ABSTRACT
The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. Thus, the objective of this work was to evaluate the effect of blocking oocyte meiosis with the addition of forskolin, an efficient inhibitor of nuclear maturation, in in vitro maturation (IVM) medium. Forskolin was added to the IVM medium for 6 h at concentrations of 0.1 mM, 0.05 mM or 0.025 mM, then the oocytes were allowed to mature in drug-free medium for 18 h. The oocytes were assessed for the stage of nuclear maturation, the activity and distribution of mitochondria, oocyte ultrastructure, the number of viable cells and the apoptosis rate. After forskolin treatment, the oocytes were fertilized in vitro and cultured for 7 days. On day 7, the blastocyst rate, the ultrastructure, the number of intact cells and the apoptosis rate of the blastocysts were measured. No differences were observed for the stage of nuclear maturation of the oocyte, the mitochondrial activity and distribution, the blastocyst rate or total number of intact cells. However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (P < 0.05). We conclude that all the experimental groups reached the MII stage after the addition of forskolin and that the highest concentration of forskolin caused cellular degeneration without harming embryo production on the 7th day.
Subject(s)
Blastocyst/drug effects , Colforsin/pharmacology , Fertilization in Vitro/methods , Oocytes/drug effects , Animals , Blastocyst/cytology , Cattle , Cells, Cultured , Embryonic Development/drug effects , Female , Fertilization in Vitro/veterinary , Male , Meiosis/drug effects , Oocytes/cytology , Time Factors , Vasodilator Agents/pharmacologyABSTRACT
The objective of this study was to evaluate the potential of bovine IVF blastocysts at different stages of embryonic development in establishing ESC-like. Furthermore, blastocysts cultured in medium containing (10%) and (2.5%) fetal calf serum (FCS) were compared to determine if the serum concentration during in vitro culture alters the blastocyst's potential to establish ESC-like culture. It was observed that only ICM's from expanded blastocysts adhered to the monolayer (n=160) independent of the concentration of serum used during IVF culture. There were no observable differences in potential to establish ESC-like in embryos cultured with 2,5% or 10% FCS . The bFGF didn´t seems to be required for maintenance of bovine ESC-like regardless of culture conditions. Blastocysts and colonies in primary culture and after the first passage were positive for Oct4, Nanog, SSEA-3 and TRA-1-81. Expanded blastocysts gave rise to ESC-like colonies, and the addition of LIF was sufficient to maintain cells undifferentiated in culture.
