Influence of the age of breeders and storage time of eggs from broiler breeders incubated in multi-stage and single-stage hatchers on the hatching and quality parameters of the 1-day-old chick

[Influência da idade das reprodutoras e do tempo de armazenamento dos ovos de reprodutoras de frangos de corte incubados em incubadoras de múltiplos estágios e de estágio único na eclosão e nos parâmetros de qualidade do pinto de um dia]

ABSTRACT

The objective of the study was to evaluate the influence of breeder age and storage time of broiler breeder eggs, as well as the impact of single-stage and multi-stage incubators on hatchability parameters and day-old chick quality. 16,128 fertile eggs from the Cobb 500 strain were stored for different periods (3, 6, 9 and 12 days) and subjected to two types of incubators (single-stage and multi-stage). Eggs from young breeders (30 weeks) showed a higher percentage of vessels in the chorioallantoic membrane, especially in single-stage incubators. Embryo weight was higher in eggs from older breeders (60 weeks) and in multi-stage incubators. Hatchability was higher for eggs from young breeders and for the single-stage system, with lower rates of moisture loss and late mortality. It is concluded that eggs from young breeders are more tolerant to storage and that breeder age negatively affects incubation parameters, especially in multi-stage incubation systems, indicating that the choice of incubation system and storage time should be adapted to the breeder age to optimize hatch rate and chick quality.

Keywords:
incubation; storage; hatchability; quality; age of the breeder

RESUMO

O objetivo do estudo foi avaliar a influência da idade das reprodutoras e do tempo de armazenamento dos ovos de matrizes de corte, bem como o impacto de incubadoras de estágio único e múltiplo sobre os parâmetros de eclosão e a qualidade dos pintinhos de um dia. Foram armazenados 16.128 ovos férteis da linhagem Cobb 500 por diferentes períodos (três, seis, nove e 12 dias), sendo posteriormente submetidos a dois tipos de incubadoras (estágio único e múltiplo). Os ovos de reprodutoras jovens (30 semanas) apresentaram maior percentual de vasos na membrana corioalantoide, principalmente em incubadoras de estágio único. O peso do embrião foi maior em ovos de reprodutoras mais velhas (60 semanas) e em incubadoras de estágio múltiplo. A eclodibilidade foi maior para ovos de reprodutoras jovens e para o sistema de estágio único, com menores taxas de perda de umidade e mortalidade tardia. Conclui-se que os ovos de reprodutoras jovens são mais tolerantes ao armazenamento e que a idade das reprodutoras afeta negativamente os parâmetros de incubação, principalmente em sistemas de incubação de estágio múltiplo, indicando que a escolha do sistema de incubação e do tempo de armazenamento deve ser adaptada à idade das reprodutoras para otimizar a taxa de eclosão, a qualidade e uniformidade dos dos pintainhos.

Palavras-chave:
incubação; armazenamento; eclodibilidade; qualidade; idade da matriz

INTRODUCTION

In recent years, the global increase in animal protein consumption has generated significant growth in Brazilian livestock farming, with a growing consumer preference for chicken protein. The Brazilian Association of Animal Protein (ABPA) projects a record scenario for domestic chicken meat consumption in 2025 (Chicken…, 2025), estimating a per capita consumption of 47.8 kg. This phenomenon is indicative of the continuous development of poultry farming, maintaining an average growth rate of 8% per year, largely driven by technological integration, consolidating the Brazilian poultry model as a global reference (Evolution…, 2023).

Technology plays a crucial role in poultry farming, especially in the incubation phase, where hatcheries represent a fundamental technological innovation to increase productivity. Developed to maximize production, these systems exhibit high survival rates and accelerate embryonic development (Boleli et al., 2016).

Incubation automates factors such as humidity, temperature, and egg movement, enabling a higher number of eggs to be incubated while reducing labor needs. Single-stage incubation involves embryos at the same development stage, whereas multi-stage incubation accommodates embryos at different stages in the same machine.

In commercial hatcheries, storing fertilized eggs before incubation is a standard method to synchronize hatchings and organize operations in poultry production. It allows for some adaptability to needs and makes hatching synchronization easier (Tona et al., 2022). However, both negative and positive effects could result from storing eggs before incubation. The hatchability rates and chick quality are negatively correlated with the amount of time eggs are stored since the embryonic development is significantly influenced by the mechanical, morphological, and physical properties of eggs (Oke et al., 2024). Day-old chicken quality can be connected to egg quality, which is influenced by several factors, including breeder age, and the hatchery system. One of the most significant hatchery changes around the world during the last years is the migration from multi-stage to single-stage systems (Molenaar et al., 2010).

Once thought to be only advantageous because of the higher level of biosecurity, single stage systems are now recognized as more efficient in meeting embryos requirements as well, multiple stage systems have a window from 28-32 hours, with single stage systems from 12-24, with significant impact on chick quality (Araújo et al., 2016).

Incubation is essential to meet the growing demand for chicken meat, with Brazil leading global production, providing nutritious food for the entire world (Evolution..., 2023). This process exemplifies how technology drives efficiency, quality and sustainability in the industry, being fundamental to feeding a growing global population. Brazilian poultry farming, with its innovative approach, stands out as a driving force on the world stage, ensuring food security and a promising future for the animal protein industry.

In this context, with the aim of investigating the influence of the age of the breeders and the storage time of eggs from broiler breeders, as well as the impact of different incubation conditions in multi- and single-stage incubators on hatching and hatching parameters quality of the 1-day-old chick, this project aims to explore the determining factors for the success of incubation in the poultry industry. Through the analysis of relevant data and information, we seek to understand how technology and management practices can be optimized to ensure high hatching rates, survival and quality of chicks, thus contributing to the continuous advancement of poultry farming and meeting the growing demand for chicken meat in a sustainable and efficient way.

ETHICAL ASPECTS

The research was submitted to the Ethics Committee on Animal Use of the Parana Federal University, and approved under the number 05/2012.

MATERIAL AND METHODS

The experiment was conducted at the Poultry Experimentation Laboratory of the Federal University of Parana - Palotina Sector and in a commercial hatchery. 16128 fertile eggs were separated from breeders of the Cobb 500 lineage, half from breeders at the beginning of laying (30 weeks) and half from breeders at the end of laying (60 weeks). The eggs were collected on the same day and stored according to the experimental design in the hatchery's egg storage room (19 - 21º C and 60 to 65% RH). The experimental design in the hatchery was completely randomized in a 2x2x4 factorial scheme (2 breeder ages: 30 and 60 weeks vs 2 incubator models: multi-stage and single-stage vs 4 storage periods: 3, 6, 9 and 12 days) with 16 treatments and 6 replications. Multi-stage and single-stage incubators with a profile of 8,000 ppm carbon dioxide (CO₂) were used in the first 10 days of incubation. The CO₂ level was used during the first ten days of the incubation period and after this period all eggs were subjected to a level of 4,000 ppm CO₂ until transfer on the eighteenth day and 3,000 ppm until hatching.

Before incubation, all eggs from each treatment were weighed and distributed into 6 replications containing 168 eggs each (1008 eggs/treatment). The experimental unit was considered the floor of the incubation cart containing 4 trays of 42 eggs.

At 11 days of incubation, the vascularization of the chorioallantoic membrane of 12 eggs per treatment was evaluated according to the methodology used by Verhoelst et al. (2011). Eggs with 11 days of incubation, a period in which the chorion-allantoic membrane is completely adhered to the eggshell, were carefully emptied and filled with formaldehyde to allow the erythrocytes to attach to the blood vessels. Subsequently, the eggs were divided into strips transverse to the long axis of the egg, which were divided into 8 segments each to obtain images from each segment. At the same age, the albumen was separated from the yolk sac and the pH was measured using a Gehaka PG 2000 digital pH meter.

Shells from 6 eggs per treatment were used to count the number of shell pores using the method of Rahn et al. (1981). The samples were boiled for 10 minutes in 5% aqueous NaOH solution to remove the cuticle and external and internal shell membranes. They were then washed in water and dried at room temperature. The samples were then stained in an aqueous solution of methylene blue (1%) for 2 minutes, washed quickly in water and dried at room temperature. The average number of pores contained in 4 areas of 25mm² per region of the shell was counted under a stereo-microscope, using a checkered micro-metric grid.

To evaluate moisture loss, when transferring eggs from incubators to hatchers, between the 18th and 19th day, the experimental units containing 168 eggs each were weighed individually. After hatching, on the 21st day, the hatched chicks were counted and subtracted from the total number of eggs incubated to calculate the percentage of hatching, dead and eliminated. The eliminated chicks were classified (late, crooked neck, leg problems, sticky, small, bruised, dead, black navel, inflamed navel, exposed viscera).

After hatching, embryo-diagnosis was carried out on the eggs that did not hatch from each repetition, which will be classified into mortality (0 to 4 days, 5 to 10 days, 11 to 17 days and 18 to 21 days), contaminated eggs, cracked eggs, pecked alive and pecked dead. At hatching, 6 chicks from each treatment were randomly sacrificed, which were weighed and measured. After sacrifice by cervical dislocation, the heart, breast, intestine, liver and yolk sac were extracted, which were weighed.

Statistical analysis of the data was carried out using the SAS GLM procedure (SAS, 1998). The data was subjected to analysis of variance. Cases of interaction between age, incubator and storage period. The effect of age and incubator was evaluated using the F test of analysis of variance and the effect of storage period using regression analysis. In all statistical procedures, a 5% significance level was adopted.

RESULTS

At 11 days of incubation, an interaction was observed between matrix age and egg storage period for egg weight and chorioallantoic membrane vascularization (% of vessels) and a significant interaction between matrix age and incubator for chorioallantoic membrane vascularization. For the pH measured inside the egg, there was an interaction between the 3 factors studied (Table 1). In Tables 2, 3 and 4 and Fig. 1 and 2 are the results regarding the breakdown of the interaction of the factors involved.

In Table 1 the embryo weight was affected independently by the factors evaluated. The embryo from 60-week-old dams showed greater weight than that from 30-week dams. Eggs incubated in multiple stages when compared to those incubated in single stages also present heavier embryos. On the other hand, embryo weight decreased linearly with the increasing storage period (Fig. 1).

Table 1
Incubation parameters of eggs from 30- and 60-week-old breeders stored for 3, 6, 9, and 12 days in single and multiple incubation systems at 11 days of incubation

	Egg Weight, g	pH	Embryo Weight, g	% Vessels
Age
30 weeks	62.11±0.37	7.73±0.02	5.39b±0.05	11.66±0.42
60 weeks	66.28±0.39	7.58±0.03	5.53a±0.06	8.61±0.32
Incubator
Single Stage	64.69±0.41	7.86±0.02	5.33b±0.05	8.65±0.35
Multiple Stage	63.74±0.46	7.45±0.03	5.59a±0.06	10.88±0.41
Storage
3 days	64.62±0.59	7.64±0.05	5.77±0.07	10.47±0.51
6 days	64.60±0.67	7.66±0.04	5.54±0.08	9.12±0.49
9 days	64.19±0.52	7.61±0.05	5.34±0.09	9.68±0.73
12 days	63.47±0.67	7.70±0.04	5.20±0.06	10.13±0.57
CV (%)	5.59	2.59	8.52	22.93
ANOVA
Age	0.001	0.001	0.007	0.001
Incubator	0.844	0.001	0.001	0.001
Storage	0.981	0.630	0.001*	0.514
Interactions
Age x Incubator	0.663	0.027	0.641	0.036
Age x Storage	0.002	0.775	0.211	0.004
Incubator x Storage	0.567	0.959	0.505	0.601
Age x Incubator x Storage	0.787	0.002	0.653	0.911

• ^a-b
  Mean values with different superscripts in a column differ. * Linear effect

Figure 1
Effect of eggs stored for 3, 6, 9, and 12 days on embryo weight at 11 days of incubation. Ŷ = 5,93752-0,06354x, R²: 0,98.

The analysis of interactions revealed that eggs from 30-week-old breeders exhibited a higher percentage of blood vessels in the chorioallantoic membrane, regardless of the incubation system, compared to eggs from 60-week-old breeders. Furthermore, multi-stage incubation increased the percentage of vessels in eggs from 30-week-old breeders, whereas the incubation system did not influence this parameter in eggs from 60-week-old breeders (Table 2).

Table 2
Breakdown of the interaction between incubation system (single and multiple stage) and breeder age (30 and 60 weeks)

Age	Single Stage	Multiple Stage	Effect
	Membrane Vascularization, %
30 weeks	9.63Ab	13.08Aa	0.001
60 weeks	8.05Ba	9.13Ba	0.184
Effect	0.047	0.002

• ^A-B
  Mean values with uppercase superscripts in a column differ.
• ^a-b
  Mean values with lowercase superscripts in a row differ.

In the breakdown of the interaction between breeder age and storage period, it was found that eggs from 30-week-old breeders and stored for more than 3 days showed greater vascularization of the chorioallantoic membrane compared to eggs from 60-week-old breeders (in accordance with shown in Table 3). Regarding the storage period, a quadratic behavior was observed for eggs from 60-week-old breeders. According to the derivation of the estimated equation, the highest percentage of vessels was observed at 3.4 days (as illustrated in Fig. 2).

Table 3
Breakdown of the interaction between the age of the matrices (30 and 60 weeks) and storage period (3 to 12 days)

	Egg Storage time
	3 days	6 days	9 days	12 days	Effect
Membrane Vascularization, %
Age
30 weeks	10.61A	11.37A	12.24A	11.92A	0.634
60 weeks	10.40 A	8.06B	7.30 B	8.33B	Quadratic
Effect	0.622	0.024	0.004	0.011

• ^A-B
  Mean values with uppercase superscripts in a column differ.

Figure 2
Effect of eggs stored for 3, 6, 9, and 12 days on vascular fraction (%) of the chorioallantoic membrane from 60-week-old breeders’ eggs. Ŷ = 14.48533-0.63893x+0.0938x². R²: 0.99

For egg weight, at 11 days of incubation, the breakdown of the interaction of breeder age and storage period showed a higher weight of eggs from breeders aged 60 weeks compared to those aged 30 weeks, with the exception when eggs were stored for 9 and 12 days, where there was no difference between the ages of the breeders (Table 4).

Table 4
Breakdown of the interaction between breeder age (30 and 60 weeks) and storage period (3 to 12 days) for egg weight at 11 days of incubation

	Egg storage time
	3 days	6 days	9 days	12 days	Effect
Egg weight (11 days)
Age
30 weeks	62.19B	61.69B	63.64A	60.66 A	0.922
60 weeks	67.16A	67.51A	64.54A	65.96A	0.843
Effect	0.044	0.014	0.775	0.070

• ^A-B Mean values with uppercase superscripts in a column differ.

In the breakdown of the triple interaction, for pH values, single-stage incubation, regardless of the age of the matrix, led to an increase in albumen pH when compared to incubation in a multiple-stage system (Table 5).

Table 5
Breakdown of the interaction between breeder age (30 and 60 weeks), incubation system (single and multi-stage), and storage period (3 to 12 days) for pH measurements

	pH
	30 Weeks
	3 days	6 days	9 days	12 days	Effect
Single Stage	7.86A	7.87A	7.93A	7.96A	0.953
Multi-Stage	7.61B	7.53B	7.56B	7.56B	0.882
Effect	0.0196	0.001	0.027	0.033
	60 Weeks
	3 days	6 days	9 days	12 days	Effect
Single Stage	7.91A	7.83A	7.64A	7.86A	0.344
Multi-Stage	7.15B	7.42B	7.32B	7.45B	0.217
Effect	0.006	0.018	0.023	0.041

• ^A-B
  Mean values with uppercase superscripts in a column differ.

The pore count in the eggshell was not influenced by any factor studied. Moisture loss and the percentage of hatched chicks considered rejects were affected by the 3 factors. On the other hand, the hatching percentage was higher (P<0.05) for 30-week-old breeders and higher (P<0.05) for single-stage incubation. For the storage period, there was only a difference when comparing 3 with 12 days of storage, with the lowest percentage of hatching with 12 days of storage (Table 6).

Moisture loss in eggs from 30-week-old breeders was higher during multi-stage incubation. For 60-week-old breeders, moisture loss was higher only in eggs incubated in multi-stage systems and stored for 6 days. The storage period had a quadratic effect on moisture loss in eggs from 30-week-old breeders to both incubation systems, whereas for 60-week-old breeders incubated in multi-stage systems, the effect was linear and decreasing. For eggs incubated in single-stage systems at 60 weeks, storage duration did not influence moisture loss. (Table 7).

Table 6
Incubation parameters of eggs from 30- and 60-week-old breeders stored for 3, 6, 9, and 12 days and incubated in single and multiple incubation systems

	Pores (mm2)	Moisture Loss (%)	Hatchability (%)	Low quality (%)
Age
30 weeks	14.29±0.74	10.25±0.12	84.43a±0.61	2.20±0.25
60 weeks	14.26±0.57	9.69±0.17	67.71b±0.66	2.46±0.24
Incubator
Single Stage	13.78±0.73	9.41±0.09	77.26a±1.37	1.68±0.19
Multiple Stage	14.68±0.58	10.54±0.16	74.87b±1.36	2.98±0.26
Storage
3 days	13.36±0.82	9.30±0.16	77.28a±1.75	2.55±0.28
6 days	13.26±0.86	10.20±0.23	76.73ab±1.94	1.38±0.19
9 days	15.00±1.03	10.01±0.17	76.46ab±2.04	2.54±0.35
12 days	15.48±0.93	10.39±0.23	73.80b±2.05	2.85±0.45
CV (%)	28.80	9.75	28.65	59.10
ANOVA
Age	0.909	0.001	0.001	0.034
Incubator	0.864	0.001	0.007	0.001
Storage	0.843	0.001	0.037	0.001
Interactions
Age x Incubator	0.919	0.632	0.376	0.092
Age x Storage	0.752	0.013	0.237	0.087
Incubator x Storage	0.792	0.001	0.310	0.431
Age x Incubator x Storage	0.836	0.001	0.487	0.027

• ^a-b
  Mean values with different superscripts in a column differ.

Table 7
Breakdown of the interaction between breeder age (30 and 60 weeks), incubation system (single and multiple stage), and storage period (3 to 12 days)

	Moisture Loss (%)
	30 weeks
	3 days	6 days	9 days	12 days	Effect
Single Stage	9.20B	10.23B	9.53B	9.65B	Quadratic
Multiple Stage	10.16A	11.08A	11.14A	11.04A	Quadratic
Effect	0.035	0.029	0.010	0.030
	60 weeks
	3 days	6 days	9 days	12 days	Effect
Single Stage	9.22A	8.56B	9.61A	9.27A	0.733
Multiple Stage	8.62A	10.95A	9.75A	11.57A	Linear
Effect	0.098	0.016	0.996	0.060

• ^A-B
  Mean values with uppercase superscripts in a column differ.

For 30-week-old breeders, the percentage of low-quality poultry hatching was higher when incubated in multiple stages and stored for 3, 9 and 12 days. For eggs from 30-week-old breeders incubated in a single stage, there was a decreasing linear effect of storage on the percentage of discarded chicks, whereas for the multiple stage the effect was quadratic, with the lowest value obtained when the eggs were stored for 9 days. For 60-week-old breeders, the incubation system or egg storage did not affect the percentage of scraps (Table 8).

Table 8
Breakdown of the interaction between breeder age (30 and 60 weeks), incubation system (single and multiple), and storage period (3 to 12 days)

	Low quality poultry hatching (%)
	30 weeks
	3 days	6 days	9 days	12 days	Effect
Single Stage	1.54B	1.17A	1.04B	0.71B	Linear
Multiple Stage	4.08A	1.55A	2.97A	4.61A	Quadratic
Effect	0.002	0.753	0.034	0.001
	60 weeks
	3 days	6 days	9 days	12 days	Effect
Single Stage	2.19A	0.86A	2.74A	3.16A	0.092
Multiple Stage	2.45A	1.92A	3.42A	2.91A	0.152
Effect	0.812	0.843	0.858	0.769

• ^A-B
  Mean values with uppercase superscripts in a column differ.

In Table 9, the age of the breeder affected chick weight and yolk percentage, with the highest values for 60-week-old breeders, while eggs incubated in a single-stage incubation system resulted in the greatest length of chicks. and a higher percentage of heart and chest when compared to multiple stage.

Table 9
Organ biometrics of broiler chicks post-hatching from eggs of 30- and 60-week-old breeders stored for 3, 6, 9, and 12 days in single and multiple incubation systems

	Length, cm	Chick, g	Liver, %	Intestine, %	Breast, %	Heart, %	Yolk, %
Age
30 weeks	19.02b±0.10	46.05b±0.53	2.74±0.04	3.33±0.06	3.51±0.11	0.86±0.02	9.77b ±0.34
60 weeks	19.27a±0.08	47.85a±0.48	2.75±0.04	3.20±0.08	3.46±0.10	0.84±0.02	11.66a±0.38
Incubator
Single Stage	19.55a±0.06	47.59±0.48	2.74±0.04	3.21±0.07	3.79a±0.08	0.92a±0.02	10.61±0.35
Multiple Stage	18.74b±0.09	46.32±0.55	2.76±0.04	3.32±0.08	3.18b±0.11	0.78b±0.02	10.84±0.42
Storage
3 days	19.24±0.14	46.44±0.64	2.79±0.05	3.34±0.11	3.51±0.15	0.85±0.03	10.55±0.43
6 days	19.15±0.13	46.25±0.75	2.72±0.05	3.29±0.08	3.23±0.16	0.83±0.03	10.26±0.61
9 days	19.13±0.12	47.16±0.77	2.75±0.07	3.29±0.13	3.55±0.13	0.83±0.03	11.30±0.60
12 days	19.07±0.15	48.03±0.79	2.72±0.05	3.13±0.06	3.67±0.15	0.89±0.03	10.78±0.53
CV (%)	2.67	7.63	10.04	15.41	18.52	15.81	23.98
ANOVA
Age	0.002	0.001	0.891	0.095	0.744	0.165	0.001
Incubator	0.001	0.389	0.672	0.663	0.001	0.001	0.802
Storage	0.895	0.561	0.619	0.904	0.403	0.267	0.766
Interactions
Age x Incubator	0.487	0.705	0.993	0.731	0.449S	0.198	0.439
Age x storage	0.547	0.721	0.893	0.551	0.956	0.856	0.859
Incubator x storage	0.322	0.705	0.993	0.731	0.658	0.792	0.946
Age x Incubator x storage	0.723	0.721	0.893	0.851	0.805	0.866	0.914

• ^a-b
  Mean values with different superscripts in a column differ.

The age of the older breeder had a negative impact on the percentage of contaminated eggs and fertility. As for initial mortality, up to 5 days of embryonic age, there was an interaction between the age of the matrix and the incubation system. In multi-stage incubation, initial embryonic mortality was higher for eggs from 60-week-old breeders compared to 30-week-old breeders, only when incubated in multiple stages. For the single stage, there was no effect of the age of the matrix. On the other hand, when comparing the incubation system for both ages, an effect was observed only for 30-week eggs, where single-stage incubation increased initial mortality (Table 10).

Table 10
Breakdown of the interaction between incubation system (single and multiple) and sow age (30 and 60 weeks)

Age	Single Stage	Multiple Stage	Effect
	Mortality 1 to 4 days
30 weeks	31.70Aa	22.45Bb	0.012
60 weeks	26.71Ab	30.50Aa	0.039
Effect	0.229	0.003

• ^A-B
  Mean values with uppercase superscripts in a column differ.
• ^a-b
  Mean values with lowercase superscripts in a row differ.

DISCUSSION

There was an individual interaction of the 3 factors evaluated on the embryo weight at 11 days. It was observed that the embryos were heavier when originating from 60-week-old dams and when incubated in multiple-stage machines (Table 1). The higher weight of embryos originating from old breeders was already expected since the chick also tends to be larger, which corroborates the work of some authors (Almeida et al., 2008; Rocha et al., 2008) who state that there is a natural positive relationship between the increase in the weight of the eggs and consequently the chick with the increase in the age of the breeders. This increase in weight is derived from the greater interval between ovulations in the older mother and the deposition of the same amount of yolk constituents in a smaller number of follicles, concentrating this content and increasing the weight of the yolk (Rocha et al., 2008).

Ideal water loss provides ideal chick yield and helps to achieve good quality chicks (Nowaczewski et al., 2016). So, water loss during egg storage may be responsible for embryo dehydration, reducing embryo weight, as also occurred in the present study. According to Boleli (2003), a prolonged period of storage of eggs from older breeders leads to dehydration of the eggs due to the lower density of the albumen, as it facilitates the evaporation and diffusion of water and gases between the interior of the egg and the environment, and to the largest diameter of the eggshell pores. Contrary to what happens with older breeders, prolonging the storage time of eggs from younger breeders can contribute to better embryonic development by reducing the density of the albumen, which is denser in this egg, making breathing and moisture loss more difficult (Macari and Gonzales, 2003).

There was an interaction between the 3 factors studied on the percentage of moisture loss. During unfolding, it was observed that on all storage days evaluated and regardless of the age of the matrix, the moisture loss of the egg is greater when incubated in the multi-stage machine, contributing to cracking. The single-stage incubator allows for more controlled water loss from the egg to the environment, as it maintains an internal humidity profile according to the embryonic age and egg characteristics (Molenaar et al., 2010; Araújo et al., 2016). Therefore, the problems involved with storage can be accentuated by the incubator, as demonstrated in the present study.

There was a quadratic effect of the storage period on the moisture loss of eggs from 30-week-old breeders incubated in both a single-stage and multi-stage incubator, where the highest percentage of moisture loss occurred between 8 and 11 days of storage. For the 60-week-old breeders, there was a linear decreasing effect of the storage of eggs incubated in multiple stages.

To minimize gas exchange and moisture loss, which can impair early embryonic development and lower subsequent hatching rates, breeders begin the laying process by producing eggs with thick shells, few pores, and dense albumen (Araujo et al., 2017).

Regarding pH, according to Salahi et al. (2012), incubators with better air exchange control also promote the elimination of CO₂, which raises the pH of the albumen. In the single-stage incubator, air renewal inside the incubator is more efficient, allowing greater elimination of CO₂ into the environment. Regarding the pH measured inside the egg, there was an interaction between the 3 factors studied. During splitting, it was observed that regardless of the age of the matrix, the pH of the albumen is higher when the eggs are incubated in a single-stage machine in all storage periods evaluated.

The CAM (embryonic chorioallantoic membrane) is an extra-embryonic organ that develops through the fusion of the chorion with the vascularized allantoic membrane around the fourth day of embryonic development. Its main function is the exchange of gas and nutrients (Ribatti, 2010). Its higher percentage in young eggs shows that gas and nutrient exchange is better in this egg, especially when incubated in a single-stage incubator where incubation parameters are more controlled. Single-stage incubation with moderate levels of hypercapnia is an efficient tool for hatcheries to adopt when trying to improve chick quality (Fernandes et al., 2017).

In relation to the vascularization of the chorioallantoic membrane (% of vessels) there were 2 significant interactions, between the age of the mother and incubator and between the age of the mother and egg storage period. In the breakdown of the interaction between incubator and breeder age, for eggs from 30-week-old breeders there was greater vascularization in eggs that were incubated in the multi-stage incubator. However, when compared between the ages of the breeder, the 30-week breeder had a higher percentage of vascularization regardless of the incubator. Still referring to the vascularization of the egg, in the breakdown of the interaction between the age of the matrix and the storage period, there was no significant effect between the ages of the matrix of eggs stored for 3 days, after this period the 60-week matrix has lower vascularization (Table 2). And the eggs from the 60-week-old matrix showed a quadratic effect of egg vascularization with the highest percentage of vessels observed at 3.4 days.

Elibol et al. (2002a) and Zakaria et al. (2005), found that, regardless of the strain evaluated (Cobb500 and Ross308), there was greater hatchability of eggs when coming from batches of younger breeders compared to those from older breeders, which also occurred in the present study. Eggshell porosity plays a crucial role in the rate of weight loss and gas exchange, particularly in larger eggs, which are more susceptible to evaporation and gas transfer (Stewart et al., 2013). All of this is accentuated in the multi-stage machine, since, in this incubator, ventilation performs a double function of refrigeration and gas exchange, not being completely efficient in either function (Calil, 2007). Furthermore, it was also observed that hatchability decreased when eggs were stored for more than 9 days. It is known that the duration of the egg storage period has an inverse correlation with hatchability due to greater embryonic mortality. This occurs because, during storage, even at a cooling temperature where cell development is practically at a standstill, there is cellular metabolism occurring, even if very slowly. and these cellular modifications of the egg cannot be recovered (Boleli, 2003).

The single-stage incubation program is based on the idea that the embryo must develop under constant temperature conditions, without any fluctuations, which may have been positive in inducing gene expression of characteristics such as breast and heart weight. Since, some studies have demonstrated that temperature fluctuations for these characteristics were harmful (Leksrisompong et al., 2007; Oviedo-Rondón et al., 2009).

Late mortality (18 to 21 days) and “dead pecked” eggs were significantly higher in multi-stage incubators. This corroborates French (2010), who states that the increase in the birth window present in this incubator, due to the incubation of eggs originating from matrices of different ages, leads to damage in the maturation of the heart and lungs in embryos that are not well developed and, if the chick is hatching, it may suffer from hypoxia leading to an increase in dead pecked eggs. This occurs due to the increased supply of CO₂ in the final stage to stimulate hatchability.

Initial mortality is generally related to handling errors prior to incubation, such as thermal shock, since these eggs are stored at a cooling temperature and are then incubated at much higher temperatures. In the single-stage incubator, gradual preheating is carried out from 6 to 12 minutes to avoid thermal shock for these embryos (Elibol et al., 2002b), but for eggs from young breeders, which are smaller and therefore more sensitive to thermal shock (Fiuza et al., 2006), perhaps this management alone is not enough, since eggs from 30-week-old breeders that were incubated in the single-stage machine in the present study also had high averages of initial mortality. However, for multi-stage incubation, it was observed that initial embryonic mortality was higher for eggs from 60-week-old breeders compared to those from 30-week-old breeders.

In addition to the age of the breeder, egg storage can also alter other components of the egg, as verified by Epicurious (How…, 2024), a shrinkage of the cuticle and a decrease in its thickness, resulting from drying and chemical modification, culminating in an increase in the number of cracks on the cuticle surface. This explains the result of the present study, where, for eggs from 30-week-old breeders, incubated in a multi-stage incubator, the number of cracked eggs increased only after 9 days of storage, since this egg has a denser shell thickness.

CONCLUSION

The results of this study highlight the importance of the age of breeders, the storage time of eggs, and the type of incubator on chick quality and hatching rates. Eggs from young breeders show greater tolerance to storage compared to those from older breeders. The age of the breeder is identified as the primary factor negatively influencing incubation parameters, especially when eggs are incubated in multi-stage incubators. The choice of incubator, particularly between single-stage and multi-stage systems, plays a crucial role, emphasizing the need to carefully manage these variables to optimize embryonic development and ensure chick viability.

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FUNDING

This research received no external funding.

DATA AVAILABILITY STATEMENT

The research data are available upon request.

Corresponding author:

[e-mail]

Editor-chefe:

Marcelo Resende de Souza

Editor-científico:

Antônio de Pinho Marques Jr.

Publication Dates

• Publication in this collection
  28 Sept 2026
• Date of issue
  2026

History

• Received
  20 Oct 2025
• Accepted
  04 Apr 2026