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).
Whole-animal metabolic rates 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).Blaxter, K. 1989. Energy metabolism in animals and man. Cambridge University Press. 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.
Closed respirometry 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.
Open respirometry For an open-system, design constraints include washout characteristics of the animal chamber and sensitivity of the gas analyzers.Lighton, J. R. B. 2008. Measuring metabolic rates: A manual for scientists. Oxford University Press. . 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.
Calculations 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 removed prior to the oxygen analyzer, a suitable equation is
VO2 = \frac {\mathit{FR} \cdot (\mathit F_{in}O2 - \mathit F_{ex}O2)} {1 - \mathit F_{ex}O2}
For an open system with downstream flow meter, water and CO2 removed prior to the oxygen analyzer, a suitable equation is
VO2 = \frac {\mathit{FR} \cdot (\mathit F_{in}O2 - \mathit F_{ex}O2)} {1 - \mathit F_{in}O2}
where FR is the volumetric flow rate adjusted to STP (see Standard conditions for temperature and pressure) FinO2 is the fractional amount of oxygen present in the incurrent air stream (the baseline or reference), and FexO2 is the fractional amount of oxygen present in the excurrent air stream (what the animal has consumed relative to baseline per unit time).
For example, values for BMR of a 20 g mouse (Mus musculus) might be FR = 200 mL/min, and readings of fractional concentration of O2 from an oxygen analyzer are FinO2 = 0.2095, FexO2 = 0.2072. The calculated rate of oxygen consumption is 0.58 mL/min or 35 mL/hour. Assuming an enthalpy of combustion for O2 of 20.1 joules per milliliter, we would then calculate the heat production (and therefore metabolism) for the mouse as 703.5 J/h.
Respirometry equipment For open flow system, the list of equipment and parts is long compared to the components of a closed system, but the chief advantage of the open system is that it permits continuous recording of metabolic rate. The risk of hypoxia is also much less in an open system.
Pumps for air flow Vacuum Pump: a pump is needed to push (i.e., upstream location) or pull (i.e., downstream location) air into and through the animal chamber and respirometry flow-through system. Subsample pump: To pull air through the analyzers, a small, stable, reliable pump is used.
Flow meter and flow controllers Bubble flow meters: A simple, yet highly accurate way to measure flow rates involves timing movement of bubbles of soap film up glass tubes between marks of known volume. The glass tube is connected at the bottom (for push systems) or at the top (for pull systems) to the air stream. A small rubber pipette bulb attached at the base of the tube acts as both a reservoir and delivery system for the soap bubbles. Operation is simple. First, wet the glass surface along the path bubbles travel (e.g., press the bulb so that copious amounts of soap are pushed up the glass by the air flow) to provide a virtually friction-free surface. Second, pinch the bulb so that one clean bubble is produced. With a stopwatch in hand, record the time required for the bubble to travel between marks on the glass. Note the volume recorded on the upper mark (e.g., 125 = 125 ml), divide the volume by the time required to travel between marks and the result is the flow rate (ml/s). These instruments can be purchased from a variety of sources, but they may also be constructed from appropriate-sized, glass volumetric pipettes. Acrylic flow meters : Under some circumstances of high flow rates we may use simple acrylic flow meters (0–2.5 liters/min) to control the flow rates through the metabolic chambers. The meters are located upstream from the metabolic chambers. The flow meters are simple to use but should be calibrated twice daily for use in the respirometry system: once before recording begins (but after the animal has been sealed inside the chamber!!) and again at the end of the recording (before the animal is removed from the chamber). Calibration must be done with a bubble flow meter because the calibration marks on the acrylic meters are only approximate. For proper calibration of flow rates remember that both barometric pressure and temperature of the air streaming through the flow meter (which we assume to be equal to room temperature) must be recorded. Mass flow meters: The equations required for calculating rates of oxygen consumption or carbon dioxide production assume that the flow rates into and out of the chambers are known exactly. We use mass flow meters which have the advantage of yielding flow rates independent of temperature and air pressure. Therefore, these flow rates can be considered to be corrected to standard conditions (Standard Temperature Pressure). We only measure and control flow at one location—downstream from the chamber. Therefore, we must assume that the inflow and outflow rates are identical. However, during construction of the respirometry system, flow rate must be measured at all steps, across all connections, to verify integrity of flow. Needle valves: Mass flow meters may be purchased with mass flow controllers which permit setting flow rates. These are expensive, however. Respirometry research often will attempt to measure more than one animal at a time, which would necessitate one chamber per animal and thus controlled flow through each chamber. An alternative and more cost-effective method to control flow would be via stainless steel or carbon steel needle valves. Needle valves plus mass flow meters provides a cost-effective means to achieve desired flow rates. The valves cost about $20.
Tubing and chambers Tubing and connections : Various kinds of tubing can be used to connect the components of the respirometry system to and from the animal chamber. A variety of kinds of flexible tubing may be used, depending on the characteristics of the system. Acetyl, Bev-A-Line, Kynar, nylon, Tygon tubing and connectors may be used in regions of the system where oxidizing atmospheres are low (e.g., background levels of ozone only); Teflon tubing would be recommended if there is an expectation for appreciable amounts of ozone to be present because it is inert to ozone. Teflon tubes are more costly and lack flexibility.