oxidative enzymes

Effects of deer velvet extract from Formosan sika deer on the
embryonic development and anti-oxidative enzymes mRNA expression
in mouse embryos
Shih-Lin Cheng a
, Yi-Lin Lai b
, Ming-Che Lee b
, Perng-Chih Shen b
,
Shyh-Shyan Liu c
, Bing-Tsan Liu b,n
Q1
a
Graduate Institute of Bioresources, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, ROC
b
Department of Animal Science, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, ROC
c
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, ROC
a r t i c l e i n f o
Article history:
Received 22 October 2013
Received in revised form
19 March 2014
Accepted 4 April 2014
Keywords:
Formosan sika deer velvet
Reactive oxygen species
Mouse embryo
Antioxidation
Anti-oxidative enzymes
a b s t r a c t
Ethnopharmacrological relevance: The deer velvet or its extracts has been widelyQ2 used in clinic. It has
been used in promoting reproductive performances and treating of oxidation and aging process. The aim
of this study is to investigate the effects of velvet extract from Formosan sika deer (Formosan sika deer;
Cervus nippon taiouanus, FSD) velvet on mouse embryonic development and anti-oxidant ability in vitro.
Materials and methods: Mouse 4-cells embryos were divided into 16 groups for 72 h in vitro incubation.
The embryonic development stages and morphology were evaluated every 12 h in experimental period.
The quantitative real time PCR was used to measure the CuZn-SOD, GPx and CAT mRNA expression of the
blastocysts.
Results: The 4-cells embryos of hydrogen peroxide (HP) groups did not continue developing after oxidant
stress challenged. The blastocyst developmental rate (90.0–90.4%, P>0.05) and normal morphological
rate (84.4–85.1%, P>0.05) of the 1% and 2% DV extract groups were similar to those in the control group
(90.7% and 88.8%, respectively). The embryos challenged by HP (5, 10 and 25 μM) and subsequently
incubated in mHTF medium with 1% and 2% of deer velvet (DV) extracts were able to continue
development; the blastocyst developmental rate of these groups were similar to that in the control
group. The relative mRNA expression of the focused anti-oxidative enzymes in the mouse embryos did
not signiﬁcantly differ among the designed DV treatment groups (P>0.05).
Conclusion: The FSD velvet extract in adequate concentration could promote anti-oxidative enzymes
mRNA expression followed the challenge of hydrogen peroxide, relieve the mouse embryo under
oxidative stress, and maintain the blastocyst developmental ability in vitro.
& 2014 Published by Elsevier Ireland Ltd.
1. Introduction
There are many types of free radicals in a living system. Such
free radicals could be produced during metabolism processing
in vivo, although most molecules in vivo are non-radical. Reactive
oxygen species (ROS) including hydrogen peroxide (H2O2, HP) and
free radicals (such as superoxide anions and hydroxyl radicals)
could be involved in cell damaging. Oxidative damage could occur
in all aerobes including plant, animals and aerobic bacteria while
being exposed to ROS concentrations higher than normal and
low antioxidant defenses system (Halliwell, 2006). In addition, the
research showed a direct relationship between high HP concen-
tration and elevated fragmentation grade or apoptosis in mouse
embryos (Yang et al., 1998). High HP level could induce zona
pellucida dissolution, cytoplasmic shrink, loss of ooplasm micro-
tubule dynamics (OMD) and cortical granule functions in mouse
embryo (Goud et al., 2008; Trimarchi et al., 2000). Furthermore,
HP could induce the increase in abnormal embryo occurring rate,
resulting in embryo necrosis, embryo implantation failure, and
endometriosis (Guérin et al., 2001). Several antioxidant enzymes
could protect oocyte and embryos against peroxidative damage by
superoxide dismutase (SOD), catalase (CAT) and glutathione perox-
idase (GPx) in vivo (Cebral et al., 2007). The correlation had been
observed among mRNA, protein and enzyme activity levels (Forsberg
et al., 1996), which indicated that the antioxidant enzymes were
regulated primarily at the pre-translational level. There are many
kinds of method for evaluation of internal antioxidant enzymes gene
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/jep
Journal of Ethnopharmacology
http://dx.doi.org/10.1016/j.jep.2014.04.006
0378-8741/& 2014 Published by Elsevier Ireland Ltd.
n
Correspondence to: No.1, Shuehfu Rd., Neipu, Pingtung 91201, Taiwan, ROC.
Tel.: þ886 87703202x6199; fax: þ886 87740148.
E-mail address: ﬂea957@gmail.com (B.-T. Liu).
Please cite this article as: Cheng, S.-L., et al., Effects of deer velvet extract from Formosan sika deer on the embryonic development and
anti-oxidative enzymes mRNA expression in mouse embryos. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.
jep.2014.04.006i
Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

expression such as real time RT-PCR, western blotting, immunohis-
tochemistry and fluorescence in situ hybridization in cell or tissue. And
yet, among them only real time RT-PCR is an efficient and precise
quantitative method for estimating the transcript levels of genes
expression in mouse oocytes or embryos (Jeong et al., 2005).
Deer velvet (DV) has been used in traditional Chinese medicine
(TCM) or health foods for over 2000 years (Zhou et al., 2009), and
it has been recorded in traditional Chinese medicine classic
written by Li Shi-Zhen about 500 years ago (Tseng et al., 2012).
TCM herbalists believe that DV is able to nourish the kidney Yin-
tonify, invigorate the spleen, strengthen bones and muscles, and
promote blood circulation etc. (Zhou et al., 2009). Various carbo-
hydrates, amino acids, lipids, sterols and minerals can be obtained
from DV, especially in the upper section (Bubenik et al., 2005; Li et
al., 2007; Sunwoo et al., 1995). Many reports have indicated that
DV or its extract contains many functional ingredients including
epidermal growth factor, insulin like growth factor, glycosamino-
glycan, and some water insoluble compounds such as phospholi-
pids and long-chain fatty acids (Hou et al., 2012; Ji et al., 2009).
There have been a number of reports on DV or its extract bio-
medical functions and activities toward the immune system (Zha
et al., 2013), bone metabolism (Tseng et al., 2012), anti-
inflammatory (Dai et al., 2011), reproductive performance (Kim
et al., 2012; Xu et al., 2010) and anti-oxidation (Wang et al., 2004)
effects on experimental animals. Most of animal model studies
indicated that DV could promote reproductive functions, such as
promoted cockscomb growth (Li et al., 1989), increased serum
testosterone concentrations and spermatogenesis (Bae, 1975), and
sex glands growth (Bae, 1976). In addition, DV has been used
experimentally in preventing and treating of oxidation (Wang et
al., 2004), and aging process (Liu et al., 2010) in mice in vivo. The
aim of this study is to investigate the effects of velvet extract from
Formosan sika deer (FSD) velvet on mouse embryonic develop-
ment and evaluate the antioxidant ability by antioxidant enzyme
mRNA expression in vitro.
2. Materials and methods
2.1. Maintaining and management of experimental animals
Six weeks old female ICR; Bltw CD-1 strain mice purchased
from BioLASCO, Taiwan Co., Ltd. were accustomed their new
environment for at least 1 week before the experiment. They were
maintained in an automatic light/dark cycle controlled room (300–
400 lx, 12 L/12 D). Temperature and relative humidity were kept in
2272 1C and 5575% respectively. The animal care and manage-
ment were performed in accordance with the guidebook for the
care and use of laboratory animals (Yu, 2005).
2.2. Preparations of deer velvet extract
The FSD velvet samples were harvested 75 days in growing
period in National Pingtung University of Science and Technology,
Taiwan. The fresh velvet samples were divided into tip, upper,
middle and basal sections (Kim et al., 1999). The upper section
(100 g) was sliced and grinded into 2–3 mm pieces by Osterizer 12
speed blender (Oster, Model: 6641), and mixed with 500 mL of
cold 20% alcohol (v:v, Merck, 1.00983.2500), and stirred with a
magnetic stir bar for 16–18 h at 4 1C. After a stirring process, the
DV extract solution was centrifuged (5000g) for 20 min at 4 1C, and
the insoluble components were discarded. For removing the
residual alcohol from the DV extract an air exhausting system
was used for 24 h at roomQ3 temperature. Finally, the prepared DV
extract stock solutions were sterilized by passing through 0.22 μm
filters (Millipore Corp, Carrigtwohill, Ireland), and Q4stored at
À20 1C (Chen, 2001; Horng, 2003). The extract recovery ratio
was 72%.
2.3. Superovulation and embryo collection
Each mouse was intraperitioneally (IP) injected with 10 IU of
chorionic gonadotrophin (eCG, Sigma G4877). 10 IU of human
chorionic gonadotrophin (hCG, Sigma C1063) was given 48 h
followed IP injection to induce superovulation (Nagy et al.,
2003). Immediately after receiving hCG, the female mice were
placed into cages containing intact male mice for breeding and
checked for vaginal plugs at the following day, and then trans-
ferred to new cages where they were group-caged (4–5 mice per
cage) for 50–54 h until embryos collection. Mice were sacrificed by
a cervical dislocation method at 50–54 h after hCG injection. 4-
cells embryos were flushed out with mHTF medium from oviduc-
tal ampullae. The cumulus–corona cells of 4-cells embryo were
removed by a narrow-bore pilled glass Pasteur pipette in mHTF
medium and subsequently placed into mHTF medium drops
covered with mineral oil in 35 mm diameter plastic culture dish
(150255; Nunc, Roskilde, Denmark), and then equilibrated at 37 1C
in a humidified atmosphere of 5% CO2 in air for further
experiment.
2.4. Evaluation of embryo development and morphology
Evaluation of the embryo stages was observed by a light
microscope (Leica MZ75) every 12 h. They were classified accord-
ing to descriptions defined Q5by Nagy et al. (2003). The following
stages were performed: 8-cells embryo: intact zona pellcide (ZP)
with 8 blastomere and no cytoplasmic vesicles; morula: com-
pacted blastomere with an intact ZP; blastocyst stage: including
early blastocysts (with initial blastocele), expanded, hatching and
hatched blastocysts.
The fragmented and/or lysed embryos that did not continue
developing were recorded as arrested embryos. The arrested
embryos were classified into the following types. Type І: the
blastomeres of embryo displayed fully lyse, necrotic and/or frag-
ment. Type ІІ: the blastomeres of embryo showed partially lyse or
fragment. Type ІІІ: the embryo displayed few lyse or fragment
blastomeres and/or cytoplasmic vesicles. Type IV: blastocyst smal-
ler than control, with no intact blastomeres, two or more
blastoceles and/or vesicles in blastomeres were considered as
morphologic abnormal embryo.
2.5. Experimental design
The 4-cells embryos were randomly divided into different
treatment groups and incubated in the incubator for 72 h at
37 1C with a humidified atmosphere of 5% CO2 in air. After embryo
stages observation, only blastocysts were placed into RNAlater
solution (Applied Biosystems, AM7020) and stored in À80 1C until
for antioxidant gene expression assay.
2.5.1. Effects of FSD velvet extract and HP challenge on embryonic
development and anti-oxidative enzyme mRNA expression
In control group A: embryos were cultured in the mHTF
medium; DV extract groups: embryos were cultured in the mHTF
medium containing 1% (B group, DV 1), 2% (C group, DV 2) and 4%
(D group, DV 4) DV extract. HP challenge groups: embryos were
cultured in 5 μM (E group, HP 5), 10 μM (F group, HP 10) and
25 μM (G group, HP 25) HP contained mHTF medium for 1 h.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
S.-L. Cheng et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎2
jep.2014.04.006i

Following the 1 h HP challenge, embryos were washed in mHTF
medium to removed HP, and then cultured in mHTF medium for
72 h.
2.5.2. Effects of FSD velvet extract combined with HP on embryonic
development and anti-oxidative enzymes mRNA expression
Embryos were cultured in mHTF medium containing HP (5, 10
and 25 μM) for 1 h oxidative challenge and then cultured in mHTF
medium supplemented with 1%, 2% and 4% of DV extract. The
following groups were performed: 5 μM HP and 1% DV extract (H
group, HP5þDV1), 10 μM HP and 1% DV extract (I group,
HP10þDV1), 25 μM HP and 1% DV extract (J group, HP25þDV1),
5 μM HP and 2% DV extract (K group, HP5þDV2), 10 μM HP and 2%
DV extract (L group, HP10þDV2), 25 μM HP and 2% DV extract (M
group, HP25þDV2), 5 μM HP and 4% DV extract (N group,
HP5þDV4), 10 μM HP and 4% DV extract (O group, HP10þDV4),
and 25 μM HP and 4% DV extract (P group, HP25þDV4).
2.6. Analysis of anti-oxidative enzymes mRNA expression
2.6.1. RNA isolation and reverse transcription
Total RNA was isolated from mice blastocysts by an RNA
extraction kit (absolutely RNAs
total RNA microprep kit; Strata-
gene, 400753). RNA concentrations were measured by a spectro-
photometer. Only high concentrated RNA was sampled for first-
strand cDNA reverse transcription. RNA reverse transcription was
performed using a reverse transcription kit (AccessQuick™
RT-PCR
System; Promega, A-1700) according to the manufacturer's
instructions. First-strand cDNA was stored at À80 1C for quanti-
tative real time PCR (qRT-PCR) analysis.
2.6.2. Analysis of quantitative real time PCR
Specific primers of the copper, zinc superoxide dismutase
(CuZn-SOD), GPx, CAT and β-actin for the qRT-PCR used in this
study are shown in Table 1. For each RT-PCR reaction, 2 μl of cDNA
was mixed with 12.5 μl SYBR Premix Ex Taq (2X)(TaKaRa, RR0412),
1 μl of primer and ddH2O to final reaction volume of 25 μl in total
per well. The qRT-PCR was conducted with the following program:
95 1C for 5 s, 64 1C for 20 s, and 72 1C for 10 s followed by 40
cycles. All samples were analyzed twice and the geometric means
of the Ct values were further used for mRNA expression profiling.
The geometric mean of two housekeeping genes β-actin was used
for normalizing the target gene. The delta Ct (ΔCt) values were
calculated as the difference between target gene and geometric
mean of the reference genes: (ΔCt¼Cttarget ÀCthousekeeping gene) as
described by Pfaffl (2001).
3. StatisticalQ8 analysis
The data were expressed as mean7S.E. The significant differ-
ences were first analyzed by one way analysis of variance (ANOVA)
from the statistical package for the social science (SPSS 10.0).
Duncan's multiple range test was used to detect differences
between the treatment means, and Po0.05 was considered
statistically significant.
4. Results
4.1. Effects of FSD velvet extract and hydrogen peroxide on
developmental ability and anti-oxidative enzymes mRNA expression
of the ICR mouse embryos
The blastocyst development rate in groups B (1% DV) and C (2%
DV) was close to that of the control group A (90.0–90.7%), while
only 73.675.05% of the mouse embryos in the group D (4% DV)
(P>0.05, Table 2). The embryos in groups E (5 μM HP), F (10 μM
HP) and G (25 μM HP) following 1 h HP challenged; the mouse
embryos were arrested. The embryos in groups E and F after
oxidative stress led to the partial lysed of mouse blastomeres,
whereas resulted in complete lysed Q6in group G (Table 4). The
morphologically normal embryo ratio in groups B and C was
similar to that in group A (84.4–88.8%). The morphologically
normal embryo ratio in group D was not significant lower than
that in control group (65.9 vs. 88.8%, P>0.05). Only a few of
embryos in group D displayed type ІI arrested (1.1%) and abnormal
blastocyst (6.6%) during the culture period, whereas 26.4% of
embryos exhibited type ІІI abnormalities which were cultured in
mHTF medium supplemented with 4% DV (Table 4).
Using SYBR Green I as a real time quantification system to
estimate the relative antioxidant enzymes mRNA expressions of
the Cu, Zn-SOD, GPx and CAT in mouse embryos, the mRNA
expression in the control group was set as a baseline value of
1.00 and compared with the intensity of expression in the
experimental groups. In groups E, F and G, embryos cultured in
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
Table 1
The primer sequences of anti-oxidative enzymes used in real time polymerase chain reaction (RT-PCR).
Gene Primer Product size (bp) Reference
CuZn-SOD Sence 50
-AAggCCgTgTgCgTgCTgAA-30
246 Mouatassim et al., 1999
Antisence 50
-CAggTCTCCAACATgCCTCT-30
GPx Sence 50
-CCTCAAgTACgTCCgACCTg-30
Antisence 50
-CAATgTCgTTgCggCACACC-30
CAT Sence 50
-gCAgATACCTgTgAACTgTC-30
Antisence 50
-gTAgAATgTCCgCACCTgAG-30
β-actin Sence 50
-TgCgTgACATCAAAgAgAAg-30
197 Steuerwald et al., 2000
Antisence 50
-gATgCCACAggATTCCATA-30
CuZn-SOD: CuZn superoxide dismutase; GPx: glutathione peroxidase; CAT: catalase; β-actin: internal control.
Table 2
Effects of hydrogen peroxide and FSD velvet extract on the in vitro developmental
ability of the mouse embryos.
Treatment
groups1
N2
No. (%) of embryos developed to
8-cells Compacted
morula
Blastocyst
A, Control 107 104 (97.271.06)a
100 (93.571.71)a
97 (90.772.23)a
B, DV1 94 90 (95.772.36)a
90 (95.772.36)a
85 (90.473.02)a
C, DV2 90 88 (97.872.50)a
88 (97.872.50)a
81 (90.073.98)a
D, DV4 91 84 (92.372.64)a
82 (90.173.16)a
67 (73.675.05)a
E, HP5 50 4 ( 8.076.39)b
0 ( 0.070.00)b
0 ( 0.070.00)b
F, HP10 53 0 ( 0.070.00)b
0 ( 0.070.00)b
0 ( 0.070.00)b
G, HP25 91 0 ( 0.070.00)b
0 ( 0.070.00)b
0 ( 0.070.00)b
a, b
Means7S.E. with different superscripts in the same column are significantly
different (Po0.05).
1
The unit for DV and HP is percentage and μM, respectively.
2
Numbers of examined 4-cells stage embryos.
S.-L. Cheng et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3
jep.2014.04.006i

HP challenge presented arrested blastomeres, which prevented
them from developing into blastocysts, and could not apply for
relative anti-oxidative enzymes mRNA expressions analysis. The
embryos in C and D groups which cultured in DV exhibited higher
focused relative anti-oxidative enzymes mRNA expressions than
those in control group (Table 6).
4.2. Effect of deer velvet extract combined with hydrogen peroxide
on developmental ability and anti-oxidative enzyme mRNA
expression of the ICR mouse embryos in vitro
Following 1 h culture in HP, the mouse embryos were trans-
ferred to mHTF medium containing DV extract for continuous
development to the blastocyst stage. The blastocyst development
rates of the embryos cultured with 1% or 2% DV extract were close
to those of the control group. However, the rate of blastocyst
development in mouse embryos subjected to the combination of
more highly concentrated HP and DV extract was lower than that
of the other groups (Table 3). Especially, the rates of blastocyst
development in M, O and P groups were significantly lower than
those of the control group (76.9%, 76.9%, and 65.8%, respectively,
vs. 90.7%; Po0.05). The mouse embryos in H to P groups did not
present Type I abnormal embryos; data is not shown in Table 5.
The rate of normal development to blastocyst was the lowest
(43.0%) among the group which was cultured with 25 μM HP and
4% DE (group P).
The mRNA expression of SOD in the D, N, O and P groups (1.69,
1.73, 1.71, and 1.53 times that of the control group, respectively)
was significantly higher than that in J group (0.55, Po0.05)
(Table 6). The differences in the mRNA expressions of GPx and
CAT among the mouse embryos did not reach statistical signifi-
cance, ranging between 0.59 and 4.48 times that of the control
group (P>0.05).
5. Discussions
Several exogenous factors or culture conditions can alter the
metabolism of mammalian embryos in vitro, resulting in an
increase in ROS production and subsequent oxidative attacks
(Wasserman and Fahl, 1997), which may adversely affect embryo-
nic development (Cebral et al., 2007; Maître et al., 1993). In this
study, mouse 4-cells embryos were unable to continue develop-
ment after being challenged with HP for 1 h (5, 10 and 25 μM of
HP). These results were similar to those previously described by
Cebral et al. (2007); however, the previous reported that the
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
Table 3
Effects of the hydrogen peroxide combined with FSD velvet extract on the in vitro developmental ability of the mouse embryos.
Treatment groups1
N2
No. (%) of embryos developed to
8-cells Compacted morula Blastocyst
A, control 107 104 (97.271.06)a
100 (93.571.71)a
97 (90.772.23)a
H, HP5þDV1 76 74 (97.471.75)a
74 (97.471.75)a
72 (94.772.31)ab
I, HP10þDV1 78 74 (94.972.13)a
74 (94.972.13)a
70 (81.773.15)abc
J, HP25þDV1 80 78 (97.571.77)a
78 (97.571.77)a
72 (90.074.07)abc
K, HP5þDV2 78 77 (98.770.83)a
76 (97.472.71)a
73 (93.674.71)abc
L, HP10þDV2 78 74 (94.972.89)a
74 (94.972.89)a
70 (89.773.25)abc
M, HP25þDV2 78 73 (93.672.36)a
71 (91.072.57)a
60 (76.975.96)c
N, HP5þDV4 78 71 (91.074.67)a
71 (91.074.67)a
62 (79.575.52)abc
O, HP10þDV4 78 71 (91.076.29)a
70 (89.776.26)a
60 (76.976.49)bc
P, HP25þDV4 79 65 (82.376.04)a
62 (78.577.05)a
52 (65.877.13)c
a, b, c
Means7S.E. with different superscripts in the same column are significantly different (Po0.05).
1
2
Table 4
Effects of the FSD velvet extract and hydrogen peroxide on the abnormality of the
mouse embryos in vitro.
Treatment groups1
N2
Normal (%) No. (%) of arrested/abnormal embryos
Type I Type II Type III Type IV
A, control 107 95 (88.8)a
0a
0a
11 (10.3)a
1 (0.9)a
B, DV1 94 80 (85.1)ab
0a
0a
9 (9.6)a
5 (5.3)a
C, DV2 90 76 (84.4)ab
0a
0a
9 (10.0)a
5 (5.6)a
D, DV4 91 60 (65.9)ab
0a
1 (1.1)a
24 (26.4)a
6 (6.6)a
E, HP5 50 0 (0.0)d
0a
50 (100)b
– –
F, HP10 53 0 (0.0)d
0a
53 (100)b
– –
G, HP25 91 0 (0.0)d
91
(100)b
– – –
a, b
Values with different superscripts in the same column are significantly different
(Po0.05). Type I: full lysed, necrotic or fragmented embryos; Type II: partially
lysed/ fragmented blastomeres and/or cytoplasmic vesicles; Type III: embryos with
some lysed/ fragmented blastomeres and embryo arrest at 4-cells to CM stage
period; Type IV: abnormal cavitation have two or more blastoeles in inner cell mass
at blastocyst.
1
2
Table 5
Effects of the hydrogen peroxide combined with FSD velvet extract on the
abnormality of the mouse embryos in vitro.
Treatment groups1
N2
Normal (%) No. (%) of arrested/abnormal embryos
Type II Type III Type IV
A, control 107 95 (88.8)a
0a
11 (10.3)a
1 (0.9)a
H, HP5þDV1 76 71 (93.4)a
0a
4 (5.3)ab
1 (1.3)a
I, HP10þDV1 78 67 (85.9)ab
0a
8 (10.3)ab
3 (3.8)a
J, HP25þDV1 80 66 (82.5)ab
0a
8 (10.0)ab
6 (7.5)ab
K, HP5þDV2 78 70 (89.7)ab
0a
5 (6.4)ab
3 (3.8)a
L, HP10þDV2 78 63 (80.8)ab
0a
8 (10.3)ab
7 (9.0)ab
M, HP25þDV2 78 55 (70.5)bc
0a
18 (23.1)b
5 (6.4)ab
N, HP5þDV4 78 59 (75.6)ab
0a
16 (20.5)ab
3 (3.8)a
O, HP10þDV4 78 54 (69.2)abc
0a
18 (23.1)b
6 (7.7)a
P, HP 25þDV 4 79 34 (43.0)c
8 (10.1)a
27 (34.2) b
10 (12.7)b
a, b, c
Values with different superscripts in the same column are significantly
different (Po0.05). Type I: full lysed, necrotic or fragmented embryos; Type II:
partially lysed/ fragmented blastomeres and/or cytoplasmic vesicles; Type III:
embryos with some lysed/ fragmented blastomeres and embryo arrest at 4-cells
to CM stag period; Type IV: abnormal cavitation have two or more blastoceles in
inner cell mass at blastocyst.
1
2
Numbers of examined 4-cell stage embryos.
jep.2014.04.006i

embryotoxicity occurred only when HP in culture medium
exceeded 60 μM, which inhibited embryonic growth in vitro
(Zhang et al., 2005). Following the addition of FSD velvet extract
into the culture medium, we discovered an increase in blastocyst
development rates (65.8–94.7%) during incubation followed HP
challenged (Table 3) compared to the HP alone groups (Table 2),
demonstrating that some constituents in FSD velvet extract were
capable of neutralizing or mitigating the influence of oxidative
stress on the developmental competence of mouse embryos
in vitro.
However, an increase in HP concentration was accompanied by
concurrent increase in the percentage of abnormal embryos. In
this study, we used ethanol as the extraction solvent, due to its
miscibility with water-soluble and fat-soluble substances, such as
proteins, fatty acids, vitamins and steroids (Gropper et al., 2009).
Fat-soluble vitamins A (retinol palmitate) and E (α-tocopherol)
have been shown to maintain the grade 1–2 quality embryos of
bovine embryos produced in vitro and promote higher growth
rates in early, expanded, and hatched blastocysts (Olson and
Seidel, 1995; Shaw et al., 1995). Furthermore, the addition of 1%
alcohol to the culture solution, or oral administration to animals
with alcohol was shown to negatively affect embryonic develop-
ment and increase the incidence of abnormal embryos (Cebral et
al., 2001; Wiebold and Becker, 1987). The stock solution of FSD
velvet extract presented a residual alcohol concentration of 8%,
and the alcohol concentrations in the culture medium of the DV
extract treated group D was 0.32%, which may account for the
reduction in embryonic growth.
The intracellular expression of anti-oxidative enzymes is
genetically regulated, which is further modulated by the intensity
of oxidative stress. It is possible that the metabolites of O2 within
cells act as signals triggering the expression of anti-oxidative
enzymes (Barnett and Bavister, 1996; Maître et al., 1993). The
defense mechanisms of cells or tissue are activated by oxidative
stress, in which Cu/Zn-SOD and Mn-SOD are the first catalysts
involved in the conversion of O2

into HP (Hanukoglu, 2006), which
attacks target cells (Fujii et al., 2005). The inability of cells to resist
this attack leads to aging and various diseases in tissue and organs
(Agarwal et al., 2005). However, as an unstable oxide (Hanukoglu,
2006), HP requires GPx and CAT detoxification, for which the
expression of the three aforementioned anti-oxidative enzymes or
their genes is a crucial indicator of oxide removal (Chun et al.,
1994; Guérin et al., 2001). In this study, the mouse embryos
cultured in mHTF medium supplemented with DV extract did
not significantly improve SOD mRNA expression. However, we
found the mouse embryos in most of groups, which cultured in
mHTF medium supplemented with DV extract exhibited higher
ratio of mRNA expression of GPx and CAT (1.25–2.12 times
compare to SOD) than that of SOD in group A (Table 6). Peltola
et al. (1996) indicated that GPx and CAT are able to reduce HP into
H2O, O2 and oxidized glutathione (GSSG). In this case, FSD velvet
extract could be able to fortify the defense mechanisms of mouse
embryos against oxidative stress as well as enhance their devel-
opmental competence.
Supplying senescence-accelerated mouse (SAMP8) with feed
containing 2% FSD velvet for four to six weeks was shown to
significantly reduce the H2O2 levels in the plasma of male mice
and the liver of female mice, even though the expression of
antioxidant enzyme genes in the liver was Q7not substantially
enhanced (Huang, 2008). Ji et al. (2009) indicated that the DV
extract contains several unidentified substances, such as peptides,
which are capable of removing oxides. Liu et al. (2010) showed
that DV extract could reduce serum malondialdehyde (MDA)
concentration, increase the serum SOD, GPx and CAT concentra-
tions, and enhance the antioxidant ability of ALX-induced diabetic
mice. Recent reports demonstrated that DV or its extract contained
active components, in which GSH, polypeptides and monoamine
oxidases had an inhibitory effect on oxidation (Hao et al., 2012;
Tian et al., 2009; Wang et al., 2010). These cases suggest that DV
extract might possess with antioxidant capacity, whether admi-
nistered orally or provided directly to cells can participate in redox
and mitigate oxidative stress.
6. Conclusions
This study demonstrates that adequate concentration of the
deer velvet extract from Formosan sika deer is capable of enhan-
cing the mRNA expression of anti-oxidative enzymes in mouse
embryos, mitigating oxidative stress in mouse embryos, and
enhancing the developmental competence of blastocysts.
Acknowledgments
The authors would like to thank Prof. Ming-Huei Liao, Dept. of
Veterinary Medicine, NPUST, Taiwan, R.O.C. for kindly providing
facilities and technical supports for anti-oxidative enzyme mRNA
expression analysis.
References
Agarwal, A., Gupta, S., Sharma, R.K., 2005. Role of oxidative stress in female
reproduction. Reproductive Biology and Endocrinology 3, 338–347.
Bae, D.S., 1975. Studies on the effects of velvet on growth of animals I. Effects of
velvet of different levels on weight gain, feed efficiency and development of
organs of chickens. Korean Journal of Animal Science 17, 571–576.
Bae, D.S., 1976. Study on the effect of velvet on growth of animals. II. Effect of velvet
on the growth internal organs and blood picture of chicken. Korean Journal of
Animal Science 18, 342–348.
Barnett, D.K., Bavister, B.D., 1996. Inhibitory effect of glucose and phosphate on the
second cleavage division of hamster embryos: is it linked to metabolism?
Human Reproduction 11, 177–183.
Bubenik, G.A., Miller, K.V., Lister, A.L., Osborn, D.A., Bartos, L., Kraak, G.J.V.D., 2005.
Testosterone and estradiol concentrations in serum, velvet skin, and growing
antler bone of male white-tailed deer. Journal of Experimental Zoology Part A,
Comparative Experimental Biology 303, 186–192.
Cebral, E., Rettori, V., Gimeno, M.A.F., 2001. Impact of chronic low-dose ethanol
ingestion during sexual maturation of female mice in in vitro and in vivo
embryo development. Reproductive Toxicology 15, 123–129.
Cebral, E., Carrasco, I., Vantman, D., Smith, R., 2007. Preimplantation embryotoxicity
after mouse embryo exposition to reactive oxygen species. Biocell 31, 51–59.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
Table 6
Effects of FSD velvet extract with or without hydrogen peroxide on the relative
mRNA expression of the anti-oxidative enzymes of the mouse blastocysts in vitro.
Treatment
groups1
Relative mRNA expression
SOD GPx#
CAT#
GPx/
SOD#
CAT/
SOD#
GPxþCAT/
SOD#
A, control 1.00ab
1.00 1.00 1.00 1.00 2.00
B, DV1 0.93ab
1.77 1.19 1.83 1.25 3.08
C, DV2 1.24ab
2.51 2.30 2.03 2.12 4.15
D, DV4 1.69a
1.49 3.18 0.81 2.05 2.86
H, HP5þDV1 0.94ab
1.29 2.82 1.40 2.89 4.29
I, HP10þDV1 1.30ab
1.86 4.21 1.36 3.65 5.01
J, HP25þDV1 0.55b
0.59 1.17 0.72 1.44 2.48
K, HP5þDV2 1.15ab
1.54 3.19 1.24 2.82 4.05
L, HP10þDV2 1.29ab
1.64 3.04 1.18 2.37 3.55
M, HP25þDV2 1.40ab
2.98 3.88 1.44 3.83 5.27
N, HP5þDV4 1.73a
1.35 3.37 0.81 1.80 2.61
O, HP10þDV4 1.71a
2.04 4.48 1.19 2.77 3.96
P, HP25þDV4 1.53a
1.61 1.78 1.09 1.12 2.21
a, b
Values with different superscripts in the same column are significantly different
(Po0.05). SOD: superoxide dismutase. GPx: glutathione peroxidase. CAT: catalase.
1
#
Values within the same column are not significantly different (P0.05).
S.-L. Cheng et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 5
jep.2014.04.006i

Chen, R.N., 2001. The Effect of Antler Extract on the Proliferation of Antler Cells
(Master thesis). Tunghai University, Taichung, Taiwan, R.O.C.
Chun, Y.S., Kim, J.H., Chung, K.S., 1994. Effects of superoxide dismutase on the
development of preimplantation mouse embryos. Theriogenology 41, 511–520.
Dai, T.Y., Wang, C.H., Chen, K.N., Huang, I.N., Hong, W.S., Wang, S.Y., Chen, Y.P.,
Kuo, C.Y., Chen, M.J., 2011. The antiinfective effects of velvet antler of Formosan
sambar deer (Cervus unicolor swinhoei) on Staphylococcus aureus-infexted mice.
Evidence-Based Complementary and Alternative Medicine 2011 (9 pages).
Forsberg, H., Borg, L.A., Cagliero, E., Eriksson, U.J., 1996. Altered levels of scavenging
enzymes in embryos subjected to a diabetic environment. Free Radical
Research 24, 451–459.
Fujii, J., Iuchi, Y., Okada, F., 2005. Fundamental roles of reactive oxygen species and
protective mechanisms in the female reproductive system. Reproductive
Biology and Endocrinology 3, 43–52.
Goud, A., Goud, P.T., Diamond, M.P., Gonik, B., Abu-Soud, H.M., 2008. Reactive
oxygen species and oocyte aging: role of superoxide, hydrogen peroxide, and
hypochlorous acid. Free Radical Biology and Medicine 44, 1295–1304.
Gropper, S.S., Smith, J.L., Groff, J.L., 2009. Advanced Nutrition and Human Metabo-
lism, 5th ed. Thomson Learning, USA.
Guérin, P., Mouatassim, El.S., Menezo, Y., 2001. Oxidative stress and protection
against reactive oxygen species in the pre-implantation embryo and its
surroundings. Human Reproduction Update 7, 175–189.
Hanukoglu, I., 2006. Antioxidant protective mechanisms against reactive oxygen
species (ROS) generated by mitochondrial P450 systems in steroidogenic cells.
Drug Metabolism Reviews 38, 171–196.
Halliwell, B., 2006. Reactive species and antioxidants. Redox biology is a funda-
mental theme of aerobic life. Journal of Plant Physiology 141, 312–322.
Hao, J., Li, S.F., Zhou, R., Wang, J.Y., Tian, S.J., 2012. Supercritical CO2 extraction of
monoamine oxidase inhibitor from freezn-dried powder of antler velvet. In:
Proceedings of the 10th International Symposium on Supercritical Fluids. May
13–16. San Francisco, CA, USA.
Hou, Z.H., Zhang, Y., Jin, C.A., Zhao, C.D., Sun, X.D., Cui, S.H., 2012. Research progress
on chemical constituents of antler. Special Wild Economic Animal and Plant 2,
58–60.
Horng, R.W., 2003. The Effects of Antler and Serum Factors on the Muscle
Development (Master thesis). Tunghai University, Taichung, Taiwan, R.O.C.
Huang, T.Y., 2008. Effect of the Velvet Antler of Formosan Sika Deer in the
Antioxidative Ability in Senescence-Acclerated Mice (Master thesis). Tunghai
University, Taichung, Taiwan, R.O.C.
Ji, J.X., Qian, J., Huang, F.J., Wu, W.T., Gao, X.D., 2009. Study on the active principles
and pharmacological actions of hairy antler. Chinese Journal of Biochemical
Pharmaceutics 30, 141–143.
Jeong, Y.J., Choi, H.W., Shin, H.S., Cui, X.S., Kim, N.H., Gerton, G.L., Jun, J.H., 2005.
Optimization of real time RT-PCR methods for the analysis of gene expression
in mouse eggs and preimplantation embryos. Molecular Reproduction and
Development 71, 284–289.
Kim, H.S., Lim, H.K., Park, W.K., 1999. Antinarcotic effects of the velvet antler water
extract on morphine in mice. Journal de Pharmacologie 66, 41–49.
Kim, J.H., Yang, Y.I., Ahn, J.H., Lee, J.G., Lee, K.T., Choi, J.H., 2012. Deer (Cervus
elaphus) antler extract suppresses adesion and migration of endometriotic cells
and regulates MMP-2 and MMP-9 expression. Journal of Ethnopharmacology
140, 391–397.
Li, F.L., Bao, L.H., Yang, B.F., Wu, X.C., Li, W.H., 1989. Effect of pantocrine on chicken
development. Journal of Harbin Medical University 23, 386–387.
Li, Z.H., Wu, L.M., Yao, Y.X., Wang, Q.K., 2007. Comparison of amino acid contents in
different products of Cervus nippon temminck velvet antler. Amino acids and
Biotic Resources 29, 16–18.
Liu, Y., Wang, Z.Y., Zhou, L.P., 2010. Anti-hyperglycemic and anti-aging effects of
deer antler velvet extract diabetic mice induced by alloxan. Journal of Northeast
Forestry University 38, 97–98.
Maître, B., Jornot, L., Junod, A.F., 1993. Effects of inhibition of catalase and super-
oxide dismutase activity on antioxidant enzyme mRNA levels. The American
Journal of Physiology 265, 636–643.
Mouatassim, S.E., Guérin, P., Ménézo, Y., 1999. Expression of genes encoding
antioxidant enzymes in human and mouse oocytes during the final stages of
maturation. Molecular Reproduction and Development 5, 720–725.
Nagy, A., Gertsentein, M., Vintersten, K., Behringer, R., 2003. Manipulating the
Mouse Embryo: A Laboratory Manual, third ed. Cold Spring Harbor, New York,
USA.
Olson, S.E., Seidel, G.E., 1995. Vitamin E improves development of bovine embryos
produced in vitro. Theriogenology 43, 289.
Peltola, V., Huhtaniemi, I., Metsa-ketela, T., Ahotupa, M., 1996. Induction of lipid
peroxidation during steroidogenesis in the rat testis. Endocrinology 137,
105–112.
Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-
time RT-PCR. Nucleic Acids Research 29, 45–50.
Shaw, D.W., Farin, P.W., Washburn, S.P., Britt, J.H., 1995. Effect of retinol palmitate
on ovulation rate and embryo quality in superovulated cattle. Theriogenology
44, 51–58.
Steuerwald, N., Cohen, J., Herrera, R.J., Brenner, C.A., 2000. Quantification of mRNA
in single oocytes and embryos by real-time rapid cycle fluorescence monitored
RT-PCR. Molecular Reproduction and Development 6, 448–453.
Sunwoo, H.H., Nakano, T., Hubson, R.J., Sim, J.S., 1995. Chemical composition of
antlers from wapiti (Cervus elaphus). Journal of Agricultural and Food Chemistry
43, 2846–2849.
Tian, Z.M., Zhang, L.Z., Gong, X.C., Wei, J.M., Wu, W.R., Wu, Y.H., 2009. Effects of
different temperature and time on the activity of GSH in fresh Cornu Cervi
Pantotrichum. Hubei Agricultureal Sciences 48, 1461–1463.
Trimarchi, J.R., Liu, L.P., Smith, J.S., Keefe, D.L., 2000. Noninvasive measurement of
potassium efflux as an early indicator of cell death in mouse embryos. Biology
of Reproduction 63, 851–857.
Tseng, S.H., Sung, H.C., Chen, L.G., Lai, Y.J., Wang, K.T., Sung, C.H., Wang, C.C., 2012.
Effects of velvet antler with blood on bone in ovariectomized rats. Molecules
17, 10574–10585.
Wang, L.J., Chen, X.G., Piao, X., 2004. Effect of pilose antler oral liquod (PAOL) on
antioxidant enzymes and MDA. Pharmacology and Clinics of Chinese Materis
Medica 20, 33–34.
Wang, H., Huang, T.B., Gao, K.X., Sun, H., Gao, Z.L., 2010. Preparation and purifican-
tion of velvet antlers peptides and its antioxidant activies. Chemical Journal of
Chinese University 31, 2390–2395.
Wasserman, W.W., Fahl, W.E., 1997. Functional antioxidant responsive elements.
Proceedings of the National Academy of Sciences of United States of America
94, 5361–5366.
Wiebold, J.L., Becker, W.C., 1987. In-vivo and in-vitro effects of ethanol on mouse
preimplantation embryos. Journal of Reproduction and Fertility 80, 49–57.
Xu, L.P., Li, J.G., Wu, F.F., Zang, C.C., Xu, Y.P., Jin, L.J., 2010. Effects of deer antler velvet
power prepared by auxiliary agents-mediated micro-shear technology and
ultrafine comminution technology on reproductive performance of Kunming
mice. Journal of Northeast Forestry University 38, 93–96.
Yang, H.W., Hwang, K.J., Kwon, H.C., Kim, H.S., Choi, K.W., Oh, K.S., 1998. Detection
of reactive oxygen species (ROS) and apoptosis in human fragmented embryos.
Human Reproduction 13, 998–1002.
Yu, J.U.L., 2005. A Guidebook for the Care and Use of Laboratory Animals, third ed.
Council of Argiculture, Taiwan, R.O.C..
Zhang, X., Sharma, R.K., Agarwal, A., Falcone, T., 2005. Effect of pentoxifylline in
reducing oxidative stress-induced embryotoxicity. Journal of Assisted Repro-
duction and Genetics 22, 415–417.
Zha, E.H., Li, X.X., Li, D.D., Guo, X.S., Yue, X.Q., 2013. Immunomodulatory effects of a
3.2 kDa polypeptide from velvet antler of Cervus nippon Temminck. Interna-
tional Immounopharmacology 16, 210–213.
Zhou, R., Wang, J.Y., Li, S.F., Liu, Y., 2009. Supercritical fluid extraction of monoamine
oxidase inhibitor from antler velvet. Separtation and Purification Technology
65, 275–281.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
jep.2014.04.006i

oxidative enzymes

Recommended

Recommended

More Related Content

What's hot

What's hot (17)

Similar to oxidative enzymes

Similar to oxidative enzymes (20)

Recently uploaded

Recently uploaded (20)

oxidative enzymes