title: Hand-made cloned buffalo (Bubalus bubalis) embryos: comparison of different media and culture systems.
abstract: Hand-made cloning (HMC) has proved to be an efficient alternative to the conventional micromanipulator-based technique in some domestic animal species. This study reports the development of an effective culture system for in vitro culture of zona-free cloned buffalo (Bubalus bubalis) embryos reconstructed using adult skin fibroblast cells as nucleus donor. Cleavage and blastocyst rates observed were 52 and 0% in modified Charles Rosenkrans 2 (mCR2), 61 and 4.6% in modified Synthetic Oviductal Fluid (mSOF), and 82 and 40.3% in Research Vitro Cleave (RVCL; Cook, Australia) medium, respectively. Similarly, higher blastocyst rates (24.5 +/- 4.1%) were observed when zona-free parthenotes were cultured in RVCL medium. Culturing zona-free cloned buffalo embryos on flat surfaces (FS) yielded significantly higher (p < 0.05) blastocyst rates than Well of the Wells (WOW) or microdrops (MD). Furthermore, development in WOW was found to be significantly better than MD culture. The quality of HMC blastocysts was examined using differential staining. This study establishes the application of zona-free nuclear transfer procedures for the production of hand-made cloned buffalo embryos and the development of efficient culture system and appropriate media requirements for enhancing their preimplantation development.
authors.affiliation: Embryo Biotechnology Laboratory, Animal Biotechnology Center, National Dairy Research Institute, Karnal-132001, India. drriazshah@gmail.com
authors.forename: Riaz A
authors.initials: RA
authors.lastname: Shah
authors.forename: Aman
authors.initials: A
authors.lastname: George
authors.forename: Manoj K
authors.initials: MK
authors.lastname: Singh
authors.forename: Dharmendra
authors.initials: D
authors.lastname: Kumar
authors.forename: Manmohan S
authors.initials: MS
authors.lastname: Chauhan
authors.forename: Radhaysham
authors.initials: R
authors.lastname: Manik
authors.forename: Prabhat
authors.initials: P
authors.lastname: Palta
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Singla
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001755:Blastocyst / Q000502:physiology; D002020:Buffaloes / Q000196:embryology*; D000001:Calcimycin / Q000494:pharmacology; D019976:Cloning, Organism / Q000379:methods*; D003470:Culture Media; D046149:Embryo Culture Techniques; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000196:embryology / Q000502:physiology*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D010312:Parthenogenesis / Q000187:drug effects / Q000502:physiology
chemical_list: D000900:Anti-Bacterial Agents; D003470:Culture Media; D000001:Calcimycin