The cuticle in reptile eggshell has not been formally defined and often, diverse nomenclature is used to describe this layer e.g. covering layer (44, 45); cover (46); cuticle (15, 47). It also has been argued that the reptilian cuticle is not equivalent to the cuticle present on avian eggshells (48); however, a formal comparison has never been done. To date, few reports exist on the cuticle in non-avian eggs. This missing information likely results from the general scarcity of studies about egg formation and composition in non-avian reptiles. In reptile species, cuticle is more commonly observed on calcified eggshells of some turtles and geckos, and less often on soft shells as those of snakes and lizards. With the exception of crocodiles (48), cuticle has been found on eggs of all reptile groups including turtles, snakes, lizards and geckos ( Table 1 ), and shows large variation in structure ( Figure 4 ). In crocodiles, organic material was observed plugging the pores of American alligator (Alligator mississippiensis) eggs (48). This consisted of crystalline nanospheres, which covered the surface of the shell for a few days after oviposition, but then disappeared, perhaps due to acidic erosion inside the nest. Despite the similarities with the pore plugs formed by the cuticle of avian eggs, it has been argued that this material should not be considered a true cuticle (48). In other reptiles, cuticles vary in thickness from 2 to 50 um ( Table 1 ) and can appear as a homogeneous amorphous layer, a mixed layer containing crystals or spherulitic granules (e.g., Sceloporus virgatus; Figure 4 ) (52).

Eggshell cuticles have also been identified in fossilized eggs of a non-avian theropod dinosaur (53) and enantiornithines (54, 55), an extinct group of avialans from the Mesozoic. One remarkably preserved enantiornithine egg was discovered as a single unlaid egg, within the abdominothoracic cavity of the mother. It exhibits a distinctive cuticle layer enriched in phosphorous and consisting of spherical nanostructures of calcium phosphate (55) similar to those observed in modern bird cuticles. This discovery supports in favor of the presence of cuticle nanospheres in the common ancestor of birds.

Due to its commercial importance, most studies of egg formation have focused on the chicken (Gallus gallus), and consequently we know more about the reproductive physiology of this bird than any other avian species. Therefore, here we have highlighted the deposition of the cuticle in the chicken reproductive system. Egg formation is initiated by ovulation (release of an ovarian follicle into the proximal oviduct), and the forming egg sequentially acquires its compartments as it transits different segments ( Figure 2 ). Following mineralization of the calcified shell in the uterus (shell gland pouch), the cuticle layer is deposited on its surface. The cuticle constituents are secreted by non-ciliated secretory cells during the last 2 h before egg expulsion (oviposition) (4, 56, 57). The normal endocrine events which regulate ovulation and oviposition are necessary for deposition of the cuticle (57). Uterine transcriptomic analysis in chickens laying eggs with different degrees of cuticle deposition suggests that at least two categories of genes are responsible for controlling the production of cuticle (58). Cuticle deposition and oviposition are mediated by both clock genes, which regulate the timing of cellular events to ensure that cells respond appropriately to their environment, and immediate early genes, which are critical in the activation of cellular processes by external factors (58). The lack of differences in gene expression between the uterus of hens laying eggs with the best and worst cuticle deposition indicated that the genetic variability of the trait could lie outside the oviduct (58). In another recent study, transcriptomic analysis has demonstrated that the physiological state of the uterus regulates eggshell quality and egg ultrastructure. Specific genes (PTGDS, PLCG2, ADM and PRLR) are predicted to play a critical role in cuticle deposition by modulating uterine secretion rhythm and function (2).

Cuticle deposition is distinct from other events in egg formation. Since termination of shell mineralization occurs before cuticle deposition, the cuticle is not contiguous with the organic matrix of the eggshell (27), instead it is a specific secretion which is distinct from other events in eggshell calcification (56). Avian eggs exhibit variability in shell coloration/pigmentation due to the presence of blue-green biliverdin and red-brown protoporphyrin pigments deposited into the outer surface of a developing egg in the shell gland (59); however, no direct correlation has been established between deposition of shell pigment and cuticle formation. Most of the shell pigment is located in the outer calcified layers, with only 13–20% found in the cuticle and, although pigment deposition and cuticle deposition are temporally close (60, 61), pigment deposition occurs earlier; it is almost complete an hour before the expected time of oviposition. According to genetic studies, there is no connection between the genes controlling shell pigment deposition and cuticle formation, and neither event is dependent on the other (56). In at least one avian species (tinamou), the glossy appearance of the eggshell is produced by an extremely smooth cuticle, composed of calcium carbonate, calcium phosphate, proteins and pigments (62).

There have been several anatomical investigations of the oviductal morphology in reptiles (63–65). However, these studies have not provided detailed information about eggshell formation. Unlike birds, reptiles possess an oviduct undifferentiated into separate anatomical regions. The exception to this rule are crocodilians, which have an oviduct demarcated into six regions, homologous to that of birds (66). Thus, in chelonians and squamates, the oviduct is not specialized for the production of different eggshell components and the proteinaceous membrane and calcified shell are both deposited in the homogeneous uterus (64). Furthermore, the entire process from ovulation to shell deposition is much longer in reptiles compared to the average 24 hours in the fowl (67). Deposition of cuticle may initiate several days after ovulation, concurrent with a change in shape of the fibrous shell membrane and its adoption of peaks and valleys. It has been hypothesized that the superficial deposition of minerals and organic matter might be initiated, at least in part, by the reorganization of the shell fibers and subtle chemical changes in the oviduct (67).

The term “cuticle” a distinct layer on the outer surface of the calcified or fibrous shell, of organic or mixed composition, and of oviductal origin. It is also defined as shell accessory material (SAM) on external surface of eggshell in some studies on bird species including Mandarin duck (Aix galericulata), domestic turkey (Meleagris gallopavo) and goose (Anser anser) (68, 69). The cuticle of chicken eggs is a relatively thin layer with variable thickness (0.5-12 μm), with patchy and uneven distribution on the eggshell surface (20, 70, 71). It is more complete in freshly laid eggs and when dry, has a glossy appearance (20). Variable cuticle thickness is observed in eggs of many other avian species (15). For example, the approximate thickness of the cuticle layer varies considerably between the White Pelican (Pelecanus onocrotalus) (130 μm), Japanese quail (Coturnix japonica) (10 μm), Greater Flamingo (Phoenicopterus ruber roseus) (110 μm), and Humboldt Penguin (Spheniscus humboldti) (45 μm) (15). On the other hand, the cuticle is absent in eggs from some clades such as parrots, petrels and pigeons (42). It has been proposed that environmental or reproductive selection pressures are responsible for divergent cuticle features in eggs of different species (42).

The structure and morphology of the cuticle can be evaluated by scanning and transmission electron microscopy (SEM, TEM) ( Figures 3 , 5 and 6 ) (20). As the cuticle dries and hardens upon exposure to the environment, it displays micro-cracks and micro-fissures on its outer surface, as visualized by SEM ( Figure 5 ) (15, 72, 73). The chicken cuticle is composed of two layers: inner (vesicular) and outer (non-vesicular) layers identified by TEM ( Figure 6 ). The inner layer is composed of vesicles ranging from approximately 50 to 500 nm that are deposited during the final phase of eggshell calcification (termination) (20). Each vesicle is composed of a core and mantle with electron lucent and electron dense properties, respectively (74). The non-vesicular outer cuticle is more compact and homogenous; it is a water-insoluble layer with non-mineralized components (15). TEM imaging also reveals that cuticle thickness varies widely in chicken eggs, even being completely absent in some regions (20, 70).

In avian species, the inner cuticle is composed of vaterite crystals, but is organized as nanospheres on others (42). Many species lack nanospheres in their cuticle, but this nano structuring is hypothesized to have been present in eggs of the avian ancestor (42).

Currently, our knowledge about composition of reptilian cuticles is very limited. Early studies showed that the cuticle in squamates (a monophyletic group comprising lizards, snakes and amphisbaenians) contains primarily neutral hydrophobic amino acids, soluble proteins, which vary greatly in composition, and negligible amounts of lipids (75). More recently, carboxylated carbohydrates and sulfated mucins have been reported in the cuticles of eggs of the Argentine black and white tegu (Salvator merianae) (49). The presence of phosphorous is a clear attribute of the cuticle layer in birds. The function of phosphorous is not entirely clear, although it has been suggested that the presence of phosphate terminates eggshell growth because it competes with carbonate for calcium during eggshell mineralization (49). However, in order to interpret the significance of phosphate/phosphorus in cuticle, it will be important to distinguish between inorganic phosphate (i.e. as hydroxyapatite (68) and that present in phosphorylated cuticle proteins such as OCX-32 (5, 7). Large concentrations of phosphorous have also been detected in the cuticles of non-avian reptiles (45, 76).

All evidence points to the uterus (shell gland) as the oviduct segment where cuticle production and its deposition occurs (56, 78–80). It is observed that material accumulating in the epithelial and tubular gland cells of the chicken shell gland lining before oviposition, such as dermatan sulfate, has disappeared from these cells after oviposition (79). In quail, an unidentified protein found on the shell and in the shell gland also accumulates towards the end of egg formation and is largely depleted after oviposition (80). OCX-32, a significant component of the cuticle, disappeared from the shell gland secretory cells when an egg was laid normally, but not when the egg was laid prematurely without a cuticle (56). Furthermore, OCX-32-positive cells are not found in the vagina (56). Finally, a study in which eggs were removed from the oviduct just before oviposition also concluded that the cuticle was formed in the uterus (78).

Thus, there is strong evidence that the cuticle is deposited at the very end of egg formation, not least because it is the last layer of the egg structure. Previous studies on the relative timing of cuticle deposition and oviposition had only allowed an estimate to within 4 h pre-oviposition (80). A more recent study narrowed this interval, demonstrating that pigments, where present, were laid down an hour before oviposition and the cuticle secretion therefore occurred less than an hour in advance of oviposition (56). Even with synchronized oviposition, reducing the estimate of cuticle deposition in proximity to oviposition is difficult, due to natural variability in timing between individuals.

There is limited information on the control of cuticle deposition. Clearly, as we will see in a later section (4.2), genetics controls a relatively large part of the variance in cuticle deposition (25, 56); however, there is much less information concerning the importance and nature of non-genetic factors. Mild stress in hens, such as relocation to pens from cages, does reduce cuticle coverage, although the effect is relatively small (56). The effect of hen age remains controversial. In a longitudinal study, where up to 100 hens from both layer and broiler breeder lines were followed from peak of lay to 50 weeks of age, there was no statistically significant effect of hen age on cuticle coverage, although brown eggshell pigment was clearly reduced in older birds (81). Moreover, no change in cuticle deposition in hens up to 70 weeks of age was observed in a commercial flock of Leghorn hens laying white eggs (82). Another study using white eggs laid by Hy-Line CV22 hens observed no significant change in cuticle amide content between 16 and 70 weeks of hen age (20). On the other hand, there was a significant decrease in cuticle amide content in both white and brown eggs from Lohmann hens over 21 to 66 weeks of age (17). Therefore, the influence of hen age upon cuticle deposition remains unclear, but there may be distinctions between different lines of hens, egg color and possibly rearing/caging systems.

Moreover, physiological control of cuticle deposition is not well understood. Inducing premature oviposition of the egg with gonadotropin releasing hormone (GnRH) at the top of the endocrine cascade resulted in a normal cuticle after oviposition, whilst conversely the induction of oviposition by arginine vasotocin (AVT) or prostaglandin F2alpha (PGF2α) at the bottom of the endocrine cascade that induces oviposition does not (56). Thus, there are indications that a number of hormones or factors might induce cuticle secretion and deposition if they occur in the correct order and the most proximate endocrine signals on their own are not sufficient, but definitive evidence remains elusive (56).

It is intriguing that the standard measurements of eggshell quality, which are very important selection tools to improve production parameters in the poultry industry, do not include cuticle quality. Nonetheless, various methods have been evaluated to assess cuticle quality i.e. the amount or degree of coverage on the eggshell surface and chemical composition, including cuticle staining, Attenuated total reflection Fourier transform infrared spectrometry (ATR-FTIR), tryptophan fluorescence and eggshell surface contact angle (hydrophobicity).

The outer eggshell cuticle hosts microbes but the nature of the endogenous egg microbiome remains unclear. For example, it is controversial whether the interior of the egg is sterile, although this is a frequent assumption. There is a distinction between the two main mechanisms of egg contamination: vertical transmission (transovarian route) during eggshell formation in the oviduct is contrasted with horizontal transmission (trans-shell penetration) that occurs following oviposition (86). Notably, the cuticle hosts commensal microbes from maternal origin that interact with environmental microbes. The combination from these microbial origins will form the cuticle/eggshell microbiota, which is hypothesized to participate in egg defense against harmful pathogens. The abundance and the phylogenetic diversity of the cuticle microbiota depends on many factors including wettability (presence of moisturizing factors, 3.1), surface structure (2.3) and biochemistry of the eggshell (2.4; 3.2), in addition to the climate, nesting environment (nature of the nest material) and parental care (peculiarities of incubation conditions and parental presence). Hence, the composition of the egg microbiome may vary across large ecological scales and result from multifactorial variables.

Once the cuticle is exposed to microbes, including both Gram-positive and Gram-negative strains, the probability of trans-shell infection post-oviposition is affected by local environmental conditions (especially when high temperature meets high relative humidity), the exposure period and the nature of eggshell microbiota (38–40). The subsequent contamination of the internal egg components by Gram-negative microbes, as frequently observed, is then dependent on the capacity for bacteria to penetrate the eggshell (motile capacity of non-clustering bacteria, moisture), followed by their resistance to the egg white antimicrobial activities, high pH and viscous nature (36)

Thus, the eggshell microbiota is an inheritance from both the maternal microbiota and the environment. At laying time, the egg surface is moist but will progressively dehydrate before and during incubation. The surface characteristics of the egg, the loss of the moisture layer, the availability of nutrients, and the presence of antimicrobial molecules (3.2) are thought to dictate the phylogenetic composition of the eggshell microbiota that will survive on the eggshell surface during incubation. All these interacting factors are crucial to influence the probability of trans-shell contamination of the egg. However, the dominant factor that is invariably associated with hatching failure in both wild and domestic species, is pathogen pressure from the environment and the subsequent bacterial load on the eggshell (38, 39, 87–91). Noticeably, the eggs of different avian species can support different bacterial densities and diversity, but hatching success remains similar between avian species, independent of incubation specificities (parental or remotely from parents). Such an observation suggests a complex but finely balanced regulation between all intrinsic and external components of egg incubation, in order to maintain hatching success at similar levels in all species (88). It also supposes adaptive mechanisms that have evolved in response to differences in climate and habitat.

The eggshell microbiome can theoretically participate in egg defense via multiple indirect and synergistic effects. Most hypotheses of such an activity are based on general mechanisms that have been described in other biological systems. However, compared to many biological systems, it is noteworthy that whereas there is a complex feedback between the microbiota and cellular immunity of the host, such cellular mediators are absent at the acellular eggshell surface.

The first mechanism by which the cuticle microbiota may participate in limiting pathogen establishment is the competition between commensal microbiota and environmental pathogens for adhesion to the eggshell surface. The microbiota from maternal origin may form a biofilm thereby limiting the accessibility of eggshell seeding sites for other microorganisms. Indeed, adhesion has been shown to be critical to understanding competition within microbial communities (139) and it is likely that such a competitive exclusion is occurring at the eggshell surface. A recent study demonstrated that removal of cuticle significantly increased number of adhering Salmonella Typhimurium cells on the outer eggshell surface indicating role of cuticle in modulating bacterial adherence (34). The cuticle layer experiences a sequential deposition of microbes, initially from the reproductive tract/cloacal segment and then from the external environment (93, 95, 96). These bacterial communities are thought to form the initial microbiota that will adhere to the eggshell surface and saturate adhesion sites, thereby blocking adhesion of opportunistic pathogens to the eggshell. In certain ecosystems, the formation of biofilms has been shown to confer an advantage to seeding bacteria that exploit the nutritive potential of organic particles that are present locally (140). Indeed, the competition for available nutrients is a potential additional mechanism by which eggshell bacterial core species may remain dominant. Generally, the uptake of nutrients by bacteria is constrained by the size of organic particles and their solubility in water. Therefore, the efficiency of assimilation of nutrients by microorganisms will depend on the secretion of microbial oxidoreductases and hydrolytic enzymes (141) that trigger the breakdown of organic particles to aid assimilation. Some cooperative interactions between microorganisms forming the dominant species may also exist (123), but have not yet been described for the eggshell microbiota. A third mechanism relies on the production of antimicrobial substances or metabolites including acidifying substances (lactic acid, acetic acid, formic acid, etc.) (142) that can act as potentiators of antimicrobial proteins and peptides (143), or antimicrobial molecules (ethanol, fatty acids, hydrogen peroxide and bacteriocins) (142). The presence of these substances in the microbial layer depends on the nature of the organisms characterizing the eggshell microbiome.

The cuticle affects eggshell wettability, water vapor conductance and regulates ultraviolet reflectance in various ground-nesting species.

An intact cuticle forms pore plugs that occlude the respiratory pores and is an effective physical barrier against microbial penetration (5, 17, 28, 29). The pore openings on the surface of the eggshell are covered by the proteinaceous cuticle, which extends into pores up to 50 μm to restrict bacterial entry (29, 177). The pore plugs permit water/gas exchange (35, 177) while physically impeding bacterial passage/penetration and restricting access to the egg interior. In addition to this physical defense, the eggshell cuticle components also function as a chemical barrier. Various cuticle proteins including OCX-32, OCX-36 and lysozyme C possess antimicrobial activity (24–26, 30–33). Moreover, a recent study showed that removal of cuticle using bleach treatment significantly increased the number of adhering Salmonella Typhimurium cells on the eggshell surface as compared to those with an intact cuticle (34). This evidence suggests that, in addition to functioning as a barrier to microbial migration through the respiratory pores, the cuticle also functions to reduce bacterial adherence (34).

Many environmental factors, through their direct influence on the gaseous environment of incubation, or indirectly, e.g. influencing the growth of pathogens that can contaminate the egg, have the potential to affect the presence of cuticle on eggs. A few lines of evidence already suggest that the cuticle is an evolutionarily labile structure that can vary greatly in relation to the nesting environment. Across Sauropsida, the clade that includes all amniotes except mammals, rigid eggshells evolved convergently (178, 179), thus, any external shell component would have also certainly been independently acquired and/or lost. A few decades ago, Board (1982) noted the presence of cuticle on eggs of certain species nesting in wet environments and suggested that these were an adaptation for waterproofing eggs in those nests (10). More recently, D’Alba et al. (42) provided the first comparative study considering the association between the eggshell cuticles and nesting ecology (42). This study suggested that the presence of cuticle is an ancestral trait that has been lost multiple times in birds but conserved in species that nest in hydric environments, where wet incubation sites expose eggs to a higher risk of embryo asphyxiation and microbial infection.

Exposure to solar radiation could also influence the presence of cuticle on eggs, as they prevent harmful ultraviolet wavelengths from reaching the embryo (155, 156). This effect could be particularly important in species that reproduce in exposed nests (birds), which receive more sunlight. In buried eggs, as in most non-avian reptiles, moist and acidic environments may increase the occurrence of eggshell corrosion (146) Thus, the cuticle could be an adaptation to counter this effect. Indeed, eggshells in these types of nests are characterized by cuticular layers (44) However, for the most part, the effect of environmental factors on evolution of cuticle deposition in reptiles is largely unknown, meaning that considerable work will be necessary to test these hypotheses.

Conventional cages are the most common production system for table eggs in many jurisdictions; however, many countries such as the United States and Canada are in the process of supplementing or replacing conventional cages with alternative housing systems such as free-run, free-range and enriched system (180, 181). In Europe, the use of conventional cages for laying hen has been banned since 1 January 2012 (182). This is mainly due to hen welfare concerns, arising from an increased interest in retail egg production systems from the consumers, egg producers, legislators, consumer groups as well as animal welfare organizations (181, 183). Conventional cages provide good health, hygiene, and low mortality, but restrict normal behaviors such as movement, stretching and wing-flapping; however, hen aggression and nesting behavior are modified in cage-free systems (184, 185). Previous study has shown higher cuticle coverage in free range, barn systems and cage eggs (180). This is in agreement with a recent study where better cuticle quality and lower Salmonella adherence was observed in eggs from free range systems as compared to enriched and free run (34). This observation could reflect lower bird stress levels in free range systems; however independent stress biomarkers were not measured in this study. Cuticle deposition requires a normal endocrine cascade and is susceptible to environmental stressors (56, 186). Birds under stress release adrenaline, which accelerates the transit time of the egg through the reproductive tract, leading to early termination of cuticle deposition in the shell gland (56, 186). Furthermore, animal handling also affects behavior and productivity in hen production systems (187–189). Poor egg production and quality is more prevalent in farms where the animals are roughly handled and frightened of humans (187, 188) due to the physiological changes associated with elevated stress (188, 190)

Studies in chicken have investigated the genetic contribution to cuticle variability, usually expressed as heritability. In general, the level of heritability is moderate, lying between 0.26 and 0.53 for a range of strains of hen from broiler breeders to layer strains producing both brown and white eggs (27, 85, 191). There was no evidence of negative or positive genetic correlation with production traits; in other words, the genes for determining cuticle deposition were not linked to traits such as egg laying rate, egg weight, eggshell breaking strength, and body weight at laying age etc. (27, 85, 191). This was not the case for the genetic correlation between the cuticle and egg shell color, where a link was observed. This interaction was different between lines and may reflect differences in the relationship between cuticle deposition and pigment deposition, which at least for a commercial brown egg layer, were independent events (55). However, that may not be true in every line. In terms of the Minolta L* value of an egg which measures luminance from light to dark or a* on the green-red axis there was evidence for a significant positive correlation of darkness and redness with cuticle deposition in a Rhode Island red (RIR) line (191). However, when a range of lines with different egg colors was compared there was non-significant genetic correlation in a RIR line between pigment and cuticle deposition, a positive genetic correlation in a white and tinted egg layer and a negative relationship in a white rock brown egg layer (85). So clearly, the relationship between pigment and cuticle varies between lines and may reflect simply differences in the timing and amount of deposition of both cuticle and pigment. Potentially the most troublesome relationship is the positive correlation observed in white egg layers between the Minolta b* value and cuticle deposition, which might result in a yellower and less white appearance when there is more cuticle (85). The most significant genetic correlation is that with hen age, which demonstrates that the cuticle quality measured in early eggs is valid for selection for those laid later in life, and therefore, only one measurement needs to be made (85).

Egg quality parameters, such as eggshell color, shell breaking strength, eggshell thickness, etc., decrease with hen age, which determines the end of productive life of a flock (12, 192). Some studies have shown that the thickness of the cuticle is also dependent on hen age and has been reported to decrease with increasing age of the hen (17, 20, 180, 193, 194); however, this aspect remains controversial. Older hens at the end of the laying cycle (about 70 weeks) lay eggs with a lesser amount and patchier distribution of cuticle. This greatly increases eggshell permeability to water and can make eggs more susceptible to trans-shell bacterial contamination (20). Similarly, other studies have confirmed a poor cuticle coverage in eggs from older hens (37, 83). Additionally, many recent studies have also confirmed an age-related decline in cuticle deposition (17, 20, 180, 194). For example, completeness of cuticle coverage was higher in eggs from the 44-week-old flock as compared to 64- or 73-week-old flock (180). Another study validated less cuticle on eggs laid by hens at 60 weeks of age as compared to those from 25-week-old hens (194). These studies used cuticle protein staining to validate an age-related diminishment in cuticle. Furthermore, a combination of both cuticle staining and infrared spectroscopy (ATR-FTIR used for measurement of cuticle chemical composition, see 2.4, 2.6) confirmed that cuticle coverage and chemical composition are dependent on hen age (17, 20). Eggs from younger hens (up to 48-week-old) had better cuticle quality and higher protein signals as compared to older hens (up to 70-week-old) (17). However, previous findings showed that there is no effect of hen age on the cuticle coverage and a greater diversity occurs in cuticle deposition among hens (82). These findings are consistent with a recent study that did not observe an age-related decline in the cuticle coverage. In this study, cuticle deposition was evaluated in hens from 24-50-week of age (81), while most of the previous studies observed an age-related decline beyond 50-week of hen age (17, 20, 180, 194). A major limitation of former studies (17, 20, 194) is that cuticle measurements were not carried out on eggs from the same hens. Observations from different birds from different flocks and at different ages increases a possibility of finding conflicting evidence due to enhanced variability. Future studies where eggs from individual hens are followed for a long duration is recommended to determine if cuticle deposition and its chemical composition decline with age (81). This is particularly important, since laying persistency is a major trait currently being developed further in laying hens. The “long life” layer, which will be capable of producing 500 eggs in a laying cycle of 100 weeks, is on the horizon (192).

There are natural variations in the amount/thickness of cuticle present on eggshell surface and its presence/stability is affected by factors including egg freshness, and commercial washing (17, 20, 27, 83).