Reprod Dom Anim doi: 10.1111/rda.
12445
ISSN 0936–6768
Alpha-Linolenic Acid Supplementation in Tris Extender Can Improve
Frozen–Thawed Bull Semen Quality
A Kaka1,2, H Wahid1, Y Rosnina1, N Yimer1, AM Khumran1, AA Behan3 and M Ebrahimi4
1
Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Malaysia; 2Sindh Agriculture
University, Tando jam, Pakistan; 3Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Malaysia;
4
Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Malaysia
Content
The study was conducted to evaluate the effects of a-linolenic
acid (ALA) on frozen–thawed quality and fatty acid compo-
sition of bull sperm. For that, twenty-four ejaculates obtained
from three bulls were diluted in a Tris extender containing 0
(control), 3, 5, 10 and 15 ng/ml of ALA. Extended semen was
incubated at 37°C for 15 min, to allow absorption of ALA by
sperm cell membrane. The sample was chilled for 2 h, packed
into 0.25-ml straws and frozen in liquid nitrogen for 24 h.
Subsequently, straws were thawed and evaluated for total
sperm motility (computer-assisted semen analysis), membrane
functional integrity (hypo-osmotic swelling test), viability
(eosin-nigrosin), fatty acid composition (gas chromatography)
and lipid peroxidation (thiobarbituric acid-reactive substances
(TBARS)). A higher (p < 0.05) percentage of total sperm
motility was observed in ALA groups 5 ng/ml (47.74
07)
and 10 ng/ml (44.90
0.7) in comparison with control
(34.53
3.0), 3 ng/ml (34.40
2.6) and 15 ng/ml (34.60

2.9). Still, the 5 ng/ml ALA group presented a higher (p <
0.05) percentage of viable sperms (74.13
0.8) and sperms
with intact membrane (74.46
09) than all other experimen-
tal groups. ALA concentration and lipid peroxidation in post-
thawed sperm was higher in all treated groups when compared
to the control group. As such, the addition of 5 ng/ml of ALA
to Tris extender improved quality of frozen–thawed bull
spermatozoa.
Introduction
The spermatozoa, a highly polarized and specialized
cell, loose its repairing, growth and cell division abilities
during maturation. Therefore, sperms are cryopreserved
to prolong their life, which usually decreases or arrests
the metabolism of sperm (Yoshida 2000). Cryopreser-
vation consists of freezing and thawing processes, which
decreases sperm viability and changes membrane struc-
ture, properties and cause death of sperms (Watson
1995, 2000). The main cause of impairment of mem-
brane function is the transformation of lipids into a
rigid structure during cryopreservation (Watson 2000).
Polyunsaturated fatty acids (PUFA) are major constit-
uents of the lipids present in the cell membrane (Poulos
et al. 1986; Conquer et al. 1999). PUFA maintain the
structure and function of cell membrane during freezing
and thawing (Robinson et al. 2006).
Studies conducted earlier with dietary fatty acids and
their alternative sources have produced positive effects on
sperm during cryopreservation. Docosahexanoic acid
(DHA) has increased motility in human sperms (Conquer
et al. 1999) and bull semen (Gholami et al. 2010). Tuna
and fish oil (contains n-3 fatty acids) also improved
motility, normal acrosome, fatty acid composition and
© 2014 Blackwell Verlag GmbH
normal morphology of boar sperm (Rooke et al. 2001)
and ram (Samadian et al. 2010) and sperm parameters of
buck (Dolatpanah et al. 2008).
In vitro addition of PUFA decreased motility and
increased lipid peroxidation of human sperm (Koppers
et al. 2010) while oleic acids (OA), linoleic acid (LA),
arachidonic acid (AA), palmitic acid (PA), ALA and
oleic acid (OA) improved total sperm motility, progres-
sive motility, viable sperms and sperms with intact
membrane in chilled and frozen–thawed sperm of boars
(Hossain et al. 2007) and bulls (Keirnan et al. 2012).
ALA is an n-3 fatty acids (PUFA) group consists of 18
carbon items and three double bond (18:3n-3) (Keirnan
2012). This study was conducted to evaluate the effects
of addition of ALA to the Tris extender during
cryopreservation process on frozen–thawed bull sperm
parameters, fatty acids composition and lipid peroxida-
tion of plasma membrane.
Material and Methods
Animals
Three fertile bulls of Brangus-simmental cross-breed
were selected from Universiti Putra Malaysia farm
(UPM) (2o 90 18.36″ N, 101o 430 49.61″ E). Bulls were
approximately 3–4 years of age and 620 to 650 kg of
weight. The body condition score (BCS) of the bulls
ranged from 4 to 6 (1; thin to 9; obese) (Eversole et al.
2009). All bulls were kept at similar management and
feeding. Brachiaria decumbens, and palm kernel cake
(PKC) containing approximately 16% crude protein
and 2.6% crude fat were given at a rate of 3 kg/bull/day.
Mineral licks and water were provided ad libitum.
Semen collection and experimental design
Twenty-four ejaculates were collected using an electro-
ejaculator (Electro jac 5, Ideal Instruments Neogen
Company, Lexington, KY, USA) twice a week. Ejacu-
lates were transported at 37°C in Coleman cooler box to
the laboratory to evaluate samples for further process-
ing. Ejaculates with general motility percentage of ≥70%
and normal morphological and viability percentage of
≥80% were used in the study.
Each ejaculate was diluted in Tris extender (Tris
buffer 3.51 g, fructose 1.25 g, citric acid 1.97 g, glycerol
7% and egg yolk 20% per 100 ml) with different levels
of ALA (C18:3n-3) (Sigma Chemical Co., St. Louis,
MO, USA) such as 0, 3, 5, 10 and 15 ng/ml of extender.
As the fatty acids are insoluble in water, 0.05% ethanol
2 A Kaka, W Haron, R HJ Yusof, N Yimer, K AM, AA Behan and M Ebrahimi
was used as solvent (Nasiri et al. 2012). The samples
were diluted in test tubes and incubated in a water bath
at 37°C for 15 min for absorption of ALA by a sperm
cell membrane. After that, samples were chilled at 5°C
for 2 h as previously described by Memon et al. (2012),
then packed in 0.25 ml straws with 20 9 106 sperm/
straws and stored in liquid nitrogen for 24 h. The stored
samples were thawed in a water bath at 37°C for 30 s
(Yoshida 2000) and then evaluated for total sperm
motility, viability, plasma membrane, fatty acid com-
position and lipid peroxidation.
Post-thawed semen evaluation
Total sperm motility was assessed using computer-
assisted semen analyser (CASA) (Hamilton Thorne
bioscience version 12.12c, Beverly, MA, USA). 10 ll
of semen diluted in 0.85% NaCl, placed on pre-warmed
slide and covered with a cover slip, then slides were
placed in the CASA and motility was recorded. Sperm
viability was assessed using eosin-nigrosin stain (Evans
and Maxwell 1987). Sperm viability was assessed at 400x
magnification of light microscopes (Nikon Eclipse 50i,
Tokyo, Japan), by counting at least 200 spermatozoa.
Sperms with white and pink colour were considered live
and dead, respectively (Memon et al. 2012).
Hypo-osmotic swelling test (HOST) was used to
assess change in functional plasma membrane integrity
by plasma membrane functional integrity as described
by Revell and Mrode (1994). Briefly, 100 ll of post-
thawed semen was added in 1 ml of HOST solution
(fructose 13.51 g, trisodium citrate 7.35 g into distilled
water 1000 ml; osmolarity of 150 mOsm/kg) and incu-
bated for 60 min at 37°C. Afterwards, 15 ll of solution
was placed in pre-warmed slide covered with coverslip,
and sperms were evaluated under a light microscope at
400x magnification (Nikon Eclipse 50i, Tokyo, Japan).
The spermatozoa that swellen in response to the test
solution were considered as having functional intact
membrane. At least 200 spermatozoa per slide were
counted and expressed in percentage.
Fatty acid analysis
Fatty acids were extracted from semen samples using a
Folch et al. (1957) method which modified by Rajion
et al. (1985) as described by Ebrahimi et al. (2012) using
chloroform: methanol 2:1 (v/v). The extracted fatty
acids were transmethylated to their fatty acid methyl
Table 1. Post-thawed bull sperm characteristics for different concentration of ALA
Sperm parameters 0 3
Motility 34.53
3.0 b
Membrane Functional integrity 63.13
1.6b
Viability 62.67
1.4bc
ALA: a-linolenic acid.
Values are mean
SEM.
a,b,c
Values with different superscripts within raw show significant difference at p < 0.05.
esters (FAME) using 0.66N potassium hydroxide
(KOH) in methanol and 14% methanolic boron triflu-
oride (BF3) (Sigma Chemical Co.,) according to the
methods by Association of Official Analytical Chemists
(AOAC, 1990). The FAME were separated using
Agilent 7890A gas chromatography (Agilent Technolo-
gies, Palo Alto, CA, USA) using a 30 m 9 0.25 mm ID
(0.20 lm film thickness) Supelco SP-2330 capillary
column (Supelco, Inc., Bellefonte, PA, USA). One
microlitre of FAME was injected by an autosampler
into the chromatograph, equipped with a flame ioniza-
tion detector (FID). The injector temperature was
programmed at 250°C, and the detector temperature
was 300°C. The column temperature programme initi-
ated runs at 100°C, for 2 min, warmed to 170°C at
10°C/min, held for 2 min, warmed to 220°C at 7.5°C/
min, and then held for 10 min to facilitate optimal
separation. The identification of the peaks was made by
comparison of equivalent chain lengths with those of
authentic fatty acid methyl esters (37 Component
FAME mix, Supelco, Bellefonte, PA, USA). Peak areas
were determined automatically using the Agilent Gas
Chromatography Chemstation software (Agilent Tech-
nologies) as described by Ebrahimi et al. (2013).
Lipid peroxidation (LP) test
Lipid peroxidation was measured using thiobarbituric
acid-reactive substances (TBARS) according to the
method by Mercier et al. (1998). 500 ll of thawed
semen was mixed with TBRAS solution and then heated
in a water bath at 95°C for 60 min until the develop-
ment of a pink colour. After cooling, 1 ml of distilled
water and 3 ml of n-butyl alcohol were added to the
extracts and vortexes. The mixtures were centrifuged at
3500 g for 10 min. Absorbance of supernatant was read
against an appropriate blank at 532 nm using a spec-
trophotometer (Secomam, Domont, France). The mal-
ondialdehyde (MDA) was calculated from a standard
curve of 1, 1, 3, 3-tetraethoxypropane and expressed as
nmol/3 9 108 sperm.
Statistical analysis
Results are presented as mean
standard error of the
mean (SEM). Data of sperm cytological characteristics,
fatty acid and MDA were analysed using the general
linear model (GLM) procedure of SAS 9.2 version.
Differences between means were analysed by the
ALA concentration, (ng/ml)
5 10 15
34.40
2.6 b
47.73
0.7a
44.90
0.7a
34.60
2.9b
65.93
2.6b 74.46
0.9a 65.20
0.8b 65.40
2.5b
67.26
2.4b 74.13
0.8a 65.40
0.6bc 62.20
1.9c
© 2014 Blackwell Verlag GmbH
Alpha-Linolenic Acid and Bull Semen Cryopreservation 3

Table 2. Post-thawed fatty acid composition of sperm in control group (without ALA) and ALA-supplemented group at different levels

ALA concentration, (ng/ml)

Fatty acid 0 3 5 10 15

C14:0 0.50
0.02 0.48
0.01 0.50
0.01 0.51
0.01 0.50
0.01
C16:0 25.27
0.36 25.54
0.07 25.57
0.08 25.36
0.06 25.36
0.07
C16:1 2.95
0.05 2.98
0.02 2.98
0.02 3.00
0.01 3.00
0.04
C17:0 0.38
0.08 0.52
0.15 0.55
0.20 0.72
0.21 0.47
0.07
C18:0 7.99
0.19 8.03
0.14 8.07
0.18 7.78
0.14 8.02
0.24
C18:1n-9 42.00
0.83 41.48
0.22 41.37
0.46 40.80
0.39 41.17
0.39
C18:2n-6 16.41
0.66 15.90
0.37 15.78
0.45 16.22
0.43 15.57
0.58
C18:3n-6 0.66
0.08 0.45
0.02 0.46
0.03 0.66
0.12 0.45
0.03
C18:3n-3 0.36
0.01c 0.58
0.12bc 0.77
0.08ab 1.01
0.10ab 1.44
0.08a
C20:4n-6 1.59
0.31 1.96
0.02 1.93
0.02 1.94
0.03 1.88
0.04
C20:5n-3 0.70
0.05 0.87
0.01 0.87
0.01 0.88
0.01 0.86
0.01
C22:5n-3 0.59
0.14 0.64
0.06 0.63
0.04 0.60
0.05 0.63
0.02
C22:6n-3 0.61
0.10 0.56
0.13 0.52
0.17 0.53
0.05 0.64
0.06
SFA 34.13
0.37 34.56
0.20 34.68
0.16 34.37
0.22 34.35
0.33
MUFA 44.94
0.84 44.47
0.23 44.36
0.44 43.80
0.38 44.17
0.35
PUFA 20.93
0.64 20.97
0.36 20.96
0.56 21.83
0.51 21.49
0.66
n-6PUFA 18.66
0.64 18.32
0.39 18.18
0.48 18.82
0.55 17.91
0.65
n-3PUFA 2.27
0.11c 2.65
0.20bc 2.79
0.17b 3.01
0.06b 3.58
0.13a

ALA: a-linolenic acid.

SFA: saturated fatty acids: sum of (C14:0+ C16:0+C17:0+ C18:0).
MUFA: monounsaturated fatty acids: sum of (C16:1+C18:1n-9).
PUFA: polyunsaturated fatty acids: sum of (C18:2n-6+C18:3n-6+C18:3n-3+C20:4n-6+C20:5n-3+C22:5n-3+C22:6n-3).
n-6PUFA: sum of (C18:2n-6+C18:3n-6+C20:4n-6).
n-3PUFA: sum of (C18:3n-3+C20:5n-3+ C22:5n-3+C22:6n-3).
Values are mean
SEM.
a,b,c
Values with different superscripts within rows show significant difference p < 0.05.

Lipid peroxidation Discussion

20.00
MDA nm/3x108

ab
15.00 ab
10.00
b b
5.00
0.00
0 3 5 10
ng/ml
Fig. 1. MDA production in frozen–thawed bovine semen supple-
mented with different levels of ALA in Tris extender (nmol/3 x 108
sperm). Values are means
1 standard error bar. Values with
different superscripts differ significantly at p < 0.05
Duncan’s test. Values were considered to be statistically
significant when p < 0.05.
Results
The mean percentage of post-thawed sperm parameters
is presented in Table 1. There was a significant
(p < 0.05) effect of ALA on post-thawed sperm motility,
membrane integrity and viability. Overall results
showed that total sperm motility was higher at 5 and
10 ng/ml ALA, while membrane integrity and viability
were higher at 5 ng/ml ALA. The fatty acid composi-
tions of sperm with different levels of ALA are presented
in Table 2. The results showed that increasing the
concentration of ALA in the sperm was parallel to the
increase in the level of ALA concentration in the
extender. A significant difference was observed among
treated groups and control group (p < 0.05). Lipid
peroxidation results are presented in Fig. 1. The lipid
peroxidation was increased with increasing the ALA
concentration in the extender.
a
It is accepted that considerable damage occurs to the
spermatozoa during freezing and thawing (Yousef et al.
2003). Cryopreservation changes the lipid composition
of sperm membrane sperm, motility, viability and
15 acrosome status due to physical and chemical stress on
spermatozoa (Schiller et al. 2000; O’Connell et al. 2002;
Martinez-Soto et al. 2013). It is stated in many studies
that lipid composition of the sperm plasma membrane
has important consequence on the functional charac-
teristics of spermatozoa (Lenzi et al. 2000; Gulaya et al.
2001; Lessig et al. 2004; Aksoy et al. 2006; Tavilani
et al. 2008). PUFA maintain membrane fluidity (Lenzi
et al. 2002); due to presence of bonds, PUFA in the
sperm membrane are prone to peroxidation (Alvarez
et al. 1987).
In the current study, total sperm motility was higher
in groups 5 ng/ml and 10 ng/ml ALA in comparison
with control, 3 ng/ml and 15 ng/ml. In addition, 5 ng/
ml ALA group presented a higher percentage of viable
sperms and sperms with functional intact membrane
than all other experimental groups. The improvement in
total motility, viability and membrane functional integ-
rity may be due the increase in membrane fluidity and
resistance against cooling and freezing by ALA. Takah-
ashi et al. (2012) reported that addition of palmitic acid
and linoleic acid improved frozen–thawed sperm motil-
ity and viability of bull semen.
Another report indicated that addition of soft gels
that contain n-3, n-6 and n-9 fatty acids decreased
motility of bull sperms in cooled and frozen semen
(Kandelousi et al. 2013). The difference in this result
may be due the concentration and source of PUFA; in

© 2014 Blackwell Verlag GmbH

4 A Kaka, W Haron, R HJ Yusof, N Yimer, K AM, AA Behan and M Ebrahimi

previous study, soft gels were used, which contain all
type of unsaturated fatty acids, while in current study,
only single ALA was used. Other cause of difference in
results may be solvent used to dissolve fatty acids, and
polyethylene glycol (PEG) has a detrimental effect on
sperm (Kandelousi et al. 2013) but ethanol used in the
current study has no detrimental effect on sperm
viability during cryopreservation (Nasiri et al. 2012).
Fatty acids are important to improve the fluidity of
membrane, prevent sperm damage triggered by ice
crystal during freezing and improve fertilization ability
of sperm (Maldjian et al. 2005). Variation in concen-
trations of PUFA affects the flexibility and compress-
ibility of membrane, which decreases sperm movement
(Neuringer et al. 1988; Castellano et al. 2010). In the
current study, ALA and n-3 PUFA were higher in ALA-
supplemented groups than in the control revealing that
the plasma membrane was capable of absorbing ALA
effectively. ALA absorption increased parallel as the
ALA concentration increased. Similar results are
reported after using DHA by Nasiri et al. (2012), and
Maldjian et al. (2005) reported the occurrence of fatty
acid absorption by sperm membrane. The effects of fatty
acids may vary due to factors such as methods of
addition, sources, type of fatty acids and concentration
of fatty acids (Castellano et al. 2010; Kandelousi et al.
2013).
The MDA is increased with the overproduction of
reactive oxygen species (ROS) which oxidizes sperm
membrane fatty acids and in result affects sperm quality
parameters (Sanocka and Kurpisz 2004). In the current
study, all ALA-supplemented groups produced higher
MDA compare to the control group, which is similar to
ROS production by PA, OA and ALA in chilled semen
(Keirnan et al. 2012). The results showed that chilled
and frozen sperm quality was improved with 5 ng/ml.
Considering the result of sperm parameters, lipid
peroxidation and fatty acid composition of sperm cells,
it would be concluded that the optimum level of ALA to
be added for superior semen quality is 5 ng/ml.
Acknowledgements
Asmatullah Kaka wishes to acknowledge the support of Sindh
Agriculture University Tandojam Pakistan for awarding scholarship
under the project ‘Strengthening of Sindh Agriculture University’ to
pursue his PhD. Special thanks to Universiti Putra Malaysia for
allowing the use of animals and laboratory facilities.
Conflict of interest
The authors would like to declare that there is no conflict of interest
regarding publications of this article.
Author contributions
Asmatullah Kaka wrote the article; H. Wahid and Y. Rosnina checked
the article; N. Yimer helped in writing the paper; A.M. Khumran
helped in writing and checking the paper; Atique Ahmed Behan
checked language of the paper; and M. Ebrahimi helped in the analysis
of data.

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Submitted: 28 Mar 2014; Accepted: 21 Sep
2014
Author’s address (for correspondence): H
Wahid, Department of Veterinary Clinical
Studies, Faculty of Veterinary Medicine,
Universiti Putra Malaysia, 43400, UPM,
Serdang, Selangor Darul Ehsan, Malaysia.
E-mail: wahidh@upm.edu.my