Decreased lipid granules of In vitro produced bovine embryos with low concentrations of forskolin

This paper proposes the chemical induction of lipolysis addition of forskolin on the sixth day of embryo culture, in order to increase the rate of survival of IVP embryos. Eight replicates were performed using 1.519 oocytes, which were washed and transferred to drops of IVM in TCM 199 supplemented with 10% FCS and remained in an oven with 5% CO2 in air, at a temperature of 38.5 ° C and humidity absolute for 24 h. Semen was selected by Percoll gradient obtaining a concentration adjusted to 2x106 spermatozoa / mL. Oocytes were incubated for 24 h in incubator with 5% CO2 in air, at a temperature of 38.5 ° C and absolute humidity. Presumptive zygotes were cultured in SOF and 2.5% FBS. The embryos were kept in an oven with 5% CO2, 5% O2 and 90% N2 at a temperature of 38.5 ° C and absolute humidity D to 6 (0 = D FIV), which were divided time into four groups for the addition of Forskolin® (F-6886): Control (group cultured in the absence of forskolin for seven consecutive days); F 2,5μM (group cultivated with 2,5μM forskolin for 24 hours); F 5μM (group cultivated with 5μM forskolin for 24 hours); F 10μM (group cultivated with 10μM forskolin for 24 hours). Apoptosis was analyzed using the TUNEL technique (deoxinucleotil terminal transferase Uracil Nick End Labeling), and the lipid content analysis was performed with Sudan Black B (S-0395). Statistical analysis Data were analyzed using ANOVA using SAS PROC GLM (SAS Inst., Inc., Cary, NC, USA). Sources of variation in the model including treatment and replicas were regarded as fixed and random effects, respectively. Data are presented as mean and standard least squares error. For all analyzes was adopted the significance level of 5%. There was no difference in blastocyst formation rate: control (37.0 ± 4.0); 2,5μM F (38.6 ± 4.0); F 5μM (40.7 ± 4.0); F 10μM (31.4 ± 4.0). While all groups treated with forskolin showed differences on lipid measurement: control (50.6 ± 1,1ab); 2,5μM F (46.2 ± 1,1c ); F 5μM (49.9 ± 1,2b ); 10μM F (53.9 ± 1,2a ); total number of whole cells: control (140.1 ± 10,7ac); F 2,5μM (173 ± 9,0b ); F 5μM (120.6 ± 11,5c ); F 10μM (157.0 ± 13,9ab) and cell apoptosis: control (12.1 ± 3.5a ); 2,5μM F (16.7 ± 4,1b ); F 5μM (11.1 ± 6,5a ); 10μM F (14.2 ± 4,6ab) (P u003c0.05). It was concluded that forskolin is a lipolytic agent effective even at low concentrations (F 2.5 mM), leading to the formation of blastocysts with more cells, comparing it to the control group. However, the concentration of 5 uM forskolin induced programmed cell death at the lowest rate.