Synergistic effect of alanine and glycine on bovine embryos cultured in a chemically defined medium and amino acid uptake by vitro-produced bovine morulae and blastocysts.

Abstract

The effect of glycine and alanine on the development of 2- to 4-cell bovine embryos, and amino acid uptake by bovine morulae and blastocysts, were examined through the use of a chemically defined medium. Bovine embryos at 2- to 4-cell stages were prepared by in vitro maturation and fertilization and cultured in a synthetic oviduct fluid medium (SOFM) containing polyvinyl alcohol (PVA) instead of BSA in order to examine the effect of amino acids. Morulae or blastocysts obtained from culture in SOFM containing BSA were cultured for 10 h in SOFM containing PVA to determine amino acid uptake. The combination of essential and nonessential amino acids with or without glutamine improved development to the blastocyst stage over that observed in the control (34% or 32% vs. 19%, respectively; p < 0.05). The optimal supplemental concentrations were 5 mM for alanine and 10 mM for glycine. At these concentrations, development to blastocysts was enhanced by the addition of alanine or glycine independently (35% or 36% vs. 26%, respectively; p < 0.05); the combined addition of alanine and glycine greatly (p < 0.01) improved proportions of blastocysts (45% vs. 26%) and hatched blastocysts (10% vs. 2%) compared to those obtained in the control. Addition of glycine with or without alanine to culture medium significantly increased cell number per blastocyst over that in the control (137 +/- 5 or 131 +/- 5 vs. 106 +/- 4, respectively; p < 0.01). Bovine morulae and blastocysts depleted aspartate, serine, and glutamate at a highly significant rate (p < 0.001) and arginine at a significant rate (p < 0.05), and produced alanine at a highly significant rate (p < 0.001) when cultured in medium containing 20 essential and nonessential amino acids. Serine, asparagine, glycine, alanine, and glutamine were highly (p < 0.001) produced by bovine morulae and blastocysts cultured in a medium containing essential amino acids without glutamine. These results indicate that alanine and glycine in a defined medium synergistically improve development of in vitro-produced bovine embryos, and also that bovine morulae and blastocysts prefer aspartate, glutamate, serine, and arginine and produce glutamine and several nonessential amino acids (serine, asparagine, alanine, and glycine).