Performance of Broilers Submitted to High CO2 Levels During Incubation Combined with Temperature Fluctuations at Late Post-Hatch

Incubation represents around 1/3 of the life cycle of broilers of modern strains; therefore, the appropriate management of this period is crucial to ensure the quality of the neonate. An experiment evaluated the effect of carbon dioxide concentrations during incubation on the live performance, heart morphology, and differential leukocyte count in the blood of broilers submitted to fluctuating temperatures between 35 and 42 days of age. In total, 2,520 fertile eggs were distributed according to a completely randomized design with four CO2 concentrations (4,000; 6,000; 8,000 and 10,000 ppm) during the first ten days of incubation, after which all eggs were incubated at the same CO2 level (4,000 ppm). After hatching, male chicks were placed in the experimental broiler houses, and at 35 days of age, 25 birds from each level of CO2 were separated and placed in cages to study the effect of cyclic temperature variations up to 42 days of age on their live performance, heterophil:lymphocyte ratio, and heart morphology. At 42 days of age, blood was collected for hematology and two birds per replicate were sacrificed and evaluated for ascites score and heart and liver relative weights. Heart morphology was assessed by analyzing digital images. In this experiment, hypercapnia during incubation and fluctuating temperatures during the growout did not affect mortality, ascites score, heart and liver relative weights, or heart characteristics (p>0.05). However, heterophill:lymphocyte ratio increased (p<0.05) with increasing carbon dioxide concentrations during incubation. It was concluded that high CO2 levels during incubation did not influence the resistance of broilers to fluctuating temperatures during the last week of age.


INtRODUCtION
Genetic selection in the poultry industry has produced incredibly fast-growing broilers, and this is already observed during embryonic development.In order to achieve the highest productivity and production efficiency, it is essential to fully understand the requirements of the developing embryo during incubation (De Smit et al., 2008).
Since the development of the first incubation machine, commercial hatcheries of the early 20 th century underwent many changes that improved their efficiency and reliability.More recently, single-stage incubation was introduced.In single-stage incubators,all eggs within the incubator are set at the same developmental phase (same embryonic age).In this type of incubation system, labor costs are reduced, and the equipment can be cleaned and disinfected at the end of each hatching cycle in order to minimize microbial contamination risks (Hulet, 2007).Furthermore, single-stage incubators can precisely meet temperature, relative humidity, and CO 2 concentration requirements of the embryo during the different phases of embryonic development (Bennett, 2010).These conditions will create the ideal environment for embryonic development (Everaert et al., 2007).
It has been demonstrated that different CO 2 levels may play a role in the embryo development (Walsberg, 1980;De Smit et al., 2006;Everaert et al., 2008).High CO 2 levels early in incubation, similar to situation in nature, may have beneficial effects.Several recent studies investigated the effect of hypercapnia, i.e. high CO 2 concentrations, on broiler performance (Buys et al., 1998;De Smit et al., 2006;Everaert et al., 2008).An increase in CO 2 during early incubation is reported to improve hatchability, accelerate pre-and postnatal growth, and hatching time (De Smit et al., 2006;Tona et al., 2007).
Although these results are promising, it is necessary to understand the best level and period to apply this management practice (De Smit et al., 2006), and understand the physiological changes induced by hypercapnia.Everaert et al. (2007) evaluated the use of high CO 2 levels during the second half of incubation and did not find any differences in the live weight of 7-d-old chicks.However, other studies showed that high CO 2 levels during the first 10 days of incubation increased embryo body weight and early hatchability (De Smit et al., 2008).Therefore, it is believed that the best age of CO 2 exposure is during the formation of chorioallantoic membrane (CAM) (from 5 to 11 days of incubation), resulting in increased CAM vascularity and better oxygen transport capacity in later stages (Druyan etet al., 2012).
This period of critical hypoxia may trigger structural changes in the lungs and heart, predisposing birds to ascites (Decuypere et al., 2000).Consequently, the incubation period may potentially improve broiler growth, metabolism and health.For instance, Buys et al. (1998) considered that embryos exposed to higher CO 2 levels during incubation may have lower incidence of ascites during the rearing phase.
Modern broiler strains are selected for fast growth and high feed efficiency, and therefore, more oxygen is required to meet their high metabolic rate.A high incidence of subclinical heart disease was demonstrated in fast-growing broilers, as well as right ventricle hypertrophy (Baghbanzadeh & Decuypere, 2008).Therefore, as a consequence of the underdevelopment of the pulmonary and cardiovascular systems of these birds (De Smit et al., 2008), birds suffer ascites, with mortality occurring between 5 and 6 weeks of age, although the etiology dates back to the embryonic period (Coleman & Coleman, 1991).The adaptation to the environment depends on a mechanism called epigenetic adaptation.Chickens may be habituated to tolerate thermal stress during the pre-natal period by the epigenetic adaptation mechanism (Nichelmann et al., 2002).
The influence of early hypercapnia on postnatal thermal tolerance needs to be further studied.For this reason, the objective of this study was to evaluate different CO 2 levels during the first 10 days of incubation and its relationship with exposure to high temperature of broilers between 35 and 42 days of age on live performance, heart morphology, and blood leukocyte counts.

MAtERIAL AND MEtHODS
The experiment was conducted at the Experimental Poultry House from the Federal University of Paraná, Sector of Palotina, Palotina, state of Paraná, Brazil.All animal husbandry and biological material collection procedures were approved by the local Ethics Committee on the use of experimentation animals.In total, 2,016 fertile eggs were distributed according to a completely randomized design into four CO 2 incubation levels (4,000; 6,000; 8,000; or 10,000 ppm) in four single-stage incubators.Eggs were incubated in hatchery belonging to a poultry company of the state of Paraná, Brazil, and the incubators were not ventilated until CO 2 has reached the desired level.The other variables (temperature and O 2 level) followed the standards of the company.The different CO 2 levels were applied during the first ten days of incubation, after which all eggs were submitted to the same CO 2 level (4,000 ppm).
After hatching, 840 Cobb male chicks were randomly assigned to same treatments used in the incubation phase (four treatments) with seven replicates of 30 birds each.Feed and water were supplied ad libitum.Diets based on corn and soybean meal were formulated according to the chemical composition of feedstuffs and nutritional recommendations adopted by the company.A lighting program of 24 hours of light was applied during the entire experimental period.
At 35 days of age, 100 birds with aproximately 2,300 kg average weight were housed in metal cages under controlled temperature and humidity and distributed according to the CO 2 levels to which they were submitted during incubation, with five replicates of five birds each.Birds were submitted to 12 hours at 25° C, which was maintained during the night, and 12 hours at 32-35° C during the day.The thermal oscillation is one of the main environmental factors that is negatively correlated with ascites in broilers.
At 42 days of age, all birds in each experimental unit and feed residues were weighed for the determination of body weight gain, feed intake, and feed conversion from 35 to 42 days of age.Two birds by replicate (10 birds per treatment) were randomly selected, and their blood was collected for total and differential leukocyte counts in blood smears, as described by Campo and Dávila (2002).In this methodology, 100 leukocytes, including granular (heterophil, eosinophil and basophil) and non-granular (lymphocyte and monocyte) leukocytes, were counted and the heterophil/lymphocyte ratio was then calculated.
Birds were weighed and sacrificed.The heart and liver of each bird was collected and weighed.Hearts were cross-sectioned below the left and right atrioventricular valves and a second cross section was made at the stem of the papillary muscle.A crosssectional image of a 4-mm section of each heart was digitally recorded using an image analysis software (Image Proplus 4.1).Heart right wall thickness (RWT), left wall thickness (LWT), ventricular septum (VS), and heart circumference (CH) were determined.
Data were submitted to analysis of variance, using SAS software package (GLM procedure) (SAS, 1998).
The assumption of normality was previously checked.When CO 2 level effect was detected, a regression analysis was run, in which linear and quadratic polynomial models were analyzed.Models were selected by the fitted coefficient of determination (fit-R2); by the significance of regression and the lack of fitness (tested by the F test); by the significance of regression coefficients (tested by the F test).Maximum and minimum points of the quadratic model were estimated by derivation of the fitted coefficient of the regression.The significance level was set at 5% for all statistics procedures.

RESULtS AND DISCUSSIONS
Hypercapnia during incubation and cyclic environmental temperatures from 35 to 42 days of age had no effect on performance (Table 01), heart and liver relative weights, or on heart morphology measures (Table 02).The fact that CO 2 levels did not significantly affect body weight gain, relative liver weight, or heart morphology in broilers suggests that the embryos exposed to high CO 2 levels during incubation coped better with the effects of temperature fluctuations.It was concluded that the hypercapnia during incubation does not influence these characteristics.
One of physical factor studied in the incubation process is the CO 2 level.The high metabolic rate of embryos of modern broiler strains requires ventilation of the incubator to remove the heat, which may lead to excessive removal of CO 2 .However, recent studies questioned the need of high CO 2 levels during incubation.The assumed objective of providing high CO 2 levels is to allow the embryo to develop adaptive mechanisms in order to obtain good quality hatchlings and increase the resistance of broilers to stressors during the rearing period (Everaert et al., 2007;De Smit et al., 2008;Everaert et al., 2012).Adaptation to environment conditions depends on a mechanism called epigenetic adaptation.Chickens can be conditioned to thermal stress tolerance during the incubation by the epigenetic adaptation mechanism (Nichelmann et al., 2002).
Several studies showed that a gradual increase of CO 2 levels from 7,000 to 15,000 ppm during the first 10 days of incubation in an airtight incubator accelerated embryonic development and improved hatchability (De Smit et al., 2008).Yet, many single-stage programs allow the damper to open in response to CO 2 levels at or above the set point and provide excessive amounts of fresh air that destabilize the thermal environment.This may interfere with physiological responses of broilers exposed to adverse temperature conditions.However, in the present study, no changes in the heart morphology that could be characterized as ascites were observed.
On the other hand, hypercapnia in initial phase of incubation may have a direct effect on the angiogenesis.Systemic acidosis, resulting from hypercapnia or induced by other factors, also increases angiogenesis (Everaert et al., 2008).The growth of the vascular network in the chorioallantoic membrane increases its O 2 diffusing in a sigmoid manner, parallelly to the change in O 2 uptake from the embryo (Dzialowski et al., 2002;Chan and Burggren, 2005).This leads to the hypothesis that early antenatal changes caused by temporal hypoxia may make natural critical hypoxia later during incubation less severe, and thereby reducing the sensitivity to ascites.Likewise, Decuypere et al. (1998) reported that broilers incubated at high CO 2 levels presented lower mortality due to ascites only during the first 2 weeks of age.Hassanzadeh et al. (2002) showed that eggs incubated at relatively high carbon dioxide concentration hatched earlier than those incubated at regular levels and the resulting broilers presented lower incidence of ascites during the rearing period.Bahadoran et al. (2010) investigated the influence of hypoxia during the early stage of incubation and in a low altitude incubator until hatching on incidence of ascites in broiler chickens.The results indicated that early prenatal hypoxia due to high altitude may change the endocrine functions of embryos, enhance embryo growth, shorten the hatching window, and decrease the incidence of ascites in broiler chickens.
This was not confirmed in our study.However, there is little information regarding the effects of CO 2 levels during incubation on physiological response of broiler chickens exposed to fluctuating temperatures or to poor air quality (dust concentration, carbon dioxide levels, and oxygen levels).The incidence of ascites in broilers is also influenced by other factors.Broilers may not be able to adapt to the higher oxygen demands related to environmental stress.Additionally, if broilers with lung and/or heart capacity deficiencies encounter factors that interfere with oxygen transport, breathing ability, or cardiac output, they could become more susceptible to ascites (McGovern et al., 2001).
Relative to organ weights, our results are consistent with those obtained by De Smit et al., (2008), who did not find any significant differences in the heart weight of embryos exposed to high CO 2 levels (up to 7,000 ppm) during the first 10 days of incubation.Ascites in fast-growing broilers is usually associated with an increase in the weight, volume, and area of the right ventricle of the heart.When the right ventricle enlarges as a result of increasing blood pressure and increasing blood flow, the volume of blood the right ventricle can hold also increases.The ratio of right ventricle to total ventricle mass is a gross indicator of ascites (Julian et al., 1986).Image analysis proved to be a valid technique for quantifying the area of broiler hearts (McGovern et al., 2001).However, these authors consider that the technique should be standardized to obtain better accuracy.This requires the atria side of the heart crosssection to be placed upward and the heart shape should be maintained as natural as possible.Therefore, differences in results may be attributed to technique variations.
With reference to indicators of heat stress, Al-Murrani et al. (1997) mentioned that the heterophil/ lymphocyte ratio is widely variable among individuals, but according to Maxwell (1993), this ratio is a more reliable indicator of mild to moderate stress than plasma corticosterone concentration.In the present study, heterophil and lymphocyte counts and the H:L ratio were significantly affected by treatments (Table 03).There was a linear increase in the H:L ratio of broilers submitted to cyclic variation of temperature during the last week of life due to an increase in heterophil counts and decrease in lymphocyte counts as CO 2 incubation levels increased in incubation.This suggests that higher CO 2 levels during incubation did not increase the resistance of broilers to heat stress.During stress, the hormone cortisol is released, increasing heterophil and decreasing lymphocyte counts (Perea et al., 1997).The H/L ratio has become widely accepted as a reliable and accurate physiological indicator of the stress response in chickens (Gross & Siegel, 1983).The differential leukocyte count shows that the normal heterophil: lymphocyte ratio is about 1:2.Nevertheless, this ratio increases when birds are submitted to stress conditions by increasing the number of circulating heterophils.Furthermore, Thaxton & Siegel (1982) and Miller & Qureshi (1991), reported that when broilers are exposed to high temperatures, ranging between 32.2 and 43 ° C for short periods or cycles of constant high temperatures, their immune response is weakened due to the involution of the lymphatic system, including atrophy of the thymus, Bursa of Fabricius, spleen, and pancreas.The release of corticosterone also may cause the involution of lymphoid tissues (thymus, bursa of Fabricius, and spleen) and the suppression of humoral and cell-mediated immunity (Rosales et al., 1989).Gross & Siegel (1993) suggested that heterophil to lymphocyte ratio values above 0.8 indicate high stress levels.

CONCLUSION
Although high CO 2 levels during incubation influenced some development parameters of the studied broilers, hypercapnia during incubation did not improve broilers' tolerance to fluctuating temperatures during the last week of rearing, as shown by the heterophil:lymphocyte ratio results.Other effects of hypercapnia during the incubation must be investigated, mainly on the development of the immune system and of the gastrointestinal tract.

Table 1 -
Effect of CO 2 concentrations during incubation and temperature fluctuations from 35-42 days of age on the live performance of broilers.

Table 2 -
Effect of CO 2 concentrations during incubation and temperature fluctuations from 35-42 days of age on relative heart weight (RHW) and relative liver weight (RLW), heart right wall thickness (RWT), left wall thickness (LWT), ventricular septum (VS) and circumference heart (CH) of broiler chicken.
SEM: Standard Error Mean; NS: Not Significant Performance

Table 03 -
Effect of CO 2 concentrations during incubation and temperature fluctuations between 35 and 42 days of age on heterophil:lymphocyte ratio (H/L), heterophil counts (H) and lymphocytes counts of broilers.