Post on 05-Apr-2018
8/2/2019 Hwang ok
1/11
Animal Reproduction Science 100 (2007) 107117
Temporal effects of-tocopherol and l-ascorbicacid on in vitro fertilized porcine
embryo development
Mohammad Shamim Hossein, Md Abul Hashem, Yeon Woo Jeong,
Myeong Seop Lee, Sue Kim, Ji Hye Kim, Ok Jae Koo,Seon Mi Park, Eu Gine Lee, Sun Woo Park, Sung Keun Kang,
Byeong Chun Lee , Woo Suk Hwang
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University,
56-1 Sillim-dong, Gwanak-gu, Seoul 151-742, Republic of Korea
Received 12 May 2006; received in revised form 17 June 2006; accepted 21 June 2006
Available online 24 July 2006
Abstract
The susceptibility of embryos to reactive oxygen species (ROS) varies in different stages of embryo
development. The present study evaluated temporaleffects of-tocopherol andl-ascorbic acid on the porcine
embryo development, and investigated whether a single or twice supplements of these two antioxidants at
a divided concentrations favors the embryo development. In order to determine temporal effects of -
tocopherol and/or l-ascorbic acid, 100M -tocopherol or 200M l-ascorbic acid were supplemented to
the North Carolina State University (NCSU)-23 embryo culture media at 0, 48, 96 and 120 h of culture. In
another set of experiments, the concentration was divided into two equal halves, i.e., 50 M -tocopherol
and 100M l-ascorbic acid, and supplemented twice at 0 and 48, 0 and 96, or 48 and 96 h of culture.
Supplementing culture media with 100M -tocopherol for the entire culture period of 168 h or starting
from the 48 h of culture yielded higher blastocyst percentage compared with the control or starting fromthe 96 or 120 h of culture. l-Ascorbic acid (200M) alone or together with -tocopherol (100M) with
a single supplement did not affect the frequency of blastocyst formation or number of cells in blastocyst.
l-Ascorbic acid with a divided supplements yielded higher blastocyst percentage compared with the control.
No synergistic effect was observed on embryo development at a single supplement of these antioxidants.
Although, at divided supplements higher blastocyst percentage was observed compared with control group,
no further beneficial effect was observed compared with -tocopherol or l-ascorbic acid alone. Our results
demonstrated that the embryotrophic effects of-tocopherol and/or l-ascorbic acid, in terms of frequency
Corresponding author. Tel.: +82 2 880 1247; fax: +82 2 884 1902.
E-mail address: bclee@snu.ac.kr (B.C. Lee).
0378-4320/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2006.06.013
mailto:bclee@snu.ac.krhttp://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013mailto:bclee@snu.ac.kr8/2/2019 Hwang ok
2/11
108 M.S. Hossein et al. / Animal Reproduction Science 100 (2007) 107117
of blastocyst formation and number of cells in blastocyst, depends on the concentration and supplementation
timing.
2006 Elsevier B.V. All rights reserved.
Keywords: -Tocopherol; l-Ascorbic acid; Porcine; IVF; Embryo culture
1. Introduction
Aerobic organism produces a range of reactive oxygen species (ROS) during metabolic pro-
cess (Nasr-Esfahni and Johnson, 1991; Gardner and Lane, 2002). The oocytes and embryos
also produce endogenous ROS by various enzymatic actions and metabolic pathways (Riley and
Behrman, 1991; Harvey et al., 2002). ROS participates in various cell processes especially in
tissue remodeling, hormone signaling, steriodogenesis and germ cell function at a physiolog-
ical concentration. However, it modifies normal cell functions, endanger cell survival, or bothwhen the critical concentration is overwhelmed (Droge, 2002). Therefore, ROS must be con-
tinuously inactivated in order to keep only a small amount necessary to maintain normal cell
functions.
Mechanisms for detoxification of and protection against ROS exit in in vivo system. These
include enzymes such as superoxide dismutase which produces hydrogen-peroxide from super-
oxide radicals, and catalase and glutathione peroxidase which decompose hydrogen-peroxide to
water (Guerin et al., 2001; Oyawoye et al., 2003). In addition, there are low molecular weight
antioxidants which act as ROS scavengers such as l-ascorbic acid, uric acid, glutathione and
tocopherols (McEvoy et al., 2000; Sturmey and Leese, 2003). Both the follicular and oviductal
fluid is rich in oxygen scavengers which protect oocytes and embryos from oxidative damage
(Gardiner and Reed, 1995; Lapointe et al., 1998). When oocytes and embryos are cultured in
vitro, they are deprived from these natural defense systems. Therefore, it is crucial to protect them
against oxidative stress during in vitro culture by adding antioxidant into the culture media to
optimize embryo production.-Tocopherol is the predominant lipid-soluble antioxidant in animal cells. It protects cells from
oxygen radicals in vivo and in vitro (Chow, 1991). Ascorbic acid is a water-soluble vitamin which
has been considered to be the most important antioxidant in extracellular fluids (Buettner, 1993;
Rose and Bode, 1993). Adding ascorbic acid into the culture media prevents follicular apoptosis
in rat and mouse follicles, and also improves the blastocyst production in mouse ( Eppig et al.,2000; Tilly and Tilly, 1995). Ascorbic acid acts synergistically with -tocopherol under some
conditions by regenerating tocopherol from tocopheroxyl radicals, the products of tocopherol and
free radical interaction (Chow, 1991).
Rapid changes on the cell physiology and embryonic metabolism intervene during the early
embryonic development (Gardner and Lane, 2002). The zygote has a low metabolic activity
and exhibits low levels of oxygen consumption whereas after blastocoel formation embryo
metabolism significantly increases (Harvey et al., 2002; Leese, 1995). So, the level of ROS
production may vary in different developmental stages as ROS produces along with metabolic
process. The susceptibility of embryos to ROS also varies according to developmental stages
(Ali et al., 2003). Thus, the requirement of antioxidant supplement into culture medium maynot be the same at different stages of development. There are reports about embryotrophic effect
of l-ascorbic acid and -tocopherol (Olson and Seidel, 2000; Wang et al., 2002; Kitagawa et
al., 2004) but no information is available about temporal effects of antioxidant supplement into
http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.0138/2/2019 Hwang ok
3/11
M.S. Hossein et al. / Animal Reproduction Science 100 (2007) 107117 109
the culture medium. Accordingly, the present study was undertaken to evaluate temporal effects
of-tocopherol and l-ascorbic acid on in vitro fertilized porcine embryo development, and to
investigate whether a single or two time supplement at divided concentrations favors the embryo
development.
2. Materials and methods
2.1. Recovery and culture of oocytes
Ovaries were collected from gilts at a local slaughterhouse and transported to the laboratory in
physiological saline supplemented with 100 U/ml penicillin G (SigmaAldrich Corp., St. Louis,
MO) and 100 mg/ml streptomycin sulphate (SigmaAldrich) at around 3035 C within 2 h of
collection. Cumulusoocyte complexes (COCs) from follicles 36 mm in diameter were aspirated
using an 18-gauge needle attached to a 10 ml disposable syringe (Kimetal.,2004). Compact COCswere selected and cultured in tissue culture medium (TCM)-199 (Invitrogen, Carlsbad, CA),
supplemented with 10 ng/ml epidermal growth factor (EGF; SigmaAldrich), 4 IU/ml pregnant
mare serum gonadotropin (PMSG) and hCG (Intervet, Boxmeer, Netherland) and 10% porcine
follicular fluid (pFF). The pFF was aspirated from 3 to 7 mm follicles from the prepubertal
gilt ovaries. After centrifugation at 150 g for 30 min, supernatant was collected and filtered
sequentially through 1.2 and 0.45m syringe filters (Gelman Sciences, Ann Arbor, MI). Prepared
pFF was stored at 20 C until use. Each group of 50 COCs was cultured in 500 l TCM-199
incubated at 39 C in a humidified atmosphere of 5% CO2 in 95% air. After culturing for 22 h,
COCs were washed three times and cultured in PMSG- and hCG-free TCM-199 medium for
another 22 h.
2.2. In vitro fertilization and culture
Frozen semen was thawed at 39 C for 1 min in a water bath, diluted in 10 ml Dulbeccos
PBS (Invitrogen) supplemented with 0.1% BSA (SigmaAldrich), 75g/ml potassium penicillin
G and 50g/ml streptomycin sulphate and centrifuged twice at 350 g for 3 min. Then, the
sperm pellet was suspended in modified Tris-buffered medium (mTBM) to give a concentra-
tion of 2 107 sperm/ml. The mTBM consisted of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2,
20 mM Tris (T-1410; SigmaAldrich), 11 mM glucose, 5 mM sodium pyruvate and 8 mg/ml BSA(A-6003; SigmaAldrich). At 44 h of maturation culture, oocytes were freed from cumulus cells
by repeated pipetting in 0.1% hyaluronidase (SigmaAldrich) in TCM-199 medium and washed
three times with pre-equilibrated mTBM. After washing, 2025 oocytes were placed in 45 l
drops of the mTBM (fertilizing drop) covered with pre-warmed paraffin oil and 5 l sperm sus-
pension added to each fertilization drop to give a final sperm concentration of 2 106 sperm/ml.
After co-incubation of gametes for 6 h, the presumptive zygotes were washed and transferred
into in vitro culture (IVC) medium. The basic IVC medium was North Carolina State Univer-
sity (NCSU)-23 medium supplemented with 0.4% BSA (SigmaAldrich) (Kim et al., 2004).
Oocytes were cultured in NCSU-23 medium containing 0.5 mM pyruvate and 5.0 mM lac-
tate instead of 5.5 mM glucose at 39
C, 5% CO2, 5% O2 and 90% N2 atmosphere. At 96 hafter culture, the IVC medium was supplemented with 10% FBS (Invitrogen). The cleavage
rate and formation of blastocysts were evaluated under a stereomicroscope at 48 and 168 h of
culture.
8/2/2019 Hwang ok
4/11
110 M.S. Hossein et al. / Animal Reproduction Science 100 (2007) 107117
2.3. Differential staining
The quality of blastocysts was assessed by differential staining of the inner cell mass (ICM)
and trophectoderm (TE) cells according to the modified staining procedure of Thouas et al.
(2001). Briefly, hatched blastocysts were used as such and non-hatched blastocysts were treatedwith 0.25% pronase (w/v, SigmaAldrich) for 5 min to dissolve the zonae pellucidae. After
rinsing in NCSU-23 medium, zona-free blastocysts were stained with 0.01% (w/v) bisbenz-
imide for 1 h. After rinsing in NCSU-23 medium, blastocysts were treated with 0.04% (v/v)
Triton X-100 (SigmaAldrich) for 3 min followed by treatment with 0.005% (w/v) propidium
iodide (SigmaAldrich) for 10 min. After rinsing with NCSU-23 medium, stained blastocysts
were mounted on glass slides under a cover slip and examined under an inverted micro-
scope (Nikon Corp., Tokyo, Japan) equipped with epifluorescence. The ICM nuclei labeled
with bisbenzimide appeared blue and TE cell nuclei labeled with both bisbenzimide and pro-
pidium iodide appeared pink. Any blastocysts without dual stain were excluded from the
study.
2.4. Experimental design
-Tocopherol and l-ascorbic acid were purchased from SigmaAldrich Corp. The drugs were
added into the NCSU-23 culture medium according to experimental designs. -Tocopherol was
first dissolved in 95% ethanol as a 2000 stock solution, stored in at 4 C wrapping with
aluminum foil and then diluted as a 10 concentration prior to each experiment. Ethanol
at a concentration of 0.05%, the concentration used to dissolve the -tocopherol, has no
effect on embryo development (Olson and Seidel, 2000). In order to determine the temporal
effects of -tocopherol and/or l-ascorbic acid, 100M -tocopherol and 200M l-ascorbic
acid was added to the culture media at 0, 48, 96 and 120 h of culture. To evaluate the
effects of two time supplement of -tocopherol and/or l-ascorbic acid at a lower concentra-
tion, the total concentration was divided into two equal halves, i.e., 50 M -tocopherol and
100M l-ascorbic acid and supplemented twice at 0 and 48, 0 and 96, or 48 and 96 h of
culture.
2.5. Statistical analysis
In each experimental group, oocytes were randomly distributed. Each experiment was repeatedsix times. All data were subjected to one-way ANOVA followed by Tukey test to determine
differences among experimental groups using GraphPad Version 4.0 (Motulsky, 2003). Statistical
significance was determined when a P value was less than 0.05.
3. Results
3.1. Temporal effects of-tocopherol on in vitro fertilized porcine embryo development and
quality
Supplementing NCSU-23 with 100M -tocopherol for the entire culture period of 168 h(16.2%) or at 48 h of culture (17.3%) significantly increased the number of blastocyst percentage
compared with the control (10.5%) or supplementing at 96 h (9.6%) or 120 h (9.9%) of culture
(Table 1). Increased number (P < 0.05) of ICM, TE and total cells was observed when-tocopherol
8/2/2019 Hwang ok
5/11
M.S. Hossein et al. / Animal Reproduction Science 100 (2007) 107117 111
Table 1
Temporal effects of 100M -tocopherol on in vitro fertilized porcine embryo development and cell allocation to inner
cell mass (ICM) and trophectroderm (TE) in blastocysts
Duration
(h)
Embryo development Cell numbers (meanS.E.M.)
No. ofoocytes
No. oftwo-cell
embryos (%)a
No. ofblastocysts
(%)a
No. ofevaluated
blastocysts
ICM TE Total
Control 277 174 (62.8) 29 (10.5) a 18 11.2 0.3 a 30.1 0.8 a 41.2 1.1 a
0168 265 170 (64.2) 43 (16.2) b 26 12.9 0.4 bc 38.8 1.4 b 51.6 1.8 b
48168 272 185 (68.0) 47 (17.3) b 29 13.7 0.5 c 39.3 1.6 b 53.1 2.0 b
96168 260 175 (67.3) 25 (9.6) a 19 11.3 0.3 ab 31.4 1.0 a 42.7 1.1 a
120168 262 168 (64.1) 26 (9.9) a 17 11.4 0.4 ab 31.3 1.1 a 43.8 1.4 a
Within the same column, values with different letters (ac) were significantly different (P < 0.05).a Percentage of the number of oocytes.
was supplemented for the entire culture period of 168 h (12.9, 38.8 and 51.6, respectively) or at
48 h of culture (13.7, 39.3 and 53.1, respectively) compared with other experimental groups
(11.211.4, 30.131.4 and 41.243.8, respectively).
3.2. Effects of two time supplements of-tocopherol at a divided concentration on in vitro
fertilized porcine embryo development and quality
Higher blastocyst percentage (P < 0.05) was observed in all two time -tocopherol sup-plemented groups at a divided concentration (16.618.1% versus 9.4%) compared to the
control (Table 2). The increased number of total cells (54.057.2 versus 41.0), ICM
(13.814.5 versus 10.9) and TE (40.242.7 versus 30.1) cells was observed in the treat-
ment groups compared with the control. There was no difference in the frequency of blasto-
cyst formation and number of cells in blastocyst among different -tocopherol supplemented
groups.
Table 2
Effects of twotime supplement of-tocopherol at a divided concentrationon in vitrofertilized porcine embryo developmentand cell allocation to inner cell mass (ICM) and trophectroderm (TE) in blastocysts
Supplement
(h)
Embryo development Cell numbers (meanS.E.M.)
No. of
oocytes
No. of
two-cell
embryos (%)a
No. of
blastocysts
(%)a
No. of
evaluated
blastocysts
ICM TE Total
Control 220 170 (77.3) 22 (9.4) a 16 10.9 0.6 a 30.1 1.4 a 41.0 2.0 a
0 and 48 268 196 (73.1) 48 (18.1) b 26 13.8 0.6 b 40.2 2.4 b 54.0 3.0 b
0 and 96 280 212 (75.7) 48 (17.1) b 30 14.5 0.5 b 42.7 1.9 b 57.2 2.4 b
48 and 96 289 215 (74.4) 49 (16.6) b 32 13.9 0.5 b 40.9 2.2 b 54.9 2.7 b
Within the same column, values with different letters (a and b) were significantly different (P < 0.05). Total concentration
of-tocopherol (100M) were divided into two equal halves (50 and 50 M) and supplemented to the culture media at
different time.a Percentage of the number of oocytes.
8/2/2019 Hwang ok
6/11
112 M.S. Hossein et al. / Animal Reproduction Science 100 (2007) 107117
Table 3
Temporal effects of 200M ofl-ascorbic acid on in vitro fertilized porcine embryo development and cell allocation to
inner cell mass (ICM) and trophectroderm (TE) in blastocysts
Duration
(h)
Embryo development Cell numbers (meanS.E.M.)
No. ofoocytes
No. oftwo-cell
embryos (%)a
No. ofblastocysts
(%)a
No. ofevaluated
blastocysts
ICM TE Total
Control 252 170 (67.5) 24 (9.5) 14 11.3 0.4 30.7 1.3 42.0 1.6
0168 288 187 (64.9) 26 (9.0) 17 11.8 0.3 33.7 0.7 45.4 0.8
48168 270 185 (68.5) 28 (10.4) 24 11.6 0.3 33.1 0.5 44.8 0.7
96168 230 149 (64.8) 22 (9.6) 18 11.5 0.3 32.4 0.8 43.9 0.9
120168 240 167 (69.6) 24 (9.4) 19 11.9 0.4 33.5 1.8 45.4 2.1
a Percentage of the number of oocytes.
3.3. Temporal effects ofl-ascorbic acid on in vitro fertilized porcine embryo development
and quality
Supplementing 200M l-ascorbic acid into culture medium had no effect on the frequency
of blastocyst formation compared with the control (Table 3). No significant difference was
observed in the number of total cells (43.945.4 versus 42.0), ICM (11.511.9 versus 11.3)
and TE (32.433.7 versus 30.7) cells in treatment groups compared with the control.
3.4. Effects of two time supplement ofl-ascorbic acid at a divided concentration on in vitro
fertilized porcine embryo development and quality
Two time supplement of l-ascorbic acid at a divided concentration increased (P < 0.05) the
frequency of blastocyst formation (14.018.2% versus 8.9%) compared with the control (Table 4).
The increased number (P < 0.05) of total cells (52.458.2 versus 41.4), ICM (13.915.6 versus
11.1) and TE (38.542.6 versus 30.3) cells was observed in treatment groups compared with the
control. There were no differences in the frequency of blastocyst formation and number of cells
among l-ascorbic supplemented groups.
Table 4
Effects of two time supplement ofl
-ascorbic acid at a divided concentration on in vitro fertilized porcine embryodevelopment and cell allocation to inner cell mass (ICM) and trophectroderm (TE) in blastocysts
Supplement
(h)
Embryo development Cell numbers (meanS.E.M.)
No. of
oocytes
No. of
two-cell
embryos (%)a
No. of
blastocysts
(%)a
No. of
evaluated
blastocysts
ICM TE Total
Control 226 160 (70.8) 20 (8.9) a 16 11.1 0.5 a 30.3 1.0 a 41.4 1.5 a
0 and 48 268 196 (73.1) 46 (17.2) b 28 13.9 0.4 b 38.5 1.8 b 52.4 2.1 b
0 and 96 280 210 (75.0) 51 (18.2) b 32 15.6 0.5 b 42.6 1.7 b 58.2 2.1 b
48 and 96 279 208 (74.6) 44 (14.0) b 32 14.0 0.5 b 39.9 2.1 b 53.9 2.5 b
Within the same column, values with different letters (a and b) were significantly different (P < 0.05). Total concentration
ofl-ascorbic acid (200M) were divided into two equal halves (100 and 100M) and supplemented to the culture drop
at different time.a Percentage of the number of oocytes.
8/2/2019 Hwang ok
7/11
M.S. Hossein et al. / Animal Reproduction Science 100 (2007) 107117 113
Table 5
Temporal effects of simultaneous supplement of 100M -tocopherol and 200M l-ascorbic acid on in vitro fertilized
porcine embryo development and cell allocation to inner cell mass (ICM) and trophectroderm (TE) in blastocysts
Duration
(h)
Embryo development Cell numbers (meanS.E.M.)
No. ofoocytes
No. oftwo-cell
embryos (%)a
No. ofblastocysts
(%)a
No. ofevaluated
blastocysts
ICM TE Total
Control 186 130 (69.1) ab 19 (10.2) 14 12.9 0.5 32.4 0.7 45.1 1.0
0168 230 145 (63.0) b 22 (9.6) 11 11.6 0.5 30.9 1.4 42.5 1.5
48168 254 182 (71.7) a 38 (14.9) 22 12.1 0.3 32.1 0.1 44.2 0.9
96168 225 151 (67.1) ab 25 (11.1) 18 12.1 0.4 28.9 1.3 41.0 1.4
120168 230 150 (65.7) ab 27 (10.4) 16 11.9 0.3 29.8 1.7 41.7 1.9
Within the same column, values with different letters (a and b) were significantly different (P < 0.05).a Percentage of the number of oocytes.
3.5. Temporal effects of simultaneous supplement of-tocopherol andl-ascorbic acid on in
vitro fertilized porcine embryo development and quality
Simultaneous supplement of 100M -tocopherol and 200M l-ascorbic acid at a single
time did not improve the frequency of blastocyst formation (9.614.9% versus 10.2%) compared
with the control (Table 5). No significant difference was observed in the number of total cells
(41.044.2 versus 45.1), ICM (11.612.1 versus 12.9) and TE (28.932.1 versus 32.4) cells in
treatment groups compared with the control.
3.6. Effects of simultaneous two time supplements of-tocopherol andl-ascorbic acid at a
divided concentration on in vitro fertilized porcine embryo development and quality
Two time supplements of-tocopherol andl-ascorbic acid at a divided concentration improved
(P < 0.05) the number of blastocyst percentage (17.221.7% versus 10.3%) compared with the
control (Table 6). The increased number (P < 0.05) of total cells (55.462.4 versus 42.9), ICM
(16.117.3 versus 12.0) and TE (39.345.1 versus 30.9) cells was observed in treatment groups
compared with the control.
Table 6Effects of simultaneous two time supplement of-tocopherol and l-ascorbic acid at a divided concentration on in vitro
fertilized porcine embryo development and cell allocation to inner cell mass (ICM) and trophectroderm (TE) in blastocysts
Supplement
(h)
Embryo development Cell numbers (meanS.E.M.)
No. of
oocytes
No. of
two-cell
embryos (%)a
No. of
blastocysts
(%)a
No. of
evaluated
blastocysts
ICM TE Total
Control 223 160 (71.7) 23 (10.3) a 16 12.0 0.5 a 30.9 1.6 a 42.9 2.1 a
0 and 48 227 165 (72.7) 39 (17.2) b 28 16.5 0.6 b 41.7 2.1 b 58.2 2.4 b
0 and 96 230 169 (73.5) 50 (21.7) b 34 17.3 0.5 b 45.1 1.8 b 62.4 2.1 b
48 and 96 238 169 (71.0) 46 (19.3) b 32 16.1 0.4 b 39.3 1.3 b 55.4 1.6 b
Within the same column, values with different letters (a and b) were significantly different ( P < 0.05). -Tocopherol
(50M) and l-ascorbic acid (100M) were simultaneously supplemented to the culture drop at different time.a Percentage of the number of oocytes.
8/2/2019 Hwang ok
8/11
114 M.S. Hossein et al. / Animal Reproduction Science 100 (2007) 107117
4. Discussion
The present study was undertaken to evaluate the suitable timing and duration of antioxidant
supplement into the in vitro fertilized porcine embryo culture medium to improve embryo devel-
opment and its quality. Two well-known antioxidants, lipidophillic -tocopherol and hydrophilicl-ascorbic acid, were tested for their embryotrophic effects. Our results demonstrated that both
the -tocopherol and l-ascorbic acid have embryotrophic effects, alone or together, depending
on supplement timing, duration and concentration. In the previous studies, concentrations of-
tocopherol used in in vitro embryo production were varied from 50 to 800M and l-ascorbic
acid up to 400M (Wang et al., 2002; Kitagawa et al., 2004). Kitagawa et al. (2004) reported
that 100M -tocopherol had embryotrophic effect on porcine IVF derived embryos. Indeed,
concentrations more than 400M -tocopherol induced a dose-dependent decrease in blastocyst
development and blastocyst cell number (Wang et al., 2002). Thus, in the present study, we used
100M of-tocopherol to evaluate its temporal effects on porcine embryo development. In addi-
tion, 200M l-ascorbic acid was selected because higher concentration than 200M producedtoxicity in mouse embryos (Wang et al., 2002) and bovine oocytes (Dalvit et al., 2005).
The antioxidant activity of-tocopherol in preventing free radical-initiated peroxidative tissue
damages is accepted by most investigators and is believed to be the primary free radical scav-
enger in mammalian cell membranes (Rose and Bode, 1993; Droge, 2002). Large amount of-tocopherol is present in the ovary and follicular fluid which implies its diversified actions on
the reproductive system (Attaran et al., 2000). Adding -tocopherol to the bovine embryo culture
medium improved developmental competence of embryo to theblastocyst stage (Olson andSeidel,
2000). In the present study, -tocopherol at a concentration of 100M had beneficial effects on
porcine IVF derived embryo development. With a single supplement at 0 or 48 h of the culture
period and twice supplement with divided concentrations (at 0 and 48, 0 and 96, or 48 and 96 h),
supplementing -tocopherol produced significantly higher blastocyst percentage compared with
the control. Porcine embryos contain a much higher concentration of fatty acids compared with
other mammalian embryos (McEvoy et al., 2000; Sturmey and Leese, 2003), they might be more
susceptible to lipid peroxidation. As lipid peroxidation occurs as a chain reaction, the reaction
expands to surrounding phospholipids. Therefore, -tocopherol can stop this chain reaction and
eventually lead to more blastocyst formation. Ascorbic acid is a potent direct antioxidant which
provides protection against endogenous oxidative DNA damage (Fraga et al., 1991). It has been
supplemented in IVM and IVC medium with much debate on its embryotrophic effects. Tatemoto
et al. (2001) used 250Ml
-ascorbic acids during IVM of porcine oocytes and reported that itdid not improve the developmental competence of porcine embryos after IVF. Whereas, Wang et
al. (2002) reported that adding ascorbic acid into an embryo culture medium significantly affects
embryo development in a dose dependant manner. Our study demonstrated that supplementing
200M l-ascorbic acid into embryo culture media as a single supplement for any duration neither
improved the frequency of blastocyst formation nor the blastocyst quality. However, when the
concentration was divided into two equal halves and added at 0 and 48, 0 and 96, or 48 and 96 h,
higher blastocyst percentage was observed compared with the control. Our results suggest the
importance of concentration and duration ofl-ascorbic acid for embryotropic effects.
The functional interrelation between -tocopherol and micronutrients, notably the l-ascorbic
acid has long been recognized (Chow, 1991). However, in the present study, no synergistic effectof -tocopherol and l-ascorbic acid on embryo development was observed. Although, when
these two oxidants were added twice at a divided concentration, higher blastocyst percentage was
observed compared with the control, but no further development was observed compared with
8/2/2019 Hwang ok
9/11
M.S. Hossein et al. / Animal Reproduction Science 100 (2007) 107117 115
-tocopherol or l-ascorbic acid alone. It is suggested that antioxidant therapy may have negative
and undesirable effects if a safety threshold dosage of antioxidants is surpassed. Likewise our
study, Olson and Seidel (2000) reported that co-supplemention of-tocopherol and l-ascorbic
acid at a concentration of 100M each has deleterious effect on bovine. In a recent study, Dalvit
et al. (2005) stated that when the maturation medium for bovine oocytes was supplemented with-tocopherol and ascorbic acid together, the number of blastocyst percentage was diminished
significantly compared with not only the control but also with -tocopherol or ascorbic acid
alone. Thus, the reason why we did not obtain the synergistic effect of-tocopherol and ascorbic
acid at a single supplement could be high concentrations of the two antioxidants that surpassed
the safety threshold dosage of antioxidants.
The ROS production by early embryos and their susceptibility to ROS varies with the stages
of development. ATP production in the early embryo relies mostly on glycolysis, with less than
30% being produced via oxidative phosphorylation (Trimarchi et al., 2000; Harvey et al., 2002).
This suggests that more anaerobic metabolism of the early embryos could help to minimize the
production of ROS by the mitochondria, and thus reduce the risk of oxidative stress. In this regard,no embryotrophic effect of antioxidants was observed in the present study; neither l-ascorbic
acid nor -tocopherol had improved the cleavage rate at single or a divided supplement. Cleavage
rate was even lower when they were supplemented together at 0 h of culture. This indicates that
before the two cells stage embryo does not produce much ROS or they are more stable to the
deleterious effect of ROS or both. Embryo metabolism increases sharply as the cell number
increases in it. Because ROS production is related with metabolism, it is likely to produce
more ROS at advance stage of embryonic development. There is report that mouse blastomere
(Nonogaki et al., 1994) and bovine 916 cells stage embryos are vulnerable to ROS compared
to other stages (Ali et al., 2003). In the present study, no improvement in blastocyst frequency
was observed when -tocopherol was supplemented after 96 h of culture. This result suggest that
irreversible damage to the cell organelles might occur before this period that antioxidant cannot
recovered when added at/after 96 h of culture. Thus, antioxidant is recommended to supplement
into the culture media within first 48 h of culture.
5. Conclusion
Embryotrophic effects of-tocopherol were observed when media was supplemented a single
time with 100M or two times with 50M depending supplementing timing. On the other hand,
200Ml
-ascorbic acid alone or with 100 M -tocopherol had no effect on porcine embryonicdevelopment with a single time supplement. Simultaneous two time supplements of-tocopherol
and l-ascorbic acid with a divided concentration supported better porcine embryo development
and increase the embryo quality compared with control, no further beneficial effect was observed
compared with -tocopherol and l-ascorbic acid. So, we recommend to supplement porcine
embryo culture medium with 100M -tocopherol within first 48 h of culture at a single
supplement, or 50M -tocopherol or 100M l-ascorbic acid at twice supplements at 0 and
96 h of culture.
Acknowledgements
This study was supported by grants from the Korean Ministry of Science and Technology (Top
Scientist Fellowship). The authors acknowledge a graduate fellowship provided by the Ministry
of Education through BK21 program.
8/2/2019 Hwang ok
10/11
116 M.S. Hossein et al. / Animal Reproduction Science 100 (2007) 107117
References
Ali, A.A., Bilodeau, J.F., Sirard, M.A.,2003. Antioxidantrequirements for bovine oocytes varies during in vitro maturation,
fertilization and development. Theriogenology 59, 939949.
Attaran, M., Pasqualotto, E., Falcone, T., Goldberg, J., Miller, K., Agarwal, A., Sharma, R.K., 2000. The effect of follicular
fluid reactive oxygen species on outcome of in vitro fertilization. Int. J. Fertil. 45, 314320.
Buettner, G.R., 1993. The pecking order of free radicals and antioxidants: lipid peroxidation, -tocopherol, and ascorbate.
Arch. Biochem. Biophys. 300, 535543.
Chow, C.K., 1991. Vitamin E and oxidative stress. Free Radic. Biol. Med. 11, 215232.
Dalvit, G., Llanes, S.P., Descalzo, A., Insani, M., Beconi, M., Cetica, P., 2005. Effect of alpha-tocopherol and ascorbic
acid on bovine oocyte in vitro maturation. Reprod. Domest. Anim. 40, 9397.
Droge, W., 2002. Free radicals in the physiological control of cell function. Physiol. Rev. 82, 4795.
Eppig, J.J., Hosoe, M., OBrien, M.J., Pendola, F.M., Requena, A., Watanabe, S., 2000. Conditions that affect acquisition of
developmental competence by mouse oocytes in vitro: FSH, insulin, glucose and ascorbic acid. Mol. Cell. Endocrinol.
163, 109116.
Fraga, C.G., Motchnik, P.A., Shigenaga, M.K., Helbock, H.J., Jacob, R.A., Ames, B.N., 1991. Ascorbic acid pro-
tects against endogenous oxidative DNA damage in human sperm. Proc. Natl. Acad. Sci. U.S.A. 88, 11003110036.
Gardiner, C.S., Reed, D.J., 1995. Synthesis of glutathione in the preimplantation mouse embryo. Arch. Biochem. Biophys.
318, 3036.
Gardner, D.K., Lane, M., 2002. Development of viable mammalian embryos in vitro: evolution of sequential media.
In: Cibelli, J., Lanza, R.P., Campbell, K.H.S., West, M.D. (Eds.), Principles of Cloning. Academic Press, USA, pp.
187208.
Guerin, P., Mouatassim, S.E., Menezo, Y., 2001. Oxidative stress and protection against reactive oxygen species in the
pre-implantation embryo and its surroundings. Hum. Reprod. Update 7, 175189.
Harvey, A.J., Kind, K.L., Thompson, J.G., 2002. REDOX regulation of early embryo development. Reproduction 123,
479486.
Kim, H.S., Lee, G.S., Hyun, S.H., Lee, S.H., Nam, D.H., Jeong, Y.W., Kim, S., Kang, S.K., Lee, B.C., Hwang, W.S.,
2004. Improved in vitro development of porcine embryos with different energy substrates and serum. Theriogenology61, 13811393.
Kitagawa, Y., Suzuki, K., Yoneda, A., Watanabe, T., 2004. Effects of oxygen concentration and antioxidants on the in
vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentationin porcine embryos.
Theriogenology 62, 11861197.
Lapointe, S., Sullivan, R., Sirard, M.A., 1998. Binding of a bovine oviductal fluid catalase to mammalian spermatozoa.
Biol. Reprod. 58, 747753.
Leese, H.J., 1995. Metabolic control during preimplantation mammalian development. Hum. Reprod. Update 1, 6372.
McEvoy, T.G., Coull, G.D., Broadbent, P.J., Hutchinson, J.S., Speake, B.K., 2000. Fatty acid composition of lipids in
immature cattle, pig and sheep oocytes with intact zona pellucida. J. Reprod. Fertil. 118, 163170.
Motulsky, H., 2003. GraphPad Prisom Version 4.00 for Windows GraphPad Software. San Diego, California, USA, 176
pp.
Nasr-Esfahni, M.H., Johnson, M.H., 1991. The origin of reactive oxygen species in mouse embryos cultured in vitro.
Development 113, 551560.
Nonogaki, T., Noda, Y., Goto, Y., Kishi, J., Mori, T., 1994. Developmental blockage of mouse embryos caused by fatty
acids. Assist. Reprod. Genet. 11, 482488.
Olson, S.E., Seidel Jr., G.E., 2000. Culture of in vitro-produced bovine embryos with Vitamin E improves development
in vitro and after transfer to recipients. Biol. Reprod. 62, 248252.
Oyawoye, O., Abdel Gadir, A., Garner, A., Constantinovici, N., Perrett, C., Hardiman, P., 2003. Antioxidants and reactive
oxygen species in follicular fluid of women undergoing IVF: relationship to outcome. Hum. Reprod. 18, 2270
2274.
Riley, J.C., Behrman, H.R., 1991. In vivo generation of hydrogen peroxide in the rat corpus luteum during luteolysis.
Endocrinology 128, 17491753.
Rose, R.C., Bode, A.M., 1993. Biology of free radical scavengers: an evaluation of ascorbate. FASEB J. 7, 11351142.Sturmey, R.G., Leese, H.J., 2003. Energy metabolism in pig oocytes and early embryos. Reproduction 126, 197204.
Tatemoto, H., Ootaki, K., Shigeta, K., Muto, N., 2001. Enhancement of developmental competence after in vitro fertiliza-
tion of porcine oocytes by treatment with ascorbic acid 2-O-alpha-glucoside during in vitro maturation. Biol. Reprod.
65, 18001806.
8/2/2019 Hwang ok
11/11
M.S. Hossein et al. / Animal Reproduction Science 100 (2007) 107117 117
Thouas, G.A., Korfiatis, N.A., French, A.J., Jones, G.M., Trounson, A.O., 2001. Simplified technique for differential
staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts. Reprod. Biomed. Online 3,
2529.
Tilly, J.L., Tilly, K.I., 1995. Inhibitors of oxidative stress mimic the ability of follicle-stimulating hormone to suppress
apoptosis in cultured rat ovarian follicles. Endocrinology 136, 242252.
Trimarchi, J.R., Liu, L., Porterfield, D.M., Smith, P.J., Keefe, D.L., 2000. Oxidative phosphorylation dependent and-independent oxygen consumption by individual preimplantation mouse embryos. Biol. Reprod. 62, 18661874.
Wang, X., Falcone, T., Attaran, M., Goldberg, J.M., Agarwal, A., Sharma, R.K., 2002. Vitamin C and Vitamin E supple-
mentation reduce oxidative stress-induced embryo toxicity and improve the blastocyst development rate. Fertil. Steril.
78, 12721277.