The goal of this study was to modify three steps method for measuring protection rate of amino acids which has smaller size than nylon bag pores. To prepare the protected amino acids by the Schiff base method, each of the essential amino acids (methionine and lysine) in the aqueous solvent were brought to isoelectric pH and then reacted with the same molar ratio of aldehydes the reaction was refluxed for 30 minutes to 6 days (depending on the type of aldehyde used). The three-step in vitro method was used to estimate rumen insoluble protein in the rumen. In the manufacture of protected amino acid ligands, compounds with methionine amino acid had higher efficiency than lysine compounds and also glutaraldehyde ligands showed higher production efficiency compared to benzaldehydes. The glutaraldehyde lysine ligand showed the lowest release after 2 h of incubation in the rumen medium. At time 4 the methionine benzaldehyde ligand had the lowest release. Benzaldehyde had the lowest release at time 6 and 8. According to the results of this table, Lysine benzaldehyde had better resistance to different phases and methionine benzaldehyde had the lowest release rate compared to the other ligands. Based on this study, it can be concluded that the use of chemical methods to protect amino acids can be applied.

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