title: Piglets produced from cloned blastocysts cultured in vitro with GM-CSF.
abstract: In general, pig embryos established by somatic cell nuclear transfer (SCNT) are transferred at the one-cell stage because of suboptimal embryo culture conditions. Improvements in embryo culture can increase the practical application of late embryo transfer. The goal of this study was to evaluate embryos cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro, and to track the in vivo developmental competency of SCNT-derived blastocysts from these GM-CSF embryos. The receptor for GM-CSF was up-regulated in in vitro-produced embryos when compared to in vivo-produced cohorts, but the level decreased when GM-CSF was present. In vitro fertilized (IVF) embryos, supplemented with GM-CSF (2 or 10 ng/ml), showed a higher frequency of development to the blastocyst stage compared to controls. The total cell numbers of the blastocysts also increased with supplementation of GM-CSF. Molecular analysis demonstrates that IVF-derived blastocysts cultured with GM-CSF exhibit less apoptotic activity. Similarly, an increase in development to the blastocyst stage and an increase in the average total-cell number in the blastocysts were observed when SCNT-derived embryos were cultured with either concentration of GM-CSF (2 or 10 ng/ml). When SCNT-derived embryos, cultured with 10 ng/ml GM-CSF, were transferred into six surrogates at Day 6, five of the surrogates became pregnant and delivered healthy piglets. Our findings suggest that supplementation of GM-CSF can provide better culture conditions for IVF- and SCNT-derived embryos, and pig SCNT-derived embryos cultured with GM-CSF in vitro can successfully produce piglets when transferred into surrogates at the blastocyst stage. Thus, it may be practical to begin performing SCNT-derived embryo transfer at the blastocyst stage.
authors.affiliation: Division of Animal Sciences, University of Missouri, Columbia, Missouri 65211, USA. leekil@missouri.edu
authors.forename: Kiho
authors.initials: K
authors.lastname: Lee
authors.forename: Bethany K
authors.initials: BK
authors.lastname: Redel
authors.forename: Lee
authors.initials: L
authors.lastname: Spate
authors.forename: Jennifer
authors.initials: J
authors.lastname: Teson
authors.forename: Alana N
authors.initials: AN
authors.lastname: Brown
authors.forename: Kwang-Wook
authors.initials: KW
authors.lastname: Park
authors.forename: Eric
authors.initials: E
authors.lastname: Walters
authors.forename: Melissa
authors.initials: M
authors.lastname: Samuel
authors.forename: Clifton N
authors.initials: CN
authors.lastname: Murphy
authors.forename: Randall S
authors.initials: RS
authors.lastname: Prather
mesh_terms: D000818:Animals; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary*; D017931:DNA Primers / Q000235:genetics; D046149:Embryo Culture Techniques / Q000379:methods* / Q000662:veterinary*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism / Q000494:pharmacology*; D016014:Linear Models; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D027724:Reproductive Techniques, Assisted / Q000662:veterinary; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D034421:Sus scrofa / Q000196:embryology*
chemical_list: D017931:DNA Primers; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor