Econstructed embryos cultured for 6 and 30 h after activation were surgically transferred

Econstructed embryos cultured for 6 and 30 h after activation were surgically transferred to the oviducts of the estrous surrogate mother by feeding a 14 cm Tom cat catheter (Tyco Healthcare Group LP, MA, USA) through the fimbriae at 0 and 9 h after the first standing estrus was exhibited, respectively. Pregnancy was detected at approximately 23 days after activation using an ultrasound scanner (HS-101V, Honda Electonics Co., Ltd., Yamazuka, Japan).Microsatellite AnalysisParentage analysis was performed in piglets produced by SCNT and the surrogate recipient to confirm the genetic identity of the SCNT piglets with the donor cells. The isolated genomic DNA samples obtained from each newborn piglet (ear tissue) and recipient (ear tissue) were used for microsatellite analysis and sent to a company that specializes in parentage verification for swine (Shanghai GeneCore BioTechnologies Co., Ltd.). Microsatellite analysis of the genomic DNA was performed using 11 porcine-specific microsatellite markers (S0026, S0070, S0155, S0226, SW122, SW24, SW72, SW830, SW840, SW857, and SW936) labeled with the fluorescent dye carboxyfluorescein (FAM).Solvent Yellow 14 biological activity Nuclear TransferSCNT was performed as previously described [23,25]. After culturing for 38 h to 42 h, oocytes with expanded cumulus cells were briefly treated with 0.1 (w/v) hyaluronidase and denuded of cumulus cells using a finely drawn glass capillary pipette. Oocytes extruding the first polar body with uniform cytoplasm were cultured in NCSU23 medium supplemented with 0.1 mg/mL demecolcine, 0.05 M sucrose, and 4 mg/mL bovine serum albumin (BSA) for 0.5 h to 1 h. The oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm using a bevelled pipette (approximately 20 mm in diameter) in Tyrode’sCloning of Banna Miniature Inbred PigTable 1. Effects of different donor cells on the development of SCNT embryos of Banna miniature inbred pig.Donor cell type Fetal 125-65-5 price fibroblast Newborn fibroblast Adult fibroblastNo. of embryos (Repeats) 895(10) 843(9) 1279(10)No. of cleavage ( ) 667(74.367.7) 655(76.467.1) 780(62.167.9)a a bNo. of blastocyst ( ) 254(27.465.1)a 186(21.863.5)a 168(13.564.8)b*Values with different superscript letters within a column are significantly different (a,bP , 0.05). doi:10.1371/journal.pone.0057728.tStatistical AnalysisFor proportional 10457188 data, the differences between groups were analyzed for variance using the StatviewH software package. The level of significance was set at P,0.05.no significant difference in body weight was observed among the three groups (P.0.01). These weights of the fetal, newborn, and adult fibroblast groups were significantly greater than that of the control groups (P,0.01).Results Effect of the Donor Cell Type on the Development of Embryos Derived from SCNTThe effect of the donor cell type on the development of embryos derived from SCNT was investigated (Table 1). The cleavage rate and blastocyst formation rate of the reconstructed embryos did not significantly differ between the fetal (74.3 and 27.4 ) and newborn (76.4 and 21.8 ) fibroblast groups (P.0.05), but both groups exhibited significantly higher 1326631 rates than the adult fibroblast group (61.9 and 13.0 ; P,0.05). Our results showed that the blastocysts derived from the fetal fibroblasts had more ICM cells, TE cells, total cells and TCM/total cell than those derived from the adult fibroblasts; however, no significant difference was observed among the three groups (P.0.05; Table 2).DNA Parentage.Econstructed embryos cultured for 6 and 30 h after activation were surgically transferred to the oviducts of the estrous surrogate mother by feeding a 14 cm Tom cat catheter (Tyco Healthcare Group LP, MA, USA) through the fimbriae at 0 and 9 h after the first standing estrus was exhibited, respectively. Pregnancy was detected at approximately 23 days after activation using an ultrasound scanner (HS-101V, Honda Electonics Co., Ltd., Yamazuka, Japan).Microsatellite AnalysisParentage analysis was performed in piglets produced by SCNT and the surrogate recipient to confirm the genetic identity of the SCNT piglets with the donor cells. The isolated genomic DNA samples obtained from each newborn piglet (ear tissue) and recipient (ear tissue) were used for microsatellite analysis and sent to a company that specializes in parentage verification for swine (Shanghai GeneCore BioTechnologies Co., Ltd.). Microsatellite analysis of the genomic DNA was performed using 11 porcine-specific microsatellite markers (S0026, S0070, S0155, S0226, SW122, SW24, SW72, SW830, SW840, SW857, and SW936) labeled with the fluorescent dye carboxyfluorescein (FAM).Nuclear TransferSCNT was performed as previously described [23,25]. After culturing for 38 h to 42 h, oocytes with expanded cumulus cells were briefly treated with 0.1 (w/v) hyaluronidase and denuded of cumulus cells using a finely drawn glass capillary pipette. Oocytes extruding the first polar body with uniform cytoplasm were cultured in NCSU23 medium supplemented with 0.1 mg/mL demecolcine, 0.05 M sucrose, and 4 mg/mL bovine serum albumin (BSA) for 0.5 h to 1 h. The oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm using a bevelled pipette (approximately 20 mm in diameter) in Tyrode’sCloning of Banna Miniature Inbred PigTable 1. Effects of different donor cells on the development of SCNT embryos of Banna miniature inbred pig.Donor cell type Fetal fibroblast Newborn fibroblast Adult fibroblastNo. of embryos (Repeats) 895(10) 843(9) 1279(10)No. of cleavage ( ) 667(74.367.7) 655(76.467.1) 780(62.167.9)a a bNo. of blastocyst ( ) 254(27.465.1)a 186(21.863.5)a 168(13.564.8)b*Values with different superscript letters within a column are significantly different (a,bP , 0.05). doi:10.1371/journal.pone.0057728.tStatistical AnalysisFor proportional 10457188 data, the differences between groups were analyzed for variance using the StatviewH software package. The level of significance was set at P,0.05.no significant difference in body weight was observed among the three groups (P.0.01). These weights of the fetal, newborn, and adult fibroblast groups were significantly greater than that of the control groups (P,0.01).Results Effect of the Donor Cell Type on the Development of Embryos Derived from SCNTThe effect of the donor cell type on the development of embryos derived from SCNT was investigated (Table 1). The cleavage rate and blastocyst formation rate of the reconstructed embryos did not significantly differ between the fetal (74.3 and 27.4 ) and newborn (76.4 and 21.8 ) fibroblast groups (P.0.05), but both groups exhibited significantly higher 1326631 rates than the adult fibroblast group (61.9 and 13.0 ; P,0.05). Our results showed that the blastocysts derived from the fetal fibroblasts had more ICM cells, TE cells, total cells and TCM/total cell than those derived from the adult fibroblasts; however, no significant difference was observed among the three groups (P.0.05; Table 2).DNA Parentage.