Both this human and the truck are powered by burning fuel, we call this process combustion or oxidation. The human converts fats and carbohydrates in the fries to energy, energy for movement and other physiological processes to sustain life. The truck, on the other hand, uses cooking oil to move its pistons and send power to the wheels. The truck does its combustion in a series of sudden explosions. In the human, living cells do the breakdown in sequential biochemical steps, but the outcome is the same. A chemical fuel plus enough oxygen yields carbon dioxide and water vapor and heat.

Now how do we measure the output of our combustion engines? One way is called direct calorimetry. The process involves directly measuring heat produced during combustion reactions. It is relatively easy burning a small object in a well-insulated chamber to estimate its energy content. This approach can also work for small animals, provided the airflow is well controlled. However, direct calorimetry becomes a major challenge when larger animals are concerned. An alternative to measure the output of our combustion engines is called indirect calorimetry or respirometry. Instead of heat release, we measure O2 consumed and CO2 produced during oxidation. For the oxidation of any fuel, there is a fixed relationship between oxygen, CO2, and heat. So, it’s possible to measure the oxygen and CO2 and then calculate the heat production.

How do we measure oxygen consumption and CO2 production? Well, the law of conservation of mass states that matter can neither be created nor destroyed. Thus, the difference between O2 and CO2 in, and O2 and CO2 out, must be due to the animal that’s inside the box or the metabolic chamber. We have to measure O2 and CO2 concentrations of incurrent air and excurrent air. The animal changes excurrent O2 and CO2 concentrations. These changes are referred to as delta O2 and delta CO2.

Where in this process do we measure airflow rates? Well, air flows into the chamber with a known composition at a measured flow rate. The animal removes O2 and adds CO2 and water vapor. This changes the excurrent flow rate. Thus, flow rate in is never equal to the flow rate out. Flow rate out can change with the removal of water vapor as well as the removal of CO2. Flow rate also depends on temperature and pressure, both of which should be known. It is imperative to know where the flow rate is measured, either incurrent or excurrent.

How do we calculate the following variables? Oxygen consumption we call VO2 and carbon dioxide production we call VCO2. We need to know the flow rate and the changes in oxygen – we call that delta O2. And changes in carbon dioxide – this is called delta CO2. In this case, O2 is expressed in fractional concentration. This is a unitless value, moles of oxygen divided by moles of air equals FO2. And the same applies for CO2. Mass balance states that VO2 is the product of flow rate and the fractional change in oxygen. And delta oxygen is the difference between the incurrent and the excurrent fractional oxygen concentrations. Then, VO2 is the difference between the incurrent VO2 and the excurrent VO2, which in turn is the difference between the product of the incurrent flow rate and the incurrent oxygen and the product of the excurrent flow rate and the excurrent oxygen. These same principles apply to VCO2. In short, the changes caused by the animal are determined by the air composition in and the air composition out. Recall that flow rate is key and in theory you’ll need to know the flow rates both incurrent and excurrent. But in reality, usually only one flow meter is used. You can either measure FRi or FRi.

So how do we account for the unknown flow rates? Well, if flow rate FR is measured upstream from the animal chamber, we call this push mode. We would start with the equations earlier derived for VO2. Incorporating incurrent and excurrent flow rates and fractional O2 values, and we do the same thing for CO2. These equations can now be combined reflecting fractional changes for incurrent and excurrent air. Substituting for FRe (the excurrent flow rate) requires a fairly elaborate algebraic derivation and is beyond the scope of this video. But the final equations correct for the unknown excurrent flow rate. If flow rate FR is measured downstream from the animal chamber, we call this pull mode. Again, start and rearrange the equations as done earlier. But now substitute for FRi, which is the unknown. This results in a similar elaborate algebraic derivation, but now the equations are correct for the unknown incurrent flow rate. Specifically, these VO2 and VCO2 equations look relatively similar, but the substitutions of Fri versus FRe result in different algebraic derivations. When FRi is known, the derived VO2 equation incorporates excurrent fractional oxygen in the equations. When FRe is known, it’s the incurrent fractional oxygen that’s incorporated into the equations. These seemingly subtle differences yield reliable calculations.

If only one gas sample can be measured at a time, how can incurrent and excurrent values both be measured? This is done by using a bypass channel to circumvent the animal chamber. This is also known as a baseline. This periodically sends incurrent air flow to the analyzer. This is air that’s untouched by the animal, and it allows us to measure the composition of the incurrent air. Switching between incurrent and excurrent air streams can be done manually or automated electronically.

And what about multiple animals? We can add a common airflow generator. Then we feed equivalent incurrent flows into each animal chamber. We also add a bypass or a baseline. These also have equivalent excurrent flow rates. Now we add a common gas analyzer chain and a switching mechanism, also known as a multiplexer. This feeds individual excurrent air flows in a sequence to the analyzers with an interleaved baseline. Now we have all the components we need for indirect calorimetry, or respirometry. An air source, a pump or pressurized air, flow measurement and control, a switching device to switch between the animal and baseline, animal chambers, a gas analyzer chain to measure H2O, CO2, and oxygen, and importantly, a user interface for data acquisition and system control.

The next step is to configure these components into a basic respiratory system. Start with the source to generate the airflow. Use a scrubber to eliminate unwanted trace gases, CO2, and water vapor. Control the rate of airflow. Feed the airflow into a baseline to switch to an animal chamber or to bypass to measure the incurrent air. This practice is called baselining. Measure water vapor. Remove water vapor from the airflow. Measure CO2, then remove CO2 from the airflow, and finally, measure O2. Add an acquisition and control interface, acquire O2 data, acquire CO2 data, and acquire H2O data. Always acquire airflow rate data simultaneously. Acquire data on baselining status, either chamber or baseline. Finally, add automated control to the baselining. And now you have the basic configuration of a respirometry system.