Theriogenology 85 (2016) 954959

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Theriogenology
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Trehalose improves semen antioxidant enzymes activity,

post-thaw quality, and fertility in Nili Ravi buffaloes
(Bubalus bubalis)
Sajid Iqbal a, Syed Murtaza Hassan Andrabi b, Amjad Riaz a, *,
Aneela Zameer Durrani c, Nasim Ahmad a
a

Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore, Pakistan

Animal Reproduction Laboratory, Animal Sciences Institute, National Agricultural Research Centre, Islamabad, Pakistan
c
Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore, Pakistan
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 29 May 2015
Received in revised form 5 November 2015
Accepted 6 November 2015

Our objectives were to study the effect of trehalose in extender on (1) antioxidant enzymes
prole during cryopreservation (after dilution, before freezing, and after thawing), (2)
in vitro quality (after thawing), and (3) in vivo fertility of Nili Ravi buffalo (Bubalus bubalis)
bull spermatozoa. Semen samples (n 20) from four buffalo bulls were diluted in Triscitric acidbased extender having different concentrations of trehalose (0.0, 15, 30, 45,
and 60 mM) and frozen in French straws. At post dilution, prole of sperm catalase (U/mL)
was higher (P < 0.05) in extenders containing 15, 30, and 45 mM of trehalose as compared
to control. Although proles of superoxide dismutase (U/mL) and total glutathione (mM)
were higher (P < 0.05) in extenders containing 15 and 30 mM of trehalose as compared to
control. At prefreezing, sperm catalase, superoxide dismutase, and total glutathione proles were higher (P < 0.05) in all the treatment groups as compared to control. At post
thawing, the proles of catalase and total glutathione were higher (P < 0.05) in extender
containing 30-mM trehalose as compared to other treatment groups and control. Whereas,
prole of superoxide dismutase was higher (P < 0.05) in extenders containing 30, 45, and
60 mM of trehalose as compared to control and 15mM group. Post thaw total sperm
motility (%) was higher (P < 0.05) in extender containing 30-mM trehalose as compared to
control and 15 and 60-mM groups. Although sperm progressive motility (%), rapid velocity
(%), average path velocity (mm/s), straight line velocity (mm/s), curvilinear velocity (mm/s),
plasma membrane (structural and functional, %), acrosome (%), and DNA (%) integrity were
higher (P < 0.05) in extender containing 30 mM trehalose as compared to other treatment
groups and control. The fertility rates (61% vs. 43%) were higher (P < 0.05) in buffaloes
inseminated with semen doses cryopreserved in extender containing 30 mM of trehalose
than the control. It is concluded that addition of 30-mM trehalose in extender improves
the semen antioxidant enzymes activity, post thaw quality, and fertility in Nili Ravi
buffaloes.
2016 Elsevier Inc. All rights reserved.

Keywords:
Buffalo semen
Trehalose
Cryopreservation
Antioxidant enzyme activity
Semen quality
Fertility

1. Introduction

* Corresponding author. Tel.: 92 333 5262325; fax: 92 42 99211461.

E-mail address: dramjadriaz@uvas.edu.pk (A. Riaz).
0093-691X/$ see front matter 2016 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2015.11.004

Cryopreservation is a technique adopted for the propagation of superior germplasm of dairy animals [1]. The
success of this procedure primarily depends on the number
of structurally and functionally viable spermatozoa [2].

S. Iqbal et al. / Theriogenology 85 (2016) 954959

Cryopreservation reduces semen antioxidant enzymes

prole and fertility through generation of reactive oxygen
species (ROS) [3]. High contents of saturated and polyunsaturated fatty acids particularly in buffalo sperm predominantly make them vulnerable to the injuries tempted
by excessive ROS release [46]. The presence of higher
prole of polyunsaturated fatty acids in sperm needs an
effective antioxidant system to counter the per oxidative
activity [7,8]. Nature has gifted spermatozoa with an
intracellular and extracellular antioxidant defense mechanism, which includes enzymatic and nonenzymatic
systems [9,10]. The enzymatic antioxidant defense system
mostly comprises of superoxide dismutase, catalase, and
glutathione peroxidase and reductase.
Catalase catalyzes the dissociation of H2O2 into H2O and
O2 [11], reduces the oxidative stress, and nally enhances
sperm motility [12,13]. The physiological role of superoxide
dismutase is to prevent the lethal effect of ROS by dismutation of O2 to H2O2, ultimately exert positive effect on
membrane integrity during the cryopreservation process
[14]. Glutathione belongs to the tripeptide thiols group and
physiologically helps in protection of cell by reducing
oxidative stress [15] through elimination of H2O2 [16].
The antioxidant defense systems of mammalian
spermatozoa are predominantly of cytoplasmic in nature.
Spermatozoa dispose of their cytoplasm during maturation
stage of spermatogenesis. Consequently, mammalian
spermatozoa are decient of adequate reserves of natural
antioxidants against the detrimental effects of ROS and lipid
peroxidation (LPO) during cryopreservation [7,8,17]. Therefore, the consequences of oxidative stress can be reduced by
adding antioxidants in semen extender. It has been documented that addition of antioxidants in extender enhanced
the post-thaw sperm parameters (viability, motility, and
fertility) in bovine [18], ovine [19], and caprine [20].
Trehalose is a nonreducing disaccharide sugar, and
when used at higher doses in extender, it reduces intracellular ice crystal formation during cryopreservation
[2123]. Moreover, trehalose has the antioxidative property at lower doses and protects the spermatozoa by
reducing LPO [24,25]. Literature regarding sperm antioxidant enzymes prole and use of trehalose as an antioxidant
in water buffalo bulls is scarce. Therefore, the objectives of
present study were to study the effect of trehalose in
extender on (1) antioxidant enzymes prole during cryopreservation (after dilution, before freezing, and after
thawing), (3) in vitro quality (after thawing), and (3) in vivo
fertility of buffalo bull spermatozoa.
2. Materials and methods
2.1. Chemicals
The chemicals used in the preparation of extender
including trehalose (T0167) were procured from Sigma
Aldrich Chemical Co., USA.
2.2. Selection of animals
Present study was conducted at Semen Production Unit,
Qadirabad, Sahiwal, Pakistan (173 M, 73 74 E, 30 31.15 N).

955

Four mature donor Nili Ravi buffalo bulls of similar age

(6 years) were selected. Bulls were individually housed and
maintained under uniform feeding and managemental
conditions. Good quality seasonal green fodder was provided
with 10% body weight of bulls. Fixed amount of concentrate
(3 kg/day) was offered to each bull and water ad libitum.
2.3. Semen collection and initial evaluation
Articial vagina maintained at 42  C was used to collect
the semen. Two consecutive ejaculates were collected once
in a week from each animal for 5 weeks (replicate). Each
ejaculate was shifted to water bath maintained at 37  C in
the laboratory within a minute. After collection percentage,
motility was evaluated under phase-contrast microscope
(Olympus, Japan) at magnication  200. Each semen
sample was evaluated for sperm concentration spectrophotometrically (IMV, France) [26]. Ejaculates having
greater than 65% visual motility and greater than 500  106
sperm/mL were selected for further processing.
2.4. Semen extension and freezing
Tris-citric acidbased extender (Tris 199.80 mM, citric
acid 69.75 mM, fructose 55.56 mM, egg yolk 20% [vol:vol],
glycerol 7% [vol:vol], benzyl penicillin 1000 [IU/mL], and
streptomycin sulfate 1000 [mg/mL]; pH 6.9) was used in the
present study. Semen samples of individual animal were
diluted with the extender containing different concentrations of trehalose (0, 15, 30, 45, and 60 mM; osmotic
pressure 264, 273, 293, 307, and 322 mOsmol/kg, respectively) to a nal concentration of 50  106 spermatozoa/mL
at 37  C. Cooling of extended semen samples to 4  C was
done in 2 hours and equilibration at 4  C for 4 hours. Semen
samples were packed and sealed in 0.54 mL French straws
at 4  C. After equilibration, semen-lled straws were frozen
in liquid nitrogen vapors for 10 minutes. Finally, straws
were dipped and stored in liquid nitrogen (196  C). To
evaluate post-thaw sperm quality parameters, two straws
per treatment were thawed at 37  C in water bath for
30 seconds. Post-thaw semen quality parameters were
evaluated after at least 24 hours of storage.
2.5. Sperm antioxidant enzymes prole
Antioxidant enzymes prole (catalase, superoxide dismutase, and total glutathione) was determined during
different stages of cryopreservation (after dilution, before
freezing, and after thawing). Pooled ejaculates of each bull
were analyzed for antioxidant enzymes prole. Thawed
semen sample (120 mL) were centrifuged at 1600  g for
5 minutes to remove the seminal plasma. After discarding
the supernatant, 360 mL of 1% Triton X-100 solution was
added into the precipitate. The suspension was incubated
for 20 minutes and was subsequently centrifuged at 25  C
for 30 minutes at 4000  g. The precipitate was resuspended, and nally, the supernatant was collected
containing crude extract of enzymes in the sperm [27]. The
antioxidant enzymes (catalase, superoxide dismutase,
and total glutathione) prole was analyzed through ELISA
kit. Catalase enzyme prole was analyzed through the

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S. Iqbal et al. / Theriogenology 85 (2016) 954959

commercial kit Oxiselect STA-339 (Cell Biolabs, USA),

superoxide dismutase with SigmaAldrich 19160 kit, and
total glutathione (peroxidase and/or reductase) with
Oxiselect STA-312 (Cell Biolabs, USA).
2.6. Post-thaw sperm evaluation
Post-thaw sperm quality assays were performed at
Animal Reproduction Laboratory, Animal Sciences Institute,
National Agricultural Research Centre, Islamabad, Pakistan.
2.6.1. Sperm motility parameters, velocity distribution, and
kinematics
Post-thaw sperm motility parameters, velocity distribution, and kinematics were analyzed through computerassisted sperm analysis (CASA; CEROS, version 12.3;
Hamilton-Thorne Biosciences, USA). The CASA analysis was
carried out at standard setting as follows: Standard: frame
rate: 60 Hz; minimum contrast: 56; minimum cell size: ve
pixels; average path velocity (VAP) cutoff: 20.0 mm/s; Prog.
Min VAP: 80.0 mm/s; straight line velocity cutoff: 0.0 mm/s.
Frozen-thawed semen sample (7 mL) was placed on a
prewarmed glass slide, and then, the slide was positioned
in a portable MiniTherm Stage (37  C). CASA analysis was
carried out at 10x objective. CASA total motility (%), progressive motility (%), rapid velocity (%), medium velocity
(%), slow velocity (%), VAP (mm/s), straight line velocity
(mm/s), curvilinear velocity (mm/s), amplitude of lateral
head displacement (mm/s), and beat cross frequency (Hz)
were recorded. At least 200 spermatozoa per sample were
recorded for evaluation of CASA parameters.
2.6.2. Sperm viability and plasma membrane integrity
After thawing, sperm viability and plasma membrane
integrity (structurally and functionally) was determined
with supravital-hypo osmotic swelling test (HOST). The
HOST solution composed of sodium citrate 0.735 g, fructose
1.351 g, and 100 mL distilled water (osmolarity 150
mOsmol/kg). Aliquots of frozen-thawed semen samples
(50 mL) were mixed with HOST solution (500 mL) and
incubated at 37  C for 40 minutes. After incubation, 5 mL of
semen was mixed with 5 mL of Eosin (0.5% tri-sodium citrate
dihydrate [wt:vol] 2.92%) on a prewarmed glass slide. A
cover slip was placed on the mixture and analyzed under
microscope (phase contrast, magnication:  400). Two
hundred spermatozoa per sample were counted for
estimation of structural and functional integrity of plasma
membrane. Spermatozoa displaying clear heads and
swollen tails were designated as structurally and functionally viable, whereas, sperm with pink heads and unswollen
tails were classied as nonviable [28,29].
2.6.3. Sperm acrosome integrity
To assess acrosomal integrity, thawed semen sample
(500 mL) was mixed with 50 mL of a 1% solution of formal
citrate (1 mL of 37% formaldehyde solution and 2.92-g
tri-sodium citrate dihydrate, dissolved in 100-mL distilled
water). Five mL of the previously mentioned mixture
was placed on a glass slide, covered with a cover slip,
and evaluated under a phase-contrast microscope (oil
immersion, magnication:  1000). The percentage of

spermatozoa having normal acrosome was recorded by

counting 200 spermatozoa [26].
2.6.4. Sperm DNA integrity
Acridine orange staining method was followed to
determine sperm DNA integrity. Two hundred sperm per
sample were counted under the epiuorescence microscope (480/550 nm). Spermatozoa with normal DNA content (double stranded) shined green, whereas spermatozoa
that shined red uorescence were considered with
damaged DNA (single stranded) [30].
2.7. In vivo fertility
Based on the statistical analysis of data, best results on
semen antioxidant enzymes prole and post-thaw parameters were found with 30-mM trehalose in extender. Therefore, articial insemination doses were prepared with and
without the addition of 30-mM trehalose in semen extender
from one of the four buffalo bulls for fertility trial. A total of
200 inseminations (100 of treatment and 100 of control)
were performed in eld in District Sialkot, Pakistan. All the
inseminations were performed at 24 hours after onset of
heat. The inseminated buffaloes were examined per rectum
for pregnancy diagnosis at 60 days after insemination.
2.8. Statistical analysis
The present experiment was repeated ve times. Results
were expressed as the mean  standard error of the mean.
Means were analyzed by two factor factorial (bull and
treatment) model of ANOVA, followed by Duncans multiple range test to dene statistically the differences in all
parameters among all groups using the SPSS software
package (version 16.0; SPSS, Chicago, IL, USA). Differences
within values of P < 0.05 were considered to be statistically
signicant. Pregnancy rates were evaluated using the chisquare test.
3. Results
The data on effect of trehalose in extender on antioxidant enzymes (catalase, superoxide dismutase, and total
glutathione) prole during cryopreservation (after dilution,
before freezing, and after thawing) of Nili Ravi buffalo bull
spermatozoa are presented in Table 1. At post dilution,
prole of sperm catalase (U/mL) was higher (P < 0.05) in
extenders containing 15, 30, and 45 mM of trehalose as
compared to control. Although proles of superoxide dismutase (U/mL) and total glutathione (mM) were higher
(P < 0.05) in extenders containing 15 and 30 mM of
trehalose as compared to control. At prefreezing, sperm
catalase, superoxide dismutase, and total glutathione proles were higher (P < 0.05) in all the treatment groups as
compared to control. At post thawing, the proles of catalase and total glutathione were higher (P < 0.05) in
extender containing 30-mM trehalose as compared to
other treatment groups and control. Whereas, prole of
superoxide dismutase was higher (P < 0.05) in extenders
containing 30, 45, and 60 mM of trehalose as compared to
control and 15-mM group.

S. Iqbal et al. / Theriogenology 85 (2016) 954959

957

Table 1
Effect of trehalose in extender on antioxidative prole (catalase, superoxide dismutase, and total glutathione) during cryopreservation (after dilution, before
freezing, and after thawing) of Nili Ravi buffalo bull spermatozoa (n 4 bulls and n 20 ejaculates).
Stage of
cryopreservation
After dilution

Before freezing

After thawing

Concentration
of trehalose (mM)

Sperm antioxidative prole

Catalase (U/mL)e

0
15
30
45
60
0
15
30
45
60
0
15
30
45
60

3.98
5.29
5.44
4.89
4.45
4.71
8.66
8.98
8.78
7.71
1.52
1.82
3.68
1.58
1.92

















Total glutathione (mM)e

Superoxide dismutase (U/mL)e

0.37
0.30ab
0.58a
0.28ab
0.37bc
0.18c
0.15ab
0.10a
0.31a
0.75ab
0.06b
0.12b
0.49a
0.04b
0.19b

52.37
71.61
75.52
59.13
68.38
51.76
65.04
65.52
65.33
65.53
41.23
41.77
50.50
49.80
49.96

















2.43
3.46a
3.80a
5.20b
4.0a
0.41b
2.76a
2.78a
2.84a
3.04a
0.81b
2.08b
0.80a
0.71a
0.85a

76.73
81.93
85.04
81.44
77.97
55.17
61.61
65.36
63.16
63.04
37.31
39.40
40.52
39.45
37.93

















4.86c
2.10ab
0.68a
0.91abc
1.52bc
0.38c
1.42b
1.31a
0.90ab
1.47b
0.19d
0.85b
0.25a
0.27b
0.46c

Values lacking a common superscript in a column indicate signicant (P < 0.05) difference among the groups at a given stage.
e
Values are mean  standard error of the mean.

freezing, and after thawing), (2) in vitro quality (after

thawing), and (3) in vivo fertility of Nili Ravi buffalo bull
spermatozoa. Cryopreservation is a major cause of oxidative stress to sperm. Consequently, it exerts an irreparable
injury to the sperm organelles and alteration in enzymatic
prole, accompanied with reduced sperm motility, functional membrane integrity, and fertilizing ability [31,32].
Superoxide dismutase, catalase, and glutathione
peroxidase and reductase are considered as the major
defenders of the sperm membranes against ROS and LPO
[13]. In the present study, it was found that sperm antioxidant (catalase, superoxide dismutase, and total glutathione) proles were signicantly improved at post
dilution and prefreezing with the addition of trehalose in
extenders. At post thaw, activities of these enzymes were
signicantly improved with the addition of 30 mM
trehalose in extender as compared to control. This
improvement in sperm antioxidant enzymes prole
indicates the benecial effect of trehalose in extender
during cryopreservation. Moreover, these results are

The data on effect of trehalose in extender on post-thaw

quality parameters of Nili Ravi buffalo bull spermatozoa are
presented in Table 2. Post thaw total sperm motility (%) was
higher (P < 0.05) in extender containing 30-mM trehalose as
compared to control and 15 and 60-mM groups. Although
sperm progressive motility (%), rapid velocity (%), average
path velocity (mm/s), straight line velocity (mm/s), curvilinear
velocity (mm/s), plasma membrane (structural and functional, %), acrosome (%), and DNA (%) integrity were higher
(P < 0.05) in extender containing 30-mM trehalose as
compared to other treatment groups and control.
The fertility rates (61% vs. 43%) were higher (P < 0.05) in
buffaloes inseminated with semen doses cryopreserved in
extender containing 30 mM of trehalose than the control.
4. Discussion
The present study for the rst time has investigated the
effect of trehalose in extender on (1) antioxidant enzymes
prole during cryopreservation (after dilution, before

Table 2
Effect of trehalose in extender on post-thaw CASA parameters (motilities, velocity distribution, and kinematics) and plasma membrane, acrosome and DNA
integrity of Nili Ravi buffalo bull spermatozoa (n 4 bulls and n 20 ejaculates).
Semen quality parameters

Concentration of trehalose (mM)e

0

Total motility (%)

Progressive motility (%)
Rapid (%)
Medium (%)
Slow (%)
Average path velocity (mm/s)
Straight line velocity (mm/s)
Curvilinear velocity (mm/s)
Amplitude of lateral head displacement (mm)
Beat cross frequency (Hz)
Plasma membrane integrity (%)
Acrosome integrity (%)
DNA integrity (%)

53.74
9.45
17.10
41.90
15.28
67.90
54.76
101.04
6.24
18.29
24.90
59.25
91.45

15














2.19bc
0.39b
1.21bc
2.36
2.36
1.19b
1.15b
1.91b
0.21a
0.72
0.66d
0.27d
0.25c

55.03
11.05
16.90
38.35
14.30
67.12
54.50
97.36
5.40
17.12
31.10
61.35
92.20

30














3.38bc
0.61b
1.08bc
2.53
1.70
1.40b
1.03b
2.35b
0.14b
0.53
0.48b
0.36b
0.29b

66.78
16.95
24.56
42.42
11.72
74.24
60.01
108.53
5.35
16.97
38.65
69.30
97.55

45














4.06a
0.81a
1.75a
2.77
1.62
1.41a
1.27a
2.18a
0.11b
0.62
0.43a
0.16a
0.11a

Values lacking a common superscript in a row indicate signicant (P < 0.05) difference among the groups.
e
Values are mean  standard error of the mean.

60.28
10.85
17.59
42.88
16.87
66.83
54.23
98.19
5.23
17.47
31.65
60.65
91.75

60














3.08ab
0.47b
1.19b
2.33
2.14
1.05b
1.03b
1.85b
0.11b
0.93
0.51b
0.45bc
0.21bc

49.13
9.35
13.45
35.77
13.80
64.81
52.38
95.48
5.52
17.92
27.75
60.05
91.60















4.39c
0.82b
1.32c
3.21
2.91
1.21b
1.24b
1.75b
0.28b
0.75
0.66c
0.60cd
0.21bc

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S. Iqbal et al. / Theriogenology 85 (2016) 954959

comparable with the previous studies conducted in bull

[27] and ram [3335].
Motility is generally considered to be one of the most
signicant features related with the fertilizing ability of
sperm and is a countenance of viability and structural
integrity. In addition, a positive correlation of CASA parameters was found with in vitro fertility in bulls [36].
Results of the present study on post-thaw motility parameters, velocity distribution, and kinematics were
signicantly improved with the addition of 30-mM trehalose in semen extender. Previous studies conducted on
bovine [27], caprine [25], ovine [34], and swine [37] semen
reported a signicant improvement in post-thaw motility
with the addition of 25, 50, and 100-mM trehalose in
semen extender. Furthermore, with increasing the concentration of trehalose, negative effects were detected on
sperm motility [20,25]. In our case, best results on postthaw CASA parameters were obtained with 30 mM of
trehalose, which is lower in comparison with the previous
studies [27,37,38]. The differences with these studies might
be due to the extender composition or species or both.
Present study also revealed that addition of trehalose
increased the osmolarity of extender in a dose dependent
manner. It is pertinent to mention that the optimum osmolarity of extender for cryopreservation of buffalo bull
semen is 285 to 295 mOsmol/kg [30]. It is, therefore, suggested that the cryoprotective effect of trehalose is associated with the osmotic effect and specic exchanges with
the membrane phospholipids [27].
Sperm plasma membrane is susceptible to ROS and LPO
attack during cryopreservation, and its damage results in
compromised fertility [39]. Supravital HOST evaluates the
viable spermatozoa with intact plasma membrane. Results
of the present study indicate that the addition of trehalose
at a concentration of 30 mM in semen extender signicantly increases the number of spermatozoa with intact
plasma membrane at post thawing. Our results are in
accordance with the previous studies on bull and Angora
buck semen [20,27]. The protective effect of trehalose is
through stabilizing sperm plasma membrane, modulating
membrane uidity, and lowering the intracellular ice
crystal formation [38].
Presence of normal acrosome is important for successful
acrosome reaction and ultimately fertilization [2]. In the
present study, there was a signicant improvement in
acrosome integrity with the addition of 30-mM trehalose.
Our results are in accordance with the previous studies
conducted on Shiba goat and Angora goat and bull semen
[25,27,38].
The process of freezing and thawing alters sperm DNA
structure [40]. Normal range of DNA integrity is 97% to 99%
in bulls with high fertility [41]. Results of the present study
reported a lower percentage (2.5%) of DNA damage with
the addition of 30-mM trehalose in semen extender. These
results are in agreement with the previous study conducted
in bovines [42].
In the present study, fertility rates were signicantly
higher with semen cryopreserved in extender containing
30-mM trehalose. Overall conception rates of the present
study with varying sperm concentration falls within the
range reported earlier in buffalo [4345]. However, Anzar

et al. [46] found a lower conception rate in buffaloes as

compared to the present study. This difference might be due
to technical knowhow and geoclimatic conditions [44,45].
4.1. Conclusions
It is concluded that addition of 30-mM trehalose in
extender improves the semen antioxidant enzymes activity,
post thaw quality, and fertility in Nili Ravi buffaloes.
Acknowledgments
The authors are thankful to Dr. Tasneem Akhtar (Chief
Research Ofcer, Buffalo Research Institute Pattoki, District
Kasur, Pakistan) for nancial assistance in procurement of
ELISA test kits. Authors are also grateful to Dr. Mustafa
Numan Bucak, Associate Professor, Department of Reproduction and Articial Insemination, Faculty of Veterinary
Medicine, Selcuk University, Konya, Turkey for providing
trehalose.
References
[1] Anzar M, Graham EF. Effect of ltration on post-thaw quality of bull
semen. Theriogenology 1995;43:43949.
[2] Bailey JL, Bilodeau JF, Cormier N. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J Androl
2000;21:17.
[3] Bilodeau JF, Blanchette S, Gagnon C, Sirad MA. Levels of antioxidant
defenses are decreased in bovine spermatozoa after a cycle of
freezing and thawing. Mol Reprod Dev 2000;55:2828.
[4] Uysal O, Bucak MN. Effects of oxidized glutathione, bovine serum
albumin, cysteine and lycopene on the quality of frozen-thawed
ram semen. Acta Vet Brno 2007;76:38390.
[5] Andrabi SMH. Factors affecting the quality of cryopreserved buffalo
(Bubalus bubalis) bull spermatozoa. Reprod Domest Anim 2009;44:
55269.
[6] Alvarez JG, Storey BT. Differential incorporation of fatty acids into
and peroxidative loss of fatty acids from phospholipids of human
spermatozoa. Mol Reprod Dev 1995;42:33446.
[7] Aitken RJ, Fisher H. Reactive oxygen species generation and human
spermatozoa: the balance of benet and risk. Bioessays 1994;16:
25967.
[8] Alvarez JG, Storey BT. Role of glutathione peroxidase in protecting
mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res 1989;23:7790.
[9] Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid
peroxidation and production of hydrogen peroxide and superoxide
in human spermatozoa. Superoxide dismutase as major enzyme
protectant against oxygen toxicity. J Androl 1987;5:33848.
[10] Lewis SEM, Boyle PM, McKinney KA. Total antioxidant capacity of
seminal plasma is different in fertile and infertile men. Fertil Steril
1995;64:86870.
[11] Hugo A. Catalase. In: Hans UB, editor. Methods of enzymatic analysis. New Yolk: Academic Press; 1974. p. 6734.
[12] Upreti GC, Jensen K, Munday R, Duganzich DM, Vishwanath R,
Smith JF. Studies on aromatic amino acid oxidase activity in ram
spermatozoa: role pyruvate as an antioxidant. Anim Reprod Sci
1998;51:27587.
[13] Bilodeau JF, Blanchette SI, Gagnon C, Sirard MA. Thiols prevent H2O2
mediated loss of sperm motility in cryopreserved bull semen.
Theriogenology 2001;56:27586.
[14] Lasso J, Noiles E, Alvarez J, Storey B. Mechanism of superoxide
dismutase loss from human sperm cells during cryopreservation. J
Androl 1994;15:25565.
[15] Irvine DS. Glutathione as a treatment for male infertility. Rev Reprod
1996;1:612.
[16] Meister A, Anderson ME. Glutathione. Annu Rev Biochem 1983;52:
1160.
[17] Storey BT. Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa. Mol Hum Reprod
1997;3:20313.

S. Iqbal et al. / Theriogenology 85 (2016) 954959

[18] Sarozkan S, Bucak MN, Tuncer PB, Ulutas PA, Bilgen A. The inuence
of cysteine and taurine on microscopic-oxidative stress parameters
and fertilizing ability of bull semen following cryopreservation.
Cryobiology 2009;58:1348.
[19] Aisen EG, Medina VH, Venturino A. Cryopreservation and postthawed fertility of ram semen frozen in different trehalose
concentrations. Theriogenology 2002;57:18018.
[20] Atessahin A, Bucak MN, Tuncer PB, Kzl M. Effects of anti-oxidant
additives on microscopic and oxidative parameters of Angora goat
semen following the freeze-thawing process. Small Rumin Res
2008;77:3844.
[21] Leibo SP, Songsasen H. Cryopreservation of gametes and embryos of
non-domestic species. Theriogenology 2002;57:30326.
[22] Purdy PH. A review on goat sperm cryopreservation. Small Rumin
Res 2006;63:21522.
[23] Bykleblebici S, Tuncer PB, Bucak MN, Eken A, Sarzkan S,
Tasdemir U, et al. Cryopreservation of bull sperm: effects of
extender supplemented with different cryoprotectants and antioxidants on sperm motility, antioxidant capacity and fertility results.
Anim Reprod Sci 2014;150:7783.
[24] Chen Q, Haddad GG. Role of trehalose phosphate synthase and
trehalose during hypoxia from ies to mammals. J Exp Biol 2004;
207:31259.
[25] Tuncer PB, Tasdemir U, Buyukleblebici S, Ozgurtas T, Coskun E,
Erol H, et al. Effects of different doses of trehalose supplementation
in egg yolk extender in frozenthawed Angora buck semen. Small
Rumin Res 2013;113:3839.
[26] Ansari MS, Rakha BA, Ullah N, Andrabi SMH, Iqbal S, Khalid M, et al.
Effect of exogenous glutathione in extender on the freezability of
Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa. Anim Sci Pap
Rep 2010;28:23544.
[27] Hu JH, Zan LS, Zhao XL, Li QW, Jiang ZL, Li YK, et al. Effects of
trehalose supplementation on semen quality and oxidative stress
variables in frozen thawed bovine semen. J Anim Sci 2010;88:
165762.
[28] Tartaglionea CM, Ritta MN. Prognostic value of spermatological
parameters as predictors of in vitro fertility of frozen-thawed bull
semen. Theriogenology 2004;62:124552.
[29] Akhter S, Ansari MS, Rakha BA, Andrabi SMH, Khalid M, Ullah N.
Effect of low density lipoproteins in extender on freezability and
fertility of buffalo (Bubalus bubalis) bull semen. Theriogenology
2011;76:75964.
[30] Mughal DH, Ijaz A, Yousaf MS, Rehman H, Aleem M, Zaneb H, et al.
Assessment of optimal osmotic pressure of citrate egg yolk extender
for cryopreservation of buffalo bull (Bubalus bubalis) semen. J Anim
Plant Sci 2013;23:9648.
[31] Bucak MN, Tuncer PB, Sarzkan ve S, Ulutas PA. Comparison of the
effects of glutamine and an amino acid solution on post-thawed ram
sperm parameters, lipid peroxidation and anti-oxidant activities.
Small Rumin Res 2009;81:137.

959

[32] Bucak MN, Sarzkan S, Tuncer PB, Ulutas ve PA, Akcadag HI. Effect
of antioxidants on microscopic semen parameters, lipid peroxidation and antioxidant activities in Angora goat semen
following cryopreservation. Small Rumin Res 2009b;81:905.
[33] Aisen E, Quintana M, Medina V, Morello H, Venturino A. Ultramicroscopic and biochemical changes in ram spermatozoa cryopreserved with trehalose-based hypertonic extenders. Cryobiology
2005;50:23949.
[34] Bucak MN, Atessahin A, Varsl O, Yuce A, Tekin N, Akcay E. The inuence of trehalose, taurine, cysteamine and hyaluronan on ram
semen: microscopic and oxidative stress parameters after freeze
thawing process. Theriogenology 2007;67:10607.
[35] Bucak MN, Atessahin A, Yuce A. Effect of anti-oxidants and oxidative
stress parameters on ram semen after the freezethawing process.
Small Rumin Res 2008;75:12834.
[36] Kathiravan P, Kalatharan J, John Edwin M, Veerapandian C. Computer automated motion analysis of crossbred bull spermatozoa and
its relationship with in vitro fertility in zona-free hamster oocytes.
Anim Reprod Sci 2008;104:917.
[37] Hu JH, Li QW, Li G, Jiang ZL, Bu SH, Yang H, et al. The cryoprotective
effect of trehalose supplementation on boar spermatozoa quality.
Anim Reprod Sci 2009;112:10718.
[38] Aboagla EM, Terada T. Trehalose-enhanced uidity of the goat
sperm membrane and its protection during freezing. Biol Reprod
2003;69:124550.
[39] Andrabi SMH, Ansari MS, Ullah N, Afzal M. Effect of non-enzymatic
antioxidants in extender on post-thaw quality of buffalo (Bubalus
bubalis) bull spermatozoa. Pak Vet J 2008;28:15962.
[40] Anzar M, He L, Buhr MM, Kroetsch TG, Pauls KP. Sperm apoptosis in
fresh and cryopreserved bull semen detected by ow cytometry and
its relationship with fertility. Biol Reprod 2002;66:35460.
[41] Bochenek M, Smorag Z, Pilch J. Sperm chromatin structure assay of
bulls qualied for articial insemination. Theriogenology 2001;56:
55767.
[42] Bucak MN, Tuncer PB, Sarzkan S, Baspnar N, Taspnar M, oyan K,
et al. Effects of antioxidants on post-thawed bovine sperm and
oxidative stress parameters: antioxidants protect DNA integrity
against cryodamage. Cryobiology 2010;61:24853.
[43] Tahir MN, Bajwa MA, Latif M, Mushtaq M, Shah MH. Effects of
insemination dose and season on conception rates in buffaloes. Pak
Vet J 1981;1:1613.
[44] Andrabi SMH, Ahmed N, Abbas A, Anzar M. Effect of two different
antibiotic combinations on fertility of frozen buffalo and Sahiwal
bull semen. Pak Vet J 2001;21:1669.
[45] Andrabi SMH, Siddique M, Ullah N, Khan LA. Effect of reducing
sperm numbers per insemination dose on fertility of cryopreserved
buffalo bull semen. Pak Vet J 2006;26:179.
[46] Anzar M, Farooq U, Mirza MA, Shahab M, Ahmed N. Factors affecting
the efciency of articial insemination in cattle and buffaloes in
Punjab. Pak Vet J 2003;23:10613.