Effects of Copper and Superoxide Dismutase Content of Seminal Plasma on Buffalo Semen Characteristics
S. Asri Rezaii
To investigate the effects of copper and superoxide
dismutase (SOD) content of seminal plasma on buffalo semen characteristics,
54 semen samples collected from buffalo bulls by a bovine artificial vagina
were used. Semen characteristics (motility, viability, morphology, concentration
and volume) were recorded. Seminal plasma was harvested by centrifugation
and kept frozen until analysis. Seminal plasma copper content was determined
by atomic absorption procedure and SOD was measured by using a kit. The
mean total copper value of seminal plasma was recorded as 2.51 ±
0.04 mg kg-1 (Mean ± SEM) and the mean total SOD values
was 39.02 ± 0.81 IU mL-1. To reduce the range of variability,
the data were categorized according to their motility records in 3 groups
of Excellent (Ex, >90% motile, n = 33), Good (Go, 80-89% motile, n
= 15) and Moderate (Mo, <79% motile, n = 6). The mean motility, viability,
copper and SOD values in Ex group was recorded as 92.24 ± 0.51%,
94.00 ± 0.48%, 2.56 ± 0.04 mg kg-1 and 39.52
± 0.57 IU mL-1, respectively. These values were 81.66
± 0.62%, 85.26 ± 0.95%, 2.38 ± 0.11 mg kg-1
and 36.48 ± 1.51 IU mL-1 in Go group and 71.66 ±
1.05%, 77.00 ± 2.94%, 2.55 ± 0.10 mg kg-1 and
50.66 ± 2.51 in Mo group, respectively. The mean copper value in
Ex group was highly (r = 0.600) correlated with SOD and correlated with
sperm motility (r = 0.372) and viability (r = 0.363), while, in Go group
it was highly correlated (r = 0.945) with SOD and sperm viability (r =
0.652) and in Mo group it was correlated (r = 0.874) with semen volume
only. The mean SOD values in Ex group was highly correlated with sperm
motility (r = 0.492) and viability (r = 0.490) and mean copper values,
in Go group, it was highly correlated whit sperm viability (r = 0.659)
and mean copper values and in Mo group it had no significant correlations
with semen parameters. These results suggest that copper and SOD content
of the buffalo seminal plasma have an influence on the sperm motility
and viability which are the most important factors in semen fertility.
Copper appears to be involved in spermatozoa mobility and it may also
act on the pituitary receptors which control the release of LH. In the
seminal fluid, the level of copper appears to fall in cases of azoospermia
and to increase in oligo- and asthenozoospermia (Pleban and Mei, 1983;
Skandhan, 1992) but the findings of different authors are somewhat contradictory.
It is true that the concentrations in the ejaculate vary considerably
from one day to the next and that they also vary in different fractions
from a single ejaculate. The toxic effect of copper on spermatozoa has
often been reported. Copper reduces the oxidative processes and glucose
consumption, which reduces or abolishes mobility (Skandhan, 1992).
The production of free radicals, either by extracellular lipid peroxidation
or in situ by the way of oxidative phosphorylation in the mitochondria,
could damage the nuclear membrane (Vishwanath and Shannon, 1997).
Superoxide dismutase (SOD) scavenges both extracellular and intracellular
superoxide anion and prevents lipid peroxidation of the plasma membrane.
In order to act against H2O2, it must be conjugated
with catalase or glutathione peroxidase (GPx). SOD also prevents premature
hyperactivation and capacitation induced by superoxide radicals before
ejaculation (Agarwal and Prabakaran, 2005).
SODs are ubiquitous metalloproteins that catalyze the dismutation of
superoxide anion into hydrogen peroxide and molecular oxygen. Appreciable
amounts of SOD and much lower concentrations of GPx and catalase have
been reported in ejaculated ram sperm (Abu-Erreish et al., 1978).
A correlation between SOD activity and the time of motility loss has been
found in human sperm, after exposure to H2O2 (Martí
et al., 2003).
Copper is necessary for many enzymes like the Cu-Zn-superoxide-dismutase
(SOD), which is involved in cell protection against free (oxygen) radicals.
Elevated copper concentrations reduce oxidative processes and glucolysis
that may cause immotility and reduced viability (Pesch et al.,
Human seminal plasma possesses both superoxide dismutase (SOD)-like and
catalase-like activities. However, little is known about the putative
source of these key antioxidant activities in human semen. Zini et
al. (2002) reported that catalase-like and SOD-like activities (two
major antioxidant activities) are primarily derived from post-epididymal
(i.e., seminal vesicle, prostate) secretions.
The findings on the seminal plasma catalase and SOD activities and total
antioxidant capacity (TAC) are controversial. Sanocka et al. (1996)
showed a significant elevation in intracellular activity of SOD and decreasing
in catalase activity in infertile samples. Zini et al. (2002) study
showed that seminal plasma activity of SOD in infertile men is significantly
greater than in fertile men while catalase activity is not different between
The seminal plasma of mammals has been reported to contain large amounts
of superoxide dismutase (SOD) activity, much higher than that found in
other extracellular fluids. Spermatozoa have also been reported to contain
SOD activity (Peeker et al., 1997).
Sikka et al. (1995) reported an association between leukocytospermia
and ROS has been found to correlate with increased chemokine (Interleukin
8; IL-8) and decreased SOD activity.
Toxic effects of copper on seminal plasma are manifested in the decrease
of motile spermatozoa percentage and in decrease of malformed sperm cells
(Massanyi et al., 2005).
In boar semen, copper can inhibit enzymes having functional sulfidryl
groups, bind to and affect the confirmation of nucleic acids, disrupt
pathways of oxidative phosphorylation, although the precise response depends
upon the individual properties of metal (Massanyi et al., 2003a).
There is little information available about copper and SOD contents in
the buffalo seminal plasma. This study was carried out to: (1) estimate
the copper and superoxide dismutase contents of the seminal plasma in
buffalo bulls, (2) test whether any correlation exists between these parameters
and semen characteristics.
MATERIALS AND METHODS
Animals: Fifty four semen samples were collected by a bovine artificial
vagina from sexually mature buffalo bulls (4-5 years old) kept in The
Buffalo Breeding Center Northwest of Iran, Urmia (37 ° 33` N, 45 °
4` E) during the summer and autumn of 2007.
Semen evaluation: Immediately after collection, the ejaculate
was placed in a 37 °C water bath and the volume was recorded. Semen
motility was evaluated immediately after collection. Gross motility was
scored from 0 to 5 on a wet mount of neat semen at 100x magnification
(0 = cells present without motion; 5 = very rapid dark swirls). The percentage
of progressively motile spermatozoa was estimated by microscopic examination
at x400 magnification on a pre-warmed slide (37 °C) according to procedure
of Ax et al. (2000). Sperm concentration was measured using spectrophotometer
(Vital Scientific- IMV, The Netherlands) and the percentage of viable
spermatozoa was estimated by viewing 200 spermatozoa under x1000 magnification
using eosin-nigrosin staining method of Barth (2007). The semen samples
were cooled at room temperature and transported to the laboratory within
Preparation of seminal plasma: Fresh semen was centrifuged (Clements,
model 2000, Sydney, Australia) at 5,000 rpm for 10 min. The supernatants
were transferred into 1.5 mL tubes, re-centrifuged to eliminate the remaining
cells and kept frozen (-20 °C) until analysis within a week.
Data analysis: The obtained data was analyzed by using SPSS software
(version 11.5 for Windows; SPSS Inc., Chicago, IL, USA) computer program.
Results are quoted as arithmetic mean ± standard error of mean
(SEM) and significance was attributed at p<0.05.
Pearson`s correlation coefficient (two tailed) test was used to examine
the correlation between all the parameters of the semen. The comparison
of the semen parameters and copper and SOD contents of the seminal plasma
in groups of samples was carried out by Tukey HSD test.
The results of the semen evaluation in Table 1 shows
the total copper content of the seminal plasma was recorded as 2.51 ±
0.04 mg kg-1, while, the superoxide dimutase value was 39.92
± 0.81 IU mL-1. In order to have a better insight of
these results and make the range of variations narrower, the samples were
categorized in three groups of Excellent (Ex, >90% motile) (n = 33),
Good (Go, 80-89% motile) (n = 15) and Moderate (Mo, <79% motile) (n
= 6) according to their progressive motility rates. The mean values for
progressive motility were recorded as 92.24 ± 0.51% in Ex, 81.66
± 0.62% in Go and 71.66 ± 1.05% in Mo groups, which were
significantly different. The mean copper value in Ex group (2.56 ±
0.04 mg kg-1) was positively correlated with sperm progressive
motility and viability (p = 0.033 for both) and highly positively correlated
with seminal plasma SOD values (p = 0.000). The mean copper value in Go
group (2.38 ± 0.11 mg kg-1) was highly positively correlated
with sperm viability and SOD values (p = 0.008 and p = 0.000), but in
Mo group (2.55 ± 0.10 mg kg-1) had a positive significant
correlation with semen volume (p = 0.022) only (Table 2).
The observed negative correlations of the mean copper value in this group
(Mo) with sperm gross and progressive motility, viability, concentration
and seminal plasma SOD values were not statistically significant.
|| Semen characteristics of the buffalo semen (Mean ±
SEM) (n = 54)
||The comparison of the characteristics of three groups
of seminal plasma samples
|Mean values denoted by different letter(s) (a,
b and c) are significantly different (p<0.05)
The copper content of the semen has been reported to have an effect on
spermatozoa number and motility (Skandhan, 1992) and also on prevention
of lipid peroxidation in spermatozoa membrane via the activity of superoxide
dismutase. This study was performed to investigate the copper content
of the seminal plasma in water buffalo bulls and its correlation with
superoxide dismutase, that is reported to be involved in its structure
(Pesch et al., 2006) and with other parameters used for semen evaluation.
The mean total copper value of the buffalo seminal plasma in this study
was recorded as 2.51 ± 0.04 mg kg-1. Since the specific
gravity for seminal plasma in this study was recorded as 1030 mg, by conversion
of the unit, the total copper content of one litter of the seminal plasma
may be a little less (2.43 mg L-1) which does not affect the
results noticeably. The mean copper value in Ex group (2.56 ± 0.04
mg kg-1) which was higher but not significantly different with
the other two (Go and Mo) groups, was positively correlated with sperm
progressive motility and viability and highly positively correlated with
seminal plasma SOD values. The mean copper value in Go group (2.38 ±
0.11 mg kg-1) was highly positively correlated with sperm viability
and SOD values, but in Mo group (2.55 ± 0.10 mg kg-1)
had a positive significant correlation with semen volume only. The observed
negative correlations of the mean copper value in this group with sperm
gross and progressive motility, viability, concentration and seminal plasma
SOD values were not statistically significant. This may be the effect
of a small number of samples in this group.
Massányi et al. (2003b) determined the seminal concentrations
of copper and other trace elements in different animals. They observed
that the seminal copper concentration was significantly higher in ram
(2.49 ± 0.18 mg kg-1) and fox (2.16 ± 0.53 mg
kg-1 than that in bull (1.64 ± 0.21 mg kg-1),
boar (1.64 ± 0.28 mg kg-1) and stallion (0.86 mg kg-1).
Klemmt and Scialli (2005) stated that human seminal fluid chemical concentrations
are typically similar to or lower than blood concentrations, although
some antimicrobial agents achieve higher concentrations in semen than
in blood. This may be an important factor when adding supplementary copper
to the animals` ration.
A positive correlation of copper content of buffalo seminal plasma with
sperm motility observed in this study is in agreement with the report
of Skandhan (1992) and Massanyi et al. (2005) for the sperm motility
but not for the sperm concentration.
Pant and Srivastava (2003) reported that there was no significant difference
in copper levels among the different infertile categories. They observed
a positive correlation between copper and fructose (r = 0.81, r = 0.72,
p<0.05) in oligoasthenospermic and azoospermic men, respectively. This
is in agreement with our results for Moderate (Mo) group although we did
not further categorized this group.
The total SOD activity observed in buffalo seminal plasma here was 39.92
± 0.81 IU mL-1. Nair et al. (2006) reported SOD
activity in seminal plasma of buffalo bulls in India as 0.86 ±
0.03 U mg-1 protein and it increased to 1.91 ± 0.02
U mg-1 after 72 h storage at refrigerator temperature which
is very different with our results. The SOD activity in seminal plasma
has been reported to be 27 ± 10.8 U mL-1 in the canine
(Cassani et al., 2005) and 5.89 ± 0.96 in normal human subjects
(Khosrowbeygi and Zarghami, 2007).
Siciliano et al. (2001) and Hsieh et al. (2002) showed
that there is no significant difference in seminal plasma or sperm SOD
activity between normozoospermic and oligo-or asthenozoospermic males
and also Khosrowbeygi and Zarghami (2007) observed that activities of
SOD did not correlate significantly with sperm motility and concentration.
This is in agreement with our results of correlations between SOD and
sperm concentrations and contradictory to the results of its correlation
with sperm motility in Ex group.
Vishwanath and Shannon (1997) reviewed the details of the peroxidation
reactions in semen storage and described the exact role of copper and
SOD in these reactions.
In mammals there are three SOD isoenzymes, the cytosolic dimeric CuZn-SOD,
the mitochondrial matrix Mn-SOD and the secretory tetrameric extracellular
SOD (EC-SOD). Plasma also contains EC-SOD with reduced heparin-affinity
resulting from truncations and other modifications of the carboxyterminal
ends. Since the substrate, the superoxide anion radical, crosses membranes
poorly, the SOD isoenzymes must exert distinct protective roles in their
respective compartments (Peeker et al., 1997). In the Peeker et
al. (1997) study the occurrence and distribution of CuZn-SOD, Mn-SOD
and EC-SOD in human seminal plasma and spermatozoa were investigated.
The activity of the cytosolic CuZn-SOD of spermatozoa was exceptionally
high, but the activity of the mitochondrial Mn-SOD was low. The content
of the secretory EC-SOD was very low. Prostate gland appears to be the
main source of CuZn-SOD according to the split ejaculate study. The activity
in the final seminal plasma is ~10% of that of prostate gland cytosol.
Some apocrinic secretion may occur in the prostate gland epithelium, which
might explain the presence of CuZn-SOD in the seminal plasma (Peeker et
Cassani et al. (2005) stated that increased SOD activity in seminal
plasma, when there is a decreased activity in spermatozoa, could be attributed
to the enzyme loss from damaged spermatozoa. The increase of the plasma
membrane permeability in this type of spermatozoa seems to permit the
loss of the enzyme as was detected in spermatozoa with altered plasma
membrane permeability as a consequence of the cryopreservation process.
This may be the case for a significantly higher (50.66 ± 2.51 versus
39 ± 0.57 IU mL-1) level of SOD in our Mo group.
Nissen and Kreysel (1983) reported that SOD detected in human seminal
plasma inhibits lipid peroxidation and there is a good relationship between
SOD-activity and sperm motility; a similar effect has been reported by
Skandhan (1992). This is in agreement with our results in Ex group.
Lewis et al. (1995) stated that superoxide dismutase (SOD) activity
in seminal plasma appears to have no correlation with percentage motility
in infertile men. This does not apply to our results of Ex group but is
in agreement with the results of Go and Mo groups.
These observations reveal that copper and SOD content of seminal plasma
are important parameters correlated with sperm motility and viability,
which are considered to be the most important factors in semen fertility
after being inseminated to the females.
It can be concluded that copper and superoxide dismutase content of the
seminal plasma in buffalo bulls have an effect on sperm motility and viability.
Low levels of copper in seminal plasma may be observed in the semen samples
of poor quality, while its high levels, accompanied with high levels of
SOD, might be indicative of some cell damage.
We would like to thank the authorities of the Buffalo Breeding Center
in Northwest of Iran for their support and the supply of buffalo semen.
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