Effect of Hypercapnia During Incubation and Broiler Breeder Age on Development of the Gastrointestinal Tract in Embryos and Hatchlings

ABSTRACT

Exposure to increasing concentrations of CO₂ in the first 10 days of incubation may have effects on the development of bird cardiac and respiratory organs. Moreover, the age of breeders can influence hatching performances. This study aimed to investigate the effect of exposure to increasing concentrations of CO₂ in the first 10 days of incubation on the morphophysiological development of the digestive system of embryos and chicks from broiler breeders aged 31 and 41 weeks. A total of 860 fertile eggs from the Cobb strain were distributed in a completely randomized design, in a 2 x 2 factorial arrangement, with 2 different gaseous environments (Control (C) - no increase in CO₂ concentration and, Hypercapnia (CO₂) - a gradual increase in CO₂ concentration until reaching 1% on the 10th day) and 2 different broiler breeder ages (31 and 41 weeks). Half of the eggs were obtained from 31-week-old breeders, and the other half from 41-week-old breeders. Compared to the control group, incubation in an atmosphere with 1% CO₂ led to an increase in villus heights in the duodenum, jejunum, and ileum of the embryos, with a reduction in villus density in the same segments. Chicks from 41-week-old broiler breeders showed higher villus heights in the duodenum, jejunum, and ileum at day 1 post-hatching, and lower villus density at 7 days. It was concluded that the incubation of fertile eggs in hypercapnia conditions could positively affect the small intestine of embryos and post-hatch chicks.

Keywords:
Carbon dioxide; hatching; intestinal mucosa; intestine; poultry

INTRODUCTION

Despite the great technological development already achieved in artificial incubation, some aspects related to the gaseous environment are still being investigated to determine epigenetic effects and increase the efficiency of the process (Decuypere & Bruggeman, 2007; Druyan et al., 2018; Okur et al., 2022). Previous studies have shown that a gradual increase in CO₂ concentrations up to the first 10 days of embryonic development positively impacts incubation, resulting in reduced hatching time, improved hatchability, reduced embryonic mortality and changes in bird development during and after hatching (De Smit et al., 2006, 2008; Bruggeman et al., 2007; El-Hanoum et al., 2019; Kroetz Neto et al., 2023).

Exposure to increasing concentrations of CO₂ in the first 10 days of incubation induces a reduction in albumen pH, stimulates the vascularization of the chorioallantoic membrane, and increases the levels of thyroid hormones and corticosterone (Bruggeman et al., 2007; Tona et al., 2007; De Smit et al., 2006, 2008; Verhoelst et al., 2011; El-Hanoum et al., 2019). Fernandes et al. (2017) demonstrated the effects of hypercapnia in the first 10 days of incubation on the density of blood vessels in the chorioallantoic membrane, with an increase in the relative weight of the liver and changes in the morphological aspects of the heart, such as an increase in the thickness of the right ventricular wall. CO₂ exposure did not affect chick body temperature and blood pressure (Rocha et al., 2020), but reduced the ventilatory response in chicks after hatching (Szdzuy & Mortola, 2008). Although many studies show effects on different systems, few researches, if any, describe the effect of hypercapnia on the development of the gastrointestinal tract.

Another important factor is the age of the broiler breeder, which affects the weight of eggs and chicks, albumen quality (Tona et al., 2004), the development of the embryo (Peebles et al., 2001), and the conductance of the shell (Peebles et al., 1987). These and other variables can also be affected under hypercapnia

Considering:

1. that embryonic development is characterized not only by the growth of organic systems in isolation, but rather by an interaction between them;

2. that the embryonic environment can modulate the development of such systems (Burggren et al., 2016) and;

3. the angiogenic effect on the chorioallantoic membrane of hypercapnia during early incubation (Verhoelst et al., 2011);

this research was developed to test the hypothesis that exposure to CO₂ in up to 10 days of incubation would stimulate the development of the gastrointestinal tract of embryos and post-hatch chicks.

MATERIAL AND METHODS

Experimental design

Research on animals was conducted according to the standards of the Institutional Committee on Animal Use (Protocol 025330/12).

This study was conducted in Jaboticabal, São Paulo, Brazil (21°14’05” South latitude, 48°17’09” West longitude, average altitude of 615.01 m).

A total of 860 Cobb500^® eggs (430 from 31-week-old breeders and 430 from 41-week-old breeders) obtained from a commercial hatchery (Globoaves, Itirapina, São Paulo, Brazil) were weighed (60.0 and 66.5 g, for broiler breeders aged 31 and 41 weeks, respectively), numbered, and distributed in a completely randomized design in a 2 x 2 factorial arrangement with 5 replications of 43 eggs. The factorial arrangement includes 2 incubator gaseous environments and 2 breeder ages. The eggs were equally distributed in 2 vertical incubators equipped with humidity and temperature controllers (Premium Ecológica, Belo Horizonte, Minas Gerais, Brazil), in which there was either no increase in CO₂ concentration (Control, C), or a gradual increase in CO₂ concentration until reaching 1% on the 10th day (Hypercapnia, CO₂), as shown in Figure 1. The increase in CO₂ concentration was obtained through the gradual injection of CO₂ in the incubator. The incubators’ CO₂ concentration was monitored using a computerized system and CO₂ sensors (Vaisala GMW21D, Waarloos, Belgium).

Figure 1
CO₂ concentrations (%) in the incubators. (C: control and CO₂: increase in CO₂ concentration up to 1% in the first 10 days of incubation).

Incubator temperature and air humidity were maintained at 37.5˚C and 60%, respectively, until 21 days, and eggs were turned 45˚ every hour until the 18^th day of incubation. The two incubators were maintained under normal ventilation conditions, with openings open throughout the incubation. After hatching, the chicks were housed in cages (1.0 m wide x 0.60 deep x 0.40 m high) in an air-conditioned chamber with a temperature of 32 ºC and a continuous light program until 7 days of age. The diet contained 22.48% crude protein and 2960 kcal of metabolizable energy/kg, following the recommendations of Rostagno et al. (2011).

Egg Weight Loss, Hatchability, Chick Body Weight, and Chick Quality

Egg weight loss was calculated as EWL (%) = [(W₀-W_F)/ W₀] × 100, where W₀ is egg weight at setting and W_F is egg weight at the 18th day of incubation. Hatchability was calculated as the percentage of fertile eggs set that hatched. After hatching, chicks were weighed, and chick quality was evaluated according to Tona et al. (2003).

Allometric Growth of Digestive System Organs

The allometric growth of digestive system organs was evaluated in embryos at 16, 18 and 20 days of incubation, and in chicks at 1, 4, and 7 days after hatching (after fasting for 12 hours), using five embryos and five chicks from each treatment at each age. Embryos and chicks were euthanized by decapitation and the digestive system was separated into yolk sac, proventriculus, gizzard, intestine, and liver, with each organ being washed and weighed. The body weight of the embryos and chicks was obtained by discounting the weight of the yolk sac from the total weight of the live bird. The relative weight (%) of each organ was obtained through the ratio between the weight of the organ and the body weight of the embryo or chick (discounting the yolk sac).

Intestinal Light Microscopy

Samples from the duodenum were collected in the proximal descending portion of the duodenal loop, from the jejunum, posterior to the distal portion of the duodenal loop, and from the ileum, in the region cranial to the cecum. The samples were collected from 5 birds of each treatment at 16, 18, and 20 days of incubation, and at 1, 4, and 7 days after hatching, the same birds used in the evaluation of organ allometric development. The samples were fixed in Bouin’s solution, dehydrated in an increasing series of ethanol (70%, 80%, 90%, and 100%), and included in paraffin. Five histological sections with 5 micrometers each were made per sample, and they were stained with hematoxylin and eosin. The histological sections’ images were obtained using an image capture system (Leica QWin; Leica Imaging Systems, Wetzlar, Germany).

The analyzes were carried out using the Image J^® software (Rasband, 2004). The height of villi in the first stage was measured according to Uni et al. (2003), at 16, 18, and 20 days in embryos, and at 1, 4, and 7 days in chicks, with 30 readings/intestinal segment.

Intestinal Scanning Electron Microscopy

The density of intestinal villi was evaluated at 20 days of incubation, and at 1, 4, and 7 days after hatching in 5 birds of each treatment, using scanning electron microscopy in the same regions of the small intestine mentioned previously. The samples were processed according to the methodology described by Maiorka et al. (2003), in the same birds used in the evaluation of organ allometric development and intestinal light microscopy. Villus density per area was expressed as the number of villi/mm².

Statistical analysis

The results are presented as means with the standard error of the mean. All statistical analyses were performed using the SAS software (SAS Institute, 2001). The data were analyzed using Analysis of Variance (ANOVA), and in case of significant differences, the means were compared by the Tukey test at 5% probability (p<0.05).

The statistical model used was:

$Y_{ijk}=\mu +{\alpha }_i+{\beta }_j+{\left(\alpha \times \beta \right)}_{ij}+{\epsilon }_{ijk},$

in which Y_ijk is the response variable measured for different incubator gaseous environments (i) and breeder ages (j) in replication k, µ is the general constant, αi is the fixed effect of incubator gaseous environments, β_j is the fixed effect of breeder ages, α × β)_ij is the interaction between incubator gaseous and breeder ages, and ε_ijk is the random error term.

Comparisons were made after verifying the homogeneity of the variance between treatments using the Levene test (Petrie & Watson, 2006), and the normality using the Cramér-von Mises test (Sprent, 1989).

RESULTS

Data analysis showed no interaction between hypercapnia and broiler breeder age for all parameters analyzed. Table 1 shows data on egg weight loss, hatchability, chick weight and quality score, with no difference between control and CO₂ treatments (p>0.05). For breeder ages, the lowest egg weight loss (p=0.0498) and highest chick weight (0.0007) were for breeders aged 41 weeks. Exposure to CO₂ did not influence the weight of the yolk sac and the relative weight of the digestive system organs, with only an increase in embryo weight being observed at 16 days of incubation (p=0.011) with incubation in hypercapnia, with no observed effect after hatching (data not shown).

Table 1
Average values of egg weight loss (EWL, %), hatchability (%), day-old chick body weight (CBW, g), average chick quality score (CQE) and chicks with quality score=100 (100CQE, %), in eggs from 31- and 41-week-old breeders incubated without increase in CO₂ concentration (C) and with gradual increase in CO₂ concentration until reaching 1% on the 10th day (CO₂).

Factors	EWL	Hatchability	CBW	CQE	100CQE
CO2 concentration
C	9,61	90,06	42,57	97,32	77,50
CO2	9,48	92,38	43,09	97,73	79,50
Breeder age (weeks)
31	9,95 a	92,13	41,35 b	98,00	78,50
41	9,14 b	90,31	44,31 a	97,05	78,50
SEM*	0,40	1,94	0,94	0,61	3,98
p value
CO2	0,7515	0,2565	0,4758	0,5058	0,6437
Breeder age	0,0498	0,3680	0,0007	0,1343	1,0000
CO2 x Breeder age	0,7730	0,6466	0,9772	0,4466	0,4897

• *SEM: Standard error of the mean.
• Means followed by different letters differ according to Tukey’s test at 5% (p<0,05).

In comparison to 31-week-old broiler breeders, eggs from 41-week-old broilers resulted in higher body weight (p=0.036, 0.047, and 0.024, respectively) and yolk weight (p<0.001, 0.003, and 0.037, respectively) at 16, 18 and 20 days of incubation. Moreover, eggs from 41-week-old broiler breeders resulted in a higher chick weight 1, 4, and 7 days after hatching (p=0.002, <0.001 and 0.005), and a higher relative liver weight at 1 day after hatching (p=0.013), when compared to eggs from 31-week-old broiler breeders (Figure 2).

Figure 2
Effect of broiler breeder age (31 and 41 weeks) on the weight of embryos and yolk at 16, 18 and 20 days of incubation and weight of chicks free of the yolk sac and liver at 1, 4 and 7 days after hatching. Asterisks indicate a significant difference between groups (p<0.05).

Incubation at 1% CO₂ resulted in an increase in the height of the intestinal villi in the duodenum at 20 days of incubation (p<0.001) and 1 day after hatching (p=0.001), and at 16 days of incubation in the jejunum (p<0.001) and ileum (p=0.026) (Figures 3, 4 and 5). Villus of embryos and chicks from 41-week-old breeders showed higher height in the duodenum at 20 days of incubation (p=0.001) and 1 day after hatching (p=0.018). In the jejunum, increased height of villi was observed at 1 day after hatching (p=0.008), and in the ileum at 1 and 7 days after hatching (p=0.025 and 0.022, respectively) for chicks from 41-week-old breeders (Figures 3, 4 and 5).

Figure 3
Effect of incubation in control (C) and increase in CO₂ concentration up to 1% in the first 10 days of incubation (CO₂) (A) and effect of breeder age (31 and 41 weeks) (B) on height of the villi in the duodenum, jejunum and ileum of embryos at 16, 18 and 20 days. Asterisks indicate a significant difference between groups (p<0.05).

Figure 4
Effect of incubation in control (C) and increase in CO₂ concentration up to 1% in the first 10 days of incubation (CO₂) (A) and broiler breeder age (31 and 41 weeks) (B) on the height of the villi in the duodenum, jejunum and ileum of chicks at 1, 4, and 7 days after hatching. Asterisks indicate a significant difference between groups (p<0.05).

Figure 5
Photomicrographs of cross-sections of intestinal segments of embryos at 16 days of incubation. A, C, and E represent the duodenum, jejunum, and ileum, respectively, of embryos incubated without increase in CO₂ concentration, and B, D, and F represent the duodenum, jejunum, and ileum, respectively, of embryos incubated with a gradual increase in CO₂ concentration until reaching 1% on the 10th day. (- = 100 µm).

Villus density was also influenced by incubation in hypercapnia. In comparison to the control group, the group incubated in 1% CO₂ showed lower villus density in the duodenum in chicks 1 day after hatching (p<0.001). In the jejunum, this effect was observed in embryos with 20 days (p=0.015) of incubation and in chicks at 4 and 7 days after hatching (p=0.004 and 0.015, respectively). In the ileum, the lowest density was observed in embryos with 20 days of incubation (p=0.022) and in chicks on the 4th day after hatching (p=0.031) (Figure 6 and 7). Chicks from 41-week-old broiler breeders showed lower villus density in the duodenum, jejunum, and ileum at 7 days (p=0.014, 0.001, and 0.008) compared to chicks from 31-week-old breeders.

Figure 6
Effect of incubation in control (C) and with an increase in CO₂ concentration up to 1% in the first 10 days of incubation (CO₂) (A) and of breeder age (31 and 41 weeks) (B) on villus density in the duodenum, jejunum and ileum of chicks at 1, 4 and 7 days after hatching. Asterisks indicate a significant difference between groups (p<0.05).

Figure 7
Photomicrographs of the surface of intestinal segments of embryos at 20 days of incubation. A, C, and E represent the duodenum, jejunum, and ileum, respectively, of embryos incubated without increase in CO₂ concentration, and B, D, and F represent the duodenum, jejunum, and ileum, respectively, of embryos incubated with a gradual increase in CO₂ concentration until reaching 1% on the 10th day. ( - = 50 µm).

DISCUSSION

Incubation with increased CO₂ concentration during the early stages can improve hatchability (De Smit et al., 2006, 2008; El-Hanoun et al., 2019) or not affect it (Bruggeman et al., 2007, Fernandes et al., 2016; Kroetz Neto et al., 2023). In our study, no improvement in hatchability was observed with hypercapnia, regardless of the breeder age, showing that the effect of incubation in hypercapnia on hatchability can be influenced by other factors, such as genetics (De Smit et al., 2008). Most research on incubation in hypercapnia so far has used non-ventilated incubators (De Smit et al., 2006, 2008; Fernandes et al., 2016). These can influence humidity conditions, resulting in differences in egg weight loss. In our work, incubators were kept ventilated and there was no difference in egg weight loss. Chick weight and quality were not influenced by incubation in hypercapnia, in line with other studies with a pattern of hypercapnia similar to ours (De Smith et al., 2006, 2008; Fernandes et al., 2016; Kroetz Neto et al., 2023).

Total embryo weight (16^th day incubation) in hypercapnia was similar to the findings reported in the literature (De Smit et al., 2006, 2008; Tona et al., 2007). However, regarding the relative weight of the gastrointestinal tract organs, no statistical differences were found when comparing the embryos and chicks from the control and hypercapnia groups. The exception was the lower relative weight of the liver observed in embryos incubated in hypercapnia at 16 days of age. Studies confirm the absence of an effect of incubation in hypercapnia on the relative weight of various organs such as heart, liver, gizzard, intestine, spleen, bursa and lungs (De Smit et al., 2008; Maatjens et al., 2014; Tong et al., 2015; Fernandes et al., 2017). Thus, in this study, the findings show that the weight of the organs of the gastrointestinal tract is also not influenced by incubation in hypercapnia.

Despite the lack of effects on the relative weight of the intestine, the increase in CO₂ concentration determined a change in villi height. The higher the villi, the lower the density per square area, indicating higher surface absorption. The effects of incubation in hypercapnia on villi development may be associated with increases in plasma T3 and corticosterone concentrations, as evidenced by De Smit et al. (2006). Such hormones accelerate the rate of embryo development (Decuypere et al., 1991). These hormones are involved in the growth and functional maturation of various organs in embryos of avian species, including the intestine (Black, 1978). There have been reports of a relation between thyroid hormones and cell differentiation in the intestine of chick embryos (Moog, 1961).

The effects on villi development of CO₂ during incubation at a 1% concentration could also be attributed to the angiogenesis stimulus that occurs in conditions of hypercapnia. Verhoelst et al. (2011) reported higher vascularization of the chorioallantoic membrane in conditions of hypercapnia, supporting the idea that villi growth can be affected by increasing oxygen supply. Vasculogenesis occurs at the beginning of embryonic development; thus, the effect on intestinal villi development seems to be dependent of blood vessels and oxygen supply (Pardanaud et al., 1989; Risau, 1997).

The observed results regarding villus height and villus density suggest that hypercapnia of up to 1% in the first 10 days of incubation may improve nutrient absorption capacity in the small intestine.

The hypothesis that exposure to CO₂ would affect the digestive system differently depending on the age of the broiler breeder has not been confirmed, since there was no interaction between the factors studied. This shows that the results obtained with hypercapnia are independent from the age of the broiler breeder. On the other hand, broiler breeder age influenced some studied parameters; for instance, the lower percentage of weight loss in eggs from older broiler breeders (Iqbal et al., 2016). Embryos and newly hatched chicks presented higher weight and higher yolk weight when coming from eggs from older breeders as compared to young ones, confirming the results obtained by Bray & Iton (1962), that evidenced the positive correlation between egg weight and embryo weight, especially at the end of incubation.

El Sabry et al. (2013) observed higher villi height and villus surface area in the jejunum of chicks from old broiler breeders compared to young ones. Schaefer et al. (2006) also reported a higher villus surface area in all segments of the small intestine in birds from older broiler breeders, compared to young ones. These results suggest that chicks from older broiler breeders have a greater ability to absorb nutrients.

CONCLUSIONS

In conclusion, incubation in hypercapnia can affect morphological characteristics in the small intestine, by increasing villus height and reducing density, regardless of the age of the broiler breeder.

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Publication Dates

• Publication in this collection
  13 Nov 2023
• Date of issue
  2023

History

• Received
  02 May 2023
• Accepted
  06 Sept 2023