The formation and the fine structure of the components (cuticle, porous calcitic shell, shell membranes and limiting membrane) of the integument of the hen's egg were studied in detail with Scanning Electron Microscopy or, when appropriate, Transmission Electron Microscopy, in order to build up a detailed picture of the physical, antimicrobial systems of the egg pre- and post-oviposition. This phase of the study revealed that (i) the limiting membrane is present on the egg in the magnum, (ii) the interstitial spaces of the shell membranes in the oviduct are filled with amorphous material which probably condenses to form the mantle on the individual fibres of the membranes and (iii) the cuticle at oviposition is immature and mechanically weak. The immature cuticle is an ineffective barrier to bacterial penetration for a few minutes post-oviposition. When the components of the integument were considered as resistances in series, the mature cuticle - especially that on brown eggs - was shown to be the major barrier to the movement of water in the liquid but not the vapour state. Consequently this structure plays an important role in protecting the egg contents from infection. Work needs to be done in order to overcome this resistance - differences in partial pressure caused by immersing warm eggs in a cold solution overcame the cuticle's resistance. The efficacy of this method was enhanced by reducing the surface tension or "degassing" the water. Of the many methods used to assess cuticle quality, staining was the only one that would appear to be of any use at the moment in a breeding programme that sought improvements in the quality and hence effectiveness of the cuticle as a barrier to infection. The porosity of the shell, as measured by GH2O, was not associated with water uptake by eggs. Nevertheless the pore contributed to the waterproofing, and hence antimicrobial defence of the egg, by imposing such a resistance to water movement that the osmotic potential of the albumen to pull water across the shell membranes was negated. The shell membranes proved to be ineffective barriers to microbial movement. The contribution of the limiting membrane to the egg's defence could not be assessed with the techniques used in this study. The environment within the shell membranes was selective for Gram-negative bacteria. Indeed the membranes appeared to offer a haven for microorganisms that had passed across the shell, especially in infected eggs held under conditions recommended for hatching eggs before transfer to incubation temperature. In practice viable organisms persisted in these structures whereas contaminants of the underlying albumen died out especially with incubation temperatures approaching the body temperature of the hen. Heavy microbial challenges (>106/egg) to the shell membranes resulted in heavy contamination of the albumen within two days of infection. It was deduced that contaminants of the albumen failed to multiply unless they made contact with the yolk. The above observations were discussed in the context of practices that should be considered by hatchery managers who seek to interrupt the transfer of Salmonella from the breeding to the rearing flock by egg treatment. The identification of the immature cuticle state directs attention to the management and hygiene of nest boxes and the primary role of the cuticle in the eggs antimicrobial defence. It suggests that breeding programmes ought to be concerned with the quality of the cuticle throughout the laying cycle. The influence of temperature on the behaviour of contaminants in the shell membranes suggest also that treatment of eggs with germicides to control Salmonella transmission needs to be done.

The microbiological safety of avian eggs is a major concern for the poultry industry and for consumers due to the potential for severe impacts on public health. Innate immune defense is formed by proteins with antimicrobial and immune-modulatory activities and ensures the protection of the chick embryo against pathogens. The objective of this project was to identify the chicken eggshell membrane (ESM) proteins that play a role in these innate immune defense mechanisms. We hypothesized that ESM Ovocalyxin-36 (OCX-36) is a pattern recognition protein, and characterized purified ESM OCX-36. OCX-36 has antimicrobial activity against S. aureus and binds E. coli lipopolysaccharide (LPS) and S. aureus lipoteichoic acid (LTA). We additionally investigated the OCX-36 nonsynonymous single nucleotide polymorphisms (SNPs) at cDNA position 211. The corresponding isoforms (proline-71 or serine-71) were purified from eggs collected from genotyped homozygous hens. A significant difference between Pro-71 and Ser-71 OCX-36s for S. aureus LTA binding activity was observed. From these experiments, we confirmed the hypothesis that OCX-36 is a pattern recognition molecule. We also found that OCX-36 has anti-endotoxin properties and is a macrophage immunostimulator to produce NO and TNF-α. Digested OCX-36 down-regulated the expression of genes involved in LPS signaling and inflammatory responses. Moreover, OCX-36-derived peptides inhibited the production of LPS-induced pro-inflammatory mediators associated with endotoxemia in vivo. Quantitative proteomics analysis of ESMs was performed to evaluate changes in ESM protein abundance during chick embryonic development. Bioinformatics analysis revealed enrichment of proteins associated with antimicrobial and immune protection, vascularization, calcium mobilization and lipid transport, which are vital for chick embryonic development. In unfertilized eggs, protease inhibitors and antimicrobial proteins were enriched. In summary, the ESMs are enriched in proteins with antimicrobial, antioxidant and immune-modulatory properties, which aid in the development of the chick embryo and protect the embryo and unfertilized egg against pathogen invasion.

The eggshell of reptiles is essential for not only protecting the embryo, but can also serve as source of calcium for embryonic skeletal development. Whereas embryonic lepidosaurs and chelonians rely on their yolk sac for calcium during development, embryonic archosaurs mobilise eggshell calcium supply to both the embryo and the yolk sac. By the time archosaurs hatch, their residual yolk sacs have a calcium content equal or greater than at time of oviposition, which is used to support post-hatching growth. To date, no study has looked into how removal of the calcareous eggshell affects embryonic development in archosaurs. I tested how the removal of the calcareous eggshell affects embryonic and hatchling growth and biomechanic function of the skeleton in embryos and hatchlings of the American alligator (Alligator mississippiensis). Experimental eggs had their eggshell manually peeled, while control eggs were sham handled but otherwise not altered. Sampling of eggs occurred on a weekly basis until the end of incubation. Embryos, yolk sacs, and eggshells were removed and analyzed for morphological, histological and biomechanical parameters. Results show that at the time of eggshell peeling yolk sac calcium reserves were sufficient for experimental embryos to develop, but animals hatched in diminutive state. Serial clearing and staining of embryos revealed that onset of bone mineralization was similar for both treatment groups. Growth trajectory of experimental hatchlings parallelled that of control animals, without compensatory growth. Experimental hatchlings were observed to have flexible lower jaws and produced a weaker bite force than control hatchlings. Cross-sections of the mandible and femoral mid-diaphysis had a significantly reduced cross-sectional area in experimental hatchlings. I conclude that loss of the calcareous eggshell during incubation leads to severe constraint on growth and biomechanics of the alligator skeleton.

Microbial Reduction on Eggshell Surfaces by the use of Hydrogen Peroxide and Ultraviolet Light. (August 2011) Steven Michael Gottselig, B.S., Texas A&M University Chair of Advisory Committee: Dr. Craig Coufal The effect of hydrogen peroxide (H2O2) in combination with ultraviolet light (UV) as an egg sanitization process on eggshell surfaces was studied. Preliminary experiments were conducted to develop an optimized methodology for eggshell disinfection that will be an effective and efficient way to reduce microorganisms on hatching eggs. Several experiments were conducted to reduce the natural flora found on the eggshell surface. Hatching eggs were collected from White Leghorn hens housed in floor pens with nest boxes. Eggs had no adhering organic material present. Results from these experiments led to the modification of the prototype equipment as well as the treatment application methodology. Following the experiments to optimize the methodology for H2O2 spraying and UV exposure time, the methodology was applied to eggs inoculated with Salmonella Typhimurium. Eggshell crush and rub methodology was used to enumerate bacteria within the pores and membranes of the egg. The optimized H2O2 and UV combination treatment process was then applied to commercial broiler breeder hatching eggs to evaluate the effects on hatchability. Based on the parameters tested, results indicate that two applications of 3% H2O2 followed by 5 sec of UV exposure after each application produced the most consistent microbial reductions on eggshells. To enhance these effects, the addition of a 180° rotation between the two applications showed to be effective at further reducing the natural flora found on the eggshell surface. Studies using this optimal methodology on eggs inoculated with Salmonella at 9 log10 CFU/egg yielded greater than 5 log10 CFU/egg reductions. However, this methodology had little to no effect on reducing bacteria found within the pores and membranes of the eggs inoculated with Salmonella. These findings indicate that the effects of the disinfection process are largely limited to the eggshell surface. Hatch studies showed significant reductions in eggshell microbial levels under field conditions with eggs having large amounts of organic material present on the shell surface. Hatchability was maintained after treatment when compared to untreated eggs. Additional studies are needed to develop advanced equipment to apply this technology under commercial conditions.

Temperature is the most important among several factors that are needed for optimum avian embryonic development during incubation. Previous studies have implied that incubation temperature may have an impact on chick quality and post hatch performance. The use and accuracy of different methodologies for the estimation of embryonic temperature and eggshell conductance values during incubation were determined in three trials. Results suggested that air cell temperature using transponders may be used for the estimation of embryo temperature from 12 through 19 days of incubation and the subsequent calculation of eggshell conductance and specific eggshell conductance values. A final trial was conducted to further substantiate the estimation of embryo temperature using air cell readings by implanting probes alongside transponders in the air cell. It was determined that further modifications are needed to perfect this particular method.

Avian embryonic development, hatchability, and post-hatch performance are greatly affected by temperature. Therefore, a pragmatic, minimally invasive, and industrially applicable method for the precise and accurate measurement of embryonic temperature is needed. The objective of this research was to develop such a method. Preliminary research concerning air cell depth profiles and the relationships of eggshell thickness to thermal gradients across the shells of broiler hatching eggs were determined. This provided information for appropriately positioning and timing wired thermistor probe insertion into egg air cells for the practical and accurate estimation of embryo temperature. The relationship between air cell temperature readings using transponders and wired thermistor probe network assemblies were likewise determined. Embryo temperature estimation using probes was shown to be a satisfactory, but their depth should be adjusted daily by 0.042 cm after Day 12 of incubation in order to mirror transponder temperature readings.

The chicken eggshell possesses physical and chemical barriers to protect the embryo from pathogens. The avian eggshell cuticle is the outmost layer of the eggshell whose protein constituents remain largely unknown. Since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination, we hypothesize that cuticle protein components play an important role in microbial resistance. In our study, at least 47 proteins were identified by LC/MS/MS in the non-calcified cuticle layer. Similar to Kunitz-like protease inhibitor (also annotated as ovocalyxin-25, OCX-25) and ovocalyxin-32 (OCX-32) were two of most abundant proteins of the cuticle proteins. Some proteins that have antimicrobial activity were also detected in the proteomic results, such as lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor. This study represents the first comprehensive report of the cuticle proteome. Since the sequence similarity of the kunitz motif in OCX-25 is similar to that of BPTI, it is predicted that it will have the same trypsin inhibitory and antimicrobial activity against Gram-positive and/or Gram-negative bacteria. In order to test the antimicrobial property and trypsin inhibitor activity of OCX-25, cuticle proteins were extracted by 1N HCl. Antimicrobial activity was monitored using the Bioscreen C instrument; and antimicrobial activity was identified primarily against Staphylococcus aureus. Trypsin inhibitor activity was studied by using a specific trypsin assay, and the assay indicated that the cuticle proteins could inhibit the reaction of trypsin and substrate. Therefore, the current research has provided some insight into the antimicrobial and enzymatic aspects of the cuticle proteins, and its function for egg protection. Eggshell membranes are another important component of the chicken eggshell.Due to its insoluble and stable properties, there are still many questions regarding formation and constituents of the eggshell membranes. The purpose of our study was to identify eggshell membrane proteins, particularly these responsible for its structural features, by examining the transcriptome of the white isthmus during its formation. Bioinformatics tools were applied to analyze the differentially expressed genes as well as their encoded proteins. Some interesting proteins were encoded by the over-expressed genes in the white isthmus during the formation of eggshell membranes, such as Collagen X, and similar to spore coat protein SP75. These proteins may have potential applications. Our study provides a detailed description of the chicken white isthmus transcriptome during formation of the eggshell membranes; it could lead to develop the strategies to improve food safety of the table egg.

The chicken eggshell possesses physical and chemical barriers to protect the embryo from pathogens. The avian eggshell cuticle is the outmost layer of the eggshell whose protein constituents remain largely unknown. Since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination, we hypothesize that cuticle protein components play an important role in microbial resistance. In our study, at least 47 proteins were identified by LC/MS/MS in the non-calcified cuticle layer. Similar to Kunitz-like protease inhibitor (also annotated as ovocalyxin-25, OCX-25) and ovocalyxin-32 (OCX-32) were two of most abundant proteins of the cuticle proteins. Some proteins that have antimicrobial activity were also detected in the proteomic results, such as lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor. This study represents the first comprehensive report of the cuticle proteome. Since the sequence similarity of the kunitz motif in OCX-25 is similar to that of BPTI, it is predicted that it will have the same trypsin inhibitory and antimicrobial activity against Gram-positive and/or Gram-negative bacteria. In order to test the antimicrobial property and trypsin inhibitor activity of OCX-25, cuticle proteins were extracted by 1N HCl. Antimicrobial activity was monitored using the Bioscreen C instrument; and antimicrobial activity was identified primarily against Staphylococcus aureus. Trypsin inhibitor activity was studied by using a specific trypsin assay, and the assay indicated that the cuticle proteins could inhibit the reaction of trypsin and substrate. Therefore, the current research has provided some insight into the antimicrobial and enzymatic aspects of the cuticle proteins, and its function for egg protection. Eggshell membranes are another important component of the chicken eggshell.Due to its insoluble and stable properties, there are still many questions regarding formation and constituents of the eggshell membranes. The purpose of our study was to identify eggshell membrane proteins, particularly these responsible for its structural features, by examining the transcriptome of the white isthmus during its formation. Bioinformatics tools were applied to analyze the differentially expressed genes as well as their encoded proteins. Some interesting proteins were encoded by the over-expressed genes in the white isthmus during the formation of eggshell membranes, such as Collagen X, and similar to spore coat protein SP75. These proteins may have potential applications. Our study provides a detailed description of the chicken white isthmus transcriptome during formation of the eggshell membranes; it could lead to develop the strategies to improve food safety of the table egg.

The chicken eggshell possesses physical and chemical barriers to protect the embryo from pathogens. The avian eggshell cuticle is the outmost layer of the eggshell whose protein constituents remain largely unknown. Since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination, we hypothesize that cuticle protein components play an important role in microbial resistance. In our study, at least 47 proteins were identified by LC/MS/MS in the non-calcified cuticle layer. Similar to Kunitz-like protease inhibitor (also annotated as ovocalyxin-25, OCX-25) and ovocalyxin-32 (OCX-32) were two of most abundant proteins of the cuticle proteins. Some proteins that have antimicrobial activity were also detected in the proteomic results, such as lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor. This study represents the first comprehensive report of the cuticle proteome. Since the sequence similarity of the kunitz motif in OCX-25 is similar to that of BPTI, it is predicted that it will have the same trypsin inhibitory and antimicrobial activity against Gram-positive and/or Gram-negative bacteria. In order to test the antimicrobial property and trypsin inhibitor activity of OCX-25, cuticle proteins were extracted by 1N HCl. Antimicrobial activity was monitored using the Bioscreen C instrument; and antimicrobial activity was identified primarily against Staphylococcus aureus. Trypsin inhibitor activity was studied by using a specific trypsin assay, and the assay indicated that the cuticle proteins could inhibit the reaction of trypsin and substrate. Therefore, the current research has provided some insight into the antimicrobial and enzymatic aspects of the cuticle proteins, and its function for egg protection. Eggshell membranes are another important component of the chicken eggshell.Due to its insoluble and stable properties, there are still many questions regarding formation and constituents of the eggshell membranes. The purpose of our study was to identify eggshell membrane proteins, particularly these responsible for its structural features, by examining the transcriptome of the white isthmus during its formation. Bioinformatics tools were applied to analyze the differentially expressed genes as well as their encoded proteins. Some interesting proteins were encoded by the over-expressed genes in the white isthmus during the formation of eggshell membranes, such as Collagen X, and similar to spore coat protein SP75. These proteins may have potential applications. Our study provides a detailed description of the chicken white isthmus transcriptome during formation of the eggshell membranes; it could lead to develop the strategies to improve food safety of the table egg.

Em lagartos, componentes de história de vida apresentam grande plasticidade fenotípica e evoluem de maneira correlacionada. O número de ovos, por exemplo, tende a ser correlacionado positivamente com o tamanho corpóreo das fêmeas grávidas, tanto em linhagens evolutivas quanto entre indivíduos, embora alguns tipos de hábito pareçam restringir ninhadas maiores. Há indícios de que outros fatores, como componentes climáticos, ou latitude, também sejam determinantes das características de história de vida do grupo, tanto por questões de fisiologia termal quanto ecológicas. Componentes climáticos influenciam ainda diferentes aspectos da reprodução em lagartos, como por exemplo a estrutura e morfologia da casca dos ovos de Squamata, conforme indicam diferenças nestas características entre linhagens de lagartos ovíparos. Neste contexto, o presente trabalho investigou aspectos evolutivos da história de vida de lagartos da subfamília Tropidurinae, e sua relação com componentes climáticos e latitude, através de métodos filogenéticos comparativos. Observou-se que o número de ovos da ninhada é positivamente correlacionado com o tamanho corpóreo ao longo da linhagem dos tropiduríneos. Por outro lado, esse padrão não é observado no caso de hábito escansorial e utilização de frestas de rochas, em razão de possíveis compromissos envolvendo peso e forma corpórea com efeitos na sobrevivência das fêmeas. O tamanho corpóreo em tropiduríneros é maior em regiões geográficas com maiores componentes de precipitação, possivelmente relacionado a disponibilidade de alimento. Há diminuição do tamanho corpóreo com incrementos na distância da linha do Equador, até aproximadamente 1500 km. A partir desse limite, o tamanho corpóreo aumenta. Adicionalmente foi estudada a morfologia e estrutura da casca de Tropidurus torquatus, uma das espécies com distribuição mais ampla dentro da subfamília. A morfologia da casca de Tropidurus torquatus segue o padrão geral para Squamata com algumas particularidades, mas estudos comparativos futuros é que elucidarão padrões evolutivos da morfologia da casca. / In lizards, life history components have great phenotypic plasticity and evolve in a correlated way. Clutch size, for example, tends to be positively correlated to body size of gravid females, when comparing different evolutive lineages or different individuals, though some kinds of habit seems to restrict larger clutches. There are indications that other factors, such as climate components or latitude, also determine characteristics of lizard life history, as a matter of thermal physiology or ecology. Climate factors also affect different aspects of lizards reproduction, for example the structure and morphology of Squamata eggshells, as differences observed in these factors between oviparous lizards lineages indicate. In this context, the present work investigated evolutive aspects of Tropidurinae lizards life history and its relation to climate components and latitude, using comparative phylogenetic methods. It has been observed that clutch size is positively correlated to body size in the Tropidurinae lineage. On the other hand, this pattern is not observed in lizards with scansorial habits or those that inhabit rock crests, maybe due to compromises involving the weight and shape of female bodies that can affect female survival. The body size in Tropidurinae is larger in geographical regions presenting higher precipitation, possibly due to increased food availability. Female body size decreases with increased distance from the Equator until approximately 1500 km. Beyond this limit, female body size increases. Additionally, morphology and structure of eggshells were studied on Tropidurus torquatus, one of the species within the Tropidurinae group with higher latitudinal range of distribution. Tropidurus torquatus eggshell morphology follows the general pattern described for Squamata with some particularities, and future comparative studies are needed to elucidate evolutive patterns of eggshell morphology in the group.