this study was carried out in strict accordance with the recommendations of the guide for the care and use of laboratory animals of the national veterinary and 
 quarantine service ( anyang , korea ) . the protocol was approved by the committee on the ethics of animal experiments of chungbuk national university ( cheongju , 
 korea ) ( permit number : cbnua-584 - 13 - 01 ) . all surgery was performed under isoflurane anesthesia , and all efforts were made to minimize suffering . porcine ovaries were obtained from a local slaughterhouse and transported to the laboratory within 2 h in 0.9% ( w / v ) nacl solution that was supplemented with 
 penicillin - g ( 100 iu / ml ) and streptomycin sulfate ( 100 mg / l ) at 3035c . follicular fluid with oocytes was aspirated from antral follicles ( 36 mm in diameter ) 
 using an 18-gauge needle connected to a 10-ml disposable syringe and collected into a 15-ml centrifuge tube . cumulus - oocyte complexes ( cocs ) were recovered 
 under a stereomicroscope , and those with at least three layers of compact cumulus cells and a homogenous cytoplasm were selected for ivm . the selected cocs were 
 washed three times in hepes - buffered tyrode s medium containing 0.05% ( w / v ) polyvinyl alcohol ( tlh - pva ) , and transferred to 500 l of tissue culture medium 199 
 ( invitrogen , carlsbad , ca , usa ) supplemented with 0.8 g / ml zinc ( zinc supplemented group only ) , 0.6 mm cysteine , 0.91 mm sodium pyruvate , 10 ng / ml epidermal 
 growth factor , 75 g / ml kanamycin , 1 g / ml insulin , and 10% ( v / v ) pig follicular fluid . the concentration of zinc was set at 0.8 g / ml in accordance with 
 previous report . for maturation , the selected cocs were washed three times in the above described tissue culture 
 medium 199 ( ivm medium ) containing hormone supplements ( 10 iu / ml equine chorionic gonadotropin and 10 iu / ml human chorionic gonadotropin [ intervet , boxmeer , the 
 netherlands ] ) , and approximately 5060 oocytes were transferred to each well of a 4-well nunc dish ( roskilde , denmark ) containing 500 l of culture medium and 
 equilibrated for at least 2 h with 5% co2 at 39c in a humidified atmosphere . after 22 h of maturation with hormones , the cocs were washed twice and 
 cultured in hormone - free ivm medium for an additional 1820 h. fetal fibroblasts were derived and cultured as described previously . briefly , a pig fetus ( landrace  duroc 
 crossbreed ) at embryonic day 40 was isolated from the uterus of a female pig . the tissues of the fetus were cut into 0.5-mm pieces and cultured in a 
 100-mm culture dish . cells were grown at 37c in an atmosphere of 5% co2 in dulbecco s modified eagle s medium ( dmem ) containing 4.5 g / l glucose 
 ( gibco brl , grand island , ny , usa ) and supplemented with fetal bovine serum ( 10% ) , penicillin ( 100 u / ml ) , and streptomycin ( 100 g / ml ) . prior to scnt , the cells were thawed and cultured in above described dmem for 34 
 days until they reached 80% confluence . adherent cells ( passage 7~10 ) were treated with trypsin for ~1 min and used for scnt . after 4042 h of ivm , denuded oocytes were incubated for 5 min in manipulation medium ( calcium - free tlh containing 0.2% bovine serum albumin [ tlh - bsa ] ) 
 containing 5 g / ml hoechst 33342 , washed twice with fresh manipulation medium , and transferred to a drop of manipulation medium containing 5 g / ml cytochalasin 
 b. the oocytes were enucleated by aspirating the polar body and mii chromosomes using a 16-m glass pipette ( humagen , charlottesville , va , usa ) . after 
 enucleation , a trypsinized fetal fibroblast of 1415 m with a smooth cell surface was transferred to the perivitelline space of the enucleated oocyte . the 
 couplets were equilibrated with 280 mm mannitol solution containing 0.001 mm cacl2 and 0.05 mm mgso4 for 23 min and then transferred to a 
 fusion chamber containing two electrodes overlaid with 280 mm mannitol solution . membrane fusion was induced by applying an alternating current field of 2 v 
 cycling at 1 mhz for 2 sec , followed by two pulses of 160 v / mm direct current for 60 sec using a cell fusion generator ( lf201 ; nepa gene , chiba , japan ) . the oocytes were examined after 30 min , 
 and fused , normally shaped oocytes were collected . the collected scnt embryos were washed three times with ivc medium ( pzm 3 ) and cultured in 30-l microdrops of ivc medium . embryos in culture medium were 
 covered with pre - warmed mineral oil and incubated at 39c for 7 days under a humidified atmosphere of 5% o2 , 5% co2 , and 90% 
 n2 . blastocyst 
 formation was assessed on day 7 . at 4 h post - activation , scnt embryos were transferred to naturally cycling landrace  duroc crossbreed gilts on the first day of standing estrus . the reproductive tract was exposed , and the scnt embryos ( 100120 embryos per recipient ) 
 were transferred into an oviduct at the ampullary isthmic junction . if 
 fetal echoes did not correspond with the gestational age of the fetuses and if signs of fetal absorption , such as small vesicles without any detectable fetuses , 
 were observed , an abortion was considered to have occurred . data related to the rates of cleavage , blastocyst formation , litter size , and production efficiency are shown as mean  sem and were compared by student s 
 t - test . data related to pregnancy rate and pregnancy status were analyzed using chi - square tests . this study was carried out in strict accordance with the recommendations of the guide for the care and use of laboratory animals of the national veterinary and 
 quarantine service ( anyang , korea ) . the protocol was approved by the committee on the ethics of animal experiments of chungbuk national university ( cheongju , 
 korea ) ( permit number : cbnua-584 - 13 - 01 ) . all surgery was performed under isoflurane anesthesia , and all efforts were made to minimize suffering . porcine ovaries were obtained from a local slaughterhouse and transported to the laboratory within 2 h in 0.9% ( w / v ) nacl solution that was supplemented with 
 penicillin - g ( 100 iu / ml ) and streptomycin sulfate ( 100 mg / l ) at 3035c . follicular fluid with oocytes was aspirated from antral follicles ( 36 mm in diameter ) 
 using an 18-gauge needle connected to a 10-ml disposable syringe and collected into a 15-ml centrifuge tube . cumulus - oocyte complexes ( cocs ) were recovered 
 under a stereomicroscope , and those with at least three layers of compact cumulus cells and a homogenous cytoplasm were selected for ivm . the selected cocs were 
 washed three times in hepes - buffered tyrode s medium containing 0.05% ( w / v ) polyvinyl alcohol ( tlh - pva ) , and transferred to 500 l of tissue culture medium 199 
 ( invitrogen , carlsbad , ca , usa ) supplemented with 0.8 g / ml zinc ( zinc supplemented group only ) , 0.6 mm cysteine , 0.91 mm sodium pyruvate , 10 ng / ml epidermal 
 growth factor , 75 g / ml kanamycin , 1 g / ml insulin , and 10% ( v / v ) pig follicular fluid . the concentration of zinc was set at 0.8 g / ml in accordance with 
 previous report . for maturation , the selected cocs were washed three times in the above described tissue culture 
 medium 199 ( ivm medium ) containing hormone supplements ( 10 iu / ml equine chorionic gonadotropin and 10 iu / ml human chorionic gonadotropin [ intervet , boxmeer , the 
 netherlands ] ) , and approximately 5060 oocytes were transferred to each well of a 4-well nunc dish ( roskilde , denmark ) containing 500 l of culture medium and 
 equilibrated for at least 2 h with 5% co2 at 39c in a humidified atmosphere . after 22 h of maturation with hormones , the cocs were washed twice and 
 cultured in hormone - free ivm medium for an additional 1820 h. briefly , a pig fetus ( landrace  duroc 
 crossbreed ) at embryonic day 40 was isolated from the uterus of a female pig . the tissues of the fetus were cut into 0.5-mm pieces and cultured in a 
 100-mm culture dish . cells were grown at 37c in an atmosphere of 5% co2 in dulbecco s modified eagle s medium ( dmem ) containing 4.5 g / l glucose 
 ( gibco brl , grand island , ny , usa ) and supplemented with fetal bovine serum ( 10% ) , penicillin ( 100 u / ml ) , and streptomycin ( 100 g / ml ) . the adherent cells were 
 subcultured . prior to scnt , the cells were thawed and cultured in above described dmem for 34 
 days until they reached 80% confluence . adherent cells ( passage 7~10 ) were treated with trypsin for ~1 min and used for scnt . after 4042 h of ivm , denuded oocytes were incubated for 5 min in manipulation medium ( calcium - free tlh containing 0.2% bovine serum albumin [ tlh - bsa ] ) 
 containing 5 g / ml hoechst 33342 , washed twice with fresh manipulation medium , and transferred to a drop of manipulation medium containing 5 g / ml cytochalasin 
 b. the oocytes were enucleated by aspirating the polar body and mii chromosomes using a 16-m glass pipette ( humagen , charlottesville , va , usa ) . after 
 enucleation , a trypsinized fetal fibroblast of 1415 m with a smooth cell surface was transferred to the perivitelline space of the enucleated oocyte . the 
 couplets were equilibrated with 280 mm mannitol solution containing 0.001 mm cacl2 and 0.05 mm mgso4 for 23 min and then transferred to a 
 fusion chamber containing two electrodes overlaid with 280 mm mannitol solution . membrane fusion was induced by applying an alternating current field of 2 v 
 cycling at 1 mhz for 2 sec , followed by two pulses of 160 v / mm direct current for 60 sec using a cell fusion generator ( lf201 ; nepa gene , chiba , japan ) . the oocytes were examined after 30 min , 
 and fused , normally shaped oocytes were collected . the collected scnt embryos were washed three times with ivc medium ( pzm 3 ) and cultured in 30-l microdrops of ivc medium . embryos in culture medium were 
 covered with pre - warmed mineral oil and incubated at 39c for 7 days under a humidified atmosphere of 5% o2 , 5% co2 , and 90% 
 n2 . at 4 h post - activation , scnt embryos were transferred to naturally cycling landrace  duroc crossbreed gilts on the first day of standing estrus . the reproductive tract was exposed , and the scnt embryos ( 100120 embryos per recipient ) 
 were transferred into an oviduct at the ampullary isthmic junction . pregnancy was diagnosed on day 30 and was checked every 24 weeks by ultrasonography . if 
 fetal echoes did not correspond with the gestational age of the fetuses and if signs of fetal absorption , such as small vesicles without any detectable fetuses , 
 were observed , an abortion was considered to have occurred . data related to the rates of cleavage , blastocyst formation , litter size , and production efficiency are shown as mean  sem and were compared by student s 
 t - test . data related to pregnancy rate and pregnancy status were analyzed using chi - square tests .
zinc supplementation ( 0.8 g / ml ) in in vitro maturation ( ivm ) medium significantly enhances oocyte quality . in this study , we compared the 
 development of somatic cell nuclear transfer ( scnt ) embryos produced from conventional ivm ( control ) and zinc - supplemented ivm oocytes . 
 a total of 1206 and 890 
 scnt embryos were produced using control and zinc - supplemented oocytes , respectively , and then were transferred to 11 and 8 recipients , respectively . 
 five 
 control recipients and three zinc - supplemented recipients became pregnant . 
 two live piglets and eight mummies were born from two control recipients , and ten 
 live piglets and six stillborn piglets were born from three zinc - supplemented recipients . 
 the production efficiency significantly increased in the 
 zinc - supplemented group ( 0.33% vs. 3.02% ) . 
 this report suggests that zinc supplementation in ivm medium improved the production efficiency of 
 cloned pigs .