title: Hope for restoration of dead valuable bulls through cloning using donor somatic cells isolated from cryopreserved semen.
abstract: Somatic cells were isolated from cryopreserved semen of 4 buffalo bulls, 3 of which had died over 10 years earlier, and were established in culture. The cells expressed cytokeratin-18, keratin and vimentin indicating that they were of epithelial origin. The cells were used as nuclear donors for hand-made cloning for producing buffalo embryos. The blastocyst rate and quality, as indicated by apoptotic index, were comparable among embryos produced using cells obtained from fresh or frozen-thawed semen or those obtained from conventional cell sources such as skin. Examination of the epigenetic status revealed that the global level of H3K27me3 but not that of H3K9/14ac and H4K5ac differed significantly (P<0.05) among cloned embryos from different bulls. The relative mRNA abundance of HDAC1, DNMT1, P53 and CASPASE 3 but not that of DNMT3a differed in cells and in cloned embryos. Following transfer of 24 cloned embryos produced from fresh semen-derived cells to 12 recipients, one calf weighing 55 kg, which is now 6 months of age and is normal, was born through normal parturition. Following transfer of 20 embryos produced from frozen-thawed semen-derived cells to 10 recipients, 2 became pregnant, one of which aborted in the first trimester; the calf born was severely underweight (17 kg), and died 12 h after birth. The ability of cells derived from fresh and frozen-thawed semen to produce live offspring confirms the ability of these cells to be reprogrammed. Our findings pave the way for restoration of highly precious progeny-tested bulls, which has immense economic importance, and can also be used for restoration of endangered species.
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India; Cellular Reprogramming Lab., Department of Animal Physiology and Reproduction, Central Institute for Research on Buffaloes, Hisar, India.
authors.forename: Naresh L
authors.initials: NL
authors.lastname: Selokar
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
authors.forename: Monika
authors.initials: M
authors.lastname: Saini
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
authors.forename: Prabhat
authors.initials: P
authors.lastname: Palta
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
authors.forename: Manmohan S
authors.initials: MS
authors.lastname: Chauhan
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
authors.forename: Radheysham
authors.initials: R
authors.lastname: Manik
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Singla
mesh_terms: D000818:Animals; D002417:Cattle; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D002999:Clone Cells / Q000166:cytology; D019976:Cloning, Organism; D015925:Cryopreservation / Q000379:methods*; D019175:DNA Methylation / Q000235:genetics; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000166:cytology; D044127:Epigenesis, Genetic; D005786:Gene Expression Regulation; D006657:Histones / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008297:Male; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D035845:Research Embryo Creation; D012661:Semen / Q000166:cytology*
chemical_list: D006657:Histones; D012333:RNA, Messenger; D008239:Lysine