For many years, raised CO2 levels at the end of the incubation period were thought to be harmful for the developing bird embryo – this is a myth that couldn’t be further from the truth. Under natural circumstances, the level of CO2 measured in the micro-environment under the hen reaches 0.4% or 4,000 ppm at least, and often more. This represents ten times the normal level of CO2 in the outside air. In fact, when sitting on the eggs, the mother hen cuts off the airstream around the eggs to create a protected environment so that embryos can develop in the best possible conditions. Eager to better understand this process, Petersime set out to determine the optimal CO2 levels during hatching and the correct moment to raise them. 

Three essential levels

It’s long been known that in hatcheries, healthy chicks come from mastering and controlling three essential levels, in this order: 

  • Temperature: the most important environmental parameter, as a slight increase or decrease can severely affect embryonic development, hatchability and post-hatch performance.
  • CO2 and O2 levels: affect the development of the embryo’s cardiovascular system, and thus its respiration. 
  • Humidity level of the air surrounding the eggs: determines the rate at which the eggs lose water and, as a result, lose weight. 


Fundamental changes from day 18 on

Many studies have confirmed that dramatic changes occur to the chick, both physically and physiologically, from around day 18 onwards of the incubation period. The most relevant change occurs when internal pipping starts; that is when the internal eggshell membrane is perforated giving access to the air chamber. This is then followed by external pipping and continues until the chick fully emerges from the shell. The whole process is the transition from fluid to dry

In essence, the O2 and CO2 levels in the egg’s air cell change as the embryo begins to adopt convective gas exchange by inflating its lungs – in other words, breathing! Previous studies indicate that the O2 level decreases to approximately 14.2%, while the CO2 concentration rises to about 5.6%. Some might consider these levels excessive and therefore detrimental to the embryo — as they would be to humans — but this is not the case for birds, which have additional breathing capacities for a certain period of time. Indeed, for about 24 hours, starting from when internal pipping begins, the embryo uses both the vascular and pulmonary respiration systems to breathe at the same time (see image), resulting in a combined O2 uptake and therefore improved respiratory capacity

hatchery; CO2; incubation; hatching; pipping
Respiratory evolution of a chick.

Different levels of CO2 – a practical test

Scientific evidence, combined with practical observations in commercial hatcheries, confirms that a controlled higher CO2 concentration, even above 1% (10,000 ppm), in between pipping and hatching phases actually results in a narrower hatch window without negatively affecting the quality and health of the chicks. In fact, a narrow window like this is better, as it leads to more uniform day-old chicks, as well as a more efficient growth and processing of the chicks in the future. 

In partnership with a large and modern commercial hatchery (LAR Cooperativa Agroindustrial, Brazil — which produces about 350,000 chicks a day) and the State University of Ponta Grossa (Brazil), Petersime investigated the effects of higher and lower CO2 levels – 0.45% (4,500 ppm) and 0.80% (8,000 ppm) – in the hatcher. 

320 chicks were subjected to different CO2 levels from internal pipping (which can be automatically detected by Petersime’s Synchro-Hatch™ technology) until hatching, for a maximum of 24 hours. Blood samples were taken to verify their health and strength

Among the many blood parameters analysed, we looked at two measurements in particular: 

  • The number of heterophiles (antibodies that circulate in blood)
  • The lymphocyte level (a type of immune cell) 

We also checked the H:L (heterophile to lymphocyte ratio) of all the blood samples (see table). 

Variables CO2 levels during about 24h in the hatcher
  (0.45%)/(4,500 ppm) (0.80%)/(8,000 ppm)
Heterophile count 21.1 ± 5 7.5 ± 3
Lymphocyte count 78.8 ± 5 92.5 ± 3
Heterophile/Lymphocyte ratio 0.27 ± 0.07 0.08 ± 0.03

The results (and implications)

The results of the experiment showed that there are nearly three times more heterophiles when CO2 levels are at 0.45% (4,500 ppm), compared to 0.80% (8,000 ppm). 

Scientists have long known that a higher heterophile count – essentially part of the innate immune response – points to a reduced ability to fight inflammatory processes and stress. 

Lymphocyte levels, by contrast, are excellent markers for assessing the immunocompetence of birds (i.e. their ability to prevent or control infection by pathogens and parasites). Essentially, the higher lymphocyte count at 0.80% CO2 indicates a better response of the immune system. In short, chicks subjected to the higher CO2 level are clearly likely to be more robust and better able to fight disease. 

The heterophile/lymphocyte ratio (H:L) is a fitting parameter for measuring the degree of stress in chicks. Petersime’s study shows that CO2 levels at 0.45% (4,500 ppm) result in 0.27 H:L (intermediate stress) while CO2 levels of 0.80% result in 0.08 H:L (low stress). These findings suggest that it is natural for birds to experience a certain amount of CO2 stimuli during hatching for a short period of time, and that they – contrary to what some believe – even benefit from raised CO2 levels at certain stages. 

H:L ratios – what do the numbers mean?

< 0.20: low stress
0.20 - 0.50: intermediate stress
≥ 0.80: high stress


It has been suggested that an increased CO2 level during hatching causes stress, and forces hatching. Research shows that changing CO2 levels are in fact a positive stimulus – a persistent myth finally debunked! The right CO2 level at the right moment therefore helps the embryo make the switch from chorioallantoic respiration to lung ventilation only, just as it occurs in nature. And the benefits were confirmed by the hatch performance results as well: with +0.50% Hatch of Fertile for the eggs that were exposed to 0.80% CO2

About the authors
Petersime Bruno Machado Sqr
Bruno Machado Global Incubation Consultant

As a dedicated poultry business specialist, Bruno Machado holds a degree in Animal Science, an MBA in Poultry and an MBA in Business Management. He has gained deep expertise in the poultry industry, e.g. in the role of hatchery manager. In his capacity as Global Incubation Consultant at Petersime, his work covers research projects, field trials as well as customer training and support.

Petersime Eduardo Romanini Sqr (1)
Eduardo Romanini R&D Incubation Coordinator

Eduardo Romanini graduated with a PhD in Bioscience Engineering focused on incubation technologies. Eduardo joined Petersime to implement the company’s first single-stage hatchery in Brazil. As the next steps, he took on the technical and incubational support for Latin American customers, and engaged in an EU research and product development programme. Today, Eduardo coordinates Petersime’s incubation research projects and commercial trials.

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