We have examined the in vitro and in vivo development of cloned embryos produced by incorporation of fetal fibroblast into in vitro matured and enucleated cow oocytes by direct injection and by fusion. For injection, nuclei were either mechanically isolated using the microinjection needle or chemically isolated by treatment with NP-40 lysis buffer. Fetal fibroblasts were serum starved and treated with calcium ionophore before injection to induce chromatin condensation. A range of 8% to 16% of successfully injected oocytes developed to blastocysts in culture and a total of nine pregnancies resulted from transfer of cloned embryos produced by this method. Nuclear transfer by fusion resulted in 22% development to blastocysts. Unlike in mice, the embryos derived from injection did not result in viable pregnancies, which may suggest species differences. All pregnancies were terminated after 45 to 150 days from transfer. Two pregnancies resulted from transfer of cloned embryos obtained by fusion which produced two healthy female calves. The study proposes an alternative method for the production of cow cloned embryos. Further research, however, is required to optimize bovine cloning by injection.
Summary.Inheritance of the centrosome (centriole) and its behaviour during fertilization and embryogenesis of cattle is presented. The bovine embryo follows the human pattern of centriole behaviour, which is common to most animals including large mammals. Thus, most mammals obey Boveri's rule of paternal centrosomal inheritance and perpetuation, whereas the mouse is an exception to the rule, showing maternal inheritance. The sperm centrosome was traced from fertilization to the hatching blastocyst stage in the cow and its presence was confirmed at every stage of cleavage, as reported in the human. It is concluded that the bovine embryo is a more appropriate model than the mouse for research in fertilization and assisted-reproduction technology.
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