EFFECTS OF PHOSPHATE, ENERGY SUBSTRATES, AND AMINO-ACIDS ON DEVELOPMENT OF INVITRO-MATURED, INVITRO-FERTILIZED BOVINE OOCYTES IN A CHEMICALLY DEFINED, PROTEIN-FREE CULTURE-MEDIUM

Bovine oocytes that had been matured and fertilized in vitro were cultured in a simple, chemically defined, protein-free medium (mTLP-PVA). When the medium was supplemented with 19 amino acids, development to the 8-cell (14-20% vs. 38-46%), morula (0-6% vs. 27-32%), and blastocyst (0-1% vs. 9-13% ) stages 96, 144, and 192 h after insemination, respectively, was significantly greater in the absence than in the presence of glucose (5.56 mM) regardless of the presence of phosphate (1.05 mM). However, blastocyst development was difficult in medium with any combination of glucose and phosphate without amino acids. In mTLP-PVA with amino acids and different concentrations of phosphate, the highest proportions of embryos reaching the greater-than-or-equal-to 8-cell (56%), morula (44%), and blastocyst (24% ) stages were obtained at a 0.35 mM concentration. When lactate and pyruvate were omitted from mTLP-PVA (mT-PVA) supplemented with amino acids and 0-35 mM phosphate, the first cleavage was completely inhibited. Although lactate or pyruvate alone could support blastocyst development to a limited extent (10-15%), a significantly higher proportion (22%) of blastocysts was obtained in medium with both lactate (10 mM) and pyruvate (0.5 mM). When different concentrations of glucose were added to mTLP-PVA with amino acids and 0.35 mM phosphate 120 h after insemination, significantly higher proportions of blastocysts (29-32% ) and expanded blastocysts (20-23%) 192 and 216 h after insemination, respectively, were obtained at 2.78 and 5.56 mM concentrations than at 0-1.39 and 11.12 mM concentrations; however, the values for the expanded blastocysts were not significantly different from the value (15%) at 1.39 mM.