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Adenylate energy charge (AEC) has been used as a practical index of the physiological status and health in several disciplines, such as ecotoxicology and aquaculture. This study standardizes several procedures for AEC determination in penaeid shrimp that are very sensitive to sampling. We concluded that shrimp can be frozen in liquid nitrogen and then stored at -76?C for up to two years for further analysis, or freshly dissected and immediately homogenized in acid. Other cooling procedures, such as immersion in cold water or placing shrimp on ice for 15 min resulted in 50% and 73% decreases in ATP levels, and 9-fold and 10-fold increases in IMP levels, respectively. Optimal values of AEC (0.9) were obtained in shrimp recently transferred from ponds to indoor conditions, but decreased to 0.77 after one month in indoor tanks when stocked at high densities; the AEC re-established to 0.85 when the shrimps were transferred to optimal conditions (lower density and dark tanks). While the levels of arginine phosphate followed the same pattern, its levels did not fully re-establish. Comparison of different devices for sample homogenization indicated that a cryogenic ball mill mixer is the more suitable procedure.