230 EVALUATION OF APOPTOSIS IN IN VITRO-PRODUCED BOVINE EMBRYOS MATURED WITH FORSKOLIN

Abstract

The maintenance of oocytes in the germinal vesicle stage for a few hours could result in more competent oocytes for use in biotechnology. Related to this, forskolin (Sigma-Aldrich, St. Louis, MO, USA) is an efficient inhibitor of nuclear maturation because of its ability to increase the levels of intracellular cyclic adenosine monophosphate. This study aimed to show whether the use of forskolin would be able to inhibit maturation in bovine oocytes, producing a higher rate of in vitro embryos. Nellore oocytes from a slaughterhouse (n = 960) were matured in TCM-199 with Earle’s salt + 10% FCS, FSH, and LH, in a 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium with forskolin at 3 different concentrations, 0. 1 mM (n = 240), 0.05 mM (n = 240), and 0.025 mM (n = 240), whereas untreated oocytes acted as controls (n = 240). The oocytes were then cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h (Day 0) of maturation, oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the same conditions as described above. Semen was selected through Percoll gradient, and the concentration was adjusted to 2 × 106 sperm mL–1. The presumed zygotes were cultured in 90-µL droplets of SOFaa + 0.6% BSA + 2.5% FCS in a 5% CO2, 5% O2, and 90% N2 atmosphere until Day 7, when blastocysts were evaluated. Apoptosis in blastocysts was accessed through TUNEL reaction. Data were analysed by ANOVA, followed by Tukey’s test using the general linear models procedure (PROC GLM) of SAS (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. No statistical differences were observed in blastocyst production rate (n = 297): control (n = 88): 36.7% ± 3.7; 0.1 mM forskolin (n = 61): 25.1% ± 3.7; 0.05 mM forskolin (n = 70): 29.2% ± 3.7; 0.025 mM forskolin (n = 78): 32.6% ± 3.7 (P > 0.05). However, when we analysed the apoptosis rates, differences were found among groups: control: 6.0% ± 6.3a; 0.1 mM forskolin: 33.4% ± 6.3b; 0.05 mM forskolin: 27.2% ± 6.3ab; 0.025 mM forskolin: 10.0% ± 6.3ab (P < 0.05). Although there was no difference in blastocyst rate, the TUNEL technique allowed us to identify that a high dose of forskolin was detrimental for in vitro-produced bovine embryos. FAPESP: 10/50410-2.