Effects of amino acids on the development of in-vitro matured/in-vitro fertilization bovine embryos in a simple protein-free medium

Abstract

The effects of commercially available amino acids, referred to as Eagle's non-essential amino acids (NEAA) and essential amino acids (EAA), on the development of bovine embryos produced by in-vitro maturation and in-vitro fertilization (IVF) in a simple modified protein-free KSOM medium containing polyvinyl alcohol (PVA) were studied. Embryos produced by IVF were cultured in an atmosphere of 5% O2, 5% CO2 and 90% N2 at 39 degrees C for the first 40-44 h in modified KSOM. Then embryos with four or more cells were cultured in modified KSOM-PVA with different amino acids for another 6 days in experiments 1 and 2. In experiment 3, to study embryo hatching and degeneration further as criteria of medium suitability, the culture time was 8 days. In experiment 1, more blastocysts were produced (P < 0.05) in the medium with 0.5 x NEAA (37%) than with 0.25 or 0.5 x EAA (both 15%). In experiment 2, when 0.0, 0.5 and 1.0 x concentrations of NEAA were combined with 0.0 and 0.5 x concentrations of EAA, the combination 1.0 x NEAA with 0.5 x EAA resulted in the most blastocysts (39%), with 11% hatching (P < 0.05). Without NEAA, 1.0 x EAA resulted in 6% blastocysts (P < 0.05). In experiment 3, KSOM-PVA with 1.0 x NEAA plus 0.5 x EAA resulted in 41% blastocysts, similar to the 40% blastocysts in KSOM-bovine serum albumin (BSA) with the same amino acids added. When culture was continued for 2 more days, more embryos hatched in these two treatments. These results indicate that the combination of 1.0 x NEAA with 0.5 x EAA promoted blastocyst formation and hatching, but EAA alone was detrimental. With PVA successfully replacing BSA, a completely defined medium resulted, which was equal or superior to complex medium, and is potentially useful for the culture of embryos from other species, such as humans.