chemicals : all chemicals were purchased from sigma - aldrich corporation 
 ( st . louis , mo , u.s.a . ) unless stated otherwise . plant material , preparation and isolation of trans--viniferin : the leaf 
 and stem of v. amurensis were gathered on keryong mountain in daejeon , 
 korea .
botanical identification and isolation of trans--viniferin were performed by 
 professor ki - hwan bae at the herbarium of the college of pharmacy , chungnam national 
 university , korea . dried leaf and stem of v. amurensis ( 4.6 kg )
were 
 extracted using methanol ( meoh ) ( 15 l  24 hr  3 times ) at room 
 temperature , filtered and concentrated to yield an meoh extract ( 658 g ) .
trans--viniferin 
 ( 1,148 mg ) was obtained from an meoh extract after purification by silica gel column 
 chromatography .
ovary collection , recover and in vitro oocyte maturation : ovaries of 
 prepubertal gilts were collected from a commercial abattoir and transported to the 
 laboratory within 2 hr in 0.9% ( w / v ) nacl solution supplemented with penicillin - g ( 100 
 iu / ml ) and streptomycin sulfate ( 100 mg / l ) at 30 to 
 35c .
the follicular fluid with oocytes was aspirated from 3- to 6-mm antral follicles with 
 a 10-ml disposable syringe and 18-gauge needle and collected in a 
 15-ml centrifuge tube .
cumulus - oocyte complexes ( coc ) were recovered 
 under a stereomicroscope ; those with at least three layers of compact cumulus cells and 
 homogenous cytoplasm were selected for ivm .
the selected cocs were washed three times in a 
 hepes - buffered tyrode s medium containing 0.05% ( w / v ) polyvinyl alcohol ( tlh - pva ) and 
 transferred into 500 l of tissue culture medium 199 ( life technologies , 
 rockville , md , u.s.a . ) supplemented with 26 mm sodium bicarbonate , 0.91 mm sodium pyruvate , 
 0.57 mm cysteine , 10 ng / ml epidermal growth factor , 0.5 
 iu / ml porcine luteinizing hormone , 0.5 iu / ml porcine 
 follicle stimulating hormone , 10% ( v / v ) porcine follicular fluid ( pff ) , 75 
 g / ml penicillin - g and 50 
 g / ml streptomycin .
after centrifugation at 1,600  g 
 for 30 min , the supernatants were collected and filtered sequentially through 1.2- and 
 0.45-m syringe filters ( gelman sciences , ann arbor , mi , u.s.a . ) .
the selected cocs were 
 washed three times in oocyte maturation medium containing hormone supplements , and 
 approximately 5060 oocytes were transferred into each well of a 4-well nunc dish ( nunc , 
 roskilde , denmark ) containing 500 l of culture medium and equilibrated at 
 least 2 hr with 5% co2 at 39c in a humidified atmosphere . after 22 hr of 
 maturation with hormones ,
the oocytes were washed twice in a maturation medium without 
 hormone supplements and then cultured for 22 hr without hormone supplements at 39c under 5% 
 co2 in air .
assessment of nuclear maturation : after 44 hr of culture , oocytes were 
 stained with 10 g / ml hoechst 33342 in absolute alcohol , 
 visualized under epifluorescence microscopy ( 330385 nm ; at a magnification of 400 ) and 
 assessed for nuclear progression .
oocyte nuclear maturation status was classified as 
 germinal vesicle ( gv ) , metaphase i , anaphase - telophase i and metaphase ii ( mii ) according to 
 meiotic maturation stage .
measurement of ros and intracellular gsh levels : the ivm oocytes were 
 sampled 44 hr after ivm to determine intracellular ros and gsh levels .
ros and gsh levels 
 were measured by methods previously described [ 37 , 
 41 ] .
briefly , h2dcfda 
 ( 2,7-dichlorodihydrofluorescein diacetate ; invitrogen ) and celltracker blue cmf2hc 
 ( 4-chloromethyl-6.8-difluoro-7-hydroxycoumarin ; invitrogen ) were used to detect 
 intracellular ros as green fluorescence and gsh level as blue fluorescence , respectively . 

ten oocytes from each treatment group were incubated ( in the dark ) for 30 min in tlh - pva 
 supplemented with 10 m h2dcfda and 10 m celltracker . 

after incubation , oocytes were washed with d - pbs ( invitrogen , carlsbad , ca , u.s.a . ) 
 containing 0.1% ( w / v ) pva and placed into 10 l microdrops , and 
 fluorescence was observed under an epifluorescence microscope ( te300 ; nikon , tokyo , japan ) 
 with uv filters ( 460 nm for ros and 370 nm for gsh ) .
the fluorescence intensities of oocytes were analyzed with the 
 image j software ( version 1.41o ; national institutes of health , bethesda , md , u.s.a . ) and 
 normalized to the control .
we performed another gsh measurement method for more accurate 
 determination of each oocyte s gsh value .
after ivm ( 4244 hr ) , the oocytes were stripped of 
 surrounding cumulus cells by repeated pipetting , and matured oocytes ( defined as oocytes in 
 which the first pb was visualized under a stereomicroscope ) were selected for gsh 
 measurement .
briefly , mii 
 oocytes from each group were washed three times in 0.2 m sodium phosphate buffer 
 ( na2hpo4 , nah2po4 and 10 mm edta-2na , ph 7.2 ) , 
 and groups of 5060 oocytes ( per sample ) in 10 l sodium phosphate buffer 
 were transferred to 1.7-ml microfuge tubes ; 10 l of 1.25 
 mm phosphoric acid ( final concentration of 0.625 m h3po4 ) in distilled 
 water was added to each sample .
gsh concentrations in the oocytes were determined using a 
 5,5-dithio - bis-(2-nitrobenzoic acid ) ( dtnb)-gsh reductase ( gssg ) recycling assay . before 
 the assay ,
the frozen samples were thawed at room temperature , vortexed , centrifuged and 
 microscopically evaluated to ensure complete lysis of the oocytes .
the supernatants were 
 transferred to a 96-well microtiter plate , and for each sample , 700 l of 
 0.33 mg / ml nadph in 0.2 m assay buffer containing 10 mm edta ( stock buffer , 
 ph 7.2 ) , 100 l of 6 mm dtnb in the stock buffer and 180 
 l of distilled water and 1 u per sample of gssg ( sigma g3664 , 441 
 u / ml ) were added in a conical tube , mixed and immediately added to the 
 sample .
the plate was immediately placed in a microtiter plate reader , and optical density 
 was measured with a 405-nm filter ( emax , molecular devices , sunnyvale , ca , u.s.a . ) .
standard 
 curves were prepared for each assay , and gsh content per sample was determined using the 
 standard curve .
the gsh concentrations ( pm / oocyte ) were calculated by dividing the total 
 concentration per sample by the total number of oocytes present in the sample .
gene expression analysis by real - time polymerase chain reaction ( rt - pcr ) : 
 rt - pcr was performed with 120 matured cocs at a time .
after ivm , cocs were denuded by gently 
 pipetting with 0.1% hyaluronidase , and oocytes were washed three times in tlh - pva .
isolated 
 cumulus cells and cumulus - free ( or denuded ) matured oocytes were separately selected under a 
 stereomicroscope for the gene expression study .
total 
 rna was extracted using the trizol reagent ( invitrogen ) , according to the manufacturer s 
 protocol , and the total rna concentration was determined by measuring the absorbance at 260 
 nm .
first - strand complementary dna ( cdna ) was prepared by subjecting 1 g 
 of total rna to reverse transcription using moloney murine leukemia virus ( mmlv ) reverse 
 transcriptase ( invitrogen ) . to determine the conditions for logarithmic - phase pcr 
 amplification of target mrna
, 1-g aliquots were amplified using differing 
 numbers of cycles . the housekeeping gene ,
cytochrome oxidase subunit 1 ( 1a ) , was pcr 
 amplified to rule out the possibility of rna degradation and to control for the variation in 
 mrna concentrations in the rt reaction .
a linear relationship between the pcr product band 
 visibility and the number of amplification cycles was observed for the target mrnas .
the cdna was amplified in a 
 20-l pcr reaction , which contained 1 u taq polymerase ( intron bio 
 technologies , co. , ltd . , seongnam , korea ) , 2 mm dntp mix and 10 pm of each gene - specific 
 primer .
quantitative real - time pcr was performed with 1 l cdna template 
 added to 10 l 2 x sybr premix ex taq ( takara bio inc . , otsu , japan ) 
 containing specific primers at a concentration of 10 pm each .
the reactions were carried out 
 for 32 cycles , and the cycling parameters were as follows : denaturation at 95c for 30 sec , 
 annealing at 55c for 30 sec and extension at 72c for 30 sec .
all oligonucleotide primer 
 sequences are presented in table 1table 1.sequences of the oligonucleotide primers and probe used in rt - pcrgeneprimer sequencesproduct size ( bp)gene bank accession numberpcnaf : 5-cctgtgcaaaagatggagtg-3187xm_003359883r : 5-ggagagagtggagtggctttt-3bakf : 5-gcggaaaacgcctatgagta-3189xm_001928147r : 5-gcagtgatgcagcatgaagt-3baxf : 5-tgcctcaggatgcatctacc-3199xm_003127290r : 5-aagtagaaaagcgcgaccac-3dnmt1f : 5-cctctatggacggcttgagt-3185nm_001032355r : 5-ggtgcttgtccaggatgttg-3oct4f : 5-gcggacaagtatcgagaacc-3200nm_001113060r : 5-cctcaaaatcctctcgttgc-3bcl2f : 5-agggcattcagtgacctgac-3193nm_214285r : 5-cgatccgactcaccaatacc-3caspase-3f : 5-cgtgcttctaagccatggtg-3186nm_214131r : 5-gtcccactgtccgtctcaat-31-af : 5-caccgtaggaggtctaacg-3293ap_003428r : 5-gtatcgtcgaggtattccg-3. the fluorescence intensity was measured at the end of the extension phase of 
 each cycle .
the reaction cycle at which the pcr products exceeded this fluorescence intensity 
 threshold was deemed the threshold cycle ( ct ) in the exponential phase of the pcr 
 amplification .
the expression of target gene was quantified relative to that of the internal 
 control gene .
the relative quantification was based on a comparison of cts at constant 
 fluorescence intensity .
the amount of transcript present was inversely related to the 
 observed ct , and for every two - fold dilution in the amount of transcript , ct was expected to 
 increase by 1 . the relative expression ( r )
was calculated using the equation r=2[ct 
 samplect control ] . to determine a normalized arbitrary value for each gene , every data 
 point was normalized to the control gene as well as to its respective control .
parthenogenesis : for parthenogenesis ( pa ) , oocytes that reached the mii 
 stage at 44 hr of ivm were activated with two pulses of 120 v / mm dc for 60 
 sec in 280 mm mannitol solution containing 0.01 mm cacl2 and 
 0.05 mm mgcl2 .
following electrical activation , pa embryos were treated with 0.4 
 g / ml demecolcine and 5 
 g / ml cytochalasin b in in vitro 
 culture ( ivc ) medium for 4 hr , respectively . the pa embryos were washed three times with 
 embryo culture medium and cultured in 25 l microdrops ( 10 
 gametes / microdrop ) of porcine zygote medium 3 ( pzm3 ) . the embryos with cultured microdrops
were covered with pre - warmed mineral oil 
 and incubated at 39c for 168 hr under a humidified atmosphere of 5% o2 , 5% 
 co2 and 90% n2 . in all experiments ,
the culture media were renewed 
 at 48 hr ( day 2 ) and 96 hr ( day 4 ) after pa . in vitro fertilization : the ivf procedure performed was that reported by 
 kwak et al . .
for ivf , liquid 
 semen was supplied weekly by the veterinary service laboratory ( department of livestock 
 research , yong - in city , gyeonggi - do , republic of korea ) and kept at 17c for 5 days before 
 use .
the semen sample was washed twice by centrifugation with dulbecco s phosphate - buffered 
 saline ( dpbs ) supplemented with 0.1% bsa at 2,000  g for 2 min .
after 
 washing , the sperm pellet was resuspended in modified tris - buffered medium ( mtbm ) that had 
 been pre - equilibrated for 18 hr at 39c under 5% co2 .
after 44 hr of ivm , the 
 cocs were denuded by gently pipetting with 0.1% hyaluronidase and washed three times in 
 tlh - pva .
groups of 15 
 oocytes were randomly placed into 40 l microdrops of mtbm in a 35  10 mm 
 petri dish ( falcon ; bd labware , franklin lakes , nj , u.s.a . ) covered with pre - warmed mineral 
 oil . after appropriate dilution ,
5 l of the sperm suspension was added to 
 a 40 l microdrop of fertilization medium ( mtbm ) to yield a final sperm 
 concentration of 1  10 sperm / ml . just before fertilization , 
 sperm motility was assessed , and more than 80% motile sperms were used in every experiment . 
 to use stored liquid semen ,
the oocytes were 
 co - incubated with sperms for 20 min at 39c in a humidified atmosphere of 5% co2 
 and 95% air .
after 20 min co - incubation with sperm , the loosely attached sperms were removed 
 from the zona pellucida ( zp ) by gentle pipetting .
the oocytes were then washed three times 
 in mtbm and incubated in mtbm without sperm for 56 hr at 39c in a humidified atmosphere of 
 5% co2 and 95% air .
thereafter , gametes were washed three times with embryo 
 culture medium and cultured in 25 l microdrops ( 10 gametes / microdrop ) of 
 pzm3 . the embryos with cultured microdrops
hr under a humidified atmosphere of 5% o2 , 5% 
 co2 and 90% n2 .
in all experiments , the culture media were renewed 
 at 48 hr ( day 2 ) and 96 hr ( day 4 ) after ivf .
experimental design : in experiment 1 , the effect of trans--viniferin 
 treatment during ivm on oocyte nuclear maturation was examined .
oocytes were randomly 
 allocated and cultured in ivm media supplemented with different concentrations of 
 trans--viniferin ( 0 , 0.1 , 0.5 , 1.0 and 5.0 m ) for the whole culture 
 period ( 44 hr ) .
after ivm , nuclear maturation was evaluated by hoechst 33342 staining . in 
 experiment 2 ,
the effect of trans--viniferin treatment during ivm on the intracellular 
 levels of gsh and ros was examined .
oocytes were randomly allocated and cultured in ivm 
 media supplemented with different concentrations of trans--viniferin ( 0 , 0.5 and 5.0 
 m ) for the whole culture period ( 44 hr ) .
after ivm , the intracellular 
 levels of gsh and ros were evaluated . in experiment 3 ,
the effect of trans--viniferin 
 treatment during ivm on the expression of proliferating cell nuclear antigen ( pcna ) , 
 octamer - binding transcription factor 4 ( oct4 ) , dnmt1 , caspase-3 , bcl-2 homologous antagonist 
 killer ( bak ) and bcl-2associated x protein ( bax ) mrna in matured oocytes and bak , bax , 
 caspase-3 and b - cell lymphoma 2 ( bcl2 ) mrna in cumulus cells were analyzed .
the mrna 
 expression was compared in the control group and a treated ( 0.5 m ) group . 
 in experiment 4 ,
the effect of trans--viniferin treatment during ivm on subsequent 
 developmental competence in pa and ivf embryos was examined .
a one - way analysis of variance with duncan s 
 multiple - range test was used to assess nuclear maturation rate , gsh and ros levels , cleavage 
 rate , developmental rate of blastocysts and total cell numbers .
trans--viniferin treatment during ivm did not improve the nuclear maturation of oocytes in 
 the treated groups compared with the control group ( table 2table 2.effect of trans--viniferin treatment during porcine ivm on nuclear 
 maturationtrans--viniferinconcentration 
 ( m)no . of oocytes
culturedfor maturation% of oocytes at the stage ofgerminalvesiclemetaphase lanaphase andtelophase lmetaphase ll0 ( control)1205.0  1.78.3  1.02.5  1.684.2  0.80.11204.1  2.18.3  1.00.8  0.886.6  1.40.51145.1  2.16.9  1.42.6  0.985.5  0.81.01205.8  3.78.3  2.22.5  1.683.3  1.95.01204.1  2.513.3  2.43.3  1.479.2
a , b ) within a column , means without a 
 common superscript differ ( p<0.05 ) . ) .
the control group and treated groups had similar proportions of matured 
 oocytes ( mii , anaphase and telophase i stage rates : 84.2 , 86.6 , 85.5 , 83.3 and 79.2% ; 2.5 , 
 0.8 , 2.6 , 2.5 and 3.3% ; and 0 , 0.1 , 0.5 , 1.0 and 5.0 m , respectively ) . 

there were 
 significantly more immature oocytes in the 5.0 m treatment group than in 
 the 0.5 m treatment group .
the 5.0 m treatment group 
 ( 13.3% ) had an increased ( p<0.05 ) number of mi stage oocytes as compared 
 with the 0.5 m treatment group ( 6.9% ) .
there were significantly fewer 
 mature oocytes in the 5.0 m treatment group than 0.1 than in the 0.5 
 m treatment groups .
the 5.0 m treatment groups ( 79.2% ) 
 had a decreased ( p<0.05 ) number of mii stage oocytes as compared with 
 the 0.1 and 0.5 m treatment groups ( 86.6 and 85.5% ) .
a , b ) within a column , means without a 
 common superscript differ ( p<0.05 ) .
trans--viniferin treatment increased ( p<0.05 ) intracellular gsh levels 
 and decreased ( p<0.05 ) ros generation in mii oocytes after ivm ( figs . 2 and 
 3fig .
oocytes were stained with celltracker blue ( a  c ) and h2dcfda ( d  f ) to detect 
 intracellular levels of glutathione and reactive oxygen species , respectively . 

metaphase ii ( mii ) oocytes derived from the maturation medium supplemented with 0.5 
 m trans--viniferin ( b and e ) , 5.0 m 
 trans--viniferin ( c and f ) or without trans--viniferin ( a and d).fig .
3.effect of trans--viniferin in maturation medium on intracellular glutathione ( gsh ) 
 and reactive oxygen species ( ros ) levels in in vitro matured porcine 
 oocytes .
a , b values with different 
 superscripts are significantly different ( p<0.05 ) . ) .
the 0.5 and 5.0 m trans--viniferin treatment groups showed 
 significantly higher gsh levels as compared with the control group ( 0.5 and 5.0 
 m vs. control : 1.12 and 1.19 vs. 1.0 pixel / oocyte ) . additionally ,
the 
 trans--viniferin treatment groups showed significantly reduced ros levels as compared with 
 the control group ( 0.5 and 5.0 m vs. control : 0.14 and 0.13 vs. 1.0 
 pixel / oocyte ) .
the gsh reductase recycling assay results revealed that the 5.0 
 m treatment group ( 16.11 pm / oocyte ) had similar gsh levels as compared 
 with the control group ( 14.57 pm / oocyte ) ( table 
 3table 3.effect of trans--viniferin in maturation medium on intracellular glutathione 
 ( gsh ) concentration in in vitro - matured porcine oocytestrans--viniferin concentration 
 ( m)0 ( control)0.55.0gsh concentration ( pm / oocyte)(no . of oocytes 
 examined)14.57  0.25(151)16.77  0.18(129)16.11
a , b ) values with different superscripts 
 are significantly different ( p<0.05 ) . ) .
only the 0.5 m treatment group ( 16.77 pm / oocyte ) showed 
 significantly higher gsh levels as compared with the control group .
oocytes were stained with celltracker blue ( a  c ) and h2dcfda ( d  f ) to detect 
 intracellular levels of glutathione and reactive oxygen species , respectively . 

metaphase ii ( mii ) oocytes derived from the maturation medium supplemented with 0.5 
 m trans--viniferin ( b and e ) , 5.0 m 
 trans--viniferin ( c and f ) or without trans--viniferin ( a and d ) .
effect of trans--viniferin in maturation medium on intracellular glutathione ( gsh ) 
 and reactive oxygen species ( ros ) levels in in vitro matured porcine 
 oocytes .
a , b ) values with different superscripts 
 are significantly different ( p<0.05 ) .
the effects of trans--viniferin treatment during ivm on the expression of transcription 
 factors and genes involved in apoptosis and cell proliferation in mature oocytes are shown 
 in fig .
sem expression of pcna , oct4 , dnmt1 , caspase-3 , bak and bax mrna in matured 
 oocytes treated with trans--viniferin during in vitro maturation . 

p<0.05 vs. 
 control .. trans--viniferin treatment during ivm promoted higher ( p<0.05 ) 
 dnmt1 mrna expression in the 0.5 m treatment group as compared with the 
 control .
however , the expression of other genes ( pcna , oct4 , caspase-3 , bak and bax ) did not 
 significantly differ from the control .
mean  sem expression of pcna , oct4 , dnmt1 , caspase-3 , bak and bax mrna in matured 
 oocytes treated with trans--viniferin during in vitro maturation . 

the effects of trans--viniferin treatment during ivm on the expression of 
 apoptosis - related genes in cumulus cells are shown in fig . 5fig .
5.mean  sem expression of bak , bax , caspase-3 and bcl2 mrna in cumulus cell treated 
 with trans--viniferin during in vitro maturation .
p<0.05 vs. control .. trans--viniferin treatment during ivm significantly reduced 
 ( p<0.05 ) bax mrna expression of cumulus cells in the 0.5 
 m treatment group as compared with the control .
mean  sem expression of bak , bax , caspase-3 and bcl2 mrna in cumulus cell treated 
 with trans--viniferin during in vitro maturation .
no significant differences in cleavage rates among the groups were observed for both pa and 
 ivf embryos on day 2 ( table 4table 4.effect of trans--viniferin treatment during ivm on embryonic development in 
 porcine pa embryostrans--viniferinconcentration 
 ( m)no . of embryosculturedembryos developed to ( % ) total cell numberin blastocyst2cellblastocyst0 ( control)9976.7  3.750.0  5.843.1  2.10.510675.8  1.152.3  1.759.6  4.25.09881.4  2.950.3 
a , b ) within a column , means without a 
 common superscript differ ( p<0.05 ) . ) . about 7580% of pa embryos and 60% of ivf embryos cleaved in all groups 
 ( table 5table 5.effect of trans--viniferin treatment during ivm on embryonic development in 
 porcine ivf oocytestrans--viniferinconcentration 
 ( m)no . of embryosculturedembryos developed to ( % ) total cell numberin blastocyst2cellblastocyst0 ( control)14058.1  4.220.1  1.636.4  2.20.513759.6  3.126.3  3.753.6  4.05.013157.3  1.128.4 
a , b ) within a column , means without a 
 common superscript differ ( p < 0.05 ) . ) .
no significant differences in blastocyst formation were observed in pa and 
 ivf embryos among the groups at day 7 ( tables 4 
 and 5 ) .
approximately 50% of pa embryos and 
 2025% of ivf embryos developed to the blastocyst stage in all groups .
the numbers of cells 
 in the blastocysts of pa and ivf embryos increased ( p<0.05 ) in the 
 trans--viniferin treatment groups as compared with the control .
total cell numbers of pa 
 embryos increased significantly in the 0.5 and 5.0 m treatment groups 
 ( 59.6  4.2 and 60.8  4.6 ) as compared with the control group ( 43.1  2.1 ) ( table 4 ) .
ivf embryos showed similar results ; total 
 cell number increased significantly in the 0.5 and 5.0 m treatment groups 
 ( 53.6  4.0 and 47.9  3.1 ) as compared with the control group ( 36.4  2.2 ) ( table 5 ) .
a , b ) within a column , means without a 
 common superscript differ ( p<0.05 ) .  
a , b ) within a column , means without a 
 common superscript differ ( p < 0.05 ) .
in vitro swine embryo production systems are inefficient as compared with 
 in vivo systems .
incomplete cytoplasmic maturation is believed to result in abnormal fertilization , 
 including polyspermy and asynchronous pronuclear formation , which are thought to be the major reasons for poor developmental competence 
 of in vitro matured / fertilized embryos .
the addition of various antioxidants has been examined in an attempt to 
 improve the quality of in vitro produced embryos due to their protective 
 effects during culture [ 1 , 6 , 35 , 51 ] . no previous reports are available regarding the influence of 
 trans--viniferin .
we demonstrated that trans--viniferin treatment during ivm had 
 beneficial effects on oocyte maturation , increasing intracellular gsh synthesis , reducing 
 ros levels and increasing dnmt1 gene expression of oocytes ; reduced bax gene expression of 
 cumulus cells ; and increased the total cell number of blastocysts in subsequent embryonic 
 development of pa and ivf embryos .
some 
 differences were observed among the treatment groups , but there were no significant 
 differences between the treatment groups and control group .
resveratrol derivatives , including 
 trans - resveratrol-4-o--d - glucoside , transresveratrol , ( + ) -ampelopsin a , trans--viniferin , 
 cis--viniferin , -2-viniferin , gnetin h and suffruticosol a and b , have been isolated from 
 more than 70 plant species including grapes , plums and peanuts [ 24 , 39 ] . among these plants ,
trans--viniferin may have 
 antioxidant effects as a hydroxyl radical scavenger in vivo [ 25 , 29 ] . 

furthermore , trans--viniferin may inhibit glutamate - induced increases in intracellular 
 calcium ion concentrations , ros generation , changes in apoptosis - related proteins and 
 hypoxia - induced neuronal cell death in cultured neurons .
jeong et al . 
 reported that trans--viniferin protected against glutamate - induced neurotoxicity in 
 cultured cortical neurons ; pretreatment of mouse cortical neurons with 5 m 
 of trans--viniferin reduced the neuronal death induced by 500 m 
 glutamate , and glutamate - induced neuronal death is usually associated with the elevation of 
 intracellular calcium ion concentrations following nmda receptor activation .
they also demonstrated that trans--viniferin at 
 concentrations of 5.0 m showed significant inhibition of elevation of 
 glutamate - induced intracellular calcium ion concentrations in cultured cerebral cortical 
 neurons , the involvement of oxidative stress toxicity could be investigated by measurement 
 of ros accumulation and trans--viniferin ( 0.5 , 1.0 and 5.0 m ) showed 
 concentration - dependent inhibition of the glutamate - induced ros generation in cultured 
 cerebral cortical neurons .
similar antioxidative 
 effects of trans--viniferin on porcine oocytes were observed in the present study .
the 0.5 
 and 5.0 m trans--viniferin treatment during ivm effectively reduced ros 
 levels and increased gsh levels , but did not show a concentration - dependent effect .
it is 
 estimated because of the difference between cell types and whether or not artificial 
 induction of ros .
this finding was most likely due to the antioxidative activity of intracellular gsh , which 
 was increased by trans--viniferin treatment .
we inferred that trans--viniferin was 
 involved in cytoplasmic maturation rather than nuclear maturation and increased 
 intracellular gsh levels in ivm oocytes , which contributed to improve oocyte quality . 

however , no beneficial effect of trans--viniferin treatment was found during blastocyst 
 formation , even though trans--viniferin effectively reduced ros levels and increased gsh 
 levels .
many antioxidant compounds have been used to avoid oxidative stress during 
 in vitro culture .
transferrin , penicillamine , hypotaurine and taurine are 
 often added to culture media , because positive effects of these compounds on embryo 
 development have been observed [ 2 , 4 , 36 , 38 ] .
the positive effects of some plant extracts , such as 
 anthocyanin and resveratrol , have also been reported [ 29 , 51 ] . however , avoiding oxidative stress 
 during oocyte and embryo culture is a complex problem . simply adding the necessary ros 
 scavengers is insufficient , as the choice of antioxidant compounds to be used and their 
 concentrations are difficult to ascertain .
some compounds , such as thiols and vitamins , must 
 be used with care , as they too can have a negative impact on the embryo [ 32 , 43 ] .
further , 
 an excess of antioxidant compounds may have deleterious effects on the embryo .
most studies have shown that prolonged 
 experimentally induced ros production severely inhibits embryonic development .
ros concentrations increase during the two  four - cell 
 transition period in mice , indicating that an 
 increase in ros may be associated with the arrest of development at the two - cell stage . 

however , excessive reduction of ros after treatment with a high level of antioxidants has 
 toxic effects on bovine embryonic development and the viability of human cultured cells 
 [ 12 , 41 ] . 

thus , ros may play a pivotal role in the regulation of cell proliferation and embryonic 
 development .
furthermore , an appropriate level of ros may be necessary for embryonic 
 development and cell proliferation . in this study
, trans--viniferin effectively reduced 
 ros , but the level of ros was not appropriate ros for embryonic development
. the blastocyst 
 formation rate did not change , and only total cell number increased , which is a meaningful 
 result .
increased total cell number in 
 mouse embryos is associated with improved embryo quality and postimplantation developmental 
 potential .
when koo et al . 
  compared the total cell numbers of in 
 vivo derived blastocysts to in vitro derived blastocysts and 
 blastocysts derived from somatic cell nuclear transfer , total cell numbers varied from a 
 mean of 122.5 for in vivo blastocysts ( highest developmental potential ) to 
 108.2 cells for in vitro derived blastocysts and 98.0 cells for nuclear 
 transfer blastocysts ( lowest developmental potential ) in cattle . hence , the increased total cell numbers of blastocysts derived from 
 cocs exposed to trans--viniferin could reflect improved embryo quality .
gene expression patterns can indicate oocyte status and can be influenced by the culture 
 environment .
. pcna is an essential component of the dna replication and 
 repair machinery , oct4 is essential for early 
 development of mouse and human embryos [ 5 , 22 ] , dnmt1 is involved in dna methylation and cell 
 proliferation   and caspase-3 , bak , bax and bcl2 
 are associated with apoptosis [ 15 , 21 ] .
activated caspase-3 cleaves numerous 
 proteins , triggering biochemical cascades that lead to cell death .
trans--viniferin ( 5.0 
 m ) significantly blocked the glutamate - induced decrease in bcl-2 and 
 increase in bax expression in cultured cerebral cortical neurons . in the current study , apoptosis - related gene ( caspase-3 , bak and 
 bax ) expression in oocytes was not altered .
in cumulus cells , bax gene expression was 
 significantly decreased in the treatment groups ( p<0.05 ) . during oocyte 

maturation , cumulus cells support oocytes via small molecule transport . reducing apoptosis 
 of cumulus cells
dnmt1 
 accumulates in oocytes during the growth phase and is in the cytoplasm of mature mii 
 oocytes , where it maintains dna methylation and might also be involved in maintaining 
 imprints .
dna methylation gradually increases during germ cell development before 
 fertilization [ 9 , 53 ] .
epigenetic markers , such as genomic methylation , regulate gene expression and 
 development [ 19 , 30 ] .
interfering with the proper establishment of methylation can result in 
 tumorigenesis and death [ 11 , 16 , 31 ] .
therefore , maintaining 
 appropriate methylation levels are important for embryonic development . increased dnmt1 
 expression in mii oocytes maintains high dna methylation before fertilization .
in other 
 words , trans--viniferin treatment improved cytoplasm quality by increasing dnmt1 
 expression .
supplementation with trans--viniferin during ivm of porcine oocytes could help cytoplasmic 
 maturation by increasing intracellular gsh concentrations and decreasing ros levels , leading 
 to appropriate gene expression .
but , trans--viniferin did not affect nuclear maturation , 
 and it even showed adverse effects at a high concentration .
improvement of oocyte 
 cytoplasmic quality might cause an increase of total cell number of blastocysts during 
 subsequent in vitro development . however , trans--viniferin treatment 
 during ivm did not improve the subsequent blastocyst formation rate , and it was unclear how 
 trans--viniferin affected the oocyte cytoplasm .
the mechanism underlying the effects of 
 trans--viniferin during ivm should be determined to identify ways to improve developmental 
 competence during ivp .
 abstracttrans--viniferin is a naturally occurring polyphenol belonging to the stilbenoid family 
 that has been isolated from vitis amurensis , one of the most common wild 
 grapes in asia . 
 we investigated the effects of trans--viniferin on in 
 vitro maturation ( ivm ) and developmental competence after in 
 vitro fertilization ( ivf ) or parthenogenesis ( pa ) . 
 we observed that 
 trans--viniferin treatment during ivm did not improve nuclear maturation rates of oocytes 
 in any group , but significantly increased ( p<0.05 ) intracellular 
 glutathione ( gsh ) levels and reduced reactive oxygen species ( ros ) levels in the 0.5 
 m treatment group . 
 trans--viniferin treatment during ivm of recipient 
 oocytes promoted higher ( p<0.05 ) expression of dna methyltransferase-1 
 ( dnmt1 ) mrna in the 0.5 m treatment group as compared with the control 
 group . 
 however , the expression of essential transcriptional and apoptosis - related genes 
 did not significantly differ from that of the control . in cumulus cells , 
 pro - apoptosis 
 gene expressions were changed as apoptosis decreased . 
 oocytes treated with 
 trans--viniferin during ivm did not have significantly different cleavage rates or 
 blastocyst formation rates after pa , but total cell numbers were significantly higher 
 ( p<0.05 ) in the 0.5 and 5.0 m treatment groups 
 compared with those in the control group . 
 ivf embryos showed similar results . in 
 conclusion , these results indicate that trans--viniferin treatment during porcine ivm 
 increased the total cell number of blastocysts , possibly by increasing intracellular gsh 
 synthesis , reducing ros levels , increasing dnmt1 gene expression of oocytes and decreasing 
 pro - apoptosis gene expressions of cumulus cells . 
MATERIALS AND METHODS
RESULTS
DISCUSSION