title: Production of hand-made cloned buffalo (Bubalus bubalis) embryos from non-viable somatic cells.
abstract: Use of non-viable somatic cells for hand-made cloning (HMC) can enable production of cloned animals from tissues obtained from elite or endangered dead animals. Buffalo skin fibroblast cells were rendered non-viable by heat treatment and used for HMC. Although fusion (93.6 ± 1.72 vs 67.1 ± 2.83%) and cleavage (90.3 ± 1.79 vs 65.8 ± 1.56%) rate was lower (P < 0.001) than that for controls, blastocysts could be successfully produced. However, blastocyst rate (34.1 ± 2.43 vs 6.9 ± 2.18%, P < 0.001) and total cell number of blastocysts (TCN, 221.3 ± 25.14 vs 151.1 ± 21.69, P < 0.05) were lower and apoptotic index (4.8 ± 1.06 vs 10.9 ± 1.21) was higher (P < 0.001) than that of controls. In another experiment, ear tissue of slaughterhouse buffaloes was preserved in mustard oil at room temperature for 48 h following which somatic cells were harvested by enzymatic digestion and used for HMC. Although fusion (96.8 ± 1.48 vs 84.2 ± 3.19%), cleavage (89.6 ± 3.59 vs 77.2 ± 3.99%), and blastocyst rate (36.9 ± 7.45 vs 13.1 ± 6.87%) were lower (P < 0.01), TCN (223.0 ± 27.89 vs 213.3 ± 28.21) and apoptotic index (3.97 ± 0.67 vs 5.22 ± 0.51) of blastocysts were similar to those of controls. In conclusion, HMC can be successfully used for production of blastocysts from non-viable cells and from cells obtained from freshly slaughtered buffaloes. This can pave the way for the restoration of farm or wild animals by HMC if somatic cells could be obtained within a few hours after their death.
authors.affiliation: Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: E K A
authors.initials: EK
authors.lastname: Duah
authors.affiliation: Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: S K
authors.initials: SK
authors.lastname: Mohapatra
authors.affiliation: Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: T J
authors.initials: TJ
authors.lastname: Sood
authors.affiliation: Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: A
authors.initials: A
authors.lastname: Sandhu
authors.affiliation: Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: S K
authors.initials: SK
authors.lastname: Singla
authors.affiliation: Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: M S
authors.initials: MS
authors.lastname: Chauhan
authors.affiliation: Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: R S
authors.initials: RS
authors.lastname: Manik
authors.affiliation: Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India. prabhatpalta@yahoo.com.
authors.forename: P
authors.initials: P
authors.lastname: Palta
mesh_terms: D000818:Animals; D002020:Buffaloes / Q000196:embryology*; D002452:Cell Count; D016923:Cell Death; D002470:Cell Survival; D019976:Cloning, Organism / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology*; D035845:Research Embryo Creation / Q000379:methods*; D012867:Skin / Q000166:cytology; D013194:Staining and Labeling; D013696:Temperature
keywords: Cloned embryos; Cloning; Nuclear transfer; SCNT