INTRODUCTION

Embryonic development (ED) depends in the first instance on the pores of the shell that allow the diffusion of oxygen (O ₂ ) and carbon dioxide (CO ₂) between the external environment of the egg and the embryo's blood (^{Cordeiro and Hincke, 2016}). This gas exchange develops through the chorioallantoic membrane (^{John, 2017}), which is supplied by blood vessels and whose function is similar to the placenta of mammalian fetuses (^{Koyama and Tennyson, 2016}). The primary function of the respiratory system is to transport O ₂ and CO ₂, between the environment and the tissues (^{D'Alba et al., 2017}); therefore, respiration is regulated to meet metabolic demands, supplying O₂ and eliminating CO ₂ (^{Okur, 2019}). The partial pressure of oxygen (pO2) and the partial pressure of carbon dioxide (pCO2) in the air chamber are a stimulus for the embryo to perform the pecking (^{Deeming, 2016}).

^{Gildersleeve and Boeschen (1983)} carried out an experiment with turkey eggs, where they added CO₂ levels higher than atmospheric concentrations, in order to stimulate ED during the beginning of the incubation period, although ED was stimulated, no differences in hatchability between eggs incubated in ranges of atmospheric CO₂ to 1% CO₂ during the first 2 days of incubation, so that the temperature of the shell, together with the concentration of CO₂, affects the body weight of the chick at birth (^{Maatjens et al., 2014a}; ^{Maatjens et al., 2014b}).

^{De Smit et al (2006)} y ^{De Smit et al (2008)} modified the ventilation conditions, to raise CO₂ during the first 10 days of the ED, using two reproductive strains, 45 and 60 weeks old; with different levels of susceptibility to ascites syndrome (AS). The CO₂ levels resulted in 1 and 1.5%, so the ED10 of higher CO₂ levels resulted in higher absolute and relative body weight (to the egg weight) than the ED10 to ED18; they had accelerated growth, elevated levels of corticosterone and plasma T3, and higher pCO2 in the air chamber; the hatching window was smaller in a time of 10 to 15 h, and the chick weight was greater with respect to normal ventilation. Likewise, ^{García et al (2013)} restricted ventilation during the first 10 days of the ED and found an improvement in the incubation parameters.

During the last phase of development, the chicken embryo varies its O₂ consumption with the ambient temperature (^{Deeming, 2016}). During the 2nd phase of ED, once the embryo produces its own metabolic heat, it removes CO₂ in a range of 0.05 to 0.3% (^{D'Alba et al., 2017}), so it also depends on the diffusion of gases to through the pores (^{Mortola and Labbe, 2005}). Thus, a hypoxic condition can limit ED, limit beak and foot development, develop heart hypertrophy, changes in heart rhythm, pericardial and pulmonary edema, changes in hemoglobin and AS; since in the last stage of ED, the phase where the hypoxic condition occurs, the embryo consumes up to 60 % more O₂ (^{Burggren and Elmonoufy, 2017}; ^{John, 2017}; ^{Itani et al., 2018}). Another factor that can play an important role in ED is altitude; since shell decreasing conductance. At this stage, the embryo needs energy from anaerobic metabolism (^{Huang et al., 2017}). Blood glucose and tissue glycogen reserves are what provide this energy, necessary for hatching (^{Fathollahipour et al., 2018}). It is known that there are differences in embryos metabolism with different ages and commercial lines, so if it is intended to optimize the incubation conditions, it is necessary to deepen their requirements (^{Huang et al., 2017}).

The objective of the present investigation was to determine incubation parameter effect with two levels of CO₂ and two egg weights.

MATERIAL AND METHODS

The experiment followed institutional and national guidelines for the care and use of animals; all procedures were approved by the Ethics Review Committee of the University of Colima. The study was carried out with 600 fertile eggs of commercial Cobb 500 breeder, 41 weeks old and weighing 65 and 70 g. The eggs were placed in two commercial single-stage incubators (HatchTech; Gildetrom 25.3905 TB., Veenendaal, Netherlands), with a capacity of 4800 eggs/each. The machines, which have a temperature sensor (± 0.1ºF), a humidity sensor [± 1% relative humidity (RH)] and a CO₂ sensor (± 100 ppm). The eggs were turned at an angle of 45° and then 90° every hour.

The air circulation was horizontal and laminar, through perforated radiators, which cause pressure differences, for better air distribution and a uniform flow through each egg mass, from top to bottom and from front to back. The same machines functioned as hatchers, and in both, the routine management of an incubator plant was carried out, from the reception of the egg to the removal of the hatchery machines.

RESULTS

In table 1, it is observed that the highest percentage of hatchability was with the concentration of 3000 ppm of CO₂, and with 65 g in egg weight. The CO₂ levels considered in the experiment did not affect the weight of the chicken; therefore, the treatments with 70 g eggs obtained heavier chickens. The lowest percentage in moisture loss with respect to CO₂ concentration was recorded at 4000 ppm and without statistical differences (P <0.05), between eggs with 65 and 70 g. The largest size of the chicken with respect to the CO₂ concentration was observed at 3000 ppm and without statistical differences between eggs with 65 and 70 g.

In Table 1, it is observed that the combination between CO₂ concentrations and egg weights did not show differences within the group (65 and 70 g, respectively); but it occurred between groups, with the lowest chicken weight (P <0.05), in the treatment that combines 3000 ppm of CO₂ with 65 g eggs. Regarding chicken size, the result with the largest size was observed in the combination of 3000 ppm of CO₂, with eggs of 65 and 70 g. Blood glucose concentration did not show differences in both factors. The highest percentage of hematocrit with respect to CO₂ concentration was recorded at 4000 ppm and without statistical differences (P <0.05), between eggs with 65 and 70 g. The highest concentration of plasma proteins with respect to the concentration of CO₂, was recorded at 4000 ppm. The result with the highest percentage of hematocrit was recorded in the combination of 4000 ppm of CO₂, with eggs of 70 g.

DISCUSSION

Hatchability (it is the ability of the egg to hatch) improves with a lower concentration of CO₂ (^{Fathollahipour et al., 2018}). In the present work, it was found that the lower concentration of CO₂ improves hatchability; so the integrity of the shell plays an important role for gas exchange. Then, when the embryo reaches day 18 of incubation, lung respiration begins, where thyroid hormone plays an important role in the lung surfactant development (^{Hamidu et al., 2018}). The process of pulmonary respiration contributes O2 to the chicken embryo (^{Deeming, 2016}). However, the lung respiration maturation without having the shell intact, and with the increase in the metabolic demand for O2 at the end of incubation, cause an increase in pCO2 and a decrease in pO₂ in the air chamber, (^{Flores-Santin et al., 2018}), activating the birth trigger mechanism (^{Ramachandran and McDaniel, 2018}). So the normal ED of the chicken depends on the air changes that take place through the chorioallantoic membrane (^{John, 2017}), which, together with shell pores, carries out the exchanges of O₂ and CO₂ between the blood and the environment (^{Deeming, 2016}).

The shell is the main responsible for the difference between pCO₂ and water vapor, where the O₂ flow is affected by the shell and the inner membrane (^{Ramachandran and McDaniel, 2018}); so it may be one of the main reasons why hatchability was not effective at a higher level of CO₂. In the same way, the conductance of the gas in the shell depends on the ratio of the area of the pore to its length; in the end, this relationship is equivalent to shell thickness (^{Cordeiro and Hincke, 2016}).

In the present study, it was observed that by having a lower concentration of CO₂ in the environment, hatching was improved; since a hypoxic condition was not created at the end of the incubation process and pO₂ and pCO₂ in the air chamber were not affected. When embryos are subjected to hypoxic conditions during the incubation process, the body growth, beak and legs is inhibited; as mentioned (^{Burggren and Elmonoufy, 2017}). In the experiment, chicken weight was not affected by the different CO₂ concentrations, but the chickens that came from eggs with 70 g were heavier. In this regard, it is known that chicken weight is directly related to egg weight (^{D'Alba et al., 2017}). The chicken size was greater when the CO₂ concentration was 3000 ppm, these data suggest that if the embryos develop in environmental conditions with a low O₂ concentration, they lose weight during pecking and hatching (^{Ramachandran and McDaniel, 2018}). Therefore, embryos in dense conditions of ambient O₂ gain weight. This implies that embryos in O2- rich environmental settings are better metabolically prepared to initiate pecking and birth (^{Deeming, 2016}).

In the study, chicken size was used as a quality criterion to determine the productive performance of the flock; on the other hand, the glycogen content in body tissues is important in the physiological requirement at the time of initiating the pecking (^{Maatjens et al., 2014}a; ^{Maatjens et al., 2014b}). Similarly, it directly influences embryo survival; thus, in the study, the CO2 conditions did not affect the blood glucose levels in the chickens, which may condition birds’ vitality on the farm.

The hematocrit level showed a slightly higher numerical trend in the treatment with a higher concentration of CO₂, information similar to that reported by ^{Scheele et al. (2003)} who quantified elevated hematocrit values as a result of an increase in the erythropoietic response. The plasma protein concentration was lower when the CO₂ conditions were kept at 4000 ppm. These data suggest that in CO₂-rich environments, the erythropoietic response is used more (^{Ramachandran and McDaniel, 2018}), and as a consequence the concentration of plasma proteins is reduced, as observed in the present study.

During chicken ED, commercial incubators handle concentrations of 4000 ppm of CO₂; but with the results obtained, it is suggested to lower said concentration to 3000 ppm, mainly to improve hatchability.

CONCLUSION

3000 ppm CO₂ concentrations improve hatchability, moisture loss and chick size; it also has low levels of plasma proteins.