Genetic markers associated with bone composition in Rhode Island Red laying hens.

BACKGROUND: Bone damage has welfare and economic impacts on modern commercial poultry and is known as one of the major challenges in the poultry industry. Bone damage is particularly common in laying hens and is probably due to the physiological link between bone and the egg laying process. Previous studies identified and validated quantitative trait loci (QTL) for bone strength in White Leghorn laying hens based on several measurements, including bone composition measurements on the cortex and medulla of the tibia bone. In a previous pedigree-based analysis, bone composition measurements showed heritabilities ranging from 0.18 to 0.41 and moderate to strong genetic correlations with tibia strength and density. Bone composition was measured using infrared spectroscopy and thermogravimetry. The aim of this study was to combine these bone composition measurements with genotyping data via a genome-wide association study (GWAS) to investigate genetic markers that contribute to genetic variance in bone composition in Rhode Island Red laying hens. In addition, we investigated the genetic correlations between bone composition and bone strength. RESULTS: We found novel genetic markers that are significantly associated with cortical lipid, cortical mineral scattering, medullary organic matter, and medullary mineralization. Composition of the bone organic matter showed more significant associations than bone mineral composition. We also found interesting overlaps between the GWAS results for tibia composition traits, particularly for cortical lipid and tibia strength. Bone composition measurements by infrared spectroscopy showed more significant associations than thermogravimetry measurements. Based on the results of infrared spectroscopy, cortical lipid showed the highest genetic correlations with tibia density, which was negative (- 0.20 ± 0.04), followed by cortical CO3/PO4 (0.18 ± 0.04). Based on the results of thermogravimetry, medullary organic matter% and mineral% showed the highest genetic correlations with tibia density (- 0.25 ± 0.04 and 0.25 ± 0.04, respectively). CONCLUSIONS: This study detected novel genetic associations for bone composition traits, particularly those involving organic matter, that could be used as a basis for further molecular genetic investigations. Tibia cortical lipids displayed the strongest genetic associations of all the composition measurements, including a significantly high genetic correlation with tibia density and strength. Our results also highlighted that cortical lipid may be a key measurement for further avian bone studies.

Bone quality and composition are influenced by egg production, layer line, and oestradiol-17ß in laying hens.

Keel bone fractures are a serious animal welfare problem in laying hens. The aim of the current study was to assess the influence of egg production, oestradiol-17ß, and selection for high laying performance on bone quality. Hens of two layer lines differing in laying performance (WLA: 320 eggs per year, G11: 200 eggs per year) were allocated to four treatment groups. Group S received a deslorelin acetate implant that suppressed egg production. Group E received an implant with the sexual steroid oestradiol-17ß. Group SE received both implants and group C did not receive any implant. In the 63rd week of age, composition and characteristics of the tibiotarsi were assessed using histological analysis, three-point bending test, thermogravimetric analysis, infrared spectroscopy, and two-dimensional X-ray diffraction, respectively. Non-egg-laying hens showed a higher total bone area and a higher relative amount of cortical bone compared to egg-laying hens. Hens of layer line G11 showed a higher relative amount of medullary bone and a higher degree of mineralization of the cortical bone compared to hens of layer line WLA. These differences in bone composition may explain different susceptibility to keel bone fractures in non-egg-laying compared to egg-laying hens as well as in hens of layer lines differing in laying performance. The effect of exogenous oestradiol-17ß on bone parameters varied between the layer lines indicating a genetic influence on bone physiology and the way it can be modulated by hormone substitution.

Antimicrobial defenses of table eggs: Importance of antibacterial proteins in egg white as a function of hen age in an extended production cycle.

The importance of egg natural defences to prevent bacterial contamination and their relation with hen age in extended production cycles were evaluated. Egg-white from eggs of different hen age groups (up 100-weeks-old) and lines (Hy-Line white and brown) were inoculated with Gram-positive Staphylococcus aureus or Gram-negative Salmonella Typhimurium, ranging from 103-106 CFU/mL. Our results show that concentrations of egg-white lysozyme and, particularly, ovotransferrin are important to modulate bacterial survival in a dose-dependent matter. Depending on protein concentration, their effect ranges from bactericidal to bacteriostatic, with a threshold for bacterial contamination that depends also on hen age and line. The concentrations of lysozyme and ovotransferrin increased with hen age (up to 2 and 22 w/w% of total protein, respectively), and eggs laid by older hens exhibited the greatest potential to prevent the growth of the highest Salmonella inoculum (106 CFU/mL). Salmonella-penetration experiments demonstrated that non-contaminated eggs display significantly higher concentrations of antimicrobial proteins. However, eggs from older hens needed a higher concentration of these proteins (>20% ovotransferrin) to prevent bacterial contamination, showing that antimicrobial protein concentrations in egg-whites was not the only factor influencing bacterial contamination. Finally, this study demonstrated that egg-white of eggs produced by old hens are less prone to contamination by Salmonella.

No evidence that selection for egg production persistency causes loss of bone quality in laying hens.

BACKGROUND: The physiological adaptations that have evolved for egg laying make hens susceptible to bone fractures and keel bone damage. In modern laying hen breeds, longer periods of egg laying could result in a greater risk of poor bone quality, and selection for increased egg production has frequently been stated to be a cause. However, the existing literature does not support this hypothesis. To test the hypothesis that egg production is associated with quality, breaking strength and density of bone, genetic correlations between these traits were estimated in White Leghorn and Rhode Island Red breeds. Genetic correlations of cortical and medullary bone material chemical properties with bone quality were also estimated, in order to identify methods to improve bone quality with appropriately targeted measurement of key traits. RESULTS: Estimates of heritability for bone quality traits were moderate (0.19-0.59) for both White Leghorn and Rhode Island Red breeds, except for the keel bone trait, which had a heritability estimate equal to zero. There was no evidence for genetic or phenotypic relationships between post-peak egg production and bone quality. In the White Leghorn breed, the estimate of the genetic correlation between pre-peak production/age at first egg and bone quality was significant and negative (- 0.7 to - 0.4). Estimates of heritability of thermogravimetric measurements of tibial medullary bone mineralisation were significant (0.18-0.41), as were estimates of their genetic correlations with tibia breaking strength and density (0.6-0.9). CONCLUSIONS: The low genetic correlation of post-peak egg production with bone quality suggests that selection for increased persistency of egg production may not adversely affect bone quality. Onset of puberty and mineralisation of the medullary bone, which is a specialised adaptation for egg laying, were identified as important factors associated with the quality of the skeleton later during egg production. These are traits for which genetic, as well as environmental and management factors can positively impact the overall quality of the skeleton of laying hens.

An eQTL in the cystathionine beta synthase gene is linked to osteoporosis in laying hens.

BACKGROUND: Skeletal damage is a challenge for laying hens because the physiological adaptations required for egg laying make them susceptible to osteoporosis. Previously, we showed that genetic factors explain 40% of the variation in end of lay bone quality and we detected a quantitative trait locus (QTL) of large effect on chicken chromosome 1. The aim of this study was to combine data from the commercial founder White Leghorn population and the F2 mapping population to fine-map this QTL and understand its function in terms of gene expression and physiology. RESULTS: Several single nucleotide polymorphisms on chromosome 1 between 104 and 110 Mb (galGal6) had highly significant associations with tibial breaking strength. The alternative genotypes of markers of large effect that flanked the region had tibial breaking strengths of 200.4 vs. 218.1 Newton (P < 0.002) and, in a subsequent founder generation, the higher breaking strength genotype was again associated with higher breaking strength. In a subsequent generation, cortical bone density and volume were increased in individuals with the better bone genotype but with significantly reduced medullary bone quality. The effects on cortical bone density were confirmed in a further generation and was accompanied by increased mineral maturity of the cortical bone as measured by infrared spectrometry and there was evidence of better collagen cross-linking in the cortical bone. Comparing the transcriptome of the tibia from individuals with good or poor bone quality genotypes indicated four differentially-expressed genes at the locus, one gene, cystathionine beta synthase (CBS), having a nine-fold higher expression in the genotype for low bone quality. The mechanism was cis-acting and although there was an amino-acid difference in the CBS protein between the genotypes, there was no difference in the activity of the enzyme. Plasma homocysteine concentration, the substrate of CBS, was higher in the poor bone quality genotype. CONCLUSIONS: Validated markers that predict bone strength have been defined for selective breeding and a gene was identified that may suggest alternative ways to improve bone health in addition to genetic selection. The identification of how genetic variants affect different aspects of bone turnover shows potential for translational medicine.

Change in the chicken eggshell cuticle with hen age and egg freshness.

For a fuller understanding of the functionality of the eggshell cuticle, we conducted a detailed study using a wide array of analytical techniques (scanning and transmission microscopy), energy dispersive x-rays, and attenuated total reflection-Fourier transform infrared spectroscopy to analyze the structure, morphology, and chemical composition of this organic coating. This study shows that the cuticle has a compositional gradation with an outer part richer in proteins and an inner part richer in sulfated polysaccharides and phosphates. It also shown that the cuticle composition, thickness, and degree of coverage are highly dependent on hen age and egg freshness. During the course of the first laying year, the thickness and degree of glycosylation of the cuticle decreases with hen age, and at the end of the laying cycle, the cuticle is significantly depleted in lipids. There are also well-defined compositional changes in the cuticle of freshly laid eggs as time passes and there is a notable increase in the permeability of the eggshell after 24 h due to cuticle drying. We discuss how these changes in the cuticle can affect the food safety of eggs in relation to the risk of trans-shell contamination by bacteria (i.e., Salmonellosis).

Genetics of tibia bone properties of crossbred commercial laying hens in different housing systems.

Osteoporosis and bone fractures are a severe problem for the welfare of laying hens, with genetics and environment, such as housing system, each making substantial contributions to bone strength. In this work, we performed genetic analyses of bone strength, bone mineral density, and bone composition, as well as body weight, in 860 commercial crossbred laying hens from 2 different companies, kept in either furnished cages or floor pens. We compared bone traits between housing systems and crossbreds and performed a genome-wide association study of bone properties and body weight. As expected, the 2 housing systems produced a large difference in bone strength, with layers housed in floor pens having stronger bones. These differences were accompanied by differences in bone geometry, mineralization, and chemical composition. Genome scans either combining or independently analyzing the 2 housing systems revealed no genome-wide significant loci for bone breaking strength. We detected 3 loci for body weight that were shared between the housing systems on chromosomes 4, 6, and 27 (either genome-wide significant or suggestive) and these coincide with associations for bone length. In summary, we found substantial differences in bone strength, content, and composition between hens kept in floor pens and furnished cages that could be attributed to greater physical activity in pen housing. We found little evidence for large-effect loci for bone strength in commercial crossbred hens, consistent with a highly polygenic architecture for bone strength in the production environment. The lack of consistent genetic associations between housing systems in combination with the differences in bone phenotypes could be due to gene-by-environment interactions with housing system or a lack of power to detect shared associations for bone strength.

Research Note: Changes in eggshell quality and microstructure related to hen age during a production cycle.

We have studied in detail the changes that occur in eggshell structure and composition during a production cycle in order to better understand the deterioration of eggshell quality with hen age (at 33, 45, and 67 wk). To study changes in eggshell ultrastructure and microstructure characteristics (mammillary density, palisade layer thickness, size, and orientation of calcite crystals) and the cuticle composition, we used complementary analytical techniques such optical and electron microscopy, X-ray diffraction, and infrared spectrometry. The marked decrease in eggshell breaking strength from 5.8 Kg at 33 wk to 4.4 Kg at 67 wk (25% reduction) could not be solely explained by the modest reduction in eggshell thickness (6-10% reduction) and seems to be associate to abrupt changes in eggshell ultra- and microstructure characteristics (i.e., decreased mammillary density; increased size of crystal units), occurring in older hens. Particularly, the decrease in mammillary density reduces the attachment points of the eggshell mineral to the membranes and therefore should negatively impact eggshell mechanical properties. Also, the observed increase in the calcite crystal size making the shell could also reduce the cohesion of crystals and eggshell resistance against impacts. Additionally, there was a decrease in the amount of cuticle and internal egg quality parameters (egg albumen height) with hen age that could have a negative impact in egg safety and quality.

Relationship between Bone Quality, Egg Production and Eggshell Quality in Laying Hens at the End of an Extended Production Cycle (105 Weeks).

(1) Background: Nowadays the industry aims to improve lay persistency for extended cycles (100 weeks or longer) to make egg production more sustainable. However, intensive egg production challenges hen health, inducing severe osteoporosis and the incidence of bone fractures. In this study, the relationship between bone quality and egg production, and/or eggshell quality, was evaluated at the end of an extended laying cycle of 100 weeks, comparing groups of hens with different production and eggshell quality parameters; (2) Methods: Quality parameters of egg (as weight, egg white height), eggshell (as thickness, weight, breaking strength, elasticity and microstructure) and tibiae bone (weight, diameter, cortical thickness, ash weight, breaking strength, medullary bone) were determined; (3) Results: Hens from groups with a high egg production and good eggshell quality have poorer bone quality (lower ash weight and lesser amount of medullary bone). However, Pearson's correlation analysis shows no clear relationship between bone and egg/eggshell parameters. (4) Conclusions: Bone and egg production/eggshell quality are independent and can be improved separately. Medullary bone has an important contribution to bone mechanical properties, being important to accumulate enough bone medullary bone early in life to maintain skeletal integrity and eggshell quality in old hens.

Changes with age (from 0 to 37 D) in tibiae bone mineralization, chemical composition and structural organization in broiler chickens.

Broiler chickens have an extreme physiology (rapid growth rates) that challenges the correct bone mineralization, being an interesting animal model for studying the development of bone pathologies. This work studies in detail how the mineralization, chemistry, and structural organization of tibiae bone in broiler chickens change with age during the first 5 wk (37 D) from hatching until acquiring the final weight for slaughter. During the early growth phase (first 2 wk), the rapid addition of bone tissue does not allow for bone organic matrix to fully mineralize and mature, and seems to be a critical period for bone development at which bone mineralization cannot keep pace with the rapid growth of bones. The low degree of bone mineralization and large porosity of cortical bone at this period might be responsible of leg deformation and/or other skeletal abnormalities commonly observed in these birds. Later, cortical bone porosity gradually decreases and the cortical bone became fully mineralized (65%) at 37 D of age. At the same time, bone mineral acquires the composition of mature bone tissue (decreased amount of carbonate, higher crystallinity, Ca/P = 1.68). However, the mineral part was still poorly organized even at 37 D. The oriented fraction was about 0.45 which means that more than half of apatite crystals within the mineral are randomly oriented. Mineral organization (crystal orientation) had an important contribution to bone-breaking strength. Nevertheless, locally determined (at tibia mid-shaft) bone properties (i.e., cortical thickness, crystal orientation) has only a moderate correlation (R2 = 0.33) with bone breaking strength probably due to large and highly heterogeneous porosity of bone that acts as structural defects. On the other hand, the total amount of mineral (a global property) measured by total ash content was the best predictor for breaking strength (R2 = 0.49). Knowledge acquired in this study could help in designing strategies to improve bone quality and reduce the incidence of skeletal problems in broiler chickens that have important welfare and economic implications.