Validated quantitative trait loci for eggshell quality in experimental and commercial laying hens.

Compromised eggshell quality causes considerable economic losses for the egg industry. Breeding for improved eggshell quality has been very challenging. Eggshell quality is a trait that would greatly benefit from marker-assisted selection, which would allow the selection of sires for their direct contribution to the trait and would also allow implementation of measurements integrating a number of shell parameters that are difficult to measure. In this study, we selected the most promising autosomal quantitative trait loci (QTL) affecting eggshell quality on chromosomes 2, 3, 6 and 14 from earlier experiments and we extended the F2 population to include 1599 F2 females. The study was repeated on two commercial populations: Lohmann Tierzucht Rhode Island Red line (n = 692 females) and a Hy-Line White Plymouth Rock line (n = 290 progeny tested males). We analyzed the selected autosomal QTL regions on the three populations with SNP markers at 4-13 SNPs/Mb density. QTL for eggshell quality were replicated on all studied regions in the F2 population. New QTL were detected for eggshell color on chromosomes 3 and 6. Marker associations with eggshell quality traits were validated in the tested commercial lines on chromosomes 2, 3 and 6, thus paving the way for marker-assisted selection for improved eggshell quality.

Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?

In the past 50 years, selection starting initially at the breed level and then using quantitative genetics coupled with a sophisticated breeding pyramid, has resulted in a very productive hybrid for a variety of traits associated with egg production. One major trait currently being developed further is persistency of lay and the concept of the "long life" layer. Persistency in lay however cannot be achieved without due consideration of how to sustain egg quality and the health and welfare of the birds in longer laying cycles. These multiple goals require knowledge and consideration of the bird's physiology, nutritional requirements, which vary depending on age and management system, reproductive status and choice of the selection criteria applied. The recent advent of molecular genetics offers considerable hope that these multiple elements can be balanced for the good of all in the industry including the hens. The "long life" layer, which will be capable of producing 500 eggs in a laying cycle of 100 weeks, is therefore on the horizon, bringing with it the benefits of a more efficient utilisation of diminishing resources, including land, water, raw materials for feed as well as a reduction in waste, and an overall reduced carbon footprint.

Enhancing the egg's natural defence against bacterial penetration by increasing cuticle deposition.

The cuticle is a proteinaceous layer covering the avian egg and is believed to form a defence to microorganism ingress. In birds that lay eggs in challenging environments, the cuticle is thicker, suggesting evolutionary pressure; however, in poultry, selection pressure for this trait has been removed because of artificial incubation. This study aimed to quantify cuticle deposition and to estimate its genetic parameters and its role on trans-shell penetration of bacteria. Additionally, cuticle proteins were characterised to establish whether alleles for these genes explained variation in deposition. A novel and reliable quantification was achieved using the difference in reflectance of the egg at 650 nm before and after staining with a specific dye. The heritability of this novel measurement was moderate (0.27), and bacteria penetration was dependent on the natural variation in cuticle deposition. Eggs with the best cuticle were never penetrated by bacteria (P < 0.001). The cuticle proteome consisted of six major proteins. A significant association was found between alleles of one of these protein genes, ovocleidin-116 (MEPE), and cuticle deposition (P = 0.015) and also between alleles of estrogen receptor 1 (ESR1) gene and cuticle deposition (P = 0.008). With the heritability observed, genetic selection should be possible to increase cuticle deposition in commercial poultry, so reducing trans-generational transmission of microorganisms and reversing the lack of selection pressure for this trait during recent domestication.

Adhesion mechanisms in European whitefish Coregonus lavaretus eggs: is this a survival mechanism for high-energy spawning grounds?

This study examined the potential biochemical and mechanical structures that may contribute to egg adhesion in European whitefish Coregonus lavaretus. Experiments showed that eggs from a population of C. lavaretus from Loch Eck remained non-adhesive in a solution chemically similar to ovarian fluid but became adhesive seconds after contact with water. Examination of the ultrastructure of the chorion showed that the morphology changed significantly after contact with water, with nodule-like protuberances attached to connective filaments on the surface present in water-hardened but not non-water hardened eggs. Biochemical analysis showed the presence of Chain A, RNase ZF-3e proteins in the chorion of water-hardened but not non-water hardened eggs. Histochemical staining of the chorion of C. lavaretus eggs showed that the externa, but not the interna, stained positively for the presence of glycoproteins. From these results, it was concluded that C. lavaretus from Loch Eck possess both anatomical and biochemical adhesive mechanisms that have been undocumented in this species so far.

Structural and physical changes in the hen's eggshell in response to the inclusion of dietary organic minerals.

1. An experiment was designed to determine whether the inclusion of sodium selenite, Sel-Plex or Sel-plex + Bioplex in the diet exerted any influence on structural and functional properties of the eggshell at 22, 38, 58 and 72 weeks age. 2. Significant age effects were demonstrated for egg weight, dynamic stiffness and total shell thickness. 3. The eggshells from birds fed on the Sel-Plex + Bioplex treatment were statistically stronger than those produced by the birds on sodium selenite. 4. From mid-lay onwards the shells laid by birds fed on the Sel-Plex + Bioplex treatment displayed, at ultrastructural level, the crystal modification termed 'confluence' together with early fusion of the palisade columns which comprised the bulk of the shell. 5. At all periods of lay the shells of birds fed on sodium selenite, Sel-Plex and the control diet demonstrated the presence of a variety of crystalline aggregates localised between the forming palisade columns.

Genetic variation in eggshell crystal size and orientation is large and these traits are correlated with shell thickness and are associated with eggshell matrix protein markers.

The size and orientation of calcium carbonate crystals influence the structure and strength of the eggshells of chickens. In this study, estimates of heritability were found to be high (0.6) for crystal size and moderate (0.3) for crystal orientation. There was a strong positive correlation (0.65) for crystal size and orientation with the thickness of the shell and, in particular, with the thickness of the mammillary layer. Correlations with shell breaking strength were positive but with a high standard error. This was contrary to expectations, as in man-made materials smaller crystals would be stronger. We believe the results of this study support the hypothesis that the structural organization of shell, and in particular the mammillary layer, is influenced by crystal size and orientation, especially during the initial phase of calcification. Genetic associations for crystal measurements were observed between haplotype blocks or individual markers for a number of eggshell matrix proteins. Ovalbumin and ovotransferrin (LTF) markers for example were associated with crystal size, while ovocleidin-116 and ovocalyxin-32 (RARRES1) markers were associated with crystal orientation. The location of these proteins in the eggshell is consistent with different phases of the shell-formation process. In conclusion, the variability of crystal size, and to a lesser extent orientation, appears to have a large genetic component, and the formation of calcite crystals are intimately related to the ultrastructure of the eggshell. Moreover, this study also provides evidence that proteins in the shell influence the variability of crystal traits and, in turn, the shell's thickness profile. The crystal measurements and/or the associated genetic markers may therefore prove to be useful in selection programs to improve eggshell quality.

New hypotheses on the function of the avian shell gland derived from microarray analysis comparing tissue from juvenile and sexually mature hens.

Activation of the shell gland region of the avian oviduct is mediated by ovarian steroids. To understand more extensively how shell glands are maintained and function, we have compared gene expression in the shell glands from juvenile and laying hens using a chicken cDNA microarray. Average expression profiles of juvenile and sexually mature shell glands were compared resulting in the identification of 266 differentially regulated genes. Reverse transcription quantitative polymerase chain reaction confirmed expression differences. The differentially expressed genes included several with known involvement in shell gland function, including ion transport and shell matrix proteins. There were also many unpredicted differentially expressed genes, and for some we propose hypotheses for their functions. These include those encoding (a) osteoprotegerin, a decoy death receptor for receptor activator of nuclear factor NFkB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), that in the shell gland, may prevent apoptosis and/or may have an endocrine effect by preventing RANKL's action on bone osteoclasts that mobilize stored calcium; (b) prostatic acid phosphatase (ACPP) and prostate stem cell antigen (PSCA) that could play a role in sperm physiology within the shell gland; (c) urea transporter (SLC14A2) that could provide a novel anti-microbial defence; (d) bactericidal/permeability-increasing protein-like 2 (BPIL2), and other potential anti-microbials that have not previously been documented in the chicken. These new hypotheses, if borne out experimentally, will lead to a greater understanding of shell gland function including the processes involved in eggshell formation and anti-microbial activity.

Microcracks in eggs.

An experimental program of mechanical testing has shown that microcracks initiate in hens' eggs at loads less than that necessary to cause total structural failure. In contact loading, computational modeling and numerical analysis show that very high stress levels develop on the inner surface of the eggshell as it conforms to a hard contact surface. This causes a series of microcracks to initiate at the inner surface of the shell and radiate out from the load site. A series of concentric circumferential microcracks also develop beneath the cuticle from the edge of the contact zone. Calculations relating to the eggshells' dynamic response indicate that microcracks have a little effect on the structural stiffness and resonant frequencies of the egg. As a result, microcracks are unlikely to be detected by online crack detection systems, which rely on mechanical excitation. Eggs in retail outlets are likely to contain microcracks as a result of insults experienced during the collection, grading, and packing processes. Because the eggshell forms the first line of defense against potentially pathogenic microorganisms entering the egg contents, microcracks could potentially compromise egg safety.

Probability of an egg cracking during packing can be predicted using a simple non-destructive acoustic test.

1. The aim of this investigation was to test the predictive power of the dynamic stiffness measurement to identify eggs which are most likely to crack under field conditions. 2. A representative sample of eggs (n = 1660) was collected from the front of the cages in a commercial battery unit. Egg weight, % damping and dynamic stiffness (Kdyn) were recorded using an acoustic crack detection device. Intact eggs were marked and replaced in the front of the cages. These eggs were subsequently passed through online collection, grading and packing machinery, along with a volume of unmarked eggs. At the end of packing the acoustic test was repeated on the marked eggs, and these were subsequently categorised as being either intact (0) or cracked (1). 3. A logistic regression of the probability of cracking vs Kdyn revealed that as the Kdyn measurement decreases below 15,000 N/m there is a rapid increase in the probability that an egg will crack during routine handling. 4. Additional variables (visit, egg weight,% damping and position in the house (battery [1 to 7], side [1, 2] and tier [1 to 8]) were also fitted to the model but only egg weight, visit and tier effects significantly improved the model fit. 5. This study confirms that the dynamic stiffness measurement can predict the probability of an egg cracking in the field and with high precision. As this measurement also has a high heritability, it could be incorporated into breeding programmes, where it would offer an excellent method to improve eggshell quality and reduce the incidence of cracked eggs.

The influence of line and laying period on the relationship between different eggshell and membrane strength parameters.

We investigated the correlations between different eggshell and membrane strength parameters and their changes over time. Eggshell and membrane characteristics were measured for 2 different lines of laying hens, Hisex Brown and Bovans Brown, from wk 33 up to wk 78. Several parameters were investigated: 2 parameters concerning total eggshell strength (static and dynamic stiffness), 1 structural parameter (i.e., eggshell thickness), 2 parameters describing the material characteristics of the shell [dynamic Young's modulus (EM) and nitrogen content], and 3 parameters to measure the membrane characteristics (attachment force breaking strength and nitrogen content). This study shows that the correlations among parameters change during the laying period and among lines. The EM of the eggshell increased during the laying period for the Hisex line. No difference was observed in case of the Bovans line. In general, a negative correlation was obtained between eggshell thickness and the EM. The correlation between static and dynamic stiffness increased during lay. Finally, the attachment force and breaking strength of the membranes declined during the laying period.

Effect of environmental enrichment during the rearing phase on subsequent eggshell quality in broiler breeders.

This study set out to investigate if access to an enriched environment during the rearing phase influences subsequent production and eggshell quality in broiler breeders. Broiler breeder pullets were reared under standard rearing conditions (control treatment) or under standard conditions with an environmental enrichment of bales of plastic-wrapped wood shavings (enriched treatment). At 19 weeks of age both groups were transported to a laying facility and housed in standard pens according to treatment type. Egg weight, shape, shell thickness, non-destructive deformation, breaking strength, stiffness and ultrastructural quality were compared at beginning of lay (25 weeks old), peak production (31 weeks old), mid-lay (45 weeks old) and end of lay (57 weeks old). Production and hatchery data were also collected throughout lay. Significant treatment-age interactions were detected for non-destructive deformation, breaking strength and stiffness, such that birds which had received prior enrichment went on to maintain a better shell quality with age. The measurements of total shell thickness, mammillary thickness and effective shell thickness all showed a trend for a treatment-age interaction. Significant age effects were found for weight, shape index, shell thickness, breaking strength and stiffness. All production and hatchery data also demonstrated significant changes with age. It is concluded that early enrichment with bales of wood shavings may provide a novel approach to the maintenance of eggshell quality at a time when calcium metabolism begins to reduce in efficiency.

Supplementation of a corn-soybean meal diet with manganese, copper, and zinc from organic or inorganic sources improves eggshell quality in aged laying hens.

An experiment was conducted to evaluate the effects on eggshell quality of supplementing the diet of laying hens with a combination of Zn, Mn, and Cu from organic or inorganic sources. Three groups of hens, aged 32, 60, and 69 wk were fed a corn-soybean basal diet containing 32.6 mg/kg Zn, 24.7 mg/kg Mn, and 4.95 mg/kg Cu or a basal diet supplemented with 30-30-5 and 60-60-10 mg/kg of Zn, Mn, and Cu, respectively. Addition of Zn, Mn, and Cu in combination increased their concentrations in egg yolk and slightly decreased egg weight during some sampling from aged hens. The Zn, Cu, and Mn dietary supplementation did not affect the following measures of eggshell quality: percentage eggshell, eggshell index (shell weight per unit surface area), and eggshell stiffness. However, there was a small increase in elastic modulus of the eggs from one group of the older hens due to supplementation with Zn, Mn, and Cu. More consistently we observed improved breaking strength and fracture toughness (resistance to fracture) in the two oldest groups of hens at 1, 5, and 9 wk after introduction of the experimental diets. The effect was observed regardless of the source of trace elements.

Transmission electron microscopy of the vertical crystal layer and cuticle of the eggshell of the domestic fowl.

1. Eggshell pieces (1 cm2) were decalcified using a solution of EDTA (200 g/litre, pH 6.9 to 7.0) in paraformaldehyde (2 g/litre) and glutaraldehyde (0.5 g/litre) in phosphate buffer. 2. They were prepared for transmission electron microscopy (TEM). 3. TEM identified a vertically aligned matrix associated with the vertical crystal layer (VCL). It is hypothesised that the vertical orientation of calcite crystals in the VCL is closely linked to this vertical matrix. 4. TEM also revealed the presence of a 2-layered cuticle, the inner layer containing vesicles which were absent in the outer. 5. Cuticular vesicles contain hydroxyapatite and are thought to play a role in the termination of shell formation. The current paper presents data relating to microbial apatitic systems that strengthen this hypothesis.

Organic matrix morphology and distribution in the palisade layer of eggshells sampled at selected periods during lay.

1. 1 cm2 pieces of eggshells from a commercial battery flock were plasma etched to remove the outer shell membranes. 2. They were decalcified using EDTA (200 g/l, pH 6.9 to 7.0) in paraformaldehyde (20 g/l) and 25% gluteraldehyde (20 ml in 0.98 l) in phosphate buffer, then prepared for light and transmission electron microscopy. 3. Light microscopy revealed a differential distribution of matrix material within all 3 regions of the palisade layer at the beginning of lay. 4. Transmission electron microscopy revealed a more even distribution of matrix at the beginning of lay, although morphological differences were observed. At the end of lay all 3 regions showed an increase in % matrix and vesicles/10 cm2 of micrograph compared to the middle and beginning of lay periods. 5. It is hypothesised that matrix vesicles are involved in the regulation of the physiochemical environment within the forming eggshell and that the decline in shell quality associated with the end of lay is related to a concomitant change in matrix quality.