Respirometry

Respirometry is a general term that encompasses a number of techniques for obtaining estimates of the rates of metabolism of vertebrates, invertebrates, plants, tissues, cells, or microorganisms via an indirect measure of heat production (calorimetry). Respirometry is a general term that encompasses a number of techniques for obtaining estimates of the rates of metabolism of vertebrates, invertebrates, plants, tissues, cells, or microorganisms via an indirect measure of heat production (calorimetry). The metabolism of an animal is estimated by determining rates of carbon dioxide production (VCO2) and oxygen consumption (VO2) of individual animals, either in a closed or an open-circuit respirometry system. Two measures are typically obtained: standard (SMR) or basal metabolic rate (BMR) and maximal rate (VO2max). SMR is measured while the animal is at rest (but not asleep) under specific laboratory (temperature, hydration) and subject-specific conditions (e.g., size or allometry), age, reproduction status, post-absorptive to avoid thermic effect of food). VO2max is typically determined during aerobic exercise at or near physiological limits. In contrast, field metabolic rate (FMR) refers to the metabolic rate of an unrestrained, active animal in nature. Whole-animal metabolic rates refer to these measures without correction for body mass. If SMR or BMR values are divided by the body mass value for the animal, then the rate is termed mass-specific. It is this mass-specific value that one typically hears in comparisons among species. Respirometry depends on a 'what goes in must come out' principle. Consider a closed system first. Imagine that we place a mouse into an air-tight container. The air sealed in the container initially contains the same composition and proportions of gases that were present in the room: 20.95% O2, 0.04% CO2, water vapor (the exact amount depends on air temperature, see dew point), 78% (approximately) N2, 0.93% argon and a variety of trace gases making up the rest (see Earth's atmosphere). As time passes, the mouse in the chamber produces CO2 and water vapor, but extracts O2 from the air in proportion to its metabolic demands. Therefore, as long as we know the volume of the system, the difference between the concentrations of O2 and CO2 at the start when we sealed the mouse into the chamber (the baseline or reference conditions) compared to the amounts present after the mouse has breathed the air at a later time must be the amounts of CO2/O2 produced/consumed by the mouse. Nitrogen and argon are inert gasses and therefore their fractional amounts are unchanged by the respiration of the mouse. In a closed system, the environment will eventually become hypoxic. For an open-system, design constraints include washout characteristics of the animal chamber and sensitivity of the gas analyzers. However, the basic principle remains the same: What goes in must come out. The primary distinction between an open and closed system is that the open system flows air through the chamber (i.e., air is pushed or pulled by pump) at a rate that constantly replenishes the O2 depleted by the animal while removing the CO2 and water vapor produced by the animal. The volumetric flow rate must be high enough to ensure that the animal never consumes all of the oxygen present in the chamber while at the same time, the rate must be low enough so that the animal consumes enough O2 for detection. For a 20 g mouse, flow rates of about 200 ml/min through 500 ml containers would provide a good balance. At this flow rate, about 40 ml of O2 is brought to the chamber and the entire volume of air in the chamber is exchanged within 5 minutes. For other smaller animals, chamber volumes can be much smaller and flow rates would be adjusted down as well. Note that for warm-blooded or endothermic animals (birds and mammals), chamber sizes and or flow rates would be selected to accommodate their higher metabolic rates. Calculating rates of VO2 and/or VCO2 requires knowledge of the flow rates into and out of the chamber, plus fractional concentrations of the gas mixtures into and out of the animal chamber. In general, metabolic rates are calculated from steady-state conditions (i.e., animal's metabolic rate is assumed to be constant). To know the rates of oxygen consumed, one needs to know the location of the flow meter relative to the animal chamber (if positioned before the chamber, the flow meter is 'upstream,' if positioned after the chamber, the flow meter is 'downstream'), and whether or not reactive gases are present (e.g., CO2, water, methane, see inert gas). For an open system with upstream flow meter, water (e.g., anhydrous calcium sulfate) and CO2 (e.g., a carbon dioxide adsorbent like Ascarite II, a registered trademark of the Arthur H. Thomas Co.) removed prior to oxygen analyzer, a suitable equation is For an open system with downstream flow meter, water and CO2 removed prior to oxygen analyzer, a suitable equation is