title: Developmental potential of bovine hand-made clone embryos reconstructed by aggregation or fusion with distinct cytoplasmic volumes.
abstract: Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure being one of its potential limiting factors. The aim of this study was to determine the effect of the fusion of hemicytoplasts or aggregation of hemiembryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by hand-made cloning (HMC), parthenogenesis or by in vitro fertilization (IVF). One or two enucleated hemicytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemiembryos were in vitro cultured in the well-of-the-well (WOW) system, being allocated to one of six experimental groups, on a per WOW basis: single clone or parthenote hemiembryos (1 x 50%); aggregation of two (2 x 50%), three (3 x 50%), or four (4 x 50%) clone or parthenote hemiembryos; single clone or parthenote embryos (1 x 100%); or aggregation of two clone or parthenote embryos (2 x 100%). Control zona-intact parthenote or IVF embryos were in vitro cultured in four-well dishes. Results indicated that the increase in the number of aggregated structures within each WOW was followed by a linear increase in cleavage, blastocyst rate, and cell density. The increase in cytoplasmic volume, either by fusion or by aggregation, had a positive effect on embryo development, supporting the establishment of pregnancies and the birth of a viable clone calf after transfer to recipients. However, embryo aggregation did not improve development on a hemicytoplast basis, except for the aggregation of two clone embryos.
authors.affiliation: Center of Agroveterinarian Sciences, Santa Catarina State University, Lages, SC, Brazil.
authors.forename: Eduardo de Souza
authors.initials: ES
authors.lastname: Ribeiro
authors.forename: Renato Pereira da Costa
authors.initials: RP
authors.lastname: Gerger
authors.forename: Lain Uriel
authors.initials: LU
authors.lastname: Ohlweiler
authors.forename: Ivens
authors.initials: I
authors.lastname: Ortigari
authors.forename: Joana Cláudia
authors.initials: JC
authors.lastname: Mezzalira
authors.forename: Fabiana
authors.initials: F
authors.lastname: Forell
authors.forename: Luciana Relly
authors.initials: LR
authors.lastname: Bertolini
authors.forename: José Luiz
authors.initials: JL
authors.lastname: Rodrigues
authors.forename: Carlos Eduardo
authors.initials: CE
authors.lastname: Ambrósio
authors.forename: Maria Angélica
authors.initials: MA
authors.lastname: Miglino
authors.forename: Alceu
authors.initials: A
authors.lastname: Mezzalira
authors.forename: Marcelo
authors.initials: M
authors.lastname: Bertolini
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D019976:Cloning, Organism; D003593:Cytoplasm; D004622:Embryo, Mammalian / Q000166:cytology*; D053652:Nuclear Transfer Techniques; D010312:Parthenogenesis