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ORIGINAL ARTICLE
Lycopene and resveratrol improve post-thaw bull sperm
parameters: sperm motility, mitochondrial activity and DNA
integrity
M. N. Bucak
1
, M. B. Ataman
1
,N.Bas
ßpınar
2
, O. Uysal
3
,M.Tas
ßpınar
4
, A. Bilgili
5
,C.
€
Ozt€
urk
1
,
S
ß.G
€
ung€
or
1
,M.E.
_
Inanc
ß
3
& E. Akal
6
1 Department of Reproduction and Artificial Insemination, Veterinary Faculty, Selcuk University, Konya, Turkey;
2 Department of Biochemistry, Veterinary Faculty, Selcuk University, Konya, Turkey;
3 Department of Reproduction and Artificial Insemination, Veterinary Faculty, Ankara University, Ankara, Turkey;
4 Department of Medical Biology, Medical Faculty, Yuzuncu Yil University, Van, Turkey;
5 Department of Pharmacology and Toxicology, Veterinary Faculty, Ankara University, Ankara, Turkey;
6 Department of Reproduction and Artificial Insemination, Veterinary Faculty, Ondokuz Mayıs University, Samsun, Turkey
Keywords
antioxidants —cryopreservation —DNA
damage —oxidative stress —sperm
Correspondence
Prof. Dr Mustafa N. Bucak, Department of
Reproduction and Artificial Insemination,
Faculty of Veterinary Medicine,
Selcuk University,
Konya, Turkey.
Tel.: +903322233589;
Fax: +903322410063;
E-mail: mustafanbucak@yahoo.com
Accepted: April 29, 2014
doi: 10.1111/and.12301
Summary
We focussed on evaluating the protective effect of lycopene and resveratrol on
post-thaw bull sperm and oxidative stress parameters. Nine ejaculates for each
bull were used in the study. Each ejaculate, splitted into three equal aliquots and
diluted at 37 °C with base extenders containing lycopene (1 910
3
gml
1
)
and resveratrol (1 mM), and no antioxidant (control), was cooled to 5 °C and
then frozen. Frozen straws were thawed in a water bath for evaluation. The sup-
plementation of the semen extender with lycopene and resveratrol increased the
percentages of post-thawed computer-assisted sperm analysis (CASA) motility
(55.8 3.8 and 61.9 4.0%) and progressive motility (38 2.4 and
37 8.8), compared with the controls (50.7 2.65 and 33.3 3.74%, respec-
tively, P<0.05). Resveratrol provided a higher ALH (4.3 0.1), in comparison
with the control (3.9 0.3, P<0.05). The supplementation of the semen exten-
der with lycopene and resveratrol produced a higher mitochondrial activity
(24.6 2.9 and 30.1 6.5% respectively), compared with that of the control
(11.8 9.5%, P<0.05). It was determined that both antioxidants resulted in a
lower percentage of sperm with damaged DNA than that of the control
(P<0.05). Sperm motion characteristics except for ALH, acrosome integrity,
sperm viability and oxidative stress parameters were not affected by the adding
of lycopene and resveratrol.
Introduction
The membranous structures of the spermatozoon (plasma
membrane, outer acrosomal membrane and mitochondrial
membrane) are highly sensitive to the freeze–thawing pro-
cess. These structures being composed of 65–70% thermo-
dynamic phospholipids (fatty acids) result in an
irreversible phase change from the liquid phase into the gel
phase in the event of the cooling of the membranes (Wat-
son, 1995, 2000). The occurrence of such a phase change
brings about a change in the kinetics of the intramembra-
nous enzymes and results in decreased post-thaw viability.
The instability caused by these changes leads to cold shock
and oxidative stress-induced damage in the cell. Further-
more, the abundance of unsaturated phospholipids in the
membrane structure causes susceptibility to lipid peroxida-
tion, and free radicals generated by lipid peroxidation lead
to cellular damage during the long-term storage of sperm
cells (Watson, 1995; Holt, 2000a,b). Spermatozoa and the
seminal plasma contain several antioxidants, which pro-
vide protection against the toxic effects of free radicals
(Alvarez & Storey, 1983; Nissen & Kreysel, 1983; Jeulin
et al., 1989). However, following the freeze–thawing pro-
cess, this antioxidant system fails in protecting spermato-
zoa against oxidative damage and the toxic effects of free
radicals (Bilodeau et al., 2000; Chatterjee et al., 2001;
Gadea et al., 2004). Due to these reasons, with an aim to
reduce oxidative damage and the toxic effects of free radi-
cals during the freeze-thawing of sperm, semen extenders
are supplemented with various antioxidant compounds,
©2014 Blackwell Verlag GmbH 545
Andrologia 2015, 47, 545–552
and thereby, post-thaw sperm parameters are improved
(Alvarez & Storey, 1983; Uysal & Bucak, 2007; Bucak et al.,
2010). Lycopene is the most abundant carotenoid in toma-
toes and red fruits and is considered the most efficient
antioxidant of all carotenoids (Di Mascio et al., 1989).
Lycopene is most likely involved in the quenching of sin-
glet molecular oxygen and the trapping of peroxy radicals
and thereby contributes to the protection of cells and tis-
sues against the harmful effects of lipid peroxidation. The
antioxidant activity of lycopene is mostly catalytic (Stahl &
Sies, 1996, 2003; Velmurugan et al., 2004). Studies have
shown that lycopene can improve sperm motility, mem-
brane integrity (Uysal & Bucak, 2007) and DNA damage
(Zini et al., 2010). Resveratrol (3,5,40-trihydroxystilbene),
a nonflavonoid polyphenol found mainly in grapes, plays
an important role as an antioxidant and acts as an effective
scavenger of free radicals (superoxide anion, hydroxyl radi-
cal and metal-induced radicals) (Leonard et al., 2003; Will-
cox et al., 2004). Resveratrol also exhibits a protective
effect against lipid peroxidation (LPO) and DNA damage
caused by free radicals in sperm cells (Revel et al., 2001;
Branco et al., 2010; Collodel et al., 2011).
The potential effects of lycopene and resveratrol-supple-
mented extenders on the sperm parameters of bulls follow-
ing the freeze–thawing process have not been investigated
before. We focussed on evaluating the protective effect of
lycopene and resveratrol on sperm motility, motion kine-
matics, viability, acrosome integrity, mitochondrial acti-
vity, DNA damage and oxidative stress parameters.
Materials and methods
Animals and semen collection
Three Holstein bulls (3–4 years of age) were housed at a
private dairy farm in Konya and maintained under uni-
form feeding and housing conditions. A total number of
30 ejaculates (10 ejaculates for each bull) were collected
from the bulls with the aid of an artificial vagina twice a
week, according to standard AI procedures. Ejaculates con-
taining spermatozoa with >80% forward progressive
motility and concentrations higher than 1.0 910
9
sper-
matozoa ml
1
were used in the study. Nine ejaculates for
each bull were used in the study. Immediately after collec-
tion, the ejaculates were immersed in a warm water bath at
34 °C until their assessment in the laboratory. Semen
assessment was performed within approximately 20 min.
Semen processing
The volume of the ejaculates was measured in a conical
tube graduated at 0.1-ml intervals and the sperm concen-
tration using a haemocytometer. Sperm motility was
estimated using phase-contrast microscopy. A Tris-based
extender (Tris 297.58 mM, citric acid 96.32 mM, fructose
82.66 mM, egg yolk 15% (v/v), glycerol 5% (v/v), pH 6.8)
was used as the base extender (freezing extender). Each
mixed ejaculate was split into three equal aliquots and
diluted at 37 °C with base extenders containing lycopene
(1 910
3
gml
1
), resveratrol (1 mM), and no antioxi-
dant (control), to a final concentration of approximately
60 910
6
spermatozoa ml
1
, in a 15-ml plastic centrifuge
tube. Diluted semen samples were aspirated into 0.25-ml
(medium-sized) French straws, sealed with polyvinyl alco-
hol powder, and equilibrated at 5 °C for 3 h. After equili-
bration, the straws were frozen in liquid nitrogen vapour,
4 cm above the liquid nitrogen, for 15 min and plunged
into liquid nitrogen for storage. After stored for 1 month,
the frozen straws were thawed individually at 37 °C for
20 s in a water bath for microscopic evaluation.
Evaluation of microscopic sperm parameters
Analysis of computer-assisted sperm analysis (CASA) motil-
ity and kinematic parameters
The Sperm Class Analyzer (SCA
â
) CASA system
(Microptic S.L., Barcelona, Spain) was used to analyse
sperm motility and various kinematic parameters. A 5-ll
sample of diluted semen was put onto a pre-warmed slide
covered with a coverslip and sperm motility characteris-
tics were determined with a 109objective at 37 °C. The
following motility values were recorded: motility (%),
progressive motility (%), total sperm motility, VAP (aver-
age path velocity, lms
1
), VSL (straight linear velocity,
lms
1
), VCL (curvilinear velocity, lms
1
), ALH
(amplitude of lateral head displacement, lm) and LIN
(linearity index (LIN =(VSL/VCL) 9100). For each
evaluation, seven microscopic fields, each including at
least 250 cells, were analysed.
Assessment of sperm plasma membrane integrity (viability)
This assessment was performed by staining with a sperm
viability kit (SYBR-14/PI Molecular Probe: L 7011 Invi-
trogen, Carlsbad, CA, USA). The staining protocol was
modified from a study of Garner & Johnson (1995). A
working solution of SYBR-14 was diluted 1 : 10 with
DMSO (Applichem A3006), then divided into equal
aliquots (30 ll) after being filtered through a 0.22-lm
Millipore Millex-GV filter, and stored at 20 °C. Propi-
dium iodide (PI) was dissolved in distilled water at a
concentration of 2 mg ml
1
, divided into equal aliquots
(30 ll) after being filtered through a 0.22-lm Millipore
Millex-GV filter and stored at 20 °C. Thawed straws
were diluted 1 : 3 with Tris stock solution without glyc-
erol and egg yolk, and then 30 ll of the diluted semen
was mixed with 6 ll of SYBR-14 and 2.5 ll of PI. The
546 ©2014 Blackwell Verlag GmbH
Andrologia 2015, 47, 545–552
Influence of antioxidants on bull sperm freezing M. N. Bucak et al.
sample was gently mixed, incubated at 37 °C in the dark
for 20 min and then added 10 ll of Hancock’s solution
(Schafer & Holzmann, 2000) for semen fixation. A wet
mount was made using a 2.5-ll drop of the sample
placed directly onto a microscope slide and covered
by a cover slip. At least 200 spermatozoa per sample
were examined at 10009magnification under a fluores-
cence microscope (Leica DM 3000 Microsystems GmbH,
Ernst-Leitz-Straße, Wetzlar, Germany; excitation at 450–
490 nm, emission at 520 nm) to assess sperm membrane
integrity. Sperm cells displaying green–red or red colori-
sation were considered to have membrane damage, while
those displaying green colorisation were considered to
have an intact membrane.
Assessment of sperm acrosome integrity
Sperm acrosome status was assessed using fluorescein iso-
thiocyanate conjugated to Arachis hypogaea (peanut)
(L7381 FITC-PNA, Sigma-Aldrich Co., St. Louis, MO,
USA) and by PI staining as described by Nagy et al.
(2003) with modifications. 120 lg of FITC-PNA was
added to 1 ml of PBS for the preparation of the staining
solution, then divided into equal aliquots (100 ll) after
being filtered and stored at 20 °C. Thawed straws were
diluted 1 : 3 with Tris stock solution without glycerol
and egg yolk, and then 60 ll of the diluted semen was
mixed with 10 ll of FITC-PNA and 2.5 ll of PI. The
sample was gently mixed, incubated at 37 °C in the dark
for 20 min and added 10 ll of Hancock’s solution (Scha-
fer & Holzmann, 2000) for semen fixation. A wet mount
was made using a 2.5-ll drop of the sample placed
directly onto a microscope slide and covered by a cover
slip. At least 200 sperm cells per sample were examined
at 10009magnification under a fluorescence microscope
(Leica DM 3000; excitation at 450–490 nm, emission at
520 nm) to assess sperm acrosome integrity. Spermatozoa
displaying bright green or patchy green fluorescence were
considered as acrosome nonintact or damaged, whereas
cells which did not display green fluorescence in the acro-
some cap were regarded as acrosome intact.
Assessment of sperm mitochondrial activity
Sperm mitochondrial activity was assessed with a staining
protocol modified from Garner et al. (1997). A stock
solution of 5,50, 6,60-tetrachloro-1,10, 3,30tetraethyl-benz-
imidazolylcarbocyanine iodide (1.53 mM) (T3168 JC-1,
Invitrogen) was prepared in DMSO solution and then
divided into equal aliquots (100 ll) after being filtered
and stored at 20 °C. Thawed straws were diluted 1 : 3
with Tris stock solution without glycerol and egg yolk,
and then 300 ll of the diluted semen was mixed with
2.5 ll JC-1 and 2.5 ll PI. The sample was gently mixed,
incubated at 37 °C in the dark for 20 min and added
10 ll of Hancock’s solution (Schafer & Holzmann, 2000)
for semen fixation. A wet mount was made using a 2.5-ll
drop of the sample placed directly onto a microscope
slide and covered by a cover slip. At least 200 sperm cells
per sample were examined at 10009magnification under
a fluorescence microscope (Leica DM 3000; excitation at
450–490 nm, emission at 520 nm) to assess mitochon-
drial activity. A high level of yellow/orange fluorescence
associated with the sperm midpiece (where the mitochon-
dria are located) indicated high mitochondrial activity.
Mitochondria with low activity stained green.
Assessment of sperm DNA damage
Sperm DNA damage was investigated using the single cell
gel electrophoresis (COMET) assay, which is generally
performed under neutral conditions. Our method was
similar to several protocols applied in previous studies
and involved few modifications (Duty et al., 2002; Li
et al., 2008). The straws were thawed by gentle shaking in
a37°C water bath for 10 s, and centrifuged once at 600
gfor 10 min at 4 °C. The remaining sperm cells were
washed with PBS (Ca
2+
and Mg
2+
free).
Each pre-cleaned slide was pre-coated with a layer of
1% normal melting point agarose in PBS (Ca
++
and
Mg
++
free) and then dried at room temperature. Approx-
imately 100 000 sperm cells (18 ll) were mixed with
0.75% low melting point agarose (50 ll) at 37 °C, and
this suspension was poured onto the first agarose layer.
The slides were allowed to solidify for 20 min at 4 °C.
The coverslips were removed, and the slides were
immersed in freshly prepared cold lysis buffer. The slides
were then incubated at 37 °C in lysis buffer with
20 lgml
1
of proteinase K for 2 h. The slides were
removed from the lysis buffer, drained and placed into a
horizontal electrophoresis unit filled with fresh neutral
electrophoresis buffer at 4 °C for a 20-min incubation to
allow the DNA to unwind. Electrophoresis was per-
formed at room temperature, at 25 V for 20 min. Fol-
lowing electrophoresis, the slides were air-dried, stained
with 50 llof8llml
1
ethidium bromide, and covered
with a coverslip.
The images of 200 randomly chosen nuclei per sample
were analysed visually. Observations were made at a mag-
nification of 4009using a fluorescence microscope
(Olympus, Japan). Damage was detected by a tail of frag-
mented DNA that migrated from the sperm head, causing
a ‘comet’ pattern, whereas whole sperm heads, without a
comet, were not considered as damaged.
Oxidative stress parameters
Briefly, thawed semen samples were centrifuged at 800 g
for 20 min at 4 °C to separate the cells from the diluted
©2014 Blackwell Verlag GmbH 547
Andrologia 2015, 47, 545–552
M. N. Bucak et al. Influence of antioxidants on bull sperm freezing
seminal plasma, and spermatozoa were washed twice with
PBS at 800 9gfor 20 min. After centrifugation, the
supernatant was discarded and the pellet was completed to
500 ll with PBS. Subsequently, the sperm suspension was
transferred into a 2-ml beaker filled with ice water and
sonicated with a probe (Bandelin Sonopuls, Bandelin Elec-
tronic HeinrichstraBe, D-12207, Gerate-Typ:UW 2070,
Pro-Nr. 51900037369.004, Berlin) for 10 s on ice, such that
the process was repeated six times at intervals of 30 s to
separate the sperm head and tail. For LPO analysis, 10 ll
of 0.5 mMBHT (butyl-hydroxytoluene) was added to
120 ll of the homogenate samples and stored at 86 °C
until analysis. The remaining homogenate was centrifuged
at 8000 gfor 15 min at +4°C and the supernatant was col-
lected and stored at 86 °C for AOP analysis.
Determination of lipid peroxidation (LPO) level
LPO level was determined using commercial kits of LPO-
586TM Oxis Research (OxisResearch
TM
, Bioxytech, CA,
92202, USA) by spectrophotometry (UV 2100 UV-VIS
Recording Spectrophotometer Shimadzu, Japan). The
assay is based on the reaction of a chromogenic reagent,
N-methyl–2- phenylindole with MDA and 4-hydroxyalke-
nals (LPO) at 45 °C. One molecule of either MDA or
4-hydroxyalkenal reacts with two molecules of N-methyl–
2-phenylindole in acetonitrile, to yield a stable chromo-
phore with maximal absorbance at 586 nm. The results
are expressed as lmol for 10
9
cells ml
1
.
Determination of Total Antioxidant Activity (AOP)
AOP activity was determined with an AOP-490
TM
Oxis
Research kit (OxisResearch
TM
, Bioxytech) by spectropho-
tometry. The assay was based on the reduction of
Cu++ to Cu+by the combined action of all the antioxi-
dants present in the sample. A chromogenic reagent,
bathocuproine (2,9-dimethyl-4,7-diphenyl-1,10-phenan-
throline), selectively forms a 2 : 1 complex with Cu+,
which has a maximum absorbance at 490 nm. A standard
of known uric acid (a water soluble antioxidant) concen-
tration is used to create a calibration curve. The results
are expressed as mmol for 10
9
cells ml
1
.
Statistical analysis
The study was replicated nine times. Results are expressed
as mean SEM. Sperm motility, motion characteristics
and abnormality were analysed by analysis of variance,
followed by Tukey’s post hoc test to determine significant
differences between the groups. Sperm with damaged
DNA were evaluated by the chi-square test. Differences
with values of P<0.05 were considered to be statistically
significant. Statistical analyses were performed by using
the SPSS 11.5 package program.
Results
As shown in Table 1, the supplementation of the semen
extender with lycopene and resveratrol increased the per-
centages of CASA motility (55.8 3.8 and 61.9 4.0%)
and progressive motility (38 2.4 and 37 8.8%), com-
pared with the controls (50.7 2.65 and 33.3 3.74%,
respectively, P<0.05), following the freeze–thawing pro-
cess. As shown in Table 2, no significant differences were
observed between the groups for sperm motion character-
istics (P>0.05), except for the ALH value. Resveratrol
provided a higher ALH (4.3 0.1 lm), in comparison
with that of the control group (3.9 0.3 lm, P<0.05).
Lycopene and resveratrol produced a higher mitochon-
drial activity (24.6 2.9 and 30.1 6.5% respectively),
compared with that of the control group (11.8 9.5%,
Table 3, P<0.05). No significant differences were
observed between the groups for sperm acrosome integ-
rity and viability (Table 3, P>0.05). In the comet assay,
it was determined that both antioxidants reduced DNA
damage and resulted in a lower percentage of sperm with
damaged DNA than that of the controls (Table 3,
P<0.05). The effects of the two antioxidants on LPO
and antioxidant activity in thawed bull sperm are shown
in Table 4. Supplementation of the semen extender with
antioxidants did not significantly affect LPO level and
AOP activity in comparison with the control group
(P>0.05).
Discussion
Long-term sperm storage (freezing) results in membrane
deterioration due to membrane phase transitions occur-
ring in the highly specialised regions of the sperm plasma
membrane and leads to the modification of sperm func-
tions (Maxwell & Watson, 1996). Increases in cryodamage
and the sensitivity of sperm to oxidative stress after the
freeze–thawing process, results in a reduction in sperm
motility, viability, functional membrane integrity and
DNA integrity, and antioxidant enzyme activity during
the freeze–thawing process (Hammerstedt, 1993; Bucak
et al., 2010). Frozen-thawed semen is more easily
Table 1 Mean (SEM) sperm motilities in frozen-thawed bull semen
Groups
CASA
motility (%)
Progressive
motility%
Control 50.7 2.65
a
33.3 3.74
a
Lycopene 1 910
3
gml
1
55.8 3.8
b
38.0 2.4
b
Resveratrol 1 mM61.9 4.0
c
37.0 8.8
a,b
P–*–*
*P<0.05. Different superscripts within the same column demon-
strate significant differences.
548 ©2014 Blackwell Verlag GmbH
Andrologia 2015, 47, 545–552
Influence of antioxidants on bull sperm freezing M. N. Bucak et al.
peroxidized than fresh sperm. Intracellular antioxidant
capacity fails to provide protection against oxidative dam-
age and the potential toxic effects of free radicals following
freeze–thawing (Trinchero et al., 1990; Rosato et al., 2012).
This study was performed to investigate whether lyco-
pene or resveratrol would provide protection against tem-
perature shock and oxidative damage during the
cryopreservation of bull sperm. Both resveratrol and lyco-
pene increased the post-thaw sperm motility and mito-
chondrial activity. The axosome and associated dense fibres
of the midpiece of sperm cells are covered by mitochondria,
which generate energy from intracellular ATP stores that
are responsible for sperm motility (Garner & Hafez, 1993).
Cryopreservation can induce axonemal damage, resulting
in the decrease of sperm motility, mitochondrial membrane
potential and morphological-functional integrity as a result
of ATP depletion (De Lamirande & Gagnon, 1992; Cum-
mins et al., 1994). Sperm motility is important for the
actual penetration of the cumulus cells and zona pellucida
of the ovum (Garner & Hafez, 1993). A strong correlation
has been reported to exist between the motility of sperm
and high mitochondrial activity (Kasai et al., 2002; Marti-
nez-Pastor et al., 2004). In the present study, the supple-
mentation of the semen extender with resveratrol before
cryopreservation may act as an effective scavenger of free
radicals and membrane cryoprotector (Willcox et al.,
2004), significantly improving sperm motility and led to
high mitochondrial activity. Resveratrol reaches peroxi-
dized rigid membranes and increases membrane fluidity,
such that it interacts more efficiently with radicals in the
disordered lipid bilayer (Brittes et al., 2010). Owing to this
action, resveratrol increases sperm motility and acts against
LPO, preserving sperm chromatin and plasma membranes
(Collodel et al., 2011). The results obtained for motility
and mitochondrial activity in the present study are in con-
trast to those previously reported for frozen–thawed ram
and human sperm, where resveratrol supplementation did
not provide any improvement in post-thaw sperm motility
(Garcez et al., 2010; Meamar et al., 2012; Silva et al., 2012)
and mitochondrial activity (Silva et al., 2012). The dispar-
ity between the results of the present study compared with
previous studies was probably attributed to the different
species, extender composition and antioxidant doses used
in the indicated previous studies. On the other hand, the
resveratrol findings reported by Meamar et al. (2012) for
human sperm viability and motion characteristics are in
agreement with those obtained in the present study, such
that no statistically significant difference was determined
between the groups, except for the ALH value. ALH (mean
width (lm) of the head oscillation as the sperm swims)
may have a great impact on the conception rates achieved
Table 2 Mean (SEM) sperm motion charac-
teristics in frozen–thawed bull semen Groups VAP (lms
1
) VSL (lms
1
) VCL (lms
1
) ALH (lm) LIN (lms
1
)
Control 78 7.45
a
65.1 7.2
a
110.6 8.8
a
3.9 0.3
a
58.8 2.5
a
Lycopene
1910
3
gml
1
83.1 3.7
a
69.6 4.9
a
113.5 3.5
a
4.0 0.1
ab
61.3 3.7
a
Resveratrol 1 mM83.7 7.9
a
67.4 7.6
a
119.2 7.6
a
4.3 0.1
b
56.4 3.3
a
P–––*–
–, no significant difference (P>0.05).
*P<0.05. Different superscripts within the same column demonstrate significant differences.
Table 3 Mean (SEM) flourescent staining
parameters in frozen–thawed bull semen Groups Sperm viability (%)
Acrosome
integrity (%)
High mitochondrial
activity (%)
Damaged
DNA%
Control 31.7 3.9
a
42.2 4.0
a
11.8 9.5
a
11.8
a
Lycopene
1910
3
gml
1
36.2 6.4
a
48.7 4.4
a
24.6 2.9
b
6.5
b
Resveratrol 1 mM40.3 5.9
a
47.26 8.0
a
30.1 6.5
b
3.6
c
P––**
–, no significant difference (P>0.05).
*P<0.05. Different superscripts within the same column demonstrate significant differences.
Table 4 Mean (SEM) LPO levels (lM910
9
) and AOP (mM910
9
)
activities in frozen–thawed bull semen
Groups LPO (lM910
9
) AOP (mM910
9
)
Control 37.0 10.2
a
28.8 8.2
a
Lycopene 1 910
3
gml
1
23.6 12.0
a
24.1 3.0
a
Resveratrol 1 mM37.1 9.6
a
30.7 13.1
a
P––
–, no significant difference (P>0.05). Different superscripts within
the same column demonstrate significant differences.
©2014 Blackwell Verlag GmbH 549
Andrologia 2015, 47, 545–552
M. N. Bucak et al. Influence of antioxidants on bull sperm freezing
with the application of insemination techniques using fre-
ezed–thawed human sperm (Freour et al., 2010).
As shown in the Tables, the supplementation of the
semen extender with lycopene was proven to improve
sperm motility and high mitochondrial activity after
thawing. It has been proposed that lycopene displays an
antioxidative function in cells by donating its electrons to
oxygen free radicals; thus, quenching and neutralising
them before they can damage cells (Di Mascio et al.,
1989). The current finding of sperm motility was in
agreement with those reported for ram sperm (Uysal &
Bucak, 2007), which suggested the maintenance of motil-
ity with cryopreservation using a low dose of lycopene.
The comet assay is a widely applied technique for mea-
suring and analysing DNA breakage in individual cells
(Ostling & Johanson, 1984). It has also been proven as a
valid technique for evaluating the capacity of antioxidants
to protect the integrity of genetic material in biological
studies (Heaton et al., 2002; Novotna et al., 2007; Tuncer
et al., 2010). Furthermore, some authors suggest that
sperm DNA integrity is a more objective marker of sperm
function as opposed to sperm parameters such as motility
(Twigg et al., 1998; Rajesh et al., 2002). In this study, an-
tioxidants maintained DNA integrity, compared with the
controls. These results obtained for DNA were in agree-
ment with studies on human sperm following incubation
(Zini et al., 2010) and the liquid storage of rabbit semen
(Rosato et al., 2012), which suggested that DNA damage
was diminished with lycopene supplementation. It was
hypothesised that the dose-dependent effect of lycopene
leads to opposing outcomes (Uysal & Bucak, 2007). In a
previous study on resveratrol, the protective effect against
DNA damage was obtained at a much higher concentra-
tion (10 mM) for human sperm (Branco et al., 2010).
However, recently it has been reported that resveratrol is
highly toxic for humans at concentrations above 15 lM
(Collodel et al., 2011; Meamar et al., 2012). Although the
antioxidants used in the present study are free radical
scavengers (Leonard et al., 2003; Stahl & Sies, 2003), nei-
ther achieved any success in preventing LPO formation
and maintaining AOP, when compared with the controls.
The findings obtained in the present study for LPO are in
agreement with those reported for bull (Bucak et al.,
2010, 2012) and ram (Coyan et al., 2011) sperm, indicat-
ing no decrease in the LPO level in the presence of vari-
ous antioxidants for the frozen state. In addition to this
report, we could state that antioxidants are not an influ-
ential factor in the prevention of LPO following the
freeze–thawing of mammalian sperm.
In conclusion, the results of this study demonstrated the
impact of antioxidants against the cryoinjury of bovine
sperm in vitro. The results obtained suggest that the sup-
plementation of the semen extender with resveratrol and
lycopene offers protection for sperm motility, high mito-
chondrial activity and DNA integrity.
Supplementation of the semen extender with antioxi-
dants did not improve LPO level and AOP activity fol-
lowing the freeze–thawing process. This study highlights
the differential effect of resveratrol and lycopene on
sperm survival and DNA integrity, in the light of previous
studies, and makes a contribution to ruminant semen
technology to obtain high fertility.
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