title: Chemically assisted somatic cell nuclear transfer without micromanipulator in the goat: effects of demecolcine, cytochalasin-B, and MG-132 on the efficiency of a manual method of oocyte enucleation using a pulled Pasteur pipette.
abstract: The present study aimed to facilitate widespread application of a previously described manual method of somatic cell nuclear transfer (SCNT) by investigating the effects of demecolcine (a microtubule-depolymerizing chemical), cytochalasin-B (a microfilament-depolymerizing chemical: 2.5μg/ml for 15min) and MG-132 (a proteasome inhibitor chemical) on the (i) incidence of cytoplasmic protrusion of MII chromosomes, (ii) improvement of manual oocyte enucleation, and (iii) in vitro and in vivo developmental competence of SCNT embryos in the goat. Following in vitro maturation, around 65% of goat oocytes contained a characteristic cytoplasmic protrusion of MII-chromosomes. Treatment with demecolcine (0.4μg/ml for 30min) significantly increased this rate to 92.2±4.5%. Treatment with MG-132 (2μM for 30min) could not improve this rate when used alone (61.4±11.5%), but when combined with demecolcine (86.4±8.1%). Treatment with cytochalasin-B completely suppressed this rate whenever used, either alone (7.7±5.1%) or in combination with demecolcine (3.9±1.3%). In a direct comparison, there was no significant difference in quantity and quality of embryos propagated by the manual vs. micromanipulation-based methods of SCNT (cleavage: 85.3±4.5 vs. 89.5±8.9%, blastocyst: 19.5±4.3 vs. 24.3±4.4%, grade 1 and 2 blastocyst: 33.8±7.1 vs. 29.5±6.3%, total cell count: 125±11.1 vs. 122±10.5, respectively). Furthermore, development to live kids at term was not significant between the two SCNT methods. From both technical and economical points of view, the overall in vitro and in vivo efficiency of this manual method of SCNT proved it a simple, fast and efficient alternative for large scale production of cloned goats.
authors.affiliation: Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran. Electronic address: smhosseini@royaninstitute.org.
authors.forename: S M
authors.initials: SM
authors.lastname: Hosseini
authors.affiliation: Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Hajian
authors.affiliation: Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Forouzanfar
authors.affiliation: Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
authors.forename: S
authors.initials: S
authors.lastname: Ostadhosseini
authors.affiliation: Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
authors.forename: F
authors.initials: F
authors.lastname: Moulavi
authors.affiliation: Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
authors.forename: H R
authors.initials: HR
authors.lastname: Ghanaei
authors.affiliation: Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Medicine, ACECR, Tehran, Iran.
authors.forename: H
authors.initials: H
authors.lastname: Gourbai
authors.affiliation: Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 19395-4644, Tehran, Iran.
authors.forename: A H
authors.initials: AH
authors.lastname: Shahverdi
authors.affiliation: Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran; Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Medicine, ACECR, Tehran, Iran; Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 19395-4644, Tehran, Iran.
authors.forename: A D
authors.initials: AD
authors.lastname: Vosough
authors.affiliation: Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran. Electronic address: mh.nasr-esfahani@royaninstitute.org.
authors.forename: M H
authors.initials: MH
authors.lastname: Nasr-Esfahani
mesh_terms: D000818:Animals; D002467:Cell Nucleus; D019976:Cloning, Organism / Q000379:methods; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology*; D003703:Demecolcine / Q000494:pharmacology*; D006041:Goats; D007976:Leupeptins / Q000494:pharmacology*; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D050257:Tubulin Modulators; D003571:Cytochalasin B; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D003703:Demecolcine
keywords: Chemically assisted enucleation; Cytochalasin-B; Demecolcine; Goat; MG-132; Manual oocyte enucleation; SCNT