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Article

The Impact of Eggshell Thickness on the Qualitative Characteristics of Stored Eggs Produced by Three Breeds of Laying Hens of the Cage and Cage-Free Housed Systems

by
Vjaceslavs Kocetkovs
1,*,
Vitalijs Radenkovs
2,3,*,
Karina Juhnevica-Radenkova
2,
Dmitrijs Jakovlevs
3 and
Sandra Muizniece-Brasava
1
1
Faculty of Food Technology, Latvia University of Life Sciences and Technologies, LV-3004 Jelgava, Latvia
2
Processing and Biochemistry Department, Institute of Horticulture, LV-3701 Dobele, Latvia
3
Division of Smart Technologies, Research Laboratory of Biotechnology, Latvia University of Life Sciences and Technologies, LV-3004 Jelgava, Latvia
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2022, 12(22), 11539; https://doi.org/10.3390/app122211539
Submission received: 26 October 2022 / Revised: 8 November 2022 / Accepted: 11 November 2022 / Published: 14 November 2022
(This article belongs to the Special Issue Biochemical Composition of Food)
Browse Figures
Review Reports Versions Notes

Abstract

:
The study aimed to compare the physical-chemical attributes of table eggs from three laying hen breeds housed in the cage and cage-free conditions and to characterize the morphological characteristics of the eggshell interior. A morphological and elemental analysis performed by scanning electron microscope coupled with energy dispersive X-ray spectroscopy revealed no abnormalities in the structural integrity of eggshells. The thickness of the eggshell varied in the range from 356.2 to 366.4 µm, with no statistically significant differences between the values. Eggshell membrane thickness was between 20.0 and 59.9 µm, with eggs derived from cage-housed hens, i.e., H/LS/CCE and H/HN/CCE having thinner membrane layers. The results revealed no direct relationships between eggshell and membrane thickness and physical-chemical parameters’ change. However, the presence of thick and long spider-like microcracks on the eggshell surface of eggs from cage-free housed hens H/D/BWE was the main factor that presumably contributed to substantial weight loss during 36 days of egg storage. A noticeable decline in eggshell-breaking strength along with the enlargement of air cells was observed in eggs produced under an enriched cage system H/LS/CCE after 28 days. In contrast, the minor changes in air cell size occurred in eggs from cage-free housed laying hens H/D/BWE. Protein quality indicators such as albumen height and Haugh units were well correlated with each other, and the intensity of their changes during egg storage, to a greater extent, was found to be storage time-dependent. No significant depletion of egg albumen was revealed during the first 15 days of egg storage. According to the United States Department of Agriculture, the quality corresponded to grade A (reasonably firm). However, after 18 days of storage, Haugh unit values were lower than 60, corresponding to grade B (weak and watery). The most apparent reduction in the Haugh unit was observed in eggs produced by enriched cage H/HN/CCE and cage-free H/D/BWE hens. The egg quality was storage time-dependent, and their deterioration rate was primarily associated with the genetic background of laying hens and housing conditions.

1. Introduction

Due to their high nutritional value, hen eggs are one complete commodity, and their market share is steadily increasing. Over the past ten years, burgeoning demand for eggs resulted in impressive global production growth [1]. According to FAOSTAT 2022, the world production of eggs increased by 29.2%, from 67.0 million metric tons in 2012 to 86.6 million in 2020 [2]. In industrialized countries, roughly 30% of eggs are processed at breaker plants that produce liquid egg products and a relatively large number of solid eggshell waste [3]. While most eggs are sold through the retail channel, equating to about 65% of the total sale, the remaining 5% represents a marriage sorted out at the initial processing stage due to non-compliance with the quality standards.
Scientific evidence reveals that eggs are an excellent source of essential amino acids and provide such groups of bioactive compounds as micro and macroelements and vitamins that represent significant importance for human health and functionality [4,5,6,7]. Moreover, readily digested and assimilated nutrients make eggs essential in human nutrition [7]. On the other hand, the availability of fatty acids, especially representatives of unsaturated ones, along with high moisture content and water activity, make eggs highly perishable [8,9]. According to Commission Regulation (EC) No 589/2008, the shelf-life of eggs at an ambient temperature and relative air humidity of 50% is defined as 28 days after the laying date [10]. During this time, albumen as a freshness indicator undergoes substantial depletion, making grade A eggs with reasonably firm albumen move to grade B with watery albumen. In addition, irreversible changes in protein quality contribute to the increase in amino acid and fatty acid susceptibility to oxidation processes and loss of antioxidant and antimicrobial activities [8,11]. Therefore, eggs of such quality are no longer suitable for fresh consumption. However, they can further be utilized as a raw material for producing liquid whole egg products, taking into account the issue of microbiological safety [12].
The loss of moisture and CO2 through the eggshell pores and microcracks is the main factor leading to pH rise and, eventually, egg quality deterioration [13]. A change in pH triggers a cascade of reactions, such as degradation of the ovomucin-lysozyme complex, liquefaction of albumen, and weakening of the vitelline membrane [14]. Meanwhile, the changes in eggs’ physical and chemical features are more intensive in the case of the thinner eggshell layer [15,16]. This statement was further reinforced by Veldsman et al. [17] and Ahmed et al. [3], indicating the importance of eggshell thickness and structural integrity as a physical barrier to preserve bird eggs from moisture loss and protect them from contamination by pathogenic microorganisms. The nutritional value of eggs and structural integrity of the eggshell are determined by bird age and genotype [18]. The importance of a balanced diet for laying hens has been highlighted by Stefanello et al. [19] and Qiu et al. [20], indicating the relevance of mineral sources used in the diet of laying hens derived from inorganic compounds such as carbonates, phosphates, oxides, and sulfates in the formation of eggshells [19,20]. The presence of an adequate amount of trace minerals in the diet makes the eggshell structure more rigid and robust enough to prevent failure during packing and transportation. Along with nutrition, the matter of poultry housing conditions is highlighted by Islam et al. [21], revealing that laying hens face high-stress circumstances during conventional housing, resulting in substantially higher cholesterol levels and weakening the eggshell.
The storage of eggs in air-conditioned rooms at relatively low temperatures and appropriate humidity delays the ageing processes of eggs and maintains their quality [22]. This statement is consistent with the observation made by Eke [13], revealing that the process of moisture evaporation can be delayed by refrigerated storage. However, ensuring such conditions in practice seems not economically feasible, particularly when transporting eggs over long distances to countries with warm climates. The limited information on the factors that, apart from storage conditions, could facilitate egg ageing during storage [16,17,23,24] promoted the design of this study, focusing on the evaluation of the eggshell thickness produced by laying hens under conventional and cage-free housed systems. It is hypothesized that the presence of a denser and thicker eggshell membrane and the eggshell itself could contribute to the delay of water movement across the shell and prevent the dehydration of the egg interior components.
Following the hypothesis, the study aimed to investigate eggshells’ morphology and structural features derived from hens of three laying breeds and draw a line between their thickness and physical-chemical properties’ change during 36 days of storage.

2. Materials and Methods

2.1. Screening of Physical Attributes of Hen Eggs

Fresh eggs were obtained from the company JSC “Balticovo” (Iecava, Latvia) from three laying hen breeds: Lohmann Sandy (42 weeks old, enriched cage housing), H&N Coral (41 weeks old, enriched cage housing), H&N Coral (41 weeks old, cage-free housing), and Dekalb (43 weeks old, cage-free housing). To evaluate the egg physical-chemical properties’ change as a function of storage time, 180 eggs from each hen breed were collected within 4 h of being laid. Harvested eggs were packed individually per 16 pc in cardboard packaging with a perforated lid. The total number of harvested eggs amounted to 720 eggs. At the initial stage of storage, 16 eggs from each group were taken and immediately delivered to the laboratory of JSC “Balticovo” for the screening of quality attributes. At the same time, the remaining part of the eggs was placed in a humidity and temperature-controlled storage room for subsequent storage. The eggs were stored under controlled conditions at 20 °C with 50% relative air humidity. The length of the experiment was 36 days. To elucidate the changes in quality indicators, eggs’ physical-chemical properties were analyzed once every four days, i.e., 1, 4, 8, etc., (except for 8–11, 11–15, and 15–18 days) during 36 days of storage. In the successive days of storage, 16 eggs per breed were taken to ensure an average sample.
At selection, eggs were weighed and cracked onto a flat surface. The heights of the inner thick albumen and Haugh unit were measured with an electronic albumen height digital Haugh tester ORKA (Bountiful, UT, USA) as proposed by Eisen et al. [25]. The pH of the albumen was measured immediately after laying with a pH-meter Jenway 3510 Benchtop pH Meter (Barloworld Scientific, Staffordshire, UK) according to ISO 1842:1991. The shell thickness was measured using a micrometer Mitutoyo 223–101 (Kawasaki, Japan). Shell strength was determined utilizing the FUTURA Egg-Shell-Tester V. 2.0 (Lohne, Germany) tester equipped with an aluminum compression disc 7.62 cm in diameter attached to the unit and an egg holder. The egg was placed so that the disc contacted the eggs from the large end of the egg. Dry matter was analyzed using a Shimadzu MOC–120H (Shimadzu Corporation, Tokyo, Japan) moisture analyzer.
The trials were conducted in a commercial layer house JSC “Balticovo” in 2022. The diets and drinking water were offered ad libitum. The ingredients and chemical composition of the basal diet are presented in Table 1. Diets were in mash form. Laying hens were housed in layer cages, the size of which were L: 2.4 × W: 0.6 × H: 0.5 m (0.14 m2 per hen), and in cage-free conditions.

2.2. Scanning Electron Microscope (SEM) and Energy Dispersive Spectroscopy (EDX)

The eggshell specimens were taken using a Mira3 scanning electron microscope by Tescan Orsay Holding, a.s. (Brno, Czech Republic). The eggshell specimens (n = 10 eggs × 5 replicates) were washed with ultra-high purity water and then dried under ambient conditions at 20 ± 1 °C for two days. The dried eggshells were manually broken into small pieces of 0.4 × 0.4 cm, mounted onto SEM pin stubs using double-sided adhesive carbon discs, and coated with a gold-palladium 15 nm thick layer using Leica EM ACE600 vacuum sputter coater (Leica Microsystems, Wien, Austria). The eggshell and membrane thickness measurements were completed at three regions of the egg, i.e., the large end, equator, and small end. The conditions were adjusted to operate under high vacuum mode utilizing the Secondary Electron (SE) and Back-scattered Electron (BSE) detectors. The morphology of the eggshell was examined, raising magnification up to 2000× for a detailed dimension measurement and elemental composition analysis operating at 15 kV acceleration voltage. An elemental composition analysis has been conducted employing INCA x-act LN2-free Analytical Silicon Drift Detector with PentaFET® Precision energy-dispersive X-ray spectrometer by Oxford Instruments Inc. (Abingdon, United Kingdom). A 15 mm working distance with dead time from 40 to 60% was adjusted.

2.3. Statistical Analysis

The data analysis was performed using arithmetical values and standard deviations. Microsoft Excel v16 software was used. The impact of factors and their interaction, the significance effect (p-value), was examined with a two-way analysis of variance (ANOVA).

3. Results and Discussion

3.1. Morphological and Elemental Analysis of Eggshell Components by Scanning Electron Microscopy and Energy Dispersive Spectroscopy

It has been documented that the composition of mineralized eggshells forms roughly 96% of calcium carbonate (calcite). In comparison, the remaining 4% represents such minor components as magnesium, phosphorus, and various trace elements located mainly in the organic matrix [26]. From the outside direction to the inside, the eggshell comprises histological layers such as outer and inner shell membranes, mammillary layer, palisade, and cuticle layers (Figure 1A–C). The eggshell membrane represents significant importance since it provides the shell foundation and prevents microbial invasion [27]. The eggshell membrane is located between the albumen fraction and eggshell, and attached to the latter with its organic-rich rounded surface structures and mammillary knobs [28]. The eggshell membrane is represented as a fibrous network consisting of collagen type I, dermatan sulfate, chondroitin sulfate, and glucosamine [29]. The discovery of the eggshell membrane as a source of multiple health-promoting compounds makes it practical and safe for treating pain and stiffness associated with knee osteoarthritis [30].
The presence of a high vacuum, though, in the SEM system used to prevent electrical discharge in the gun assembly and to allow the electrons to travel within the instrument unimpeded caused a slight detachment of the eggshell membrane from the eggshell. However, we successfully measured the eggshell and membrane thickness. As seen, the thickness of the eggshell varied in the range from 356.2 to 366.4 µm, with the eggshell derived from eggs of cage hens H/LS/CCE having a thinner layer, and the eggshell derived from eggs of cage-free hens H/D/BWE having a thicker layer (Table 2). It is worth noting, though, no statistically significant differences (p ≥ 0.05) were observed between the eggshell thicknesses of four eggs. The observed values are consistent with Sales et al. [27], reporting the range of chicken eggshell thickness from 240 to 370 µm. The authors also indicated that the eggshell thickness of domestic hen eggs was not uniform over the whole egg surface, i.e., thickness at the poles was thicker than at the equator. Therefore, its thickness must be measured within the large end, equator, and small end of the egg. However, Fecheyr-Lippens et al. [31] reported a nearly similar thickness of the chicken eggshell layer. In turn, statistically significant differences (p ≤ 0.05) were observed between the eggshell membrane thickness, which was found in the range from 20.0 to 59.9 µm. Similarly, as in the case of eggshell thickness, cage eggs produced from H/LS/CCE hens demonstrated a tangibly thinner layer of eggshell membrane, while cage-free eggs derived from H/D/BWE hens had a thicker membrane. Thinner eggshell membranes observed in caged hens are most likely associated with the deficiency of minerals, especially Cu. This element is reported to act as a promoter and component of crucially essential enzymes involved in the formation of the eggshell membrane, such as Cu-containing amine oxidase [20].
The relevance of the palisade layer thickness and the organization of calcite crystals have been highlighted by Stefanello et al. [19], as these factors strongly contribute to the strength of the eggshell. However, as seen (Figure 1A–C), the calcite columns in all four eggs had a wedge-shaped structure, and no significant difference was observed between the samples. Furthermore, on a more detailed microscopic examination of eggshell specimens, no prominent large and uniform type B mammillary knobs were seen, confirming the lack of endometrial tissue damages of those documented to take direct involvement in the inhibition of the processes of ion transmission and the crystallization of eggshell formation [32].
The egg’s integrity and physical appearance make the first representation to the consumer. If the product does not meet perceived expectations, consumer confidence de-creases [33]. The quality of the eggshell is essential to the processing industries because structurally, whole eggs will arrive at the consumer in the best condition. It has been reported that eggs injured during routine handling and transport to retail outlets cause major financial losses to the egg industry [34]. Therefore, according to visual inspection techniques, defective eggs that demonstrate even minor signs of cracking are sorted out already at the initial processing stage. Unfortunately, the ovoscope, as a frequently used instrument for assessing the quality of eggs, has certain limitations in detecting microcracks, and occasionally defective eggs reach retailers and eventually consumers. In particular, the abundance of long and thick cracks on the outer eggshell surface is reported to promote egg quality deterioration and endanger consumer health due to a risk of microbial cross-contamination, especially when eggs are stored at room temperature [35,36].
For a more detailed examination of eggshell microcracks, backscattered electrons (BSEs) imaging accomplished by the SEM system was utilized, allowing phase separation between dense and thin surface regions, thereby identifying amorphic regions. BSE imaging revealed obvious microcracks on the outer surface of the eggshell, as illustrated in Figure 2A–C.
The availability of thick and long microcracks was observed on the surface of the eggshell of eggs derived from enriched cage-housed hens H/LS/CCE, followed by cage-free housed hens H/D/BWE. The thickness values corresponded to an average of 1.41 and 1.48 µm, respectively. In both cases, spider-like cracking was seen on the surface of the eggshells. The least number of cracks, as well as their thickness, were found on the surface of the eggshell of eggs derived from cage-free H/HN/BCE and cage H/HN/CCE hens, corresponding to 0.67 and 0.91 µm, respectively. On the eggshell surface of eggs obtained from cage hens H/HN/CCE, obvious hairline cracks could be seen. Interestingly, Khabisi et al. [37] highlighted the presence of hairline cracks on the surface of the eggshells that had promoted the most substantial weight loss and decrease in hatchability compared to star-cracked eggs. It is worth noting that from the total number of eggs investigated (n = 50), the eggs derived from cage-free hens of H/HN/BCE represented no apparent signs of eggshell surface cracking.
The EDS mapping analyses of the entire eggshell from four egg samples revealed almost similar profiles and composition of elements (data not represented). In general, six base elements, i.e., Na, Mg, P, S, Ca, and O, were detected in the eggshell (Figure 3). As indicated, an intensive signal corresponding to the S element was deposited predominantly on the membrane layer (on average, 0.94% per weight). The availability of S was due to the presence of sulfur-containing amino acids in the collagen-rich fraction, such as cystine (9–10%) and methionine (3–4%) [38].
During the elemental mapping analysis, the presence of P was revealed (on average, 1.50% per weight), mainly in the form of calcium phosphate (Ca3(PO4)2), as reported by Li et al. [39]. This statement can be reinforced by the same uniform distribution pattern of Ca and O elements as P throughout mammillary and palisade layers of the eggshell. It is worth noting that during the EDS mapping analysis, the presence of P with high signal intensity was also observed in the outer part of the eggshell. Meanwhile, the same intense deposition of Mg near the inner and outer surfaces of the eggshell (on average, 0.92% per weight) was revealed. The presence of Mg in the form of magnesium carbonate (MgCO3) salt was reported by Borhade and Kale [40]. This observation can be supported by the porous nature of the eggshell and the outstanding solubility of magnesium salts and phosphates rather than calcium salts by the alcohol mixtures and aqueous acid [41]. The levels of O, C, and Ca elements were the highest among all the elements, corresponding to 48.3, 30.1, and 19.8% distribution by weight, respectively. The availability of C (carbon) as the principal element observed along with Ca and O is explained by the double-sided adhesive carbon disc applied for the fixation of eggshell specimens. Previous studies with an EDS mapping analysis of eggshell interior revealed the availability of calcium carbonate (CaCO3), which makes up 97% of the total amount of elements [42].

3.2. Changes in Physical-Chemical Attributes of Non-Processed Eggs during Storage under Ambient Conditions

An initial evaluation of the main quality attributes of eggs uncovered statistically significant (p ≤ 0.05) differences in selected criteria values (except for pH value) during 36 days of shelf-life under an ambient temperature of 20 ± 1 °C and relative air humidity of 50%. The most remarkable change in the physical factors of the eggs during the storage time appeared to be egg weight (Figure 4). A 4.1% decrease in egg weight was observed after 11 days of storage compared to the initial value of each egg sample. The results are consistent with Khan et al. [43], indicating a reasonably similar percentage reduction after 11 days of egg storage at 21 °C. Weight loss after 28 days of storage was found to be in the range of 5.3 to 12.2%, which is fairly higher than that reported by Jones et al. [44] for unwashed eggs kept for four weeks under 22 °C temperature. It is worth noting that the most significant weight reduction was observed for eggs produced by cage-free housed laying hens H/D/BWE, with the thickness of their eggshell highlighted in the previous experiment as the thickest. In turn, the eggs of H/LS/CCE and H/HN/BCE produced under the enriched cage and cage-free housing systems demonstrated the most negligible weight loss. As part of the subsequent storage for 36 days, the weight loss ranged from 7.7 to 13.3%. As with 28 days of storage, eggs of H/D/BWE showed remarkable weight loss, and H/LS/CCE and H/HN/BCE were the lowest.
Egg weight loss during storage is associated primarily with the loss of water and CO2 through the eggshell and eggshell membrane. Furthermore, it is worth noting that the observation made by Grashorn [45] highlights a direct relationship between the thickness of eggshells and weight loss. Unfortunately, though, no such interconnection was supported by the results of this study, indicating possibly other mechanisms of water movement from the eggs to the surrounding environment. This statement can be reinforced by Rocculi et al. [46]. The authors indicated that the elongation of air cells could increase moisture loss in eggshells during storage, making it easier for vapors to escape from the eggs.
Indeed, the remarkable enlargement of air cells was noticed already after 11 days of egg storage, corresponding to an increase within the range from 18.7 to 53.8%, with eggs from cage-housed hens H/HN/CCE having the most negligible increase, and the eggs derived from cage-housed hens H/LS/CCE the highest (Figure 5). It is worth noting that the observed values did not exceed the maximum value of 6.0 mm, implying that the quality of eggs still complies with the regulations defined by the Commission Regulation (EC) No 589/2008 and belongs to class A [10].
However, after 28 days of egg storage, the size of air cells was exponentially enlarged by an average of 81.5% (for all tested eggs), and the observed mean values corresponded to 5.8 mm, respectively. Meanwhile, an additional 17 days of egg storage did not lead to exceeding a critical value of 6.0 mm. However, further storage of eggs negatively affected the enlargement of air cells, reaching an average value of 8.1 mm on day 36. Considering this observation solely, the maximum shelf-life of the selected eggs could be defined as 28 days, which is also in line with EC regulations No 589/2008 [10]. No apparent relationship between the weight loss and air cells can be seen after 11 or 28 days of egg storage.
It is believed that the increase in air cell space and moisture and CO2 loss promote the weakening of the eggshell structure and disrupt its integrity. Indeed, an increase in the space between the outer and inner membranes leads to the loss of eggshell strength. However, the results of this experiment revealed ambiguous data regarding this criterion, indicating a relative fluctuation in eggshell-breaking strength as a function of storage time (Figure 6).
For instance, on the 11th day of storage of eggs only, a 2.5% reduction in the eggshell-breaking strength was observed in eggs derived from cage-free housed laying hens H/HN/BCE, while an up to 20% increase was revealed in eggs produced by cage-free housed hens H/D/BWE. The most apparent reduction in the eggshell strength was observed in eggs produced under an enriched cage system H/LS/CCE after 28 days of storage, corresponding to a 13.5% decrease (42.4 N). The observed value, however, does not differ significantly (p ≥ 0.05) from that indicated by Zita et al. [47]. After 36 days of storage, minor changes took place in the structure of eggs obtained from cage-free housed laying hens H/D/BWE, as only a 5.6% increase in their strength was observed compared with the initial value. It was expected that egg shrinkage would harm eggshell integrity, though data obtained confirmed no direct relationship between weight loss and breaking strength values after 11 or 28 days of egg storage.
The albumen height was the main indicator of egg quality to be considered critically during the evaluation, since a direct relationship between albumen height and egg freshness was repeatedly documented [48,49]. Furthermore, the pH shift from acidic to alkaline caused by water and CO2 loss led to albumen liquefaction and viscosity decline [8]. The results of this study revealed a gradual decline in albumen height over 36 storage days (Figure 7). As shown, the reduction in albumin height after 11 days of egg storage varied from 18.6 to 35.5%, with eggs from cage-free housed laying hens H/HN/BCE having the lowest reduction and eggs from cage-free eggs housed hens H/D/BWE the highest. However, the values found still complied with the criteria framed in the EC regulation. The observed initial albumen height values are slightly lower than those reported by [9,14,48], perhaps due to the difference in breeds of laying hens. The most significant reduction in albumen height was found in eggs from enriched cage-housed hens H/HN/CCE, while no significant differences were revealed for the other three laying hens.
The most notable reduction in albumen height was uncovered during the following storage of eggs for an extra 17 days, reaching a reduction of 48.6% of the initial height. It is worth noting that the percentage decrease is consistent with those noted by Drabik et al. [50], indicating a reduction of 45.4% of the initial albumen height for eggs stored for 28 days under 21 °C temperature.
To sufficiently characterize the freshness of the stored eggs, the Haugh unit was calculated following the methodology proposed by Raymond Haugh in 1937 as one of the crucial markers next to the eggshell thickness and eggshell strength [51]. At the start of egg storage, the Haugh unit approximated 68.3%, and the observed value was in line with data indicated by Grashorn [45] (Figure 8).
However, analogous to albumen height, the value of the Haugh units was greatly influenced by storage time, particularly during the first 14 days of storage. On the 11th day of egg storage, the percentage reduction corresponded to 20.3%. A reasonably similar decrease in Haugh units was reported by Xu et al. [36], revealing a decrease of 24.9% after 11 days of untreated egg storage under 25 °C temperature. Up to the 15th day of storage, the eggs corresponded to grade A as proposed by the United States Department of Agriculture (USDA) [52]. A further decrease in Haugh unit values was also observed; however, it was less apparent than at the initial storage stage. After 22 days of storage, the percentage decline reached 30.7% in comparison with the initial value. The most apparent decline in the Haugh unit was observed in eggs produced by enriched cage H/HN/CCE and cage-free H/D/BWE laying hens. Further storage for up to 36 days revealed the most noticeable Haugh unit loss after 28 days of storage, which corresponded to a 36.9% loss. The observed Haugh unit’s reduction was less pronounced than that reported by Drabik et al. [50], identifying almost a 50% loss from the initial Haugh unit value. As expected, the values of Haugh units were well correlated with albumen height; however, no direct relationship was confirmed between protein quality and thickness of the eggshell and its membrane.
Based on the data presented in the work of Cotterill et al. [53], it is apparent that after laying eggs, a significant part of CO2 has already been lost, which leads to a substantial rise in the albumen pH. The rise of albumen pH is considered a natural process since it ensures the high antimicrobial activity of albumen against certain pathogenic bacteria [54]. However, with the progression in pH rise, the enzymes destabilizing the ovomucin-lysozyme complex and degradation of clusterin and ovalbumin become more active. An increase in their activity leads to solubilization and deterioration in the gelling properties of albumen [8]. As reported by Rizzi, the most intense liquefication of albumen was observed at pH 9.0 [55].
It was observed that the pH of albumen on the 1st day of egg storage varied in the range from 8.9 to 9.3, with the albumen derived from eggs of cage-free housed hens H/NH/BCE having the lowest value and albumen derived from eggs of cage-free hens H/D/BWE the highest (Figure 9). The availability of thick microcracks was confirmed in a previous experiment with the SEM analysis for eggshells derived from H/D/BWE eggs, which may be the leading cause of extensive CO2 loss.
The change in albumen pH, similar to other quality features of eggs, was affected by storage time and, to a lesser extent, thickness of the eggshell and membrane. The most noticeable increase in the albumen pH was observed after 11 days of egg storage, corresponding to an average 2.3% rise from the initial value. At this stage, the albumen had undergone perhaps the most substantial depletion. The highest value of albumen pH was observed in eggs produced by enriched cage H/HN/CCE and cage-free H/HN/BCE hens, which was also in line with losses of albumen height and Haugh unit values. As part of the subsequent storage, the pH of albumen fluctuated for some eggs: higher for some, and lower for others. Furthermore, the nonlinear increase in albumen pH as a function of storage time was also highlighted by Drabik et al. [56]. Relative fluctuations in the pH values confirmed the loss of CO2 and equilibrium of the carbonate-bicarbonate buffer system that perhaps pushed toward the repeated production of CO2 and pH decrease [57].

4. Conclusions

A structural analysis of eggshells performed by scanning electron microscope coupled with energy dispersive X-ray spectroscopy revealed no substantial deviations in the morphology of eggshells. The thickness of the eggshell and membrane varied in the range from 356.2 to 366.4 µm and from 20.0 to 59.9 µm, respectively. No statistically significant difference was observed between the thickness of the eggshells tested. Furthermore, the eggs derived from cage-housed laying hens demonstrated substantially thinner layers of eggshell membranes. The lack of essential micronutrients such as Cu in the diet of hens under cage-housed conditions is a possible cause of poor egg membrane development. From the total number of eggs investigated (n = 50), the eggs derived from cage-free hens of H/HN/BCE represented no apparent signs of eggshell surface cracking, which likely contributed to the preservation of water and CO2 loss during 36 days of storage. The most significant changes in the quality of eggs occurred after 28 days of storage, especially with such parameters as weight and breaking strength. Eggs obtained from cage-free housed hens H/D/BWE showed the most significant weight loss, and eggs from the cage and cage-free housed hens H/LS/CCE and H/HN/BCE the lowest, respectively. It was expected that egg shrinkage would harm eggshell integrity. However, data obtained revealed no direct relationship between weight loss and breaking strength values after 11 or 28 days of egg storage. It is hypothesized that the main factor contributing to the weakening of eggshell strength was the availability of long and thick spider-like microcracks on the eggshell surface of H/LS/CCE eggs. Protein quality indicators such as albumen height and Haugh units were well correlated with each other, and the intensity of their reduction during egg storage to a greater extent was found to be storage time-dependent. No substantial depletion of egg albumen was observed during the first 15 days of egg storage. The quality of eggs still complied with the regulations outlined by the United States Department of Agriculture quality standards and belonged to grade A (reasonably firm). However, after 18 days of storage, the albumen had undergone the most substantial depletion since Haugh unit values dropped below 60, making eggs enter the B grade (weak and watery). The most apparent reduction in the Haugh unit was revealed in eggs produced by enriched cage H/HN/CCE and cage-free H/D/BWE housed hens, and these values were well correlated with albumen pH values. The EDS mapping analyses of the entire eggshell from four egg samples revealed almost similar profiles and composition of elements (data not represented). In general, six base elements, i.e., Na, Mg, P, S, Ca, and O, were detected in the eggshell.
Overall, the quality of eggs was found to be storage time-dependent, and the rate of their deterioration was primarily associated with the genetic background of the laying hens and housing conditions rather than with the structure of the eggshell.

Author Contributions

Conceptualization, V.K. and V.R.; data curation, V.K. and V.R.; formal analysis, V.R., V.K. and D.J.; investigation, V.R., V.K. and D.J.; methodology, V.R., V.K., D.J. and S.M.-B.; resources, V.K.; software, V.R.; visualization, V.K., V.R. and D.J.; writing—original draft, V.K. and V.R.; writing—review and editing, V.K., V.R. and K.J.-R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets and analysis of the study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Applsci 12 11539 g001 550
Figure 1. Radial or cross-sectional scanning electron microscopy micrographs of rigid laying hen eggshells showing tightly abutting shell units. The fibrous shell membrane has pulled away from the calcareous layer. Red arrows indicate: ShMb_O—outer shell membrane; ShMb_I—inner shell membrane; Cp—cuticle pore; PsL—palisade layer; MaL—mammillary layer; CuL—cuticle layer. Note: SEM micrographs were taken at the initial stage of storage (0 days). (A)—eggs from cage-free housed laying hens (H/HN/BCE); (B)—eggs from cage-free housed laying hens (H/D/BWE); (C)—eggs from enriched cage-housed laying hens (H/LS/CCE); (D)—eggs from enriched cage-housed laying hens (H/HN/CCE).
Figure 1. Radial or cross-sectional scanning electron microscopy micrographs of rigid laying hen eggshells showing tightly abutting shell units. The fibrous shell membrane has pulled away from the calcareous layer. Red arrows indicate: ShMb_O—outer shell membrane; ShMb_I—inner shell membrane; Cp—cuticle pore; PsL—palisade layer; MaL—mammillary layer; CuL—cuticle layer. Note: SEM micrographs were taken at the initial stage of storage (0 days). (A)—eggs from cage-free housed laying hens (H/HN/BCE); (B)—eggs from cage-free housed laying hens (H/D/BWE); (C)—eggs from enriched cage-housed laying hens (H/LS/CCE); (D)—eggs from enriched cage-housed laying hens (H/HN/CCE).
Applsci 12 11539 g001
Applsci 12 11539 g002 550
Figure 2. The external surface of the eggshell of laying hens captured by scanning electron microscope showing longitudinal hairline and spider-like cracks (indicated by the yellow L letter with width values). Note: Scanning electron micrographs were taken at the initial stage of storage (0 days). (A)—eggs from cage-free housed laying hens (H/HN/BCE); (B)—eggs from cage-free housed laying hens (H/D/BWE); (C)—eggs from enriched cage-housed laying hens (H/LS/CCE); (D)—eggs from enriched cage-housed laying hens (H/HN/CCE).
Figure 2. The external surface of the eggshell of laying hens captured by scanning electron microscope showing longitudinal hairline and spider-like cracks (indicated by the yellow L letter with width values). Note: Scanning electron micrographs were taken at the initial stage of storage (0 days). (A)—eggs from cage-free housed laying hens (H/HN/BCE); (B)—eggs from cage-free housed laying hens (H/D/BWE); (C)—eggs from enriched cage-housed laying hens (H/LS/CCE); (D)—eggs from enriched cage-housed laying hens (H/HN/CCE).
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Figure 3. Elemental mapping analysis (A) and energy dispersive spectroscopy (B) of the eggshell derived from eggs of DeKalb laying hens aged 42 weeks and housed under cage-free conditions. Note: S—sulfur; P—phosphorous; Mg—magnesium; Ca—calcium; C—carbon; O—oxygen.
Figure 3. Elemental mapping analysis (A) and energy dispersive spectroscopy (B) of the eggshell derived from eggs of DeKalb laying hens aged 42 weeks and housed under cage-free conditions. Note: S—sulfur; P—phosphorous; Mg—magnesium; Ca—calcium; C—carbon; O—oxygen.
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Figure 4. Changes in egg weight (g) during 36 days of storage. Note: H/LS/CCE—eggs from enriched cage-housed laying hens; H/HN/CCE—eggs from enriched cage-housed laying hens; H/HN/BCE—eggs from cage-free housed laying hens; H/D/BWE—eggs from cage-free housed laying hens. Means within the same day of storage with different small letters (a, b) are significantly different at p < 0.05, while means within the same hen breed with different capital letters (A, B, C) are significantly different at p < 0.05.
Figure 4. Changes in egg weight (g) during 36 days of storage. Note: H/LS/CCE—eggs from enriched cage-housed laying hens; H/HN/CCE—eggs from enriched cage-housed laying hens; H/HN/BCE—eggs from cage-free housed laying hens; H/D/BWE—eggs from cage-free housed laying hens. Means within the same day of storage with different small letters (a, b) are significantly different at p < 0.05, while means within the same hen breed with different capital letters (A, B, C) are significantly different at p < 0.05.
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Applsci 12 11539 g005 550
Figure 5. Changes in egg air cell size (mm) during 36 days of egg storage. Note: H/LS/CCE—eggs from enriched cage-housed laying hens; H/HN/CCE—eggs from enriched cage-housed laying hens; H/HN/BCE—eggs from cage-free housed laying hens; H/D/BWE—eggs from cage-free housed laying hens. Means within the same day of storage with different small letters (a, b) are significantly different at p < 0.05, while means within the same hen breed with different capital letters (A–D) are significantly different at p < 0.05.
Figure 5. Changes in egg air cell size (mm) during 36 days of egg storage. Note: H/LS/CCE—eggs from enriched cage-housed laying hens; H/HN/CCE—eggs from enriched cage-housed laying hens; H/HN/BCE—eggs from cage-free housed laying hens; H/D/BWE—eggs from cage-free housed laying hens. Means within the same day of storage with different small letters (a, b) are significantly different at p < 0.05, while means within the same hen breed with different capital letters (A–D) are significantly different at p < 0.05.
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Figure 6. Changes in eggshell-breaking strength (N) during 36 days of egg storage. Note: H/LS/CCE—eggs from enriched cage-housed laying hens; H/HN/CCE—eggs from enriched cage-housed laying hens; H/HN/BCE—eggs from cage-free housed laying hens; H/D/BWE—eggs from cage-free housed laying hens. Means within the same day of storage with different small letters (a, b, c) are significantly different at p < 0.05, while means within the same hen breed with different capital letters (A–D) are significantly different at p < 0.05.
Figure 6. Changes in eggshell-breaking strength (N) during 36 days of egg storage. Note: H/LS/CCE—eggs from enriched cage-housed laying hens; H/HN/CCE—eggs from enriched cage-housed laying hens; H/HN/BCE—eggs from cage-free housed laying hens; H/D/BWE—eggs from cage-free housed laying hens. Means within the same day of storage with different small letters (a, b, c) are significantly different at p < 0.05, while means within the same hen breed with different capital letters (A–D) are significantly different at p < 0.05.
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Applsci 12 11539 g007 550
Figure 7. Changes in albumen height (mm) during 36 days of egg storage. Note: H/LS/CCE—eggs from enriched cage-housed laying hens; H/HN/CCE—eggs from enriched cage-housed laying hens; H/HN/BCE—eggs from cage-free housed laying hens; H/D/BWE—eggs from cage-free housed laying hens. Means within the same day of storage with different small letters (a, b) are significantly different at p < 0.05, while means within the same hen breed with different capital letters (A–D) are significantly different at p < 0.05.
Figure 7. Changes in albumen height (mm) during 36 days of egg storage. Note: H/LS/CCE—eggs from enriched cage-housed laying hens; H/HN/CCE—eggs from enriched cage-housed laying hens; H/HN/BCE—eggs from cage-free housed laying hens; H/D/BWE—eggs from cage-free housed laying hens. Means within the same day of storage with different small letters (a, b) are significantly different at p < 0.05, while means within the same hen breed with different capital letters (A–D) are significantly different at p < 0.05.
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Figure 8. Changes in Haugh units during 36 days of egg storage. Note: H/LS/CCE—eggs from enriched cage-housed laying hens; H/HN/CCE—eggs from enriched cage-housed laying hens; H/HN/BCE—eggs from cage-free housed laying hens; H/D/BWE—eggs from cage-free housed laying hens. Means within the same day of storage with different small letters (a, b) are significantly different at p < 0.05, while means within the same hen breed with different capital letters (A–F) are significantly different at p < 0.05.
Figure 8. Changes in Haugh units during 36 days of egg storage. Note: H/LS/CCE—eggs from enriched cage-housed laying hens; H/HN/CCE—eggs from enriched cage-housed laying hens; H/HN/BCE—eggs from cage-free housed laying hens; H/D/BWE—eggs from cage-free housed laying hens. Means within the same day of storage with different small letters (a, b) are significantly different at p < 0.05, while means within the same hen breed with different capital letters (A–F) are significantly different at p < 0.05.
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Figure 9. Changes in albumen pH during 36 days of egg storage. Note: H/LS/CCE—eggs from enriched cage-housed laying hens; H/HN/CCE—eggs from enriched cage-housed laying hens; H/HN/BCE—eggs from cage-free housed laying hens; H/D/BWE—eggs from cage-free housed laying hens. The lack of small and capital letters on the plot indicates no statistically significant variations in the pH values between storage days and hen breed.
Figure 9. Changes in albumen pH during 36 days of egg storage. Note: H/LS/CCE—eggs from enriched cage-housed laying hens; H/HN/CCE—eggs from enriched cage-housed laying hens; H/HN/BCE—eggs from cage-free housed laying hens; H/D/BWE—eggs from cage-free housed laying hens. The lack of small and capital letters on the plot indicates no statistically significant variations in the pH values between storage days and hen breed.
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Table
Table 1. Composition of feed base formula used for feeding laying hens.
Table 1. Composition of feed base formula used for feeding laying hens.
IngredientH/LS/CCE, H/HN/CCE
H/HN/BCE		H/D/BWE
IngredientContent, %
Corn11.0015.00
Wheat47.0036.00
Triticale0.004.00
Barley4.0014.00
Soybean press cake12.977.96
Sunflower seeds10.809.40
Oat2.001.80
Sunflower/Rape oil1.501.00
Other composition/premix10.739.65
Note: H/LS/CCE—Lohmann Sandy (hens aged 42 weeks, enriched cage housing, cream eggs); H/HN/CCE—H@N Corol (hens aged 41 weeks, enriched cage housing, cream eggs); H/HN/BCE—H@N Corol (hens aged 41 weeks, cage-free housing, cream eggs); and H/D/BWE—DeKalb (hens aged 43 weeks, cage-free housing, cream eggs).
Table
Table 2. Mean size values of eggshell and membrane thickness and surface microcracks captured by scanning electron microscopy at 0 days of eggs’ storage produced by laying hens of various origins and housing systems.
Table 2. Mean size values of eggshell and membrane thickness and surface microcracks captured by scanning electron microscopy at 0 days of eggs’ storage produced by laying hens of various origins and housing systems.
Kuts
H/HN/BCE 		Kieku
H/D/BWE		 Litegg
H/LS/CCE		 Madona H/HN/CCE
ESSMBWMCESSMBWMCESSMBWMCESSMBWMC
Average size, µm362.6 a53.2 b0.67 c366.4 a59.9 a1.48 a356.2 a20.0 c1.41 a362.4 a21.4 c0.91 b
STDEV22.95.30.2217.78.50.1419.64.20.1811.55.30.10
Note: Values are means ± SD values of n = 50. Means within the same attribute with different superscript letters (a, b, c) are significantly different at p ≤ 0.05. SD—standard deviation; ES—size of eggshell; SMB—size of eggshell membrane; WMC—width of surface microcracks.
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Kocetkovs, V.; Radenkovs, V.; Juhnevica-Radenkova, K.; Jakovlevs, D.; Muizniece-Brasava, S. The Impact of Eggshell Thickness on the Qualitative Characteristics of Stored Eggs Produced by Three Breeds of Laying Hens of the Cage and Cage-Free Housed Systems. Appl. Sci. 2022, 12, 11539. https://doi.org/10.3390/app122211539

AMA Style

Kocetkovs V, Radenkovs V, Juhnevica-Radenkova K, Jakovlevs D, Muizniece-Brasava S. The Impact of Eggshell Thickness on the Qualitative Characteristics of Stored Eggs Produced by Three Breeds of Laying Hens of the Cage and Cage-Free Housed Systems. Applied Sciences. 2022; 12(22):11539. https://doi.org/10.3390/app122211539

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Kocetkovs, Vjaceslavs, Vitalijs Radenkovs, Karina Juhnevica-Radenkova, Dmitrijs Jakovlevs, and Sandra Muizniece-Brasava. 2022. "The Impact of Eggshell Thickness on the Qualitative Characteristics of Stored Eggs Produced by Three Breeds of Laying Hens of the Cage and Cage-Free Housed Systems" Applied Sciences 12, no. 22: 11539. https://doi.org/10.3390/app122211539

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