Early zygotes are suitable recipients for bovine somatic nuclear transfer and result in cloned offspring.
Cloning by somatic cell nuclear transfer (SCNT) subverts sperm-mediated fertilization that normally leads to physiological activation of the oocyte. Therefore, artificial activation is required and it is presently unclear what developmental consequences this has. In this study, we aimed to improve cattle cloning efficiency by utilizing a more physiological method of activating SCNT reconstructs. We carried out in vitro fertilization (IVF) of zona-intact bovine oocytes before SCNT. We removed the zona pellucida 4 h after insemination, stained the fertilized eggs with Hoechst 33342 and mechanically removed both male and female chromatin. The enucleated pre-activated cytoplasts were fused with male adult ear skin fibroblasts ("IVF-NT" group). Chemically activated SCNT embryos, produced according to our standard operating procedure for zona-free SCNT, served as controls. After 7 days, in vitro development to blastocysts of morphological grade 1-3 or grade 1-2 was very similar in both groups (39 vs 40% and 20 vs 21% respectively). However, post-implantation development was improved after sperm-mediated activation. Across four replicate runs, pregnancy establishment at day 35 was significantly higher for IVF-NT than for control SCNT embryos (30/49 = 61 vs 17/41 = 42% respectively; P < 0.05). Development into calves at term or weaning was also higher in the IVF-NT group compared with control SCNT (9/49 = 18 vs 3/41 = 7% and 6/49 = 12 vs 3/41 = 7%; P = 0.11 and 0.34 respectively).
Schurmann
Anita
A
AgResearch Ltd, Ruakura Research Centre, Reproductive Technologies, East Street, Hamilton, New Zealand.
Wells
David N
DN
Oback
Björn
B
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary*; D004624:Embryo Transfer; D005260:Female; D005307:Fertilization in Vitro; D005347:Fibroblasts; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D009866:Oogenesis; D011247:Pregnancy; D011256:Pregnancy Outcome; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015053:Zygote
D007476:Ionophores; D047428:Protein Kinase Inhibitors; D015759:Ionomycin; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine