Hussain and Salam / Intl. R. J. of Pharmaceuticals (2011), Vol. 01, Issue 01, pp.

21-26 ISSN 2048-4143

Research Paper

A Study on the Cryopreservation of Stallion Semen with

Alpha Lipoic Acid
Dr. Jawad Hussain1, Abdul Salam2 and Dr. Ali Gohar3
1
School of Animal, Rural and Environmental Sciences, Nottingham Trent University, England, United Kingdom
2
Department of Chemistry, Loughborough University, England, United Kingdom
3
University of Veterinary and Animal Sciences, Lahore, Pakistan
1
E-Mail: jawadhussain787@yahoo.com

Abstract
Horses have the lowest fertility rate in all of the domesticated species. In order to accomplish successful conception,
artificial insemination, the assisted reproductive technique is the best procedure. Sperm quality during cryopreservation
dramatically diminishes because of the oxidative stresses which results from the production of reactive oxygen species
(ROS) during sperm metabolism. The aim of this study was to investigate if antioxidant alpha lipoic acid (ALA)
supplementation increases the efficacy of fertility Parameters in cryopreserved stallion semen. For this research, a different
concentrations / inclusion level of Alpha lipoic acids (0.0, 1.0, 0.5, 0.1, 0.05, 0.025 and 0.125 mmol / ml) was used in the
semen of two adult Equus caballus. Post thawed semen analysis (Motility, vitality, Plasma membrane integrity, Acrosomal
integrity and morphology of the semen) was conducted. The results indicate that there is no positive effect of ALA on
fertility parameter of the post thawed stallion semen. In conclusion, ALA like in other bovine species, do not appear to be
cryoprotectant to stallion semen.

Keywords: Artificial Insemination, Sperm Quality, Cryopreservation, Alpha Lipoic Acids (ALA)

1. Introduction
Horses have the lowest fertility rate in all the domesticated
species which might be possible, as horses are mostly
bread selectively for their conformation and performance
characteristics rather than for their fertility
accomplishment (Baczynska et al, 2007 and Blomfield,
2010). The aim of this study was to investigate if
antioxidant alpha lipoic acid (ALA) supplementation
increases the efficacy of fertility Parameters in
cryopreserved stallion semen. Artificial insemination (AI)
is considered one of the modern way of breed
improvement which is use for the genetic improvement
and improved production potential of horses (Stout, 2006).
In the process of (AI), chilled or cryopreserved semen is
used (Morel, 1999). Advantages of AI comprise the
reduced risk of the spread of both venereal and non-
venereal disease such as equine infectious anaemia, equine
influenza and strangles (Aurich et al, 1997 and
Blanchard et al, 2003).
During cryopreservation, osmotic and thermal shocks
induce reactive oxygen species (ROS). A small amount of
ROS is imperative for normal function of spermatozoa, but
when the balance between ROS production and
neutralization is disrupted, an excess of ROS leads to
oxidative stress affecting the fluidity of membrane and
integrity of DNA in sperm nucleus (Aitkin, 1999). These
changes diminish the post-thaw sperm quality and
subsequent fertility.
The seminal plasma of mammals contains an antioxidant
defence system comprising of enzymatic and non-
enzymatic antioxidants. However during the storage
procedure semen is diluted by the addition of extender to
produce multiple doses from a single ejaculate. This
dilution decreases the concentration of natural antioxidants
in semen, leading to the production of ROS in supra-
physiologic limits.

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 Hussain and Salam / Intl. R. J. of Pharmaceuticals (2011), Vol. 01, Issue 01, pp. 21-26
All cellular components including lipids, proteins, nucleic
acid and sugars are potential targets for ROS. Therefore,
the amount of ROS should be limited to the minimum
level in order to maintain the normal cell function
(Agarwal et al, 2003). The excessive ROS can be
neutralized by the restoration of antioxidant defence
system of semen. This can be obtained through the
addition of suitable antioxidants in semen extender. The
addition of antioxidants such as vitamin E and cysteine to
semen extender improved the longevity and quality of
chilled and frozen-thawed semen in several species
(Bilodeau et al, 2001).
However, there is no data regarding the effect of α-lipoic
acid (ALA) on horse spermatozoa. ALA has been proved
to be a potent antioxidant and capable of scavenging
hydroxyl radicals, hypochlorous acid, peroxynitrite, singlet
oxygen, and superoxide (Packer et al, 2001). ALA has also
the potential to regenerate other antioxidants such as
vitamin C (Ibrahim et al, 2008) and vitamin E (Packer et
al, 2001). In spite of these promising results, the use of
ALA in semen extenders used for cryopreservation is not
common and recent knowledge is unable to find literature
that evaluates the potential effect of ALA on frozen-
thawed spermatozoa.
2. Material and Method
A total of two adult equine stallions (9 years old Welsh
section C) were used in this study. These stallions had
clinically normal reproductive tract and were donating
semen of acceptable quality for artificial insemination.
Semen from the stallions was collected with the help of
artificial vagina. Immediately after collection of semen
from stallions, the ejaculates were shifted to a water bath at
37o C and subjected to gross (volume and colour) and
microscopic evaluation for Estimation of progressive
motility, percentage motility, and morphology). Ejaculates
having mass motility between 50%- 60% were used in
research experiments. After evaluation, the semen was
extended in Egg Yolk Citrate extender at 37° C within 10
minutes after collection as per Khan & Ijaz , (2007). The
composition of Egg Yolk Citrate extender was as (Tris
24.20gm, Citric Acid 13.40gm, Fructose 10gm, Glycerol
70ml, Egg Yolk 200ml and distilled water up to 1000 ml )
respectively. Different inclusion levels (0.0, 1.0, 0.5, 0.1,
0.05, 0.025 and 0.125 mmol/ml) of ALA were used. For
the above mentioned inclusion levels, (0.0), (0.0206),
(0.0103), (.0021), (.0011), (.0006) and (.0003) grams of
ALA was taken in each centrifuge tube. Then 20 micro
litters of ethanol were added in all of the centrifuge tubes
respectively. These tubes were kept at water bath at 37° C
to allow the ethanol to evaporate so that a crystalline layer
of ALA deposited at the bottom of all test tubes except
control.
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ISSN 2048-4143
Then semen (3 ml) was added to all of these centrifuge
tubes and allowed to centrifuge them in a centrifugation
machine at 2100 rpm for 15 minutes. After centrifugation,
the supernatant was discarded from each of the centrifuge
tube and 03 ml of the egg yolk extender was added in all of
the tubes respectively. In order to cool the semen from 37o
C to 4o C, the test tubes were shifted from water bath to
cold cabinet and kept there for 2 hours. After equilibration,
straws (0.5 ml) were filled with semen and sealed just
before cooling at 4o C in cold cabinet as per the method
established by Rasul et al, (2001). After adjusting the
temperature to 4o C, the semen was kept for a further of 4
hours for the spermatozoa to acquire equilibration. At the
time of analysis, at least 02-04 straws of semen from each
treatment were thawed at 37° C for 30 seconds to perform
the semen Quality parameters.
2.1. Semen Concentration
Semen concentration was measured before freezing. For
this purpose, 10 µl of semen was mixed with formal citrate
in a pre-warmed Eppendorf. The dilution rate of semen
and citrate solution was kept 1:10. Haemocytometer was
cleaned with ethanol and a small drop of solution was put
on the haemocytometer. Then a clean cover slip was
placed over it and observed the slide under the Olympus
BX 51 microscope to determine the concentration.
2.2. Pre and Post Thawed Semen Motility
Pre- thawed motility was determined by putting a fresh
drop of semen on a pre-warmed slide and observed under
phase contrast microscope (40x). For post-thawed motility,
semen straws (of each treatment) were thawed in water
bath at 37o C for 30 seconds. A drop (10 µL) from each of
the thawed semen was placed on a pre-warmed glass slide
and covered with a cover slip. Percentage motility was
assessed under a phase-contrast microscope at 40x. A
minimum of three fields were observed for percentage
motility and considered as a single data point.
2.3. Vitality of Spermatozoa
To find out the live dead ratio of semen 20 µL of semen
with 20 µL of stain was mixed in preheated (37o C)
Eppendorf. Then a drop (10 µL) of mixture was put on a
clean slide to make a thin smear and observed under a
phase contrast microscope at 100X (oil emersion lens)
for unstained heads of spermatozoa
(live) and stained heads of spermatozoa (dead).
2.4. Acrosomal Integrity
A 500 µL portion of each semen sample was fixed in 50
µL of 1% formal citrate containing 2.9 % (w/v) tri-sodium
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 Hussain and Salam / Intl. R. J. of Pharmaceuticals (2011), Vol. 01, Issue 01, pp. 21-26 ISSN 2048-4143

citrate dihydrate. Two hundred spermatozoa were counted
under a phase contrast microscope (100X) for their
acrosomal integrity (NAR). Acrosome abnormalities like
absent, ruffled and swollen acrosomes were observed
(Rasul et al, 2001) but were counted in negative
consideration.
plasma membrane integrity starts from 01% and ends at
03% whereas Figure 4 shows 98.75% to 100%
normal apical ridges of acrosomes which means that ALA
did not produce worse effect on the acrosomes of equine
sperm.

2.5. Plasma Membrane Integrity

Plasma membrane integrity was assessed using the hypo

osmotic swelling test (HOST) assay. The hypo osmotic
swelling test was performed by first mixing 500 µL
HOST-solution in glass tube with 50 µL of each semen
sample and incubating the mixture for 45 min at 37 °C in
an incubator (Ijaz et al, 2009). After incubation, one drop
(~ 5 µl) of the treated mixture was examined in a thin
slide-cover slip preparation under a phase contrast
microscope (400x). Two hundred spermatozoa were
counted per sample, and the number of spermatozoa
showing clear characteristic swelling of the tail, an
indication of an intact plasma membrane, was recorded.
The mean of three observations was considered as a single Figure 1. Shows Effects of Different Doses of ALA on Different
data point. Interval Motility after Thawing of Semen.

3. Results
The fresh semen was first examined for its motility and
concentration. The motility of fresh semen for horse 1 and
horse 2 was 60% and 50% respectively. Fresh semen of
horse 1 and 2 had shown 81.5% and 80% vitality
respectively. Semen concentration was determined by
haemocytometer. The concentration of semen for the horse
1 and horse 2 was 12 million/ml and 11.50 million/ ml
respectively. After cryopreservation of stallion semen,
different parameters were analyzed. It was found that
motility of the semen was reduced with increasing
concentration of ALA. The detail results of research are
explained in Table 1.
Figure 2. Live Dead and ALA Different Concentration
Different concentration of alpha lipoic acids and their
effects on the sperm motility after cryopreservation are
shown in the Figure 1. The result shows that, as compare
to the fresh semen, the ultimate motility of the sperm after
cryopreservation’s with ALA decreases.

It was also observed that sperm viability (Live/dead) with

different concentration of alpha lipoic acids was also
changing which shows the overall ineffectiveness of ALA
on the viability of stallion semen (Figure 2).

Figures 3, 4, and 5 show the plasma membrane integrity,

acrosomal integrity and morphology of the stallion sperm
which were cryopreserved with ALA.

In Figure 3, for different concentration of alpha lipoic

acids in equine semen, the lowest value for mean positive Figure 3. Mean +Ve HOST and Different Concentration of ALA

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 Hussain and Salam / Intl. R. J. of Pharmaceuticals (2011), Vol. 01, Issue 01, pp. 21-26 ISSN 2048-4143

Figure 4. Mean Normal Apical ridge Percentage % in 200 Sperms Figure 5. Different Morphological Conditions of Stallion Sperms

Table 1. Shows the Overall Results of the ALA Efficacy on Different Fertility Parameter

% Mean HOST Mean Normal

% Mean %Mean No of Abnormal
Animals Treatment Positive in 200 Apical Ridge %
Motility Vitality Sperm out of 200
Sperms in 200 sperms
A/01=0 grams/control 6.75% 12.5% 03=1.5% 200=100% 41=20.5%
B/02=0.0206g 1.5% 04.5 02=01% 199=99.5% 52=26%
C/03=0.0103g 4.25% 0% 06=03% 198=99% 52=26%
Horse-1 D/04=0.0021g 0.25% 02% 05=2.5% 200=100% 52=26%
E/05=0.0011g 3% 04% 02=01% 191=95.5% 45=22.5%
F/06=0.0006g 2.5% 05% 0=0% 200=100% 41=20.5%
G/07=0.0003g 0.25% 0% 04=02% 200=100% 48=24%
A/01=0 grams/control 4.5% 15.5% 02=01% 200=100% 40=20%
B/02=0.0206g 0.75% 5.5% 0=0% 200=100% 46=23%
C/03=0.0103g 1% 02.5% 0=0% 198=99% 50=25%
Horse-2 D/04=0.0021g 3.75% 04% 03=1.5% 199=99.5% 36=18%
E/05=0.0011g 0% 0.5% 06=03% 200=100% 40=20%
F/06=0.0006g 0% 0% 05=2.5% 195=97.5% 45=22.5%
G/07=0.0003g 0% 0% 01=0.5% 199=99.5% 39=19.5%

Similarly Figure 5, shows the abnormality of sperm treated
with ALA with mean percentage range from 20.2 to
25.5%. The total no. of sperms is further investigated for
thick neck, large head, and double neck and tail percentage
respectively.
4. Discussion
Fertility is a binomial variable which determine the
conception rate of mare Stallion (Graham & Moc'e, 2005).
Breeding management, history and age can also contribute
in the mare pregnancy rate (Colenbrander et al, 2003).
Fertility and spermatozoa viability has been assessed by
various assays in different studies (Graham & Moc'e,
2005). Some stallion may exhibit low fertility due to
having defective spermatozoa parameter; these defects can
be examined and eliminated via laboratory assays (Graham
& Moc'e, 2005). Pregnancy itself is the best tool to assess
the fertility of stallion (Samper et al, 2007). Spermatozoa
their selves are highly vulnerable to oxidative stress-
induced damage because their plasma membrane contain
large amount of poly unsaturated fatty acids (Alvarez &
Storey, 1995) and their cytoplasm contain low
concentrations of scavenging enzymes (Aitken & Fisher,

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 Hussain and Salam / Intl. R. J. of Pharmaceuticals (2011), Vol. 01, Issue 01, pp. 21-26
1994). It is interesting that the seminal plasma of mammals
is well capable with a set of antioxidant defence
mechanism (Sikka, 1996). This antioxidant defence
mechanism is composed of enzymatic antioxidants such as
super-oxide dismutase (Alvarez et al, 1987),
the glutathione peroxidase/glutathione reductase
(GPx/GRD) system (Chaudiere et al, 1984) and catalase
(Jeulin et al, 1989) and the non enzymatic antioxidants
such as ascorbate (Fraga et al, 1991), urate (Thiele et al,
1995), α-tocoperol (Aitken & Clarkson, 1988), pyruvate
(De-Lamirande & Gagnon, 1992), glutathione
(Lenzi et al, 1994), taurine and hypotaurine (Alvarez &
Storey, 1983).
This study was conducted to find out the effect of
cryopreservation of semen with alpha lipoic acid in
stallion. Evaluation of efficacy of ALA as an antioxidant
for equine sperm motility, vitality, morphology, acrosomal
integrity and plasma membrane integrity (HOS) was also a
part of this project. The results of our study are as follows;
The motility of fresh semen for horse 1 and horse 2 was
60% and 50% respectively. This motility was reduced
dramatically after addition of different inclusion levels of
ALA in semen extender as shown in Table 1.
Similarly plasma membrane integrity, acrosomal integrity,
spermatozoa vitality did not show any significant
improvement as compared to control. Our findings are
disappointing showing the overall ineffectiveness of ALA
as an antioxidant for improving the post-thawed quality of
equine semen.
5. Conclusion
In conclusion, results obtained from this study show
that the motility and viability of stallion semen
was decreased after cryopreservation with alpha
lipoic acids which might be due to the morphological
abnormalities of the sperm, particularly large headed
acrosome which may be the reason of DNA damage. It
should be noted that DNA damage also affects the
fertility, pregnancy rate and the embryonic development of
the animal (Ibrahim et al, 2008). The second reason
may be the increased acidy produced by the alpha
lipoic acid which can cause the immobilization of
spermatozoa. Further more, LA can be reduced to the more
potent dihydrolipoic acid (DHLA) (Navari-Izzo et al,
2002), which regenerates the stock of reduced
antioxidants.
Due to its multi-functional nature, it is possible that the
benefits of LA could be expressed in more concrete
experimental settings beyond the purpose of the
presentstudy, or in more complex media formulations,
such as those used for in-vitro fertilization (IVF).
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ISSN 2048-4143
Direct addition of DHLA could be a suitable option for
future experiments, since that strategy would not rely on
the ability of sperm cells to convert LA to DHLA, but
media formulation must be taken into an account that
metal cations (e.g. iron, copper etc) must be absent. In
order to achieve a synergistic effect, supplementing the
medium with several antioxidants such as reduced
glutathione could be a level headed approach.
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