Accepted Manuscript Use of Aloe Vera-based extender for chilling and freezing collared peccary (Pecari tajacu) semen A.L.P. Souza, G.L. Lima, G.C.X. Peixoto, A.M. Silva, M.F. Oliveira, A.R. Silva PII:
S0093-691X(16)00008-X
DOI:
10.1016/j.theriogenology.2016.01.007
Reference:
THE 13475
To appear in:
Theriogenology
Received Date: 14 January 2015 Revised Date:
4 January 2016
Accepted Date: 4 January 2016
Please cite this article as: Souza A, Lima G, Peixoto G, Silva A, Oliveira M, Silva A, Use of Aloe Verabased extender for chilling and freezing collared peccary (Pecari tajacu) semen, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.01.007. 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.
1
ACCEPTED MANUSCRIPT 1
Use of Aloe Vera-based extender for chilling and freezing collared peccary (Pecari
2
tajacu) semen
3
ALP Souzaa, GL Limaa, GCX Peixotoa, AM Silvaa, MF Oliveiraa, AR Silvaa*
4 5
a
6
do Semi-Árido (UFERSA), Mossoró, RN, Brazil
7
*Corresponding author. Tel.: 55 84 33178374. E-mail address: [email protected]
8
(AR Silva).
SC
9 Abstract
M AN U
10
RI PT
Laboratory of Animal Germplasm Conservation - LCGA, Universidade Federal Rural
As an alternative for the conservation of collared peccary semen, this research
12
aims at evaluating the use of Aloe vera (AV) extract as a cryoprotectant for semen
13
chilling and freezing. Five ejaculates were divided in two aliquots that were diluted in
14
Tris plus egg yolk—EY (20%) or AV extract (20%)—and chilled at 5 ºC. In both
15
treatments, an adequate semen conservation was achieved and values closer to 40%
16
motile sperm with viability and osmotic response ranging from 20 to 40%, and normal
17
morphology of 80% were found after 36 h of storage. Moreover, 12 other ejaculates
18
were diluted in Tris plus EY (20%) or AV extract (5, 10, or 20%) and glycerol (3%).
19
Samples were frozen in liquid nitrogen and thawed after one week. After thawing, all
20
the treatments containing EY or AV provided similar values for sperm morphology,
21
viability, osmotic response, membrane integrity, sperm motility, ALH, BCF, and rapid,
22
low, and static subpopulations, but the highest values for STR and the lowest values for
23
VCL were found using 20% AV (P < 0.05). In conclusion, we demonstrate that Aloe
24
vera extract at a 20% concentration could be used as an alternative substitute to egg
AC C
EP
TE D
11
2
ACCEPTED MANUSCRIPT 25
yolk in the formulation of Tris extenders for collared peccaries’ semen chilling or
26
freezing.
27 28
Keywords: Tayassu tajacu; sperm chilling; sperm freezing; Aloe vera; cryoprotectant.
30
RI PT
29 1. Introduction
31
With the advent of semen conservation, the possibility of germplasm storage
33
enables posterior manipulation and use of assisted techniques such as in vitro
34
fertilization or artificial insemination [1]. Despite this, the decrease in temperature rates
35
during semen chilling and freezing procedures is critical to maintain the sperm quality
36
after rewarming, with the sperm sensitivity varying depending on the species [2].
M AN U
SC
32
To reduce the cryoinjuries, several substances have been used as cryoprotectants,
38
and the hen’s egg yolk has showed satisfactory results for both domestic [3, 4, 5] and
39
wild species [6, 7, 8]. Egg yolk presents beneficial effects on sperm cryopreservation as
40
a protectant of the plasma membrane and acrosome against temperature-related injury
41
[3]. It is believed that the phospholipids, cholesterol, and low-density lipoproteins
42
present in egg yolk may be factors that provide protection to the sperm against cold
43
shock during the freeze–thaw process [4]. However, because it is a product of animal
44
origin, there is the possibility of bacterial contamination [9], which contributes to the
45
difficulty of exchanging semen among different regions or countries [5]. Furthermore,
46
egg yolk can interfere with microscopic observations or biochemical assays, as it
47
contains granular material of the same size and shape as the sperm [10].
AC C
EP
TE D
37
48
Several authors search for an alternative medium that is free of animal products
49
for semen conservation. In this sense, the Aloe vera appears as an alternative from
3
ACCEPTED MANUSCRIPT
vegetal origin for this purpose, as it constitutes some substances that can act as the
51
conventional cryoprotectants [11]. Along the years, Aloe vera has been used in the
52
cosmetic and toiletry industry due to its curative and therapeutic properties [12]. In
53
addition, several studies show the positive action of Aloe vera extract on
54
spermatogenesis [13]. The use of Aloe vera extract for an effective semen conservation,
55
however, is restricted to the caprine semen chilling [14] and ovine semen freezing and
56
artificial insemination [15]. Therefore, it presents a potential as a good alternative for
57
sperm cryoprotection and should be better explored and extrapolated to other species,
58
including wild animals.
SC
RI PT
50
The collared peccary (Pecari tajacu) is a wild species whose meat and pelt has
60
great international demand, but the number of individuals in some of its natural habitats,
61
such as the Caatinga and Atlantic Forest, is critically decreasing due to poaching, which
62
has necessitated them to be bred under captivity [16]. The development of assisted
63
techniques that make the conservation of collared peccary viable besides the
64
implementation of germplasm banks is very important for their multiplication, which
65
has generated some studies on its semen conservation. It was demonstrated that their
66
semen could be chilled at 17 ºC for only 24 h by using the Beltsville Thawing Solution
67
as extender [17]. At freezing, however, several studies demonstrated that the sperm
68
quality could be efficiently preserved using Tris [18] or coconut water extenders [6] and
69
both plus egg yolk. In order to present an alternative cryoprotectant for the conservation
70
of its semen, this research aims at evaluating the use of Aloe vera extract as a
71
cryoprotectant for the peccaries’ semen chilling and freezing.
AC C
EP
TE D
M AN U
59
72 73 74
2. Materials and methods
4
ACCEPTED MANUSCRIPT
The ethics committee of the Universidade Federal Rural do Semi-Árido—
76
UFERSA has approved the experimental protocols and the animal care procedures
77
adopted (process no. 23091.0253/114). The reagents used in this study were obtained
78
from Sigma-Aldrich (St. Louis, MO, United States), except when cited.
79 80
2.1. Animals and semen collection
81
RI PT
75
Samples were obtained from 12 sexually mature male collared peccaries with an
83
average (SD) age and weight of 40.7 ± 1.6 mo and 22.5 ± 2.8 kg, respectively. Five of
84
the individuals served as semen donors for the semen chilling and freezing, being
85
subjected to two electroejaculation sessions on a 30-day interval. The other seven
86
animals were used as semen donors for only the semen freezing.
M AN U
SC
82
The animals belonged to the Center of Multiplication of Wild Animals,
88
UFERSA, located in the northeast of Brazil (Mossoró, RN, Brazil; 5 °10=S, 37 °10=W).
89
The climate is typically semiarid, with an average annual temperature of 27 °C. The
90
animals were isolated from the females at 6 months before the commencement of the
91
study and maintained under a 12-h natural photoperiod. They were maintained outdoors
92
in groups of six in paddocks (20 m to 3 m) with a covered area of (3 to 3 m) and fed
93
sow food and fruits (with water available ad libitum).
EP
AC C
94
TE D
87
Animals were fasted 12 h before the experiments began. They were then
95
physically restrained using a hand net and anesthetized using intravenous administration
96
of propofol (Propovan®, Cristalia, Fortaleza, Brazil), which was given as a bolus (5
97
mg/kg) [19]. They were kept in lateral recumbency, and the semen was collected with
98
an electroejaculator (Autojac®, Neovet, Campinas, SP, Brazil) that was connected to a
99
12 V source. The stimulatory cycle comprised 10 stimuli in each voltage, starting from
5
ACCEPTED MANUSCRIPT
5 V, followed by a voltage increase in steps from 1 V to 12 V. Each electrical stimulus
101
lasted 3 s, with intermittent breaks of 2 s. The stimuli cycle was maintained for a 10 min
102
duration from the beginning of the procedure. The electroejaculator probe was 15 x 1.3
103
cm, and it was inserted 12 cm into the rectum. Semen was collected into plastic tubes
104
and immediately evaluated [18].
RI PT
100
105 106
2.2. Experimental design
SC
107
The execution of the study was divided into two steps. At first, we verified the
109
effect of Aloe vera (AV) extract on the short-term preservation at 5 ºC of collared
110
peccaries’ semen by comparing it with the egg yolk (EY), both at a 20% concentration
111
added to the Tris extender. Afterward, we verified the effect of AV extract at different
112
concentrations (5, 10, and 20%) added to the Tris extender for the collared peccaries’
113
semen freezing. In both experiments, we used the Tris-based extender formulation
114
previously described for collared peccaries [18].
TE D
M AN U
108
115
2.3. Extraction of Aloe Vera
EP
116 117
The plant Aloe vera was cultured in a sandy clay soil type at the UFERSA
AC C
118 119
campus, located in the northeast of Brazil (Mossoró, RN, Brazil; 5 °10=S, 37 °10=W),
120
under a typical semiarid climate, with an average annual temperature of 27 °C. In order
121
to obtain the crude extract of Aloe vera, we extracted the parenchyma of its leaves as a
122
colorless gel. It was filtered through a sieve; then, it was packed in a glass container till
123
its use.
124
6
ACCEPTED MANUSCRIPT 125
2.4. Semen chilling
126 Semen samples that were collected after electroejaculation of 5 individuals were
128
divided into two aliquots. The first was diluted in Tris plus EY (20%), and the other was
129
diluted in Tris plus AV extract (20%), reaching a concentration of 100 x 106 sperm/mL.
130
Samples were evaluated immediately after dilution (0 h) for sperm total motility,
131
viability, osmotic response, and morphology. Then, samples were stored in the water
132
jacket at 27°C and equilibrated for 40 min to reach 15°C in a biological incubator
133
(Quimis, Diadema, SP, Brazil). Furthermore, the incubator was adjusted to establish a
134
temperature at 5 ºC for 30 min. Samples were rewarmed at 37 ºC and reevaluated every
135
12 h till 36 h.
M AN U
SC
RI PT
127
136 137
2.5.Semen freezing
TE D
138
Semen was frozen according to a methodology previously developed for
140
peccaries [4]. Samples derived from 12 individuals were divided into 4 aliquots. These
141
were diluted in Tris extender supplemented with EY (20%), as a control group, or AV
142
extract at 5, 10, or 20%. All the dilutions were conducted at room temperature (27 °C),
143
and the samples were then equilibrated for 40 min to reach 15°C in a biological
144
incubator (Quimis, Diadema, SP, Brazil). Furthermore, the incubator was adjusted to
145
establish a temperature at 5 ºC for 30 min. At that point, the sample was added to the
146
extender with 6% glycerol (also at 5ºC), which resulted in a final concentration of 3%
147
glycerol in the extender and 100 x 106 sperm /mL. Finally, the samples were packed in
148
0.25-mL plastic straws (IMV Technologies; L’Aigle, France) that were placed
149
horizontally in an insulated box for 5 min, at 5 cm above the nitrogen (N2) vapors, and
AC C
EP
139
7
ACCEPTED MANUSCRIPT 150
then plunged into liquid N2 for storage at −196 ºC, following a fast freezing rate at −40
151
ºC/min. After 1 week, frozen straws were thawed by immersion in a water bath at 37°C
152
for 1 min [6]. Samples were evaluated for sperm motility parameters, viability,
153
morphology, osmotic response, and membrane integrity.
155
RI PT
154 2.6. Semen evaluation
156
Fresh ejaculates were evaluated for aspect, color, and volume. The sperm
158
concentration was determined using a Neubauer counting chamber [20]. After
159
electroejaculation, rewarming, and freezing–thawing, samples were evaluated for sperm
160
morphology, viability, and osmotic response. Bengal Rose–stained smears were
161
prepared to evaluate sperm morphology using light microscopy (1000×), counting 200
162
cells per slide [21]. Sperm viability was determined by analyzing a slide stained with
163
bromophenol blue under light microscopy (400×), counting 200 cells per slide [22].
164
Functional integrity of the sperm membrane was verified by evaluating the sperm
165
osmotic response under a hypo-osmotic swelling (HOS) test, using distilled water (0
166
mOsm/L) as the hypo-osmotic solution. A total of 200 sperms were examined, and
167
those with a swollen coiled tail were considered as presenting a functional membrane.
168
[23]. For the evaluation of the plasma membrane integrity in frozen–thawed samples,
169
we used a fluorescent solution that contained fluorophores diacetate 6-carboxy-
170
fluorescein (C-FDA) and propidium iodide (PI). Samples were incubated for 10 min and
171
evaluated by epifluorescence microscopy (Episcopic Fluorescent attachment “EFA”
172
Halogen Lamp Set, Leica, Kista, Sweden). For each sample stained with CFDA/PI, 200
173
sperms were counted and classified as having or not having an intact membrane (based
174
on color) [24].
AC C
EP
TE D
M AN U
SC
157
8
ACCEPTED MANUSCRIPT 175 176
2.7. Computer-assisted sperm assessment
177 Sperm motility parameters were analyzed by computer-assisted sperm
179
assessment (IVOS 7.4G, Hamilton-Thorne ResearchTM, Beverly, MA, USA) in fresh,
180
rewarmed, and frozen–thawed semen. The settings of the instrument were validated for
181
collared peccaries semen before analysis, including temperature 37 ºC; 60 frames/s;
182
minimum contrast, 45; straightness threshold, 30%; low-velocity average pathway
183
(VAP) cutoff, 10 µ/s; and medium VAP cutoff, 30 µ/s. Five independent and
184
nonconsecutive microscopic fields were randomly selected and scanned. The following
185
endpoints were analyzed: number of counted cells, total motility (%), VAP (µm/s),
186
velocity straight line (VSL; µm/s), curvilinear velocity (VCL; µm/s), amplitude of
187
lateral head (ALH; µm), beat cross frequency (BCF; Hz), straightness (STR;%), and
188
linearity (LIN;%). According to low VAP cutoff (LVV) and medium VAP cutoff
189
(MVV), the overall sperm population was subdivided into four categories: rapid, with
190
VAP > MVV; medium, with (LVV < VAP 0.05).
ACCEPTED MANUSCRIPT
Table 2: Values (means ± SEM)* for sperm morphology, viability, osmotic response and plasma membrane integrity in collared peccaries (Pecari tajacu) frozen-thawed semen (n=12) diluted in Tris plus egg yolk or Aloe vera extract in different concentrations. AV 20%
AV 10%
AV 5%
Normal morphology (%)
76.6 ± 1.4
66.9 ± 4.2
67.6 ± 2.7
72.2 ± 3.2
Viability (%)
25.1 ± 2.9
23.3 ± 5.5
21 ± 5.5
20.7 ± 2.4
Osmotic response (%)
34.8 ± 3.7
31.4 ± 5.6
30.8 ± 4.9
28.7 ± 4.8
Membrane integrity (%)
27.5 ± 3.1
27 ± 3.7
27.6 ± 4.2
24.7 ± 3.6
AC C
EP
TE D
M AN U
SC
RI PT
EY 20%
ACCEPTED MANUSCRIPT
Table 3: Values (means ± SEM) for sperm kinetic motility parameters provided by computerized analysis in collared peccaries (Pecari tajacu) frozen-thawed semen (n=12) diluted in Tris plus egg yolk (EY) or Aloe vera (AV) at different concentrations (20, 10, 5%). AV 20%
AV 10%
Total motility (%)
27.1 ± 5.03
46.4 ± 3.9
40.5 ± 7.6
35.7 ± 7
Velocity straight line (µm/s)
24.3 ± 3.6a
20.5 ± 4.7ab
14.9 ± 1.5b
18.4 ± 2.3ab
Velocity average curvilinear (µm/s)
86.8 ± 7.9a
64.7 ± 8.9b
67.3 ± 3.1ab
69 ± 6.3ab
Velocity average pathway (µm/s)
37.8 ± 4.8a
28.6 ± 5.0ab
24.8 ± 1.9b
28.2 ± 2.8ab
Linearity (%)
32.1 ± 2.1ab
33.7 ± 2.4a
26 ± 1.2c
30.5 ± 1.7b
Straightness (%)
64.2 ± 1.9b
71.6 ± 2.6a
60.5 ± 2.1b
64.5 ± 2.8b
Amplitude lateral head (µm)
6.4 ± 0.6
6.6 ± 0.3
5.8 ± 0.3
6.6 ± 0.3
Beat cross frequency (Hz)
34.8 ± 2.0
35.1 ± 1.7
38.7 ± 1.4
37.4 ± 1.4
Rapid (%)
10.1 ± 5.3
6.4 ± 0.7
5.3 ± 1.3
10.2 ± 4.9
Medium (%)
9.7 ± 2.8b
30.4 ± 3.9a
27.7 ± 6.3a
18.2 ± 5.6ab
Slow (%)
7.3 ± 1.9
9.6 ± 1.5
7.5 ± 1.4
7.3 ± 1.7
53.3 ± 4.1b
59.4 ± 8.2
60.3 ± 9.2
Static (%)
SC
M AN U
73 ± 6.5
EP
Superscript low case letters indicate significant differences among treatments (P
S0093-691X(16)00008-X
DOI:
10.1016/j.theriogenology.2016.01.007
Reference:
THE 13475
To appear in:
Theriogenology
Received Date: 14 January 2015 Revised Date:
4 January 2016
Accepted Date: 4 January 2016
Please cite this article as: Souza A, Lima G, Peixoto G, Silva A, Oliveira M, Silva A, Use of Aloe Verabased extender for chilling and freezing collared peccary (Pecari tajacu) semen, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.01.007. 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.
1
ACCEPTED MANUSCRIPT 1
Use of Aloe Vera-based extender for chilling and freezing collared peccary (Pecari
2
tajacu) semen
3
ALP Souzaa, GL Limaa, GCX Peixotoa, AM Silvaa, MF Oliveiraa, AR Silvaa*
4 5
a
6
do Semi-Árido (UFERSA), Mossoró, RN, Brazil
7
*Corresponding author. Tel.: 55 84 33178374. E-mail address: [email protected]
8
(AR Silva).
SC
9 Abstract
M AN U
10
RI PT
Laboratory of Animal Germplasm Conservation - LCGA, Universidade Federal Rural
As an alternative for the conservation of collared peccary semen, this research
12
aims at evaluating the use of Aloe vera (AV) extract as a cryoprotectant for semen
13
chilling and freezing. Five ejaculates were divided in two aliquots that were diluted in
14
Tris plus egg yolk—EY (20%) or AV extract (20%)—and chilled at 5 ºC. In both
15
treatments, an adequate semen conservation was achieved and values closer to 40%
16
motile sperm with viability and osmotic response ranging from 20 to 40%, and normal
17
morphology of 80% were found after 36 h of storage. Moreover, 12 other ejaculates
18
were diluted in Tris plus EY (20%) or AV extract (5, 10, or 20%) and glycerol (3%).
19
Samples were frozen in liquid nitrogen and thawed after one week. After thawing, all
20
the treatments containing EY or AV provided similar values for sperm morphology,
21
viability, osmotic response, membrane integrity, sperm motility, ALH, BCF, and rapid,
22
low, and static subpopulations, but the highest values for STR and the lowest values for
23
VCL were found using 20% AV (P < 0.05). In conclusion, we demonstrate that Aloe
24
vera extract at a 20% concentration could be used as an alternative substitute to egg
AC C
EP
TE D
11
2
ACCEPTED MANUSCRIPT 25
yolk in the formulation of Tris extenders for collared peccaries’ semen chilling or
26
freezing.
27 28
Keywords: Tayassu tajacu; sperm chilling; sperm freezing; Aloe vera; cryoprotectant.
30
RI PT
29 1. Introduction
31
With the advent of semen conservation, the possibility of germplasm storage
33
enables posterior manipulation and use of assisted techniques such as in vitro
34
fertilization or artificial insemination [1]. Despite this, the decrease in temperature rates
35
during semen chilling and freezing procedures is critical to maintain the sperm quality
36
after rewarming, with the sperm sensitivity varying depending on the species [2].
M AN U
SC
32
To reduce the cryoinjuries, several substances have been used as cryoprotectants,
38
and the hen’s egg yolk has showed satisfactory results for both domestic [3, 4, 5] and
39
wild species [6, 7, 8]. Egg yolk presents beneficial effects on sperm cryopreservation as
40
a protectant of the plasma membrane and acrosome against temperature-related injury
41
[3]. It is believed that the phospholipids, cholesterol, and low-density lipoproteins
42
present in egg yolk may be factors that provide protection to the sperm against cold
43
shock during the freeze–thaw process [4]. However, because it is a product of animal
44
origin, there is the possibility of bacterial contamination [9], which contributes to the
45
difficulty of exchanging semen among different regions or countries [5]. Furthermore,
46
egg yolk can interfere with microscopic observations or biochemical assays, as it
47
contains granular material of the same size and shape as the sperm [10].
AC C
EP
TE D
37
48
Several authors search for an alternative medium that is free of animal products
49
for semen conservation. In this sense, the Aloe vera appears as an alternative from
3
ACCEPTED MANUSCRIPT
vegetal origin for this purpose, as it constitutes some substances that can act as the
51
conventional cryoprotectants [11]. Along the years, Aloe vera has been used in the
52
cosmetic and toiletry industry due to its curative and therapeutic properties [12]. In
53
addition, several studies show the positive action of Aloe vera extract on
54
spermatogenesis [13]. The use of Aloe vera extract for an effective semen conservation,
55
however, is restricted to the caprine semen chilling [14] and ovine semen freezing and
56
artificial insemination [15]. Therefore, it presents a potential as a good alternative for
57
sperm cryoprotection and should be better explored and extrapolated to other species,
58
including wild animals.
SC
RI PT
50
The collared peccary (Pecari tajacu) is a wild species whose meat and pelt has
60
great international demand, but the number of individuals in some of its natural habitats,
61
such as the Caatinga and Atlantic Forest, is critically decreasing due to poaching, which
62
has necessitated them to be bred under captivity [16]. The development of assisted
63
techniques that make the conservation of collared peccary viable besides the
64
implementation of germplasm banks is very important for their multiplication, which
65
has generated some studies on its semen conservation. It was demonstrated that their
66
semen could be chilled at 17 ºC for only 24 h by using the Beltsville Thawing Solution
67
as extender [17]. At freezing, however, several studies demonstrated that the sperm
68
quality could be efficiently preserved using Tris [18] or coconut water extenders [6] and
69
both plus egg yolk. In order to present an alternative cryoprotectant for the conservation
70
of its semen, this research aims at evaluating the use of Aloe vera extract as a
71
cryoprotectant for the peccaries’ semen chilling and freezing.
AC C
EP
TE D
M AN U
59
72 73 74
2. Materials and methods
4
ACCEPTED MANUSCRIPT
The ethics committee of the Universidade Federal Rural do Semi-Árido—
76
UFERSA has approved the experimental protocols and the animal care procedures
77
adopted (process no. 23091.0253/114). The reagents used in this study were obtained
78
from Sigma-Aldrich (St. Louis, MO, United States), except when cited.
79 80
2.1. Animals and semen collection
81
RI PT
75
Samples were obtained from 12 sexually mature male collared peccaries with an
83
average (SD) age and weight of 40.7 ± 1.6 mo and 22.5 ± 2.8 kg, respectively. Five of
84
the individuals served as semen donors for the semen chilling and freezing, being
85
subjected to two electroejaculation sessions on a 30-day interval. The other seven
86
animals were used as semen donors for only the semen freezing.
M AN U
SC
82
The animals belonged to the Center of Multiplication of Wild Animals,
88
UFERSA, located in the northeast of Brazil (Mossoró, RN, Brazil; 5 °10=S, 37 °10=W).
89
The climate is typically semiarid, with an average annual temperature of 27 °C. The
90
animals were isolated from the females at 6 months before the commencement of the
91
study and maintained under a 12-h natural photoperiod. They were maintained outdoors
92
in groups of six in paddocks (20 m to 3 m) with a covered area of (3 to 3 m) and fed
93
sow food and fruits (with water available ad libitum).
EP
AC C
94
TE D
87
Animals were fasted 12 h before the experiments began. They were then
95
physically restrained using a hand net and anesthetized using intravenous administration
96
of propofol (Propovan®, Cristalia, Fortaleza, Brazil), which was given as a bolus (5
97
mg/kg) [19]. They were kept in lateral recumbency, and the semen was collected with
98
an electroejaculator (Autojac®, Neovet, Campinas, SP, Brazil) that was connected to a
99
12 V source. The stimulatory cycle comprised 10 stimuli in each voltage, starting from
5
ACCEPTED MANUSCRIPT
5 V, followed by a voltage increase in steps from 1 V to 12 V. Each electrical stimulus
101
lasted 3 s, with intermittent breaks of 2 s. The stimuli cycle was maintained for a 10 min
102
duration from the beginning of the procedure. The electroejaculator probe was 15 x 1.3
103
cm, and it was inserted 12 cm into the rectum. Semen was collected into plastic tubes
104
and immediately evaluated [18].
RI PT
100
105 106
2.2. Experimental design
SC
107
The execution of the study was divided into two steps. At first, we verified the
109
effect of Aloe vera (AV) extract on the short-term preservation at 5 ºC of collared
110
peccaries’ semen by comparing it with the egg yolk (EY), both at a 20% concentration
111
added to the Tris extender. Afterward, we verified the effect of AV extract at different
112
concentrations (5, 10, and 20%) added to the Tris extender for the collared peccaries’
113
semen freezing. In both experiments, we used the Tris-based extender formulation
114
previously described for collared peccaries [18].
TE D
M AN U
108
115
2.3. Extraction of Aloe Vera
EP
116 117
The plant Aloe vera was cultured in a sandy clay soil type at the UFERSA
AC C
118 119
campus, located in the northeast of Brazil (Mossoró, RN, Brazil; 5 °10=S, 37 °10=W),
120
under a typical semiarid climate, with an average annual temperature of 27 °C. In order
121
to obtain the crude extract of Aloe vera, we extracted the parenchyma of its leaves as a
122
colorless gel. It was filtered through a sieve; then, it was packed in a glass container till
123
its use.
124
6
ACCEPTED MANUSCRIPT 125
2.4. Semen chilling
126 Semen samples that were collected after electroejaculation of 5 individuals were
128
divided into two aliquots. The first was diluted in Tris plus EY (20%), and the other was
129
diluted in Tris plus AV extract (20%), reaching a concentration of 100 x 106 sperm/mL.
130
Samples were evaluated immediately after dilution (0 h) for sperm total motility,
131
viability, osmotic response, and morphology. Then, samples were stored in the water
132
jacket at 27°C and equilibrated for 40 min to reach 15°C in a biological incubator
133
(Quimis, Diadema, SP, Brazil). Furthermore, the incubator was adjusted to establish a
134
temperature at 5 ºC for 30 min. Samples were rewarmed at 37 ºC and reevaluated every
135
12 h till 36 h.
M AN U
SC
RI PT
127
136 137
2.5.Semen freezing
TE D
138
Semen was frozen according to a methodology previously developed for
140
peccaries [4]. Samples derived from 12 individuals were divided into 4 aliquots. These
141
were diluted in Tris extender supplemented with EY (20%), as a control group, or AV
142
extract at 5, 10, or 20%. All the dilutions were conducted at room temperature (27 °C),
143
and the samples were then equilibrated for 40 min to reach 15°C in a biological
144
incubator (Quimis, Diadema, SP, Brazil). Furthermore, the incubator was adjusted to
145
establish a temperature at 5 ºC for 30 min. At that point, the sample was added to the
146
extender with 6% glycerol (also at 5ºC), which resulted in a final concentration of 3%
147
glycerol in the extender and 100 x 106 sperm /mL. Finally, the samples were packed in
148
0.25-mL plastic straws (IMV Technologies; L’Aigle, France) that were placed
149
horizontally in an insulated box for 5 min, at 5 cm above the nitrogen (N2) vapors, and
AC C
EP
139
7
ACCEPTED MANUSCRIPT 150
then plunged into liquid N2 for storage at −196 ºC, following a fast freezing rate at −40
151
ºC/min. After 1 week, frozen straws were thawed by immersion in a water bath at 37°C
152
for 1 min [6]. Samples were evaluated for sperm motility parameters, viability,
153
morphology, osmotic response, and membrane integrity.
155
RI PT
154 2.6. Semen evaluation
156
Fresh ejaculates were evaluated for aspect, color, and volume. The sperm
158
concentration was determined using a Neubauer counting chamber [20]. After
159
electroejaculation, rewarming, and freezing–thawing, samples were evaluated for sperm
160
morphology, viability, and osmotic response. Bengal Rose–stained smears were
161
prepared to evaluate sperm morphology using light microscopy (1000×), counting 200
162
cells per slide [21]. Sperm viability was determined by analyzing a slide stained with
163
bromophenol blue under light microscopy (400×), counting 200 cells per slide [22].
164
Functional integrity of the sperm membrane was verified by evaluating the sperm
165
osmotic response under a hypo-osmotic swelling (HOS) test, using distilled water (0
166
mOsm/L) as the hypo-osmotic solution. A total of 200 sperms were examined, and
167
those with a swollen coiled tail were considered as presenting a functional membrane.
168
[23]. For the evaluation of the plasma membrane integrity in frozen–thawed samples,
169
we used a fluorescent solution that contained fluorophores diacetate 6-carboxy-
170
fluorescein (C-FDA) and propidium iodide (PI). Samples were incubated for 10 min and
171
evaluated by epifluorescence microscopy (Episcopic Fluorescent attachment “EFA”
172
Halogen Lamp Set, Leica, Kista, Sweden). For each sample stained with CFDA/PI, 200
173
sperms were counted and classified as having or not having an intact membrane (based
174
on color) [24].
AC C
EP
TE D
M AN U
SC
157
8
ACCEPTED MANUSCRIPT 175 176
2.7. Computer-assisted sperm assessment
177 Sperm motility parameters were analyzed by computer-assisted sperm
179
assessment (IVOS 7.4G, Hamilton-Thorne ResearchTM, Beverly, MA, USA) in fresh,
180
rewarmed, and frozen–thawed semen. The settings of the instrument were validated for
181
collared peccaries semen before analysis, including temperature 37 ºC; 60 frames/s;
182
minimum contrast, 45; straightness threshold, 30%; low-velocity average pathway
183
(VAP) cutoff, 10 µ/s; and medium VAP cutoff, 30 µ/s. Five independent and
184
nonconsecutive microscopic fields were randomly selected and scanned. The following
185
endpoints were analyzed: number of counted cells, total motility (%), VAP (µm/s),
186
velocity straight line (VSL; µm/s), curvilinear velocity (VCL; µm/s), amplitude of
187
lateral head (ALH; µm), beat cross frequency (BCF; Hz), straightness (STR;%), and
188
linearity (LIN;%). According to low VAP cutoff (LVV) and medium VAP cutoff
189
(MVV), the overall sperm population was subdivided into four categories: rapid, with
190
VAP > MVV; medium, with (LVV < VAP 0.05).
ACCEPTED MANUSCRIPT
Table 2: Values (means ± SEM)* for sperm morphology, viability, osmotic response and plasma membrane integrity in collared peccaries (Pecari tajacu) frozen-thawed semen (n=12) diluted in Tris plus egg yolk or Aloe vera extract in different concentrations. AV 20%
AV 10%
AV 5%
Normal morphology (%)
76.6 ± 1.4
66.9 ± 4.2
67.6 ± 2.7
72.2 ± 3.2
Viability (%)
25.1 ± 2.9
23.3 ± 5.5
21 ± 5.5
20.7 ± 2.4
Osmotic response (%)
34.8 ± 3.7
31.4 ± 5.6
30.8 ± 4.9
28.7 ± 4.8
Membrane integrity (%)
27.5 ± 3.1
27 ± 3.7
27.6 ± 4.2
24.7 ± 3.6
AC C
EP
TE D
M AN U
SC
RI PT
EY 20%
ACCEPTED MANUSCRIPT
Table 3: Values (means ± SEM) for sperm kinetic motility parameters provided by computerized analysis in collared peccaries (Pecari tajacu) frozen-thawed semen (n=12) diluted in Tris plus egg yolk (EY) or Aloe vera (AV) at different concentrations (20, 10, 5%). AV 20%
AV 10%
Total motility (%)
27.1 ± 5.03
46.4 ± 3.9
40.5 ± 7.6
35.7 ± 7
Velocity straight line (µm/s)
24.3 ± 3.6a
20.5 ± 4.7ab
14.9 ± 1.5b
18.4 ± 2.3ab
Velocity average curvilinear (µm/s)
86.8 ± 7.9a
64.7 ± 8.9b
67.3 ± 3.1ab
69 ± 6.3ab
Velocity average pathway (µm/s)
37.8 ± 4.8a
28.6 ± 5.0ab
24.8 ± 1.9b
28.2 ± 2.8ab
Linearity (%)
32.1 ± 2.1ab
33.7 ± 2.4a
26 ± 1.2c
30.5 ± 1.7b
Straightness (%)
64.2 ± 1.9b
71.6 ± 2.6a
60.5 ± 2.1b
64.5 ± 2.8b
Amplitude lateral head (µm)
6.4 ± 0.6
6.6 ± 0.3
5.8 ± 0.3
6.6 ± 0.3
Beat cross frequency (Hz)
34.8 ± 2.0
35.1 ± 1.7
38.7 ± 1.4
37.4 ± 1.4
Rapid (%)
10.1 ± 5.3
6.4 ± 0.7
5.3 ± 1.3
10.2 ± 4.9
Medium (%)
9.7 ± 2.8b
30.4 ± 3.9a
27.7 ± 6.3a
18.2 ± 5.6ab
Slow (%)
7.3 ± 1.9
9.6 ± 1.5
7.5 ± 1.4
7.3 ± 1.7
53.3 ± 4.1b
59.4 ± 8.2
60.3 ± 9.2
Static (%)
SC
M AN U
73 ± 6.5
EP
Superscript low case letters indicate significant differences among treatments (P
