Early Development of IVM/IVF Bovine Embryos Cultured with or without Somatic Cells in a Simple Serum-free Medium with Different Concentrations of CO2 and O2

Abstract

The effect of various O2 and CO2 mixtures on development of bovine embryos produced by in vitro maturation and in vitro fertilization (IVM/IVF) was examined in a newly formulated simple KSOM medium versus a complex Menezo B2 serum-free medium and in co-culture. Numbers of cells comprising blastocysts were also counted using Hoechst 33342 stain. Following routine IVM/IVF procedures oocytes and zygotes were cultured for 40 to 44 h in various media. Then 2- to 8-cell embryos had cumulus cells removed and were alotted randomly for continued culture to the same experimental culture media, but with various gas conditions. In Experiment 1, the percentage of embryos developing to and beyond morula stages in 5, 10 and 20% O2 with 5% CO2 in humidified air was 22, 15 and 12%, respectively, with the 22 versus 12% being different (P<0.05). Blastocyst cell number in the 5 and 10%) O2 treatments was higher than in 20% O2 (P<0.05). Also, the simple KSOM and complex Menezo B2 media were compared and more embryos developed into blastocysts (10%) in KSOM medium than in B2 medium (5%; P<0.05), but cell number did not differ (P>0.05). In Experiment 2, the proportions of embryos developing into blastocysts in 5 and 10% CO2 were 15 and 6% (P<0.05), respectively, and in 5 and 10% O2 were 15 and 7%, respectively (P<0.05). Mean blastocyst cell number (82) was highest with 5% CO2 and 10% O2 even though more blastocysts were obtained with 5% CO2 and 5% O2 (P<0.05). In Experiment 3, embryos were co-cultured on monolayers of bovine oviduct epithelial cells (BOEC) or buffalo rat liver cells with KSOM medium in 5 and 10% O2 and 5% CO2. Treatment effects did not differ except 10% O2 was superior to 5% O2 when considering both morulae and blastocysts (41 vs 33%; P<0.05). These experiments indicate that the O2-CO2 concentrations can affect the development of bovine embryos produced by IVM/IVF when cultured in a simple serum-free medium with and without co-culture and that the simple KSOM medium can serve as a defined medium to study required supplementation for culturing IVM/IVF bovine oocytes. © 1994, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT. All rights reserved.