Single stage incubators and Hypercapnia during incubation affect the vascularization of the chorioallantoic membrane in broiler embryos

Single stage incubators and Hypercapnia during incubation affect the vascularization of the chorioallantoic membrane in broiler embryos J. I. M. Fernandes,∗,1 C. Bortoluzzi,∗,2 J. M. Schmidt,∗ L. B. Scapini,∗ T. C. Santos,† and A. E. Murakami† ∗

Laboratory of Poultry Experimentation, Federal University of Parana – Palotina, 85950-000, Parana, Brazil; and † Department of Animal Science, State University of Maringa, 87020-900, Parana, Brazil CO2 (4,000 ppm). Eggs that were incubated in multiplestage incubators presented a lower percentage of vessels in the chorioallantoic membrane, lower yolk absorption by the embryo, wall depth of the right ventricle, and greater humidity losses in the eggs when compared to eggs in the single-stage incubators. The eggs submitted to hypercapnia, between 5,000 and 6,000 ppm of CO2 , had a higher percentage of vessels in the chorioallantoic membrane; the embryos originating from these eggs had higher weight, with higher relative weight of the liver. However, the same levels reduced the yolk absorption. Single-stage incubation with moderate levels of hypercapnia is an efficient tool to be adopted by the hatcheries when attempting to improve chick quality.

Key words: hypercapnia, angiogenesis, incubators 2016 Poultry Science 00:1–6 http://dx.doi.org/10.3382/ps/pew274

INTRODUCTION

fusion. The effects of hypercapnia or the high concentration of CO2 on angiogenesis in the CAM are not well known, although systemic acidosis during hypercapnia or induced by other mechanisms can have a direct effect on the increase in the vascularization of the CAM (Everaert et al., 2008). The CAM is a highly vascularized extra-embryonic membrane that is located on the internal side of the fertilized eggshell. During embryogenesis, the blood vessels proliferate and differentiate in an arteriovenous system, which allows for gas exchange with the external environment (Verhoelst et al., 2011). Within 44 h of incubation, the heart begins to beat and the mechanic force of the blood stream influences the beginning of the vascular network formation in the chorioallantoic membrane. At approximately 5 d of incubation, the development of the chorioallantoic membrane begins, and at d 8, it becomes the primary source of oxygen capture during the greatest part of embryonic development (Tullett and Deeming, 1982). The increase in the vascular network of the CAM may have an important impact on the embryo’s metabolism and consequently affect the post-hatching growth and development; the embryos adapted to moderate hypoxia conditions may have increased capillary

Embryonic development is a dynamic process determined by the embryo’s genetic potential and the environmental conditions in which it develops (Decuypere and Bruggeman, 2007). Currently, egg incubation in single-stage incubators is a highly controlled process during which temperature, humidity, oxygen, and CO2 concentrations can be monitored in compliance with the specific requirements of the embryo during all developmental phases (Bennett, 2010). Changes in the environment during embryogenesis can induce changes in the development of certain regulatory physiological systems, which will cause permanent phenotypic changes in the embryos (Druyan et al., 2012). For example, angiogenesis in the chorioallantoic membrane (CAM) may be changed under different gas concentrations during distinct periods of embryogenesis, which consequently affects the ability of oxygen dif C 2016 Poultry Science Association Inc. Received September 3, 2015. Accepted July 5, 2016. 1 Corresponding author: [email protected] 2 Current address: Department of Poultry Science, The University of Georgia, Athens, GA, USA, 30602

1

Downloaded from http://ps.oxfordjournals.org/ at Cornell University Library on October 4, 2016

ABSTRACT Incubation management can have direct effects on neonate health and consequently affect post-hatching development. The effects of incubation in multiple and single stage incubators with different concentrations of CO2 were evaluated in terms of the vessel density in the chorioallantoic membrane, hatching, heart morphology, and body development of the neonate up to the tenth day. A total of 2,520 fertile eggs were used and distributed in a completely randomized design with 4 levels of CO2 in 4 single-stage incubators (4,000; 6,000; 8,000; and 10,000 ppm) and a control treatment based on multiple-stage incubation, totaling 5 treatments. The levels of CO2 were used during the first 10 d of the incubation period, and after this period, all eggs were submitted to the same level of

2

FERNANDES ET AL.

MATERIAL AND METHODS Experimental Design The local Ethics Committee on the use of experimentation animals approved all animal husbandry and biological material collection procedures. The experiment was conducted in a commercial hatchery, in which 2,520 fertile eggs from Cobb 500 broiler breeders at 38 wk of age were used. The experimental design involving the incubators was completely randomized, with 4 levels of CO2 (4,000, 6,000, 8,000, and 10,000 ppm) in the single-stage incubators and a control treatment based on the multiple-stage incubator, totaling 5 treatments with 504 eggs each treatment. The eggs for each treatment were weighed and distributed in 12 trays containing 42 eggs per tray. The dampers of each single-stage incubator remained closed, and it was kept a minimum ventilation rate, allowing the CO2 level to gradually increase from 0.4% (4,000 ppm) to 1% (10,000 ppm). The desired CO2 levels were maintained until the tenth d of incubation, and after this period, all eggs were subjected to the same level (4,000 ppm). The temperature and RH patterns were maintained the same between single-stage incubators, but not between singleand multiple-stage incubators. When the eggs were placed into the incubators, the CO2 level was 900 ppm, and its concentration increased 1,000 ppm per day. When the desired level was reached, the damper was automatically opened, and this level was kept using sensors inside of each incubator. Temperature and relative humidity (RH) were maintained constant among the single stage-incubators, according to the profile established at the beginning of the experiment (96.5◦ F to 100.4◦ F; 40% RH), to reach the

specific pattern for each phase of the embryonic development. On the other hand, in the multiple-stage incubator, the temperature and RH (98.5◦ F; 50% RH) were not altered until the moment in which the eggs were transferred to the hatcher. The CO2 level in this machine was maintained between 3,000 and 5,000 ppm, because of the presence of eggs in different embryonic developmental stages.

Measurements and Sampling On d 11 of incubation, the pH of the internal content of 24 eggs from each treatment was measured. Through an orifice where the air chamber is located, the pHmeter was carefully inserted into the egg in order to measure the pH of the albumen. The yolk, albumen, and the embryo were removed carefully. The eggs were filled with formalin for retention of the erythrocytes into the vessels of the CAM (Verhoelst et al., 2011). After a fixation of 24 h, each eggshell with CAM adhered was cut and fragments from the equatorial region were analyzed in a stereomicroscope (Stereoscopic Zoom Microscope, Motic China Group Co., Ltda.). From each egg, five digital images (6.7 mm2 /image) were obtained using a Moticam 2500 camera (Motic China Group Co., Ltda). The images were analyzed in the Image J software to determine the percentage of vessels in each image. Each image was split into color channels and the blue channel was used; the image was then adjusted to a threshold OTSU (Figure 1 - A and B). An area inside each picture without shades or background was selected and the particles analyzed in order to determine the percentage of black areas, which were considered as vessels in order to estimate the vascular fraction, % (Figure 1 - C). At 18 d of incubation, all eggs from each treatment were weighed for calculation of the weight loss of each egg and were then transferred to the hatcher. The eggs were transferred to single-stage hatchers (4,000 ppm of CO2, 96.5 to 97.2◦ F and 40 to 45% RH), or multiplestage hatchers (98.2◦ F and 50% RH). After hatching, the number of chicks that were born was determined in order to calculate the hatching percentage. Chicks from each treatment (20 birds) were randomly sacrificed by cervical dislocation, after which they were weighed and measured. From these same chicks, the intestine, liver, yolk sac, and heart were removed and weighed individually. The relative weight of each organ was obtained by the following equation: Relative weight = (organ weight/body weight) x 100. The hearts were sectioned transversely in the atrioventricular groove and were photographed by using the stereomicroscope. The heart morphology was evaluated by using a computerized image analyzer, Image Proplus 5.2, of the M´ıdia Cibertecnics. The heart circumference, wall depth of the right and left ventricles (3 points), and the interventricular septum also were measured.

Downloaded from http://ps.oxfordjournals.org/ at Cornell University Library on October 4, 2016

development, increasing their oxygen-transporting capacity, which positively influences the growth development post hatch (Druyan et al., 2012). Another factor is the fact that CO2 is highly soluble in the albumen of the egg. The higher CO2 level in the incubator may liquefy the albumen early on, thereby allowing for higher nutrient absorption from the eggshell (Everaert et al., 2011) and its circulation through the embryo. Some studies showed that the progressive increase in CO2 concentrations from 7,000 to 15,000 ppm during the first 10 d of incubation accelerated embryonic development and improved egg hatching (De Smit et al., 2006). We hypothesize that single-stage incubation and a higher level of CO2 during incubation could improve the quality of the neonate, by increasing the vascularization of the chorioallantoic membrane. Therefore, the objective of this study was to evaluate the effects of incubation in multiple- and single-stage incubators with different CO2 concentrations on the formation of the vascular network in the CAM of the embryo, hatching, and the body and organ development of newly hatched chicks.

3

HYPERCAPNIA DURING INCUBATION OF BROILER EMBRYOS

Statistical Analysis For all statistical analysis performed, each egg and/or each chick was considered as an experimental unit. Data were analyzed by PROC GLM of SAS (SAS Institute, 2002). First of all, the normality of the residues and the variance homoscedasticity were checked. When an effect was observed in the CO2 levels, a regression analysis was performed to evaluate the means through linear and quadratic polynomial models. The comparison among the different CO2 levels in the single- and multiple- (control) stage incubators was performed by the Dunnett’s test (contrast analysis). For all statistical procedures, the significance level of 5% was adopted.

RESULTS AND DISCUSSION According to the obtained results (Table 1), egg weight at the start of incubation, as well as the pH of the albumen, or the egg/embryo ratio was similar among treatments (P > 0.05). However, at 11 d of incubation, an effect (P < 0.05) was observed on the other evaluated parameters. The contrast analysis (Dunnett’s test) revealed that the level of 6,000 ppm of CO2 in the

single-stage incubator decreased (P < 0.05) the embryo weight and increased (P < 0.05) the blood vessel density in the CAM at 11 d when compared to the eggs in the multiple-stage incubator. Besides, among the CO2 levels in the single-stage incubators, a quadratic effect was observed (P < 0.05) for the embryo weight and the vascular fraction of the CAM; and according to the equation, the highest embryo weight and vessel density were obtained at the levels of 5,417 and 5,156 ppm of CO2 , respectively (Figure 2). Druyan et al. (2012) considered the expected changes in gas diffusion and blood transport ability to be more efficient in embryos that develop under hypoxic conditions, which can be related to greater CAM vascularization and, consequently, a greater supply of oxygen for the embryo. Druyan et al. (2012) observed a significant increase in the vessel density of the CAM of embryos that were incubated in environments with lower O2 concentration from 5 to 12 d, which is in agreement with our results. The stimulatory effect of hypercapnia and systemic acidosis on angiogenesis was already described by Everaert et al. (2008). These authors highlighted the increase in the vascular network as something that

Table 1. Effect of multiple- and single-stage incubation with different CO2 levels on the embryo weight, albumen pH, and percentage of blood vessels in the chorioallantoic membrane of fertile eggs at 11 d of incubation. Treatments MS SS – SS – SS – SS –

4,000 6,000 8,000 10,000

Mean Contrast Regression CV(%)

Embryo weight1 , g

Egg weight, g

pH

Embryo weight/ Egg weight

Vessels percentage2

16.04∗ 15.25 15.12∗ 15.97 15.59

60.73 60.99 60.02 61.07 61.74

7.02 6.91 7.08 7.21 7.23

26.46 25.05 25.23 26.26 25.33

19.41∗ 22.66 24.82∗ 24.08 18.83

15.59 P < 0.05 Quadratic 7.66

60.92 NS NS 6.11

7.09 NS NS 7.42

25.66 NS NS 8.71

21.96 P < 0.05 Quadratic 22.12

MS: multiple stage; SS: single stage. 1ˆ Y = 15,98225-0,00024537x+0,00000002264553x2 ; R2 : 0,99. 2ˆ Y = 19,02975 + 0,00208x-0,0000002017036x2 ; R2 : 0,99. ∗ P < 0.05 Significant difference in the contrasts MS × SS (Dunnet’s test).

Downloaded from http://ps.oxfordjournals.org/ at Cornell University Library on October 4, 2016

Figure 1. Images in blue channel version. A) Image obtained with a blue filter. B) Image obtained after applying the OTSU type correction factor. C) Image obtained of B selection for the calculation of % of black corresponding vessels.

4

FERNANDES ET AL.

can be attributed to a lower pH in the albumen of eggs incubated under higher CO2 levels, although they recommended more refined studies to clarify this relation. Low pH levels have a stimulatory effect on the expression of the vascular growth factors VEGF and bFGF, the main regulators of angiogenesis in the CAM. Also, Druyan et al. (2012) observed higher expression of genes that codify these vascular growth factors in embryos that were exposed to periods of controlled hypoxia from 5 to 12 d of the embryonic development. After the twelfth day of incubation, it is hypothesized an increasing tolerance to CO2 , however, the pysiological mechanisms behind this time-dependent tolerance are not yet understood but may be related to the increasing buffering capacity of the embryo with age against acidosis caused by high levels of dissolved CO2 . (Bruggeman et al., 2007; Everaert et al., 2008). In the present study, the eggs were incubated in different CO2 concentrations in the period between the first and tenth d, and the pH of the albumen content of the egg was measured on the eleventh d of incubation. Thus, it is possible that an acid-base balance was established after the gas condition had been standardized, which may explain the increase in the pH, although not significant, that was obtained as a function of the increased CO2 concentrations (Table 1). No significant difference was observed for the hatching rate regardless of the incubator model and CO2 con-

Table 2. Effect of multiple- and single-stage incubation with different CO2 levels on the hatchability and weight loss of fertile eggs. Treatments MS SS – SS – SS – SS –

4000 6000 8000 10000

Mean Contrast Regression CV, %

Weight loss of fertile eggs, %

Hatchability, %

11,4 8,2∗ 9,3∗ 8,4∗ 9,7∗

91,9 89,9 90,6 91,5 91,9

9,40 P < 001 NS 16,89

91,20 NS NS 2,12

MS: multiple stage; SS: single stage. ∗ P < 0.05 Significant difference in the contrasts MS × SS (Dunnet’s test).

centration. These results differ from the research developed by De Smith et al. (2008), as they observed a significant effect when the embryo was exposed during the first 10 d of incubation to gradual increases of CO2 up to 1% (10,000 ppm) on hatching rates without losses in newly hatched chick quality. The weight loss of the egg was greater (P < 0.05) for eggs that were incubated in multiple-stage incubators when compared to the losses that occurred in eggs that were incubated in the single-stage incubators (Table 2). In those machines, the heat that was generated by older embryos may have contributed to acceleration of the observed

Downloaded from http://ps.oxfordjournals.org/ at Cornell University Library on October 4, 2016

Figure 2. Blood vessels density in the chorioallantoic membrane (ve: vessels density) A) Multiple stage B) 4,000 ppm C) 6,000 ppm D) 8,000 ppm E) 10,000 ppm.

5

HYPERCAPNIA DURING INCUBATION OF BROILER EMBRYOS Table 3. Effects of multiple- and single-stage incubation with different levels of CO2 on the relative and absolute organ weight of chicks at one d of age. Chicken weight, g

Chicken length, cm

Heart, %

Intestine, %

Liver1 , %

Yolk2 , %

4000 6000 8000 10000

45.13 46.28 45.25 46.68 45.55

18.69 19.13 18.88 19.47 19.02

0.79∗ 0.88∗ 0.84 0.83 0.85

30.98 32.99 31.11 30.80 33.78

27.47∗ 28.49 30.25∗ 27.85 28.43

10.00∗ 7.45∗ 8.63 8.42 9.19

Mean Contrast Regression CV(%)

45.78 NS NS 5.88

19.03 0.059 NS 4.60

31.93 0.06 NS 12.89

28.50 0.01 Quadratic 9.38

8.74 0.04 Quadratic 25.31

Treatments MS SS – SS – SS – SS –

0.84 NS NS 13.59

MS: multiple stage; SS: single stage. ∗ P < 0.05 Significant difference in the contrasts: 1 MS × 4,000 ppm; 2 MS × 6,000 ppm; 3 MS × 4,000 ppm (Dunnet’s Test). 1ˆ Y = 27,28712 + 0,00067167∗ x-0,0000000591386x2 ; R2 : 0,22. 2ˆ Y = 9,72127 - 0,00067341x + 0,00000006362356x2 ; R2 : 0,82.

Treatments MS SS – SS – SS – SS –

4000 6000 8000 10000

Mean Contrast Regression CV(%)

RV, μ m 0.85∗ 0.88 1.04∗ 0.94∗ 0.96∗ 0.94 P < 0.05 NS 20.86

IS1 , μ m

LV, μ m

CC, μ m

1.91 1.90 1.91 2.08 2.02

1.41 1.42 1.51 1.57 1.47

27.05 27.49 25.92 27.04 26.67

1.96 NS linear 12.42

1.47 NS NS 19.70

26.84 NS NS 5.09

MS: multiple stage; SS: single stage. ∗ P < 0.05 Significant difference in the contrasts: MS × SS – 6,000 ppm; MS × SS – 8,000 ppm; MS × SS – 10,000 ppm (Dunnet’s Test). 1ˆ Y = 1,79171 + 0,00002602, R2 : 0,61.

water losses, thereby resulting in greater losses in the egg weight. A significant effect (P > 0.05) was not observed for the incubator model and the CO2 concentration of the single-stage incubator on chick weight and the relative weight of the heart and intestine. The chickens from the single-stage incubator were longer and had a higher relative weight of the liver and a higher yolk absorption rate when compared to chicks from the multiple-stage incubator (Table 3). When the effects among the CO2 levels were analyzed, a quadratic effect of hypercapnia (P < 0.05) was observed on the relative weight of the liver and yolk (Table 3). The relative weight of the liver may be related to the embryo’s ability to absorb and to metabolize nutrients from the yolk. It was observed that the highest relative weight of the liver and lowest residual yolk were at the levels of 5,678 and 5,292 ppm of CO2 , respectively (Table 3). The increased residual yolk in chicks that were incubated in multiple-stage incubators or under high CO2 concentrations in the single-stage incubators shows that the embryos did not adequately utilize their energy reserves. A large part of the energy that is needed for embryonic development is obtained from yolk fat deposition. When hypoxic conditions are maintained for longer periods of time, the amount of oxygen can be in-

sufficient for the energy metabolism and, consequently, compromise the embryonic development. Regarding the morphologic aspects of chicks’ hearts, the single-stage incubator with CO2 concentrations above 4,000 ppm increased (P < 0.05) the wall depth of the right ventricle in comparison to the multiplestage incubator (Table 4). The interventricular septum increased linearly (P < 0.05) with the increase of CO2 concentrations in the regression analysis. However, the wall depth of the left ventricle and the cardiac circumference were not affected by hypercapnia. Villamor et al. (2004) showed that hypoxia during incubation increased the cardiac area and the left cardiac wall depth. These cardiac changes may influence the susceptibility of broilers to metabolic diseases such as ascites and sudden death syndrome. Studies that target incubation conditions are very promising. The multiple-stage incubators, although still widely used by the industry, decrease the vessel density of the chorioallantoic membrane, increase humidity losses of the embryos, and decrease the yolk absorption. Therefore, it is possible to conclude that single-stage incubation with moderate levels (between 5,000 and 6,000 ppm of CO2 ) of hypercapnia is an efficient tool to be used during the incubation process to improve the neonate quality. More studies must be conducted

Downloaded from http://ps.oxfordjournals.org/ at Cornell University Library on October 4, 2016

Table 4. Effect of multiple- and single-stage of incubation with different CO2 levels on the thickness of right ventricular wall (RV), interventricular septum (IS), thickness of left ventricular wall (LV), and cardiac circumference (CC) of chickens at one d of age.

6

FERNANDES ET AL.

in order to clarify the effects of hypercapnia on later organ development of broilers, and under heat stress conditions.

REFERENCES

Downloaded from http://ps.oxfordjournals.org/ at Cornell University Library on October 4, 2016

Bennett, B. 2010. The advances of single-stage versus multi-stage incubation. In. Hatch. Prac. 20:373–384. Bruggeman, V., A. Witters, L. De Smit, M. Debonne, N. Everaert, B. Kamers, O. M. Onagbesan, P. Degraeve, and E. Decuypere. 2007. Acid-base balance in chicken embryos (gallusdomesticus) incubated under high CO2 concentrations during the first 10 days of incubation. Resp. Physiol. Neurobiol. 159:147–154. De Smit, L., V. Bruggman, J. K. Tona, M. Debonne, O. Onagbesan, L. Arckens, J. De Baerdemaeker, and E. Decuypere. 2006. Embryonic developmental plasticity of the chick: Increased CO2 during early stages of incubation changes the developmental trajectories during prenatal and postnatal growth. Comp. Bioch. and Physiol. a-Molecular & Integrat. Physiol. 145:166–175. De Smit, L., V. Bruggeman, M. Denonne, J. K. Tona, B. Kamers, N. Everaet, A. Wiiters, O. Onagnesan, L. Arckens, J. De Baerdemaeker, and E. Decuypere. 2008. The effect of nonventilation during early incubation on the embryonic development of chicks of two commercial broiler strains differing in ascites susceptibility. Poult. Sci. 87:551–552. Decuypere, E., and V. Bruggeman. 2007. The endocrine interface of environmental and egg factors affecting chick quality. Poult. Sci. 86:1037–1042.

Druyan,, S., E. Levi, D. Shinder, and T. Stern. 2012. Reduced O2 concentration during CAM development—Its effect on physiological parameters of broiler embryos. Poult. Sci. 91:987–997. Everaert, N., L. De Smit, M. Debonne, A. Witters, B. Kamers, E. Decuypere, and V. Bruggeman. 2008. Changes in acid-base balance and related physiological responses as a result of external hypercapnia during the second half of incubation in the chicken embryo. Poult. Sci. 87:362–367. Everaert, N., E. Willemsen, E. Willems, L. Franssens, and E. Decuypere. 2011. Acid-base regulation during embryonic development in amniotes, with particular reference to birds. Resp. Physiol. Neurobiol. 178:118–128. SAS Institute Inc. 2002. Software and services: system for Windows, version 8.0, software Cary. Tullett, S. G., and D. C. Deeming. 1982. The relationship between eggshell porosity and oxygen consumption of the embryo in the domestic fowl. Comp. Biochem. Physiol. A Comp. Physiol. 72:529–533. Verhoelst, E., B. Ketelaere, V. Bruggeman, E. Villamor, E. Decuypere, and J. Baerdemaeker. 2011. Development of a fast, objective, quantitative methodology to monitor angiogenesis in the chicken chorioallantoic membrane during developmentInt. Int. J. Devel. Biol. 55:85–92. Villamor, E., C. G. A. Kessels, K. Ruijtenbeek, R. J. V. Suylen, J. Belik, J. G. R. D. Mey, and C. E. Blanco. 2004. Chronic in ovo hypoxia decreases pulmonary arterial contractile reactivity and induces biventricular cardiac enlargement in the chicken embryo. Am J Physiol Regul Integr Comp Physiol. 287:642–651.