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Theriogenology 73 (2010) 1267–1275

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Effects of different cryoprotective agents on ram

sperm morphology and DNAintegrity
Z. Nur a,*, B. Zik b, B. Ustuner a, H. Sagirkaya a, C.G. Ozguden b
a
Department of Reproduction and Artificial Insemination, Uludag University, Veterinary Faculty, Turkey
b
Department of Histology and Embryology, Uludag University Veterinary Faculty, Bursa, Turkey
Received 31 July 2009; received in revised form 28 November 2009; accepted 27 December 2009

Abstract
This study investigates the effects of glycerol, 1,2 propanediol, sucrose, and trehalose on post-thaw motility, morphology, and
genome integrity of Awassi ram semen. Ejaculates of thick consistency with rapid wave motion (>+++) and >70% initial motility were
pooled. Sperm were diluted to a final concentration of 1/5 (semen/extender) in 0% cryoprotectant, 6% glycerol, 6% 1,2 propanediol,
62.5 mM sucrose or 62.5 mM trehalose using a two-step dilution method. The equilibrated semen was frozen in 0.25-ml straws. Semen
samples were examined for sperm motility, defective acrosomes (FITC-Pisum sativum agglutinin (FITC PSA)), DNA integrity
(acridine orange staining (AO)) and apoptotic activity (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
(TUNEL) and Caspase-3 activity) at four time points: after dilution with extender A, after cooling to 5 8C, after equilibration and post-
thaw. Freezing and thawing procedures (cooling at 5 8C, dilution, equilibration, and thawing) had negative effects on motility
(P < 0.001), acrosome integrity (P < 0.001), and DNA integrity as determined by AO (P < 0.001) and TUNEL (P < 0.001) assays.
There were positive correlations between sperm with defective acrosomes and apoptotic (AO- and TUNEL-positive) spermatozoa. In
contrast, a significant negative correlation was found between sperm motility and defective acrosomes and AO- and TUNEL positivity
(P < 0.01). The cryopreservation process acts as an apoptotic inducer in ram semen; all cryoprotectants used in the present study
allowed apoptosis to some extent, with negative effects on sperm morphology and DNA integrity. The glycerol group performed better
than the propanediol, sucrose, trehalose, and control groups in terms of post-thaw sperm motility but not DNA integrity.
# 2010 Elsevier Inc. All rights reserved.

Keywords: Ram semen; Cryopreservation; Cryoprotective agent; DNA integrity

1. Introduction maturity of the cell [6], the cryoprotectant used [7–9],

During the freeze-thawing process, mammalian
sperm are exposed to temperature changes that lead
to physical and chemical stress, changes in the plasma
membrane lipid composition [1,2], reduced head size
[3], and externalization of phosphatidylserine residues
[4,5]. These sperm alterations are dependent on the
* Corresponding author. Tel./fax.: 90 224 2941345.
E-mail address: nurzek@uludag.edu.tr (Z. Nur).
and the cooling and freeze-thawing rates [4,10–13].
Although several researchers have developed dif-
ferent extenders and protocols for freezing ram semen,
in general, fertility results are not comparable to those
obtained with fresh semen and natural mating [13–15].
These reductions in fertilization capacity have typically
been attributed to a reduced rate of sperm motility and
freeze-thaw-induced morphological [1,13,15,16] and
genomic [16,17] abnormalities.
The process of fertilization involves complex
biochemical and physiological events that cannot be
measured solely by routine semen evaluation. The

0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2009.12.007
1268 Z. Nur et al. / Theriogenology 73 (2010) 1267–1275
traditional evaluation of ejaculate quality has been
based primarily on routine semen analyses (i.e.,
motility, morphology, and acrosomal integrity), but
these routine semen evaluations have a limited capacity
for predicting the potential fertility of ejaculate [18].
Therefore, advanced techniques for semen evaluation
(e.g., in vitro fertilization, cervical mucus penetration,
DNA, and plasma membrane integrity) should be
implemented to increase the odds of accurate identi-
fication of high-quality sperm [19–21]. Several tech-
niques have been proposed to study sperm DNA
abnormalities [22]. Those in current use are the TUNEL
technique, which allows for the evaluation of sperm
DNA fragmentation [23,24]; the Comet technique,
which is another means of evaluating DNA integrity
[16,41]; caspase activity assays [5,21,25]; Annexin V
staining, which provides information regarding the
translocation of phosphatidylserine (PS) [22]; and DNA
staining by acridine orange (AO), which differentiates
between single- and double-stranded DNA based on
their respective colors under fluorescence [10,22].
Although many studies have examined the effects of
cryoprotectants on the outcomes of routine spermato-
logical evaluations, few studies have focused on the
effects of cryoprotectants on apoptotic manifestations in
mammalian semen; most of the studies that have been
conducted have shown that cryopreservation is asso-
ciated with apoptotic induction in human [5,10] and bull
[17] semen. This study aims to explore the effects of the
inclusion of various cryoprotectants (glycerol, 1,2
propanediol, sucrose and trehalose) in the Tes-Tris
(TEST)-based extender on the morphology, DNA
integrity, and apoptotic activity of freeze-thawed
Awassi ram semen.
2. Materials and methods
2.1. Chemicals
THAM (Tris(hydroxymethyl)aminomethane), Dex-
tran B (150000-200000), glycerol, 1,2 propanediol,
sucrose, trehalose, AO, PBS tablets, and poly-L-lysine
were purchased from Sigma (Sigma Chemical Co., St.
Louis, MO, USA). Triton X-100 (10% stock solution)
(11332481001) and an In Situ Cell Death Detection Kit
were purchased from Roche (Roche Diagnostics
GmbH, Mannheim, Germany). Proteinase K (003011)
and antibody diluents were purchased from Zymed
(Zymed, San Francisco, California, USA). Rabbit
polyclonal active Caspase–3 antibody (235412) was
purchased from Calbiochem (Calbiochem, La Jolla,
CA, USA). Bovine anti-rabbit fluorescein (FITC) (Sc:
2365) and mounting medium (Sc: 24941) were
purchased from Santa Cruz Biotechnology Inc. (Santa
Cruz, CA, USA). All other chemicals were purchased
from Merck (Merck & Co., Inc., Darmstadt, Germany).
2.2. Semen collection and cryopreservation
Ten Awassi rams that were 3–5 yr of age and
maintained at Uludag University, Faculty of Veterinary
Medicine in Bursa, Turkey, were used during the
breeding season. Semen was collected by electrically
stimulated ejaculation (Ruakura Ram Probe Plastic
Products, Hamilton, New Zealand, probe length 12 cm,
diameter 2.5 cm, 12 V) five times with a one-day inter-
semen collection interval [7,26]. To collect semen, rams
were restrained physically and a lubricated probe was
inserted into the rectum with downward pressure on the
front of the probe, so the electrodes rested on the upper
portion of the ampullary region. An electrical stimula-
tion was applied for 4–8 s. The electrostimulation was
stopped briefly (3–4 s) while further massage was
applied with the probe. This cycle was repeated until a
1–2 ml sample of semen was collected (usually 3–4
electrostimulations). Collected semen was placed in a
warm water bath (30 8C) and immediately evaluated for
consistency, wave motion (0–5 scale), and percentage of
motile spermatozoa (0–100%) [7]. Ejaculates with a
thick consistency, rapid wave motion (2–5 on a 0–5
scale), and >70% initial motility were pooled. Pooled
semen were then diluted with a TRIS-based extender
(20% egg yolk (v/v)) to a final concentration of 1/5
(semen/extender) in 0% cryoprotectant, 6% glycerol,
6% 1,2 propanediol, 62.5 mM sucrose or 62.5 mM
trehalose using a two-step dilution method [7]. Briefly,
pooled ejaculates were diluted to a ratio of 1:2 (semen/
extender) with extender A (no cryoprotectant, 375
mOsm) and cooled to 5 8C within 2 h. The cooled
semen was then divided into five groups and diluted to a
ratio of 1:1 (semen/extender) with extender B (400
mOsm) with one of four cryoprotectants (6% glycerol,
6% 1,2-propanediol, 62.5 mM sucrose or 62.5 mM
trehalose). In addition, the control group was diluted
with extender B with no cryoprotective agent. Extender
B was added in five steps at 5 min intervals and
equilibrated at 5 8C for 2 h. The osmotic pressure of the
extenders was determined using a freezing point
depression osmometer (Advanced 3D3 Single Sample
Osmometer, Advanced Instrument, Inc., Norwood, MA,
USA) before adding cryoprotectants and egg yolk to the
extender. Equilibrated semen samples were placed into
0.25 ml straws and frozen at 3 8C/min from +5 8C to -
8 8C and at 25 8C/min from -8 8C to -120 8C in liquid
 Z. Nur et al. / Theriogenology 73 (2010) 1267–1275
nitrogen vapor using the Nicool Plus PC freezing
machine (Air Liquide, Marne-la-Vallée Cedex 3,
France). They were then plunged into liquid nitrogen
at -196 oC, where they were stored for at least one
month. At least three straws from each group of pooled
ejaculates were thawed at 37 8C for 30 s in a water bath
to evaluate post-thaw semen characteristics.
2.3. Semen evaluation
All semen parameters were assessed at the following
four time points: after dilution with extender A, after
cooling at 5 8C, after equilibration, and after thawing.
All semen was frozen by the same person, and each of
the studied semen parameters was measured by the
same person on each occasion throughout the study.
Sperm motility was assessed subjectively using a
phase-contrast microscope (400x) with a warm slide
(38 8C) [7].
2.3.1. Fluorescein lectin staining assay (FITC-
conjugated Pisum sativum agglutinin (FITC PSA))
Acrosome integrity was assessed using FITC-
conjugated Pisum sativum agglutinin [27]. Briefly,
20 ml of diluted semen was re-suspended in 500 ml PBS
and centrifuged at 2000 rpm for 20 min; the supernatant
was then discarded. The spermatozoa pellet was re-
suspended in 250 ml PBS. One drop of resuspended
spermatozoa was smeared on a glass microscope slide
and air dried. Air-dried slides were fixed with acetone at
4 8C for 10 min, and the slides were covered with FITC
PSA solution (50 mg/ml in PBS solution) in the dark for
30 min. Stained slides were rinsed with PBS solution,
covered with glycerol, and examined under a fluores-
cence microscope. At least 100 spermatozoa per smear
were evaluated for acrosome integrity.
2.3.2. Acridine-Orange staining assay
Sperm DNA integrity was assessed using the AO
fluorescence method [10]. Air-dried slides were fixed
overnight in freshly prepared Carney’s solution (three
parts methanol and one part glacial acetic acid) and
allowed to air dry for a few minutes. Dried slides were
stained for 3 min with AO. The stained slides were
evaluated immediately under a fluorescence micro-
scope. Normal DNA content showed green fluorescence
over the head region, while DNA abnormalities showed
varying fluorescence (from yellow-green to red). At
least 100 spermatozoa per smear were evaluated for
DNA abnormalities (10). Sperm cells with changes in
fluorescence from yellow-green to red were recorded as
sperm with abnormal DNA content.
1269
2.3.3. TUNEL assay
For the TUNEL technique, we used the In Situ Cell
Death Detection Kit with fluorescein (Roche Diagnos-
tics GmbH, Mannheim, Germany) according to the
manufacturer’s protocol with slight modifications. In
brief, one drop of re-suspended spermatozoa was
smeared on a glass slide and fixed with 10%
formaldehyde for 20 min at room temperature. The
slides were washed in PBS and stored at 4 8C. Upon
removal from storage, samples were washed again in
PBS (three times for 5 min each). They were then
treated in a humidified chamber with proteinase K for
10 min at room temperature, washed with PBS, treated
with 3% H2O2 in distilled water for 10 min at room
temperature and washed again with PBS. The slides
were permeabilized with 0.1% Triton X-100 for 5 min
on ice.
The permeabilized slides were incubated in the dark
at 37 8C for 1 h with the TUNEL reaction mixture,
which contained terminal deoxynucleotidyl transferase
(TdT) plus dUTP label. After labeling, samples were
washed with PBS and analyzed immediately via
fluorescence microscopy. Negative (omitting TdT from
the reaction mixture) and positive (using DNase I, 1 mg/
ml, for 10 min at room temperature) controls were
included in each trial. At least 100 sperm were
evaluated to determine the percentage of TUNEL-
positive sperm. Each microscopic field was evaluated
first under fluorescence microscopy (40 magnifica-
tion) to determine the number of reactive sperm and
then under phase-contrast microscopy to determine the
total number of sperm per field.
2.3.4. Immunocytochemistry assay
A rabbit polyclonal active Caspase–3 antibody was
used to detect active caspase in ram semen, as described
by Marti et al. [25] with some modifications. Briefly,
20 ml of diluted semen was resuspended in 1000 ml PBS
and centrifuged at 2000 rpm for 20 min; the supernatant
was then discarded. The semen pellet was permeabi-
lized and fixed with 1000 ml of an ethanol:acetic acid
(3:1) solution at room temperature for 15 min. One drop
of re-suspended spermatozoa was smeared on a poly-L-
lysine slide, and the slides were allowed to air dry. After
washing three times with PBS, slides were incubated
with PBS containing 5% bovine serum albumin (BSA)
for 45 min at 37 8C to block non-specific sites.
After washing with PBS, the slides were incubated
first with a primary antibody (1/50 dilution) for 1 hour at
37 8C and then with a bovine anti-rabbit fluorescein-
labeled IgG secondary antibody (Santa Cruz, 1/400
dilution) for 90 min; both antibodies were diluted with
1270 Z. Nur et al. / Theriogenology 73 (2010) 1267–1275

antibody buffer (antibody diluents). Slides were washed 3. Results

and desiccated in the dark at room temperature. After
adding one drop of mounting medium, the samples were
covered with a cover slip. As controls for primary
antibody specificity, all immunofluorescence labeling
experiments were also carried out with no primary
antibodies.
All fluorescence examinations were performed with
an epifluorescence microscope (BX51, Olympus Inc.,
Japan) with a multiple fluorescence filter (U-DM-DA/
FI/TX2) at 460–490 nm excitation and 530 nm barriers.
At least 100 spermatozoa were examined per slide
(100 objective).
2.4. Statistical analyses
Data were analyzed by analysis of variance
(ANOVA) using the General Linear Model (GLM)
procedure. The model included fixed effects of
cryoprotectants and random effects of pooled ejacu-
lates (replicates). Means of obtained semen parameters
were analyzed using Tukey’s test. Repeated measures
ANOVA (using GLM procedures) were conducted to
compare the results at different stages of the
cryopreservation process. Spearman’s correlation
coefficient was used to assess the relationship between
semen motility, defective acrosomes and apoptotic
spermatozoa (AO- and TUNEL-positive). All data were
analyzed using the SPSS statistical package (SPSS 10.0
for Windows; SPSS, Chicago, IL, USA). Differences
were considered significant at P-values of less than
0.05.
Table 1 shows the differences in the percentages of
motility, defective acrosomes (FITC PSA), and AO,
TUNEL, and Caspase-3 activities of the diluted, cooled
at 5 8C, equilibrated, and thawed ram semen from the
different cryoprotectant groups.
Sperm motility was progressively reduced by cool-
ing and the freeze-thaw process (Table 1; P < 0.05).
The motility values of equilibrated semen in the sucrose
and trehalose groups were significantly lower than those
of the glycerol, 1,2 propanediol, and control groups
(P < 0.05). Post-thaw semen motility values in the
glycerol and 1,2 propanediol groups were better than
semen motility values in the sucrose, trehalose and
control groups (P < 0.05).
Acrosome integrity was assessed using FITC PSA
(Fig. 1). Acrosome integrity was negatively affected by
the freeze-thaw process. Post-thaw defective acrosome
rates were higher than those of diluted, cooled and
equilibrated spermatozoa (P < 0.05). The percentage of
defective acrosomes in the equilibrated and thawed
spermatozoa was not affected by any of the cryopro-
tectants, as compared to the control group (P < 0.05).
As shown in Fig. 1, AO-stained ram spermatozoa
with undamaged DNA showed green fluorescence,
while damaged DNA displayed a spectrum of yellow-
orange to red fluorescence. Post-thaw semen demon-
strated the highest rate of damaged DNA (assessed via
acridine orange staining) and showed significantly more
damage than the diluted, cooled to 5 8C and equilibrated
semen (P < 0.05) Post-thaw sperm chromatin integrity

Table 1
The mean (x̄  Sx̄) of studied sperm parameters in the function of cryoprotectant.
Stage Cryoprotectant n Motility Defected Acrosome Apoptotic spermatozoa
(%) x̄  Sx̄ FITC-PSA (%) x̄  Sx̄
AO (%) Tunnel (%) Caspase-3
x̄  Sx̄ x̄  Sx̄ (%) x̄  Sx̄
A
After dilution - 5 79.0  1.9 5.6  0.8 1.4  0.5 1.7  0.6 3.6  1.3
A
At 5 8C - 5 67.0  2.0 9.0  1.6 1.0  0.6 3.8  1.0 1.0  0.3
Equilibrated Glycerol 5 60.0  2.2 a 12.9  0.8 a 1.2  0.4 ab
2.0  0.6 a
0.6  0.2 a

1,2 Propanediol 5 57.0  2.0 ab 10.2  0.9 a 0.6  0.4 a

0.9  0.3 a
0.2  0.2 a

Sucrose 5 48.0  1.2 c 11.2  2.2 a 2.0  0.5 b

2.6  1.2 a
0.8  0.4 a

Trehalose 5 53.0  1.2 b 9.5  2.5 a 0.8  0.4 a

2.1  1.0 a
0.8  0.4 a

Control 5 53.0  1.2 b 9.9  0.8 a 1.0  0.3 ab

1.2  0.6 a
0.4  0.2 a

Post-thaw Glycerol 15 51.2  1.9 a 59.7  3.3 a 5.7  0.7 a

6.9  0.8 a
NO
1,2 Propanediol 15 35.0  3.0 a 52.9  3.8 a 4.5  0.6 a
6.1  0.8 ab
NO
Sucrose 15 5.1  1.2 b 53.4  2.8 a 4.8  0.5 a
5.9  1.3 ab
NO
Trehalose 15 8.0  1.6 b 54.3  2.9 a 5.3  0.8 a
6.6  0.6 ab
NO
Control 15 3.7  0.8 b 55.2  2.8 a 4.5  0.6 a
4.4  0.6 b
NO
a,.b,.c: Values with different superscripts in the same column for same stage are significantly different (P<0.05).
FITC-PSA: FITC- conjugated Pisum Sativum Agglutinin, AO: Acridine Orange, NO: Not observed
 Z. Nur et al. / Theriogenology 73 (2010) 1267–1275 1271

values of cryoprotectant supplemented groups were
similar to control group values (P > 0.05).
The results of the TUNEL assay demonstrated that a
large proportion of spermatozoa with DNA fragmenta-
tion exhibited green fluorescence (Fig. 1). This was not
the case for undamaged spermatozoa. The post-thaw
percentage of apoptotic spermatozoa was 6.0%, which
was significantly different from the diluted, cooled, and
equilibrated semen (P < 0.05). All cryoprotectants used
in this study triggered some degree of apoptosis. When
comparing the cryoprotectants and control groups, the
mean numbers of TUNEL-positive spermatozoa were
not affected by cryoprotectant type in equilibrated
semen. Also, post-thaw TUNEL-positive spermatozoa
were not affected by cryoprotectant type, with the
exception of the glycerolized groups (P < 0.05).
Three different sperm labeling patterns were found
in diluted semen after Caspase-3 immunostaining
assays, with localization in the apical, equatorial and
tail regions (Fig. 1). Caspase activity, as assessed by

Fig. 1. Representative patterns of ram sperm as observed with FITS-PSA, AO, TUNEL and Caspase-3 staining.
Row 1: FITC-PSA stained spermatozoa with intact and defected acrosome (X40).
Row 2: A intact, B defected DNA after AO staining (X40).
Row 3: A: DNA’se applied slide, B: Apoptotic spermatozoa under fluorescence microscopy, and C: View of B under phase contrast microscopy
(X40).
Row 4: Immunocytochemistry detection of Caspase-3 in fresh ram semen. A: Intact and apoptotic spermatozoa, B: Apoptotic spermatozoa (X100).
DA: Defected Acrosome, IA: Intact Acrosome, AP: Apoptotic Spermatozoa, IS: Intact Spermatozoa.
1272 Z. Nur et al. / Theriogenology 73 (2010) 1267–1275

Table 2
Effect of freezing protocol, cryoprotectant on post-thaw semen parameters.
DF Motility (%) Defected Acrosome Apoptotic Spermatozoa
FITC-PSA (%)
AO (%) Tunnel (%) Caspase-3 (%)
Stage 1 *** *** *** *** ***
Cryoprotectant 4 *** NS NS NS *
Stage*criyoprotectant 4 *** NS NS NS *
*p < 0.05; *** P < 0.001; NS Not Significant.
FITC-PSA: FITC- conjugated Pisum Sativum Agglutinin; AO: Acridine Orange.

Table 3 semen characteristics and DNA integrity of spermato-

Correlation coefficient (r) between the results of studied semen
parameters.
Defected Apoptotic spermatozoa
acrosome
PSA (%) AO (%)
Motility (%) 0. 617* 0.438*
Defected acrosome PSA (%) 0.663*
Apoptotic spermatozoa (AO)
*Correlation is significant at the (P < 0.01).
FITC-PSA: FITC- conjugated Pisum Sativum Agglutinin; AO: Acri-
dine Orange.
immunostaining, decreased in cooled and equilibrated
semen. However, none of the post-thawed spermatozoa
showed positive caspase activity.
Freezing and thawing procedures (cooling, dilution,
equilibration, and thawing) had negative effects on
motility (P < 0.001), defective acrosome FITC PSA
(P < 0.001), and apoptotic cell ratios, as determined by
AO (P < 0.001) and TUNEL (P < 0.001) assays. There
was a significant interaction between the time point and
cryoprotectant use for sperm motility (P < 0.001) and
Caspase-3 activity (P < 0.05) (Table 2).
The results of the Pearson correlation tests are shown
in Table 3. A significant negative correlation was found
between sperm motility and defective acrosomes, AO-
and TUNEL-positivity (P < 0.01). In addition, there
were positive correlations between defective acrosomes
and apoptotic (AO- and TUNEL-positive) spermatozoa.
4. Discussion
The freeze-thaw process is detrimental to mamma-
lian sperm viability, DNA, and functional integrity
[5,7,17]. More specifically, ram semen has proven to be
more difficult to cryopreserve than semen of other farm
animals [28,29]. Various extenders and cryoprotective
agents have been developed for the cryopreservation of
ram semen [7,28–30]. In the present study, we evaluated
the effects of different cryoprotectants on post-thaw
zoa frozen in a TRIS-based extender.
The mean percentages of sperm motility, defective
acrosomes, and apoptotic spermatozoa, as measured by
AO, TUNEL, and Caspase-3, in the diluted ejaculates
Tunnel (%)
were 79.0%, 5.6%, 1.4%, 1.7%, and 3.6%, respectively.
0.302* These data are in agreement with previous reports
0.500* concerning semen motility, defective acrosomes, and
0.490*
DNA integrity in rams [7,25,31].
Despite advances in the cryopreservation of mam-
malian spermatozoa, there has been less success with
ram spermatozoa than with bull spermatozoa [26].
Cryopreservation of spermatozoa reduces motility
[7,28,29,32]. In addition, acrosome [7,10,28,29,32],
plasma membrane [1,7] and DNA [10,25,32] integrities
are affected. The results of the present study show that
freeze-thawing procedures (cooling to 5 8C, dilution,
equilibration, and thawing) negatively affect motility
(P < 0.001). The rates of defective acrosomes
(P < 0.001) and apoptotic cell ratios, as determined
by AO (P < 0.001) and TUNEL (P < 0.001), were also
negatively affected.
The beneficial effect of cryoprotectant-supplemen-
ted media on post-thawed mammalian semen has been
reported in many studies [7,9,13,28–30]. Although
glycerol is the first and most commonly used
cryoprotectant for semen freezing media [15,33], the
presence of glycerol lowers the quality and fertilizing
capacity of semen [13,28,29].
In the present study, the motilities of equilibrated
semen in the sucrose and trehalose groups were
significantly lower than those of the glycerol, 1,2
propanediol, and control groups (P < 0.05). For post-
thaw semen motility, glycerol- and 1,2 propanediol-
containing groups had better values than the sucrose,
trehalose and cryoprotectant-free groups (P < 0.05).
Soylu et al. [7] reported that media containing glycerol
and 1,2-propanediol protect post-thaw motility effec-
tively when compared to sucrose, trehalose, and control
groups. The presence of high concentrations of
trehalose in freezing media improves post-thaw semen
 Z. Nur et al. / Theriogenology 73 (2010) 1267–1275
characteristics, but in low concentrations, the agent
yields little improvement [34,35]. Woelders et al. [8]
compared two different sugars (2 M trehalose and 2 M
sucrose) in extenders with high osmolality and found
that sucrose performed significantly better than treha-
lose at the highest cooling rate for post-thaw bull semen.
Extender composition assists in stabilizing cells
during the freezing and thawing process [5,7]. Post-
thaw sperm recovery was significantly better when
sperm were frozen in hypertonic extender (400 mOsm)
compared to low osmolality (350 and 375 mOsm) [7].
The osmolality of Extender B in the present study was
400 mOsm. Post-thaw sperm motility was similar to the
results reported by Soylu et al. [7] for glycerol- and 1,2
propanediol-containing groups and the cryoprotectant-
free group frozen with an extender at 400 mOsm.
Cryopreservation induces premature capacitation or
acrosome reactions [36]. In our study, acrosome
integrity was affected by the freeze-thaw process.
Post-thaw defective acrosome rates were higher than
those of diluted, cooled and equilibrated spermatozoa.
The presence of glycerol and 1,2-propenendiol in the
semen freezing extender reduces acrosomal and
morphological integrity [28,29]. The percentage of
defective acrosomes of equilibrated and thawed
spermatozoa were not affected by the type of
cryoprotectant used compared to the control group
(P < 0.05). Soylu et al. [7], using Giemsa stain for
acrosome evaluation and media with different osmol-
ality, reported that media containing glycerol and 1,2
propanediol did not protect post-thaw acrosome
integrity compared to sucrose, trehalose, and control
samples.
TUNEL and AO assays revealed that the freeze-
thawing procedures also caused deterioration of DNA
integrity. For TUNEL and AO staining, chromatin
injury was the highest in post-thaw semen and differed
significantly from diluted, cooled to 5 8C, and
equilibrated spermatozoa (P < 0.05). All cryoprotec-
tants used in this study triggered apoptosis to some
degree (P > 0.05). For post-thaw semen, a comparison
across the various groups showed that the mean values
of TUNEL-positive spermatozoa were not affected by
cryoprotectant type, with the exception of the glycer-
olized group.
Many studies have been carried out by varying the
amount of glycerol used, the timing protocol for adding
glycerol to extenders and the exclusion of glycerol from
freezing media [7,9,28,29]. Glycerol protects sperma-
tozoa from cryoinjury by removing intracellular water
and increasing extracellular osmolality (tonicity).
However, the presence of glycerol in semen extender
1273
reduces motility (at 30 8C and 5 8C), fertilizing
capability following intracervical insemination and
acrosomal integrity by accelerating the acrosome
reaction [28,29]. Post-thaw TUNEL results of the
present study indicate that the presence of glycerol in
freezing media reduces DNA integrity (P < 0.05). This
finding may partly explain the observation of reduced
fertilizing capability in semen frozen with media
containing glycerol.
Cooling and the freeze-thaw process induce certain
molecular changes in ram sperm related to capacitation
and the acrosome reaction [1]. It was observed that
Caspase-3 immunostaining assays uniquely label the
apical and equatorial regions of the acrosome cap and
that caspase activity is decreased after cooling and
equilibration. Several studies have demonstrated that
the active form of Caspase-3 is extremely weak and is
rapidly turned over to its proenzyme form [37,38].
Surprisingly, none of the post-thawed spermatozoa
showed positive caspase activity. This finding is
contrary to data reported in humans [5] and bulls
[17] for post-thaw sperm caspase activity. It could be
hypothesized that subjecting sperm to cooling-freezing
stress could lead to the breakdown of the acrosomal
plasma membrane and the subsequent release of all
cellular contents, including caspases [1,25,39].
Ram sperm acrosomes are more sensitive in terms of
cryosurvival than are those of other farm animals
[28,29]. In several studies of ram semen, the freeze-
thaw process results in a rate of acrosome defects of 45–
65% [7,28,29,31]. These reports could explain the
differences in caspase-related findings between ram
semen and semen of other animals.
Sperm DNA damage has been associated with poor
semen quality [40,41]. Statistically significant negative
correlations have been found between sperm motility
and parameters such as defective acrosomes, AO, and
TUNEL. Although there was a statistical correlation
between AO- and TUNEL-positive spermatozoa, the
investigation of DNA integrity using the TUNEL assay
gave better results than AO staining. Sun et al. [42] and
Lopes et al. [43] reported that poor-quality semen has a
greater percentage of spermatozoa with DNA fragmen-
tation than good-quality semen. In our study, there was a
positive correlation between sperm with defective
acrosomes and apoptotic (as determined through both
AO and TUNEL) spermatozoa.
In conclusion, the freeze-thaw process is detrimental
to post-thaw ram semen viability as well as DNA and
functional integrity. Although the cryoprotectants used
in this study negatively affected sperm motility, sperm
morphology and DNA integrity, the addition of glycerol
1274 Z. Nur et al. / Theriogenology 73 (2010) 1267–1275
was more successful than 1,2 propanediol, sucrose,
trehalose and the control solution for maintaining post-
thaw motility. Although all cryoprotectants used in the
study triggered apoptosis to some degree, the presence
of some cryoprotectants in the freezing media is
obligatory for maintaining post-thaw cryosurvival of
ram semen. Additional studies using new combinations
of glycerol with sugars such as sucrose or trehalose
should be performed, as it is possible that the
detrimental effect of glycerol on sperm morphology
and DNA integrity might be overcome by combining
glycerol with other sugars for semen cryopreservation.
In addition, the results of the present study show that
measuring Caspase-3 activity was not a useful tool for
assessing apoptosis after cooling and thawing for ram
semen; however, the AO and TUNEL methods might be
effective means by which to assess apoptosis in ram
semen.
Acknowledgments
This work was supported by a grant (TOVAG
105O649) from TUBITAK TURKIYE.
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