Eggshell Cuticle Research Articles

Investigating cold plasma jet effectiveness for eggshell surface decontamination

Ensuring the safety of eggs, a valuable source of high-quality protein and essential vitamins, is critical to prevent foodborne illness. Cold plasma, an emerging green technology, generates various reactive species that effectively inactivate microorganisms, attracting global attention for its potential in food safety. This study aims to explore the application of cold plasma as a chemical-free, non-thermal approach for decontaminating egg surfaces and to assess its potential as an alternative to conventional egg-washing methods. Various operation conditions of a cold plasma jet device, including power levels (300–400 W), exposure times (20–60 s), distances between the nozzle and eggshell (1–3 cm), airflow rates (30–35 L/min), and feed gases (nitrogen, air (20 %–65 % relative humidity), helium and air mixtures), were examined to decontaminate hen eggshells inoculated with Escherichia coli and Salmonella enterica bacteria. The results showed a maximum log reduction and deactivation efficiency of 1.94 and 98.74 % for E. coli, and 1.11 and 92.20 % for Salmonella, after a 60 s treatment of egg surface using a cold plasma device set at 1 cm distance, 400 W power, and 35 L/min airflow with 65 % relative humidity. Moreover, our findings indicated no significant differences in egg quality, eggshell cuticle chemical composition, and cuticle coverage between untreated eggs and those treated with cold plasma. This suggests the potential of this non-chemical, non-thermal emerging technique to be commercialized as a substitute for conventional washing methods.

Application of novel engineered water nanostructures techniques for eggshell surface decontamination

Hen eggs play a vital role in the human diet and their safety is critical to avoid the risk of foodborne illness for consumers. Engineered Water Nanostructures (EWNS) generated via an electrospray system is a novel and nanotechnology-based technique, which can be utilized for inactivating bacteria. The primary objective of this work was to assess this technique’s effectiveness as a chemical-free and non-thermal approach for decontaminating egg surfaces and investigate its potential as an alternative to the conventional method of washing eggs. The results showed that under EWNS operating conditions of 5 min of exposure time, 1 μL/min/needle of water flow rate, and 9.0 kV/cm of electric field strength, the highest inactivation efficiency of 97.6 and 80.4% were obtained for decontaminating egg surfaces contaminated by E. coli and Salmonella spp., respectively. Results also proved no significant difference in physical properties, chemical components, and eggshell cuticle coverage of EWNS-treated eggs compared to unwashed eggs.

Cold atmospheric pressure air plasma jet disinfection of table eggs: Inactivation of Salmonella enterica, cuticle integrity and egg quality

Eggshell cuticles are first lines of defense against egg-associated pathogens, such as Salmonella enterica serovar Enteritidis (SE). Infections from eggs contaminated with this strain remain a significant risk. In addition, changes in the cuticle are closely related to changes in egg safety. The emerging non-thermal atmospheric pressure plasma technology enables a high rate of microbial inactivation at near-ambient temperatures, making it ideal for food safety applications. This study examines the effects of a cold atmospheric pressure air plasma jet (CAAP-J) on eggshell cuticle and egg quality whilst inactivating SE. Shell eggs inoculated with SE (7 log10 cfu/egg) were used as the samples to test the decontamination performance of the device. The tests were conducted using an industrial CAAP-J with different power levels (600–800 W), exposure times (60–120 s), at a fixeddistance of 20 mm from the plasma jet and an air flow rate of 3600 L/h. It was found that the best results were obtained after 120 s at maximum plasma power (800 W). Subsequent to the implementation of this plasma procedure, it was determined that no viable cells could be detected. After CAAP-J treatment, the temperature remains below 50.5 °C, thereby minimizing the risk of altering egg quality. All specific measurements (egg white pH, yolk pH, yolk color, HU, and eggshell breaking strength) have shown that CAAP-J treatment has no negative effect on egg quality. No changes in eggshell cuticle quality after CAAP-J treatment was confirmed through scanning electron microscope (SEM).

Comparative N-Glycoproteomic Investigation of Eggshell Cuticle and Mineralized Layer Proteins.

The eggshell cuticle layer (ECL) and eggshell mineralized layer (EML) contain amounts of glycoproteins and proteoglycans. However, there were few comprehensive reports about the effect of post-translational modifications on protein structure and function which requires investigation. Therefore, we used comparative N-glycoproteomics to study glycoproteins in the ECL and EML. We identified a total of 272 glycoproteins in this experiment and found that glycoproteins located in EML were more than that in ECL. Moreover, they showed distinct functional difference between both layers. As N-glycosylation of ovocleidin-17 and ovocleidin-116 in the EML affected eggshell mineralization, some glycoproteins located in ECL, like ovotransferrin and ovostatin-like, possessed antibacterial activity. The several regulated glycoproteins in the EML may pertain to the regulation of mineralization, while glycosylated proteins in the ECL may contribute to molecular adhesion and defense against microbial invasion. This study provides new insights into the eggshell matrix protein contents of the ECL and EML.

New insights into the relationships between egg maternal components: the interplays between albumen steroid hormones, proteins and eggshell protoporphyrin

Recent studies have shown that the egg yolk maternal components, which are a mixture of substances that can affect the developing embryo, do not act separately but are interconnected and co-adapted. Surprisingly, no study to date has focused on the associations between maternally derived albumen steroids and albumen and eggshell compounds with pleiotropic effects. Eggshell pigment protoporphyrin (PROTO IX) should provide primary antimicrobial protection for eggs, but as a proven pro-oxidant, it may compromise female fitness. Abundant albumen proteins ovotransferrin (OVOTR) and lysozyme (LSM) have been shown to have antimicrobial, antioxidant, immunoregulatory and growth-regulatory roles. To investigate associations between albumen steroids and OVOTR, LSM and eggshell cuticle PROTO IX, we used chicken eggs with differently pigmented eggshells. We found that albumen steroid hormones were strongly intercorrelated. In addition, we revealed that albumen LSM and testosterone (T) were positively associated, while a negative association was found between albumen LSM and pregnenolone (P5). Eggshell cuticle PROTO IX was negatively associated with the concentration of albumen 17α-hydroxypregnenolone (17-OHP5). Finally, of all the hormones tested, only the concentration of albumen 17-OHP5 correlated negatively with egg volume and varied with eggshell colour and chicken breed. Although experimental evidence for the effect of maternal albumen steroids on avian developing embryo is still scarce, our study is the first to highlight co-variation and potential co-adjustment of maternally derived albumen steroids, proteins and eggshell cuticle pigment suggesting similar allocation mechanisms known for yolk maternal compounds with the potential to influence the avian embryo and offspring phenotype.

Examination of the impact of eggshell cuticle and membranes on Salmonella Enteritidis or Typhimurium recovery from inoculated and stored eggs

The effect of altering eggshell cuticle, as well as the impact of eggshell membranes and egg contents, on inoculated Salmonella recovery was examined. Four experiments were performed with Experiments 1 and 2 utilizing Salmonella Enteritidis and Experiments 3 and 4 utilizing both Salmonella Enteritidis and Typhimurium. Eggs were inoculated with 10 5 to 10 6 CFU of Salmonella after the following treatments were applied: mechanically stripped cuticle, chemically stripped cuticle, eggshells empty of egg contents, inoculation into the air cell, and unaltered positive control. Post-inoculation eggs were stored at room temperature (22 to 25°C) and eggshells were sampled at 1, 6, and 24 h for recovery of Salmonella . Mechanically and chemically stripping eggshell cuticle from this study did not impact Salmonella recovery compared to unaltered positive control eggs. Eggshells with contents removed had significantly higher recovery of Salmonella over 24 h storage, as well as eggs that were inoculated in the air cell (P≤0.05). For all experiments, Salmonella recovery decreased as holding times increased (1 to 24 h), which is consistent with literature where inoculated eggs were stored at room temperature. Results from Experiment 3 indicated that S. Typhimurium had higher recovery for all treatments and holding times compared to S. Enteritidis, except for eggshells without contents with 100% recovery at 1 h for both serotypes, and recovery of both serotypes was similar in Experiment 4. Overall, this study highlights the importance of eggshell cuticle, membranes, and albumen on the integrity of egg contents and the recovery of inoculated Salmonella from eggshells.

Properties, Genetics and Innate Immune Function of the Cuticle in Egg-Laying Species.

Cleidoic eggs possess very efficient and orchestrated systems to protect the embryo from external microbes until hatch. The cuticle is a proteinaceous layer on the shell surface in many bird and some reptile species. An intact cuticle forms a pore plug to occlude respiratory pores and is an effective physical and chemical barrier against microbial penetration. The interior of the egg is assumed to be normally sterile, while the outer eggshell cuticle hosts microbes. The diversity of the eggshell microbiome is derived from both maternal microbiota and those of the nesting environment. The surface characteristics of the egg, outer moisture layer and the presence of antimicrobial molecules composing the cuticle dictate constituents of the microbial communities on the eggshell surface. The avian cuticle affects eggshell wettability, water vapor conductance and regulates ultraviolet reflectance in various ground-nesting species; moreover, its composition, thickness and degree of coverage are dependent on species, hen age, and physiological stressors. Studies in domestic avian species have demonstrated that changes in the cuticle affect the food safety of eggs with respect to the risk of contamination by bacterial pathogens such as Salmonella and Escherichia coli. Moreover, preventing contamination of internal egg components is crucial to optimize hatching success in bird species. In chickens there is moderate heritability (38%) of cuticle deposition with a potential for genetic improvement. However, much less is known about other bird or reptile cuticles. This review synthesizes current knowledge of eggshell cuticle and provides insight into its evolution in the clade reptilia. The origin, composition and regulation of the eggshell microbiome and the potential function of the cuticle as the first barrier of egg defense are discussed in detail. We evaluate how changes in the cuticle affect the food safety of table eggs and vertical transmission of pathogens in the production chain with respect to the risk of contamination. Thus, this review provides insight into the physiological and microbiological characteristics of eggshell cuticle in relation to its protective function (innate immunity) in egg-laying birds and reptiles.

Effects of genotype and age on eggshell cuticle coverage and color profile in modern laying hen strains

The aim of this research was to investigate the effects of laying hen genotype and age on eggshell cuticle deposition. A total of 4,320 brown eggs were obtained from 3 modern hen strains (A, B, and C), currently used worldwide for commercial egg production, at different intervals of age (20–30, 40–50, and 60–70 wk). Four samplings of 120 randomly collected eggs were carried out for each genotype/interval of age. Eggs were individually weighed and cuticle blue staining was used to assess quality and degree of cuticle coverage. On each egg, the eggshell color profile was assessed before and after staining using the CIE L*a*b* system and these values were used to calculate ΔE*ab. A 4-point scale visual score (VS) system was also applied to estimate the degree of cuticle coverage after staining (0 = no coverage, 1 = partial coverage, 2 = total coverage - low degree, 3 = total coverage - high degree). The effects of genotype and age and their interaction on eggshell color attributes were assessed by means of factorial ANOVA, while omnibus Chi-Square and Chi-squared Automatic Interaction Detector algorithm were applied for the analysis of VS data. Overall, both genotype and age affected the eggshell color profile as well as the degree of cuticle coverage. Hen strain A showed better cuticle deposition in comparison with B and particularly C one, being ΔE*ab values significantly higher. The VS evaluation revealed that eggs with impaired cuticle coverage degree increased with the hen age (23, 34, and 37%, respectively for 20–30, 40–50, and 60–70 wk; P < 0.05). However, a significant interaction between genotype and age was observed: transition from early to late hen age resulted in a significantly different pattern of ΔE*ab changes in each genotype. The classification tree analysis confirmed that the hen genotype has a greater effect than the age on cuticle deposition. In conclusion, considering the importance of the cuticle in table egg production, these results highlight the crucial role exerted by the genotype on eggshell cuticle coverage.

Slightly acidic electrolyzed water as an alternative disinfection technique for hatching eggs

Conventional chemical disinfectants used for egg disinfection could result in toxic residue and endanger hatchability, chick quality, and pullet growth performance. Slightly acidic electrolyzed water (SAEW) is known as a novel disinfectant for egg sterilization due to its high efficiency and no residue. In this study, a comprehensive assessment of slightly acidic electrolyzed water and benzalkonium bromide solution (BBS) used in the disinfection channel was conducted to assess the microbial count, eggshell quality, and hatchability concomitantly. The results show that the sterilization efficiency of SAEW increased with an increase in available chlorine concentration (ACC), spraying volume, and sterilization duration. SAEW with an ACC of 150 mg/L and 10,000 mg/L benzalkonium bromide solution had the same sterilization rates of approximately 86.2% at a spraying volume of 0.5 mL/egg and sterilization duration of 180 s. Neither had significant effect on eggshell strength or thickness. The eggshell cuticle quality in the benzalkonium bromide group was significantly higher than the control group (no disinfection) and the 150 mg/L SAEW group. The embryo weight, relative embryo weight, hatchability, and embryonic mortality in the SAEW group had no significant differences of those in the benzalkonium bromide group. SAEW should be more popular because of its simple preparation, low cost, and no residue. Our results indicate SAEW is an alternative disinfectant for the sterilization of hatching eggs instead of conventional chemical disinfectants, such as benzalkonium bromide, and give a recommendation is using SAEW as a disinfectant with 150 mg/L ACC, 0.5 mL/egg spray volume, and disinfection for 180 s in the novel disinfection channel.

Cuticle deposition duration in the uterus is correlated with eggshell cuticle quality in White Leghorn laying hens

The cuticle formed in the uterus is the outermost layer as the first defense line of eggshell against microbial invasions in most avian species, and analyzing its genetic regulation and influencing factors are of great importance to egg biosecurity in poultry production worldwide. The current study compared the uterine transcriptome and proteome of laying hens producing eggs with good and poor cuticle quality (GC and PC, the top and tail of the cuticle quality distribution), and identified several genes involved with eggshell cuticle quality (ESCQ). Overall, transcriptomic analysis identified 53 differentially expressed genes (DEGs) between PC versus GC group hens, among which 25 were up-regulated and 28 were down-regulated. No differences were found in the uterine proteome. Several DEGs, including PTGDS, PLCG2, ADM and PRLR related to uterine functions and reproductive hormones, were validated by qPCR analysis. Egg quality measurements between GC and PC hens showed GC hens had longer laying interval between two consecutive ovipositions (25.64 ± 1.23 vs 24.94 ± 1.12 h) and thicker eggshell thickness (352.01 ± 23.04 vs 316.20 ± 30.58 μm) (P < 0.05). Apart from eggshell traits, other egg quality traits didn’t differ. The result demonstrated eggshell and cuticle deposition duration in the uterus is one of the major factors affecting ESCQ in laying hens. PTGDS, PLCG2, ADM and PRLR genes were discovered and might play crucial roles in cuticle deposition by regulating the uterine muscular activities and secretion function. The findings in the present study provide new insights into the genetic regulation of cuticle deposition in laying hens and establish a foundation for further investigations.

Impact of Different Layer Housing Systems on Eggshell Cuticle Quality and Salmonella Adherence in Table Eggs.

The bacterial load on the eggshell surface is a key factor in predicting the bacterial penetration and contamination of the egg interior. The eggshell cuticle is the first line of defense against vertical penetration by microbial food-borne pathogens such as Salmonella Enteritidis. Egg producers are increasingly introducing alternative caging systems into their production chain as animal welfare concerns become of greater relevance to today’s consumer. Stress that is introduced by hen aggression and modified nesting behavior in furnished cages can alter the physiology of egg formation and affect the cuticle deposition/quality. The goal of this study was to determine the impact of caging systems (conventional, enriched, free-run, and free-range), on eggshell cuticle parameters and the eggshell bacterial load. The cuticle plug thickness and pore length were higher in the free-range eggs as compared to conventional eggs. The eggshells from alternative caging (enriched and free-range) had a higher total cuticle as compared to conventional cages. A reduction in bacterial cell counts was observed on eggshells that were obtained from free-range eggs as compared to the enriched systems. An inverse correlation between the contact angle and Salmonella adherence was observed. These results indicate that the housing systems of layer hens can modify the cuticle quality and thereby impact bacterial adherence and food safety.

Antibacterial activity with eggshell nano-particles activated by microwave plasma

The eggshell cuticle is the proteinaceous outermost layer of the eggshell which regulates water exchange and protects against entry of micro-organisms. Outer eggshell and cuticle protein was extracted from domestic chicken. The aim of the research is to find out the effect of the treated and untreated nano particles of egg shells with micro wave cold plasma on the effectiveness of E. coli (negative bacteria) that infect the skin and measure the diameter of bacterial inhibition zone, the eggshell has been prepared by a chemical method (sol gel) and measure the level of acidity and the PH is neutral. The result of Atomic Force Microscope (AFM) shows that the particles diameters become smaller with nano-particles solution than for egg shell tests (powder). The nano-particles solution for egg shell exposed to cold plasma. The microwave plasma used in this search has voltage "175v" and the gas flow at " 5liter /min " and frequency (2.45 GHz) at the room temperature for five minutes’ time. The E. coli bacteria were treated with nano-particles solution egg shells. The results indicated that after 24 hours of placing the bacteria in the incubator, the inhibition zone for the nano-particles eggshell that for the is greater than that for nano-particles eggshell exposed to the plasma, and in both cases there were a good response.

Antifungal Action of Ozone on Chicken Eggshell Cuticles: A Preliminary Study

ABSTRACT Ozone gas (O3) is used as an oxidizing agent and can be applied as a sanitizer to control microorganisms and degrade toxic compounds, such as pesticides and mycotoxins, in food industries. Some studies investigated the use of O3 as an agent to reduce pathogenic bacteria such as Salmonella enteritidis. The action of O3 on the eggshell cuticle and structures of filamentous fungi is still not yet well studied. In this work, scanning electron microscopy (SEM) was used to assess the hens’ eggshell structure after the exposition to O3, mainly to obtain information about the potential antifungal action. The SEM micrographs obtained shown that the application of O3 during 120 min damaged fungi hyphae adhered to the eggshell cuticle without severely compromising the external structure of the eggshell. Further studies are necessary to establish an ideal O3 treatment for the fungal decontamination of chicken eggs.

Transcriptome analysis of the uterus of hens laying eggs differing in cuticle deposition

BackgroundAvian eggs have a proteinaceous cuticle. The quantity of cuticle varies and the deposition of a good cuticle in the uterus (Shell-gland) prevents transmission of bacteria to the egg contents.ResultsTo understand cuticle deposition, uterus transcriptomes were compared between hens with i) naturally good and poor cuticle and, ii) where manipulation of the hypothalamo-pituitary-gonadal-oviduct axis produced eggs with or without cuticle. The highest expressed genes encoded eggshell matrix and cuticle proteins, e.g. MEPE (OC-116), BPIFB3 (OVX-36), RARRES1 (OVX-32), WAP (OVX-25), and genes for mitochondrial oxidative phosphorylation, active transport and energy metabolism. Expression of a number of these genes differed between hens laying eggs with or without cuticle. There was also a high expression of clock genes. PER2, CRY2, CRY1, CLOCK and BMAL1 were differentially expressed when cuticle deposition was prevented, and they also changed throughout the egg formation cycle. This suggests an endogenous clock in the uterus may be a component of cuticle deposition control. Cuticle proteins are glycosylated and glycosaminoglycan binding genes had a lower expression when cuticle proteins were deposited on the egg. The immediate early genes, JUN and FOS, were expressed less when the cuticle had not been deposited and changed over the egg formation cycle, suggesting they are important in oviposition and cuticle deposition. The uterus transcriptome of hens with good and poor cuticle deposition did not differ.ConclusionsWe have gained insights into the factors that can affect the production of the cuticle especially clock genes and immediate early genes. We have demonstrated that these genes change their expression over the period of eggshell formation supporting their importance. The lack of differences in expression between the uterus of hens laying eggs with the best and worse cuticle suggest the genetic basis of the trait may lie outside the oviduct.

Acetic acid, vinegar, and citric acid as washing materials for cuticle removal to improve hatching performance of quail eggs

The cuticle is the outmost layer of the eggshell and may affect the hatchability by modulating eggshell conductance. Three different solutions using acetic acid (AA), vinegar (V), and citric acid (CA) for cuticle removal by egg washing were developed, and the effects of cuticle removal on hatching performance of quail hatching eggs were evaluated. A total of 5,238 fresh quail hatching eggs were randomly divided into 9 treatments as follows: unwashed control, nondipped (CND); washed control, water dipped (CWD); standard control, 0.13% sodium hyperchlorite (CSH); 2% AA (AA2); 4% AA (AA4); 44.4% V (V2); 88.8% V (V4); 2% CA (CA2); and 4% CA (CA4). Overall, AA4, V4, and CA4 treatments significantly improved the hatchability of fertile eggs (95.42%, 94.16% and 95.66%, respectively) (P < 0.05) and the hatchability of CND, CWD, CSH, AA2, V2 and CA2 treatments were 90.98%, 93.00%, 92.27%, 79.44%, 90.37%, and 90.59%, respectively. The eggshell thickness and cuticle quality results showed that all AA, V, and CA solutions can effectively remove the quail eggshell cuticle, and AA4, V4 and CA4 significantly decreased eggshell thickness (P < 0.05). Microbial activity on the eggshell surface in all acid treatments was reduced significantly at day 0 of incubation (P < 0.05) and that significantly decreased than controlled treatments over the incubation period except AA2 treatment.Egg weight loss was lower for all acid treatments than that of the CND treatment (P < 0.05). There was no clear effect of treatments on chick quality. Hatch time in AA4, V4, and CA4 treatments slightly improved compared with controlled treatments (P > 0.05). There were no significant differences between treatments for chick livability and live weight at the first 21 D of life. Results of the present study indicate that cuticle removal with AA4, V4, or CA4 could effectively decrease the microbial activity on the eggshell surface during the incubation period and improve hatchability of quail hatching eggs without negative effects on hatch time and performance of quail chicks.

Concentration of egg white antimicrobial and immunomodulatory proteins is related to eggshell pigmentation across traditional chicken breeds

Eggshell colour, quality, and biosafety of table eggs are of significant commercial interest. To date, there have been few studies investigating the relationship between eggshell pigmentation and internal egg quality in commercially bred birds. Moreover, the genetic basis and mechanisms behind the effects of extrinsic factors on deposition of antimicrobial compounds in egg white and eggshell pigments are not fully understood. In the present study, we evaluate the effect of chicken breed identity, eggshell pigmentation and the role of extrinsic factors (year and breeder identity) on variability in the concentrations of 2 major egg white antimicrobial proteins (AMPs), lysozyme (LSM), and ovotransferrin (OVOTR), across 23 traditional chicken breeds. We found that chicken breed identity and eggshell pigmentation explained most variability in the concentration of egg white LSM and OVOTR. Year and breeder identity were also significant predictors of egg white LSM and OVOTR variability, and showed selective effects on the deposition of both AMPs in egg white. We also documented a positive correlation between concentration of egg white LSM and eggshell cuticle protoporphyrin in tinted and dark brown eggs, but not in brown, white, and blue eggs. We assume that a combination of both intrinsic genetic and hormonally regulated extrinsic factors is responsible for this relationship and for the variability in egg white AMPs. In this study, we demonstrate the existence of a relationship between eggshell pigmentation and egg white AMPs content in the eggs of traditional chicken breeds that may advertise the egg's antimicrobial potential and biosafety. These findings provide novel insights into the relationship between eggshell pigmentation and egg internal quality and may stimulate the recovery and exploitation of traditional chicken breeds for egg production, where the demands for egg quality and biosafety, in conjunction with animal welfare, are a priority.

Comparative study of eggshell antibacterial effectivity in precocial and altricial birds using Escherichia coli.

In this study, we compared the antibacterial effectivity of the eggs of six precocial and four altricial bird species using Escherichia coli, based on their eggshell traits. The ultrastructure of eggshell was observed using a scanning electron microscope (SEM). According to SEM results, eggs from precocial birds (chicken, turkey, quail, duck, ostrich, and goose) had cuticle on the eggshells, while eggs from altricial birds (pigeon, budgerigar, munia, and canary) did not. The environment/selection pressure may induce the divergent evolution process in eggs of precocial and altricial birds. The E. coli experiment results showed that chicken, turkey, quail, duck, and goose eggs, with a high cuticle opacity, exhibited a much lower E. coli penetration rate. In contrast, the eggs with poor (ostrich) or without (pigeon, budgerigar, munia, and canary) cuticle exhibited a higher penetration rate. It is suggested that cuticle is a main barrier against bacterial penetration in precocial birds’ eggs. Turkey and quail eggs showed the lowest E. coli contamination rate (3.33% and 2.22%, respectively), probably because of the tightly connected nanosphere structure on their cuticle. As for altricial birds’ eggs, the eggs of budgerigar, munia, and canary with small pore diameter (0.57 to 1.22 μm) had a lower E. coli penetration rate than pigeon eggs (45.56%, 66.67%, 50%, and 97.78%, respectively, P < 0.05), indicating that pore diameter played a significant role in defending against bacterial trans-shell invasion. We found that eggshell thickness and pore area decreased with egg size. The cuticle quality had no relationship with egg size, but was closely related to the bird species. The E. coli penetration rate of altricial birds’ eggs was significantly higher than that of precocial birds’ eggs, mainly because the pores are exposed on the eggshell surface and cuticle protection is absent. This study provides detailed information on the eggshell cuticle, which gives insight into the cuticle evolution process that occurred in precocial and altricial bird species. Moreover, the results of E. coli penetration may help understanding the antibacterial behavior in birds.

Fossil eggshell cuticle elucidates dinosaur nesting ecology.

The cuticle layer consisting mainly of lipids and hydroxyapatite (HAp) atop the mineralized avian eggshell is a protective structure that prevents the egg from dehydration and microbial invasions. Previous ornithological studies have revealed that the cuticle layer is also involved in modulating the reflectance of eggshells in addition to pigments (protoporphyrin and biliverdin). Thus, the cuticle layer represents a crucial trait that delivers ecological signals. While present in most modern birds, direct evidence for cuticle preservation in stem birds and non-avian dinosaurs is yet missing. Here we present the first direct and chemical evidence for the preservation of the cuticle layer on dinosaur eggshells. We analyze several theropod eggshells from various localities, including oviraptorid Macroolithus yaotunensis eggshells from the Late Cretaceous deposits of Henan, Jiangxi, and Guangdong in China and alvarezsaurid Triprismatoolithus eggshell from the Two Medicine Formation of Montana, United States, with the scanning electron microscope (SEM), electron probe micro-analysis (EPMA), and Raman spectroscopy (RS). The elemental analysis with EPMA shows high concentration of phosphorus at the boundary between the eggshell and sediment, representing the hydroxyapatitic cuticle layer (HAp). Depletion of phosphorus in sediment excludes the allochthonous origin of the phosphorus in these eggshells. The chemometric analysis of Raman spectra collected from fossil and extant eggs provides further supportive evidence for the cuticle preservation in oviraptorid and probable alvarezsaurid eggshells. In accordance with our previous discovery of pigments preserved in Cretaceous oviraptorid dinosaur eggshells, we validate the cuticle preservation on dinosaur eggshells through deep time and offer a yet unexplored resource for chemical studies targeting the evolution of dinosaur nesting ecology. Our study also suggests that the cuticle structure can be traced far back to maniraptoran dinosaurs and enhance their reproductive success in a warm and mesic habitat such as Montana and southern China during the Late Cretaceous.

Cuticle and pore plug properties in the table egg

Food safety of table eggs is vital since many pathogens can contaminate the unfertilized egg, leading to increased risk of foodborne illness for consumers. The eggshell cuticle is the first line of defense to restrict the entry of egg-associated pathogens, such as Salmonella Enteritidis. The thickness and completeness of coverage of the cuticle layer are heritable traits that are strongly associated with egg resistance to bacterial penetration. The present study characterizes the chemical composition of the eggshell cuticle and structure of pore plugs from table eggs. Eggs collected from both brown and white egg laying Lohmann flocks (early, mid, and late lay) were either unwashed or washed. Pore plugs were characterized using scanning electron microscopy (SEM), and elemental composition was determined using energy-dispersive x-ray spectroscopy (EDS). SEM observations confirmed that the plug formed by the cuticle layer within the eggshell pore remains firmly lodged throughout the commercial washing process. The eggshell thickness and cuticle pore length visualized in brown eggs was significantly higher than in white eggs in hens of all ages. EDS analysis revealed that the pore inner surface was enriched in phosphorus and chemically different from the surrounding bulk eggshell mineral. Detailed assessment of the cuticle chemical composition was performed by Fourier transform infrared spectroscopy (FTIR). Washing of eggs removed cuticle from the eggshell surface. There was a trend of lower cuticle coverage with increasing hen age for white eggs. A significant reduction in the amount of proteins and phosphates and polysaccharides was observed in the cuticle of brown unwashed eggs with hen age. In white unwashed eggs, amides and lipids decreased with hen age; by contrast, the amount of sulfate was highest at mid-lay. The results from our research will assist selective breeding programs that target cuticle integrity and pore plug stability to enhance egg resistance to pathogen penetration and improve food safety.

What Does the Eggshell Cuticle Do? A Functional Comparison of Avian Eggshell Cuticles.

The avian eggshell is a highly ordered structure with several layers (mammillae, palisades, and vertical crystal layer) composed of calcium carbonate (∼96%) and minerals within an organic matrix. The cuticle is a noncalcified layer that covers the eggshells of most bird species. Eggshells are multifunctional structures that have evolved in response to diverse embryonic requirements and challenges, including protection from microbial infection, nest flooding, and exposure to solar radiation. However, experimental evidence for these functions across diverse taxa is currently limited. Here we investigated the effects of nanosphere cuticles on (1) bacterial attachment and transshell penetration, (2) eggshell wettability, (3) water vapor conductance, and (4) regulation of ultraviolet (UV) reflectance in seven ground-nesting bird species. We found considerable interspecific variation in ultrastructure and chemical composition of cuticles. Experimental removal of the cuticle confirmed that all nanospheres were highly effective at decreasing attachment of bacteria to shell surfaces and at preventing bacterial penetration. Cuticles also greatly decreased the amount of UV reflected by eggshells. In species with particularly small nanospheres, gas exchange was reduced by the presence of cuticle. Our results support the hypothesis that microbes and solar UV radiation can cause strong selection on bird eggs but also show that we need a greater understanding about the effects of specific nesting conditions (e.g., hydric and gaseous milieu) on embryo well-being and eggshell structure variation.