Karolina Wengerska,Justyna Batkowska,Kamil Drabik
The eggshell defect as a factor affecting the egg quality after storage
egg storage,wrinkled eggshell,pimpled eggshell,severe stripe marks,cracked eggshell
The aim of the study was to assess the influence of shell defects on the quality of eggs after storage. The study material consisted of 1,800 brown-shelled eggs from cage rearing system which were candled on the day of laying to determine the shell quality. Eggs with the 6 most common shell defects (external crack, severe stripe marks, points, wrinkled, pimples, sandy) and eggs without defects (control group) were then stored for 35 days at 14°C and 70% humidity. The weight loss of eggs was monitored every 7 days, and the quality characteristics of whole eggs (weight, specific gravity, shape), shell (defects, strength, color, weight, thickness, density), albumen (weight, height, pH) and yolk (weight, color, pH) of 30 eggs from each group were analysed at the beginning (0 days) and after 28 and 35 days of storage. The changes resulting from water loss (air cell depth, weight loss, shell permeability) were also evaluated. The study showed that all investigated shell defects significantly influenced the characteristics of the whole egg during the storage, modifying traits such as specific gravity, water loss, shell permeability, albumen height and pH, as well as proportion, index and pH of the yolk. Furthermore, an interaction between time and the shell defects presence was found.
The eggshell defect as a factor affecting the egg quality after storage

The aim of the study was to assess the influence of shell defects on the quality of eggs after storage. The study material consisted of 1,800 brown-shelled eggs from cage rearing system which were candled on the day of laying to determine the shell quality. Eggs with the 6 most common shell defects (external crack, severe stripe marks, points, wrinkled, pimples, sandy) and eggs without defects (control group) were then stored for 35 days at 14°C and 70% humidity. The weight loss of eggs was monitored every 7 days, and the quality characteristics of whole eggs (weight, specific gravity, shape), shell (defects, strength, color, weight, thickness, density), albumen (weight, height, pH) and yolk (weight, color, pH) of 30 eggs from each group were analysed at the beginning (0 days) and after 28 and 35 days of storage. The changes resulting from water loss (air cell depth, weight loss, shell permeability) were also evaluated. The study showed that all investigated shell defects significantly influenced the characteristics of the whole egg during the storage, modifying traits such as specific gravity, water loss, shell permeability, albumen height and pH, as well as proportion, index and pH of the yolk. Furthermore, an interaction between time and the shell defects presence was found.

Eggs are considered one of the most valuable and lowest-cost animal products. Their richness in nutrients together with their high digestibility and accessibility have made them one of the primary animal-origin commodities in the human diet (Chung et al., 2018). The quality of table eggs is influenced by several factors such as the age of the flock, the birds' genotype, their rearing system (Sokołowicz et al., 2018), the season of the year (Simeon et al., 2018) or the length of the storage period (Tomczyk et al., 2019) The storage of table eggs in the EU is regulated by Commission Regulation (EC), (2008) No 589/2008. An important element described in the regulation is the minimum shelf life of 28 days and the quality parameters to be met by Class A eggs. These are dictated by the fact that their quality and technological suitability deteriorate with the time of storage. The shell is the inorganic part of the bird's egg with a complex and highly organised structure. Due to the presence of pores on its surface, it allows for a gas exchange between the inside of the egg and the environment. Although eggshell thickness, shape, size or porosity vary between species, the basic structure of the shell is similar in all birds (Board and Love, 1983). In the case of table eggs, the eggshell is an important factor. It not only protects the contents of the eggs but is also responsible for their marketing. Cracked and broken eggs cause economic losses at all stages of the production and distribution cycle, which can reach up to 8% to 11% of total egg production (Hamilton & Bryden, 2021). Therefore the high quality of the shell is one of the crucial elements which egg producers pay attention to. On the other hand, shell colour and appearance is a basic element, after price, influencing consumers' choice of eggs (Rondoni et al., 2020). Consumer preferences concerning shell colour vary from country to country, i.e., in Italy, the United Kingdom, Portugal and Ireland, brown-shelled eggs are the most common choice (Aygun, 2014), while in the United States, white-shelled eggs are preferred (Heng et al., 2013). This preference may be due to consumers' greater knowledge of certain types of eggs, as well as their availability on the market (Rondoni et al., 2020). The term 'shell defects' encompasses the presence of any type of abnormality resulting from the distribution of calcium carbonate in the shell (Hunton, 1995). These are noticeable both on candling of eggs and on regular inspection. The most common shell defects include external and internal cracks, pimpels, rough, corrugated and chalky shells, as well as marbling, dots, marks or the presence of so-called weak ends. Despite an incompletely understood aetiology, shell defects occur commonly in both hatching and table eggs of all poultry species. Genetic (Ball et al., 1974; Zhang et al., 2021) or environmental factors such as inadequate nutrition (Ball et al., 1973; Stefanello et al., 2014), flock diseases and stress (Cheng and Ning, 2022) are considered to be the main causes that may influence their occurrence. Despite the widespread occurrence of shell defects, the available knowledge on their impact on the quality of stored eggs is marginal, so the aim of this study was to analyse the impact of the presence of shell defects on the quality of table eggs during 35-day storage.

1,800 brown shelled table eggs from cage system were taken as experimental material. Eggs were obtained on the same day from one hen stock kept in the same building. On the laying day, the eggs were candled to verify the presence of shell defects as follows: external cracks, internal strong cracks, internal weak cracks, severe stripe marks, points, chalky, wrinkled, marbled, weak ends, pimples, and sandy. Experimental groups were established according to 6 of the most frequent defects. Then the intact eggs were treated as a control group. Then all eggs were stored for 35 days at the temperature of 14°C and humidity of 70%. Every 7 days the egg weight loss was verified, at the beginning (0 days) and after 28 and 35 days of storage the quality traits of 30 eggs per group were analysed. The proportions of particular eggshell defects and schema of the experiment are shown in Table 1. The analysis was performed using an EQM (Egg Quality Measurement, TSS, York, UK) and the Instron Mini 55 apparatus (Instron, Norwood, MA, USA). The following egg traits were evaluated: 1 Whole egg: • Weight (EW) - electronic scale with an accuracy of 0.01 g; • Specific gravity (SG) - measurement of the egg weight both in the air and submerged in water, according to Archimede's principle; • Proportions of morphological elements (ESP - eggshell, AP - albumen, YP - yolk) in the egg weight. 2 Eggshell • Shape index (SI) -a ratio of its width and length, using electronic caliper; • Strength (SS) - force required to break the shell (N), using an Instron Mini 55 device (Instron, Norwood, MA, USA); • Color (SC) - as a percentage of reflected light; • Weight (SW) - electronic scale with an accuracy of 0.01 g; • Thickness (ST) - measured at the equator using a micrometer screw; • Density (SD) - calculated based on shell area and volume, according to Shafey (2002). 3 Egg albumen • Weight (AW) - from the difference in mass of whole egg, shell and yolk; • Height (AH) - measured after the egg has been broken onto the mirror table, by contact of the EQM sensor with the surface of the dense albumen; • Haugh units (HU) - (Williams, 1992) • pH (ApH) - by pH-meter with a combined glass electrode (Elmetron, Zabrze, Poland). 4 Egg yolk • Weight (YW) - electronic balance with an accuracy of 0.01 g; • Colour (YC) - using colorimeter according to 16-point scale (YolkFanTM, DSM Nutritional Products,®); • Index (YI) - measured using an electronic caliper, estimated as a ratio of the yolk height to its diameter; • pH (YpH) - by pH-meter with a combined glass electrode (Elmetron, Zabrze, Poland). 5 Changes resulted from water loss • Air cell depth (ACD) - visually using the template; • Weight loss (WL) – as percent, based on difference between measurements in different terms; • Shell permeability (SP) (Ar et al., 1974).- The data collected during the experiment were statistically processed using SPSS 24.0 package (Armonk, NY, USA) (IBM Corp., 2016) using multifactorial analysis of variance with Tukey's multiple comparisons tests. The level of P ≤ 0.05 was taken as significant. Two factors were included in the model: time of storage (T) and defects (D), as well as the interactions between them (T × D). The exact probability of tests was given. The results were shown as the mean and standard error of the mean (SEM). Additionally, the Spearman correlation coefficients were estimated between eggshell attributes and selected traits that change during egg storage. They were analysed for all eggs as well as by particular shell defects.

Table 2 shows the characteristics of eggshells according to the shell defect present. Eggs with external cracks were characterised by the lowest shell strength. The lightest shell colour was observed in eggs with a points, while the darkest colour was in eggs with severe stripe marks. There were no significant differences between the other defects, but the highest shell strength was observed in eggs with a points and the lowest value of this trait in wrinkled shell eggs. However, no significant variation was observed between chosen defects and traits such as shape index, weight thickness and shell strength. Table 3 presents the characteristics of the whole egg and their changes during the storage period. Significant differences in egg weight were observed at the beginning of the experiment and after 28 days. In both cases, eggs without defects were the lightest. Eggs with wrinkled shells were heaviest on the first-day of the experiment, while eggs with severe stripe marks and sandy shells were heaviest after 28 days of storage. Variation in the eggs' specific gravity was observed across all analyses, but eggs with external cracks always had the lowest value of this trait. After 35 days of storage, eggs without defects and those with points had the highest specific gravity. The lowest yolk proportion at the laying day characterised intact eggs, which differed from those in which defects were observed. After 28 days of storage, no significant differences were observed between eggs with or without shell defects. The situation changed after 35 days of storage, where the lowest albumen proportion was observed in eggs from control group and those with severe stripe marks, with the highest values of this trait observed in wrinkled shell eggs. Changes in the albumen proportion were observed in both terms, after 28 and 35 days. On the 28th day of storage, eggs without defects contained significantly less albumen than those with defects. In contrast, on the 35th day, wrinkled shell eggs had considerably the lowest albumen proportion in the total egg weight. Significant differences in shell proportion were only observed after 28 days, intact eggs had higher shell proportion than eggs with shell defects. Time was the factor influencing all whole egg traits (P<0.001), while the presence of defects, did not affect only the shell percentage. The interaction of time and defect had a significant effect on specific gravity (P=0.002) and the shell proportion in egg weight (P<0.001). The changes occurring due to water loss and diffusion are shown in Table 4. After 28 days of storage, the shallowest air cell was observed in eggs without shell defects (control group) and those with severe stripe marks and points, while on the 35th storage day, it was observed in eggs without shell defects as well as those with points. The deepest air cell after both at 28 and 35 days was observed in eggs with external cracks and wrinkled shells (P≤0.05). As this change is closely related to weight loss, identical relationships were found in respect of this feature. At the same time, it should be noted that at the end of the experiment, the smallest weight loss was recorded for intact eggs and those with severe stripe marks and points. The highest percentage of weight loss in 28 days was observed in eggs with external cracks. After 28 storage shell, permeability was also highest in eggs with external cracks, while no significant differences were observed between eggs with other defects. After 35 days of storage, eggs with external cracks had the highest shell permeability, while eggs without shell defects, with severe stripe marks and points had the lowest values of this trait. Changes in albumen quality traits are shown in Table 5. Considerably lower albumen weight at the beginning of the experiment as well as after 28 days of storage was observed in eggs without defects. After 35 days no statistical differences were observed between the groups. At the beginning of the experiment, the height of albumen in the intact eggs was significantly higher compared to eggs with defects. However, this relationship was not observed at the end of the experiment, where eggs with wrinkled shells had the highest value of this trait, while eggs with external cracks had the lowest one. Haugh units were significantly higher in eggs without defects at the beginning of the experiment, but after 35 days of storage, the highest value of this trait was observed in wrinkled shell eggs. Initially, the albumen pH of eggs from the control group was significantly lower than that of eggs with defects, but after 35 days the most favourable values of this parameter were observed in external cracks eggs. After 28 days of storage, no significant differences were observed between groups in traits such as albumen height, pH and Haugh units. The presence of defects significantly affected albumen weight (P<0.001), while time considerably influenced all analysed albumen characteristics (P<0.001). The interaction of time with defect (D×T) significantly changed only the pH of the albumen (P<0.001). Changes in quality during storage also concern the yolk (Table 6). At the beginning of the experiment, intact eggs were characterised by the lowest yolk weight, while the highest value of this trait was observed in wrinkled shell eggs. Nevertheless, after both 28 and 35 days of storage, no significant differences in yolk weight were observed. Yolk colour was also analysed and showed the lowest values on the 1st day of the experiment for eggs from the control group (without defects), which differed significantly from those with shell defects. During the storage, the yolk colour changed and showed significant variation according to the presence and type of shell defect. The lightest yolk was characteristic of eggs without defects, while the darkest one of those with external cracks. Interestingly, these changes were visible after 28 days of storage, but after 35 days no significant differences were observed. The yolk index also changed during storage, yolks from eggs with wrinkled shells differed considerably from the others, showing the highest value of this trait. There was no variation in this trait among the other groups. It should be noted that the presence of shell defects had a significant effect on yolk quality parameters during egg storage (P<0.001). Time was also not insignificant in this respect, influencing considerably all yolk characteristics. A significant interaction effect of both study factors (time x shell defect) was found in case of yolk colour as well as its pH (P<0.001). In addition, Spearman correlation coefficients were determined for individual shell defects and traits that change with the storage time (Table 7). Importantly, for some of the traits analysed, there was a variation not only in the levels of significance of the correlation coefficients but also in their direction depending on the type of defect studied. In the case of the eggs analysis, regardless of the type of defect, there was a highly significant negative correlation of egg-specific gravity with weight loss during the storage period, air cell depth and changes in albumen and yolk pH. At the same time, specific gravity was positively correlated with shell characteristics (SS and ST) and Haugh units and yolk index. Shell quality parameters i.e., strength and thickness showed a strong negative correlation with egg weight loss and air cell deepening. Weight loss during the storage period showed a strong correlation with air cell depth (negative) and yolk index and pH. Correlations between albumen (Haugh units, pH) and yolk parameters (yolk index, pH) also showed highly significant relationships (P<0.01). It is worth noting that when analysing each defect separately, some of the correlations do not occur in all cases, which may suggest the influence of the defect on changes in the particular parameters analysed. To mention just one example, the effect of shell thickness on yolk pH changes was observed only in the case of eggs affected by eggshell with points. Moreover, in the case of pimples, wrinkled and external cracks, a significant negative correlation was found between weight loss during the storage period and changes in egg albumen pH. Overall, the control group eggs showed significantly less significant correlations between the characteristics analysed than the defective shell eggs.

The quality of egg shells, and thus the presence of defects on their surface, is determined by several factors related to the genetic line of the birds, their housing system, the nutrition applied or the health and physiological state of birds (Jones and Musgrove, 2005). In addition to the obvious impact of cracks affecting the subsequent distribution stages of the obtained eggs, other defects such as shape abnormalities, the presence of pimples or the weakening of particular parts of the shell are also possible. There are many causes of this phenomenon, but regardless of the aetiology, they represent a problem in the collection, packaging and subsequent distribution of the raw material (Mazzuco and Bertechini, 2014). Reduction of the eggshell strength is an undesirable phenomenon, generating, as a result of damage, serious losses for the industry, which can reach up to 11% of the total egg production (Fathi et al., 2019). Our own research in this area showed that only the presence of cracks had a significant impact on the reduction of shell strength, while the other defects did not contribute considerably to the value of these parameters. The necessity to eliminate eggs with damaged shells has driven the development of several methods for detecting defects even at the collecting or packing stage. Among the most important are acoustic methods, based on the difference in sound waves at the site of shell cracks, the image of which in the computer recording differs from eggs without cracks (So et al., 2022) and methods based on infrared (Narushin et al., 2004) or on a pressure imaging system (Yoon et al., 2012). In this context, the remaining defects are of marginal importance, which does not mean that they do not contribute to some extent to changes in the table eggs' quality. Depending on the type of defect presence, significant differences in shell colour were also found. However, it should be noted that this measurement was made using the refraction method. Although previous studies have shown a significant impact of the shell colour on its quality and the characteristics of the egg content (Drabik et al. 2020, 2021), eggs differing in the presence of shell pigments were analysed in this regard. In the case of our study, the difference was only in brown colour saturation, so shell pigments were not strictly a source of variation. Due to the refractive method used, a logical observation is the decrease in the reflectance value observed when there is a local increase in shell permeability (external crack, severe stripe marks, points). For other defects that interfere directly with the outer layer of the shell, the colour was likely influenced by the cuticle layer. The study by Samiullah et al. (2017) seems to confirm this supposition, as eggshells lacking the mucin layer showed a different colour compared to the control group (with cuticle). In the context of the quality of table eggs, it should be mentioned that, in addition to factors related to the birds rearing system, they are also affected by environmental factors and, above all, the time passed since laying day. It was also found that with the laying flock age, changes in shell structure and the formation of shell defects occur (Banaszewska et al., 2019). Regardless of the type of defect analysed, a significant effect of time was found in the change of egg weight, its specific gravity or the proportion of particular elements in the whole egg weight. Weight loss and specific gravity are among the most frequently observed and described changes occurring in eggs during the storage period (Jones et al., 2018; Brodacki et al., 2019). These processes are related to the evaporation of water from the egg contents through the shell pores (Kralik et al., 2014) and, in the case of our study, also through cracks and defects present in the shell. For the other defects, the explanation is not so obvious. It seems that the variation may be related to shell thickness, as reported by Liu et al. (2017), who found that eggs with pimples had a lower shell thickness and weight than eggs with normal shells. The reduction in shell thickness may explain the higher values of weight loss during storage. The same conclusions were also reached by Veldsman et al. (2020), who modelled the effect of shell thickness on its permeability properties under various microclimate conditions. In terms of water loss and gas exchange, it should also be mentioned that they are closely related to the water permeability of eggshells. Although it is a natural phenomenon, it is related to both the storage conditions of the raw material and the characteristics of the shell itself. A study by Araújo et al. (2017) showed that shell quality contributes significantly to changes in the values of this parameter. The authors confirmed the influence of the age of the laying hen, as well as the region of the shell (blunt end, equator and sharp end) in shaping water conductivity. Moreover, these zones differ considerably in their porosity, which affects shell permeability. The authors showed a significant correlation between water conductivity and porosity, with similar observations not confirmed for shell thickness. These observations may partly explain the changes in egg quality we observed during storage despite the similar shell thickness parameters of the eggs included in our study, as some of the defects described do not directly affect shell thickness, but may affect shell permeability to water vapour. Changes in the egg weight and thus in the air cell depth are a natural process occurring during the ageing of raw egg material (Brodacki et al., 2019). The changes in the values of both these traits may depend on the housing system of the laying hens (Vlčková et al., 2019), but especially on the storage conditions, because both lowering and increasing the temperature or humidity can considerably affect the variation in the water loss rate (Akter et al., 2014). The above characteristics can also be influenced by any action that reduces water loss from the eggs through the shell pores, i.e., coating the shell with a layer of harmless substances, e.g. citric acid (Drabik et al., 2021), glycerine (Drabik et al., 2018) or rice protein concentrates (Pires et al., 2020a). Gavril and Usturoi (2011) showed that the percentage of weight loss can be as high as 8.95%, so the possible reduction of this process is important from a consumer point of view. In our study, the greatest egg weight loss was observed in eggs with cracked shells. This is a completely natural consequence of the ruptured shell integrity and therefore greater gas exchange. During egg storage, there is a dilution of the dense fraction of the albumen through the diffusion of water from the albumen into the yolk, as well as an increase in the pH of this element (Pires et al., 2022). The changes associated with the loss of water from the albumen lead to the Haugh units reduction (Jones & Musgrove, 2005). In our study, wrinkled eggs had the best albumen quality after 35 days of storage, despite a shell with medium permeability. This change, like the vast majority of the egg content changes, is closely related to the movement of water between the particular elements of the egg as well as the egg and the external environment. Despite some ability of the egg albumen to buffer the environment, with time this ability disappears resulting in a significant increase in the pH of this element (Drabik et al., 2021). Despite the aforementioned ability to maintain the albumen pH, biochemical changes occur over time, resulting in the release of carbon dioxide dissolved in the egg content in the form of carbonic acid. As noted by Vlčková et al. (2019), the quality of the shells in this respect is not without significance due to the regulation of gas exchange intensity. The change in pH implies further changes such as the loosening of the dense albumen structure or, indirectly due to the diffusion of water from the albumen into the yolk, also the characteristics of the yolk itself. The yolk also shows changes in quality with the time of egg storage. Both time and the presence of a shell defect were found to have a significant effect on the quality changes of this element. Normally, there is an increase in yolk mass by diffusion of water from the albumen (Monira et al., 2003), which was also observed in our study. An increase in yolk globule volume leads to a stretching of the vitelline membrane and a decrease in the yolk index (Marzec et al., 2019 ). Similarly to the case of albumen, there is also an increase in pH in the yolk (Liu et al., 2016). This parameter is closely related to changes in other egg quality traits, primarily albumen, due to the important role of water movement between egg components and the external environment. Haugh units and yolk index are indicators of changes in egg quality during storage, the correlation of which appears obvious (Pires et al., 2020b). A dilution of the dense albumen fraction results in a decrease in Haugh units (Menezes et al., 2012), while diffusion of water from the albumen increases the yolk mass and decreases the yolk index. In our study, a highly significant correlation between Haugh`s units and yolk index was observed for all eggs studied. A similar trend in Japanese quail eggs was shown by Kul and Seker (2004) and Alkan et al. (2010). However, a different conclusion was reached by Debnath and Ghosh (2015), in whose study the correlation between these traits was not significant. Eggshell thickness is one of the indirect indicators of egg quality, but its thickness varies at different points throughout the eggshell (Sun et al., 2012) In the case of Japanese quails, Alkan et al. (2010) observed a negatively significant correlation between shell thickness and Haugh units. For our study, a similar trend was observed only in eggs with points defect.

Our study showed that all investigated shell defects significantly influenced the characteristics of the whole egg during storage, modifying traits such as specific gravity, water loss, shell permeability, albumen height and pH, as well as proportion, index and pH of yolk. Furthermore, an interaction between time and the shell defects presence was found. Unfortunately, few reports deal with similar topics, although it seems reasonable to inspect eggs more carefully before storage to minimise the negative effects of defects and their interaction with storage time.