A study of the antioxidant effect of alpha lipoic acids on sperm quality.

Abstract

Assisted reproductive techniques are useful in helping infertile couples achieve successful conception. Initial studies have shown that sperm cryopreservation, one step in assisted reproduction, causes a dramatic reduction in sperm quality. This has been attributed to, among other things, free radical activities. The aim of the present study was to minimize this oxidative attack by adding an antioxidant into the sperm microenvironment. Alpha lipoic acids were selected for this purpose for their efficient free radical scavenging properties and solubility in lipid and aqueous phases.
For this investigation, semen from six Boer bucks was pooled. Seminal analysis of the baseline prior to incubation of samples with different concentrations of Alpha lipoic acids (0.00625, 0.0125, 0.025, 0.05, 0.1 mmol/ml) was performed, and post-seminal analysis was conducted after a one-hour incubation. The comet assay was used to observe the effect of Alpha lipoic acids on sperm DNA integrity. Statistical analysis using an unpaired t-test with a significance level of p<0.05 was then performed.
Our results indicate that the sperm motility rate was improved after incubation with Alpha lipoic acids at a concentration of 0.02 mmol/ml. This concentration was also capable of reducing DNA damage.
In conclusion, Alpha lipoic acids renders cryoprotection to sperm, thereby improving sperm quality.

Full text

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CLINICS 2008;64:545-50
BASIC RESEARCH
I Universiti Kebangsaan Malaysia – Physiology, Fakulti Perubatan –
Malaysia .
II Universiti Kebangsaan Malaysia – Forensic Science Unit, FSKB –
Malaysia.
III Universiti Kebangsaan Malaysia – Anatomy, Faculty of Medicine –
Malaysia.
IV MARDI – Molecular Biology and Genetic Engineering Biotech Centre
– Malaysia.
V Universiti Kebangsaan Malaysia – Biomedical Sciences – Malaysia.
Email: timi@medic.ukm.my
Received for publication on April 04, 2008
Accepted for publication on May 23, 2008
A Study of tHE AntIoxIdAnt EffECt of AlpHA
lIpoIC ACIdS on SpERm quAlIty
Siti Fatimah Ibrahim,I Khairul Osman,II Srijit DasIII, Abas Mazni Othman,IV
Norzaiti Abdul Majid,V Mohd Padzil Abdul RahmanIV

doi: 10.1590/S1807-59322008000400022
Ibrahim SF, Osman K, Das S, Othman AM, Majid NA, Rahman MPA. A study of the antioxidant effect of alpha lipoic acids
on sperm quality. Clinics. 2008;63:545-50.
OBJECTIVE: Assisted reproductive techniques are useful in helping infertile couples achieve successful conception. Initial studies
have shown that sperm cryopreservation, one step in assisted reproduction, causes a dramatic reduction in sperm quality. This has
been attributed to, among other things, free radical activities. The aim of the present study was to minimize this oxidative attack
by adding an antioxidant into the sperm microenvironment. Alpha lipoic acids were selected for this purpose for their efficient free
radical scavenging properties and solubility in lipid and aqueous phases.
METHODS: For this investigation, semen from six Boer bucks was pooled. Seminal analysis of the baseline prior to incubation of
samples with different concentrations of Alpha lipoic acids (0.00625, 0.0125, 0.025, 0.05, 0.1 mmol/ml) was performed, and post-
seminal analysis was conducted after a one-hour incubation. The comet assay was used to observe the effect of Alpha lipoic acids
on sperm DNA integrity. Statistical analysis using an unpaired t-test with a significance level of p<0.05 was then performed.
RESULTS: Our results indicate that the sperm motility rate was improved after incubation with Alpha lipoic acids at a concentra-
tion of 0.02 mmol/ml. This concentration was also capable of reducing DNA damage.
CONCLUSION: In conclusion, Alpha lipoic acids renders cryoprotection to sperm, thereby improving sperm quality.
KEywORDS: Sperm. Semen. Action. Antioxidant. Oxidative stress. Alpha lipoic acids. Analysis.
INTRODUCTION
Infertility is defined as conception failure following
regular sexual activities in the absence of anti-contraceptive
means for at least one year.1 This is a global problem
affecting 15-20% of couples throughout their reproductive
life, and its prevalence has been found to increase with age.
Causes of infertility in couples are numerous and varied, but
the majority are attributable to the male partner. Currently,
the important causes of male infertility include azoospermia,
oligospermia or hypogonadism. Studies have indicated
that some infertility problems could be overcome through
assisted reproductive technology (ART).
ART is a technology consisting of either in vitro
fertilisation (IVF), intracytoplasmic sperm injection (ICSI)
or artificial insemination (AI). All of these techniques require
viable and quality sperm to be available when contraception
is required. To ensure that the cells are available, a very low
temperature storage technique known as cryopreservation
has been adopted. Although this technique allows sperm
to be obtained and stored immediately and indefinitely
when required, studies have shown that it decreases sperm
quality with every thaw. Considering most males of infertile
couples initially have low sperm quality and counts, utilizing
cryopreservation would cause even lower quality after their
sperm have already been frozen and thawed.
ICSI requires one sperm to produce fertilization. Studies
have shown that sperm from infertile males who have

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Ibrahim SF et al.
undergone cryopreservation have a high prevalence of DNA
and physical damage. Statistical data have supported this
observation, which has shown that 85 - 90% of repeated
pregnancy losses were attributable to genetic problems.2
Interestingly, DNA damage in sperm has been found to be
related to the increasing age of the male partner.3 This in
turn would make ART a non-viable option for these groups
of infertile couples. Again, for couples opting to utilize ART,
sperm cryopreservation must be improved.
Cryopreservation is a technique that involves a very
wide range of temperature changes. There has been much
speculation as to the pathophysiology of this technique, but
most cryobiologists attribute the complications to osmotic
changes and membranes damage, a component of cold shock
injury. As such, the complications are due to the production
of large quantities of free radicals during the freezing and
thawing of the sperm. Free radicals are reactive oxygen
and nitrogen species that are unstable due to the presence
of unpaired electrons on its outer valency. Due to this
instability, the molecule tends to abstract another electron
from other nearby molecules. Once an electron has been
stolen, the molecular structure will reorganize, become non-
functional, and will eventually disassemble. Examples of the
types of molecules that undergo this fate are lipids, proteins
and DNA.
Alpha lipoic acids (ALA) have been reported to have
additional functions by which they are able to regenerate
vitamin C from reduced vitamin C in the presence of
glutathione. This capability would allow more antioxidants
to be present within the biological system without requiring
increased vitamin C in the sperm media and thus shifting
semen media to an acidic condition. Addition of ALA into
the media does not cause a major shift of pH into acidic
regions because the acid is categorized as a weak acid.
Recent findings have also indicated that ALA is able to enter
the Krebs cycle, thus assisting in the production of ATP,
which is required in viable sperm.
Based on the above facts, the present study was
conducted to study the effect of ALA in DNA damage and in
improving sperm quality during cryopreservation. As such,
the results of this study may have wider clinical importance
in fertility clinics and laboratories.
MATERIALS AND METHODS
Sexually mature (more than two years old) male Boer
buck cross-species were selected from the Malaysian
Agricultural Research and Development Institute (MARDI).
Animals were housed in a normal barn and provided access
to a normal goat diet and tap water. All investigations were
conducted in accordance with the guiding principle for
the care and use of research animals from MARDI. Fresh
semen samples were collected from male adult Boer buck
cross-species through the use of the artificial vagina method.
Semen samples were diluted in tris-citric egg yolk extender
at a ratio of 1:45 (semen:extender) and placed into 0.25 ml
sperm straws. The straws were then placed horizontally
above the liquid nitrogen surface. After a freezing period of
about seven minutes in the nitrogen vapour, the straws were
plunged into nitrogen liquid and stored. For thawing, straws
were immersed for five seconds in a water bath at 35ºC.
Sperm count and motility were performed using the
Mtrack J. Imaging System and Weber Sterility Chamber. A
total of 10 ul of diluted samples was put into the chamber.
Motility and count were evaluated using a light microscope
connected to the video monitor under magnification of 100X
and 400X. At least 10 grids of the chamber were counted
during each observation, and all procedures were performed
in triplicate. Sperm were then graded based on four groups
according to intensity of movement. The grades were fast
progressive (A), slow progressive (B), non-progressive (C)
and immotile (D). Sperm baseline readings were performed
before and after one-hour incubations with ALA. This step
provided a dose- response curve for ALA before proceeding
with the incorporation of ALA into the extender. Incubation
with ALA was carried out for one-hour, using an ELISA
bottom-flat plate in 37ºC. A total of 100 ul diluted sperm was
put into the plate according to the different concentrations of
ALA used, i.e., 0.1, 0.05, 0.025, 0.0125 and 0.00625 mmol/
ml. Graphs for motility and viability rate versus different
concentrations of ALA were plotted.
DNA damage was assessed using a modified alkaline
comet assay as described by Singh et al. (2003)3. Slide
preparations for the alkaline assay consisted of low (LMA)
and normal (NMA) melting agar. Both gel mixtures were
kept at 37ºC to maintain a liquid state until use. Slides were
prepared by first applying a thin layer of 0.75% NMA to
fully frosted slides to ensure adhesion of subsequent layers.
A 100 ul drop of the agarose was spread across the length
of the slides with a coverslip. Any excess was scrapped off.
Then, the slides were allowed to solidify for 15 minutes at
4ºC. The top layer was made by mixing 75 ul of LMA with
10 ul of sperm suspension. A volume of 85 ul was then
pipetted onto the first layer, covered with a coverslip and
allowed to solidify for another 15 minutes at 4ºC.
All slide incubation buffers mentioned below were
freshly prepared. The slides that had been created previously
were gently immersed in a Coplin jar containing pre-chilled
lysing buffer for one hour. The slides were then removed
from the solution and placed in a horizontal electrophoresis
tank. The slides were then equilibrated with freshly-made
electrophoresis solution, left for 20 minutes to allow the

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Ibrahim SF et al.
DNA to unwind. Electrophoresis was conducted for 10
minutes at 25 V and 300 mA. Subsequently, the slides were
then drained and flooded with three 15-minute changes of
fresh neutralization solution and air-dried. The dried slides
were stained with ethidium bromide by placing them in the
staining solution (20 ug/ml). To ensure the staining was
even, the slides and the solution were put on a Belly Dancer
(Rocky 3D, GmbH, Germany).
Spermatozoa analyzed for comets were visualized under
a fluorescence microscope. Whole sperm heads without a
comet were considered undamaged, whereas spermatozoa
with fragmented DNA that had migrated from the sperm
head, causing a comet pattern, were considered damaged. A
total of 25 sperm cells per slide were assessed for comets.
The comets were captured with a video camera connected to
the fluorescent microscope, and the images were evaluated
quantitatively for the percentage of tail DNA using a comet
assay imaging software (Comet Assay Software Project,
CASP version 2, Free Software Foundation. Inc.)
Statistical Analysis
All analyses were performed using SPSS software
version 12. All values obtained were tested against a
normality test before analysis for the comparison between
means. Since the number (N) of data samples was less than
100, Shapiro-Wilks was chosen as the normality test. Data
were not normally distributed (p<0.05), so Kruskall Wallis
ANOVA was chosen to compare the effect of different
concentrations of ALA versus the percentage of DNA
damage. The Mann-Whitney test was used to compare
the effect of different concentrations of ALA after the
incubation period on sperm motility and vitality. All values
obtained were presented as the mean ± SEM or median
where appropriate. A probability of p<0.05 was deemed a
significant difference in each case.
RESULTS
The mean values of fresh or baseline buck semen were
within the range of normozoospermia based on WHO (1999)
recommendations, as shown in Table 1.
There was a significant change in sperm motility in all
concentration used. However, the percentages of changes were
different for each of the concentration groups. The percentage
of motile sperm increased dramatically, from 112.8%
(concentration of 0.00625 mmol/ml) to 251.0% (0.0125 mmol/
ml). The increasing phase pattern change reversed following
introduction of more concentrated solutions of ALA. This is
clearly shown in the graph in Figure 1.
The changes in sperm motility based on the different
concentrations of ALA after the incubation period of
one hour are shown in Figure 1. The results show that
sperm motility increases following an increase in ALA
concentration, until 0.025 mmol/ml is reached. Then, sperm
motility decreased, even though the ALA concentration
increased. These findings suggest that an optimal
concentration of ALA is important in determining sperm
motility.
It was also observed that sperm viability changes after
a one-hour incubation with ALA (in Figure 2). While there
was no significant change, the graph clearly showed that the
viability rate was inversely proportional to the increment in
ALA concentration.
In Figure 3, the DNA tail moment using the comet
assay in sperm cells after a one-hour incubation with ALA
is shown. The assay involved a negative control and tests.
Based on the Kruskall Wallis ANOVA, there were significant
changes between ALA concentration and average score in
Table 1 - Sperm motility grading in this study compared to
WHO (1999) recommendation
ParameterValues WHO (1999) criterion
Density
1.Grade A 26.40 ± 1.75 %50% of forward movements (Grade
A & B) or 25% or more Grade A for-
ward movement within 60 minutes
of ejaculations
2. Grade B 41.99 ± 1.98 %
3. Grade C18.25 ± 0.65 %
4. Grade D13.35 ± 0.15 %
Motility2.99 ± 0.49 um/sec
or 68.40 ± 0.53%
More than 8 um/sec or more than
50% motile
Viability 86.53 ± 0.11%More than 75% viable
The grading was done using Weber Sterility chamber under light micro-
scope. Grade A – fast progressive movement; Grade B – slow progressive
movement; Grade C – non – progressive movement; Grade D – immotile;
* motility percentage = Grade A + Grade B; **viability percentage = Grade
A + Grade B + Grade C.
Figure 1 - Dose response curve of ALA against sperm motility. Sperm
motility was assessed using light microscope with Mtrack J Imaging System
on the Weber sterility chamber. Mann-Whitney t-test was used to determine
the differences between baseline (pre) and after one-hour incubation (post).
r2 = 0.0649 *P<0.05. N=31

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DNA tail moment. This study thus showed that incubation
with ALA affected the DNA tail moment. The optimal
concentration for ALA was identified as 0.025 mmol/ml.
DISCUSSION
DNA peroxidation is a process that involves a chemical
reaction of free radicals with the nucleotide component of
sperm DNA. This reaction has a cascading effect and was
found to be responsible for nucleotide deletion, insertion,
frame shift, DNA nicks and DNA fragmentation. In order
to minimize free radical activities, use of an antioxidant to
suppress free radical formation during freezing is essential.
Antioxidants are defined as substances that protect molecules
from oxidation by being oxidized themselves. Currently,
there are many types of antioxidants. Vitamin E, C and A are
classified as non-enzymatic antioxidants, while superoxide
dismutase, catalase, glutathione and alpha lipoic acids are
grouped as enzymatic antioxidants. Enzymatic antioxidants
are generally efficient free radical scavengers, but their
activities are largely isolated to free radicals present in the
aqueous phase. An exception to this limitation is ALA, which
is said to be a universal antioxidant because it is composed
of water and lipid-soluble antioxidant.
Results of the experiments showed that the percentage of
motile sperm increased dramatically, from 112.8% (0.00625
mmol/ml) to 251.0% (0.0125mmol/ml). This implies that
increasing ALA one-fold would also improve sperm motility
with similar effect. Unfortunately, the increasing pattern
changed following the introduction of more concentrated
solutions of ALA (Figure 1). This would suggest that, even
though ALA is capable of significantly improving the sperm
motility rate, adding more ALA into the media would cause the
media pH to become slightly acidic, thus killing the sperm.
Sperm mobility is largely dependent upon three major
factors - regulation, structural integrity and energy supply.
Regulation of movements is controlled at the mid-piece,
particularly the flagellar and principal area. Each of these
locations handles a unique function of sperm movement.
The flagellar mid-piece controls the activation of motility,
while the principal mid-piece handles hyperactivation.4 The
unsaturated lipid content and saturated protein channels in
the mid-piece usually make it the first choice for free radical
attack. Addition of ALA into the extender media allows the
antioxidant (ALA) to protect these components by creating a
shield surrounding the mid-piece (aqueous layer) and within
the structure itself (lipid layer).5
The ability of ALA to create a robust shield on the cell
membrane, along with the liquid that surrounds the sperm
indirectly, enhance the ability of the sperm to tolerate higher
volumes of free radical attack. This ability will, in turn,
indirectly reduce formation of deep pores and cracks on the
sperm surface, thus ensuring structural integrity. The rate of
sperm movement is largely dependent on the availability of
its energy supply. Due to this, normal active sperm usually
have a very active functioning mitochondria, which in turn
generates high quantities of free radicals as a by-product.
To ensure constant generation of ATP, external and internal
structural integrity of the organelle must be maintained.
Since the membrane wall and the various compartments
of the organelle are high in lipid content, addition of ALA
would protect these structures from the ever-increasing free
radical species, which are a by-product of the Krebs cycle.
Sperm mitochondria ability is also dependent on the
availability of ATP-based enzymes. Addition of ALA is
thought to have assisted in the metabolism of oxidative
Figure 2 - Dose response curve of ALA against sperm vitality. Sperm vitality
was assessed using light microscope with Mtrack J Imaging System on the
Weber sterility chamber. Mann-Whitney t-test was used to determine the
differences between baseline (pre) and after one-hour incubation (post).
There is no significant differences between pre and post treatment (P>0.05).
r2 = 0.9156 N=31
Figure 3 - Percentage of tail DNA (N=31) against a range of ALA concen-
tration (0, 0.00625, 0.0125, 0.025, 0.05 and 0.1mmol/ml). Percentage of
tail DNA was done using Comet assay imaging software (CASP version
2). Kruskal Wallis ANOVA was used to determine significant differences
between ALA concentrations and percentage of DNA tail. There is a sig-
nificant differences between the range of concentration compared to control
group. *P<0.05

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Ibrahim SF et al.
decarboxylation by acting as a co-enzyme.6 The increase
in oxidative decarboxylation would increase cytochrome C
concentration and thus directly increase the mitochondria’s
membrane potential, improving regulation of mitochondria
function and its biogenesis.7 In addition to the above,
ALA has also been reported to assist the mitochondria’s
citric cycle. This in turn will increase the level of reduced
glutathione, ATP, TCA cycle enzyme and electron transport
chain complex activities.8 ALA regulation of metabolism,
increased availability of mitochondrial co-enzymes and
improvement of protection of free radicals are thought to
eventually lead to a reduced incidence of mitochondria
dysfunction, thus ensuring sufficient ATP for sperm
movement.9
Currently, sperm quality is determined through its
morphology and motility. Although this method had been
adopted by various fertility laboratories and is considered
a gold standard, recent reports have suggested that other
factors must also be taken into consideration.10 Recent
findings have suggested that the DNA fragmentation
index (DFI) was higher in idiopathic infertile men with
the normal routine semen parameters than in fertile male
subjects.11 If conception is ever achieved by these idiopathic
infertile men, it is associated with repeated early fetal loss,
and the prevalence of incomplete pregnancies is greater
than normal.12 To ensure that full conception is achieved,
assessment of sperm DNA fragmentation is required. Until
now, the best method for this assessment was the use of the
comet assay, since it has an ability to qualitatively identify
single and double-stranded DNA damage. This study was
able to confirm the fact that addition of ALA was able to
minimize DNA damage.
Our study has shown that the DNA damage score
was significantly lower than in the negative control. This
is in accordance with a past study that had found that
administration of ALA to rats prevented oxidative stress and
decreased DNA strand breaks.13 Based on the results, it can
be concluded that there was significant correlation between
sperm motility and DNA damage. Addition of ALA into the
extender media has been shown to suppress DNA damage,
particularly at 0.025 mmol/ml.
Viable sperm by definition include even those that
are non-progressively motile. Sperm viability is closely
associated with regulated homeostasis and sperm membrane
integrity. As mentioned above, unregulated free radical
activity resulting from either environmental stress or
inadequate supply of antioxidants would disrupt membrane
structure. The disruption of the lipid and protein components
would eventually lead to selective permeability capabilities
loss and, eventually, cell death. Even though ALA is known
to posses potent anti-oxidative properties, the addition
of ALA into the sperm environment would shift the pH
towards acidity. Studies have shown that the speed of sperm
immobilization is proportional to the activity of hydrogen
ions in the sperm’s environment. Sperm will be non-motile
and enter apoptosis if the pH of its environment is not
maintained within pH 4.0 and 7.5.14 These facts are clearly
depicted in this study, in which high concentrations of ALA
caused a dramatic reduction in potentially viable sperm.
Although ALA is recognized as a universal antioxidant,
with abilities to scavenge free radicals in aqueous and non-
aqueous phases, its main efficacy is derived from its ability
to minimize peroxynitrite-induced damage efficiently. This
indirect efficiency is attributed to its ability to regulate
glutathione-s-transferase (GST) activity.15 Through the up-
regulation of this enzyme, cellular and organelle membranes
would be protected from synthetic peroxides, oxidized lipids,
retinoids, and the cytotoxicity of both 4-hydroxy-2-nonenal
(4-HNE) and hydrogen peroxide.16,17
Earlier studies have conclusively indicated that hydrogen
peroxide is the main reactive oxygen species involved in
induced motility loss, impairment of sperm-oocyte fusion
and acrosomal exocytosis.18 Hence, a significant reduction
of this component would in turn enhance sperm viability.
Interestingly, a past study also found that lipoic acid may act
as a cryoprotectant while being administered one day prior to
adriamycin therapy, thus maintaining normal steroidogenesis
and spermatogenesis.19
Recently, different techniques, including COMET assay,
Terminal transferase dUTP Nick End Labelling (TUNEL)
and sperm chromatin structure assay (SCSA), have been
noted to detect DNA damage.20 The present study can open
the door for future studies to detect DNA damage in sperm
using ALA.
Results obtained from this study show that an increase
in sperm motility was caused by ALA capabilities in energy
production but that decreases could be caused by the acidity
of the environment that produces immobilization. This was
further supported by previous findings, which suggested
that sperm motility is severely compromised when acidity
is induced by HCl.
CONCLUSION
It should be noted that damage to sperm DNA may
adversely affect male fertility and contribute to poorer embryo
development and lower pregnancy rates.21 Proper knowledge
of the causes of DNA damage and the various factors related to
its integrity may be helpful in treating infertility. In summary,
based on the results of our study, it can be concluded that an
optimal concentration of ALA was able to improve sperm
motility and viability and minimize DNA damage.

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