Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep.
Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system.
Lagutina
Irina
I
Laboratorio di Tecnologie della Riproduzione, Istituto Sperimentale Italiano Lazzaro Spallanzani, CIZ srl, Via Porcellasco 7/f, 26100 Cremona, Italy.
Lazzari
Giovanna
G
Duchi
Roberto
R
Turini
Paola
P
Tessaro
Irene
I
Brunetti
Dario
D
Colleoni
Silvia
S
Crotti
Gabriella
G
Galli
Cesare
C
D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D002417:Cattle / Q000196:embryology; D002459:Cell Fusion; D002478:Cells, Cultured; D019976:Cloning, Organism; D006736:Horses / Q000196:embryology; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D012756:Sheep / Q000196:embryology; D013045:Species Specificity; D013552:Swine / Q000196:embryology; D015044:Zona Pellucida / Q000502:physiology*