TITLE: Effects of Adding Sodium Nitroprusside to Semen Diluents
on Motility, Viability and Lipid Peroxidation of Sperm
in Holstein Bulls

  ABSTRACT.BACKGROUND:
Nitric oxide (NO) that plays important role in all sexual activities of animals
is made from the amino acid L-arginine by the enzymatic action of NO synthase (NOS). NO
makes a band with sulfur-iron complexes, but due to production of steroid sexual hormones
related to the enzymes involved in this complex, NO can change the activity of these enzymes.
NO affects many cells including vein endothelial cells, macrophages and mast cells. These
cells are also found in Leydig cells; therefore, they are important source of NO in testis tissue.
Therefore, minimizing damages to sperm at the time of freezing thawing process are really important. The aim of this study was to determine the appropriate NO concentration to be added
to the freezing extender to improve the quality of thawed sperm.

ABSTRACT.MATERIALS AND METHODS:
In this experimental randomized study, sperms of four Holstein
bulls with an average age of 4 were collected twice a week for 3 weeks. They received sodium nitroprusside (SNP) in concentrations of 0, 10, 50 and 100 nmol/ml. Data analysis
was performed using the special issue and static (SAS) 98 software. Also, mean comparison was done using Duncan's multiple ranges test (P<0.05).This research was conducted
at the laboratory of Science and Research Branch, Islamic Azad University, Tehran at
spring and summer of 2013.

ABSTRACT.RESULTS:
All concentrations of SNP used was found to increase motility and viability of spermatozoa at 1, 2 and 3 hours after thawing, significantly (P<0.05), but there was no significant
difference at zero time. Different concentrations of SNP reduced the membrane lipid peroxidation level of sperm and increased acrosome membranes integrity, implying that SNP generally
improved samples membranes, especially in 50 and 100 nmol/ml concentrations.

ABSTRACT.CONCLUSION:
According to the obtained results, addition of SNP to semen diluents increases motility and viability of spermatozoa. Also, it reduces membrane lipid peroxidation level that leads to improved sperm function.

BODY.INTRODUCTION:
Fertility is very important in livestock, especially
in bulls, because obtained sperm are used
in insemination of other cows. Freezing thawing
process causes sperm damage, like loss of lipid
membrane integrity, mitochondria activity, and
acrosome membrane integrity, leading to a reduction
in motility, viability and fertility of sperm (1).  Therefore, the most important factor in freezing
thawing process is to minimize the sperm
damages. Freezing-thawing process causes physiochemical stresses on sperm membrane, leading to
decrease quality, motility, viability and fertility of
sperm (2). Two types of damages are as follows:
Production of lots of free radicals and occurrence
of peroxidation of phospholipids in sperm membrane
which increases the level of fatty acid oxidation
including malondiealdehyde (MDA) (3-5).
Nitric oxide (NO) as an active non organic molecule
is spreadable and free and non stable which
is considered as endothelium-derived relaxing factor
in veins. It is made in body from the amino acid
L-arginine by the enzymatic action of NO synthase
(NOS). After making band with sulfur-iron complexes
NO changes the activity of these enzymes.
NO is an important transmitter molecule in mammalian
cells including human and plays a main
role in physiological and pathological processes
(6, 7). Effect of NO has been observed on many
physiological activities of organs, especially male
sexual system, like sperm motility, acrosomic reaction,
chemotaxis, ability of sperm to bind to the
egg, spermatogenesis and balancing the action of
hypothalamic-pituitary-gonadal axis (8).   There are few studies about the effects of NO
on quality of bulls' sperm, especially on measurement
of membrane damage via measuring MDA
level. The aim of this study was to determine the
appropriate NO concentration to be added to the
freezing extender in order to improve the quality of thawed sperm.

BODY.MATERIALS AND METHODS:
This research was conducted at the laboratory of
Science and Research Branch, Islamic Azad University,
Tehran at spring and summer of 2013. In
this experimental randomized study, four Holstein
bulls with a mean age of four years and appropriate
quantitative and qualitative characteristics of
sperm were selected one month before the test.
Bulls (jahed research center) were kept in separate
boxes and fed according to the National Research
Council (NRC, 2000) (9).

BODY.MATERIALS AND METHODS.SPERM METHOD:

Sperm was collected twice a week for 3 weeks.
Then samples were transferred to laboratory rapidly
and incubated at 37 ̊C in a bain-marie. After
semen volume, population and sperm motility of
each bull were determined, samples were mixed,
divided in four aliquots, and diluted using Bioxell
(Bioxell Inc., USA) containing various concentrations
of sodium nitroprusside (SNP). So, four SNP
treatments including 0 (control), 10, 50 and 100
nmol/ml were studied for 6 weeks (replications).
After dilution and packing in 0.5 cc pivots, 2 pivots
of each sample were transferred to laboratory,
and quality and quantity of sperms were analyzed
using computer-aided system analysis (CASA).
Samples were then frozen using digital freezer
and kept in nitrogen tanks. At least after 24 hours,
thawed samples were incubated and motility percentage,
viability, acrosome status and MDA level
were analyzed at 0, 1, 2, and 3 hours being kept at
37 ̊C. Freezing or cry therapy is a process of longterm
preservation of cells and tissues at very low
temperatures. To perform the insemination, the
frozen sperms should reach the proper temperature.
Payout melting process was performed in a
water bath of 32-35 ̊C.

BODY.MATERIALS AND METHODS.MEASURING THE MOTILITY OF SPERMATOZOA:

Samples were placed in warm water bath at 37 ̊C
for 3 minutes, 5 μl was transferred on a slide, and
covered with a covers lip of 18×18 mm. Sperm
analysis was done using CASA at 0, 1, 2 and 3
hours after thawing.

BODY.MATERIALS AND METHODS.MEASURING VIABILITY PERCENTAGE OF SPERM:

This was carried out using eosin staining method,
while nigrosin was used here as background color.

BODY.MATERIALS AND METHODS.MEASURING THE LEVEL OF MEMBRANE LIPID PEROXIDATION:
MDA level was measured by Esterbauer and
Cheeseman method (1990) using a spectrophotometer
(spectrophotometer uv-varian-CARY50
scan visible, Thermo, USA) at 532 nm wavelength.
This method is carried out on the bases of reaction
of MDA with thiobarbituric acid (TBA), leading to
elimination of double water molecules.

BODY.MATERIALS AND METHODS.MEASURING ACROSOME INTEGRITY OF SPERM:

In fertility, determining the acrosome percentage
is a morphological method for measuring viability
of sperm after thawing. For this, sperm motility is
inhibited initially by mixing semen with Glutaraldehyde
2% in phosphate buffer (NJ, USA). This
buffer fixes the membrane and prohibits its deterioration.
About 0.5 ml of semen sample was placed on a slide and was mixed with one drop of buffer.
This mixture was scattered on slide, and slide was
observed using a contrast phase microscope with
×1000 to ×3000 magnifications.

BODY.MATERIALS AND METHODS.MEASURING SPERM MEMBRANE FUNCTIONALITY:

About 250 ml of diluted semen was incubated in
1 ml of hypo-osmotic swelling test (HOST) solution
with osmolality of 100 m Osm/kg for 40 to
60 minutes and analyzed around 400 spermatozoa
using a contrast phase microscope with ×1000 to
×3000 magnifications.

BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS:

Data were analyzed using the special issue and
static (SAS) 98 software. ANOVA was used to
compare the mean values, while Duncan's multiple
range test was carried out to analyze the difference
between control and treatment groups. The
significance level was set at P<0.05.

BODY.RESULTS.TOTAL MOTILITY AND PROGRESSIVE MOTILITY OF SPERMATOZOA:
According to results, SNP increased total motility
of sperms significantly (P<0.05) in comparison
with control group (Table 1). It increased progressive
motility in 1, 2 and 3 hours after thawing, but
at zero hour (immediately after thawing), motility
increased only by 100 nmol/ml, indicating that it
is not significant (P>0.05). Increase in mobility
by SNP was also not significant before freezing
(P>0.05). Our findings showed that there is no significant
difference in 100-nmol treatment regarding
total motility of spermatozoa (P>0.05) (Table 1).  In case of progressive motility of spermatozoa,
the first and second hours after thawing showed
the best results, while among SNP concentrations,
100 nmol showed a significant difference compared
to the control (Table 2).   Table 1  The effects of different concentration of SNP on total motility of spermatozoa at various thawing times Treatment (SNP) 0 nmol/ml 10 nmol/ml 50 nmol/ml 100 nmol/ml Before freezing 74.08 ± 1.08a 77.08 ± 1.80a 76.5 ± 1.08 a 79.75 ± 1.85 a Immediately after thawing 57.00 ± 2.06 a 50.16 ± 1.08 a 49.25 ± 1.08 b 56.08 ± 2.02 a 1 hour after thawing 45.33 ± 1.09 a 54.08 ± 2.8 b 53.75 ± 2.8 b 58.41 ± 2.10 b 2 hours after thawing 41.42 ± 1.02 a 48.33 ± 2.8 b 46.83 ± 2.3 ab 50.66 ± 2.10 b 3 hours after thawing 33.83 ± 1.03 a 41.66 ± 1.06 b 39.58 ± 1.05 b 45.08 ± 1.09 b The data are presented as mean ± SD. SNP; Sodium nitroprusside. Common letters in each row indicate no significant difference (P>0.05).   Table 2  The effects of different concentration of SNP on progressive motility of spermatozoa at various thawing times Treatment (SNP) 0 nmol/ml 10 nmol/ml 50 nmol/ml 100 nmol/ml Before freezing 39.75 ± 2.07 a 42.41 ± 2.6 a 45.91 ± 2.73 ab 47.25 ± 2.8 b Immediately after thawing 37.08 ± 1.93 a 34.25 ± 1.09 a 34.75 ± 1.09 a 40.08 ± 2.1 a 1 hour after thawing 27.41 ± 1.07 a 36.41 ± 1.7 b 35.83 ± 1.7 b 40.33 ± 2.5 b 2 hours after thawing 24.66 ± 1.04 a 29.91 ± 1.35 b 30.08 ± 1.35 b 35.33 ± 1.49 c 3 hours after thawing 20.91 ± 1.03 a 26.08 ± 1.30 b 27 ± 1.32 b 27.58 ± 1.36 b The data are presented as mean ± SD. SNP; Sodium nitroprusside. Common letters in each row indicate no significant difference (P>0.05). 

BODY.RESULTS.SPERM VIABILITY:

SNP affected sperm viability and improved this
parameter 1 hour after thawing. All concentrations
of SNP increased viability significantly (<0.05).
This increase was observed in second hour only in
10 and 50 nmol/ml concentrations. Three hour after
thawing, all concentrations increased spermatozoa
viability significantly (P<0.05). However,
none of the used treatments could increase viability
in zero treatment (Table 3).

BODY.RESULTS.LIPID PEROXIDATION:

Different treatments of SNP reduced this parameter
significantly (P<0.05) and damaged sperm
membrane at 1 and 2 hours after thawing, but
in zero time only 50 nmol caused a significant
(P<0.05) reduction (Table 4).

BODY.RESULTS.ACROSOME INTEGRITY OF SPERM:

Variance analysis of data showed that before
freezing, SNP increased this parameter significantly
(P<0.05) only in 100 nmol/ml treatment. Also immediately
after thawing, 100 nmol treatment increased
acrosome integrity significantly (P<0.05). In first hour
after thawing, 50- and 100- nmol treatments increased
this parameter, while all concentrations at second and
third hours increased this parameter compared to the
control group. The highest ratio of healthy spermatozoa
was observed in 100-nmol treatment (Table 5).

BODY.RESULTS.SPERM MEMBRANE FUNCTIONALITY:

SNP at all different hours after thawing and even
before freezing could reduce the membrane damage
significantly (P<0.05) in all treatments, while
100 nmol/ml treatment demonstrated the best results
during the first hour after thawing (Table 6).   Table 3  The effects of different concentration of SNP on viability of spermatozoa at various thawing times Treatment (SNP) 0 nmol/ml 10 nmol/ml 50 nmol/ml 100 nmol/ml Before freezing 79.66 ± 1.07 a 82.75 ± 1.5 a 82.08 ± 1.09 a 84.66 ± 1.72 a Immediately after thawing 65.00 ± 2.4 a 59.50 ± 2.08 a 59.08 ± 2.04 a 64.58 ± 2.4 a 1 hour after thawing 52.50 ± 1.07 a 60.08 ± 1.09 b 60.25 ± 1.6 b 64.75 ± 1.77 b 2 hours after thawing 48.91 ± 1.06 a 55.33 ± 1.6 b 54.33 ± 1.5 b 57.33 ± 1.69 a 3 hours after thawing 40.75 ± 1.03 a 48 ± 1.38 ab 46.5 ±1.3 b 51.33 ± 2.01 c The data are presented as mean ± SD. SNP; Sodium nitroprusside. Common letters in each row indicate no significant difference (P>0.05).   Table 4  The effects of different concentration of SNP on level of membrane lipid peroxidation at various thawing times Treatment (SNP) 0 nmol/ml 10 nmol/ml 50 nmol/ml 100 nmol/ml Before freezing 0.20 ± 0.05 a 0.19 ± 0.05 a 0.15 ± 0.01 b 0.16 ± 0.01 ab Immediately after thawing 0.17 ± 0.008 a 0.15 ± 0.005 ab 0.11 ± 0.001 b 0.14 ± 0.005 c 1 hour after thawing 0.21 ± 0.009 a 0.14 ± 0.002 b 0.14 ± 0.002 b 0.12 ± 0.001 b The data are presented as mean ± SD. SNP; Sodium nitroprusside. Common letters in each row indicate no significant difference (P>0.05).   Table 5  The effects of different concentrations of SNP on acrosome integrity of spermatozoa at various thawing times Treatment (SNP) 0 nmol/ml 10 nmol/ml 50 nmol/ml 100 nmol/ml Before freezing 75. 5 ± 0.09 a 74.16 ± 0.09 b 72.83 ± 0.08 c 78.66 ± 1.05 d Immediately after thawing 55.66 ± 0.01 a 50.00 ± 0.09 b 47.16 ± 0.08 c 56.83 ± 0.02 d 1 hour after thawing 38.33 ± 0.05 a 38.16 ± 0.04 a 39.33 ± 0.05 b 46.33 ± 0.08 c 2 hours after thawing 29.08 ± 0.04 a 33.00 ± 0.06 b 38.33 ± 0.08 c 38.33 ± 0.08 c 3 hours after thawing 20.25 ± 0.04 a 22.16 ± 0.06 b 25.16 ± 0.09 c 27.00 ± 0.2 d The data are presented as mean ± SD. SNP; Sodium nitroprusside. Common letters in each row indicate no significant difference (P>0.05).   Table 6  The effects of different concentrations of SNP on membrane functionality at various thawing times Treatment (SNP) 0 nmol/ml 10 nmol/ml 50 nmol/ml 100 nmol/ml Before freezing 74.79 ± 0.06 a 80.79 ± 0.42 b 83.87 ± 0.5 c 87.83 ± 0.61 d Immediately after thawing 68.54 ± 0.07 a 76.45 ± 0.5 b 79.54 ± 0.7 c 82.25 ± 0.75 d 1 hour after thawing 67.29 ± 0.05 a 74.37 ± 0.3 b 79.75 ± 0.5 c 84.08 ± 0.57 d 2 hours after thawing 69.37 ± 0.07 a 75.00 ± 0.43 b 79.37 ± 0.6 c 83.66 ± 0.73 d 3 hours after thawing 72.00 ± 0.07 a 75.83 ± 0.18 b 78.12 ± 0.3 c 83.95 ± 0.57 d The data are presented as mean ± SD. SNP; Sodium nitroprusside. Common letters in each row indicate no significant difference (P>0.05). 

BODY.DISCUSSION:
NO is an important biological molecule which
plays critical role in sperm physiology like sperm
chemotaxis, sperm motility, and spermatogenesis
(8). Several studies have reported sperm motility
in low concentrations of NO in mice (10), sheep
and human (11, 12). Direct and significant correlation
was seen between NO concentrations
and sedentary sperm numbers (13, 14). Some researchers
have shown that NO is the main activator
of guanylate cyclase (ubiquitous enzymes). It is
noteworthy to mention that considerable amount
of cyclic guanosine monophosphate (cGMP) obtained
from this enzyme causes acrosome reaction,
chemotaxis and sperm-egg reaction (8, 15).
In this study, increasing effects of SNP on some
parameters like total motility and progressive motility
were in agreement with some studies (15-17),
whereas some other studies have reported different
results which is due to various SNP concentrations
(11, 18-20). Previous studies have shown
that concentrations higher than nmol per liter can
reduce sperm activity due to toxicity of NO, but
NO in low concentrations increases quality parameters
of sperm. The concentrations of 50 and 100
nmol/ml are ideal which are reported by Sharma
and Aqarwal (16). SNP increased sperm viability
and improved sperm parameters which are consistent
with some studies (16, 17) and not consistent
with others (11, 19, 20). Membrane lipid peroxidation
and acrosome integrity are considered as the
best indications of a healthy sperm membrane. Our
findings showed that different concentrations of
SNP increased HOST and incubation times, while
reduced MDA level in MDA test indicating controlling
effect of NO on lipid per oxidation. Other
studies also confirmed these results (17, 21). By
studying lipid per oxidation process, we found that
membrane injures resulted in reduction in motility
and death of sperm. Disorders in balance of
oxygen free radicals and in difference mechanism
eliminating these radicals lead to reactive oxygen
species (ROS) accumulation and induction of
oxidative stress that results in damage of proteins, membrane lipids and other cell components (22).
It has been reported that role of NO in prohibition
of lipid per oxidation is associated with its ability
in reaction with alkoxy lipid radicals (LX),
lipid peroxyl (LOO) and chain-breaking oxidation
(23, 24), which is in agreement with results
of this study. Furthermore our results indicated
that SNP improved the health of acrosome integrity
compared to control, while the best effect was
achieved for 100 nmol/ml treatment, which was
in agreement with other studies (15). Researchers
have emphasized mostly on morphological changes
of sperm like twisted tail, injured membranes
and injured acrosomes (25). Apparently, injured
sperm in freezing process increases ROS production
which causes damage to other normal sperms,
but SNP reduces acrosome injury via decreasing
oxidative stress.

BODY.CONCLUSION:
Based on the obtained results, SNP may improve
motility, viability, acrosome and plasma membrane
integrity during freezing-thawing process.
Addition of SNP before freezing provides better
results for bovine semen cryopreservation than its
inclusion in the thawing extender.

Question: Compared to Control what was the result of Sodium nitroprusside 100 nmol/ml on Acrosome integrity?
Evidence: SNP affected sperm viability and improved this
parameter 1 hour after thawing. All concentrations
of SNP increased viability significantly (<0.05).
Answer: significantly increased