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Anim. Reprod., v.6, n.2, p.392-399, Apr./Jun. 2009
_________________________________________
5
Corresponding author: ivan.bustamante@ufrgs.br
Phone: +55(51)3308-6121; Fax: +55(51)3308-6124
Received: July 10, 2007
Accepted: December 11, 2008
Skim milk-egg yolk based semen extender compensates for non-enzymatic antioxidant
activity loss during equine semen cryopreservation
I.C. Bustamante Filho
1,2,3,5
, C.D. Pederzolli
2
, A.M. Sgaravatti
2
, R.M. Gregory
3,4
, C.S. Dutra Filho
2,4
,
M.I.M. Jobim
1
, R.C. Mattos
3,4
1
Laboratory of Artificial Insemination, Faculty of Veterinary, Federal University of Rio Grande do Sul (UFRGS),
91501-970, Porto Alegre, Brazil.
2
Laboratory of Inborn Errors of Methabolism, Department of Biochemistry, UFRGS, 91501-970, Porto Alegre, Brazil.
3
REPROLAB, Faculty of Veterinary, UFRGS, 91501-970, Porto Alegre, Brazil.
4
Researcher of CNPq, Brazil.
Abstract
Cryopreservation exposes spermatozoa to
stressful conditions, leading to reduced cell viability.
Several studies propose that overproduction of reactive
oxygen species and decreased antioxidant capacity of
semen may increase the damaging effects of the
technique. The objective of this work was to evaluate
the influence of a skim milk-egg yolk based semen
extender on enzymatic and non-enzymatic antioxidant
activity in equine semen cryopreservation. Fifteen
ejaculates from six fertile Criollo stallions were
cryopreserved using a commercial citrate-Hepes, egg
yolk, skim milk and glycerol extender. Activities of
catalase, glutathione peroxidase and superoxide
dismutase and total radical-trapping antioxidant
potential were assessed in raw semen, semen diluted in
extender and thawed semen. All three enzymes showed
higher activities in raw semen than in diluted or in
thawed semen (P < 0.01), but enzyme activities did not
differ significantly between diluted and thawed semen
samples (P > 0.05). Non-enzymatic antioxidant defenses
did not differ among any of the stages in the
cryopreservation process (P > 0.05). In conclusion, the
present study shows that dilution of semen with
skim milk-egg yolk based extender after centrifugation
compensates for the non-enzymatic antioxidant
protection (but not enzymatic antioxidant defense) lost
with seminal plasma removal. The absence of
correlation between seminal and antioxidant parameters
suggests that the compensation was enough for semen
protection against oxidative stress, or antioxidant
protection plays a minor role on semen from fertile
stallions.
Keywords: antioxidants, cryopreservation, equine
semen, extender, oxidative stress.
Introduction
The storage of cryopreserved spermatozoa is
associated with a reduction in cell viability and
fertilizing capacity. The quality of stored semen is
affected by handling procedures such as dilution,
centrifugation, dilution in semen extender and freezing.
These procedures are associated with the generation of
and imbalance among reactive oxygen species (ROS;
Twigg et al., 1998; Bilodeau et al., 2000; Ball et al.,
2001; Chateerjee and Gagnon, 2001; Baumber et al.,
2005).
Although oxidative stress was suggested as an
important contributor to disruption of sperm function
over 50 years ago, the importance of oxidative stress has
gained a wider understanding in the last decade (Sharma
and Agarwal, 1996). In normal physiological functions,
there is a balanced generation of ROS and antioxidant
enzymes (Kovalski et al., 1992; Plante et al., 1994;
Aitken et al., 1995). ROS have a physiological role in
signaling events controlling sperm capacitation and
induction of the acrosome reaction in many species
including equine (De Laraminde and Gagnon, 1993; De
Laraminde et al., 1993, 1997; Griveau et al., 1994;
Leclerc et al., 1997; Baumber et al., 2003). However,
overproduction of ROS and decreased antioxidant
defense activity cause low sperm motility and viability,
DNA fragmentation and protein denaturation (Aitken et
al., 1994; Halliwell and Gutteridge, 1999; Baumber et
al., 2002; Agarwal and Said, 2005; Kankofer et al.,
2005).
The cell structure of spermatozoa makes them
potentially susceptible to damage from free radicals (De
Laraminde and Gagnon, 1995; Sikka, 2004). Sperm
membranes are rich in polyunsaturated fatty acids and
can easily undergo lipid peroxidation in the presence of
ROS, leading to changes in membrane fluidity (Alvarez
and Storey, 1982), which finally results in decreased
fertilizing capacity. In addition, low cytoplasm content
remaining after spermatogenesis contributes to sperm
cell fragility, limiting the potential for DNA and protein
repair (Bustamante Filho et al.
, 2005).
To counteract oxidative damage, spermatozoa
and seminal plasma have several mechanisms to
neutralize free radicals. Enzymatic and non-enzymatic
Bustamante Filho et al. Antioxidant status and semen extender on equine semen freezing.
Anim. Reprod., v.6, n.2, p.392-399, Apr./Jun. 2009
393
antioxidant systems work synergistically to prevent
harmful effects of byproducts from aerobic metabolism
(De Laraminde and Gagnon, 1993; De Laraminde et al.,
1993). For example, mammalian semen (mainly seminal
plasma) has many compounds with non-enzymatic
antioxidant activity (e.g., ascorbic acid, α-tocopherol,
taurine and albumin; Alvarez and Storey, 1983).
However, the presence of specific antioxidant enzymes
suggests that they also play a major role in the protection
of spermatozoa against ROS. Three enzyme system
(catalase, glutathione peroxidase, and superoxide
dismutase) have superoxide radicals and hydrogen
peroxides as substrates (Alvarez and Storey, 1989; Zini et
al., 1993; Ball et al., 2000). These antioxidants act by
reducing the production of deleterious residues from
oxidative physiological metabolism. In bovine semen, a
decrease in antioxidant activity following cryopreservation
has been reported (Bilodeau et al., 2000). Furthermore,
freeze–thawing of equine and bovine spermatozoa has
been associated with an increase in ROS generation
(Bilodeau et al., 2000).
Little is known about the dynamics of
enzymatic or non-enzymatic antioxidant defense
systems during cryopreservation of stallion semen and
the influence of semen extenders. Comprehension of
their impact during procedures such as centrifugation,
removal of seminal plasma dilution and freezing might
lead to improved fertilizing capacity of semen through
use of antioxidants in semen extenders.
In the present study we investigated antioxidant
defenses status of equine semen during
cryopreservation. To accomplish that, the effect of a
routinely used semen freezing protocol was studied on total
radical-trapping antioxidant potential (TRAP), which
evaluates non-enzymatic antioxidant defenses and
activities of the antioxidant enzymes catalase, superoxide
dismutase (SOD) and glutathione peroxidase (GPx).
Materials and Methods
Animals
Six fertile Criollo stallions between six and
nine years of age were used. The stallions belong to two
stud farms in Rio Grande do Sul, Brazil and were on a
routine semen collection schedule. They were stabled
with access to an outdoor paddock from 8 AM to 6 PM
and were fed hay and a concentrate ration balanced to
provide their daily requirements for energy, protein and
micro-nutrients twice daily. Water and mineral
supplementation were freely available.
Experimental design
In the experiment, non-enzymatic antioxidant
activity and activities of superoxide dismutase, catalase
and glutathione peroxidase were monitored on semen
samples from different stages of the cryopreservation
procedure. Stallions had a phase of sexual rest for one
week before the first semen collection. A total of fifteen
ejaculates were used in the experiment (three ejaculates
of three stallions and two ejaculates of three stallions).
Freezing was performed by a standard technique
comprising dilution of semen (1:1) with FR-1
extender (raffinose, lactose, glucose, potassium
citrate and Hepes; Nutricell, Campinas, SP, Brazil) at
30°C, centrifugation at 400 x g for 10 min and
removal of 90 – 95% of the supernatant (extender plus
seminal plasma). All samples were extended to a final
concentration of 100 x 10
6
sperm/mL in FR-5 extender
(FR-1 plus skim milk, glucose, egg yolk and glycerol;
Nutricell, Campinas, SP, Brazil), reaching a final
glycerol concentration of 2.5%, packaged into 0.5 mL
straws (IMV International Corporation, Minneapolis,
MN, USA) and directly frozen 4 cm above the liquid
nitrogen surface for 20 min (Martin et al., 1979; Klug et
al., 1992; Alvarenga et al., 2005). Semen was thawed
after seven days by plunging the straw in a water-bath at
37°C during 30 s.
For the oxidative stress assays, samples were
obtained from three cryopreservation stages: (1) raw
semen, (2) extended semen prior to freezing and (3)
post-thawed semen. From each stage, 100 µL semen
samples were suspended in 600 µL of 20 mM sodium
phosphate buffer, pH 7.4 containing 140 mM KCl and
stored at -20°C.
Experimental procedures
Semen was collected with an artificial vagina
(Hannover model, Minitüb GmbH, Germany) on an
estrous Criollo mare. After collection, the gel fraction
was removed and semen was filtered through sterile
gauze. Progressive, total motility and morphology
were evaluated after collection and after thawing. In
addition, after thawing, structural and functional
integrity of spermatozoa membranes were evaluated
by fluorescent stain (CFDA + PI; Kneissl, 1993) and
hypoosmotic swelling tests (Lagares et al., 2000),
respectively.
Catalase activity was assayed using a double-
beam spectrophotometer with temperature control
(Hitachi U-2001®). Thirty microliters of semen sample
was added to 720 µL of reaction medium consisted of 20
mM H
2
O
2
, 0.1% Triton X-100, and 10 mM potassium
phosphate buffer pH 7.0. One unit is defined as 1 µmol of
hydrogen peroxide consumed per minute (read at 240 nm),
and specific enzyme activity is reported as units per
milligram protein (Aebi, 1984; Banerjee et al., 2002;
Kasahara et al., 2002; Cortassa et al., 2004;
Bustamante Filho et al. Antioxidant status and semen extender on equine semen freezing.
Anim. Reprod., v.6, n.2, p.392-399, Apr./Jun. 2009
394
Khosrowbeygi and Zarghami, 2007).
Glutathione peroxidase activity was measured
using tert-butyl-hydroperoxide as substrate (Wendel,
1981; Munz et al., 1997; Cortassa et al., 2004). Ninety
microliters of semen sample were added to an
incubation medium containing 790 µL of 100 mM
potassium phosphate buffer containing 1 mM EDTA, pH
7.7, 20 µL of 2 mM glutathione, 30 µL of 0.15 U/mL
glutathione reductase, 10 µL of 0.4 mM azide, 10 µL of
0.1 mM NADPH and 50 µL of 0.5 mM tert-butyl-
hydroperoxide. NADPH disappearance was monitored
at 340 nm using a double-beam spectrophotometer with
temperature control (Hitachi U-2001). One GPx unit is
defined as 1 µmol of nicotinamide adenine dinucleotide
phosphate-oxidase (NADPH) consumed per minute, and
specific enzyme activity is represented as units per mg
protein.
The assay of SOD activity was carried out as
described (Marklund, 1985; Silva et al., 2005; Bhandari
et al., 2007) based on the capacity of pyrogallol to
autoxidize, a process highly dependent on superoxide
radicals. The inhibition of autoxidation of this
compound occurs in the presence of SOD, whose
activity was indirectly assayed spectrophotometrically at
420 nm, using a double-beam spectrophotometer with
temperature control (Hitachi U-2001). In a quartz cuvette
were added 30 µL of semen samples, 4 µL of 30 µM
catalase, 958 µL 50 mM Tris 1 mM EDTA pH 8.2
buffer and 8 µL of 24 mM pirogalol prepared in 10 mM
HCl. A calibration curve was performed with purified
SOD as standard, in order to calculate the activity of
SOD present in the samples. Results were reported as
units of SOD/mg protein.
Non-enzymatic antioxidant defenses were
assessed by the total radical-trapping antioxidant
potential (TRAP) method (Lissi et al., 1992; Rhemrev et
al., 2000; Evelson et al., 2001), based on
chemoluminescent intensity of luminol induced by 2,2’-
azo-bis-(2-amidinopropane; ABAP) thermolysis in a
Wallac 1409 Scintilator Counter. The initial
chemoluminescence value was obtained by adding 3 mL
of ABAP 10 mM dissolved in 50 mM sodium phosphate
buffer pH 7.4, plus 10 µL of luminol (5.6 mM) to a
glass scintillation vial. Ten microliters of 300 µM
Trolox (water soluble α-tocopherol analogue) or sample
were then added to the vial, and the chemoluminescence
was monitored until it achieved the initial levels. The
time required for this to occur is called induction time,
which is directly proportional to the antioxidant
capacity of the sample. The induction time of the
sample was compared to that presented by Trolox.
Results were reported as nmol Trolox/mg protein.
Protein concentration was determined using
bovine serum albumin as standard (Lowry et al., 1951).
Statistical analysis
Statistical analysis was performed by repeated
measures ANOVA, followed by the Tukey test for
multiple comparisons when the F value was significant.
Catalase and SOD values were transformed to
logarithms to normalize (ln) the distributions. All
analyses were performed using the Graphpad Prisma 5
software. Pearson correlation coefficients were
calculated to quantify associations between semen
characteristics (motility, structural and functional
integrity of the spermatozoa membranes), TRAP
activity and enzymatic activities (catalase, SOD and
GPx). Values of P < 0.05 were considered to be
significant.
Results
Parameters for raw semen (mean ± S.D.)
were 45.7 ± 7.8 mL for semen volume, 71.3 ± 20.0%
for total motility, 57 ± 21.9% for progressive motility
and 258.3 ± 51.1 x 10
6
spermatozoa per milliliter for
sperm concentration. At post-thawing evaluation, total
motility averaged 28.7 ± 17.5%, progressive motility
averaged 18.3 ± 14.6%, membrane structural integrity
averaged 19.4 ± 12.5% and membrane functionality
averaged 24.2 ± 15.9%. The percentage of
morphologically normal spermatozoa was 54.5 ± 9.1%.
Activities of the antioxidant enzymes and of non-
enzymatic antioxidant potential (TRAP) in raw,
diluted and thawed semen are shown in Fig. 1.
Antioxidant activities for raw, extended and frozen
semen were respectively: catalase: 2.8 ± 0.75 ln U/mg
protein, 1.33 ± 0.64 ln U/mg protein, 1.25 ± 0.69 ln U/mg
protein; SOD: 0.76 ± 0.47 U/mg protein, 0.22 ± 0.25 U/mg
protein, 0.05 ± 0.14 U/mg protein; GPx: 12.75 ± 4.98 U/mg
protein, 7.26 ± 2.97 U/mg protein, 6.56 ± 1.92 U/mg
protein; TRAP: 1.58 ± 1.04 nmol Trolox/mg protein,
1.08 ± 0.67 nmol Trolox/mg protein, 1.40 ± 0.39 nmol
Trolox/mg protein.
There was a tendency for reduction of the three
enzyme activities through stages of the cryopreservation
process. Conversely, total antioxidant potential did not
differ between stages of cryopreservation. There was no
catalase or SOD activity in analysis of the extender
alone; however, glutathione peroxidase activity and TRAP
were detected (4.23 U/mg protein and 0.22 ± 0.08 nmol
Trolox/mg protein, respectively). No significant
correlations were observed between superoxide
dismutase, catalase, glutathione peroxidase and TRAP
and any of the semen variables (P > 0.05).
Bustamante Filho et al. Antioxidant status and semen extender on equine semen freezing.
Anim. Reprod., v.6, n.2, p.392-399, Apr./Jun. 2009
395
Figure 1. Activities of (A) catalase, (B) superoxide dismutase (C) glutathione peroxidase and (D) TRAP value in
raw, diluted and frozen-thawed semen. Means with different superscripts (a, b, c) differ (P < 0.05). Data are
mean ± SEM.
Discussion
The present work documented the maintenance
of non-enzymatic antioxidant defenses by skim milk-
egg yolk based extender during stallion semen
cryopreservation.
Non-enzymatic antioxidant defenses were
assessed by the total radical-trapping antioxidant
potential (TRAP) method. TRAP results were similar in
raw, diluted and frozen-thawed equine semen,
indicating a compensatory effect by semen extender on
non-enzymatic antioxidant activity after seminal plasma
removal.
Non-enzymatic antioxidant defenses comprise
a huge number of molecules, including amino acids,
peptides, proteins and vitamins bearing different
Raw Diluted Frozen Semen
semen semen semen extender
Raw Diluted Frozen Semen
semen semen semen extender
Raw Diluted Frozen Semen
semen semen semen extender
Raw Diluted Frozen Semen
semen semen semen extender
LnU catalase/mg protein
Log (x + 1) USOD/mg protein
1.6
1.2
0.8
0.4
0.0
UGPx/mg protein
2.0
1.5
1.0
0.5
0.0
nmol Trolox/mg protein
3.2
2.4
1.6
0.8
0.0
A
B
C
D
4.5
3.0
1.5
0.0
Bustamante Filho et al. Antioxidant status and semen extender on equine semen freezing.
Anim. Reprod., v.6, n.2, p.392-399, Apr./Jun. 2009
396
reactive centers (e.g., phenols, thiols) with widely
different hydrophobicities that allow the trapping of
both hydrophobic and hydrophilic radicals (Evelson et
al., 2001). These compounds share the role of
controlling the oxidative balance of tissues and plasma
with enzymatic antioxidant systems. Although equine
semen from fertile stallions rarely presents leukocytes
and high percentage of abnormal spermatozoa (the main
sources of ROS), the cryopreservation process increases
ROS generation by spermatozoa (Ball et al., 2001).
ROS release in medium by damaged cells is a potential
danger for efficiently cryopreserved spermatozoa. After
thawing the antioxidant content of medium provided by
semen extender contributes avoiding or decreasing the
risk of lipid peroxidation of sperm cell membranes.
The semen extender used in our experiment
showed an antioxidant activity (0.22 ± 0.08 nmol
Trolox/mg protein), which might be explained by its
composition. Skim milk and egg yolk are usual
components of semen extenders. However, their precise
composition is difficult to define. Both components are
susceptible to variations in its mineral, lipid and protein
content once they are influenced by animal feed, health
and management.
Recently, a proteomic approach shed more
light on egg yolk composition (Mann and Mann, 2008).
Comprising at least 116 proteins, 86 of which were
reported to occur in egg yolk for the first time, this
article describes several proteins with probable
antioxidant activity. Egg yolk consists of approximately
33% lipid and an antioxidant protection is of a
paramount importance for embryo development. The
presence of metal chelators such as yolk phovitin,
ceruloplasmin, ovalbumin and ovotransferrin remove
free metal ions which could catalyze the production of
ROS. Also, a protein similar to extracellular superoxide
dismutase and a protein similar to plasma glutathione
peroxidase may contribute to the antioxidative capacity
of yolk (Mann and Mann, 2008).
Similarly, bovine skim milk presents
antioxidant activity for protection of its high lipid
content (Taylor and Richardson, 1980). However, we
should consider two steps in skim milk preparation: (1)
fat removal also results in loss of fat soluble vitamins
(e.g., retinols and tocopherols); (2) ultra-high
temperature processing, when milk is heated for a short
time at a temperature exceeding 135°C, consequently
inactivating enzymes such as catalase, SOD and GPx.
Nevertheless, free radical scavenger activity was
identified and related to minerals as copper and zinc,
which are necessary for proper activity of scavenger
enzymes. Also, these minerals have their own
antioxidant properties (Przybylska et al., 2007). In
addition, protein denaturation exposes sulphydryl
groups (Patrick and Swaisgood, 1976; Taylor and
Richardson, 1980; Jiménez-Guzmán et al., 2002),
enhancing antioxidant capacity of proteins and peptides
in spite of its functional structure.
Activities of the scavenger enzymes catalase,
superoxide dismutase and glutathione peroxidase had
similar profiles, being higher in raw semen than in
diluted or in frozen-thawed semen. The last step (freeze-
thawing) did not reduce the activity of these enzymes,
which were stable in the extended and freeze-thawed
semen. The decrease of enzyme activity in extended
semen was 64% for catalase, 43% for glutathione
peroxidase and 78% for superoxide dismutase.
However, these enzyme activities did not differ between
extended and frozen-thawed semen, indicating that the
freezing procedure had no effect on their activity. The
decrease of enzyme activities in diluted semen observed
in this study was expected and is explained by
preparation of equine semen for cryopreservation,
which involves the removal of 90 to 95% of seminal
plasma and consequently the removal of a dominant
source of antioxidant protection (Zini et al., 1993; Ball
et al., 2000; Baumber et al., 2005). Evaluation of the
commercial extender used in this work did not detect
SOD and CAT activity and only minimal GPx activity
was found, which shows that the extender did not
compensate for loss of enzymatic antioxidant protection
caused by removal of seminal plasma.
Spermatozoa are potentially susceptible to
damage caused by excess ROS due to their high amount
of polyunsaturated fatty acids in membrane
phospholipids and to the relatively small volume of
cytoplasm. Elimination of most of the cytoplasm during
the terminal stages of spermatozoa differentiation
results in a limited defense against oxidative stress,
making the cell dependent on the antioxidant support of
seminal plasma (Baumber et al., 2005). Antioxidant
systems control the balance between production and
neutralization of ROS and protect spermatozoa
against peroxidative damage (Griveau and Le Lannou,
1997a, b).
Numerous studies have evaluated effects of
antioxidants on male fertility in several species
(Parinaud et al., 1997; Hsu et al., 1998; Bruemmer et
al., 2002; Foote et al., 2002). Although many clinical
trials demonstrated a beneficial effect of antioxidants in
selected cases of male infertility, other studies failed to
verify similar benefits. Investigators have used different
antioxidants in different combinations, making it
difficult to reach a definitive conclusion.
Deichsel et al. (2008), working with antioxidant
oral supplementation (tocopherol 300 mg/day; ascorbic
acid 300 mg/day; L-carnitin 4000 mg/day; folic acid 12
mg/day), have not found a pronounced effect on semen
quality of stallions. Conversely, Arlas et al. (2008)
found a higher total radical trapping potential in
stallions supplemented with rice oil containing gama-
oryzanol. Animals also presented an increase of total
motility and membrane functionality (HOST) on fresh
semen.
Bustamante Filho et al. Antioxidant status and semen extender on equine semen freezing.
Anim. Reprod., v.6, n.2, p.392-399, Apr./Jun. 2009
397
The addition of antioxidants to
cryopreservation extender did not improve the quality of
spermatozoa after thawing (Baumber et al., 2005).
Similar results were found (Ball et al., 2001) in equine
semen stored at 5°C. Conversely, Aurich et al. (1997)
described a protective effect of ascorbic acid on sperm
membrane integrity, in spite of a prejudicial effect on
progressive motility. As the addition of antioxidants did
not improve frozen semen quality, some authors suggest
that causes other than oxidative stress are responsible
for sperm damage (Baumber et al., 2005; Pagl et al.,
2006). This may explain the similar TRAP values
among cryopreservation stages and the absence of
correlation between TRAP values and semen variables
in our work. Pagl et al. (2006) reported that the loss of
sperm motility during cooled storage was an effect not
only of plasma membrane dysfunction but of
mitochondrial membrane dysfunction as well. Addition
of antioxidants to semen during cooled storage may
have only limited effects.
In conclusion, the present study presents
evidence that the composition of skim milk-egg yolk
based semen extender provides non-enzymatic
antioxidant factors that compensate for loss resulting
from seminal plasma removal. However, this
compensation was not observed for catalase, superoxide
dismutase and glutathione peroxidase. Since no
correlation between antioxidant and seminal parameters
was found, oxidative stress might play a minor role in
semen from fertile stallions or the non-enzymatic
antioxidant activity provided by semen extender was
enough to avoid deleterious effects caused by ROS.
Acknowledgments
This work was supported by Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) and Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES).
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