Due to logistic and practical reasons, it is sometimes necessary to use modern single stage incubators in a multi stage set up, so for instance adding trolleys with fresh eggs 2 times a week and transferring the two times a week after 18 days. This is not ideal for a good control of especially the egg shell temperature (as an "average" air temperature have to be used that is actually below optimum in the beginning and above optimum at the end of the incubation process), but it also brings the question on how the ventilation should be done.

In modern single stage machines we often run the ventilation based on a maximum allowed level of carbon dioxide in the machine, for instance 3500 ppm. This means that when the embryos are at maximum development (17-18 days) we bring in enough air to remove the carbon dioxide produced by the embryos and limit the level in the machines to 3500 ppm. However, it is questionable if this is the right way also for single stage machines if we operate them in a multi stage setting.

In principle we ventilate the machines for 3 different reasons, to remove the heat, to remove the moisture from the egg and control the moisture loss of the eggs, and to remove the produced carbon dioxide. In older multi stage machines the cooling capacity was often limited, so we often had to ventilate to remove heat. In modern single stage machines the cooling capacity is much more, and especially in a multi stage setting (not all the eggs being at the same moment at 18 days of incubation) the cooling capacity from the cooling coils will be sufficient to take out the heat. This means that the ventilation should be based on relative humidity (control of moisture loss of the eggs) or on carbon dioxide, which ever will be the first limiting factor.

Let's start with ventilation for carbon dioxide. If a machine of 100.000 eggs is used in a multi stage setting, there will normally be eggs in there of 6 different ages (setting eggs two times a week). The average age of the embryos in that machine (to determine the production of carbon dioxide) can be estimated to be approximately 10-12 days. In theory the average age will be 9 days, but to be on the safe side and because it will not always be completely balanced, lets assume the average age is 12 days. On this website there is a program

(https://www.poultryperformanceplus.com/calculations/249-theoretical-calculation-of-actual-ventilation-levels-in-an-incubator) 

which can be used to calculate the ventilation rate of a machine. In that program we can see that an embryo of 12 days produces approximately 90-91 ml of CO2 per day. With the program, we can calculate that a machine of 100.000 eggs (90% fertility) needs to ventilate 114 m3 per hour if the incoming air contains 500 ppm CO2 and we want the air in the machine to hold 3500 ppm. If we want the air in the machine to contain 2500 ppm we need to ventilate 170 m3/hr, and for 1500 ppm CO2 we need to ventilate 341 m3/hr.

But we do not only ventilate for carbon dioxide, but also for relative humidity. To get an average moisture loss of approximately 11-12% (weight loss) at 18 days, we need a relative humidity of approximately 50-60%. The eggs are losing moisture to the air, so if we do not ventilate or ventilate too little, the relative humidity in the air will go up and the moisture loss will not be sufficient. If we ventilate too much the relative humidity will go down and we lose too much moisture or the humidification system will come in, creating cold spots in the machine. 

If eggs lose 11-12% moisture in 18 days, they lose approximately 0,6% per day. If the eggs are 65 grams and the machine contains 100.000 eggs, the moisture loss will be 100.000 x 0.065 x 0.006 = 39 liter of water per day or 1.625 liter of water per hour. This water needs to be taken out of the machine by the air. 

Lets assume that we want an relative humdity in the machine of 55% with an air temperature of 37oC. The Mollier diagram tells us that air of that property holds 24.1 g of moisture per m3, so each m3 of air that leaves the machine will take out 24.1 g. The incoming air also holds moisture. If for instance the air that enters the machine is 24oC and 50% relative humidity, the Mollier diagram tells us that it contains 12.9 g of moisture per m3. This means that every m3 of air can uptake 24.1 - 12.9 = 11.2 g of moisture.

To keep the carbon dioxide on a level of 3500 ppm in our multi stage situation, we needed to ventilate 114 m3 per hour. However, this ventilation level will only take out 114 m3 x 11.2 g = 1.277 kg (liter) of moisture, where we needed to take out 1.625 liter of water. As a result the relative humdity would go up and we would not lose enough moisture from the eggs. If we reduce the carbon dioxide level to 2500 ppm, we were ventilating 170 m3/h, which would result in 170 x 11.2 = 1.904 kg (liter) of water instead of the 1.625 liter that we are looking for. If we would then ask for 55% relative humdity, the machine would start adding water to the air as we take more water out than what comes from the eggs. With 1500 ppm the air would take out 341 x 11.2 g is 3.819 liter per hour.

What this calculation shows is that if we use a single stage machine in a multi stage mode, the ventilation should be based on relative humidity control and not on carbon dioxide control. The production of carbon dioxide in a multi stage machine is not enough to be limiting for the ventilation, but the producdtion of water by the eggs is. To keep the machine in balance with a constant relative humidity and a controlled moisture loss, the relative humidity should overrule the carbon dioxide settings, as the last one will not become limiting.