The aim of the study was to determine the amino acid requirements of the in vitro-produced bovine embryo as it develops from the zygote to the blastocyst, using a two-step culture system. When added to synthetic oviduct fluid (SOF) for the first 72-h culture, Eagle's nonessential amino acids and glutamine (NeGln) significantly increased development to the 8- to 16-cell stage (Day 4 postinsemination [pi]) and subsequent blastocyst development (Day 7 pi). Glutamine alone during the first 72-h culture did not stimulate development to the 8- to 16-cell stage (p > 0.05); however, the removal of glutamine from NeGln reduced the stimulatory effects of the nonessential amino acids. Replacing glutamine with betaine (an organic osmolyte) in NeGln did not stimulate development to the 8- to 16-cell stage compared to culture in SOF, but it did improve subsequent blastocyst development, indicating an osmolytic function of glutamine during the first 72-h culture. The addition of Eagle's essential amino acids and glutamine to SOF, or to medium already containing nonessential amino acids and glutamine for the first 72-h culture, did not affect cleavage to the 8- to 16-cell stage or subsequent blastocyst development (p > 0.05). Beyond Day 4 pi, culture with 20aa (nonessential and essential amino acids and glutamine) increased blastocyst development, total cell number, and the number of cells in both the trophectoderm and inner cell mass, compared to culture with other groups of amino acids (p < 0.05). Substituting betaine for glutamine in 20aa reduced blastocyst formation, indicating a non-osmolytic function of glutamine during the second 72-h culture. Further, there was a significant negative correlation between the concentration of essential amino acids (quarter, half, or single strength) and embryo development during both the first 72-h and second 72-h culture (p < 0.01), indicating that the concentration of essential amino acids was too high during culture of the bovine embryo. This study identified the temporal and differential effects of amino acids during development of the bovine embryo from the zygote to the blastocyst.
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