Production of bovine hand-made cloned embryos by zygote-oocyte cytoplasmic hemi-complementation.
The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; hand-made cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.
Animal Reproduction Laboratory, Center of Agronomy and Veterinary Sciences (CAV), Santa Catarina State University (UDESC) , Santa Catarina, Brazil.
Joana Claudia
JC
Mezzalira
Lain Uriel
LU
Ohlweiler
Renato Pereira
RP
da Costa Gerger
Renata
R
Casali
Fabiano Koerich
FK
Vieira
Carlos Eduardo
CE
Ambrósio
Maria Angélica
MA
Miglino
José Luiz
JL
Rodrigues
Alceu
A
Mezzalira
Marcelo
M
Bertolini
D000818:Animals; D001755:Blastocyst / Q000166:cytology; D002417:Cattle; D019976:Cloning, Organism / Q000379:methods*; D003593:Cytoplasm / Q000378:metabolism*; D005260:Female; D005307:Fertilization in Vitro; D008561:Membrane Fusion; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology*; D010312:Parthenogenesis; D015053:Zygote / Q000166:cytology*