Cell apoptosis and lipid content of in vitro–produced, vitrified bovine embryos treated with forskolin

Abstract

The presence of fetal calf serum in culture medium influences embryo quality, causing a reduction in postcryopreservation survival. Forskolin has been used to induce lipolysis and increase cryotolerance, functioning as an activator of adenylate cyclase and elevating cAMP levels. In the present experiment, bovine zygotes were cultured in synthetic oviduct fluid with amino acid plus 2.5% fetal calf serum for 6 days, when forskolin was added in three concentrations: 2.5, 5, and 10 μM. Treatment with forskolin lasted for 24 hours. Blastocyst formation rate, quantification of lipid granules, total cell numbers, and apoptosis rate were evaluated. In a second assessment, embryos were vitrified, and warming, re-expansion rate, total cell numbers, and apoptosis rate were also evaluated. There was no difference due to forskolin in blastocyst formation or re-expansion rates after vitrification. However, lipid measurements were lower (control: 136.8 and F 2.5 μM: 128.5; P < 0.05), and number of cells per embryo higher (control: 140.1 and F 2.5 μM: 173.5; P < 0.05) than controls for 2.5 μM forskolin but not for higher forskolin concentrations. The number of intact cells per embryo was higher, and the rate of apoptosis was lower in fresh than in vitrified embryos (number of cells of warmed embryos, control: 104.1, F 2.5 μM: 101.3, F 5 μM: 115.4, F 10 μM: 95.1; apoptotic of fresh cells, control: 12.1%, F 2.5 μM: 16.7%, F 5 μM: 11.1%, F 10 μM: 14.2%; and apoptotic warmed embryos, control: 22.3%, F 2.5 μM: 37.3%, F 5 μM: 33.2%, F 10 μM: 30.3%; P < 0.05). It was concluded that forskolin is an effective lipolytic agent even at low concentrations, leading to formation of blastocysts with a comparatively larger number of cells.