A 30-year retrospective on the respirometric method for measuring instantaneous metabolic fuel use in fish

In 1996, based on the ideas of Max Kleiber, we proposed a respirometric method in this journal for the quantitative determination of the roles of protein, lipid and carbohydrate as substrates for fueling aerobic metabolism in fish on an instantaneous basis. Here I provide a 30-year retrospective on its performance, explaining how it works, methodological challenges, the applications for which it has been used, its strengths and potential flaws, and the important issues to be addressed going forward. The approach is based on the simultaneous measurement of the rates of O₂ consumption (ṀO₂), CO₂ excretion (ṀCO₂) and N-waste excretion (ṀN = Ṁ_amm + Ṁ_urea-N) under steady-state conditions in the whole fish when anaerobic metabolism is not occurring. These allow the calculation of the Respiratory Quotient (RQ = ṀCO₂/ṀO₂) and the Nitrogen Quotient (NQ = ṀN /ṀO₂), and from these, the fractional contributions of each of the three fuels. Principal methodological challenges arise from the difficulties of measuring ṀCO₂, and to a lesser extent ṀN, in water. To date, the approach has been used mainly to study fuel use during feeding, fasting, starvation, sustainable exercise, and at different temperatures. In general, lipid and carbohydrates have emerged as the major fuels burned in ammoniotelic fish (where ammonia is the predominant N-waste product), while protein is conserved, though protein metabolism may be more important in ureotelic fish (where urea-N is the predominant N-waste product) and air-breathers. Possible unidentified N- products of protein oxidation, the action of the anaerobic gut microbiome in generating ṀN in the absence of ṀO₂, and the ability of the gill microbiome to convert N-waste to di-nitrogen (N₂) are highlighted as potential flaws.