Use of forskolin to delay meiosis and produce in vitro bovine embryos

Abstract

The maintenance of oocytes in germinal vesicle (GV) stage for a few hours could result in more competent oocytes for use in biotechnology. This study aimed to show if the use of forskolin is able to inhibit and reverse the maturation in bovine oocytes, producing a higher rate of in vitro embryos without apoptosis rates. Eight replicates in total were performed. Nellore oocytes were matured in TCM-199 and to delay meiosis, the oocytes (n = 584) were maintained for 6 h in medium in presence of 0.025, 0.05, or 0.1 mM Forskolin. Then, the oocytes were cultured for 18 h in agent-free medium to resume meiosis. After resumption of meiosis, the oocytes (n = 336) were stained with Hoechst 33342 to evaluate the state of the nucleus: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), or degenerated or unidentified (D/U). Then (Day 0) oocytes were fertilized in human tubal fluid (Irvine, New Zealand) and the presumed zygotes were culture in SOFaa + 0.6% BSA + 2.5% FCS until Day 7, when the blastocyst (n = 177) rate was evaluated. Apoptosis in blastocysts was assessed by terminal deoxynucleotidyl transferase uracil nick-end labeling (TUNEL) reaction. Data were analysed by ANOVA, followed by Tukey test using the general linear model (PROC GLM) of SAS (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. There were no statistical differences in state of the nucleus, only in MI (Control = GV: 0.0, GVBD: 0.8, MI: 8.3a, MII: 67.7, D/U: 7.3; F 0.025 mM = GV: 2.8, GVBD: 0.7, MI: 20.8ab, MII: 67.7, D/U: 8.9; F 0.05 mM = GV: 0.0, GVBD: 4.4, MI: 15.8ab, MII: 65.9, D/U: 13.7; and F 0.1 mM = GV: 0.0, GVBD: 1.0, MI: 34.1b, MII: 50.2, D/U: 14.6; P < 0.05). There were no statistical differences in blastocyst rate (Control: 36.7, F 0.025 mM: 32.6, F 0.05 mM: 29.2 and F 0.1 mM: 25.1 – P > 0.05). But when we analysed the apoptosis rate, differences were found among groups: (Control: 12.1a, F 0.025 mM: 12.9a, F 0.05 mM: 13.5a and F 0.1 mM: 30b; P < 0.05). Although Forskolin was able to inhibit meiosis and produce embryos at the same rates as controls, the higher dosage of this drug damaged the embryos.