Theriogenology 68 (2007) 204212

www.theriojournal.com

Effect of antioxidant supplementation on semen quality and

reactive oxygen species of frozen-thawed canine spermatozoa
A. Michael a, C. Alexopoulos a,, E. Pontiki b, D. Hadjipavlou-Litina b,
P. Saratsis a, C. Boscos a,*
a

Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece

b
School of Pharmacy, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece
Received 27 November 2006; received in revised form 19 April 2007; accepted 25 April 2007

Abstract
The objective of this study was to evaluate post-thaw quality of frozen dog semen processed with diluents containing different
antioxidants. Ejaculates were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF
movement), viability, acrosomal integrity and by the hypo-osmotic swelling test. Also, superoxide production, hydroxyl radicals
and total reactive oxygen species (tROS) were determined. The pool was divided in seven aliquots, for control and test conditions,
which were processed for cryopreservation. The sperm pellets were diluted to a final concentration of 200  106 sperm/ml with
TRIS-glucose-egg yolk extender containing one of the following supplements: vitamin C (1.5 mM), NAC (N-acetyl-L-cysteine;
1.5 mM), taurine (0.6 mM), catalase (300 U/ml), vitamin E (0.3 mM) and B16 [5-(4-dimethylamino-phenyl)-2-phenyl-penta-2,4dienoic acid; 0.3 mM]. Post-thaw semen evaluation showed that mean (S.E.M.) motility was increased ( p < 0.001) after addition
of catalase (49.75  3.63 versus 39.00  2.90 in controls), whereas more spermatozoa with RSF movement were observed
( p < 0.001) after the catalase, NAC and vitamin E treatments (31.75  3.46, 28.00  3.27, 26.75  3.15, respectively, versus
17.00  2.26 in controls). Viability was increased ( p < 0.001) after addition of catalase, taurine, NAC and tocopherol
(66.00  3.03, 61.90  2.48, 60.60  1.93 and 60.50  4.12, respectively, versus 51.70  2.81 in controls). The percentage of
swollen spermatozoa was increased after addition of catalase and taurine (61.75  1.61 and 61.25  1.49, respectively, versus
55.65  1.64 in controls). Acrosomal integrity was not influenced in any case. B16 addition had adverse effects on all parameters
evaluated. None of the reactive oxygen species were significantly reduced post-thaw in antioxidant treated semen. The results
suggest that catalase had the most pronounced effect in improving post-thaw quality of canine spermatozoa.
# 2007 Elsevier Inc. All rights reserved.
Keywords: Canine sperm; Frozen semen; Antioxidants; Reactive oxygen species

1. Introduction
Artificial insemination is widely used in most
domestic animals throughout the world in our days.
* Corresponding author at: Faculty of Veterinary Medicine, St.
Voutyra 11, 546 27 Thessaloniki, Greece. Tel.: +30 2310 994471;
fax: +30 2310 994471.
E-mail address: pboscos@vet.auth.gr (C. Boscos).

Deceased author.
0093-691X/$ see front matter # 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2007.04.053

Particularly in dogs the use of frozen semen eliminates

the costly, dangerous and time-consuming animal
transportation, allows genetically superior dogs to
reproduce even if natural mating is impossible, and
allows breeders to store semen for future use. However,
cryopreserved dog semen has a short lifespan and
markedly reduced semen quality after thawing. In
combination with the difficulty of determining the
optimal insemination time and the need for intrauterine
insemination, fertility problems are encountered when

A. Michael et al. / Theriogenology 68 (2007) 204212

cryopreserved semen is used. Conception rates depend

on the post-thaw quality of canine semen and are
generally lower than those with fresh semen [1]. This
reduction is a result of lower post-thaw viability
rates and sublethal dysfunction of the surviving
spermatozoa [2].
Reactive oxygen species (ROS) generation, induced
by the cryopreservation process, can be responsible for
mammalian sperm damage [35]. ROS production has
been associated with reduction of sperm motility,
decreased capacity for spermoocyte fusion and
infertility. Spermatozoa are sensitive to lipid peroxidation due to their high content of polyunsaturated fatty
acids, and are unable of resynthesizing their membrane
components, although this may not be the sole
mechanism by which sperm function is impaired by
ROS.
Antioxidant molecules could reduce the impact of
oxidative stress, and thus improve semen quality after
thawing. The aim of antioxidant treatments should not
be the complete ROS elimination as oxidative
mechanisms play an important role in the physiological
control of mammalian sperm functions [3,611].
Whenever ROS are found in low concentrations they
act as mediators of normal sperm functions, whereas
whenever they are produced in excess they are highly
toxic to the cell. Several antioxidant agents, such as
vitamins E and C, catalase, dimethylsulphoxide,
taurine, hypotaurine and N-acetylcysteine have already
been tested in vitro or in vivo studies concerning human,
bovine, boar, rabbit and stallion semen [1218] with
controversial efficacy and usefulness. It has been shown
that canine fresh semen is protected against oxidative
stress by endogenous SOD activity and by vitamin E
oral supplementation [19,20]. However, there are no
available references relative to the possible antioxidant
protection in canine frozen semen. The aim of the
present study was to evaluate post-thaw quality of dog
semen extended with diluents containing different
antioxidants.

205

2.2. Collection and evaluation of ejaculates

One ejaculate was obtained from each dog by digital
manipulation according to the technique previously
described by Christiansen [21]. The sperm-rich
fraction was collected into a prewarmed glass
calibrated tube, its volume was recorded and it was
placed in a water bath at 37 8C. A total of 10 ejaculates
per dog were collected during this experiment at once
or twice a week basis. Each ejaculate was analysed to
determine its semen concentration, total number of
spermatozoa and sperm motility, so that adequate
semen quality was secured. Only ejaculates with
motility >75% and sperm concentration of >200 
106 spermatozoa/ml were included in this study. Sperm
concentration was determined using a Neubauer
haemocytometer, after diluting semen with 0.05%
formal saline (dilution rate 1:400). The percentage of
motile spermatozoa and the percentage of spermatozoa
showing rapid steady forward movement (RSF movement) was estimated by subjective microscopic
examination using a phase contrast microscope
supplied with heated stage at 37 8C and magnification
400 [2123].
2.3. Pooled semen examination
The five ejaculates were each time (n = 10) pooled,
in order to increase semen volume and eliminate
variability between the evaluated samples. One aliquot
was removed and was evaluated for concentration,
sperm motility, viability, acrosomal integrity and by
the hypo-osmotic swelling test (HOS test). Viability/
morphological defects and acrosomal status were
assessed by means of eosinnigrosin and Spermac
stain (Ferti Pro1, Belgium), respectively. A total of 200
spermatozoa were counted on each slide at a 1000
magnification. For the HOS test the percentage of 200
spermatozoa with coiled/swollen tails was determined
after a 45-min incubation of 0.05 ml of semen in 0.5 ml
of 60-mOsm fructose solution.

2. Materials and methods

2.4. Semen processing
2.1. Animals
Five healthy and sexually mature dogs were used in
this study: three Golden Retrievers, one Alaskan
Malamute and one crossbred, ranging between 2 and
7 years of age, weighting between 24 and 56 kg, and
with proven fertility after natural mating. The dogs were
trained for semen collection before the study and were
used routinely as semen donors.

The pooled semen was centrifuged at 700  g for

6 min and the seminal fluid was removed. In order
to facilitate the division of the sperm-rich pellet to
the necessary aliquots, it was first diluted with Ext-1
(Ext-1 composition: 2.4 g Tris, 1.4 g citric acid, 0.8 g
glucose, 100,000 iu Na-benzylpenicillin, 0.1 g streptomycin sulphate, 20 ml egg yolk, 3 ml glycerol and
distilled water to 100 ml, pH 6.4, without any

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A. Michael et al. / Theriogenology 68 (2007) 204212

antioxidants) [24,25], to obtain concentration of

800  106 spermatozoa/ml. The diluted semen was
divided in seven aliquots, of which one was control and
six were for test conditions. Each sperm aliquot was
diluted with an equal of its volume of Ext-1 in order to
achieve concentration of 400  106 spermatozoa/ml,
that was either without antioxidants (control), or each
of the six test groups was supplemented with one of the
various antioxidants in the following concentrations:
vitamin C (6 mM), NAC (N-acetyl-L-cysteine; 6 mM),
taurine (2.4 mM), catalase (1200 U/ml), vitamin E
(1.2 mM) or B16 [5-(4-dimethylamino-phenyl)-2phenyl-penta-2,4-dienoic acid; 1.2 mM]. Immediately
after dilution portion from the control aliquot was
removed in order to estimate reactive oxygen species
of fresh semen, i.e. superoxide anion production,
hydroxyl radicals and total reactive oxygen species.
The seven aliquots [one control (without antioxidants)
and six test groups (each containing one of the
antioxidants)] were cooled to 4 8C and equilibrated for
1 h. The aliquots were rediluted with an equal of their
volume of Ext-2 [same composition as Ext-1 except
that it contained 7% glycerol and 1% Equex STM paste
(Nova Chemical Sales Inc., Scituate, MA, USA),
without any antioxidants]. After all dilutions were
completed aliquots obtained a final concentration of
200  106 spermatozoa/ml and contained one of the
following supplements: control (without antioxidant),
vitamin C (1.5 mM), NAC (1.5 mM), taurine
(0.6 mM), catalase (300 U/ml), vitamin E (0.3 mM)
and B16 (0.3 mM).
The semen was frozen in 0.5 ml straws in a
programmable freezer at a freezing rate of 5 8C/min
from 4 to 15 8C, and of 20 8C/min from 15 to
100 8C [26]. Immediately after, the straws were
immersed in liquid nitrogen. The straws were thawed at
70 8C for 8 s and each aliquot was slowly diluted with a
TRIS-buffer (TRIS-buffer composition: 2.4 g Tris, 1.4 g
citric acid, 0.8 g glucose, 100,000 iu Na-benzylpenicillin, 0.1 g streptomycin sulphate and distilled water to
100 ml, pH 6.5) at 37 8C at a rate of 1:2, before the postthaw evaluation was conducted.
Samples containing extender from each group were
handled in the same way as semen aliquots and were
used in order to subtract the extender value from the
relevant semen value during the hydroxyl radical and
total reactive oxygen species determination.
Fresh semen and extender of the control group, prior
to ROS measurements, were diluted at a rate 1:6, so that
the results from fresh and frozen-thawed samples would
be comparative, referring to the same semen concentration of 66.66  106 spermatozoa/ml.

2.5. Superoxide anion radical assay

Extracellular Superoxide anion (O2) in semen was
measured by a spectro-photometric method based on
the dismutase-inhibitable reduction of cytochrome c
[2729].
Aliquots of diluted semen (0.1 ml) were incubated in
triplicate samples for 30 min at 37 8C with a solution
containing the following: 0.36 ml modified KrebsRinger phosphate buffer (KRP; Merck, Germany) with
0.04 mmol cytochrome c (type VI, from horse heart,
Sigma, Germany); 0.02 ml Tris buffer with or without
150 U SOD (from bovine erythrocytes, Fluka, Germany); 0.02 ml Tris buffer with 50 U catalase (from
bovine liver, Sigma, Germany). Incubation was
terminated by the addition of 2 ml of ice-cold KRP
containing 1 mM N-ethylmaleimide. The test tubes
were centrifuged at 600  g for 10 min.
Superoxide anion-dependent cytochrome c reduction
was calculated by subtracting the reduction occurring in
the presence of SOD from that occurring in its absence.
For the calculation, the resulting absorption (obtained
by subtracting sample absorption550468 with SOD from
sample absorption550468 without SOD) was multiplied
by 2.5 (dilution factor) and divided by 0.0242
(micromolar extinction coefficient of cytochrome c at
550468), thus giving the nanomoles of the O2
produced by 0.1 ml of semen. The results were also
expressed as nmol O2/(ml min).
2.6. Hydroxyl radical assay


Hydroxyl radical (OH ) measurement was performed

by the determination of formaldehyde, which was
produced by the oxidation of dimethylsulphoxide
(DMSO) [30,31], modified in order to measure the

existing OH in semen.

Prior to OH measurement, washing of spermatozoa
was performed. Five hundred microlitres of semen or
extender were mixed with 1000 ml of Trisfructose
citric acid buffer (TFC; Tris 3.025 g, fructose 1.25 g,
citric acid 1.7 g, distilled water 100 ml, pH 7.0) and
centrifuged at 700  g for 8 min. Then 1 ml of the
supernatant was discarded and 1 ml of TFC buffer was
added and the mixture was gently mixed by vortex.
Aliquots of washed semen and extender (0.1 ml) were
incubated in triplicate samples for 30 min at 37 8C with
the reaction mixture that contained: 500 ml phosphate
buffer (50 mM, pH 7.4), 200 ml EDTA (0.1 mM in
phosphate buffer), 200 ml DMSO (33 mM in phosphate
buffer). The reaction was stopped with 250 ml
CCl3COOH (17.5%, w/v) and the formaldehyde pro-

A. Michael et al. / Theriogenology 68 (2007) 204212

duced was measured spectro-photometrically at 412 nm

after addition of 1 ml Nash solution, at 60 8C for 10 min.
Nash solution contained 45 g CH3COONH4, 0.9 ml
CH3COOH and 0.6 ml CH3COCH2COCH3 in 100 ml
distilled water.

The OH amount was calculated by subtracting the
absorbance of the extender from the absorbance of the
relevant semen sample. The value taken was transformed in percentage (%). The difference in absorbance
of mixtures incubated, which contained phosphate
buffer, EDTA, FeCl3 (167 mM) and ascorbic acid
(10 mM), in the presence or absence of DMSO was
defined as 100%.
2.7. Total reactive oxygen species (tROS)
determination
Total reactive oxygen species (tROS) were measured spectro-photometrically with the use of luminol
(5-amino-2,3-dihydro-1,4-phthalazinedione), which is
an extremely sensitive chemiluminescent probe that
reacts with a variety of ROS. Separate test measurements of all method components were performed and
absorbance was recorded at a range of 420210 nm.
These concluded that the oxidised form of luminol
showed higher absorbance compared with its nonoxidised form with the peak absorbance being
demonstrated at 380 nm.
Aliquots (100 ml) of semen and its relevant extender in
triplicate samples were diluted in 890 ml distilled water.
Ten microlitres of luminol (1 mM in DMSO) was added
and absorbance was measured spectro-photometrically at
380 nm. The absorbance of the extender was subtracted
from the absorbance of the relevant semen sample and
results were expressed with absorbance coefficient price
e, which was computed according to the spectrophotometric equation of Lambert Beers law. This
relationship may be expressed as:
A edc
where A = absorbance; e = molar extinction coefficient;
d = pathlength in cm; c = molar concentration.
2.8. Statistical analysis
Calculation of means, standard error and statistical
analysis were performed using SPSS 14.0 for Windows
software (SPSS Inc., Chicago, IL, USA). Data (10
replicates) were subjected to one-way analysis of
variance (ANOVA) and the Duncan multiple range test
was used to define differences among treatments.
Levene test was also used to check homogeneity of

207

variances. Differences were considered statistically

significant when p < 0.05.
3. Results
The pooled fresh semen (n = 10) was of normal
appearance (white in colour with milky viscosity). The
volume of the pool was 14.73  0.76 ml (all values are
given as mean  S.E.M.) with a sperm concentration of
305.50  17.24  106 spermatozoa/ml, giving a total
number of 4416.63  192.68  106 spermatozoa.
Sperm motility, rapid steady forward (RSF) movement
and viability were 84.00  0.67%, 76.00  1.25% and
89.00  0.76%, respectively. Morphologically normal
spermatozoa were 90.30  0.81% and the primary and
secondary defects were 2.70  0.30% and 7.00 
0.70%, respectively. Intact, defected and reacted
acrosomes were 88.90  0.86%, 7.10  0.57% and
4.00  0.65%, respectively. In fresh semen, the
percentage of spermatozoa with swollen tails was
91.05  0.49%, the superoxide production was 0.179
 0.061 nmol O2/(ml min), the hydroxyl radicals
were 15.30  1.91% and price e for tROS was
23.50  2.30.
Antioxidant addition affected post-thaw semen
quality compared with the control group (mean
 S.E.M. post-thaw values are presented in Tables 1
and 2). More specific the percentages of progressively
motile spermatozoa were higher ( p < 0.001) when
catalase was added in the semen extender. Spermatozoa
showing post-thaw RSF movement were significantly
increased ( p < 0.001) after catalase, NAC, and vitamin
E treatments. Post-thaw viability was significantly
( p < 0.001) higher in samples frozen with catalase,
taurine, NAC and vitamin E compared. Additionally,
more spermatozoa remained morphologically normal
after catalase, NAC and taurine addition ( p = 0.010).
None of the antioxidant treatments influenced spermatozoal acrosome condition positively. Catalase and
taurine addition in extenders resulted in significantly
higher post-thaw percentages of spermatozoa with
swollen/coiled tails when tested by the hypo-osmotic
test ( p < 0.001). Finally from both tables a considerable negative influence on motility, viability, intact
acrosomes and swollen spermatozoa (HOS test)
percentages was shown in the B16 treated group,
compared with the control.
The ROS values were altered by the cryopreservation
process and in some cases significant differences were
observed between fresh and post-thaw semen values.
Total ROS were significantly increased after the
cryopreservation process in all tested groups compared

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A. Michael et al. / Theriogenology 68 (2007) 204212

Table 1
Mean (S.E.M.) sperm motility, rapid steady forward (RSF) movement, viability and HOS test swollen spermatozoa of frozen-thawed dog semen
after addition of various antioxidants in semen extenders
Antioxidant

Total motility (%)

RSF movement (%)

Viability (%)

Control
Vitamin C
NAC
Taurine
Catalase
Vitamin E
B16

39.00  2.90
40.00  2.99
46.00  2.87
44.25  3.06
49.75  3.63
44.50  3.52
24.75  3.00

17.00  2.26
20.75  2.79
28.00  3.27
25.50  2.66
31.75  3.46
26.75  3.15
9.75  2.46

51.70  2.81
54.20  1.75
60.60  1.93
61.90  2.48
66.00  3.03
60.50  4.12
39.80  1.93

b
ab
ab
ab
a
ab
c

cd
bc
ab
abc
a
ab
d

HOS test swollen

spermatozoa (%)
55.65  1.64
57.80  1.28
60.00  1.06
61.25  1.49
61.75  1.61
58.10  1.63
41.55  1.97

c
bc
ab
ab
a
ab
d

b
ab
ab
a
a
ab
c

Means with different letters (a, b, c, d) in the same column indicate significant differences ( p < 0.05).

Table 2
Sperm morphological characteristics and acrosome condition of frozen-thawed dog semen after addition of various antioxidants in semen extenders
(mean  S.E.M.)
Antioxidant

Morphological
normal (%)

Primary defects
(%)

Secondary
defects (%)

Control
Vitamin C
NAC
Taurine
Catalase
Vitamin E
B16

77.90  1.01
78.10  1.26
81.10  1.24
81.00  0.43
81.10  0.64
79.90  1.29
77.00  0.75

3.10  0.35
2.90  0.49
2.40  0.17
2.00  0.30
2.40  0.31
3.20  0.33
3.40  0.27

19.00  1.00
19.00  1.09
16.50  1.28
17.00  0.50
16.30  0.72
17.20  1.11
19.50  0.96

b
ab
a
a
a
ab
b

a
ab
ab
b
ab
a
a

Acrosome
intact (%)
ab
ab
ab
ab
b
ab
a

Acrosome
defected (%)

21.20  2.11
22.30  2.08
25.50  1.68
21.80  1.62
23.00  1.86
21.80  1.62
14.40  1.64

a
a
a
a
a
a
b

35.60  3.98
33.10  4.74
33.40  2.51
30.30  4.69
31.00  3.88
33.60  3.38
32.10  5.03

Acrosome
reacted (%)
a
a
a
a
a
a
a

42.10  3.73
44.60  4.06
41.10  3.57
47.90  5.17
46.00  4.04
45.40  3.24
53.50  3.81

a
a
a
a
a
a
a

Means with different letters (a, b) in the same column indicate significant differences ( p < 0.05).

with the fresh semen ( p < 0.001, p = 0.002, p < 0.001,

p = 0.001, p < 0.001, p < 0.001 and p < 0.001 for the
control, vitamin C, NAC, taurine, catalase, vitamin E
and B16 group, respectively). The amount of hydroxyl
radicals decreased after the cryopreservation process in

all groups, compared with the fresh semen OH value,
but the reduction was significant only in the control,
vitamin E and B16 groups ( p = 0.012, p = 0.009 and
p = 0.004, respectively). After semen thawing the
superoxide production decreased in all groups com-

pared with the fresh semen sample, but none of the

differences was statistically significant ( p > 0.05). The
mean (S.E.M.) post-thaw values of reactive oxygen
species in the different experimental groups are
presented in Table 3. Post-thaw no significant difference
of the tROS values was observed between antioxidant
groups and the control ( p = 0.803). After semen
thawing none of the antioxidant groups differed
significantly compared to the control ( p = 0.156), but
the hydroxyl radical percentage tended to be signifi-

Table 3
Effect of antioxidant supplementation at superoxide anion production, hydroxyl radicals and total reactive oxygen species of frozen-thawed canine
semen
Antioxidant

Superoxide anion production

(nmol/(ml min))

Hydroxyl
radicals (%)

Control
Vitamin C
NAC
Taurine
Catalase
Vitamin E
B16

0.066  0.016
0.107  0.030
0.080  0.027
0.073  0.016
0.162  0.035
0.141  0.040
0.076  0.025

9.23  1.04
13.73  2.14
12.28  1.77
12.14  2.35
11.41  2.08
8.66  1.21
7.54  1.43

b
ab
ab
b
a
ab
ab

Total reactive oxygen

species (e)
ab
a
ab
ab
ab
ab
b

Means with different letters (a, b) in the same column indicate significant differences ( p < 0.05).

136.40  14.41
97.70  18.83
105.10  20.39
99.30  20.51
107.50  10.80
110.60  17.08
116.00  24.00

a
a
a
a
a
a
a

A. Michael et al. / Theriogenology 68 (2007) 204212

cantly higher in the vitamin C treatment compared

with the B16 treated group ( p = 0.080). Spermatozoal
post-thaw superoxide production was significantly
higher when the freezing diluent contained catalase
compared with the control and the taurine treatment
( p = 0.047).
4. Discussion
ROS in the ejaculate are produced by the spermatozoa themselves and by leucocytes, which are
infiltrated into semen [3]. Spermatozoa are vulnerable
to ROS damage due to their high polyunsaturated fatty
acid content and the use of antioxidants could reduce
the negative ROS impact to spermatozoa. The present
study showed that certain antioxidants could significantly improve dog semen quality parameters after
thawing. Catalase was the most effective of the
antioxidants tested by significantly influencing almost
all of the semen quality parameters. It constitutes one of
the three enzymatic systems semen has for its own
protection against ROS damage that allows the
degradation of hydrogen peroxide (H2O2) into oxygen
and water [3]. Hydrogen peroxide is considered the
most toxic species [3234] because of its ability to cross
membranes freely and to inhibit enzyme activities and
cellular functions, thus decreasing the antioxidant
defences of the spermatozoa [34]. The non-significant
reduction of the ROS measured in the present study may
be due to the selective elimination of hydrogen peroxide
by catalase, and thus the superoxide anion production
and the hydroxyl radical percentages were left
unimpaired. Most mammalian semen (bull, ram, boar,
rabbit, human) contain little or no catalase [35]. Indeed,
in a study concerning canine semen [20] no catalase was
found in any of the seminal plasma samples. The
positive effect of catalase addition in this study may
result by covering this probable deficit. From the
significant increase of superoxide anion production of
our findings we can hypothesize that either superoxide
is not as toxic to canine semen or catalase has the ability
to protect dog spermatozoa against the damage it
causes. This may be explained by the fact that O2 is a
primary product that subsequently dismutases to H2O2,
under the influence of SOD, which is the main target of
catalase.
The next best post-thaw semen quality was obtained
with the acetylcysteine and taurine treatments. From the
addition of the thiol-containing N-acetyl-L-cysteine
(NAC) at the extender, it can be hypothesized that NAC
may be protecting semen from oxidative stress in an
indirect way without eliminating the presence of ROS,

209

because semen quality parameters were benefited (i.e.

RSF movement, viability, morphologically normal
spermatozoa), although the ROS values were not
significantly altered. Previous studies in human concerning sperm movement concluded that NAC protected semen, which was exposed to leukocytemediated oxidative stress [15]. On the contrary, thiols
added in extenders had deleterious effects on sperm
motility of bull semen after cryopreservation [36]. In
human semen when tested in vitro it was proven that
NAC had a distinct scavenging effect against ROS, with
the concentration of 1 mg/ml being the most effective
[37]. These antioxidant properties are explained by
NACs structure and chemical reaction, which is similar
to glutathiones. It is believed to work mostly as a
precursor of intracellular cysteine and glutathione and
as a stimulator of cytosolic enzymes participating to
glutathione metabolism. Also NAC may act through
direct chemical interaction with radical species and/or
ROS-dependent by-products [38].
Taurine and hypotaurine are considered important
for sperm motility and fertility [39]. Our taurine
findings are contrary to the findings from its use on
rabbit epididymal spermatozoa, where inhibition of loss
of forward motility and reduction of the rate of lipid
peroxidation were observed [12]. In hamster sperm
taurine and hypotaurine are believed to have in vivo
roles in the maintenance and stimulation of sperm
motility, as well as stimulation of capacitation and/or
acrosome reactions [40]. Sperm protection resulting
from reactive oxygen inhibition was not observed in the
present study. Taurine is also known to play an
important role in osmoregulation, ion modulation and
neurotransmission [39], properties that may explain the
limited benefits of this antioxidant in our study. The
superoxide inhibition of the taurine treated semen
compared with catalase group may be explained if
taurine behaves like hypotaurine, which is reported to
reduce superoxide production by protecting the sperm
SOD from inactivation [12]. Intracellular superoxide is
the principal inducer of peroxidation in rabbit
spermatozoa [41], resulting in SOD inactivation.
Exogenous SOD and catalase cannot transverse the
membrane and consequently cannot protect the sperm
[12]. So spermatozoa can be protected from the addition
of antioxidants (i.e. taurine), which can traverse the
sperm plasma membrane, and inhibit lipid peroxidation,
regardless of the extracellular superoxide production.
Finally, taurine is known to have a beneficial effect on
mammalian sperm characteristics by protecting the
cells against ROS accumulation when they are exposed
in aerobic conditions [40,42].

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In this study a-tocopherol was used, which is the

most potent antioxidant compound of vitamin E. Its
addition was of very limited benefit to post-thaw quality
of dog semen. The non-significant elimination of the
ROS values was expected because vitamin E is a chainbreaking and not a scavenging antioxidant [43]. This
feature results in the neutralizing of lipid radicals [44],
so it offers protection to membrane components without
influencing ROS generation [45]. It seems that in human
seminal plasma tocopherol might not be needed in large
quantities because it can be regenerated by ascorbate
[17]. Studies in various mammals (rabbit, equine,
bovine, boar and ram) aiming to improve semen quality
by addition of tocopherol had conflicting results
[20,46]. In humans oral administration of vitamin E
improved sperm function [14]. Also vitamin E gave a
significant dose-dependent suppression of malondialdehyde production of human spermatozoa without
completely preventing the peroxidation process. At the
same time, motility and the ROS generation capacity
were left unimpaired [47], which comes in agreement
with our findings. In bovine cryopreserved spermatozoa
vitamin E showed a protective effect on the plasma
membrane integrity during deep freezing [48]. In
another study concerning dogs, it was concluded that
vitamin E treatment overcame the negative effects on
semen quality that dexamethasone treatment (to mimic
stress) induced [20].
Vitamin C did not influence post-thaw semen
quality in any way and resulted in post-thaw semen
quality similar to that of the control group. Vitamin C
might have controversial from the expected results
because it may also act as a proxidant. In the presence
of transition metals vitamin C makes radicals highly
reactive and more destructive, thus generating more
free radicals [49]. Previous studies in humans showed
that ascorbate in the range of 0.020.6 mM adversely
affected sperm motility in normozoospermic and
asthenozoospermic samples [17]. Higher concentrations of vitamin C (2.5 mM) proved detrimental to
sperm motility frozen-thawed bull semen [42], but
5 mM addition exerted a significant protective effect
on lipid peroxidation of good quality bovine frozen
spermatozoa [13]. In humans, low or deficient vitamin
C levels have been associated with low sperm counts,
increased numbers of abnormal sperm, reduced
motility and agglutination. However unlike humans,
dogs and cats are capable of producing adequate
amounts of vitamin C in their bodies to meet their
metabolic needs [49]. This can possibly explain why
dog spermatozoa were not benefited from the vitamin
C treatments.

B16 [5-(4-dimethylamino-phenyl)-2-phenyl-penta2,4-dienoic acid] addition proved to be detrimental to

post-thaw semen quality and did not reduce the ROS
impact effectively, contrary to the beneficial results
when used on extended semen (unpublished data) and
the in vitro important antioxidant properties [31]. Being
used in frozen semen studies for the first time it is most
probable that in the presence of extender cryoprotectants (glycerol and/or Equex STM paste) the lipid
soluble B16 obtained spermicide characteristics.
In conclusion, from the present study it seems that
some antioxidant treatments can sufficiently protect
canine semen during cryopreservation. Our findings
showed that from all the antioxidants tested catalase
addition in semen extenders was of greater benefit to
frozen-thawed dog semen. Antioxidant addition might
also be of further benefit to spermatozoa in the female
reproductive tract, which is characterized by higher
levels of oxidative stress [50,51]. More studies are
needed in order to determine in vivo canine fertility, to
find the appropriate antioxidants and to define the most
effective concentrations, which will improve post-thaw
quality and increase fertility rates when frozen-thawed
dog semen is used.
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