Reliable methods to measure metabolite turnover rates in vivo are widely available (Hetenyi et al. 1983), and they have been used extensively in mammalian metabolic studies, especially for humans (Wolfe, 1992). In comparison, surprisingly little work has been carried out on fish (Garin et al. 1987; Weber and Zwingelstein, 1995), mainly because it has been difficult to adapt tracer techniques to aquatic animals of smaller relative body size. The bolus injection method has been used almost exclusively in fish studies (Weber and Haman, 1995) because it only requires a single catheter and can consequently be performed with a simple dorsal aortic cannula. This method has serious limitations, however, and it has been almost totally abandoned for mammals because it can only be used under steady-state conditions and each experiment only allows the calculation of a single turnover rate after analysis of a minimum of 6–10 blood samples. In mammals, metabolite turnover rates are quantified using the more versatile continuous infusion technique because it allows measurements under steady-state as well as non-steady-state conditions; also, much more information can be obtained from a single experiment because instantaneous flux can be calculated from each blood sample separately. Unfortunately, this method requires the surgical placement of two catheters – one to infuse the metabolic tracer, the other for blood sampling – and it has never been adapted to investigate the metabolism of trout, the most commonly used teleost model. The now classic dorsal aorta cannulation technique of Smith and Bell (1964), later modified by Soivio et al. (1975), revolutionized the study of fish metabolism by allowing repeated blood sampling and intravascular injections in undisturbed, non-anaesthetized fish. Over the last 30 years, dorsal aorta cannulation has been used routinely to investigate fundamental aspects of fish biochemistry and physiology. This surgical approach has provided invaluable information on the effects of various stresses including exercise, changes in water temperature, pH, salinity, oxygen content and toxicant concentration. In studies of fish metabolism, the blood concentration of key metabolites has been monitored to examine the biochemical consequences of these stresses. In this context, turnover rate is a much more informative parameter than concentration, and the temptation to use concentration changes to draw conclusions about fluxes has not always been resisted. Such extrapolation is clearly unwarranted, however, because flux and concentration do not 1157 The Journal of Experimental Biology 199, 1157–1162 (1996) Printed in Great Britain © The Company of Biologists Limited 1996 JEB0246 This paper describes a double dorsal aorta catheterization technique allowing the measurement of substrate turnover rates by continuous infusion of metabolic tracers in rainbow trout. The placement of both catheters can be performed in about 30 min with minimal surgical training. As a practical example of a routine substrate flux measurement, glucose turnover rate of resting trout was measured by primed continuous infusion of 6-[ 3 H]glucose through one of the catheters and blood sampling from the other. The animals maintained resting metabolic rate, normal blood glucose and low blood lactate concentrations throughout the experiments. Glucose isotopic steady state was achieved in less than 40 min, and mean turnover rate was 9.0±0.7 mmol kg 21 min 21 (N=8). Comparison with published glucose turnover rates measured in trout and other teleost species suggest that values previously obtained using the ‘bolus injection technique’ are underestimates of true flux rates. We conclude that the simple surgical technique presented here opens the door to the dynamic study of substrate kinetics under a variety of experimental conditions and that it can be adapted to the investigation of most metabolic substrates, including fatty acids, glycerol, amino acids and lactate, in addition to glucose. Future application of the continuous infusion technique under steady-state as well as non-steady-state conditions will add a new dimension to the general understanding of fish metabolism. Key words: substrate fluxes, metabolite replacement rate, hepatic glucose production, continuous infusion, non-steady-state kinetics, glucose, lactate, glycerol, fatty acids, amino acids, tracer methodology, Oncorhynchus mykiss. Summary CONTINUOUS TRACER INFUSION TO MEASURE IN VIVO METABOLITE TURNOVER RATES IN TROUT FRANÇOIS HAMAN AND JEAN-MICHEL WEBER* Biology Department, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5 Accepted 16 January 1996 Introduction