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Theriogenology 68 (2007) 204–212

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, Greeceb 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-2,4-

dienoic 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) 204–212 205

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 [3–5]. ROS production has

been associated with reduction of sperm motility,

decreased capacity for sperm–oocyte fusion and

infertility. Spermatozoa are sensitive to lipid peroxida-

tion 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 [3,6–11].

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 [12–18] 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.

2. Materials and methods

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.

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 move-

ment) was estimated by subjective microscopic

examination using a phase contrast microscope

supplied with heated stage at 37 8C and magnification

400� [21–23].

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 eosin–nigrosin 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.

2.4. Semen processing

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 strep-

tomycin sulphate, 20 ml egg yolk, 3 ml glycerol and

distilled water to 100 ml, pH 6.4, without any

A. Michael et al. / Theriogenology 68 (2007) 204–212206

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)-2-

phenyl-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-benzylpeni-

cillin, 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 post-

thaw 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 concen-

tration 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

[27–29].

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 Krebs-

Ringer 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, Ger-

many); 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 absorption550–468 with SOD from

sample absorption550–468 without SOD) was multiplied

by 2.5 (dilution factor) and divided by 0.0242

(micromolar extinction coefficient of cytochrome c at

550–468), 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 Tris–fructose–

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) 204–212 207

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 trans-

formed 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 mea-

sured 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 measure-

ments of all method components were performed and

absorbance was recorded at a range of 420–210 nm.

These concluded that the oxidised form of luminol

showed higher absorbance compared with its non-

oxidised 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 absorbancewas 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 spectro-

photometric equation of Lambert Beer’s law. This

relationship may be expressed as:

A ¼ 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

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 sperma-

tozoal 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 consider-

able 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

A. Michael et al. / Theriogenology 68 (2007) 204–212208

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 (%) HOS test swollen

spermatozoa (%)

Control 39.00 � 2.90 b 17.00 � 2.26 cd 51.70 � 2.81 c 55.65 � 1.64 b

Vitamin C 40.00 � 2.99 ab 20.75 � 2.79 bc 54.20 � 1.75 bc 57.80 � 1.28 ab

NAC 46.00 � 2.87 ab 28.00 � 3.27 ab 60.60 � 1.93 ab 60.00 � 1.06 ab

Taurine 44.25 � 3.06 ab 25.50 � 2.66 abc 61.90 � 2.48 ab 61.25 � 1.49 a

Catalase 49.75 � 3.63 a 31.75 � 3.46 a 66.00 � 3.03 a 61.75 � 1.61 a

Vitamin E 44.50 � 3.52 ab 26.75 � 3.15 ab 60.50 � 4.12 ab 58.10 � 1.63 ab

B16 24.75 � 3.00 c 9.75 � 2.46 d 39.80 � 1.93 d 41.55 � 1.97 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 (%)

Acrosome

intact (%)

Acrosome

defected (%)

Acrosome

reacted (%)

Control 77.90 � 1.01 b 3.10 � 0.35 a 19.00 � 1.00 ab 21.20 � 2.11 a 35.60 � 3.98 a 42.10 � 3.73 a

Vitamin C 78.10 � 1.26 ab 2.90 � 0.49 ab 19.00 � 1.09 ab 22.30 � 2.08 a 33.10 � 4.74 a 44.60 � 4.06 a

NAC 81.10 � 1.24 a 2.40 � 0.17 ab 16.50 � 1.28 ab 25.50 � 1.68 a 33.40 � 2.51 a 41.10 � 3.57 a

Taurine 81.00 � 0.43 a 2.00 � 0.30 b 17.00 � 0.50 ab 21.80 � 1.62 a 30.30 � 4.69 a 47.90 � 5.17 a

Catalase 81.10 � 0.64 a 2.40 � 0.31 ab 16.30 � 0.72 b 23.00 � 1.86 a 31.00 � 3.88 a 46.00 � 4.04 a

Vitamin E 79.90 � 1.29 ab 3.20 � 0.33 a 17.20 � 1.11 ab 21.80 � 1.62 a 33.60 � 3.38 a 45.40 � 3.24 a

B16 77.00 � 0.75 b 3.40 � 0.27 a 19.50 � 0.96 a 14.40 � 1.64 b 32.10 � 5.03 a 53.50 � 3.81 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-

Table 3

Effect of antioxidant supplementation at superoxide anion production, hydro

semen

Antioxidant Superoxide anion production

(nmol/(ml min))

Control 0.066 � 0.016 b

Vitamin C 0.107 � 0.030 ab

NAC 0.080 � 0.027 ab

Taurine 0.073 � 0.016 b

Catalase 0.162 � 0.035 a

Vitamin E 0.141 � 0.040 ab

B16 0.076 � 0.025 ab

Means with different letters (a, b) in the same column indicate significant

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-

xyl radicals and total reactive oxygen species of frozen-thawed canine

Hydroxyl

radicals (%)

Total reactive oxygen

species (e)

9.23 � 1.04 ab 136.40 � 14.41 a

13.73 � 2.14 a 97.70 � 18.83 a

12.28 � 1.77 ab 105.10 � 20.39 a

12.14 � 2.35 ab 99.30 � 20.51 a

11.41 � 2.08 ab 107.50 � 10.80 a

8.66 � 1.21 ab 110.60 � 17.08 a

7.54 � 1.43 b 116.00 � 24.00 a

differences ( p < 0.05).

A. Michael et al. / Theriogenology 68 (2007) 204–212 209

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 sperma-

tozoa 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 signifi-

cantly 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 [32–34] 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,

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 con-

cerning sperm movement concluded that NAC pro-

tected semen, which was exposed to leukocyte-

mediated 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

NAC’s structure and chemical reaction, which is similar

to glutathione’s. 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].

A. Michael et al. / Theriogenology 68 (2007) 204–212210

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 chain-

breaking 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 malondial-

dehyde 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 0.02–0.6 mM adversely

affected sperm motility in normozoospermic and

asthenozoospermic samples [17]. Higher concentra-

tions 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-penta-

2,4-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 cryoprotec-

tants (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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