Table 2.

Challenges and proposed new strategies to understand innate immune functions of the eggshell cuticle.

Topic	Current Research	Future Prospects
Characterization of cuticle proteins	Most proteomic studies that have identified and characterized the proteins responsible for the protective capabilities of the cuticle have been conducted in chicken (section 2.4).	-Eggshell cuticle proteomics analysis in a wide gamut of egg-laying species (both avian and reptile) is necessary to provide a better understanding of the link between cuticle properties/coverage and its protective function. -Comparison of cuticle proteome in bird and reptile species is necessary to understand its evolution and response to changes in habitat/environment/climate.
Cuticle quality estimation and characterization of its chemical components	Limited methods are available to measure amount or degree of coverage of the cuticle on eggshell surface in a non-destructive manner (section 2.6). Thus, available methods have limited application in commercial poultry production.	-Cuticle proteins alter the surface hydrophobicity, which can be estimated using contact angle measurements. Cuticle surface hydrophobicity has been negatively correlated with bacterial adherence. -Measurement of contact angle, by adding a droplet of deionized water to the egg surface, could be implemented in a high throughput manner in commercial hatcheries/egg grading systems to evaluate cuticle quality and select/categorize eggs based on hydrophobicity of the eggshell surface.
Cuticle coverage	Patchy distribution of cuticle (section 2.3 and 2.6)	-Cuticle coverage is not complete on surface of the eggshell in some species, while it is absent in other species (section 1 and 2.2). -The mechanism of interaction of microbial pathogens with surface antimicrobial molecules of the cuticle is not well defined. The significance of cuticle completeness in establishing biosecurity of eggs is still not completely understood. It is not clear if cuticle coverage has evolved to sufficiently plug the pores and having uniformly complete cuticle coverage is of less importance in order to protect eggs from pathogens.
Role of eggshell microbiome	Microbiota participate in egg defense by various indirect and synergistic effects (section 2.7 and 2.8)	- Most of these mechanisms are hypothetical and require further experimental study. -It may be difficult to generalize microbiome role to all egg-laying species, considering the diversity of phyla composition that is adapted to substrate modifications (structure/composition of the cuticle) and environmental changes (nesting environment and climate). - The hypothesis that the egg interior is sterile should be experimentally evaluated.
Genetic variation and potential for genetic improvement	A moderate heritability of cuticle deposition is observed in chicken, which is important for genetic progress to increase deposition of cuticle (section 4.3).	-Currently, cuticle quality is not assessed as part of the eggshell quality or egg quality assessment. -Cuticle deposition should be incorporated into breeding programs for egg and meat type birds to reduce vertical transmission or environmental contamination with pathogens in order to improve biosecurity in poultry.
Cuticle pore and plugs	Recent studies have characterized structure of cuticle pores and plugs (section 3.1.4)	-Mechanism of pore formation in eggshell is still not known, the origin of pore and plug during egg formation is still unclear. -Localization of antimicrobial proteins in cuticle pore and plug needs to be evaluated. Elucidating their mode of action will improve methods to maintain egg quality and prevent egg contamination.
-Regulation of synthesis and deposition of cuticle	-Mechanisms controlling cuticle synthesis/secretion are not well-understood (section 2.5).	-Identification and role of potential hormones or cellular signaling cascades that regulate secretion and deposition of cuticle should be investigated.
-Effect of non-genetic factors on cuticle deposition	Stress reduces cuticle deposition (section 2.5). -Effect of age is controversial, with studies showing both no effect and age-related decline of cuticle deposition (section 2.5 and 4.4).	-Studies where eggs from individual hens are followed for a longer duration are necessary to evaluate if cuticle deposition and its chemical composition decline with age. This is particularly relevant to current interest in maintaining flocks for up to 100 weeks of age. -Physiological control of the deposition of cuticle is not well understood.