R. Asadpour. 2012. Relationship between Mineral Composition of Seminal Plasma and Semen Quality in Various Ram Breeds.
Acta Scientiae Veterinariae. 40(2): 1027.
Acta Scientiae Veterinariae, 2012. 40(2): 1027.
ORIGINAL ARTICLE
Pub. 1027
ISSN 1679-9216 (Online)
Relationship between Mineral Composition of Seminal Plasma and Semen Quality
in Various Ram Breeds
Reza Asadpour
ABSTRACT
Background: Seminal plasma of mammals is a complex fluid, which serves as a carrier for the spermatozoa on their journey from the male testes to their target, the oocyte. Seminal plasma contains a variety of biochemical components, some
of which are relatively specific for the regulation of sperm function. The objective of this study was to investigate enzyme
activities aspartate-amino-transferase (AST), alkaline phosphatase (ALP), lactate-dehydrogenase (LDH) and concentrations of macroelements[ sodium (Na+) and potassium (K+) and calcium (Ca2+) ] in seminal plasma. Moreover the present
study was performed to investigate proteins in ram seminal plasma and the correlation between specific proteins and semen
characteristics in cross breed ram.
Materials, Methods & Results: Sixteen crossbred fertile rams (four Baluchi × Moghani, four Ghezel × Baluchi, four
Ghezel × Merino, and four Merino × Moghani) were used in this study. Three ejaculates of each animal were collected
during the breeding season and examined spermatozoa with light microscopy for motility, concentration, for dead sperm
and morphology. Seminal plasma was harvested by centrifugation and then subjected to analysis enzyme, mineral composition and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Total protein, AST, ALP, LDH, Na+, K+ and calcium Ca2+
were measured. Spearman’s correlation coefficient test was applied to examine the correlation between seminal plasma
protein fractions with all the parameters of the semen. Differences were considered to be statistically significant at P < 0.05.
The highest seminal plasma concentration of Na+, K+ and Ca2+ was recorded in Ghezel ×Merinos rams. The result showed
that there was a significant negative correlation between Na+ and K+ but small correlation was recorded between Na+ and
Ca2+. The highest plasma concentration of ALP was recorded in Baluchi × Moghani ram and the lowest was recorded N
in
Merino × Moghani ram (8 ± 1.25 and 3.33 ± 0.01 U/mL, respectively). The highest plasma concentration of LDH and
AST was recorded in Ghezel × Baluchi ram (4.68 ± 0.19 U/mL, 19 ± 4 U/mL respectively). Significant correlations were
found between LDH levels and sperm viability. The results obtained indicate that ram seminal plasma protein profile is
characterized by many protein bands with molecular weights, ranging from 8.81 to 95.82 kDa. Five proteins in this study
had quantitative difference between seminal plasma from crossbred rams.
Discussion: The results showed that the relative content of protein bands wasn’t significantly different by semen characteristics. Thus this study indicates that the relative content of seminal plasma proteins could not be an essential index
to evaluate ram semen quality. Also increase in the percentage of live and normal sperm corresponded to an increase in
LDH activity in the seminal fluid. It has been stated that LDH plays an important metabolic role in sperm capacitation and
fertilization. This study suggest that the cations Na+ and K+ generally establish the osmotic balance, and seminal plasma
osmolality ultimately plays an important role in the activation sperm cell also potassium ions (K+) are also intracellular
cations, and the concentrations in the seminal fluid may be an indication of sperm plasma membrane integrity. Thus some
enzyme and mineral composition could be an essential index to evaluate cross breed ram semen quality.
Keywords: minerals, rams, SDS-PAGE, seminal plasma, semen quality.
Received: September 2011
www.ufrgs.br/actavet
Accepted: December 2011
Department of Clinical Science, Faculty of Veterinary Medicine, University of Tabriz CORRESPONDENCE: R. Asadpour [r_asadpour@tabrizu.ac.ir Fax: +98 (411) 3357834]. Faculty of Veterinary Medicine, University of Tabriz. PK: 51666-16471, Tabriz, Iran.
1
R. Asadpour. 2012. Relationship between Mineral Composition of Seminal Plasma and Semen Quality in Various Ram Breeds.
Acta Scientiae Veterinariae. 40(2): 1027.
INTRODUCTION
MATERIALS AND METHODS
Seminal plasma, which is a complex mixture
secreted from the testes, epididymis and accessory
sex glands, can affect sperm morphology, motility,
acrosome reaction and fertility [29]. Despite its physiological significance, the molecular composition of
the seminal plasma is complex and the various proteins
or polypeptides present in seminal plasma are poorly
understood. In recent years, several seminal plasma
proteins have been identified, isolated and characterized. Evidence suggests that the protein composition
of seminal plasma is different among species and that
some seminal plasma proteins are associated with
fertility in various species. Homologous proteins have
also been identified in stallion [6], canine [10], goat
[25], buffalo [1] and ram [18].
Also it is known that the seminal plasma contains substances that support the sperm cells. Sodium
(Na+) and potassium (K+) cations in the seminal plasma
establish the osmotic balance, while the essential trace
elements are the components of many important enzymes [39], also calcium (Ca2+) is needed for stimulation
of steriodogenesis in leyding cells of the testis [17 ].
Lactate dehydrogenase (LDH) plays an important role
in the sperm metabolism, sperm capacitation and fertilization [12]. Alkaline phosphatase (ALP) is primary
of testicular and epididymal origin and, therefore,
suitable for differentiation of oligo - and azoospermia
[35]. Likewise, ALP, AST is essential for metabolic
processes which provide energy for survival, motility
and fertility of spermatozoa. Previous studies are generally related to the comparisons of seminal plasma
composition between males of different fertility or
the isolation and characterisation of specific seminal
proteins that could influence sperm capacitation and
fertilisation. However, the correlation between specific
seminal proteins and semen characteristics in crossbred
rams with differing fertility has not hitherto been well
studied.
Therefore the objective of this study was to
investigate enzyme activities (AST, ALP and LDH)
and concentrations of Na+, K+ and Ca 2+. Moreover the
present study was performed to investigate proteins
in ram seminal plasma and the correlation between
specific proteins and semen characteristics in cross
breed ram.
Animal and location
This trial was performed at the sheep breeding
Station, located in Tabriz, Iran. 16 crossbred fertile
rams (4 Baluchi × Moghani; 4 Ghezel × Baluchi; 4
Ghezel × Merino; 4 Merino × Moghani), 2-3 years
of age and with a live weight of 50-65 kg used in the
study. The animals were maintained under natural
photoperiod and during the trial, the rams were housed
separately from the ewes. All rams received a daily
diet of 666 g alfalfa hay, 66.5 g barley and 136.5 g of
a commercial concentrate
Semen collection and quality evaluation
Forty eight Semen samples (three ejaculates of
each animals) were collected, from the 16 fertile cross
bred rams trained to serve an artificial vagina (AV)
(temperature of 42-43°C). Prior to collection, the prepuce was wiped clean to prevent contamination of the
semen. Semen samples were collected in the mornings,
and transported to the laboratory (37°C), within 5-10
min, and placed in a water bath at 37°C. Semen volume
was estimated directly in the calibrated semen collection tube, and sperm concentration determined with
the aid of a Neubauer hemocytometer, after dilution
(1:200) of the semen sample with a 2% eosin solution.
To evaluate the sperm progressive motility, a sample
of the diluted sperm was placed under a cover slip on
a pre-warmed (37°C) slide and subjectively assessed
using a phase contrast microscope ( × 400 magnification). The percentage live sperm was determined by
evaluating 200 sperm/semen sample, (following eosin-nigrosin staining), under a light microscope ( × 400
magnification) [13]. At least 200 cells were counted
in duplicates for each sample.
Preparation of seminal plasma
The seminal plasma was separated immediately after collection. Fresh semen was centrifuged at
1500 × g for 15 min at 5°C. The supernatants were
transferred into 1.5 mL eppenddorf of tubes and recentrifuged at 14,000 × g for 10 min at 5°C to eliminate
the remaining sperm. Seminal plasma was stored at
-20°C until used.
2
R. Asadpour. 2012. Relationship between Mineral Composition of Seminal Plasma and Semen Quality in Various Ram Breeds.
Acta Scientiae Veterinariae. 40(2): 1027.
Seminal plasma compositions
Statistical analysis
Seminal plasma compositions were determined according to protocols presented in previous studies: Glucose [26], total protein [27], AST [32], ALP
[23], LDH [2]. Na+, K+ and Ca2+ were also measured
by Micro light1 based on ion-exchanger colorimetric
method [37].
Data analysis was performed using SPSS software program (version 16.0 for Windows). All values
were expressed as Mean ± standard error of mean
(S.E.M.). Spearman’s correlation coefficient test was
applied to examine the correlation between seminal
plasma protein fractions with all the parameters of the
semen. Differences were considered to be statistically
significant at P < 0.05.
SDS polyacrylamide denaturing gel electrophoresis
(SDS-PAGE)
The seminal plasma proteins were analysed
by SDS–PAGE gel electrophoresis. Seminal plasma
samples were suspended in loading buffer (4:1 v/v)
containing 50 mM dithiotreitol, 20 mM Tris, 2.5%
sodium dodecyl sulphate (SDS), 0.002% bromophenolblue and 5% glycerol, pH 6.8. Electrophoresis
was performed in 15% separating and 5% stacking
gels according to Laemmli [24] method. Each lane
was loaded with 30 mL seminal plasma. Samples
were concentrated at 70 V for 10 min; separation was
performed at 120 V for 4 h. Gels were stained with
Coomassie Brilliant Blue, G-250.
RESULTS
The results of the semen quality parameters of
sixteen cross breed rams are summarized in Table 1,
and depicted as mean ± S.E.M. Progressive motility(r
= 0.993, P < 0.01) was correlated significantly with
sperm viability. A significant negative correlations
were observed among Sperm viability, progressive
motility and sperm abnormality (r = -0.982 and r =
-0.987, P < 0.01 respectively). Semen characteristics
(semen volume, semen pH, sperm viability, sperm concentration and sperm motility,) were not significantly
among cross breed rams.
Mean values of some seminal plasma composition in four crossbreed rams are presented in Table 2.
The highest seminal plasma concentration of Na+, K+
N
and Ca2+ was recorded in Ghezel × Merinos rams. A
Image acquisition
Gel images were analysed to determine molecular weight and relative protein content using the
Total Lab TL120 computer program (v2009, USA).
Table 1. Semen characteristics in fresh ejaculates of cross breed rams with different fertility patterns.
The similar superscript letters in the rows indicate non-significant differences.
a
significant negative correlation was observed between
Na+ and K+ (r = -0.727 P < 0.05) but small (P < 0.05)
correlation was recorded between Na+ and Ca2+ (r =
0.619 P < 0.05).
The highest plasma concentration of ALP
(Mean ± S.E.M) was recorded in Baluchi × Moghani
ram and the lowest was recorded in Merino × Moghani
ram (8 ± 1.25 and 3.33 ± 0.01 U/mL, respectively).
The highest plasma concentration of LDH and AST
(Mean ± S.E.M) was recorded in Ghezel × Baluchi ram
(4.68 ± 0.19 U/mL, 19 ± 4 U/mL respectively) (Table
2). There was not correlation among, ALP and AST
and semen characteristics. Significant correlations
were found between LDH levels and sperm viability
(r = 0.721, P < 0.05).
The distribution of cross breed rams seminal
plasma proteins is shown in Figure 1. Seventeen protein
bands with different molecular weights ranging from
8.81 to 95.82 kDa were identified on the gel. (95.82,
85.35, 80.40, 74.24, 68.56, 62.89, 58.07, 50.52, 46, 34,
40.04, 33.92, 28.73, 19.04, 16.45, 13.48, 10.97, 8.81
kDa), twelve of these were presented in all samples
3
R. Asadpour. 2012. Relationship between Mineral Composition of Seminal Plasma and Semen Quality in Various Ram Breeds.
Acta Scientiae Veterinariae. 40(2): 1027.
Table 2. Least square means of concentrations of Na+, K+, Ca2+, Alcalin posphatase (ALP), Lactate dehydrogenase (LDH), Aspartate
aminotranferase (AST) and Total protein (TP) in the seminal plasma of cross breed rams.
Different superscript letters in the rows indicate significant differences P < 0.05.
a,b
Figure1. Molecular weight (kDa) of proteins of the seminal plasma (SP), the total
protein fraction (TPF), as revealed by SDS–PAGE gel electrophoresis. Gels were
stained with Coomassie Brilliant Blue. Numbers indicate the molecular weight of the
standard in kDa. Lane 1( Merinos × Moghani) Lane 2(Merinos × Moghani) Lane
3 ( Baluchi × Moghani×) Lane 4( Baluchi × Moghani) Lane 5 (Ghezel ×Baluchi)
Lane 6 ( Merinous × Moghani× ) Lane 7(Ghezel × Merinous) Lane 8 (Baluchi ×
Moghani) Lane 9 ( Meronos Moghani Lane 10( Marker).
(Figure1). Five proteins in this study had quantitative
difference between seminal plasma from cross breed
rams. Spot 2 (85.35) was present in 7 samples, spot 7
(58.07) was shown in 7 samples, spot10 ( 40.04) was
shown in 8 samples, spot 13 (19.04) was present in
8 samples and spot16 (10.97) just was shown in 2
samples. Those spots are shown in Figure 1. The 85.35
kDa, 40.04 kDa, 19.04 (Lane 1 and Lane 2) proteins
were not detected in the semen samples obtained from
Merinos × Moghani rams. The 58.07 kDa (Lane 5 and
Lane 7) protein was not detected in the semen samples
obtained from Ghezel × Baluchi and Ghezel × Merinous rams. No significant correlations were observed
between protein bands and semen characteristics.
plasma components and semen characteristics were
investigated. Spermatological parameters were in the
range of previous studies [16,20], even though minor
differences were shown in some parameters. The mean
ejaculate volume recorded for four cross breed rams,
ranged between 1.0 and 1.4 mL, with the mean sperm
concentration being between 3.3-4.7 × 109 sperm/
mL. The mean sperm motility and sperm viability in
four cross breeds rams ranged between (67-86% and
65-75% respectively). This could be a reflection of
various factors that may influence sperm production
including genetics, environmental conditions and
nutrition. This finding is in agreement with data on
several sheep breeds in the temperate climates e.g.
the Moghani rams [40], Ghezel and Mehraban breeds
[39], and Persian Karakul rams in Iran [19].
In previous studies using SDS-PAGE, in a
gradient (4-22%) of polyacrylamide gel, 20 bands
DISCUSSION
In the current study, the fertility-associated
proteins and correlation between specific seminal
4
R. Asadpour. 2012. Relationship between Mineral Composition of Seminal Plasma and Semen Quality in Various Ram Breeds.
Acta Scientiae Veterinariae. 40(2): 1027.
were found in ovine seminal plasma and the most
prominent bands were <70 kDa [3]. In the present
study, seventeen protein fractions were detected in
cross breed rams seminal plasma with molecular
masses ranging from 8.81 to 95.82 kDa and the most
prominent spots were those < 50 kDa. In the study by
Jobim et al. [18], molecular weights ranged from 15
to 115 kDa and the most prominent spots were those
< 30 kDa. The protein spots with a molecular weight
from 15 to 20 kDa accounted for 41% of the relative
intensity of the spots of the gel. Cardozo et al. [8]
observed that when the molecular weights range between 12.5 and 83.9 kDa, the protein spots <21 kDa
had the highest relative intensity using the gradient
gel. In other study by Bergeron et al. [4], SDS-PAGE
analysis of alcohol-precipitated ram seminal proteins
indicated the presence of about 25 proteins with molecular masses from 14 to 120 kDa; a group of proteins
with a molecular mass of 15-16 kDa and 22-24 kDa
was more predominant. Also this is in agreement with
those reported in the bull [28] and stallion [34]. So
we came to the conclusion that most proteins in ram
seminal plasma are below 50 kDa. The difference
of molecular weight could be influenced by season,
breeds, age and collecting and preparation methods
of seminal plasma.
Correlation between seminal plasma proteins
and fertility of the male has been reported in some
species of domestic animals such as bull [22] ram
[18] goat [36] buffalo [1] stallion and boar [7]. The
results showed that the relative content of five protein
bands (85.35, 58.07, 40.04, 19.04 and 10.97 kDa) was
significantly differences between seminal plasma cross
breed rams.
In a study by Killian et al. [22] showed that
two of proteins 26 kDa and 55kDa associated with
high fertility in bulls. However in our study protein
band 58 kDa in Lane 5 and Lane 7 was not correlation
with semen characteristics. Protein spot 10 (40 kDa)
in lane 2 may correspond to clusterin. Clusterin is the
major glycoprotein in ram retetestis fluid, with an
apparent molecular mass of approximately 40 kDa on
SDS-PAGE and an acidic isoelectric point (3.6). The
highest tissue concentrations of clusterin are present
in the testis and epididymis; Sertoli cells may be the
testicular source of clusterin[5]. It is involved in sperm
maturation [33], and possesses heparin-binding sites
[18] suggested that this protein, present in bull seminal
plasma, is related to high semen freezability. Protein
spot 13 (19 kDa, lane 2) may be TIMP-2 (tissue inhibitor of metalloproteinase-2), a potent inhibitor of
the matrix-metalloproteinases, present in the caput
epididymal fluid in the ram, boar and stallion [30]. The
TIMPs are specific inhibitors of matrix metalloproteinases (MMPs), which are generally present at the same
time as the matrix proteases. These finding suggest
that some proteins may modulate sperm function by
providing energy and protection for spermatozoa as
a complementary substance. Yue et al. [38] reported
that protein band 72.45 kDa was correlated with sperm
viability. Although the other seven protein bands of all
seminal plasma samples showed no fertility associated
change in their relative content.
LP is a dephosphorylating enzyme that is
active in many tissues including bone, liver, kidney,
intestine, lung and placenta. The majority of ALP in
rams originates from the seminal vesicles and, to a lesser extent, from the testes and epididymides. Dogan et
al. [11] showed a significant positive correlation with
concentration, AST and LDH, but not with volume
in bull seminal plasma. While, we couldn’t found the
correlation between ALP and semen characteristics.
In our study significant correlations were
N
found between LDH levels and sperm viability. This
study is in agreement with other results. Zamiri and
Khodaei [39] showed that correlation coefficient of
the LDH level with the percentage live sperm (r =
−0.51) was however smaller than the value reported
for fat-tailed Iranian rams (r = −0.75). In this study
every increase in the percentage of live and normal
sperm corresponded to an increase in LDH activity in
the seminal fluid. It has been stated that LDH plays
an important metabolic role in sperm capacitation and
fertilization [12]. Furthermore, LDH is an intracellular
enzyme and increased levels in the seminal fluid may
be an indication of the integrity of the sperm plasma
membrane.
In the present study we couldn’t found a
correlation between AST and semen characteristics.
This information is consistent with finding Pesch et
al. [31] who reported that negative correlation was
between AST enzyme and sperm volume. Thus, increasing the percentage of abnormal spermatozoa in
ejaculate causes high concentration of transaminase
enzyme in the extra cellular fluid due to sperm membrane damage and ease of leakage of enzymes from
5
R. Asadpour. 2012. Relationship between Mineral Composition of Seminal Plasma and Semen Quality in Various Ram Breeds.
Acta Scientiae Veterinariae. 40(2): 1027.
spermatozoa [15]. However, Corteel [9] reported that
the transaminase activities (AST-ALT) in semen are
good indicators of semen quality because they measure
sperm membrane stability.
Our result showed that seminal Na+, K+ concentrations were higher than the range recorded for
other fat-tailed sheep breeds [40]. In the present study,
the high levels of Na+ and K+ ions were associated with
low percentages of motile sperm, and such semen was
considered to be of lower quality. This result is contrast
to previous finding. Zamiri and Khodaei [39] showed
that low levels of Na+ and K+ ions were associated with
high percentage of motile sperm. This study suggest
that the cations Na+ and K+ generally establish the
osmotic balance, and seminal plasma osmolality ultimately plays an important role in the activation sperm
cell also potassium ions (K+) are also intracellular
cations, and the concentrations in the seminal fluid
may be an indication of sperm plasma membrane integrity. Our result showed that high levels of Ca2+ were
associated with lower percentage of motile sperm. This
is agreement with the report of Kaya et al. [21] who
showed that increasing ejaculation frequency due to
reduction Ca2+seminal plasma and decrease in sperm
motility. Garcia and Graham [14] showed that reverse
proportional correlation existed between Ca2+ content
and the motility of seminal cells.
In conclusion our results indicate that relative
content of seminal plasma could not be an essential index to evaluate ram semen quality. While some enzyme
and mineral composition could be an essential index
to evaluate cross breed ram semen quality. However,
further studies and more experimental data are needed
to construct reliable mathematical models which would
make it possible to develop a simple method for the
prediction of ram fertility.
SOURCE AND MANUFACTURER
1
PL1000B VTT, Technical Research Centre of Finland
Vuorimiehentie, Finland.
Declaration of interest. The author report no conflicts of
interest, and alone is responsible for the content and writing
of the paper.
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