Hand-Made Cloned Goat (Capra hircus) Embryos--A Comparison of Different Donor Cells and Culture Systems

摘要

Nuclear transfer is a very effective method for propagation of valuable, extinct, and endangered animals. Handmade cloning (HMC) is an efficient alternative to the conventional micromanipulator-based technique in some domestic species. The present study was carried out for the selection of suitable somatic cells as a nuclear donor and development of an optimum culture system for in vitro culture of zona-free goat cloned embryos. Cleavage and blastocyst rates were observed 72.06 plus or minus 2.94% and 0% for fresh cumulus cells, 81.95 plus or minus 3.40% and 12.74 plus or minus 2.12% for cultured cumulus cells, and 92.94 plus or minus 0.91% and 23.78 plus or minus 3.33% for fetal fibroblast cells, respectively. There was a significant (p < 0.05) increase in blastocyst production in goats when cultured on a flat surface (FS) (23.78 plus or minus 3.33%) than well of wells (WOW) (15.84 plus or minus 2.12%) and microdrops (MD) (0.7 plus or minus 0.7%). Furthermore, cleavage and blastocyst production rates were significantly (p < 0.05) more in the WOW (15.84 plus or minus 2.12%) than the MD (0.7 plus or minus 0.7%) system. The quality of HMC blastocysts was studied by differential staining. Genetic similarity was confirmed by polymerase chain reaction (PCR)-based amplification of the second exon of the MHC class II DRB gene, which gave similar bands in electrophoresis (286 bp) both in cloned embryos and donor cells. In conclusion, the present study describes that the fetal fibroblast cell is a suitable candidate as nuclear donor, and the flat surface culture system is suitable for zona-free blastocyst development by the hand-made cloning technique in the goat.