Use of Forskolin in Meiosis Inhibition to Improve in Vitro Production Bovine Embryos.

One of the most widely used techniques is inhibition or delay in the meiotic (nuclear) maturation of oocytes, which would allow a more regulated and appropriate transition from prophase I to metaphase II. Thus, the maintenance of oocytes in Germinal Vesicle (GV) stage for a few hours could result in more competent oocytes for use in biotechnology techniques. In this matter, the forskolin is an efficient inhibitor of nuclear maturation because of its ability in raising the levels of intracellular cAMP. This study aimed to show for the first time that the use of forskolin alone is able to efficiently inhibit maturation in bovine oocytes (Sigma-Aldrich, St. Louis) producing a higher rate of in vitro embryos. Nelore oocytes were matured in TCM-199 with Earle's salt + 10% FCS, FSH and LH, in 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 hours in medium in presence of 3 different concentrations of forskolin: 1mM, 0.05mM, 0.025mM. Then the oocytes were cultured for 18 hours in agent-free medium to meiosis resume, completing 24 hours of maturation. After 24 hours (day 0) of maturation, oocytes were fertilized in human tubal fluid (HTF – Irvine, New Zeland) under the same condition above. Semen was selected through Percoll gradient and the concentration adjusted to 2 x 106 sperm/mL. The presumably zygotes were culture in 90μL droplets of SOFaa + 0.6% BSA + 2.5% FCS in 5% CO2, 5% O2, 90% N2 atmosphere until day 7, when blastocyst rate was evaluated. There were made 4 replicates. Data were analyzed with ANOVA, followed by Tukey test using the general linear model (PROC GLM). The level of significance adopted was 5%. So far, no statistical differences were observed in blastocyst production rate: Control: 34.4% ± 8.1; Forskolin 1mM: 28.3% ± 8.1; Forskolin 0.05mM: 31.2% ± 8.1; Forskolin 0.025mM: 39.4% ± 8.1 (P>0.05). As this is preliminary results and have no similar data in the literature, it will be necessary more repetitions, to prove the promising results obtained with the lowest concentration of forskolin.