collected was achieved after running two non-hierar-
chical k-means cluster analyses for dissimilarities with
data coming from the sperm progressive motility and
the sperm membrane integrity in Step 3. Analyses were
set to obtain two clusters corresponding to extreme
freezability groups. The first analysis was run on the 10
ejaculates to disclose the two clusters. The second anal-
ysis was run with the cluster having the highest number
of ejaculates to reduce them, and to load all samples in
one gel for each of the nine protein patterns to be
obtained. To check the reliability of such a classifica-
tion system, based on two sperm quality parameters, the
clusters obtained were compared with two other clus-
ters based on all sperm quality parameters in Step 3,
according to Casas et al [13].
Data from the sperm quality assessment were com-
pared in each step between GFEs and PFEs through
independent sample t-tests. Means were additionally
tested for homogeneity of variances (Levene’s test).
Normalized band volumes were calculated by dividing
the volume of each band (one ejaculate sample) for the
944 I. Casas et al. / Theriogenology 74 (2010) 940 –950
corresponding volume of alpha-tubulin. Means on GFEs
and PFEs were obtained from the normalized band vol-
umes of the three replicates performed for each protein in
each step (normalized mean band volumes). Such data
was compared between GFEs and PFEs using indepen-
dent sample t-tests in each of the nine protein patterns.
Those values out of SEM were excluded from the analy-
sis. Normalized mean band volumes were additionally
tested for homogeneity of variances (Levene’s test).
3. Results
3.1. Classification of ejaculates into GFEs/PFEs
The 10 ejaculates were classified into two clusters
(A and B; Table 1) with maximal dissimilarities in
sperm progressive motility and membrane integrity in
Step 3. Equality with clusters obtained including all
sperm quality parameters in Step 3 confirmed the reli-
ability of both parameters for such classification. Ejac-
ulates number 1, 3, 7, and 9 (cluster B) were selected as
PFEs, the group having less than 40% in mean sperm
progressive motility and membrane integrity in Step 3.
With the purpose of reducing the number of ejaculates
in cluster A, the six ejaculates were submitted to an
additional analysis from which two other clusters were
obtained (C and D; Table 2). Ejaculates number 4, 5, 6,
and 10 (cluster C) were selected as GFEs, having a
mean value over 40% in sperm progressive motility and
membrane integrity in Step 3. In short, a total of eight
ejaculates were selected for the western blot assay.
3.2. Sperm quality assessment
Results from the sperm quality assessment are
shown in Table 3 for each step during cryopreservation,
with data from the four GFEs and the four PFEs se-
lected for the western blot assay. In Steps 1 and 2, all
Table 1
Classification of ejaculates into GFEs/PFEs (clusters A, B).
Parameters in Step 3 Clusters
(240 min post-thaw) A B
Ejaculate number 2, 4, 5, 6, 8, 10 1, 3, 7, 9
Sperm progressive motility (%) 42.38 11.87
Sperm membrane integrity (%) 38.80 34.09
Final cluster centres (mean values) for the two clusters of ejaculates
obtained from non-hierarchical k-means cluster analysis on the 10
ejaculates in the study. Values correspond to the sperm progressive
motility and the sperm membrane integrity in Step 3 (240 min post-
thaw). Ejaculates in cluster B (1, 3, 7, and 9) were selected as the
PFEs (poor freezability ejaculates) because of their lowest mean
values in both parameters (fewer than 40%).
Table 2
Classification of ejaculates into GFEs/PFEs (clusters C, D).
Parameters in Step 3 Clusters
(240 min post-thaw) C D
Ejaculate number 4, 5, 6, 10 2, 8
Sperm progressive motility 46.33 34.38
Sperm membrane integrity (%) 42.03 32.35
Final cluster centres (mean values) for the two clusters of ejaculates
obtained from non-hierarchical k-means cluster analysis on the six
ejaculates in cluster A from Table 1. Values correspond to the sperm
progressive motility and the sperm membrane integrity in Step 3 (240
min post-thaw). Ejaculates in cluster C (4, 5, 6, and 10) were selected
as the GFEs (good freezability ejaculates) because of their highest
mean values in both parameters (over 40%).
sperm parameters were similar between GFEs and
PFEs except ALH in Step 1, which was significantly
higher in GFEs than in PFEs (P ⫽ 0.05). In Step 3,
differences significantly increased in many sperm qual-
ity parameters, all of them showing higher values in
GFEs than in PFEs: total motility, progressive motility,
circular trips, VCL, VSL, VAP, and BCF (P ⬍ 0.01),
ALH and membrane integrity (P ⬍ 0.05). Conversely,
no significant differences (P ⬎ 0.05) between GFEs
and PFEs were observed when evaluating LIN, STR,
WOB, and the percentage of non-reacted sperm (ORT)
in either of the assessed steps.
Since HSP90AA1 has been found into cytoplasmic
droplets and in tail foldings and coilings [13,27], signifi-
cant differences in morphology between GFEs and PFEs
had to be discarded because they could have interfered on
the comparison of the expression patterns. In this sense,
no significant differences were found in the percentage of
sperm without morphologic anomalies or with proximal
droplets between GFEs and PFEs in Step 1 (Table 3).
3.3. Western blot assay
The most representative protein patterns are shown
in Figs. 1, 2, and 3 together with graphics on the
normalized mean band volumes. Data represented on
graphics are resumed in Table 4.
No significant differences were observed between
GFEs and PFEs in normalized mean band volumes for
GLUT3 in any of the three steps (Fig. 1). Conversely,
those for HSP90AA1 were significantly lower in PFEs
than in GFEs in Step 1 (P ⫽ 0.05) and Step 2 (P ⬍
0.05). This protein did not show, however, differences
between groups in Step 3 (Fig. 2). Finally, the normal-
ized mean band volumes for Cu/ZnSOD only presented
significant differences (P ⬍ 0.01) between GFEs and
PFEs in Step 3, the former being lower than the latter
(Fig. 3).
 I. Casas et al. / Theriogenology 74 (2010) 940 –950 945

Table 3
Results from the sperm quality assessment.
Parameter Cryopreservation process
Step 1 (17 °C) Step 2 (5 °C) Step 3 (240 min post-
thaw)
Sperm motility
Total motility (%)
GFEs 97.53 ⫾ 0.66 99.58 ⫾ 0.06 †80.60 ⫾ 2.32
PFEs 96.43 ⫾ 0.91 99.01 ⫾ 0.38 30.96 ⫾ 6.60
Progressive motility (%)
GFEs 74.19 ⫾ 3.70 70.03 ⫾ 1.37 †46.33 ⫾ 1.09
PFEs 71.38 ⫾ 2.14 67.68 ⫾ 1.82 11.87 ⫾ 3.38
Circular trips (%)
GFEs 40.71 ⫾ 5.58 70.47 ⫾ 3.29 †40.83 ⫾ 2.85
PFEs 35.93 ⫾ 3.23 69.68 ⫾ 4.49 16.42 ⫾ 2.78
VCL (␮m s⫺1)
GFEs 55.90 ⫾ 3.47 93.07 ⫾ 8.30 †42.80 ⫾ 1.14
PFEs 49.48 ⫾ 2.40 77.49⫾11.02 29.86 ⫾ 0.54
VSL (␮m s⫺1)
GFEs 31.79 ⫾ 3.40 37.26 ⫾ 1.53 †25.15 ⫾ 1.10
PFEs 28.78 ⫾ 1.35 31.36 ⫾ 3.37 17.49 ⫾ 0.55
VAP (␮m s⫺1)
GFEs 44.07 ⫾ 3.53 60.88 ⫾ 3.58 †33.15 ⫾ 0.88
PFEs 40.10 ⫾ 2.04 51.18 ⫾ 6.50 22.98 ⫾ 0.65
LIN (VSL/VCL) (%)
GFEs 56.70 ⫾ 3.28 40.77 ⫾ 1.70 58.68 ⫾ 1.85
PFEs 58.28 ⫾ 1.25 41.22 ⫾ 2.25 58.38 ⫾ 0.78
STR (VSL/VAP) (%)
GFEs 71.81 ⫾ 2.39 61.46 ⫾ 1.16 75.69 ⫾ 1.42
PFEs 71.92 ⫾ 1.16 61.66 ⫾ 1.81 75.90 ⫾ 0.30
WOB (VAP/VCL) (%)
GFEs 78.73 ⫾ 2.01 66.12 ⫾ 1.75 77.48 ⫾ 1.20
PFEs 81.03 ⫾ 0.65 66.56 ⫾ 1.58 76.84 ⫾ 0.77
ALH (␮m)
GFEs *2.32 ⫾ 0.07 3.94 ⫾ 0.35 *2.19 ⫾ 0.07
PFEs 2.09 ⫾ 0.06 3.45 ⫾ 0.37 1.94 ⫾ 0.02
BCF (Hz)
GFEs 6.67 ⫾ 0.24 6.37 ⫾ 0.15 †6.17 ⫾ 0.07
PFEs 6.68 ⫾ 0.22 6.09 ⫾ 0.24 5.88 ⫾ 0.03
Sperm membrane integrity (%)
GFEs 90.59 ⫾ 1.50 81.55 ⫾ 1.98 *42.03 ⫾ 0.78
PFEs 92.15 ⫾ 0.51 83.48 ⫾ 2.12 34.09 ⫾ 2.35
Sperm without morphologic anomalies (%)
GFEs 90.80 ⫾ 2.95 — —
PFEs 88.33 ⫾ 4.20
Proximal droplets (%)
GFEs 4.48 ⫾ 2.31 — —
PFEs 9.18 ⫾ 4.57
Non-reacted sperm (%) (ORT)
GFEs 95.38 ⫾ 0.66 — 89.88 ⫾ 2.03
PFEs 94.00 ⫾ 1.37 — 88.75 ⫾ 1.45
Sperm quality assessment values (mean ⫾ SEM) of the quality parameters analyzed on the eight ejaculates in the western blot assay. Data is
displayed for the four GFEs (good freezability ejaculates) and the four PFEs (poor freezability ejaculates) in cryopreservation Step 1 (refrigerated
semen, 24h at 17 °C after collection), Step 2 (30 min after adding glycerol at 5 °C) and Step 3 (240 min post-thaw). Values with superscripts are
significantly different between GFEs and PFEs for the parameter in the same step (* P ⱕ 0.05 † P ⬍ 0.01).
946 I. Casas et al. / Theriogenology 74 (2010) 940 –950

Fig. 1. Comparison of GLUT3 expression patterns between GFEs and PFEs (see Table 4). Images on the right show the most representative protein
patterns from triplicate experiment.

Although statistical significant differences were 4. Discussion

not observed, the normalized mean band volumes of
GLUT3 and Cu/ZnSOD in Step 1 tended to be higher in
PFEs than in GFEs.
Finally, graphics showed an apparent decrease of the
expression of the three proteins from the start to the end
of cryopreservation.
Quality parameters of FT sperm in this study con-
firm that even under equal cryopreservation conditions,
the degree of alterations produced by cold-shock is not
the same in sperm coming from different ejaculates, as
previously discussed by Roca et al [5] and Gutiérrez-

Fig. 2. Comparison of HSP90AA1 expression patterns between GFEs and PFEs (see Table 4). Images on the right show the most representative
protein patterns from triplicate experiment.
 I. Casas et al. / Theriogenology 74 (2010) 940 –950 947

Fig. 3. Comparison of Cu/ZnSOD expression patterns between GFEs and PFEs (see Table 4). Images on the right show the most representative
protein patterns from triplicate experiment.

Pérez et al [28]. Just from refrigerated semen, detailed parameters display, in refrigerated semen, lower values
comparison of sperm conventional parameters between on ejaculates with poor freezability.
GFEs and PFEs disclosed the presence of singularities Differences in motility parameters on refrigerated
in both groups. A significant decrease in ALH mean semen suggest that GFEs and PFEs have particularities
value in PFEs was detected in the present study and that would determine their resistance to freezing. The
Flores et al [16] similarly noted that some motility research for highly-reliable freezability predictors re-
quires an understanding of the nature of such particu-
larities. Recent studies on refrigerated semen from
Table 4 stallions point to divergences in the mitochondrial
Results from the western blot assay. membrane potential, among ejaculates with different
Protein patterns Normalized mean band volumes freezability, as one of these singular features [29].
(intensity ⫻ mm2) Despite the fact that the feature mentioned above has
GFEs PFEs not been assayed on refrigerated semen in boars, it is
GLUT3 Step 1 1.71 ⫾ 0.24 1.84 ⫾ 0.34 known that boar PFEs have lower mitochondrial activ-
GLUT3 Step 2 1.32 ⫾ 0.22 1.29 ⫾ 0.37 ity than GFEs in FT sperm [16] and, moreover, that the
GLUT3 Step 3 0.26 ⫾ 0.03 0.26 ⫾ 0.05
Cu/ZnSOD enzyme could be implicated in this fact.
HSP90AA1 Step 1 *3.09 ⫾ 0.41 2.10 ⫾ 0.26
HSP90AA1 Step 2 *0.66 ⫾ 0.13 0.33 ⫾ 0.07 Cerolini et al [30] found higher activity of the enzyme
HSP90AA1 Step 3 0.96 ⫾ 0.13 0.96 ⫾ 0.13 in low viability boar FT sperm samples, which agrees
Cu/ZnSOD Step 1 6.60 ⫾ 0.75 7.61 ⫾ 0.67 with the highest expression of the Cu/ZnSOD antioxi-
Cu/ZnSOD Step 2 0.64 ⫾ 0.06 0.52 ⫾ 0.06 dant enzyme in PFEs after cryopreservation, as re-
Cu/ZnSOD Step 3 **0.99 ⫾ 0.05 1.52 ⫾ 0.14
trieved from the present study. For that reason the
Comparison by means of normalized band volumes (mean ⫾ SEM) enzyme could be a useful marker of freezing damage in
for the nine different protein patterns in the western blot assay,
measured in pixel intensity units ⫻ mm2. Mean values are displayed
boar sperm.
in each pattern for the four GFEs (good freezability ejaculates; During cryopreservation of mammalian sperm, the
ejaculates number 4, 5, 6, and 10) and the four PFEs (poor freezabil- Cu/ZnSOD counteracts the oxidative stress coming
ity ejaculates; ejaculates number 1, 3, 7, and 9) in three steps during from uncontrolled release of reactive oxygen species
the cryopreservation protocol: Step 1 (refrigerated semen, 24h at (ROS) from damaged mitochondria to cytosol [31–35],
17 °C after collection), Step 2 (30 min after adding glycerol at 5 °C),
and Step 3 (sperm at 240 min post-thaw). Those patterns with an
so the expression of the enzyme after freezing indicates
asterisk are significantly different between GFEs and PFEs (* P ⱕ higher oxidative stress in PFEs than in GFEs. The
0.05; ** P ⬍ 0.01). higher sperm hyperactivation reported in PFEs [13]
948 I. Casas et al. / Theriogenology 74 (2010) 940 –950
could be also induced by ROS, as it occurs in capaci-
tation both in humans [36 –39] and in pigs [40,41]. In
spite of this, some authors assert that the intense ex-
pression of Cu/ZnSOD in poor viability boar sperm
samples could also be linked to a lower efficiency of the
enzyme in those samples compared to the good viabil-
ity samples [16,41,42].
The oxidative stress provokes lower sperm motility
and membrane integrity in PFEs than in GFEs, as
observed for post-thawing outcomes. Even so, the real
difference in the percentage of total motility between
both groups tends to be artificially increased in com-
puterized assessments because the software can con-
found dead sperm with non-progressive motile sperm
due to currents in the fluid where sperm is suspended.
As a result, total motility is often higher than membrane
integrity. This effect worsens after freezing and in
GFEs, because there is a large population of motile
sperm that provokes these currents, and because there is
more dead sperm to be affected by them than there is in
refrigerated semen. Because the sperm progressive mo-
tility is, in relation with total motility, closer to the
values of membrane integrity, it results a more reliable
parameter for classifying the freezability of ejaculates
[13].
In refrigerated semen, the expression of Cu/ZnSOD
and GLUT3 did not differ significantly between GFEs
and PFEs, which compromises their role in freezability
prediction. In spite of being non-significant, the PFEs
tended to higher expression of both proteins. The lack
of consistency in reporting such tendencies could be
attributed to variations observed in expression levels
among ejaculates within a freezability group, which
would require increasing the number of samples in
future studies. Although GLUT3 and Cu/ZnSOD were
only mediocre markers of the ejaculate freezability, the
tendency mentioned above could reflect differential
seminal plasma composition between GFEs and PFEs,
according to Hernández et al [43]. Additionally, given
the roles of both proteins, it could be linked to liquid
storage sensitivity, because cooling boar sperm reduces
the ATP levels and increases the oxidative stress [44].
The most important finding of the present study was
the higher expression of the HSP90AA1 protein in
GFEs before freezing and at the 5 °C during freezing,
suggesting that it interferes in the future response of
ejaculates in front of cold-shock. The HSP90AA1 is an
ubiquitous molecular chaperone that provides resis-
tance against cell oxidative stress [45] and apoptosis
[46], and mediates cell protection and protein folding
during thermal stress, among many other functions
[47,48]. For example, HSP90AA1 may also take part in
repairing changes in protamine-DNA complexes, and
hence in the chromatin structure, that are produced
during boar sperm cryopreservation [49].
Linking the protective roles of HSP90AA1 with its
lower expression in refrigerated semen in PFEs may
explain why they have less homogeneous chromatin
than GFEs in FT sperm [50]. Moreover, the PFEs are
particularly susceptible to show motility alterations in
response to cryopreservation, in special reference to
their hyperactivity [13]. Given the amplitude of works
carried by HSP90AA1, it is reasonable to suspect it
could additionally be involved in regulating motility
alterations in boar sperm during freezing, as previously
discussed [13]. Motility functions could be affected
either by the decrease in ATP that is concomitant with
lower mitochondrial potential [34] or because of struc-
tural damage in the contractile apparatus of the flagel-
lum [42].
Despite the fact that the HSP90AA1 protein pro-
vides a safeguard of multiple sperm functions, the ap-
parent decrease of its expression from the start to the
end of cryopreservation suggests that some amount of
the protein may be leaked from the sperm cytosol to the
extracellular medium. This is probably the consequence
of structural damage on sperm irrespective of the freez-
ability of the ejaculate, as it has been discussed in other
assays [13,47]. This loss similarly occurs to GLUT3
and Cu/ZnSOD, and it is mentioned by Sancho et al
[51] and Lasso et al [32], respectively.
In conclusion, the poorer expression of HSP90AA1
in boar sperm at 17 and 5 °C during cryopreservation
and the higher expression of Cu/ZnSOD after freezing,
as found in PFEs compared to GFEs, seem to be related
to the higher sensitivity of PFEs to cold-shock.
Whereas the subtle differences between GFEs and
PFEs concerning GLUT3 and Cu/ZnSOD expression
compromise the future development of both proteins as
freezability predictors, the HSP90AA1 may be most
likely confirmed as a cold-shock molecular marker in
boar refrigerated semen.
These results indicate a need for searching cold-
shock markers to develop freezability prediction tests
for ejaculates in refrigerated semen, which would have
a high-impact on boar sperm cryopreservation.
Declarations of interest
The authors declare that there is no conflict of in-
terest that would prejudice the impartiality of this sci-
entific work.
 I. Casas et al. / Theriogenology 74 (2010) 940 –950
Funding
This research work was supported by the Spanish
Government institute INIA (grant reference RZ-2008-
0001-00-00) and the MICINN ministry (grant refer-
ences TRACE-PET-2008-0043, AGL 2009-11904). It
was also funded by a research grant awarded to I.C.
from the Autonomous Government of Catalonia and the
European Social Fund.
Acknowledgements
The authors thank Selecció Batallé SA and
BioGirona SA for providing the ejaculates, and M. Puig-
mulé and E. Garcia for kind technical and scientific sup-
port.
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