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Forskolin® (F-6886) is being used to induce lipolysis and increase cryotolerance, to be an activator of adenylate cyclase, and elevating the cyclic adenosine monophosphate (cAMP) levels. The objective of this experiment was to induce the chemical lipolysis of embryos to improve vitrification and the hypothesis would be that Forskolin decrease the amount of lipid droplets, improve the production of blastocysts, and increase the survival rate after vitrification and warming. Eight random effect were performed which oocytes (N = 1172) were matured in TCM 199® supplemented with 10% of fetal bovine serum (FBS), under 5% CO2 atmosphere, at a temperature of 38.5°C and absolute humidity for 24 h. Semen was selected by Percoll gradient with a final concentration of the 2 × 106 sperm mL–1. Presumptive zygotes were cultured in SOFaa and 2.5% of FBS and were kept in an incubator with 5% CO2, 5% O2 and 90% N2 at 38.5°C and absolute humidity until Day 6, when Forskolin was added and remained until Day 7; control (group without Forskolin); F 2.5 µM (group with 2.5 µM Forskolin); F 5 µM (group with 5 µM Forskolin). On Day 7 (Day 0 = IVF) the rate of blastocyst formation was observed then they were vitrified. Apoptosis was analysed using the TUNEL technique, and the lipid content analysis was performed with Sudan Black B® (S-0395). To estimate the lipid content of embryos, 1 photo at a blastocyst group was performed and submitted to the program ImageJ 1.14 (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). The embryos were limited to obtain the area (μm2), and gray intensity mean (arbitrary units), and gray intensity per area was calculated (arbitrary units/μm2). Data were analysed by ANOVA with PROC GLM of SAS (SAS Institute, 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 rate: control (37.0 ± 4.0%), F 2.5 μM (38.6 ± 4.0%), F 5 μM (40.7 ± 4.0%). There were difference in lipids content between all groups: control (136.8 ± 2.2ab); F 2.5 μM (128.5 ± 2.2b), F 5 μM (135.6 ± 2.3c; P

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. It was suggested that the inhibition of spontaneous nuclear IVM might allow for more time to accumulate the molecules important for embryonic development. The objective of this work was to evaluate blocking oocyte meiosis with the addition of forskolin. Slaughterhouse-derived bovine Zebu ovaries were collected and carried to the laboratory. Oocytes (n = 584) with at least 3 intact layers of cumulus cells and homogeneous cytoplasm were selected for IVM. The oocytes were transferred to drops of TCM 199 plus 10% FCS and hormones. The oocytes remained in IVM medium in 3 different concentrations of forskolin (6886), 0.1, 0.05, 0.025 mM, and a control group (withouth forskolin), for 6 h. Then they were maturated for an additional 18 h in forskolin-free medium. The first period above was an attempt to block (Block) and the second to resume (Res) the oocyte meiosis. The oocytes were incubated in a humidified atmosphere with 5% CO2 at 38.5°C in an air incubator. The oocytes were assessed for the stage of nuclear maturation, to see if they were in M II. Then oocytes were in vitro fertilized (IVF) with frozen Nelore bull semen (Bos taurus indicus). Presumptive zygotes (20–30/group) were cultured in SOFaa (synthetic oviducal fluid) supplemented with 5 mg mL–1 of BSA; the embryos were kept in an incubator with 5% CO2, 5% O2, and 90% N2 at 38.5°C and absolute humidity. On Day 7 (Day 0 = IVF) the blastocyst, the number of viable cells, and apoptosis rate (terminal deoxynucleotide transferase uridine nick-end labelling) were observed. Data were analysed with ANOVA using SAS PROC GLM (SAS Inst. Inc., Cary, NC, USA). Sources of variation in the model, including treatment and replication, were respectively considered fixed and random effects. If ANOVA was significant, the contrasts of means were performed using the least-squares difference. Data are presented as the mean and the standard error of least-squares. For all analyses, we used a significance level of 5%. No differences were observed for the stage of nuclear maturation of the oocyte (N = 336; control: 67.7 ± 8.3; F 0.025 mM, Block/Res: 67.7 ± 8.9; F 0.05 mM, Block/Res: 65.9 ± 9.8; F 0.1 mM, Block/Res: 50.2 ± 8.9), the blastocyst rate (N = 584; Control: 36.7 ± 3.7; F0.025 mM, Block/Res: 32.6 ± 3.7; F0.05 mM, Block/Res: 29.2 ± 3.7; F0.1 mM, Block/Res: 25.1 ± 3.7), and total number of intact cells (N = 10–15 embryos/group; Control:140.1 ± 13.0; F0.025 mM, Block/Res: 129.9 ± 13.0; F0.05 mM, Block/Res: 139.0 ± 13.0; F0.1 mM, Block/Res: 104.4 ± 13.0; P > 0.05). However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (N = 10–15 embryos/group): Control: 12.1 ± 2.5a; F 0.025 mM, Block/Res: 12.9 ± 2.5a; F0.05 mM, Block/Res: 13.5 ± 2.5a; F 0.1 mM, Block/Res: 30.2 ± 2.5b (P