Effects of amino acids and alpha-amanitin on bovine embryo development in a simple protein-free medium

Five experiments, utilizing 3741 embryos produced in vitro, were designed to test the effects of Eagle's nonessential amino acids, and combinations of Eagle's essential amino acids and the RNA polymerase inhibitor alpha-amanitin on the development of preimplantation bovine embryos in a modified protein-free KSOM medium. Embryos were cultured in 5% O2:5% CO2:90% N2 at 39 degrees C for the first 40-44 hr in modified KSOM, and embryos with > or = 4 cells were cultured in modified KSOM-PVA with different amino acids in experiments 1-4, and with the addition of alpha-amanitin in experiment 5. In experiment 1, addition of 0.5x of the essential amino acids, with different concentrations of nonessential amino acids significantly increased hatching of blastocysts and decreased blastocyst degeneration, but increasing the nonessential amino acids from 1x to 5x, did not stimulate embryo development. In experiments 2-4, increasing only the glycine concentration, or adding each of the 12 essential amino acids singly or several in combination to the medium containing nonessential amino acids, did not significantly improve embryo development. Taurine (0.4 mM) in the modified KSOM medium reduced blastocyst degeneration. In experiment 5, alpha-amanitin (20 microM) completely inhibited further embryo development when it was added at several stages from 4-cell embryos to morulae. The study with protein-free KSOM plus amino acids provided a completely defined simple medium for culturing bovine embryos, with evidence that continuous mRNA activity and presumed protein synthesis was obligatory to meet the complex and continuous requirements for proteins by the developing blastocyst.