Changes in hen performance and selection for improved egg quality

In the early 20^th century, hens matured between at six and nine months and produced 100 to 180 eggs per annum depending on the month in which they laid their first egg (Romanoff and Romanoff, Reference ROMANOFF and ROMANOFF1949). A combination of improvements in genetics and nutrition, the use of artificial lighting programmes and appropriate rearing systems has seen the number of eggs per bird rise from 130 per annum in 1940, to 260 per annum by 1980, and to > 300 per annum by 2000.

In the 1950's, the focus of breeding companies was on increasing egg numbers and improving feed efficiency. Egg quality traits have received attention only more recently, mainly to avoid any deterioration in eggshell quality or yolk content. Brown egg laying lines, in contrast, have been selected over a shorter time period than American white lines, and so for the European market, where consumers prefer brown eggs, breeding companies continued to focus on improving egg production and feed conversion throughout the 1980's. By the early 1990's, improvements in the region of +30 eggs, +2 kg egg mass and -35 g/kg eggs/yr feed conversion were obtained by selecting for earlier sexual maturity and decreased feed consumption resulting in lower hen weight gain (Nys et al., Reference NYS, BURLOT and DUNN2008).

Concerning egg quality, the main effort of breeding companies between 1981 and 1991 was to control egg weight with hen age. Improvements in eggshell colour and thickness were considered during this period, principally to avoid any detrimental effects resulting from selection on egg number. During the next 10 years, feed efficiency was further improved by slightly lowering the hen weight but mainly by increasing the egg mass. Egg number was increased (+26 eggs) because of earlier sexual maturity, higher peak production and better persistency in lay (25 weeks with more than 90% production in 2001). The improvement in egg mass (180 g/hen/year) and reduction in feed intake (24 g/kg of eggs) was obtained without changing mean egg weight.

From 1995, as a result of the increased demand for liquid egg products but also as a consequence of the requirements from certified production systems, including higher quality products (e.g. ‘label’ appellation), breeding companies started to include the technological properties of egg white (Haugh unit; HU) in their selection programmes. Any decrease in egg yolk/white ratio which might result from selection for egg quality has been continuously controlled. From 2000, the focus has been on improved bird viability in alternative systems of management in response to the proposed European ban on the traditional layer cage which came into force on 1^st January 2012. The current focus is now on improved sustainability of production systems, through selection for improved persistency in lay over a longer laying period (more than 80 weeks). It is well established that egg quality (shell, Haugh unit) decreases with bird age, therefore a prerequisite for keeping hens in production longer will be to reduce the number of downgraded eggs by selecting for older hens (more than 65 weeks) which exhibit good eggshell and albumen quality (HU) characteristics including a lower incidence of blood spots, a criterion considered as a priority for certified alternative production.

Recently, breeding companies have started to show interest in the development of genetic markers for novel eggshell quality traits and to localise genetic variability for egg quality traits to specific regions of a chromosome using genome scans to identify loci using medium density microsatellite maps or candidate gene approaches (Tuiskula-Haavisto et al., Reference TUISKULA-HAAVISTO, HONKATUKIA, VILKKI, DE KONING, SCHULMAN and MAKI-TANILA2002; Hocking, Reference HOCKING2005; Honkatukia et al., Reference HONKATUKIA, REESE, PREISINGER, TUISKULA-HAAVISTO, WEIGEND, ROITO, MAKI-TANILA and VILKKI2005a; Dunn et al., Reference DUNN, JOSEPH, BAIN, EDMOND, WILSON, MILONA, NYS, GAUTRON, SCHMUTZ, PREISINGER and and WADDINGTON2009; Dunn, Reference DUNN2011; Dunn et al., Reference DUNN, RODRÍGUEZ-NAVARRO, McDADE, SCHMUTZ, PREISENGER, WADDINGTON, WILSON and BAIN2012). One of the great successes of this approach was for a Mendelian trait. The discovery of a mutation on chromosome 8 (SNP) in the FMO3 gene associated with fishy odour (Honkatukia et al., Reference HONKATUKIA, TUISKULA-HAAVISTO, DE KONING, VIRTA, MAKI-TANILA and VILKKI2005b) has been used for commercial elimination of this defect. It seems likely that the next advance will be the use of genome-wide breeding values for individual birds using low cost genotyping combined with large scale phenotyping of egg quality traits (Albers, Reference ALBERS2010; Dunn, Reference DUNN2011; Preisinger, Reference PREISINGER2012).

The eggshell – new insights into its formation and structure

The eggshell of the hen is a highly ordered polycrystalline structure and was first described by Von Nathusius (1821-1899) and translated and edited by Tyler (Reference TYLER1964). The development of scanning electronic microscopy in the 1970's confirmed the early observations of these authors (Simons, Reference SIMONS1970; Hamilton, Reference HAMILTON1986; Solomon, Reference SOLOMON1991, Arias et al., Reference ARIAS, FINK, XIAO, HEUER and CAPLAN1993; Nys et al., Reference NYS, HINCKE, ARIAS, GARCÍA-RUIZ and SOLOMON1999). In essence, the hen's eggshell is composed of six layers. The inner and outer shell membranes form the innermost two layers. The inner zone of the calcified part of the shell is composed of irregular cones or mammillary knobs, the tips of which are penetrated by the outer membrane fibres. The palisade layer, which gives rise to the thickest part of the calcified shell, extends beyond the fused bases of the mammillary (knob) layer and ends in a thin vertical crystal layer where the crystallite crystals are aligned perpendicular to the shell surface. The outermost layer of the shell, the cuticle, is an organic layer deposited on the surface of the vertical crystal layer. It contains a thin film of hydroxyapatite crystals in its inner zone (Dennis et al., Reference DENNIS, XIAO, AGARVAL, FINK, HEUER and CAPLAN1996), and the bulk (two-thirds) of the superficial eggshell pigments (Nys et al., Reference NYS, ZAWADZKI, GAUTRON and MILLS1991). All avian eggshells share the same mineral component, namely the trigonal phase of calcium carbonate (CaCO₃) known as calcite, which is the more stable polymorph at room temperature. The calcite crystals within the hen's eggshell show a preferred orientation (Sharp and Silyn-Roberts, Reference SHARP and SILYN-ROBERTS1984), the C axis tending to be perpendicular to the eggshell surface in the upper region of the shell (García-Ruiz and Rodríguez-Navarro, Reference GARCÍA-RUIZ and RODRÍGUEZ-NAVARRO1994). This high degree of control of size, shape and orientation of the crystals of calcite is responsible for the eggshells unique ultra structure and exceptional mechanical properties (egg breaking strength for a domestic hen egg is 35 N for a mean eggshell thickness of 0.33 mm). Recent evidence indicates that there is a strong genetic basis to the crystal size and orientation in eggs from pedigree laying lines (Dunn et al., Reference DUNN, RODRÍGUEZ-NAVARRO, McDADE, SCHMUTZ, PREISENGER, WADDINGTON, WILSON and BAIN2012), suggesting that it may be possible to incorporate these traits into selection programmes in the future.

The thickness of the eggshell, its form and size and structural elements, as well as features of the porous system varies among different species, however, the general structure of the eggshell is basically the same in all birds (Romanoff and Romanoff, Reference ROMANOFF and ROMANOFF1949; Board, Reference BOARD1982; Mikhailov, Reference MIKHAILOV1997). The eggshell forms at body temperature in a confined space, within the distal segment of the oviduct in an acellular uterine fluid that is super-saturated with calcium and bicarbonate (80 - 120 times greater than the solubility product of calcite; Nys et al., Reference NYS, ZAWADZKI, GAUTRON and MILLS1991) and which contains the organic precursors of the shell matrix. Calcium carbonate precipitates spontaneously from this saturated milieu in the form of calcite, the polymorph promoted by the presence of the organic constituents (Hernandez-Hernandez et al., Reference HERNANDEZ-HERNANDEZ, VIDAL, GOMEZ-MORALES, RODRÍGUEZ-NAVARRO, LABAS, GAUTRON, NYS and GARCÍA-RUIZ2008). The initial radial crystal growth occurs at specific seed sites on the outer shell membranes which consist of organic aggregates, and then continues outwards and upwards to form the mammillary knob layer. Subsequently, adjacent mammillae fuse to form the base of the palisade layer where radial growth is inhibited due to competition for space. Only crystals growing approximately perpendicular to the surface can continue to elongate, explaining the observation of a preferred orientation in the upper region of the shell (García-Ruiz and Rodríguez-Navarro, Reference GARCÍA-RUIZ and RODRÍGUEZ-NAVARRO1994). The anisotropic growth of calcite crystals, which is a prerequisite for this hypothesis, is due to a control of crystal morphology by the components of the organic matrix (Chien et al., Reference CHIEN, HINCKE, VALI and MCKEE2008; Hernandez-Hernandez et al., Reference HERNANDEZ-HERNANDEZ, VIDAL, GOMEZ-MORALES, RODRÍGUEZ-NAVARRO, LABAS, GAUTRON, NYS and GARCÍA-RUIZ2008). The characterisation of the eggshell matrix proteins and the organic precursors in the uterine fluid was initiated in the 1990's. These matrix proteins, including the specific uterine ovocalyxins and ovocleidins, have since been described in numerous reviews (Nys et al., Reference NYS, HINCKE, ARIAS, GARCÍA-RUIZ and SOLOMON1999; Reference NYS, GAUTRON, GARCÍA-RUIZ and HINCKE2004; Hincke et al., Reference HINCKE, WELLMAN-LABADIE, MCKEE, GAUTRON, NYS and MANN2008; Reference HINCKE, NYS, GAUTRON, MANN, RODRÍGUEZ-NAVARRO and MCKEE2012) as has the in vitro and in vivo evidence which demonstrates their role in controlling the formation of the eggshell.

The protein composition of the egg

The egg, including the shell, contains a large range of nutrients and bioactive molecules that are essential for the development of a reproductive cell into a mature chick. Until 2000, less than 15 of these proteins had been biochemically characterised in the albumen, and less than five in the yolk (Burley and Vadehra, Reference BURLEY and VADEHRA1989; Li-Chan et al., Reference LI-CHAN, POWRIE and NAKAI1995; Sugino et al., Reference SUGINO, NITODA and JUNEJA1997). Up until this point, egg-related proteins could only be studied using classical biochemical techniques although this was later expanded by the development of molecular tools in the 1990's. The functional relevance of only a fraction of these proteins had been determined, for example, of the 13 major egg white proteins identified, lysozyme had been functionally assigned as an glycoside hydrolase, ovomucoid and ovoinhibitor, cystatin as a protease inhibitor and ovotransferrin, ovoflavoprotein and avidin were functionally categorised as having either mineral- or vitamin-binding capacity (Li-Chan et al., Reference LI-CHAN, POWRIE and NAKAI1995). For the egg yolk proteins, lipoproteins (Low Density Lipoproteins, HDL), livetins and phosvitins were successfully purified and characterised. IgG-like antibodies (IgY) found in the γ-livetin fraction have since been discovered and numerous applications for these have been developed in pharmacology and diagnostics (Schade et al., Reference SCHADE, ZHANG and TERLOZO2007; Hatta et al., Reference HATTA, KAPOOR and JUNEJA2008).

The development of high-throughput methods (proteomics and transcriptomics) used in combination with the chicken genomic sequence (International Chicken Genomic Sequence Consortium, 2004) together with the development of bioinformatics tools to identify genes and ascribe function has since revealed that there are actually hundreds of minor proteins in the egg and its shell (Gautron et al., Reference GAUTRON, RÉHAULT-GODBERT, NYS, MANN and RIGHETTI2011). For example, molecular biochemistry has revealed more than 10 specific matrix proteins, the ovocalyxins and ovocleidins (Nys et al., Reference NYS, GAUTRON, GARCÍA-RUIZ and HINCKE2004), in the eggshell. However, the most surprising information has came from a recent study which examined the entire eggshell proteome and revealed the presence of 500 proteins in the eggshell matrix (Mann et al., Reference MANN, MAČEK and OLSEN2006). A transcriptomic study has revealed that there are 600 over-expressed genes in the uterus (responsible for eggshell formation) (Jonchère et al., Reference JONCHÈRE, RÉHAULT-GODBERT, HENNEQUET-ANTIER, CABAU, SIBUT, COGBURN, NYS and GAUTRON2010). The same technologies have also revealed about 200 different proteins in the egg white (Mann, Reference MANN2007; D'Ambrosio et al., Reference D'AMBROSIO, ARENA, SCALONI, GUERRIER, BOSCHETTI, MENDIETA, CITTERIO and RIGHETTI2008) and more than 800 specifically expressed genes in the magnum (Gautron et al., Reference GAUTRON, RÉHAULT-GODBERT, NYS, MANN and RIGHETTI2011), about 300 proteins in egg yolk (Mann and Mann, Reference MANN and MANN2008) and vitelline membrane (Mann, Reference MANN2008) and hundreds of specifically expressed genes in the liver and the infundibulum. Many of these proteins are currently classified according to their putative biological function using bioinformatics by analogies with molecules previously characterised in various tissues of birds or mammals (Mine and D'Silva, Reference MINE and D'SILVA2008; Rehault-Godberg et al., 2011). These putative functions include a large range of activities (cell metabolism and regulation, protein synthesis and folding, ionic transport, antimicrobial or antioxidant activities, proteases and antiproteases). This screening of egg proteins will mobilise numerous scientists to examine the putative function of these components in coming years. The following step will be to purify a selection of molecules identified as having high potential to confirm their functional properties and to provide new insights into egg-related biology and technology (embryonic development, protective or technological properties). This strategy would allow the future development of novel proteins which could be used by the pharmaceutical and food industries. Alternatively, some of these proteins might also be used as markers to select hens producing eggs of higher quality, using single nucleotide polymorphisms (SNP) of corresponding genes, in particular to reduce the risk of food-borne disease outbreaks for humans. Some success in this respect has been made with the association between ovocleidin 116, shell thickness (Dunn et al., Reference DUNN, JOSEPH, BAIN, EDMOND, WILSON, MILONA, NYS, GAUTRON, SCHMUTZ, PREISINGER and and WADDINGTON2009) and crystal orientation (Dunn et al., Reference DUNN, RODRÍGUEZ-NAVARRO, McDADE, SCHMUTZ, PREISENGER, WADDINGTON, WILSON and BAIN2012) as well as ovocalyxin 32 with crystal orientation (Dunn et al., Reference DUNN, RODRÍGUEZ-NAVARRO, McDADE, SCHMUTZ, PREISENGER, WADDINGTON, WILSON and BAIN2012), mammillary layer thickness (Dunn et al., Reference DUNN, JOSEPH, BAIN, EDMOND, WILSON, MILONA, NYS, GAUTRON, SCHMUTZ, PREISINGER and and WADDINGTON2009) and deformation (Takahashi et al., Reference TAKAHASHI, SASAKI, NIRASAWA and FURUKAWA2010).

Improvements in our knowledge relating to egg microbiology

Eggs contents are generally sterile at oviposition, but can become contaminated by micro-organisms, including spoilage organisms and pathogenic bacteria. There are two possible and thoroughly studied routes causing bacterial egg contamination: transmitted either vertically or horizontally. In the vertical or transovarian route, the egg content becomes contaminated during its formation in the ovary or oviduct. In the horizontal route, after oviposition, micro-organisms present on the eggshell surface penetrate the eggshell.

Egg contamination by Salmonella enteritidis (SE) is currently one of the most important causes of foodborne gastroenteritis in humans throughout the world. SE seems to have a special capability to colonise the hen's reproductive tract and to survive in the egg albumen (Raspoet et al., Reference RASPOET, GANTOIS, DEVLOO, PASMANS, HAESEBROUCK, DUCATELLE and VAN IMMERSEEL2011). Although studies have showed that eggs can be contaminated by SE via penetration of the eggshell (De Reu et al., Reference DE REU, GRIJSPEERDT, MESSENS, HEYNDRICKX, UYTTENDAELE, DEBEVERE and HERMAN2006a), several studies support the idea that the transovarian route may be more important (Raspoet et al., Reference RASPOET, GANTOIS, DEVLOO, PASMANS, HAESEBROUCK, DUCATELLE and VAN IMMERSEEL2011).

The eggshell surface becomes bacterially contaminated with 10⁴ to 10⁶ CFU/egg as it passes through the vent and comes into contact with the environment (De Reu et al., Reference DE REU, MESSENS, HEYNDRICKX, RODENBURG, UYTTENDAELE and HERMAN2008). Because of their tolerance for dry conditions, the microflora on the surface of the eggshell are dominated by Gram-positive bacteria. With the introduction of alternative housing systems for laying hens in the EU, recent research has focussed on the influence of housing system on contamination of the eggshell (De Reu et al., Reference DE REU, MESSENS, HEYNDRICKX, RODENBURG, UYTTENDAELE and HERMAN2008).

The internal contents generally remain sterile after oviposition because of eggs’ natural defences. Many researchers have shown that the cuticle, the shell and shell membranes are important barriers preventing micro-organisms entering the internal egg environment at this time (De Reu et al., Reference DE REU, GRIJSPEERDT, MESSENS, HEYNDRICKX, UYTTENDAELE, DEBEVERE and HERMAN2006a). In addition, the egg albumen has important antimicrobial defences (lysozyme, ovotransferrin, physico-chemical properties). Besides Salmonella (especially SE) other major contaminants of the egg contents include Gram-negative bacteria, including E. coli, Alcaligenes spp., Pseudomonas spp. and Proteus spp., and, to a lesser extent, Gram-positive bacteria such as Staphylococcus spp. and Bacillus spp. (De Reu et al., Reference DE REU, HEYNDRICKX, GRIJSPEERDT, RODENBURG, TUYTTENS, UYTTENDAELE, DEBEVERE and HERMAN2006b). It is generally considered that that Gram-negative bacteria are better equipped to overcome the antimicrobial defences of the albumen.

The microbiota of egg products depends essentially on that of the raw material (egg), the nature of the product (e.g. liquid, dried, concentrated, added salt) and is strongly influenced by the processes of transformation and stabilisation that are applied. Pasteurisation, for example, significantly reduces the number of Enterobacteriaceae (e.g. E. coli, Pseudomonas, Salmonella). The egg product industry focuses particularly on controlling heat-resistant and spore forming psychotropic bacteria, depending on the transformation and/or stabilisation processes used (Baron and Jan, Reference BARON and JAN2011).

New developments in measuring egg freshness

Freshness makes a major contribution to egg quality and grading; however, the only quantitative parameter for egg freshness evaluation provided by European Union regulation is air cell height (EU, 2003), whereas U.S. grading requirements also consider thick albumen height, expressed as HU (USDA, 1995). These indices have been questioned because of their dependence on egg weight and laying-hen age, respectively.

Chemical methods of assessing egg freshness, such as HPLC quantification of uridine and pyroglutamic acid in albumen, are available but the high natural variability in these parameters has limited their application (Rossi et al., Reference ROSSI, POMPEI and HIDALGO1995). Very promising results however can be obtained by measuring furosine in albumen, as an index of Maillard reaction development during egg aging (Hidalgo et al., Reference HIDALGO, ROSSI and POMPEI1995), and by applying low resolution proton nuclear magnetic resonance (LR-H-NMR) to evaluate the thinning phenomenon of albumen (Capozzi el al., 1999). These methods, although sensitive, both suffer from the need for specific and expensive analytical equipments, and the former is also time consuming. Better potential as a routine control analysis is found in the spectrophotometric rapid test of the reaction between albumen and 3,3’,5,5’-tetramethylbenzidine (Rossi et al., Reference ROSSI, HIDALGO and POMPEI2001), which gives a response proportional to the level of iron passing from yolk to albumen during egg storage. Karoui et al. (Reference KAROUI, KEMPS, BAMELIS, DE KETELAERE, MERTEN, SCHOONHEYDT, DECUYPERE and DE BAERDEMAEKER2006), using front-face fluorescence, observed that fluorescent spectra recorded on thick and thin egg albumen can potentially be used to differentiate between fresh and aged eggs.

Non-destructive methods for determining egg freshness in on-line sorting machines have recently been reviewed by Mertens et al. (Reference MERTENS, KEMPS, PERIANU, DE BAERDEMAEKER, DECUYPERE, DE KETALAERE and BAIN2011). Ragni et al. (Reference RAGNI, BERARDINELLI and GUARNIERI2008) has developed PLS models based on the dielectric spectra for the prediction of the air cell height and thick albumen height. Wang et al. (Reference WANG, WANG, ZHOU and LU2009) monitored the internal egg quality (HU) using an electronic nose while Kemps et al. (Reference KEMPS, DE KETELAERE, BAMELIS, MERTENS, DECUYPERE, DE BAERDEMAEKER and SCHWÄGELE2007) evaluated the possibility of combining LR-H-NMR and visible near-infrared (VIS-NIR) transmission spectroscopy for the assessment of albumen freshness. These authors concluded that the combination of both techniques did not improve the accuracy of prediction when compared to the sole VIS-NIR. Giunchi et al. (Reference GIUNCHI, BERARDINELLI, RAGNI, FABBRI and SILAGHI2008) used Fourier transformed (FT) NIR reflectance spectroscopy to predict air cell height, thick albumen height and HU, and obtained 100% eggs prediction of days of storage. Zhao et al. (Reference ZHAO, LIN, CHEN, HUANG, SUN and ZHOU2010) measured the NIR reflectance spectra of eggs and, using a support vector data description algorithm, found that this correctly discriminated 93.3% of fresh and aged eggs according to their HU. VIS-NIS transmission spectroscopy was successfully applied by Abdel-Nour et al. (Reference ABDEL-NOUR, NGADI, PRASHER and KARIMI2011) - the spectral data in this case was compared to the egg HU and albumen pH. Prediction correlation coefficients up to 0.94 were obtained in this study. In conclusion, spectroscopic techniques appear to have the potential for measuring egg freshness, potentially on the packing line.

Modifying the nutritional value of eggs

In 1934, Cruickshank observed that the hen's diet modifies the unsaturated fatty acid composition of the yolk, whilst the saturated fatty acid composition was less affected (Cruickshank, Reference CRUICKSHANK1934). Since then, there have been many attempts to increase the proportion of unsaturated fatty acids, mainly linoleic acid, and the unsaturated/saturated fatty acids (U:S) ratio. During the eighties, human nutritionists and health experts advised consumers to increase the U:S ratio in their diet by increasing their polyunsaturated fatty acid (PUFA) intake. As a consequence, several studies were undertaken in the egg sector to achieve this goal. During the nineties, the British Nutrition Foundation and the Department of Health, stressed that not all the PUFA's had the same effect and that all saturated fatty acids were atherogenic. The recommendation at that time was therefore to increase the intake of n-3 PUFA instead of n-6 PUFA in order to reach a n-6:n-3 ratio lower than 6:1. As a result, research was directed towards the enrichment of eggs with n-3 PUFA by feeding hens with vegetable or animal products rich in these specific types of fatty acid.

The addition of rapeseed or linseed oils to the hen's diet resulted in a substantial increase in the n-3 PUFA content, mainly consisting of alpha-linolenic acid in the egg yolk with a small increase in long chain n-3 PUFA's such as eicosapentaenoic (EPA) and docosahesaenoic acids (DHA) (Caston and Leeson, Reference CASTON and LEESON1990; Cherian and Sim, Reference CHERIAN and SIM1991; Meluzzi et al., Reference MELUZZI, SIRRI, TALLARICO and FRANCHINI2001).

Several attempts have been then made to enhance the proportion of long chain n-3 PUFA (mainly DHA and EPA) in the yolk by including fish oil or marine algae in the diet of laying hens (Farrell, Reference FARRELL1994; Sirri et al., Reference SIRRI, MELUZZI, TALLARICO, FRANCHINI and IAFFALDANO2001, Sirri and Meluzzi, Reference SIRRI and MELUZZI2011). However, when the yolk was enriched with these long chain n-3 PUFA's, the arachidonic acid (n-6 PUFA) content, which is synthesised from linoleic acid (n-6 PUFA), decreased due to the larger utilisation of delta-6-desaturase in the n-3 pathway rather than in the n-6 pathway.

The concept of enriched food has been applied to the enhancement of the vitamin content of eggs. In general, as the fat-soluble vitamin concentration of the feed increases, so does the vitamin content of the yolk. In this respect vitamin E has been studied most due to its important role in human health because of its antioxidant properties. It has been shown that the alpha-tocopherol (AT) content of fresh eggs can be increased by the inclusion of alpha-tocopheryl acetate (ATA) in the diet up to doses of 20 g ATA/kg feed (Surai et al., Reference SURAI, IONOV, BUZHIN and BUZHINA1995; Grobas et al., Reference GROBAS, MENDEZ, LOPEZ BOTE, DE BLAS and MATEOS2002). Above this level the AT transfer efficiency from the diet to the yolk decreases. This also happens when high levels of unsaturated fats are added to the hen's diet (Meluzzi et al., Reference MELUZZI, SIRRI, MANFREDA, TALLARICO and FRANCHINI2000; Sirri and Barroeta, Reference SIRRI and BARROETA2007).

Vitamin A is important since it is involved in numerous processes (vision, integrity of mucous membrane, reproduction, immune response, bone development). Vitamin A levels in the egg yolk can be increased using diets supplemented with beta-carotene and retinyl acetate (Naber, Reference NABER1993; Jiang et al., Reference JIANG, MCGEACHIN and BAILEY1994).

Vitamin D is important in the formation and maintenance of bone. Mattila et al. (Reference MATTILA, ROKKA, KONKO, VALAJA, ROSSOW and RYHANEN2003) increased the yolk vitamin D content by enhancing the supplementation of dietary cholecalciferol in the hen diet.

In summary, the fortification of the hen diet with PUFA and vitamins has resulted in the production of eggs with higher nutritive value which had, in turn, a direct positive impact on not only satisfying human daily nutrients requirements, but in improving the health of consumers.

The cholesterol story

The first observation of a link between blood cholesterol and death from coronary heart disease (CHD) was made a century ago, but this hypothesis was not confirmed until 50 years later by large scale epidemiological studies (Martin et al., Reference MARTIN, HULLEY, BROWNER, KULLER and WENTWORTH1986; Griffin, Reference GRIFFIN2011). Eggs contain a large amount of cholesterol (around 200 mg/egg). In the 1970s-80s it was demonstrated that a very high daily consumption of eggs significantly increased plasma LDL cholesterol in humans. This form of cholesterol is a determinant of health risk due to its regulation by cell internalisation through the LDL receptor pathway (Brown and Goldstein, Reference BROWN and GOLDSTEIN1986). The link between eggs and dietary cholesterol resulted in a dramatic reduction in egg consumption in the 1980s and, at the same time, stimulated numerous experiments to try and reduce the level of cholesterol in eggs. However, while enrichment of egg is easily achieved by modifying the hen's diet, it was soon discovered that it was much more difficult, even impossible, to decrease the cholesterol content of eggs by nutritional or alternative means (Griffin, Reference GRIFFIN1992).

Lipids and protein precursors in the yolk issue from intact lipoproteins synthesised in the liver, which have a very stable composition in the hen (Nys and Guyot, Reference NYS and GUYOT2011). Over 95% of yolk cholesterol is associated with the yolk triglyceride-rich lipoproteins, 80% being non-esterified and linked to phospholipids on the surface of very low density lipoproteins (VLDL). In otherwords, the cholesterol content of yolk is determined mainly by the cholesterol content of the VLDL, and not by the cholesterol levels in the hen's plasma (Griffin, Reference GRIFFIN1992). The proportion of yolk versus white in an egg appears to be important in this situation. The presence of free cholesterol is crucial for cell metabolism and reproductive physiology, and any drastic reduction of plasma cholesterol by pharmacological means can impair egg formation and production (Griffin, Reference GRIFFIN1992; Nau et al., Reference NAU, NYS, YAMAKAWA and REHAULT-GODBERT2010). In 2000, the scientific evidence linking dietary cholesterol with serum cholesterol and CHD was re-appraised. Moreover, it was demonstrated that dietary cholesterol exerts a relatively minor and clinically insignificant effect on serum LDL cholesterol and the risk of CHD risk compared to the role of saturated fats and other CHD risk factors (Kritchevsky, Reference KRITCHEVSKY2000; Howell, Reference HOWELL2000, Herron et al., Reference HERRON, VEGA-LOPEZ, CONDE, RAMJIGANESH, SHACHTER and FERNANDEZ2003; Griffin, Reference GRIFFIN2011). In addition, it was shown that some of the components of eggs e.g. ovomucin (Nagaoka et al., Reference NAGAOKA, MASAOKA, ZHANG, HASEGAWA and WATANABE2002) and sphingomyelin (Noh and Koo, 2003), were able to reduce the intestinal cholesterol absorption in mammals. In summary, the daily intake of one to two eggs is no longer associated either with an increased risk of cholesterolemia or CHD (McNamara, Reference MCNAMARA2002) in the majority of the human population. Hyper-responders however still need to be cautious (Knopp et al., Reference KNOPP, RETZLAFF, WALDEN, DOWDY, TSUNEHARA, AUSTIN and NGUYEN1997; Herron et al., Reference HERRON, VEGA-LOPEZ, CONDE, RAMJIGANESH, SHACHTER and FERNANDEZ2003) with eggs as with all foods, and consumers should follow the national safety recommendations provided by their country's food standard agency or the equivalent.

Advances in egg product processing and functional properties

The egg is a major multifunctional food ingredient. The proteins, lipids and lipoproteins are responsible for these functionalities, due to their biochemical and structural characteristics, and the supramolecular assemblies they form either naturally or after processing. Nowadays, the major challenge is to control these supramolecular assemblies, to understand the structure-functional relationship, to enhance the quality of existing products and to imagine innovative products.