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Theriogenology 73 (2010) 1267–1275

www.theriojournal.com

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],

and the cooling and freeze-thawing rates [4,10–13].
During the freeze-thawing process, mammalian Although several researchers have developed dif-
sperm are exposed to temperature changes that lead ferent extenders and protocols for freezing ram semen,
to physical and chemical stress, changes in the plasma in general, fertility results are not comparable to those
membrane lipid composition [1,2], reduced head size obtained with fresh semen and natural mating [13–15].
[3], and externalization of phosphatidylserine residues These reductions in fertilization capacity have typically
[4,5]. These sperm alterations are dependent on the 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
* Corresponding author. Tel./fax.: 90 224 2941345. biochemical and physiological events that cannot be
E-mail address: nurzek@uludag.edu.tr (Z. Nur). 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 2365) and mounting medium (Sc: 24941) were
based primarily on routine semen analyses (i.e., purchased from Santa Cruz Biotechnology Inc. (Santa
motility, morphology, and acrosomal integrity), but Cruz, CA, USA). All other chemicals were purchased
these routine semen evaluations have a limited capacity from Merck (Merck & Co., Inc., Darmstadt, Germany).
for predicting the potential fertility of ejaculate [18].
Therefore, advanced techniques for semen evaluation 2.2. Semen collection and cryopreservation
(e.g., in vitro fertilization, cervical mucus penetration,
DNA, and plasma membrane integrity) should be Ten Awassi rams that were 3–5 yr of age and
implemented to increase the odds of accurate identi- maintained at Uludag University, Faculty of Veterinary
fication of high-quality sperm [19–21]. Several tech- Medicine in Bursa, Turkey, were used during the
niques have been proposed to study sperm DNA breeding season. Semen was collected by electrically
abnormalities [22]. Those in current use are the TUNEL stimulated ejaculation (Ruakura Ram Probe Plastic
technique, which allows for the evaluation of sperm Products, Hamilton, New Zealand, probe length 12 cm,
DNA fragmentation [23,24]; the Comet technique, diameter 2.5 cm, 12 V) five times with a one-day inter-
which is another means of evaluating DNA integrity semen collection interval [7,26]. To collect semen, rams
[16,41]; caspase activity assays [5,21,25]; Annexin V were restrained physically and a lubricated probe was
staining, which provides information regarding the inserted into the rectum with downward pressure on the
translocation of phosphatidylserine (PS) [22]; and DNA front of the probe, so the electrodes rested on the upper
staining by acridine orange (AO), which differentiates portion of the ampullary region. An electrical stimula-
between single- and double-stranded DNA based on tion was applied for 4–8 s. The electrostimulation was
their respective colors under fluorescence [10,22]. stopped briefly (3–4 s) while further massage was
Although many studies have examined the effects of applied with the probe. This cycle was repeated until a
cryoprotectants on the outcomes of routine spermato- 1–2 ml sample of semen was collected (usually 3–4
logical evaluations, few studies have focused on the electrostimulations). Collected semen was placed in a
effects of cryoprotectants on apoptotic manifestations in warm water bath (30 8C) and immediately evaluated for
mammalian semen; most of the studies that have been consistency, wave motion (0–5 scale), and percentage of
conducted have shown that cryopreservation is asso- motile spermatozoa (0–100%) [7]. Ejaculates with a
ciated with apoptotic induction in human [5,10] and bull thick consistency, rapid wave motion (2–5 on a 0–5
[17] semen. This study aims to explore the effects of the scale), and >70% initial motility were pooled. Pooled
inclusion of various cryoprotectants (glycerol, 1,2 semen were then diluted with a TRIS-based extender
propanediol, sucrose and trehalose) in the Tes-Tris (20% egg yolk (v/v)) to a final concentration of 1/5
(TEST)-based extender on the morphology, DNA (semen/extender) in 0% cryoprotectant, 6% glycerol,
integrity, and apoptotic activity of freeze-thawed 6% 1,2 propanediol, 62.5 mM sucrose or 62.5 mM
Awassi ram semen. trehalose using a two-step dilution method [7]. Briefly,
pooled ejaculates were diluted to a ratio of 1:2 (semen/
2. Materials and methods extender) with extender A (no cryoprotectant, 375
mOsm) and cooled to 5 8C within 2 h. The cooled
2.1. Chemicals semen was then divided into five groups and diluted to a
ratio of 1:1 (semen/extender) with extender B (400
THAM (Tris(hydroxymethyl)aminomethane), Dex- mOsm) with one of four cryoprotectants (6% glycerol,
tran B (150000-200000), glycerol, 1,2 propanediol, 6% 1,2-propanediol, 62.5 mM sucrose or 62.5 mM
sucrose, trehalose, AO, PBS tablets, and poly-L-lysine trehalose). In addition, the control group was diluted
were purchased from Sigma (Sigma Chemical Co., St. with extender B with no cryoprotective agent. Extender
Louis, MO, USA). Triton X-100 (10% stock solution) B was added in five steps at 5 min intervals and
(11332481001) and an In Situ Cell Death Detection Kit equilibrated at 5 8C for 2 h. The osmotic pressure of the
were purchased from Roche (Roche Diagnostics extenders was determined using a freezing point
GmbH, Mannheim, Germany). Proteinase K (003011) depression osmometer (Advanced 3D3 Single Sample
and antibody diluents were purchased from Zymed Osmometer, Advanced Instrument, Inc., Norwood, MA,
(Zymed, San Francisco, California, USA). Rabbit USA) before adding cryoprotectants and egg yolk to the
polyclonal active Caspase–3 antibody (235412) was extender. Equilibrated semen samples were placed into
purchased from Calbiochem (Calbiochem, La Jolla, 0.25 ml straws and frozen at 3 8C/min from +5 8C to -
CA, USA). Bovine anti-rabbit fluorescein (FITC) (Sc: 8 8C and at 25 8C/min from -8 8C to -120 8C in liquid
 Z. Nur et al. / Theriogenology 73 (2010) 1267–1275 1269

nitrogen vapor using the Nicool Plus PC freezing 2.3.3. TUNEL assay
machine (Air Liquide, Marne-la-Vallée Cedex 3, For the TUNEL technique, we used the In Situ Cell
France). They were then plunged into liquid nitrogen Death Detection Kit with fluorescein (Roche Diagnos-
at -196 oC, where they were stored for at least one tics GmbH, Mannheim, Germany) according to the
month. At least three straws from each group of pooled manufacturer’s protocol with slight modifications. In
ejaculates were thawed at 37 8C for 30 s in a water bath brief, one drop of re-suspended spermatozoa was
to evaluate post-thaw semen characteristics. smeared on a glass slide and fixed with 10%
formaldehyde for 20 min at room temperature. The
2.3. Semen evaluation slides were washed in PBS and stored at 4 8C. Upon
removal from storage, samples were washed again in
All semen parameters were assessed at the following PBS (three times for 5 min each). They were then
four time points: after dilution with extender A, after treated in a humidified chamber with proteinase K for
cooling at 5 8C, after equilibration, and after thawing. 10 min at room temperature, washed with PBS, treated
All semen was frozen by the same person, and each of with 3% H2O2 in distilled water for 10 min at room
the studied semen parameters was measured by the temperature and washed again with PBS. The slides
same person on each occasion throughout the study. were permeabilized with 0.1% Triton X-100 for 5 min
Sperm motility was assessed subjectively using a on ice.
phase-contrast microscope (400x) with a warm slide The permeabilized slides were incubated in the dark
(38 8C) [7]. at 37 8C for 1 h with the TUNEL reaction mixture,
which contained terminal deoxynucleotidyl transferase
2.3.1. Fluorescein lectin staining assay (FITC- (TdT) plus dUTP label. After labeling, samples were
conjugated Pisum sativum agglutinin (FITC PSA)) washed with PBS and analyzed immediately via
Acrosome integrity was assessed using FITC- fluorescence microscopy. Negative (omitting TdT from
conjugated Pisum sativum agglutinin [27]. Briefly, the reaction mixture) and positive (using DNase I, 1 mg/
20 ml of diluted semen was re-suspended in 500 ml PBS ml, for 10 min at room temperature) controls were
and centrifuged at 2000 rpm for 20 min; the supernatant included in each trial. At least 100 sperm were
was then discarded. The spermatozoa pellet was re- evaluated to determine the percentage of TUNEL-
suspended in 250 ml PBS. One drop of resuspended positive sperm. Each microscopic field was evaluated
spermatozoa was smeared on a glass microscope slide first under fluorescence microscopy (40 magnifica-
and air dried. Air-dried slides were fixed with acetone at tion) to determine the number of reactive sperm and
4 8C for 10 min, and the slides were covered with FITC then under phase-contrast microscopy to determine the
PSA solution (50 mg/ml in PBS solution) in the dark for total number of sperm per field.
30 min. Stained slides were rinsed with PBS solution,
covered with glycerol, and examined under a fluores- 2.3.4. Immunocytochemistry assay
cence microscope. At least 100 spermatozoa per smear A rabbit polyclonal active Caspase–3 antibody was
were evaluated for acrosome integrity. used to detect active caspase in ram semen, as described
by Marti et al. [25] with some modifications. Briefly,
2.3.2. Acridine-Orange staining assay 20 ml of diluted semen was resuspended in 1000 ml PBS
Sperm DNA integrity was assessed using the AO and centrifuged at 2000 rpm for 20 min; the supernatant
fluorescence method [10]. Air-dried slides were fixed was then discarded. The semen pellet was permeabi-
overnight in freshly prepared Carney’s solution (three lized and fixed with 1000 ml of an ethanol:acetic acid
parts methanol and one part glacial acetic acid) and (3:1) solution at room temperature for 15 min. One drop
allowed to air dry for a few minutes. Dried slides were of re-suspended spermatozoa was smeared on a poly-L-
stained for 3 min with AO. The stained slides were lysine slide, and the slides were allowed to air dry. After
evaluated immediately under a fluorescence micro- washing three times with PBS, slides were incubated
scope. Normal DNA content showed green fluorescence with PBS containing 5% bovine serum albumin (BSA)
over the head region, while DNA abnormalities showed for 45 min at 37 8C to block non-specific sites.
varying fluorescence (from yellow-green to red). At After washing with PBS, the slides were incubated
least 100 spermatozoa per smear were evaluated for first with a primary antibody (1/50 dilution) for 1 hour at
DNA abnormalities (10). Sperm cells with changes in 37 8C and then with a bovine anti-rabbit fluorescein-
fluorescence from yellow-green to red were recorded as labeled IgG secondary antibody (Santa Cruz, 1/400
sperm with abnormal DNA content. 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 Table 1 shows the differences in the percentages of
covered with a cover slip. As controls for primary motility, defective acrosomes (FITC PSA), and AO,
antibody specificity, all immunofluorescence labeling TUNEL, and Caspase-3 activities of the diluted, cooled
experiments were also carried out with no primary at 5 8C, equilibrated, and thawed ram semen from the
antibodies. different cryoprotectant groups.
All fluorescence examinations were performed with Sperm motility was progressively reduced by cool-
an epifluorescence microscope (BX51, Olympus Inc., ing and the freeze-thaw process (Table 1; P < 0.05).
Japan) with a multiple fluorescence filter (U-DM-DA/ The motility values of equilibrated semen in the sucrose
FI/TX2) at 460–490 nm excitation and 530 nm barriers. and trehalose groups were significantly lower than those
At least 100 spermatozoa were examined per slide of the glycerol, 1,2 propanediol, and control groups
(100 objective). (P < 0.05). Post-thaw semen motility values in the
glycerol and 1,2 propanediol groups were better than
2.4. Statistical analyses semen motility values in the sucrose, trehalose and
control groups (P < 0.05).
Data were analyzed by analysis of variance Acrosome integrity was assessed using FITC PSA
(ANOVA) using the General Linear Model (GLM) (Fig. 1). Acrosome integrity was negatively affected by
procedure. The model included fixed effects of the freeze-thaw process. Post-thaw defective acrosome
cryoprotectants and random effects of pooled ejacu- rates were higher than those of diluted, cooled and
lates (replicates). Means of obtained semen parameters equilibrated spermatozoa (P < 0.05). The percentage of
were analyzed using Tukey’s test. Repeated measures defective acrosomes in the equilibrated and thawed
ANOVA (using GLM procedures) were conducted to spermatozoa was not affected by any of the cryopro-
compare the results at different stages of the tectants, as compared to the control group (P < 0.05).
cryopreservation process. Spearman’s correlation As shown in Fig. 1, AO-stained ram spermatozoa
coefficient was used to assess the relationship between with undamaged DNA showed green fluorescence,
semen motility, defective acrosomes and apoptotic while damaged DNA displayed a spectrum of yellow-
spermatozoa (AO- and TUNEL-positive). All data were orange to red fluorescence. Post-thaw semen demon-
analyzed using the SPSS statistical package (SPSS 10.0 strated the highest rate of damaged DNA (assessed via
for Windows; SPSS, Chicago, IL, USA). Differences acridine orange staining) and showed significantly more
were considered significant at P-values of less than damage than the diluted, cooled to 5 8C and equilibrated
0.05. 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 comparing the cryoprotectants and control groups, the
similar to control group values (P > 0.05). mean numbers of TUNEL-positive spermatozoa were
The results of the TUNEL assay demonstrated that a not affected by cryoprotectant type in equilibrated
large proportion of spermatozoa with DNA fragmenta- semen. Also, post-thaw TUNEL-positive spermatozoa
tion exhibited green fluorescence (Fig. 1). This was not were not affected by cryoprotectant type, with the
the case for undamaged spermatozoa. The post-thaw exception of the glycerolized groups (P < 0.05).
percentage of apoptotic spermatozoa was 6.0%, which Three different sperm labeling patterns were found
was significantly different from the diluted, cooled, and in diluted semen after Caspase-3 immunostaining
equilibrated semen (P < 0.05). All cryoprotectants used assays, with localization in the apical, equatorial and
in this study triggered some degree of apoptosis. When 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 zoa frozen in a TRIS-based extender.
parameters.
The mean percentages of sperm motility, defective
Defected Apoptotic spermatozoa acrosomes, and apoptotic spermatozoa, as measured by
acrosome
AO, TUNEL, and Caspase-3, in the diluted ejaculates
PSA (%) AO (%) Tunnel (%)
were 79.0%, 5.6%, 1.4%, 1.7%, and 3.6%, respectively.
Motility (%) 0. 617* 0.438* 0.302* These data are in agreement with previous reports
Defected acrosome PSA (%) 0.663* 0.500* concerning semen motility, defective acrosomes, and
Apoptotic spermatozoa (AO) 0.490*
DNA integrity in rams [7,25,31].
*Correlation is significant at the (P < 0.01). Despite advances in the cryopreservation of mam-
FITC-PSA: FITC- conjugated Pisum Sativum Agglutinin; AO: Acri-
malian spermatozoa, there has been less success with
dine Orange.
ram spermatozoa than with bull spermatozoa [26].
Cryopreservation of spermatozoa reduces motility
immunostaining, decreased in cooled and equilibrated [7,28,29,32]. In addition, acrosome [7,10,28,29,32],
semen. However, none of the post-thawed spermatozoa plasma membrane [1,7] and DNA [10,25,32] integrities
showed positive caspase activity. are affected. The results of the present study show that
Freezing and thawing procedures (cooling, dilution, freeze-thawing procedures (cooling to 5 8C, dilution,
equilibration, and thawing) had negative effects on equilibration, and thawing) negatively affect motility
motility (P < 0.001), defective acrosome FITC PSA (P < 0.001). The rates of defective acrosomes
(P < 0.001), and apoptotic cell ratios, as determined by (P < 0.001) and apoptotic cell ratios, as determined
AO (P < 0.001) and TUNEL (P < 0.001) assays. There by AO (P < 0.001) and TUNEL (P < 0.001), were also
was a significant interaction between the time point and negatively affected.
cryoprotectant use for sperm motility (P < 0.001) and The beneficial effect of cryoprotectant-supplemen-
Caspase-3 activity (P < 0.05) (Table 2). ted media on post-thawed mammalian semen has been
The results of the Pearson correlation tests are shown reported in many studies [7,9,13,28–30]. Although
in Table 3. A significant negative correlation was found glycerol is the first and most commonly used
between sperm motility and defective acrosomes, AO- cryoprotectant for semen freezing media [15,33], the
and TUNEL-positivity (P < 0.01). In addition, there presence of glycerol lowers the quality and fertilizing
were positive correlations between defective acrosomes capacity of semen [13,28,29].
and apoptotic (AO- and TUNEL-positive) spermatozoa. In the present study, the motilities of equilibrated
semen in the sucrose and trehalose groups were
4. Discussion significantly lower than those of the glycerol, 1,2
propanediol, and control groups (P < 0.05). For post-
The freeze-thaw process is detrimental to mamma- thaw semen motility, glycerol- and 1,2 propanediol-
lian sperm viability, DNA, and functional integrity containing groups had better values than the sucrose,
[5,7,17]. More specifically, ram semen has proven to be trehalose and cryoprotectant-free groups (P < 0.05).
more difficult to cryopreserve than semen of other farm Soylu et al. [7] reported that media containing glycerol
animals [28,29]. Various extenders and cryoprotective and 1,2-propanediol protect post-thaw motility effec-
agents have been developed for the cryopreservation of tively when compared to sucrose, trehalose, and control
ram semen [7,28–30]. In the present study, we evaluated groups. The presence of high concentrations of
the effects of different cryoprotectants on post-thaw trehalose in freezing media improves post-thaw semen
 Z. Nur et al. / Theriogenology 73 (2010) 1267–1275 1273

characteristics, but in low concentrations, the agent reduces motility (at 30 8C and 5 8C), fertilizing
yields little improvement [34,35]. Woelders et al. [8] capability following intracervical insemination and
compared two different sugars (2 M trehalose and 2 M acrosomal integrity by accelerating the acrosome
sucrose) in extenders with high osmolality and found reaction [28,29]. Post-thaw TUNEL results of the
that sucrose performed significantly better than treha- present study indicate that the presence of glycerol in
lose at the highest cooling rate for post-thaw bull semen. freezing media reduces DNA integrity (P < 0.05). This
Extender composition assists in stabilizing cells finding may partly explain the observation of reduced
during the freezing and thawing process [5,7]. Post- fertilizing capability in semen frozen with media
thaw sperm recovery was significantly better when containing glycerol.
sperm were frozen in hypertonic extender (400 mOsm) Cooling and the freeze-thaw process induce certain
compared to low osmolality (350 and 375 mOsm) [7]. molecular changes in ram sperm related to capacitation
The osmolality of Extender B in the present study was and the acrosome reaction [1]. It was observed that
400 mOsm. Post-thaw sperm motility was similar to the Caspase-3 immunostaining assays uniquely label the
results reported by Soylu et al. [7] for glycerol- and 1,2 apical and equatorial regions of the acrosome cap and
propanediol-containing groups and the cryoprotectant- that caspase activity is decreased after cooling and
free group frozen with an extender at 400 mOsm. equilibration. Several studies have demonstrated that
Cryopreservation induces premature capacitation or the active form of Caspase-3 is extremely weak and is
acrosome reactions [36]. In our study, acrosome rapidly turned over to its proenzyme form [37,38].
integrity was affected by the freeze-thaw process. Surprisingly, none of the post-thawed spermatozoa
Post-thaw defective acrosome rates were higher than showed positive caspase activity. This finding is
those of diluted, cooled and equilibrated spermatozoa. contrary to data reported in humans [5] and bulls
The presence of glycerol and 1,2-propenendiol in the [17] for post-thaw sperm caspase activity. It could be
semen freezing extender reduces acrosomal and hypothesized that subjecting sperm to cooling-freezing
morphological integrity [28,29]. The percentage of stress could lead to the breakdown of the acrosomal
defective acrosomes of equilibrated and thawed plasma membrane and the subsequent release of all
spermatozoa were not affected by the type of cellular contents, including caspases [1,25,39].
cryoprotectant used compared to the control group Ram sperm acrosomes are more sensitive in terms of
(P < 0.05). Soylu et al. [7], using Giemsa stain for cryosurvival than are those of other farm animals
acrosome evaluation and media with different osmol- [28,29]. In several studies of ram semen, the freeze-
ality, reported that media containing glycerol and 1,2 thaw process results in a rate of acrosome defects of 45–
propanediol did not protect post-thaw acrosome 65% [7,28,29,31]. These reports could explain the
integrity compared to sucrose, trehalose, and control differences in caspase-related findings between ram
samples. semen and semen of other animals.
TUNEL and AO assays revealed that the freeze- Sperm DNA damage has been associated with poor
thawing procedures also caused deterioration of DNA semen quality [40,41]. Statistically significant negative
integrity. For TUNEL and AO staining, chromatin correlations have been found between sperm motility
injury was the highest in post-thaw semen and differed and parameters such as defective acrosomes, AO, and
significantly from diluted, cooled to 5 8C, and TUNEL. Although there was a statistical correlation
equilibrated spermatozoa (P < 0.05). All cryoprotec- between AO- and TUNEL-positive spermatozoa, the
tants used in this study triggered apoptosis to some investigation of DNA integrity using the TUNEL assay
degree (P > 0.05). For post-thaw semen, a comparison gave better results than AO staining. Sun et al. [42] and
across the various groups showed that the mean values Lopes et al. [43] reported that poor-quality semen has a
of TUNEL-positive spermatozoa were not affected by greater percentage of spermatozoa with DNA fragmen-
cryoprotectant type, with the exception of the glycer- tation than good-quality semen. In our study, there was a
olized group. positive correlation between sperm with defective
Many studies have been carried out by varying the acrosomes and apoptotic (as determined through both
amount of glycerol used, the timing protocol for adding AO and TUNEL) spermatozoa.
glycerol to extenders and the exclusion of glycerol from In conclusion, the freeze-thaw process is detrimental
freezing media [7,9,28,29]. Glycerol protects sperma- to post-thaw ram semen viability as well as DNA and
tozoa from cryoinjury by removing intracellular water functional integrity. Although the cryoprotectants used
and increasing extracellular osmolality (tonicity). in this study negatively affected sperm motility, sperm
However, the presence of glycerol in semen extender 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, and intactness of bull sperm after freezing and thawing. Cryo-
trehalose and the control solution for maintaining post- biology 1997;35:93–105.
[9] Yildiz C, Kaya A, Aksoy M, Tekeli T. Influence of sugar
thaw motility. Although all cryoprotectants used in the supplementation of the extender on motility, viability and acro-
study triggered apoptosis to some degree, the presence somal integrity of dog spermatozoa during freezing. Theriogen-
of some cryoprotectants in the freezing media is ology 2000;54:579–85.
obligatory for maintaining post-thaw cryosurvival of [10] Hammadeh ME, Askari A, Gerorg T, Rosenbaum P, Schmidt W.
Effect of freeze-thawing procedure on chromatin stability,
ram semen. Additional studies using new combinations
morphological alteration and membrane integrity of human
of glycerol with sugars such as sucrose or trehalose spermatozoa in fertile and subfertile men. Int J Androl
should be performed, as it is possible that the 1999;22:155–62.
detrimental effect of glycerol on sperm morphology [11] Henry MA, Noiles EE, Gao D, Mazur P, Critser JK. Cryopres-
and DNA integrity might be overcome by combining ervation of human spermatozoa. IV. The effects of cooling rate
glycerol with other sugars for semen cryopreservation. and warming rate on the maintenance of motility, plasma
membrane integrity, and mitochondrial function. Fertil Steril
In addition, the results of the present study show that 1993;60:911–8.
measuring Caspase-3 activity was not a useful tool for [12] Maxwell WMC, Landers AJ, Evans G. Survival and fertility of
assessing apoptosis after cooling and thawing for ram ram spermatozoa frozen in pellets, straws and minitubes. Ther-
semen; however, the AO and TUNEL methods might be iogenology 1995;43:1201–10.
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Effect of cryoprotective diluent and method of freeze-thawing on
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Acknowledgments [14] Sanches-Partida LG, Maxwell WMC, Pateg LG, Setchell BP.
Proline and glycine betaine in the cryoprotective diluents for ram
spermatozoa. Reprod Fertil Dev 1992;4:113–8.
This work was supported by a grant (TOVAG
[15] Stanic P, Tandara M, Sonicki Z, Simunic V, Radakovic B,
105O649) from TUBITAK TURKIYE. Suchanek E. Comparison of protective media and freezing
techniques for cryopreservation of human semen. Eur J Obstet
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