Vol. 11, No. 1
37
ORIGINAL RESEARCH
Seasonal changes in antioxidant defence
systems in seminal plasma and fluids
of the boar reproductive tract
Magdalena Koziorowska-Gilun1,2, Marek Koziorowski3, Jerzy Strzeżek2,
Leyland Fraser2
Department of Animal Biochemistry and Biotechnology, University
of Warmia and Mazury, Olsztyn, 3Department of Animal Physiology
and Reproduction, University of Rzeszow, Rzeszow, Poland
2
Received: 20 September 2010; accepted: 25 February 2011
SUMMARY
This study aimed to analyze seasonal variations in the antioxidant defence
systems of the seminal plasma and fluids of the cauda epididymis and vesicular glands of the boar. The analyzed antioxidants included superoxide
dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total Lglutathione (GSH+GSSG). Seasonal changes in total protein content and total
antioxidant status (TAS) of the seminal plasma and reproductive fluids were
also analyzed. Compared with the spring-summer period, total protein content in the seminal plasma was significantly higher during the autumn-winter
period. Among the antioxidants analyzed, only SOD activity showed marked
seasonal variations, being significantly higher during the spring-summer
Corresponding author: Department of Animal Biochemistry and Biotechnology, Faculty of Animal
Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego Street 5, 10–718
Olsztyn-Kortowo, Poland; e-mail: [email protected]
1
Copyright © 2011 by the Society for Biology of Reproduction
38
Antioxidants in boar reproductive tract
period. Likewise, the fluid of the cauda epididymis exhibited greater SOD
and CAT activity during the spring-summer period, whereas TAS levels were
markedly higher during the autumn-winter period. Neither GPx activity nor
total GSH+GSSG content in the cauda epididymal fluid was significantly
affected by the seasonal periods. The vesicular gland fluid exhibited an approximately 4-fold greater level of SOD activity during the autumn-winter
period, as compared with the spring-summer period. By contrast, greater
CAT and GPx activity, and a higher level of total GSH+GSSG were observed
in the vesicular gland fluid during the spring-summer period. In conclusion,
the findings of this study indicate that seasonal variations could have varying
effects on the antioxidant defence systems in the seminal plasma and fluids
of the boar reproductive tract. Reproductive Biology 2011 11 1: 37–47.
Key words: boar, seminal plasma, reproductive tract fluids, antioxidant
enzymes, TAS
INTRODUCTION
The male reproductive system undergoes cyclic changes caused by photoperiod [5, 12]. The domestic pigs are descendants of the wild boar characterized
by a clearly seasonal reproductive pattern, with peak activity in the late autumn and winter [8, 14]. Reproductive activity of the wild boar is associated
with variations in testosterone level, being markedly higher in late autumn
and winter [5]. Even though domestic pigs are sexually active throughout
the year, their seasonal reproductive activity was not lost. Several authors
have postulated that boar semen production is influenced by many factors
among which photoperiod and temperature account for marked variations
in the semen quality [4, 10, 16, 18, 19, 21].
It has been suggested that oxidative stress, a condition associated with
the excessive production of reactive oxygen species (ROS) is the prevalent cause of fertility disorders in mammals [17]. Evidence has shown
that the fluids of the reproductive tract create a favorable environment for
spermatozoa during their transit and maturation in the epididymis and at
ejaculation [24]. Moreover, the reproductive tract fluids comprise an array
Koziorowska-Gilun et al
39
of enzymatic and non-enzymatic defense systems in which superoxide
dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) are
the principal antioxidant enzymes that constitute the bulk of the protection of spermatozoa against a ROS-mediated attack [9, 13, 20]. We are not
aware of any published studies on the effects of season on the antioxidant
capacity of the seminal plasma and reproductive tract fluids of the boar.
Therefore, the present study examined the effect of seasonal variations on
the antioxidant defence systems in the seminal plasma and fluids of the cauda
epididymis and vesicular glands of the boar.
MATERIALS AND METHODS
Collection of seminal plasma and fluids from the boar reproductive tract
Ejaculates were collected from fourteen Polish Landrace and Large White
boars (aged 18–36 months) kept at the Insemination Stations in Olecko,
Petkowice and Slawecinek (Poland). The boars were allocated into two
experimental groups: 1/ sampled during the spring-summer period (April
through September) characterized by increasing light periods, and 2/ sampled
during the autumn-winter period (October through March) characterized
by reducing light periods [18]. Fourteen ejaculates were collected during
each seasonal period: spring-summer and autumn-winter. Seminal plasma
was separated from ejaculate by centrifugation at 1000´g for 15 min at room
temperature. The recovered seminal plasma was further centrifuged at 10
000´g for 10 min at room temperature and stored at –80ºC until further
analysis.
The fluids of the cauda epididymis and vesicular glands were collected
from 24 clinically healthy, sexually mature Polish Large White and Landrace
boars (aged 12–36 months) stationed at the Experimental Research Laboratory of the Department of Physiology and Animal Reproduction, Rzeszow
(Poland), according to the previously described method [9]. Reproductive
fluids were harvested from twelve boars during each seasonal period:
spring-summer and autumn-winter. The boars used in the experiments
40
Antioxidants in boar reproductive tract
were fed with a commercial porcine ration and were kept in individual pens
under standard environmental conditions. Water was available ad libitum.
The experiments were carried out in accordance with the guidelines set out
by the Local Ethics Committee.
Biochemical analysis
Total protein content was measured in the seminal plasma and reproductive fluids using biuret protein assay [25] with serum bovine albumin
(BSA; Serum and Vaccine Production, Cracow, Poland) as a standard. All
the antioxidant activities (SOD, CAT and GPx), total GSH+GSSG (reduced
and oxidized) content and total antioxidant status (TAS) were measured
spectrophometrically in the seminal plasma and reproductive tract fluids.
The activity of CAT, GPx, total GSH+GSSG content and TAS was measured
using commercial kits, according to the manufacturer’s recommendation.
All assays were run in duplicates.
The activity of SOD was determined according to Beauchamp
and Fridovich´s method [1]. One unit (U) of SOD activity was defined as
the amount of enzyme capable of decreasing the reduction of nitroblue tetrazolium (NBT) by 50% at 25ºC (pH 10.2). SOD activity was expressed as
U/ml. A commercial kit (Sigma Aldrich, St. Louis, MO, USA) was used to
measure CAT activity. One unit (U) of catalase decomposed 1.0 micromole
of hydrogen peroxide to oxygen and water per minute at pH 7.0 at 25°C at
a substrate concentration of 50 mM hydrogen peroxide. CAT activity was
expressed as U/ml. The activity of GPx was determined using the Ransel
Glutathione Peroxidase kit (Randox Laboratories, UK). In this assay GPx
catalyzes the oxidation of GSH with cumene hydroperoxide. In the presence
of GR and NADPH, GSSG is converted into GSH with a concomitant oxidation of NADPH to NADP+. The decrease in the absorbance was measured at
340 nm at 37ºC (pH 7.2). The activity of GPx was expressed as U/ml. Total
content of L-glutathione (GSH+GSSG) was determined using the Bioxytech
GSH-400 assay kit (Oxis International, Inc. Foster City, USA). The assay
is based on the conjugation of a substitution product (thioethers), obtained
with GSH into a chromophoric thione. Total GSH+GSSG concentration, ex-
Koziorowska-Gilun et al
41
pressed as µM, was calculated from the change at an absorbance of 400 nm
at 25°C (pH 7.8). Assays were performed after deproteinization of the sample
with 5% metaphosphoric acid. Total antioxidant status (TAS) was determined
using an assay kit (Randox Laboratories, Crumlin, UK). Samples were assayed according to the instructions. This method is based on the reaction
of ABTS (2.2’-Azino-di-[3-ethylbenzthiazoline sulphonate]) with peroxidase
and H2O2 to produce a ABTS radical cation. The level of TAS, expressed as
µM, was measured from a change in ABTS in the presence of antioxidants
at 600 nm (37°C, pH 7.4). All results are expressed as the means±SEM,
using the Statistica software package (StatSoft Incorporation, Tulsa OK,
USA). Comparisons between the seasonal periods were performed using
Student´s t-test (p< 0.05).
RESULTS
Table 1 shows seasonal changes in total protein and components of the antioxidant defence systems in the seminal plasma and fluids of the cauda
epididymis and vesicular glands. Total protein content was higher (p<0.05)
in the seminal plasma during the autumn-winter period compared with
the spring-summer period. Total protein content did not differ (p>0.05)
between the seasonal periods either in the cauda epididymal fluid or
in the vesicular gland fluid.
Seminal plasma SOD activity during the spring-summer period was
higher (p<0.05) than that of the autumn-winter period. The activity
of CAT and GPx, total GSH+GSSG content and TAS level in the seminal
plasma did not vary (p>0.05) between the seasonal periods. Higher SOD
(p<0.05) activity was demonstrated in the cauda epididymal fluid during
the spring-summer period in comparison with the autumn-winter period,
and this coincided with greater CAT activity. However, the activity of GPx
and the level of total GSH+GSSG in the cauda epididymal did not show
any marked seasonal differences but TAS level during the autumn-winter
period was higher (p<0.05) than during the spring-summer period. The level
of SOD activity was approximately 4-fold higher in the vesicular gland fluid
31.9±3.5a
41.9±2.6b
22.8±1.4
24.1±0.8
125.9±1.8
128.7±2.4
autumn-winter
spring-summer
autumn-winter
spring-summer
autumn-winter
(mg/ml)
spring-summer
Season
128.0±5.5b
30.0±11.6a
214.7±25.7b
0.80±0.09a
0.10±005b
2.4±0.5b
0.70±0.09
9.0±3.1a
4.2±1.0b
0.70±0.07
18.9±6.2a
304.9±30.0a
0.18±0.03
2.1±0.3
29.8±4.0b
0.23±0.04
1.6±0.3
39.4±5.2a
(U/ml)
GPx
(U/ml)
CAT
(U/ml)
SOD
1382.2±66.6b
2313.9±85.0a
1304.0±45.0
1090.4±80.0
1628.5±145.3
1367.7±144.7
(μM)
GSH+GSSG
2070.0±100.0
2240.0±110.0
1930.0±60.0b
1650.0±110.0a
640.2±40.8
521.0±44.2
(μM)
TAS
ab
p<0.05; spring-summer vs. autumn-winter (Student´s t-test); SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; GSH+GSSG: total L-glutathione content; TAS: total antioxidant status. Ejaculates were collected from
14 boars per group (spring-summer or autumn-winter group) to analyze seminal plasma, whereas the cauda epididymal
and vesicular gland fluids were collected from 12 boars per group.
Vesicular
glands
Cauda
epididymis
Seminal
plasma
Biological
fluids
Protein
content
Table 1. Seasonal changes in total protein content and antioxidant systems (means±SEM) in seminal plasma and reproductive fluids of the boar.
42
Antioxidants in boar reproductive tract
Koziorowska-Gilun et al
43
during the autumn-winter as compared with the spring-summer period.
In contrast, greater (p<0.05) CAT and GPx activity coincided with higher
levels of total GSH+GSSG during the spring-summer period. However, TAS
levels in the vesicular gland fluid were not significantly (p>0.05) affected
by the season.
DISCUSSION
Photoperiod is known to be a main environmental signal timing the occurrence of the reproductive cycles in several mammalian species. This cyclicity
entails seasonal changes in testicular size, weight and secretion as well as
sperm production, mating activity and fertility of males [5, 27]. The domestic
pig (Sus scrofa) is characterized by seasonal changes in its reproductive activity, and has been recognized to be a polyestrous species [16]. It is well known
that the binding of certain seminal plasma proteins to the sperm plasma membrane may modulate capacitation and acrosome reaction in spermatozoa [15,
26]. Similarly to previous reports [7, 21, 22], we showed that seminal plasma
protein levels were affected by season. It is possible that the higher total
protein content in boar seminal plasma detected during the autumn-winter
period could exert protective effects on the sperm plasma membrane integrity, which is essential for the maintenance of sperm function. In accordance
with this, ejaculates collected from boars in the autumn are characterized
by better semen quality than those collected in the summer [4, 19]. Furthermore, a high correlation between the total protein content and the antioxidant
properties of the seminal plasma has been reported [20]. It has been reported
that in rams reduced protein content in the seminal plasma in the spring or
summer could attribute to impaired sperm viability [2], reaffirming that
seminal plasma proteins play an important role in sperm membrane stability [22]. It has also been demonstrated that the addition of seminal plasma
collected during the autumn or winter period to frozen-thawed ram semen
significantly improved post-thaw sperm motility [6].
The greater activity of SOD and CAT in the fluid of cauda epididymis
was found during the spring-summer period in comparison to the autumn-
44
Antioxidants in boar reproductive tract
winter period. It seems that the presence of high enzymatic antioxidant activity in the cauda epididymal fluid may be required to protect spermatozoa
against oxidative stress during epididymal storage. Poor semen quality, such
as reduced sperm concentration and increased proportions of spermatozoa
with cytoplasmic proximal droplets, was found in boars kept under warm
conditions or under short light periods for a few days [10, 16]. Moreover,
an increase in the percentage of spermatozoa with proximal cytoplasmic
droplets in the ejaculates may be explained by epididymal dysfunction, as
a consequence of elevated temperature [16]. It shoud be emphasized that ROS
production is high in damaged spermatozoa or spermatozoa with abnormal
retention of the cytoplasmic droplets [3]. Spermatozoa have limited antioxidant capacity against ROS-mediated attack and are highly dependent on their
intrinsic intracellular antioxidants and antioxidant protection of the seminal
plasma and reproductive fluids [9, 24]. The susceptibility of spermatozoa to
oxidative stress could be attributed to their relative lack of cytoplasm, limiting the available quantity of antioxidants [17]. Hence, greater SOD and CAT
activity in the cauda epididymis fluid confers higher antioxidant protection
during epididymal storage of spermatozoa in the spring or summer compared
to autumn or winter. We observed that the antioxidant protection system
of the vesicular gland fluid harvested during the spring-summer period was
also characterized by greater CAT activity and higher levels of GPx content.
Evidence has been shown that SOD constitutes the major scavenging
capacity of antioxidants in boar seminal plasma, and the level of SOD activity in the seminal plasma fluctuated during the seasons, being markedly
higher in the spring and autumn periods [7]. However, in the present study
it was found that SOD activity in the seminal plasma was significantly
higher during the spring-summer period, probably due to differences
in breed, age of boars or individual variations between boars. Even though
GPx activity in the seminal plasma was not significantly affected by seasonal changes in this study, higher GPx activity was observed in the spring
and summer periods by other authors [11]. It is noteworthy to reaffirm that
the antioxidant triad comprising SOD, CAT and GPx constitutes the first
line of defence against the adverse effects of ROS in the boar reproductive
tract [7, 9, 17, 20, 24]. Moreover, it has been demonstrated that seasonal-
Koziorowska-Gilun et al
45
dependent changes in SOD, CAT and GPx activities in the seminal plasma
affect the freezability of ram semen [14, 23].
In the current study the seasonal patterns in the antioxidant defence
system were also shown by the variations in the levels of total GSH+GSSG
and TAS in the seminal plasma and reproductive fluids. The seasonal differences observed for total GSH+GSSG in the vesicular gland fluid and TAS
in the cauda epididymal fluids also reflect the antioxidant protection in these
fluids. A decrease in the antioxidant capacity of the seminal plasma has been
associated with enhanced lipid peroxidation of the sperm membranes, resulting
in the impaired fertilizing ability of spermatozoa [3]. It is possible that the levels of TAS in the seminal plasma and reproductive fluids are influenced mainly
by the antioxidant system comprising SOD, CAT, GPx and total GSH+GSSG,
which have been shown to protect spermatozoa against lipid peroxidation.
In summary, the results of this study indicate seasonal-dependent
changes in the antioxidant defence system of the seminal plasma and fluids
of the cauda epididymis and vesicular glands. Since these changes affect
sperm function, further studies on the mechanisms of action of the antioxidant protective systems in reproductive fluids may contribute to an improvement in the technology of boar semen cryopreservation.
ACKNOWLEDGEMENTS
This study was supported by a research project (No. N N311 1686 33), development research project of the National Research and Development Centre
(No. 12001404/2008) and funds from UWM in Olsztyn No. 0103.0803.
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