title: Piglets born from vitrified cloned blastocysts produced with a simplified method of delipation and nuclear transfer.
abstract: Successful cryopreservation of porcine embryos offers a promising perspective in the fields of agriculture, animal science, and human medical research. The objective of the present work was to establish a system facilitating the cryopreservation of porcine embryos produced by somatic cell nuclear transfer (SCNT). Several key techniques including micromanipulator-based enucleation, noninvasive delipation, zona-free fusion, and activation were combined with high efficiency. After a partial zona digestion and high-speed centrifugation, 89.8+/-2.1% (mean+/-SEM) of enucleated oocytes were successfully delipated. Delipated cytoplasts were incubated for an additional 0.5 or 2 h before fusion with somatic cells. After activation and 6 days of in vitro culture, no significant difference in the rate of blastocysts per reconstructed embryo was observed between the two groups (33.1+/-1.8% and 26.0+/-4.3% for 0.5 and 2 h recovery time, respectively). Cryopreservation of the blastocysts was performed with a Cryotop device and factory-prepared vitrification and warming solutions. One hundred fifty-five vitrified SCNT embryos were transferred surgically into two recipient sows to test their developmental capacity in vivo. One recipient became pregnant and delivered six piglets. In conclusion, our simplified delipation and SCNT procedure resulted in viable piglets after vitrification and embryo transfer at the blastocyst stage.
authors.affiliation: Population Genetics and Embryology, Institute of Genetics and Biotechnology, University of Aarhus, Tjele, Denmark. Yutao.Du@agrsci.dk
authors.forename: Yutao
authors.initials: Y
authors.lastname: Du
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Peter M
authors.initials: PM
authors.lastname: Kragh
authors.forename: Yunhai
authors.initials: Y
authors.lastname: Zhang
authors.forename: Mette
authors.initials: M
authors.lastname: Schmidt
authors.forename: Ingrid B
authors.initials: IB
authors.lastname: Bøgh
authors.forename: Xiuqing
authors.initials: X
authors.lastname: Zhang
authors.forename: Stig
authors.initials: S
authors.lastname: Purup
authors.forename: M
authors.initials: M
authors.lastname: Kuwayama
authors.forename: Arne L
authors.initials: AL
authors.lastname: Jørgensen
authors.forename: Anette M
authors.initials: AM
authors.lastname: Pedersen
authors.forename: Klaus
authors.initials: K
authors.lastname: Villemoes
authors.forename: Huanming
authors.initials: H
authors.lastname: Yang
authors.forename: Lars
authors.initials: L
authors.lastname: Bolund
authors.forename: Gábor
authors.initials: G
authors.lastname: Vajta
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology*; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods*; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods; D004624:Embryo Transfer / Q000379:methods; D005260:Female; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology; D011247:Pregnancy; D011270:Pregnancy, Animal; D013552:Swine; D015044:Zona Pellucida / Q000378:metabolism