Animal Reproduction Science 125 (2011) 189–195

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

The influence of certain aminoacids and vitamins on post-thaw fish

sperm motility, viability and DNA fragmentation
E. Cabrita a,∗ , S. Ma b , P. Diogo b , S. Martínez-Páramo b , C. Sarasquete a , M.T. Dinis b
a
ICMAN-Institute of Marine Science of Andalusia, Spanish National Research Council, Av. Republica Saharaui 2, 11510 Puerto Real, Cádiz, Spain
b
CCMAR-Center for Marine Science, University of Algarve, Campus Gambelas, 8005-139 Faro, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: During cryopreservation, dilution in the extender media reduces the seminal plasma con-
Received 28 November 2010 stituents being cells more vulnerable to oxidative stress. Vitamins (C and E) and the amino
Received in revised form 24 February 2011
acids taurine and hypotaurine are powerful antioxidants naturally present in seminal
Accepted 8 March 2011
plasma. Whether their effect may improve sperm quality and reduce sperm DNA dam-
Available online 15 March 2011
age after cryopreservation in fish sperm still remains unclear. Thus, the aim of the present
work was to analyse the effect of extender supplementation with several antioxidant com-
Keywords:
ponents on post-thawed sperm motility, viability and DNA integrity of two commercial
DNA damage
Comet assay species, gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax).
Extender supplementation Sperm collected from ten to twelve individuals was cryopreserved in ten different exten-
Sperm cryopreservation ders containing: taurine and hypotaurine (1 and 10 mM), ascorbic acid (1 and 10 mM),
Antioxidants ␣-tocoferol (0.1 and 0.5 mM) or 1 ml/l of a commercial cell antioxidant supplement. Cell
viability, motility and DNA fragmentation were determined in post-thawed samples. Addi-
tion of antioxidants (vitamins and amino acids) to D. labrax and S. aurata extenders did
not significantly increase the parameters of motility (TM, PM, VCL, VSL and Lin) or via-
bility, although 1 mM taurine slightly increased the percentage of motile cells (TM) in S.
aurata. DNA fragmentation (DNA in tail and Olive tail moment) in D. labrax sperm was
higher in treatments containing vitamins than amino acids or control. However in S. aurata
sperm, antioxidants especially taurine and hypotaurine, significantly reduced both DNA
fragmentation parameters, protecting DNA against strand breaks. These results suggest a
species-specific effect depending on the type of antioxidants used.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction with vitamins. Previously, Ciereszko and Dabrowski (1995)

Antioxidants play an important role in sperm motil-
ity, integrity, metabolism and function, protecting the cells
against oxidative damage (Alvarez and Storey, 1983). Their
effects have been widely studied in mammalian sperma-
tozoa (Aitken and Baker, 2004), however little attention
has been paid to fish sperm. The positive effects of antioxi-
dants recently demonstrated by Mansour et al. (2006) and
Metwally and Fouad (2009) in fish also reported increasing
sperm antioxidant defense after dietary supplementation
∗ Corresponding author. Tel.: +34 956832612; fax: +34 956832612.
E-mail address: elsa.cabrita@icman.csic.es (E. Cabrita).
and Lee and Dabrowsky (2004), demonstrated the same
effect in rainbow trout (Oncorhynchus mykiss) and yel-
low perch, (Perca flavescens), increasing sperm antioxidant
resistance to reactive oxygen species (ROS).
Production of ROS, is involved in changes in sperm
membrane fluidity, DNA fragmentation, protein oxida-
tion, mitochondrial impairment, and consequently, in a
decrease in motility and fertility (Sanocka and Kurpisz,
2004). Moreover, Lahnsteiner et al. (2010) demonstrated
that, not only did non-enzymatic compounds protect
the spermatozoa, but that sperm in vitro incubation
with antioxidant enzymes, such as catalase, produced an
increase in motility and integrity in brown trout sperm dur-
ing short storage (Salmo trutta), suggesting the importance

0378-4320/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2011.03.003
190 E. Cabrita et al. / Animal Reproduction Science 125 (2011) 189–195
of introducing these compounds during cryopreservation.
However, to our knowledge, no studies have been per-
formed using in vitro supplementation of antioxidants in
the extender media for fish sperm cryopreservation and
analysing their effects on sperm post-thaw quality.
Cryopreservation produces chemical and osmotic
oxidative stress on sperm structure reducing sperm motil-
ity, viability and fertilizing ability due to significant ROS
generation (Ball, 2008). Recently, DNA damage has also
been associated with damage at oxidative level in fish
sperm, being one of the causes for DNA fragmentation
(Pérez-Cerezales et al., 2009). DNA strand breaks and base
oxidization are promoted during freezing–thawing and
have been described in several species (Baumber et al.,
2003; Pérez-Cerezales et al., 2009; Thomson et al., 2009),
reducing offspring viability (Fernandez-Gonzalez et al.,
2008; Pérez-Cerezales et al., 2009, 2010).
Sperm protection against oxidative stress is mainly
provided by seminal plasma, which contains several
antioxidant components. However during cryopreserva-
tion, dilution in the extender media reduces the seminal
plasma constituents being cells more vulnerable to oxida-
tive stress. Martínez-Páramo et al. (2009) demonstrated
that total antioxidant status (TAS) was undetected after
sperm dilution in the extender media in gilthead seabrem
(Sparus aurata), although it was present in seminal plasma
before dilution. Damage to sperm function was successfully
minimized in several mammalian species by the addition
of antioxidants to the extender media prior to cryop-
reservation (Bucak et al., 2007; Thuwanut et al., 2008). It
has been demonstrated that aminoacids such as taurine,
hypotaurine, proline or glutamine reduce sperm damage
due to oxidative stress, improving post-thaw motility in
several mammalian species (bull, ram, stallion and goat,
Chen et al., 1993; Sánchez-Partida et al., 1997; Kundu
et al., 2001; Bucak et al., 2007). Taurine and hypotaurine
are sulfuronic amino acids that bind with alcoxyl, peroxyl
and hydroxyl ions (OH− ) acting as free radical scavengers
(Aitken and Baker, 2004). Vitamins are well known natu-
ral antioxidants. Among the chain-breaking antioxidants
which prevent the propagation of free-radical-induced
chain reactions, ascorbic acid and ␣-tocopherol are par-
ticularly important. Both have the capacity to prevent
lipid peroxidation and to reduce sperm oxidative damage
(Halliwell and Gutteridge, 1999). Whether their effect may
improve sperm quality and reduce sperm DNA damage
after cryopreservation in fish sperm still remains unclear.
The aim of the present work was to analyse the effect
of extender supplementation with several antioxidant
components, naturally present in seminal plasma, on post-
thawed sperm motility, viability and DNA integrity of two
important commercial species, gilthead seabream (Sparus
aurata) and European seabass (Dicentrarchus labrax).
2. Materials and methods
2.1. Reagents and solutions
Antioxidants and all chemicals were purchased from
Sigma–Aldrich, (Madrid, Spain). The dual stain propidium
iodide (PI)/SYBR-14 and the ApoTarget APO-BRDU Kit (ter-
minal deoxynucleotidyl transferase–mediated dUTP nick
end-labeling) known as the TUNEL test were purchased
from Invitrogen (Madrid, Spain). All freezing extenders
were prepared one day in advance and maintained at 4 ◦ C,
and osmolarity and pH were set to 320 mOsm/Kg and 7.4,
respectively.
2.2. Sperm collection
Sperm from European seabass (Dicentrarchus labrax,
n = 10) and gilthead seabream (Sparus aurata, n = 8) was col-
lected by stripping the males during February. Only males
producing more than 4 ml of sperm were used. The gilthead
seabream were kept at the ICMAN.CSIC (Cadiz, Spain) facil-
ities and the European seabass were obtained from a fish
farm (A. Coelho e Castro Lda, Povoa de Varzim, Portugal).
Samples were stored at 4 ◦ C until freezing (for no longer
than 1 h).
2.3. Incorporation of antioxidants in the extender media
Each sperm sample was diluted in ten different exten-
ders containing 1% NaCl plus 5% DMSO (gilthead seabream)
or 10% DMSO (European seabass) with different potential
antioxidant supplements. The supplements used in both
species were: taurine and hypotaurine (1 mM and 10 mM),
ascorbic acid (1 mM and 10 mM), ␣-tocoferol (0.1 mM and
0.5 mM) or 1 ml/l of a commercial cell antioxidant supple-
ment (Sigma, A1345). Diluted sperm (1:6, sperm:extender)
was loaded into 0.5 ml straws and frozen in liquid nitro-
gen vapour (2 cm from the LN2 surface for seabream and
6.5 cm from the LN2 surface for seabass for 10 min and
15 min, respectively). Five to eight straws per treatment
were stored in a nitrogen container until use. For thaw-
ing, straws were immersed in a water bath set at 25 ◦ C for
30 s.
2.4. Standard sperm quality assays: motility and viability
Sperm motility and viability were determined in post-
thawed samples. Computer assisted sperm analysis (CASA)
was used to determine the percentage of motile cells (TM),
motile cells with progressive movement (PM), curvilinear
velocity (VCL), straight line velocity (VSL) and spermatozoa
linearity (Lin). Briefly, 1 ␮l sperm was placed in a Mak-
ler chamber under a 10 × negative phase contrast objective
(Nikon E200, Tokyo, Japan) and motility was immediately
activated with 10–20 ␮l of seawater for seabream and
seabass sperm, respectively. The above parameters were
recorded with a Basler camera (Basler A312f/c, Ahrensburg,
Germany) recording 50 frames/s and registered with ISAS
software (Proiser, Valencia, Spain) after 15 s post activation.
Cell viability was determined using PI/SYBR-14 and flu-
orescence microscopy (Nikon E200, Tokyo, Japan). The cells
were incubated with both probes for 5 min and the number
of green and red stained cells was counted. Approximately
100 cells were observed per slide and each slide was repli-
cated twice. Results were expressed as percentage of viable
cells (green stained). A total of 80–100 cells were analyzed
corresponding to the 10 treatments tested.
 E. Cabrita et al. / Animal Reproduction Science 125 (2011) 189–195
2.5. DNA integrity assays: TUNEL and comet
For DNA analysis, TUNEL and comet assays were
performed on post-thawed sperm. For TUNEL analysis,
samples were fixed in 4% paraformaldehyde in PBS (100 ␮l
of post-thawed sperm) for 1 h at room temperature and
then washed in PBS twice (1200 g, 5 min, 4 ◦ C) before being
resuspended in cold 70% ethanol and stored at −20 ◦ C
for one week before analysis. The TUNEL protocol was
performed according to instructions provided by the manu-
facturer adapted to spermatozoa by Domínguez-Rebolledo
et al. (2009) and slightly modified for fish spermatozoa.
Briefly, 7 ␮l of a cellular suspension (2 × 106 spz) were
washed twice using the wash buffer provided with the
kit, adding 50 ␮l of DNA labeling mixture after removing
the washing buffer. After 1 h at 37 ◦ C with shaking, the
cells were washed twice using the rinse buffer. Finally,
the cells were resuspended in 100 ␮l antibody solution
(FITC-Anti-BrdUTP mAb) and incubated for 30 min at room
temperature in the dark. A PI/RNase A solution (400 ␮l) was
added to all samples to counterstain and the cells were
incubated for 10 min before analysis. A positive control
(incubation of fixed cells with 200 U/ml DNase A) and a neg-
ative control (replacing DNA labeling mixture by distilled
water) were used to standardize the assay. The samples
were analyzed by flow cytometry (FacSort Plus Analyzer,
Becton-Dickinson, USA) with a 488 nm Ar-ion laser for exci-
tation and fluorescein isothiocyanate (FITC) was read with
the FL1 photodetector (530/28BP filter). Data were regis-
tered using the CellQuest Pro 3.1 software (BD Biosciences).
For each treatment, 3 subsamples were analyzed and a total
of 5000 spermatozoa from each sample were recorded. The
analysis of the flow cytometry data was carried out using
WEASEL v. 2.6 (WEHI, Melbourne, Australia).
The comet assay was performed according to the
method described by Cabrita et al. (2005) with slight
modifications introduced by Dietrich et al. (2005). After
electrophoresis, the slides were photographed using PI to
stain DNA and observe the comets. A total of 100 cells
were analyzed in the two slides performed by treatment.
The percentage of DNA fragmentation (DNAt) and Olive
tail moment (Mt) were the parameters recorded with the
software KOMET 6.0 (Andor Technology, Belfast, Ireland).
2.6. Statistics
Percentile data were normalized through arc sine trans-
formation. Significant differences between treatments and
the control were determined using One-way Anova and
identified using Student Newman Keuls test (p ≤ 0.05) with
SPSS 16 software.
3. Results
The addition of antioxidants (vitamins and aminoacids)
to gilthead seabream extender did not significantly
increase (p ≤ 0.05) the parameters of motility (TM, PM, VCL,
VSL or Lin), although 1 mM taurine slightly increased the
percentage of motile cells (58%) when compared to the
control (43%) (Fig. 1). Indeed, the supplementation of the
extender media using a commercial antioxidant produced a
191
significant decrease (p ≤ 0.05) in both total motile cells and
progressive spermatozoa (37% and 10%, respectively). In
both species, post-thaw cell viability was not improved by
the different treatments, however the commercial antiox-
idant supplementation produced a decrease in European
seabass sperm viability (34% vs. 44% in the control, Fig. 2b),
while 10 mM taurine produced the same effect in gilthead
seabream (48%) when compared with the control (58%)
(Fig. 2a).
DNA fragmentation (DNA in tail and Olive tail moment)
determined by the comet assay showed no improvement
with the addition of antioxidants to the extender media
in European seabass sperm, since higher DNA fragmen-
tation was obtained in treatments containing vitamins
(␣-tocopherol and ascorbic acid) than amino acids (taurine
and hypotaurine) or control (Fig. 3c and d). No signifi-
cant differences were found between the control (NaCl)
and the amino acids added, taurine and hypotaurine at the
concentrations tested being non-lethal for cells. However,
in gilthead seabream sperm, antioxidants significantly
reduced (p ≤ 0.05) both DNA in tail and Olive tail moment
(Fig. 3a and b) improving DNA stability. Regarding DNA in
tail (DNAt), both 1 mM and 10 mM taurine and hypotaurine
(12.3–14.1%) significantly improved (p ≤ 0.05) post-thaw
samples in comparison with the control (18.5%). The
same effect was produced by 1 mM ascorbic acid, 0.5 mM
␣-tocoferol and the commercial antioxidant supplemen-
tation. Although mean values of DNA fragmentation were
not very high in control group in this species (18.5% DNAt),
these values were also improved (p ≤ 0.05) by the previous
treatments registering a decrease of 22% of DNA fragmen-
tation.
The TUNEL analysis was performed only in samples
with lower DNA fragmentation in both species. However,
no significant differences were found between antioxidant
supplementation (taurine, hypotaurine and commercial
antioxidant) and the control group, both for gilthead
seabrem and European seabass, registering percentages
of TUNEL positive cells between 1.7 and 1.6% (control,
seabream and seabass) and 2.2–2.8% (seabream) and
2.2–3.1% (seabass) for the different treatments tested
(Fig. 4a and b, respectively).
4. Discussion
During the process of cryopreservation, sperm is
exposed to oxidative stress during dilution in the exten-
der media, cryoprotectant exposure and cooling events. All
these steps affect sperm structure reducing sperm motility,
viability and fertilizing ability due to a significant genera-
tion of reactive oxygen species (ROS) such as hydroperoxyl
and hydroxyl radicals (Ball, 2008). Excessive accumulation
of these radicals has been reported to produce DNA dam-
age in several species such as humans, horse and equine
(Donnelly et al., 2000; Baumber et al., 2003; Ball, 2008).
ROS attack DNA at sugar level resulting in fragmentation,
base loss and strand break with terminal ring-saturated
thymines, hydroxymethyl uracyl, thymine fragments and
adenine ring-opened products (Breimer and Lindahl, 1985).
Antioxidants play an important role in the protection of
sperm and can neutralize the effects induced by the cry-
192 E. Cabrita et al. / Animal Reproduction Science 125 (2011) 189–195

a 70
b 25
a
60
a a a a a
a a a 20
a
50 a a a
a a
a

PM (%)
TM (%)

40
b 15 a
b b
30
10

20

5
10

0 0
NaCl T1 T10 H1 H10 Asc1 Asc10 Toc0.1Toc0.5 Antiox NaCl T1 T10 H1 H10 Asc1 Asc10 Toc0.1 Toc0.5 Antiox

c 100 100
d

80 80
VCL (µm/s)

VSL (µm/s)
60 60

40 40

20 20

0 0
NaCl T1 T10 H1 H10 Asc1 Asc10 Toc0.1 Toc0.5 Antiox NaCl T1 T10 H1 H10 Asc1 Asc10 Toc0.1 Toc0.5 Antiox

e 80

60
Lin (%)

40

20

0
NaCl T1 T10 H1 H10 Asc1 Asc10 Toc0.1 Toc0.5 Antiox

Fig. 1. Motility parameters: (a) TM – total motility, (b) PM – progressive motility, (c) VCL – curvilinear velocity, (d) VSL – straight line velocity, (e) Lin-linerity
determined in Sparus aurata sperm cryopreserved with the addition of several potential antioxidant supplements: T1 and T10 (1 mM and 10 mM taurine),
H1 and H10 (1 mM and 10 mM hypotaurine), Asc1 and Asc10 (1 mM and 10 mM ascorbic acid), Toc0.1 and Toc0.5 (0.1 mM and 0.5 mM, ␣-tocoferol) and
antiox (commercial antioxidant supplement). Control group was cryopreserved only in 1% NaCl and DMSO. Significant differences between treatments and
control are signed by different letters (p ≤ 0.05, n = 8, S. aurata). No significant differences were found in (c), (d) and (e) figures. Results are expressed as
mean values ± SD.

opreservation procedure. Previous studies performed by
our group in gilthead seabream demonstrated a reduction
in TAS (total antioxidant potential) in post-thaw samples,
indicating a reduction in antioxidants naturally present in
seminal plasma (Martínez-Páramo et al., 2009). This may
be due to dilution of samples in the extender media reduc-
ing the capacity of sperm to withstand further cryodamage.
Recent studies in mammalian sperm demonstrated the
beneficial effects of adding antioxidants to the extender
media of several species (Bucak et al., 2007; Thuwanut
 E. Cabrita et al. / Animal Reproduction Science 125 (2011) 189–195 193

a 70
a b 70
a a a a a
60 a a 60
b a a

Cell viability (%)

Cell viability (%) 50 50 a a
a
a a a
a a
40 40
b
30 30

20 20

10 10

0 0
NaCl T1 T10 H1 H10 Asc1 Asc10 Toc0.1 Toc0.5 Antiox NaCl T1 T10 H1 H10 Asc1 Asc10 Toc0.1 Toc0.5 Antiox

Fig. 2. Cell viability determined in S. aurata (a) and D. labrax (b) sperm supplemented with antioxidants in the extender media: T1 and T10 (1 mM and 10 mM
taurine), H1 and H10 (1 mM and 10 mM hypotaurine), Asc1 and Asc10 (1 mM and 10 mM ascorbic acid), Toc0.1 and Toc0.5 (0.1 mM and 0.5 mM, ␣-tocoferol)
and antiox (commercial antioxidant supplement). Control group was frozen only in 1% NaCl and DMSO. Significant differences between treatments and
control are signed by different letters (p ≤ 0.05, n = 8, S. aurata; n = 10, D. labrax). Results are expressed as mean values ± SD.

et al., 2008) in terms of motility and cell viability. How- VCL, VSL and linearity) and cell viability were not signifi-
ever, in similar experiments other authors were not able cantly improved after sperm thawing in both species, DNA
to demonstrate an increase in post-thaw quality in sperm fragmentation and Olive tail moment were significantly
from horse and dog (Martins-Bessa et al., 2007; Ball, lower in post-thaw gilthead seabream sperm, especially
2008), showing different antioxidant action depending on with treatments incorporating taurine and hypotaurine at
species-specification, antioxidants and concentrations. In the two concentrations tested. Taurine and hypotaurine
the present work, antioxidants (amino acids and vitamins) are considered important for sperm motility and fertility
added to European seabass and gilthead seabream sperm in several species (Kundu et al., 2001; Bucak et al., 2007).
freezing media showed a different pattern of protection in Our findings for taurine are contrary those for its use in
both species. Although sperm motility parameters (TM, PM, rabbit epididymal spermatozoa, where inhibition of loss of

a 25
b
35
a
DNA fragmentation (DNAt %)

a
a a 30
DNA fragmentation (Mt)

20
b b b b
b b 25 b
b b
15
20 c c c c
c
c c
15
10

10
5
5

0 0
NaCl T1 T10 H1 H10 Asc1 Asc10 Toc0.1 Toc0.5 Antiox NaCl T1 T10 H1 H10 Asc1 Asc10 Toc0.1 Toc0.5 Antiox

c 20
d 25 a
DNA fragmentation (DNAt %)

a a
a
DNA fragmentation (Mt)

20 a
15 a a a
b b
b b b
b b 15
b a
b b
10 b
10

5
5

0 0
NaCl T1 T10 H1 H10 Asc1 Asc10 Toc0.1 Toc0.5 Antiox NaCl T1 T10 H1 H10 Asc1 Asc10 Toc0.1 Toc0.5 Antiox

Fig. 3. DNA fragmentation determined by comet assay in frozen-thawed samples from S. aurata (a and b) and D. labrax (c and d). DNA fragmentation was
assessed in terms of the percentage of DNA in tail (DNAt) and Olive tail moment (Mt). Control group (NaCl) and experimental groups were: T1 and T10
(1 mM and 10 mM taurine), H1 and H10 (1 mM and 10 mM hypotaurine), Asc 1 and Asc10 (1 mM and 10 mM ascorbic acid), Toc0.1 and Toc0.5 (0.1 mM
and 0.5 mM, ␣-tocoferol) and antiox (commercial antioxidant supplement). Significant differences between treatments and control are signed by different
letters (p ≤ 0.05, n = 8, S. aurata; n = 10, D. labrax). Results are expressed as mean values ± SD.
194 E. Cabrita et al. / Animal Reproduction Science 125 (2011) 189–195

a 5
b 5

4 4

TUNEL (+) cells(%)

TUNEL (+) cells(%)
3 3

2 2

1 1

0 0
NaCl T10 H1 Antiox NaCl T10 H1 Antiox

Fig. 4. DNA fragmentation determined by TUNEL assay in frozen-thawed samples from S. aurata (a) and D. labrax (b). Control group (NaCl) and experimental
groups were: T10 (10 mM taurine), H1 (1 mM hypotaurine), and antiox (commercial antioxidant supplement). No significant differences were found between
treatments (p ≤ 0.05, n = 5, S. aurata; n = 6, D. labrax). Results are expressed as mean values ± SD.

forward motility was observed (Alvarez and Storey, 1983).
Taurine is also known to play an important role in osmoreg-
ulation, ion modulation and neurotransmission (Wright
et al., 1986), properties that may explain the limited ben-
efits of this antioxidant in our study. However, the amine
group of taurine shows an affinity with nucleic acids and
can thus neutralize the oxidative generation of ROS, reduc-
ing DNA damage (Zhang et al., 2004; Sokól et al., 2009). In
our study this could have been the way taurine or hypotau-
rine protected DNA from gilthead seabream. However, this
effect could not be confirmed by the TUNEL analysis proba-
bly because the extent of the damage was very low. Further
studies on characterization of taurine and hypotaurine con-
centrations naturally present in spermatozoa from both
species need to be conducted to better understand why
taurine has a greater affinity for protecting seabream sperm
than seabass.
In European seabass sperm, supplementation with
either ascorbic acid (1 or 10 mM) or ␣-tocoferol (0.1 or
0.5 mM) produced an increase in DNA damage showing a
proactive oxidation effect rather than protection against
damage. Ascorbic acid in the presence of transition met-
als makes radicals highly reactive and more destructive,
thus generating more free radicals (Donnelly et al., 1999).
Previous studies in humans showed that ascorbate in the
range of 0.02–0.6 mM adversely affected sperm motility
(Donnelly et al., 1999). Higher concentrations of vitamin C
(2.5 mM) proved detrimental to sperm motility in frozen-
thawed bull semen (Beconi et al., 1993). Similar effects
were obtained by Michael et al. (2007) in canine sperm
(1.5 mM) and in our study both in seabream and seabass
sperm. However, an opposite effect was demonstrated by
Mirzoyan et al. (2006) in sturgeon sperm regarding ascorbic
acid supplementation. These authors detected an increase
in the motility rate of Russian sturgeon sperm (Acipenser
gueldenstaedti) protected with 10 mM ascorbic acid as well
as the reduction of chromosome aberrations in developing
embryos. This reduction seems to be related to a reduction
in DNA damage in post-thaw samples used in fertility trials,
thus further concentrations need to be tested before disre-
garding the effect of ascorbic acid on the extender media
for fish sperm.
Vitamin E can inhibit lipid peroxidation reaction in the
membrane by eliminating peroxyl (ROO–), alkoxyl (RO–),
and other lipid-derived radicals (Halliwell and Gutteridge,
1999). Vitamin E was more efficacious than vitamin C
in improving post-thaw motility of human spermato-
zoa (Askari et al., 1994), although Donnelly et al. (1999)
recorded an opposite effect of vitamin E. The addition
of ␣-tocopherol protected membrane integrity in chilled
boar spermatozoa (Cerolini et al., 2000), whereas a water-
soluble vitamin E analogue (Trolox) improved boar sperm
motility and mitochondrial membrane integrity during
post-thaw incubation (Peña et al., 2003). However, as
stated before, in our study no positive effect was detected
from adding ␣-tocopherol to the freezing media.
Our results, together with data reported by other
authors, suggest a species-specific effect probably depend-
ing not only on the type of antioxidant added but also on
its concentration. Further research is required in order to
select the best concentration of antioxidants or combina-
tion of antioxidants, especially ␣-tocoferol and ascorbic
acid, which could mimic the action of seminal plasma and
provide sperm with extra protection against free radical
attacks during cryopreservation.
Acknowledgments
This work was supported by the CRYOSPERM project
(FCT, PTDC/MAR/64533/2006). E. Cabrita was supported by
a Ramón and Cajal research contract (RYC-2007-01650);
S. Martínez-Páramo, was supported by FCT postdoctoral
fellowship (SFRH/BPD/48520/2008). The authors thank the
Maresa S.A (Ayamonte, Spain), A. Coelho e Castro (Povoa de
Varzim, Portugal) and Aqualvor (Alvor, Portugal) fishfarms.
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