IN-VITRO AND IN-VIVO DEVELOPMENT OF CLONED OVINE EMBRYOS USING IN-VITRO AND IN-VIVO MATURED OOCYTES

Cloning of sheep embryos by nucleus transplantation can be achieved by using in vivo matured (oviductal) oocytes and in vivo culture. Howeve r, these steps involve cumbersome procedures. Therefore, the effects o f in vivo vs. the equivalent in vitro procedures on the pre-implantati on development of cloned embryos were compared using: 1. In vivo oocyt es and in vivo culture; 2. In vivo oocytes and in vitro culture; and 3 . In vitro oocytes and in vitro culture. Selected embryos were transfe rred to recipients. Donor embryos and oviductal oocytes were collected from superovulated Merino ewes. In vivo matured oocytes were enucleat ed and fused with inserted blastomeres from donor embryos. In vitro ma tured oocytes were enucleated and allowed to age prior to blastomere i nsertion and electrofusion. Fused embryos were cultured for approximat ely 132 h either in vivo in ligated sheep oviducts or in vitro, and th ose developed beyond the eight cell stage were transferred to recipien t ewes. More in vitro than in vivo matured oocytes fused (66 vs. 43%, p < 0.05). The in vivo development to the blastocyst, morula and 8-16 cell stages were similar for cloned embryos derived from in vitro and in vivo matured oocytes, while in vitro culture development seemed to compromise development, as no blastocyst development was observed. A s et of cloned twins derived from in vivo procedures were born from seve n cloned embryos, while two out of 10 ewes receiving in vitro derived cloned embryos became pregnant but aborted after 60 days of gestation.