title: Hand-made cloned goat (Capra hircus) embryos—a comparison of different donor cells and culture systems.
abstract: Nuclear transfer is a very effective method for propagation of valuable, extinct, and endangered animals. Hand-made 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 ± 2.94% and 0% for fresh cumulus cells, 81.95 ± 3.40% and 12.74 ± 2.12% for cultured cumulus cells, and 92.94 ± 0.91% and 23.78 ± 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 ± 3.33 %) than well of wells (WOW) (15.84 ± 2.12 %) and microdrops (MD) (0.7 ± 0.7%). Furthermore, cleavage and blastocyst production rates were significantly (p < 0.05) more in the WOW (15.84 ± 2.12%) than the MD (0.7 ± 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.
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
authors.forename: Yogesh S
authors.initials: YS
authors.lastname: Akshey
authors.forename: Dhruba
authors.initials: D
authors.lastname: Malakar
authors.forename: Arun K
authors.initials: AK
authors.lastname: De
authors.forename: Manoj K
authors.initials: MK
authors.lastname: Jena
authors.forename: Shweta
authors.initials: S
authors.lastname: Garg
authors.forename: Rahul
authors.initials: R
authors.lastname: Dutta
authors.forename: Sachin Kumar
authors.initials: SK
authors.lastname: Pawar
authors.forename: Manisha
authors.initials: M
authors.lastname: Mukesh
mesh_terms: D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000166:cytology; D002970:Cleavage Stage, Ovum / Q000166:cytology; D019976:Cloning, Organism / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D046149:Embryo Culture Techniques / Q000379:methods; D047108:Embryonic Development; D005260:Female; D005802:Genes, MHC Class II; D006041:Goats / Q000196:embryology* / Q000235:genetics / Q000276:immunology; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology; D011247:Pregnancy
chemical_list: D017931:DNA Primers