Quantitative computed tomography of the eggshell of laying hens reared in cage and free-range systemsRESUMO
O objetivo deste estudo foi obter e comparar dados físicos e tomográficos de cascas de ovos de poedeiras com 60 semanas de idade, criadas em gaiolas comerciais (n=60) e no sistema free-range (n=60). As análises foram realizadas por tomografia computadorizada quantitativa (QCT), e as comparações entre grupos utilizaram o teste t de Student (P<0,05). Ovos de galinhas criadas no sistema free-range apresentaram maior peso do ovo (P≤0,001), maior peso da casca (P≤0,001) e maior espessura física da casca (P≤0,001) em comparação com os de galinhas em gaiolas. A espessura tomográfica das cascas também foi maior nos ovos do sistema free-range (P≤0,001). Contudo, a densidade da casca, determinada pela análise de TC, foi significativamente menor (P≤0,001) no grupo free-range. Foi observada uma correlação moderada entre o peso da casca e a densidade tomográfica nos ovos do sistema free-range. Este estudo utilizou a QCT para analisar os valores densitométricos da casca de ovos de galinhas poedeiras, sugerindo que essa técnica pode ser uma ferramenta útil na avaliação da qualidade da casca dos ovos.
Palavras-chave:
qualidade do ovo; sistemas de criação; tomografia computadorizada; produção avícola
Keywords:
egg quality; housing systems; computed tomography; poultry production
Palavras-chave:
qualidade do ovo; sistemas de criação; tomografia computadorizada; produção avícola
Keywords:
egg quality; housing systems; computed tomography; poultry production
Palavras-chave:
qualidade do ovo; sistemas de criação; tomografia computadorizada; produção avícola
The eggshell comprises 8-11% of the egg’s total weight and is primarily composed of calcium carbonate. It serves as a vital barrier against physical damage and microbial contamination (Clerici et al., 2006; Ketta and Tumová, 2016; López Vargas et al., 2024). Therefore, shell quality is essential from a commercial standpoint, as eggs with dirty, fragile, cracked, or misshapen shells are unsuitable for sale (Clerici et al., 2006). Among the various factors influencing shell quality, the housing system of laying hens has been shown to play a significant role in determining the physical properties of the shell (Clerici et al., 2006; Lichovnikova and Zeman, 2008; Vlcková et al., 2018). While cage housing remains a traditional practice, it has increasingly come under scrutiny for its impact on animal welfare. In contrast, the free-range egg production system allows birds access to outdoor areas, typically letting them roam during the day and sheltering them at night. This enables hens to express natural behaviors, directly influencing their welfare (Clerici et al., 2006; Vlcková et al, 2018).
Given the influence of rearing systems on eggshell characteristics, the use of advanced, non-invasive methods for assessing shell quality becomes increasingly relevant (Kawamura, et al., 2023, López Vargas et al., 2024). Quantitative computed tomography (QCT) is an imaging technique that enables highly sensitive and precise measurement of bone mineral density, representing a non-invasive method for estimating the mineral content of bone structures (Gramp et al., 1997). Few studies have applied CT to avian eggs (Milisits et al., 2013; Winkens et al., 2022; López Vargas et al., 2024), with investigations focusing on yolk and eggshell analysis. To our knowledge, there is only one CT-based study that has assessed the eggshells of laying hens (López Vargas et al., 2024). This study offers a novel contribution by not only comparing eggshell quality from hens raised in cage and free-range systems, but also by providing a more refined and detailed application of QCT for assessing eggshell thickness, delivering new insights into its potential as a precise, non-destructive tool for poultry research and industry.
A total of 120 eggs were analyzed, comprising 60 eggs from commercially caged hens and 60 from free-range hens. All hens, 60 weeks old, were Lohmann White and Lohmann Brown strains. Each strain received a specific diet formulated by the supplier to meet official nutritional guidelines, but detailed quantitative formulations were not provided by the egg supplier. Eggs were acquired from a poultry company in Igarassu City, Pernambuco. Eggs were collected at 8:00 a.m. and immediately sent for further processing and analysis. At the time of collection, the ambient temperature was 29.5 °C and the relative humidity was 78%. Computed tomography scans were performed using a Helical CT Scanner (GE High SpeedFXi, Waukesha, Wisconsin, USA), previously calibrated. Image acquisition involved 2 mm thick transverse slices with 1 mm spacing, pitch 0.8, 120 kV, and automatic mA. A hard reconstruction filter algorithm was used, with a bone window setting. Calibration phantoms for bone and water were placed ventrally to the eggs and scanned simultaneously (Figures 1A and 1B).
The CT images were quantitatively analyzed using commercial DICOM viewer software (Osirix-64-bit, version 7.0). Radiodensity of the shell was measured at three regions of interest (ROIs): one on the right side, one on the left side, and one ventrally (Figure 1C). Average radiodensity values were obtained in Hounsfield units (HU) and corrected using the calibration phantom data (Figure 1D). A density range of 100-1500 HU was applied for segmentation, and 3D reconstruction of the ROI was performed after segmenting in all slices.
After that, the eggs were sent for analysis to the Meat Laboratory of the Department of Animal Science at UFRPE, where they were weighed individually on a precision scale with a variation of 0.01g (Bel, model L 3102i). The eggshells were then washed to remove any remaining albumen and air-dried for 48 hours before weighed. Shell thickness was measured at three anatomical regions: apical (pointed end), equatorial (mid-region), and basal (blunt end) using a digital micrometer (Mitutoyo ID-61012XBS). The mean of the three measurements was used as the final thickness value for each egg.
Statistical analyses were performed using IBM SPSS Statistics, version 25. The normality of the variables was assessed using the Shapiro-Wilk test. Descriptive statistics included mean and standard deviation. Comparisons between groups were made using the Student’s t-test for independent samples. Pearson’s correlation test was used to evaluate linear associations between variables. A significance level of p < 0.05 was adopted.
The physical and tomographic characteristics of the variables analyzed for the two rearing systems are shown in Table 1. Overall, eggs from free-range hens exhibited higher egg weight, shell weight, and both physical and tomographic thickness, but lower shell density in the CT analysis. Table 2 presents the correlation test results between variables. A moderate correlation was observed between shell weight and tomographic density in the free-range group, while other correlations were weak or very weak.
Mean and standard deviation of egg weight, shell weight, shell thickness, tomographic thickness, and shell density of laying hens reared in cage and free-range systems
Pearson’s linear correlation between direct measurements and CT measurements of eggshells from laying hens reared in cage and free-range systems
Variations in eggshell quality among commercial layers may be influenced by several factors, such as genotype, bird age, oviposition timing, housing system, dietary calcium and phosphorus, among others (Ketta and Tumová, 2016). Studies have demonstrated differences in shell quality depending on housing systems, although results have been inconsistent. Pisteková et al. (2006) observed heavier shells in eggs from hens housed in cages compared to deep-litter systems. Another study found heavier shells in birds raised in unenriched cages compared to enriched cages and cage-free systems (Lichovnikova and Zeman, 2008). In contrast, Tumova et al. (2011) reported heavier shells in eggs from cage-free hens compared to enriched cage systems. Our findings using direct physical measurements showed that eggs from free-range hens had higher egg and shell weights and greater shell thickness than those from caged hens, consistent with Tumova et al. (2011). However, it is important to note that eggshell quality is also influenced by hen genotype. Brown-egg layers typically produce eggs with thicker and more resistant shells compared to white-egg layers (Roberts, 2004).
Proper eggshell quality is essential for the poultry industry. The shell serves as a physical barrier that prevents the entry of microorganisms into the egg, which is critical because contaminated eggs can be a source of zoonotic disease (Clerici et al., 2006; Ketta and Tumová, 2016; López Vargas et al., 2024). Additionally, good shell quality reduces economic losses, as broken or malformed eggs cannot be commercialized. Various techniques, both direct and indirect, have been developed to evaluate eggshell quality (Clerici et al., 2006; Ketta and Tumová, 2016).
Recent imaging studies in poultry science (in-ovo imaging) have been conducted to assess both the shell and internal components of the egg. These methods offer the major advantage of not requiring egg destruction (López Vargas et al., 2024). Eggshell strength is closely linked to its mineral composition-approximately 94% calcium carbonate, 1% magnesium carbonate, 1% calcium phosphate, and 4% organic material. Higher mineral density in osseous and mineralized structures is associated with greater resistance to fracture, while demineralized bones are more prone to breakage. Similarly, eggshells with lower mineral content are more fragile (Clerici et al., 2006). QCT provides a precise means to evaluate this density and may serve as a quality indicator. QCT is a validated technique for assessing bone mineral density in humans, animals, and biomaterials (Gramp et al., 1997). Its potential application to egg analysis has gained attention in recent studies (Milisits et al., 2013).
Winkens et al. (2022) used CT imaging to study ostrich embryo development and measured eggshell radiodensity, reporting 1828±100 HU in fertilized and 1836±101 HU in unfertilized eggs. However, they did not use a QCT phantom, which improves accuracy. Although there is a linear correlation between CT radiodensity and Hounsfield Units, parameters such as kVp, mA, reconstruction algorithm, and slice thickness may affect attenuation values and compromise measurement accuracy. Recent research used 3D computed tomography image analysis for non-destructive approach for estimating morphometric measurements of chicken eggs, however, densitometric analyses were not performed (López Vargas et al., 2024).
It is important to highlight that the hens used in each rearing system belonged to different genetic strains (Lohmann White and Lohmann Brown), which may influence eggshell characteristics such as weight and thickness. Therefore, the absence of a factorial experimental design (strain × rearing system) limits the ability to attribute the observed differences solely to the housing system. Future studies should consider factorial approaches to isolate and better understand the interaction effects between genetic background and rearing conditions.
This study is one of the few using quantitative computed tomography (QCT) to evaluate eggshell features in laying hens, offering a non-destructive, sensitive, and consistent method for measuring shell mineral density and thickness. The results showed notable differences between rearing systems, with free-range hens laying eggs with thicker shells both physically and tomographically, but with lower shell density. These findings indicate that QCT can be a useful tool for research and commercial purposes, especially in quality control and breeding programs aimed at enhancing eggshell strength. For the poultry industry, adopting QCT-based monitoring could help reduce economic losses caused by shell defects and improve food safety by maintaining shell quality. Future research should employ factorial designs to distinguish between genetic and environmental effects, including mechanical resistance testing, and investigate how dietary, and housing changes affect eggshell mineralization. Overall, this research advances the integration of advanced imaging techniques into poultry production, aiding precise nutritional and management strategies.
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ETHICAL ASPECTS
The research was submitted to the Ethics Committee on Animal Use of the Federal Rural University of Pernambuco, and approved under the number 8680290224.
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DATA AVAILABILITY STATEMENT
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
A) Sectional images show clear distinction of shell and air chamber. Calibration phantoms appear below the eggs (arrows). B) ROI selection methodology for bone and water phantoms. C) Method for selecting three regions of interest for density analysis. D) Methodology for measuring shell thickness at corresponding ROI locations.