165 CRYOPRESERVATION OF IN VITRO PRODUCED BOVINE EMBRYOS AFTER LIPID DECREASE WITH FORSKOLIN

Abstract

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 ± 2.2^ab); F 2.5 μM (128.5 ± 2.2^b), F 5 μM (135.6 ± 2.3^c; P < 0.05). The F 2.5 μM group showed the higher rate of apoptotic cells compared to other groups: control (12.1 ± 3.5%^a), F 2.5 μM (16.7 ± 4.1%^b), F 5 μM (11.1 ± 6.5%^a; P < 0.05). After vitrification, there was no difference in re-expansion: control (71.3 ± 8.9%), F 2.5 μM (73.1 ± 8.9%); F 5 μM (66.1 ± 8.9%) and apoptosis rate: control (22.3 ± 3.1%); F 2.5 μM (37.3 ± 3.8%); F 5 μM (33.2 ± 6.5%) between the groups. The Forskolin was effective at lower concentration to diminish lipids concentrations in embryos. But when we analysed the apoptotic cell, the lower concentration of Forskolin damaged embryos, but this effect could be diminished after vitrification and warming, when the drug did not increase the apoptotic cells. However, we need to study other concentrations of Forskolin.

FAPESP (2010/50410–2/2014/21289–1) is acknowledged for support.