title: Pregnancies established from handmade cloned blastocysts reconstructed using skin fibroblasts in buffalo (Bubalus bubalis).
abstract: Handmade cloning (HMC), a simple, micromanipulation-free cloning technique, has been applied for the production of cloned embryos and offspring in many livestock species. The objective of the present study was to compare the effect of donor cell type on developmental competence of HMC embryos and to explore the possibility of establishing pregnancies using these embryos in buffalo. After technical optimization of the HMC procedure for in vitro development of cloned blastocysts, various donor cells were compared for their developmental efficiency. Using buffalo fetal-, newborn-, adult fibroblasts and cumulus cells, blastocyst production rates obtained from reconstructed embryos were 24.0+/-1.8% (35/145), 33.0+/-8.0% (56/163), 21.0+/-9.3% (29/133) and 49.6+/-1.9% (77/154), respectively. Blastocyst rates were higher (P<0.05) in cumulus cell reconstructed embryos in comparison to those derived from fetal or adult fibroblasts. Pregnancy diagnosis (transrectal ultrasonography) was carried out at Day 40 of gestation. Following transfer of HMC embryos reconstructed using newborn fibroblasts 25% (2/8) buffaloes were pregnant and are at Days 201 and 94 of gestation, whereas after transfer of HMC embryos reconstructed using fetal fibroblasts, 20% (1/5) buffaloes were pregnant and are at Day 73 of gestation. In conclusion, HMC could be a simple and efficient technique for the production of cloned embryos for establishing pregnancies in buffalo.
authors.affiliation: Embryo Biotechnology Lab, Animal Biotechnology Centre, National Dairy Research Institute, Karnal 132001, India. drriazshah@gmail.com
authors.forename: R A
authors.initials: RA
authors.lastname: Shah
authors.forename: A
authors.initials: A
authors.lastname: George
authors.forename: M K
authors.initials: MK
authors.lastname: Singh
authors.forename: D
authors.initials: D
authors.lastname: Kumar
authors.forename: T
authors.initials: T
authors.lastname: Anand
authors.forename: M S
authors.initials: MS
authors.lastname: Chauhan
authors.forename: R S
authors.initials: RS
authors.lastname: Manik
authors.forename: P
authors.initials: P
authors.lastname: Palta
authors.forename: S K
authors.initials: SK
authors.lastname: Singla
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000502:physiology; D002020:Buffaloes / Q000502:physiology*; D019976:Cloning, Organism / Q000379:methods*; D047108:Embryonic Development / Q000502:physiology; D005260:Female; D005347:Fibroblasts / Q000166:cytology*; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D011247:Pregnancy; D011256:Pregnancy Outcome; D018873:Pregnancy Rate; D011270:Pregnancy, Animal; D012867:Skin / Q000166:cytology; D014019:Tissue Donors