Accepted Manuscript The Effect of Superoxide Dismutase Mimetic and Catalase on the Quality of PostThawed Goat Semen Mojtaba Shafiei, Mohsen Forouzanfar, Sayyed Morteza Hosseini, Mohammad Hossein Nasr Esfahani PII:
S0093-691X(15)00033-3
DOI:
10.1016/j.theriogenology.2015.01.018
Reference:
THE 13070
To appear in:
Theriogenology
Received Date: 24 July 2014 Revised Date:
7 January 2015
Accepted Date: 17 January 2015
Please cite this article as: Shafiei M, Forouzanfar M, Hosseini SM, Nasr Esfahani MH, The Effect of Superoxide Dismutase Mimetic and Catalase on the Quality of Post-Thawed Goat Semen, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.01.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Revised highlighted
1 2 3
The Effect of Superoxide Dismutase Mimetic and Catalase on the Quality of Post-Thawed
4
Goat Semen
RI PT
5 6 7
Mojtaba Shafiei a, Mohsen Forouzanfar a, Sayyed Morteza Hosseini b, Mohammad Hossein Nasr
9
Esfahani c *
10 11
M AN U
SC
8
a-Department of Biochemistry, Fars Science and Research Branch, Islamic Azad University, Fars, Iran.
13
b - Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive
14
Biomedicine, ACECR, Tehran, Iran
15
c - Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute
16
for Biotechnology, ACECR, Isfahan, Iran.
TE D
12
17
*Correspondence:
18 19
Mohammad H. Nasr-Esfahani. Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center,
20
Tel: + 98 3195015680, Fax: + 9831 95015687, Email: [email protected]
22 23 24 25 26 27 28 29
EP
AC C
21
Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
ACCEPTED MANUSCRIPT
Abstract:
31
MnTE is a cell permeable superoxide dismutase mimetic agent which can convert superoxide to
32
hydrogen peroxide (H2O2). Supplementation of MnTE to commercial semen extender can
33
protect sperm from superoxide but not H2O2. Therefore, we proposed that addition of catalase
34
(0.0, 200 or 400 IU/ ml) in combination with MnTE (0.1 µM) may further improve the
35
cryopreservation efficiency of goat semen in a commercially optimized freezing media like,
36
Andromed®. Therefore, ejaculates were obtained from three adult bucks twice a week during
37
breeding season and diluted with Andromed® supplemented with or without MnTE and catalase,
38
and were frozen in liquid nitrogen. Sperm parameters and reactive oxygen species (ROS)
39
contents were evaluated 2 hours after dilution (prefreezing) and following freezing/thawing. The
40
results revealed that all the treatments ,significantly (P ≤0.05) improved sperm motility, viability,
41
membrane integrity post freezing and reduced ROS content compared to control but maximum
42
improvement was obtained in MnTE+ 400 IU/ml catalase. In addition, supplementation with these
43
antioxidants significantly (P ≤0.05) increases the cleavage rate after in vitro fertilization. In conclusion,
44
the results of present study suggest that addition of antioxidant MnTE or catalase to a
45
commercial optimized media, like Andromed®, improves total motility, membrane integrity and
46
viability of goat semen samples post thawing. But the degree of improvement for these
47
parameters significantly (P ≤0.05) higher when MnTE and catalase were simultaneously added
48
to the cryopreservation media.
49
Keywords: Antioxidant, MnTE, Semen Cryopreservation, Sperm Motility, Reactive
50
Oxygen Species.
52 53 54 55 56
SC
M AN U
TE D
EP
AC C
51
RI PT
30
ACCEPTED MANUSCRIPT
1. Introduction
58
Despite advances in semen cryopreservation, a variety of injuries occur at cellular and molecular
59
levels during semen cryopreservation which may impair sperm function and fertilization
60
potential [1, 2]. Major drawbacks of semen cryopreservation are reduction of sperm viability,
61
motility, plasma membrane integrity and impaired function of the survived cell population [3].
62
These phenomena are accounted by osmotic stress, cold shock, intracellular ice crystal and
63
oxidative stress induced by reactive oxygen species (ROS) [4]. Among the aforementioned
64
factors, excessive production of ROS plays a central role in induction of cryoinjury. ROS acts as
65
a double-edged sword in many physiological phenomena. Basal levels of ROS are required for
66
normal sperm motility, capacitation and acrosome reaction while excess ROS lead to various
67
forms of cellular damages, including impairment of membrane, DNA damage and apoptosis [5,
68
6]. Somatic cells have acquired a delicate balance between ROS generation and ROS scavengers
69
but unlike most somatic cells, sperm as one of the smallest cells of the body has little cytoplasm.
70
This makes sperm susceptible to ROS injuries which can affect sperm motility [7], viability [8],
71
DNA integrity and energy metabolism [9]. Moreover, ROS can also be produced by exogenous
72
sources such as semen handling during cryopreservation process, exposure to light, oxygen and
73
shearing forces generated by centrifugation. Therefore, supplementation of semen diluents with
74
antioxidants has been advised and a wide variety of antioxidants have been used for semen
75
cryopreservation in bovine [10], water buffalo [11], stallion [12], ram [13] and goat [14].
76
Antioxidants used during semen cryopreservation can be divided into two main categories: cell
77
permeable and impermeable antioxidants. In addition, antioxidants can be categorized as
78
enzymatic, enzyme-mimetic and non-enzymatic. Recent researches have suggested that cell
79
permeable enzyme-mimetic antioxidants are considered as valuable compounds for reducing
80
intracellular ROS levels [15]. In this regard, we and others researchers have shown that addition
81
of cell permeable enzyme-mimetic antioxidants to commercially optimized cryopreservation
82
media with antioxidant activity can further improve the quality of these media [13, 14]. MnTE
83
[Manganese (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin chloride] is a manganese
84
porphyrin compound with a superoxide dismutase (SOD) mimetic activity which has been shown
85
to successfully rescue animal models for some oxidative stress-related diseases [16]. We
86
previously proposed that 0.1µM of MnTE can improve sperm membrane and acrosomal
87
integrity. Furthermore, we showed that in vitro rate of blastocyst formation following freeze–
AC C
EP
TE D
M AN U
SC
RI PT
57
ACCEPTED MANUSCRIPT
thawing of goat semen supplemented with MnTE was higher compared to its corresponding
89
control [14].
90
SOD is one of the enzymatic antioxidants which converts superoxide anion (O2.-) into hydrogen
91
peroxide (H2O2) and oxygen (O2), while catalase can convert this H2O2 into the oxygen and
92
water [17, 18]. Therefore, supplementation with MnTE is expected to increase H2O2, which is
93
relatively stable cell permeable molecule with high oxidant potential. In other words the
94
association of SOD mimetic (MnTE) and peroxide scavengers (catalase) may promote better
95
sperm freezing performance. Therefore, the aim of this study was to address whether addition of
96
catalase in combination with MnTE or alone, into commercially optimized freezing media could
97
further improve the efficiency of cryopreservation of goat semen.
98
2. Materials and methods
99
2.1. Chemicals
M AN U
SC
RI PT
88
All chemicals used in this study were purchased from Sigma Chemical CO. (St. Louis, MO,
101
USA) unless stated otherwise. MnTE was a gift from Dr Ines Batinić-Haberle (Department of
102
Radiation Oncology, Duke University Medical School, Durham, NC 27710, USA). Andromed®
103
as semen extender was obtained from Minitube, Germany.
104
2.2. Semen collection, processing and freezing-thawing
105
Semen collection and processing procedures were carried out according to Forouzanfar et al.[14].
106
In brief, ejaculates were obtained by artificial vagina from three adult Bakhtiari bucks aged 2-3
107
years, twice a week during the breeding season (September /October, 2013) in the animal farm of
108
Reproductive Biotechnology research center at Royan Institute (Isfahan, Iran latitude 32°39'N).
109
Samples from each male were kept separated and transported at 35 °C to the laboratory for
110
microscopic assessment within 30 min. Only ejaculates showing higher than 70% motile and
111
80% morphologically normal sperm and more than 1 ml in volume were used for experiments.
112
To minimize individual differences, semen samples from the three bucks in each replicate were
113
pooled. A total of 6 ejaculates from each goat were processed. For freezing, all of the treatments were
114
repeated six times with the pooled semen samples. Andromed® was used as the base freezing
115
extender. Each pooled semen sample was divided into six equal aliquots and diluted (1:20 v/v)
AC C
EP
TE D
100
ACCEPTED MANUSCRIPT
with extender containing no antioxidant (control), 0.1 µM of MnTE (Mn), 200 IU/ml catalase
117
(CAT 200), 400 IU/ml catalase (CAT 400), 0.1 µM of MnTE plus 200 IU/ml catalase (Mn+CAT
118
200), 0.1 µM of MnTE plus 400 IU/ml catalase (Mn+CAT 400). Concentration of MnTE and
119
catalase were based on our previous report [14] and Roca et .al [19], respectively. The diluted
120
semen was cooled to 4°C over a period of 2 hours and drawn into 0.5 ml French straws (Biovet,
121
L’Agile France), heat-sealed and stored at 4°C one hour for more equilibration. The straws were
122
exposed to liquid nitrogen (LN2) vapor for 12 min, plunged into LN2, and stored in LN2 until
123
thawed and used for evaluation of sperm parameters and in vitro fertilization. Thawing was
124
carried out by plunging sealed straws in a 37°C water bath for 30 seconds. The thawed samples
125
were individually evaluated by a single trained individual.
126
2.3. Semen evaluation after thawing 2.3.1. Measurement of sperm motility
SC
M AN U
127
RI PT
116
For assessment of sperm motility post thawing, four or five straws from one treatment in each
129
group were thawed and pooled. The samples were diluted with fertilization medium (Tyrode's
130
albumin lactate pyruvate medium- Fert-TALP) to final concentration of 1x106spz/ml. The
131
percentages of rapid progressive (class A) , sperm that swim fast in a straight line, slow
132
progressive (class B), sperm that move forward but tend to travel in a curved, and non-
133
progressive (class C),sperm that do not move forward despite that they move their tails, as well
134
as total motility which refers to the population of sperm that display any type of movement, were
135
assessed according to the software setting of the computer-assisted sperm analysis (CASA)
136
system (Video Test, ltd: version Sperm 2.1© 1990-2004, Russia) [20], that was set-up for goat
137
sperm evaluation. For each sample, 5 µl of sperm suspension was loaded on a pre-warmed slide
138
and then covered by a 18 x 18 mm coverslip, a minimum of 500 sperm per sample were analyzed
139
in at least three different microscopic fields.
EP
AC C
140
TE D
128
2.3.2. Assessment of sperm plasma membrane integrity
141
Sperm plasma membrane integrity was estimated by hypo-osmotic swelling test (HOST) based
142
on curled and swollen tails [21]. For this purpose 25 µl of the diluted semen (for prefreezing or
143
thawed specimen) were mixed with 200 µl of HOST solution (100 mOsm/l, 57.6 mM fructose
144
and 19.2 mM sodium citrate) for 30 min at room temperature, subsequently 5 µl of homogenized
ACCEPTED MANUSCRIPT
mixture was mounted and directly investigated using an inverted microscope (Olympus, CKX41
146
- Japan).The percentages of spermatozoa with swollen and curved tails, categorized as intact
147
plasma membrane, were estimated from recording at least 200 spermatozoa in more than five
148
different microscopic fields.
149
2.3.3. Assessment of sperm viability
150
Sperm viability was assessed using eosin-nigrosin staining which was done for prefreezing (2
151
hours post semen dilution) and post thawing experiments with the same protocol. Fifty µl of
152
diluted or thawed semen was mixed with 100 µl of eosin (1%) in a test tube for 30 second, the
153
mixture then was stained with nigrosin (10%) for a further 30 second and mixed gently. One
154
drop of stained semen was smeared on slide and allowed to air dry in a dust-free environment
155
and examined directly at a magnification of 1000× under oil immersion using light microscope.
156
Two hundred sperm were assessed for each replicates. Sperm head that were unstained were
157
classified as “live sperm”, while sperm that appeared as pink or red were considered as “dead
158
sperm”.
SC
M AN U
2.3.4. Assessment of oxidative stress in sperm using flow cytometery
TE D
159
RI PT
145
We used flow cytometery (FAC Scan; Becton Dickinson, San Jose, CA) to determine of the ROS
161
content in thawed and diluted semen as described previously [22].In brief, the samples from each
162
treatment/replicate were centrifuged at 700 x g for10 min, the supernatant was discarded and the
163
pellet was re-suspended in 1ml of phosphate buffer saline. The percentages of ROS-positive
164
spermatozoa in each treatments were determined following incubating one million sperm per ml
165
with 5µM of 2′, 7′-Dichlorofluorescin diacetate (DCF-DA) for 30 min at room temperature.
166
Regarding mechanism of ROS reaction with DCF, it is important to note that once DCF-DA
167
enters cells, it loses its ester group and upon interaction with ROS, it produces a fluorescence
168
compound. Live sperm produce a detectable amount of physiological ROS and therefore, the
169
sperm become DCF positive. In order to differentiate between the physiological amount of ROS
170
and pathological ROS production, we reduced the concentration of DCF-DA and therefore,
171
sperm producing super physiological concentration of DCF-DA were detectable at this 5µM
172
concentration of DCF-DA.
173
2.4. In vitro fertilization and embryo culture
AC C
EP
160
ACCEPTED MANUSCRIPT
In vitro fertilization (IVF) and embryo culture were performed as described by Forouzanfar et al.
175
[14]. Goat ovaries were recovered at the local slaughterhouse, placed in normal saline (0.9 %
176
Sodium Chloride) at a temperature between 25°C and 35°C and then transported to the lab within
177
2 hours. The cumulus-oocyte complexes (COCs) comprised of at least 4-10 layers of cumulus
178
cells and oocytes with a uniform cytoplasm and homogenous distribution of lipid droplets in the
179
cytoplasm were recovered by aspiration from follicles of more than 2 mm diameter on the
180
surface of the ovaries and selected for the in vitro maturation (IVM). The selected COCs were
181
washed three times in the aspiration medium (Hepes-Tissue Culture Media (H-TCM) + 10%
182
Fetal Calf Serum (FCS) + 100 IU/ml heparin), and then cultured in maturation medium (TCM-
183
199 + 10% FCS + 5 mg/ml follicle-stimulating hormone + 5 mg/ml luteinizing hormone + 0.1
184
mM cysteamin )in 5% CO2 at 39 °C and maximum humidity for 20- 22 hours. For sperm
185
preparation, five straws representing one freezing operation in each replicate were thawed,
186
pooled and washed through two-gradient (40%–80% solutions) of Pure Sperm (Nidacon;
187
Gothenburg, Sweden) to separate motile sperm with normal morphology by centrifugation (700
188
g/ 15 min-RT). Matured COCs (totally 1254 for all treatments and control) were partially
189
stripped of the cumulus cells and transferred into 100-µl drops of fertilization medium and
190
fertilized in vitro at 39°C and 5% CO2, 5% O2 and 90% nitrogen atmosphere. IVF experiments
191
were repeated three times using pooled frozen-thawed samples in each group. For IVF, sperm at
192
final concentration of 2 × 105 sperm were co-incubated with 20-25 matured oocytes/100 µl
193
droplet for 22 hours under the same gas atmosphere condition as for IVM. Presumptive zygotes
194
were mechanically denuded of their cumulus cells via pipetting and cultured in modified
195
Synthetic Oviduct Fluid (SOF) medium for the first 3 days post insemination (day 0 = day of
196
insemination). Embryos were then transported to SOF medium supplemented with 10% Charcoal
197
stripped serum and 1.5 mM glucose up to blastocyst stage. Subsequently cleavage, blastocyst and
198
hatched rates in each group were assessed on days 3, 8 and 9 post insemination, respectively.
199
2.5. Statistical analysis
200
AC C
EP
TE D
M AN U
SC
RI PT
174
The results are reported as the mean ± standard error (SE) for each experiment. The
201
analysis of variance (ANOVA) was used for treatments comparisons .One-way ANOVA with
202
the Tukey multiple comparison post-hoc test was used to compare the different stimuli. P ≤ 0.05
203
was considered statistically significant. For IVF treatments, Student’s t-test was used to compare
ACCEPTED MANUSCRIPT
204
mean, when the data from the supplemented groups (CAT 200, CAT 400 and Mn+CAT 400)
205
were pooled and compared with control group. 3. Results
207
Table 1 shows the effects of antioxidant(s) supplementation in freezing media on
RI PT
206
percentage of sperm motility parameters before freezing (2h after dilution) and after freeze-
209
thawing. There were no significant (P ≤0.05) differences in total, and class A + B motility
210
between treatments in pre-freezing conditions. Following thawing, CAT 400 and Mn+CAT 400
211
groups resulted in better maintenance of total and class A+B motility compared to control and
212
other treatments. Mn, CAT 200 and Mn+CAT 200 groups was better maintained total and class
213
A+B motility after thawing compared to control. It is of interest to note that Mn+CAT 400
214
treatment yield higher post-thaw motility compared to control and other treatments (p
S0093-691X(15)00033-3
DOI:
10.1016/j.theriogenology.2015.01.018
Reference:
THE 13070
To appear in:
Theriogenology
Received Date: 24 July 2014 Revised Date:
7 January 2015
Accepted Date: 17 January 2015
Please cite this article as: Shafiei M, Forouzanfar M, Hosseini SM, Nasr Esfahani MH, The Effect of Superoxide Dismutase Mimetic and Catalase on the Quality of Post-Thawed Goat Semen, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.01.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Revised highlighted
1 2 3
The Effect of Superoxide Dismutase Mimetic and Catalase on the Quality of Post-Thawed
4
Goat Semen
RI PT
5 6 7
Mojtaba Shafiei a, Mohsen Forouzanfar a, Sayyed Morteza Hosseini b, Mohammad Hossein Nasr
9
Esfahani c *
10 11
M AN U
SC
8
a-Department of Biochemistry, Fars Science and Research Branch, Islamic Azad University, Fars, Iran.
13
b - Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive
14
Biomedicine, ACECR, Tehran, Iran
15
c - Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute
16
for Biotechnology, ACECR, Isfahan, Iran.
TE D
12
17
*Correspondence:
18 19
Mohammad H. Nasr-Esfahani. Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center,
20
Tel: + 98 3195015680, Fax: + 9831 95015687, Email: [email protected]
22 23 24 25 26 27 28 29
EP
AC C
21
Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
ACCEPTED MANUSCRIPT
Abstract:
31
MnTE is a cell permeable superoxide dismutase mimetic agent which can convert superoxide to
32
hydrogen peroxide (H2O2). Supplementation of MnTE to commercial semen extender can
33
protect sperm from superoxide but not H2O2. Therefore, we proposed that addition of catalase
34
(0.0, 200 or 400 IU/ ml) in combination with MnTE (0.1 µM) may further improve the
35
cryopreservation efficiency of goat semen in a commercially optimized freezing media like,
36
Andromed®. Therefore, ejaculates were obtained from three adult bucks twice a week during
37
breeding season and diluted with Andromed® supplemented with or without MnTE and catalase,
38
and were frozen in liquid nitrogen. Sperm parameters and reactive oxygen species (ROS)
39
contents were evaluated 2 hours after dilution (prefreezing) and following freezing/thawing. The
40
results revealed that all the treatments ,significantly (P ≤0.05) improved sperm motility, viability,
41
membrane integrity post freezing and reduced ROS content compared to control but maximum
42
improvement was obtained in MnTE+ 400 IU/ml catalase. In addition, supplementation with these
43
antioxidants significantly (P ≤0.05) increases the cleavage rate after in vitro fertilization. In conclusion,
44
the results of present study suggest that addition of antioxidant MnTE or catalase to a
45
commercial optimized media, like Andromed®, improves total motility, membrane integrity and
46
viability of goat semen samples post thawing. But the degree of improvement for these
47
parameters significantly (P ≤0.05) higher when MnTE and catalase were simultaneously added
48
to the cryopreservation media.
49
Keywords: Antioxidant, MnTE, Semen Cryopreservation, Sperm Motility, Reactive
50
Oxygen Species.
52 53 54 55 56
SC
M AN U
TE D
EP
AC C
51
RI PT
30
ACCEPTED MANUSCRIPT
1. Introduction
58
Despite advances in semen cryopreservation, a variety of injuries occur at cellular and molecular
59
levels during semen cryopreservation which may impair sperm function and fertilization
60
potential [1, 2]. Major drawbacks of semen cryopreservation are reduction of sperm viability,
61
motility, plasma membrane integrity and impaired function of the survived cell population [3].
62
These phenomena are accounted by osmotic stress, cold shock, intracellular ice crystal and
63
oxidative stress induced by reactive oxygen species (ROS) [4]. Among the aforementioned
64
factors, excessive production of ROS plays a central role in induction of cryoinjury. ROS acts as
65
a double-edged sword in many physiological phenomena. Basal levels of ROS are required for
66
normal sperm motility, capacitation and acrosome reaction while excess ROS lead to various
67
forms of cellular damages, including impairment of membrane, DNA damage and apoptosis [5,
68
6]. Somatic cells have acquired a delicate balance between ROS generation and ROS scavengers
69
but unlike most somatic cells, sperm as one of the smallest cells of the body has little cytoplasm.
70
This makes sperm susceptible to ROS injuries which can affect sperm motility [7], viability [8],
71
DNA integrity and energy metabolism [9]. Moreover, ROS can also be produced by exogenous
72
sources such as semen handling during cryopreservation process, exposure to light, oxygen and
73
shearing forces generated by centrifugation. Therefore, supplementation of semen diluents with
74
antioxidants has been advised and a wide variety of antioxidants have been used for semen
75
cryopreservation in bovine [10], water buffalo [11], stallion [12], ram [13] and goat [14].
76
Antioxidants used during semen cryopreservation can be divided into two main categories: cell
77
permeable and impermeable antioxidants. In addition, antioxidants can be categorized as
78
enzymatic, enzyme-mimetic and non-enzymatic. Recent researches have suggested that cell
79
permeable enzyme-mimetic antioxidants are considered as valuable compounds for reducing
80
intracellular ROS levels [15]. In this regard, we and others researchers have shown that addition
81
of cell permeable enzyme-mimetic antioxidants to commercially optimized cryopreservation
82
media with antioxidant activity can further improve the quality of these media [13, 14]. MnTE
83
[Manganese (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin chloride] is a manganese
84
porphyrin compound with a superoxide dismutase (SOD) mimetic activity which has been shown
85
to successfully rescue animal models for some oxidative stress-related diseases [16]. We
86
previously proposed that 0.1µM of MnTE can improve sperm membrane and acrosomal
87
integrity. Furthermore, we showed that in vitro rate of blastocyst formation following freeze–
AC C
EP
TE D
M AN U
SC
RI PT
57
ACCEPTED MANUSCRIPT
thawing of goat semen supplemented with MnTE was higher compared to its corresponding
89
control [14].
90
SOD is one of the enzymatic antioxidants which converts superoxide anion (O2.-) into hydrogen
91
peroxide (H2O2) and oxygen (O2), while catalase can convert this H2O2 into the oxygen and
92
water [17, 18]. Therefore, supplementation with MnTE is expected to increase H2O2, which is
93
relatively stable cell permeable molecule with high oxidant potential. In other words the
94
association of SOD mimetic (MnTE) and peroxide scavengers (catalase) may promote better
95
sperm freezing performance. Therefore, the aim of this study was to address whether addition of
96
catalase in combination with MnTE or alone, into commercially optimized freezing media could
97
further improve the efficiency of cryopreservation of goat semen.
98
2. Materials and methods
99
2.1. Chemicals
M AN U
SC
RI PT
88
All chemicals used in this study were purchased from Sigma Chemical CO. (St. Louis, MO,
101
USA) unless stated otherwise. MnTE was a gift from Dr Ines Batinić-Haberle (Department of
102
Radiation Oncology, Duke University Medical School, Durham, NC 27710, USA). Andromed®
103
as semen extender was obtained from Minitube, Germany.
104
2.2. Semen collection, processing and freezing-thawing
105
Semen collection and processing procedures were carried out according to Forouzanfar et al.[14].
106
In brief, ejaculates were obtained by artificial vagina from three adult Bakhtiari bucks aged 2-3
107
years, twice a week during the breeding season (September /October, 2013) in the animal farm of
108
Reproductive Biotechnology research center at Royan Institute (Isfahan, Iran latitude 32°39'N).
109
Samples from each male were kept separated and transported at 35 °C to the laboratory for
110
microscopic assessment within 30 min. Only ejaculates showing higher than 70% motile and
111
80% morphologically normal sperm and more than 1 ml in volume were used for experiments.
112
To minimize individual differences, semen samples from the three bucks in each replicate were
113
pooled. A total of 6 ejaculates from each goat were processed. For freezing, all of the treatments were
114
repeated six times with the pooled semen samples. Andromed® was used as the base freezing
115
extender. Each pooled semen sample was divided into six equal aliquots and diluted (1:20 v/v)
AC C
EP
TE D
100
ACCEPTED MANUSCRIPT
with extender containing no antioxidant (control), 0.1 µM of MnTE (Mn), 200 IU/ml catalase
117
(CAT 200), 400 IU/ml catalase (CAT 400), 0.1 µM of MnTE plus 200 IU/ml catalase (Mn+CAT
118
200), 0.1 µM of MnTE plus 400 IU/ml catalase (Mn+CAT 400). Concentration of MnTE and
119
catalase were based on our previous report [14] and Roca et .al [19], respectively. The diluted
120
semen was cooled to 4°C over a period of 2 hours and drawn into 0.5 ml French straws (Biovet,
121
L’Agile France), heat-sealed and stored at 4°C one hour for more equilibration. The straws were
122
exposed to liquid nitrogen (LN2) vapor for 12 min, plunged into LN2, and stored in LN2 until
123
thawed and used for evaluation of sperm parameters and in vitro fertilization. Thawing was
124
carried out by plunging sealed straws in a 37°C water bath for 30 seconds. The thawed samples
125
were individually evaluated by a single trained individual.
126
2.3. Semen evaluation after thawing 2.3.1. Measurement of sperm motility
SC
M AN U
127
RI PT
116
For assessment of sperm motility post thawing, four or five straws from one treatment in each
129
group were thawed and pooled. The samples were diluted with fertilization medium (Tyrode's
130
albumin lactate pyruvate medium- Fert-TALP) to final concentration of 1x106spz/ml. The
131
percentages of rapid progressive (class A) , sperm that swim fast in a straight line, slow
132
progressive (class B), sperm that move forward but tend to travel in a curved, and non-
133
progressive (class C),sperm that do not move forward despite that they move their tails, as well
134
as total motility which refers to the population of sperm that display any type of movement, were
135
assessed according to the software setting of the computer-assisted sperm analysis (CASA)
136
system (Video Test, ltd: version Sperm 2.1© 1990-2004, Russia) [20], that was set-up for goat
137
sperm evaluation. For each sample, 5 µl of sperm suspension was loaded on a pre-warmed slide
138
and then covered by a 18 x 18 mm coverslip, a minimum of 500 sperm per sample were analyzed
139
in at least three different microscopic fields.
EP
AC C
140
TE D
128
2.3.2. Assessment of sperm plasma membrane integrity
141
Sperm plasma membrane integrity was estimated by hypo-osmotic swelling test (HOST) based
142
on curled and swollen tails [21]. For this purpose 25 µl of the diluted semen (for prefreezing or
143
thawed specimen) were mixed with 200 µl of HOST solution (100 mOsm/l, 57.6 mM fructose
144
and 19.2 mM sodium citrate) for 30 min at room temperature, subsequently 5 µl of homogenized
ACCEPTED MANUSCRIPT
mixture was mounted and directly investigated using an inverted microscope (Olympus, CKX41
146
- Japan).The percentages of spermatozoa with swollen and curved tails, categorized as intact
147
plasma membrane, were estimated from recording at least 200 spermatozoa in more than five
148
different microscopic fields.
149
2.3.3. Assessment of sperm viability
150
Sperm viability was assessed using eosin-nigrosin staining which was done for prefreezing (2
151
hours post semen dilution) and post thawing experiments with the same protocol. Fifty µl of
152
diluted or thawed semen was mixed with 100 µl of eosin (1%) in a test tube for 30 second, the
153
mixture then was stained with nigrosin (10%) for a further 30 second and mixed gently. One
154
drop of stained semen was smeared on slide and allowed to air dry in a dust-free environment
155
and examined directly at a magnification of 1000× under oil immersion using light microscope.
156
Two hundred sperm were assessed for each replicates. Sperm head that were unstained were
157
classified as “live sperm”, while sperm that appeared as pink or red were considered as “dead
158
sperm”.
SC
M AN U
2.3.4. Assessment of oxidative stress in sperm using flow cytometery
TE D
159
RI PT
145
We used flow cytometery (FAC Scan; Becton Dickinson, San Jose, CA) to determine of the ROS
161
content in thawed and diluted semen as described previously [22].In brief, the samples from each
162
treatment/replicate were centrifuged at 700 x g for10 min, the supernatant was discarded and the
163
pellet was re-suspended in 1ml of phosphate buffer saline. The percentages of ROS-positive
164
spermatozoa in each treatments were determined following incubating one million sperm per ml
165
with 5µM of 2′, 7′-Dichlorofluorescin diacetate (DCF-DA) for 30 min at room temperature.
166
Regarding mechanism of ROS reaction with DCF, it is important to note that once DCF-DA
167
enters cells, it loses its ester group and upon interaction with ROS, it produces a fluorescence
168
compound. Live sperm produce a detectable amount of physiological ROS and therefore, the
169
sperm become DCF positive. In order to differentiate between the physiological amount of ROS
170
and pathological ROS production, we reduced the concentration of DCF-DA and therefore,
171
sperm producing super physiological concentration of DCF-DA were detectable at this 5µM
172
concentration of DCF-DA.
173
2.4. In vitro fertilization and embryo culture
AC C
EP
160
ACCEPTED MANUSCRIPT
In vitro fertilization (IVF) and embryo culture were performed as described by Forouzanfar et al.
175
[14]. Goat ovaries were recovered at the local slaughterhouse, placed in normal saline (0.9 %
176
Sodium Chloride) at a temperature between 25°C and 35°C and then transported to the lab within
177
2 hours. The cumulus-oocyte complexes (COCs) comprised of at least 4-10 layers of cumulus
178
cells and oocytes with a uniform cytoplasm and homogenous distribution of lipid droplets in the
179
cytoplasm were recovered by aspiration from follicles of more than 2 mm diameter on the
180
surface of the ovaries and selected for the in vitro maturation (IVM). The selected COCs were
181
washed three times in the aspiration medium (Hepes-Tissue Culture Media (H-TCM) + 10%
182
Fetal Calf Serum (FCS) + 100 IU/ml heparin), and then cultured in maturation medium (TCM-
183
199 + 10% FCS + 5 mg/ml follicle-stimulating hormone + 5 mg/ml luteinizing hormone + 0.1
184
mM cysteamin )in 5% CO2 at 39 °C and maximum humidity for 20- 22 hours. For sperm
185
preparation, five straws representing one freezing operation in each replicate were thawed,
186
pooled and washed through two-gradient (40%–80% solutions) of Pure Sperm (Nidacon;
187
Gothenburg, Sweden) to separate motile sperm with normal morphology by centrifugation (700
188
g/ 15 min-RT). Matured COCs (totally 1254 for all treatments and control) were partially
189
stripped of the cumulus cells and transferred into 100-µl drops of fertilization medium and
190
fertilized in vitro at 39°C and 5% CO2, 5% O2 and 90% nitrogen atmosphere. IVF experiments
191
were repeated three times using pooled frozen-thawed samples in each group. For IVF, sperm at
192
final concentration of 2 × 105 sperm were co-incubated with 20-25 matured oocytes/100 µl
193
droplet for 22 hours under the same gas atmosphere condition as for IVM. Presumptive zygotes
194
were mechanically denuded of their cumulus cells via pipetting and cultured in modified
195
Synthetic Oviduct Fluid (SOF) medium for the first 3 days post insemination (day 0 = day of
196
insemination). Embryos were then transported to SOF medium supplemented with 10% Charcoal
197
stripped serum and 1.5 mM glucose up to blastocyst stage. Subsequently cleavage, blastocyst and
198
hatched rates in each group were assessed on days 3, 8 and 9 post insemination, respectively.
199
2.5. Statistical analysis
200
AC C
EP
TE D
M AN U
SC
RI PT
174
The results are reported as the mean ± standard error (SE) for each experiment. The
201
analysis of variance (ANOVA) was used for treatments comparisons .One-way ANOVA with
202
the Tukey multiple comparison post-hoc test was used to compare the different stimuli. P ≤ 0.05
203
was considered statistically significant. For IVF treatments, Student’s t-test was used to compare
ACCEPTED MANUSCRIPT
204
mean, when the data from the supplemented groups (CAT 200, CAT 400 and Mn+CAT 400)
205
were pooled and compared with control group. 3. Results
207
Table 1 shows the effects of antioxidant(s) supplementation in freezing media on
RI PT
206
percentage of sperm motility parameters before freezing (2h after dilution) and after freeze-
209
thawing. There were no significant (P ≤0.05) differences in total, and class A + B motility
210
between treatments in pre-freezing conditions. Following thawing, CAT 400 and Mn+CAT 400
211
groups resulted in better maintenance of total and class A+B motility compared to control and
212
other treatments. Mn, CAT 200 and Mn+CAT 200 groups was better maintained total and class
213
A+B motility after thawing compared to control. It is of interest to note that Mn+CAT 400
214
treatment yield higher post-thaw motility compared to control and other treatments (p
