Genotype-dependent impact of dietary vitamin D3 on laying hens.

Vitamin D3 has an integral part in calcium and phosphorus homoeostasis, which in turn plays a key role in egg production of hens. The present study aimed to investigate whether an additional vitamin D3 supplementation improves the laying performance and egg quality of hens according to their genetic potential. For this purpose, four layer lines (low performing: R11 and L68; high performing: WLA and BLA) supplemented either with 300 or 3000 IU vitamin D3 per kg feed were compared concerning serum 25-hydroxyvitamin D3 (25-OHD3), calcium, phosphorus and alkaline phosphatase (ALP), laying performance and egg quality. The higher supplementation of vitamin D3 increased 25-OHD3 serum concentrations in all genotypes, except for R11 and WLA hens in week 49, and also elevated vitamin D3 and 25-OHD3 content in the egg yolk (p < 0.05). In week 29, 3000 IU vitamin D3 decreased pooled least squares means (LSMeans) of serum calcium concentrations considering all genotypes and increased the ALP concentrations in BLA hens (p < 0.05). Considering the whole experimental period daily egg mass of R11 hens was increased by an additional vitamin D3 supplementation (p < 0.001). Regarding all genotypes and the whole experimental period the pooled LSMeans of breaking strength of eggs from hens fed 3000 IU vitamin D3 were higher than those of hens fed 300 IU (p = 0.044). In conclusion, present results give evidence that the higher vitamin D3 supplementation might have genotype-dependently beneficial effects on calcium and phosphorus homoeostasis of hens, which might improve feed efficiency in the early laying period and promote the persistence of the laying period irrespectively of genotype. The increase of serum 25-OHD3 by the higher vitamin D supplementation supported the higher transfer of vitamin D in the egg yolk and improved genotype-dependently the breaking strength of the eggshell.

High-density genotyping reveals candidate genomic regions for chicken body size in breeds of Asian origin.

Body size is one of the main selection indices in chicken breeding. Although often investigated, knowledge of the underlying genetic mechanisms is incomplete. The aim of the current study was to identify genomic regions associated with body size differences between Asian Game and Asian Bantam type chickens. In this study, 94 and 107 chickens from 4 Asian Game and 5 Asian Bantam type breeds, respectively, were genotyped using the chicken 580K single nucleotide polymorphism (SNP) array. A genome-wide association study (GWAS) and principal component analyses (PCA) were performed to identify genomic regions associated with body size related-traits such as wing length, shank length, shank thickness, keel length, and body weight. Hierarchical clustering of genotype data showed a clear genetic difference between the investigated Asian Game and Asian Bantam chicken types. GWAS identified 16 genomic regions associated with wing length (2, FDR ≤ 0.018), shank thickness (6, FDR ≤ 0.008), keel length (5, FDR ≤ 0.023), and body weight (3, FDR ≤ 0.041). PCA showed that the first principal component (PC1) separated the 2 chicken types and significantly correlated with the measured body size related-traits (P ≤ 2.24e-40). SNPs contributing significantly to PC1 were subjected to a more detailed investigation. This analysis identified 11 regions potentially associated with differences in body size related-traits. A region on chromosome 4 (GGA4) (17.3-21.3 Mb) was detected in both analyses GWAS and PCA. This region harbors 60 genes. Among them are myotubularin 1 (MTM1) and secreted frizzled-related protein 2 (SFPR2) which can be considered as potential candidate genes for body size related-traits. Our results clearly show that the investigated Asian Game type chicken breeds are genetically different from the Asian Bantam breeds. A region on GGA4 between 17.3 and 21.3 Mb was identified which contributes to the phenotypic difference, though further validation of candidate genes is necessary.

Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR.

Primordial germ cells (PGCs), the precursors of sperm and oocytes, pass on the genetic material to the next generation. The previously established culture system of chicken PGCs holds many possibilities for functional genomics studies and the rapid introduction of desired traits. Here, we established a CRISPR/Cas9-mediated genome editing protocol for the genetic modification of PGCs derived from chickens with blue eggshell color. The sequence targeted in the present report is a provirus (EAV-HP) insertion in the 5'-flanking region of the SLCO1B3 gene on chromosome 1 in Araucana chickens, which is supposedly responsible for the blue eggshell color. We designed pairs of guide RNAs (gRNAs) targeting the entire 4.2 kb provirus region. Following transfection of PGCs with the gRNA, genomic DNA was isolated and analyzed by mismatch cleavage assay (T7EI). For absolute quantification of the targeting efficiencies in homozygous blue-allele bearing PGCs a digital PCR was established, which revealed deletion efficiencies of 29% when the wildtype Cas9 was used, and 69% when a high-fidelity Cas9 variant was employed. Subsequent single cell dilutions of edited PGCs yielded 14 cell clones with homozygous deletion of the provirus. A digital PCR assay proved the complete absence of this provirus in cell clones. Thus, we demonstrated the high efficiency of the CRISPR/Cas9 system in introducing a large provirus deletion in chicken PGCs. Our presented workflow is a cost-effective and rapid solution for screening the editing success in transfected PGCs.

Estradiol-17ß Is Influenced by Age, Housing System, and Laying Performance in Genetically Divergent Laying Hens (Gallus gallus f.d.).

The estrogen estradiol-17ß is known as one of the major gonadal steroid hormones with different functions in reproduction. In this study we analyzed estradiol-17ß concentration in laying hens of four pure bred chicken laying lines at four different time intervals of the laying period (17th-19th week of age, 33rd-35th week of age, 49th-51st week of age, and 72nd week of age). The high performing white egg (WLA) and brown egg (BLA) layer lines as well as the low performing white (R11) and brown (L68) layer lines were kept in both single cages and a floor housing system. We investigated whether there were differences in estradiol -17ß concentrations between lines at different ages that could be related to selection for high egg production or phylogenetic origin of the animals, and whether there was an influence of housing conditions on estradiol-17ß. Estradiol-17ß concentrations differed between high and low performing layer lines at all time intervals studied. High performing hens showed higher estradiol-17ß concentrations compared to low performing hens. In all lines, highest estradiol-17ß concentration was measured at their 49th to their 51st week of age, whereas the peak of laying intensity was observed at their 33rd to their 35th week of age. Additionally, hens with fewer opportunities for activity housed in cages showed higher estradiol-17ß concentrations than hens kept in a floor housing system with more movement possibilities. We could show that laying performance is strongly linked with estradiol -17ß concentration. This concentration changes during laying period and is also influenced by the housing system.

Assessment of linkage disequilibrium patterns between structural variants and single nucleotide polymorphisms in three commercial chicken populations.

BACKGROUND: Structural variants (SV) are causative for some prominent phenotypic traits of livestock as different comb types in chickens or color patterns in pigs. Their effects on production traits are also increasingly studied. Nevertheless, accurately calling SV remains challenging. It is therefore of interest, whether close-by single nucleotide polymorphisms (SNPs) are in strong linkage disequilibrium (LD) with SVs and can serve as markers. Literature comes to different conclusions on whether SVs are in LD to SNPs on the same level as SNPs to other SNPs. The present study aimed to generate a precise SV callset from whole-genome short-read sequencing (WGS) data for three commercial chicken populations and to evaluate LD patterns between the called SVs and surrounding SNPs. It is thereby the first study that assessed LD between SVs and SNPs in chickens. RESULTS: The final callset consisted of 12,294,329 bivariate SNPs, 4,301 deletions (DEL), 224 duplications (DUP), 218 inversions (INV) and 117 translocation breakpoints (BND). While average LD between DELs and SNPs was at the same level as between SNPs and SNPs, LD between other SVs and SNPs was strongly reduced (DUP: 40%, INV: 27%, BND: 19% of between-SNP LD). A main factor for the reduced LD was the presence of local minor allele frequency differences, which accounted for 50% of the difference between SNP - SNP and DUP - SNP LD. This was potentially accompanied by lower genotyping accuracies for DUP, INV and BND compared with SNPs and DELs. An evaluation of the presence of tag SNPs (SNP in highest LD to the variant of interest) further revealed DELs to be slightly less tagged by WGS SNPs than WGS SNPs by other SNPs. This difference, however, was no longer present when reducing the pool of potential tag SNPs to SNPs located on four different chicken genotyping arrays. CONCLUSIONS: The results implied that genomic variance due to DELs in the chicken populations studied can be captured by different SNP marker sets as good as variance from WGS SNPs, whereas separate SV calling might be advisable for DUP, INV, and BND effects.

Genotypic and Dietary Effects on Egg Quality of Local Chicken Breeds and Their Crosses Fed with Faba Beans.

The quality of chicken eggs is an important criterion for food safety and the consumers' choice at the point of sale. Several studies have shown that egg quality can be influenced by the chickens' genotype and by the composition of the diet. The present study aimed to evaluate the effect of faba beans as a substitute for soybeans in the diet of chickens originating from traditional low-performance breeds in comparison with high-performing laying type hens and their crosses on egg quality parameters. Chickens of six different genotypes were fed either with a feed mix containing 20% faba beans with high or low vicin contents or, as a control, a feed mix containing soybeans. The genotypes studied were the local breeds Vorwerkhuhn and Bresse Gauloise, as well as commercial White Rock parent hens and their crosses. Yolk weight, Haugh units, yolk and shell color, the frequency of blood and meat spots and the composition of the eggs were significantly influenced by the genotype. The feeding of faba beans had an effect on yolk and shell color, Haugh units and shell portion, while there was no significant influence on the frequency of blood and meat spots.

Identification and Functional Annotation of Genes Related to Bone Stability in Laying Hens Using Random Forests.

Skeletal disorders, including fractures and osteoporosis, in laying hens cause major welfare and economic problems. Although genetics have been shown to play a key role in bone integrity, little is yet known about the underlying genetic architecture of the traits. This study aimed to identify genes associated with bone breaking strength and bone mineral density of the tibiotarsus and the humerus in laying hens. Potentially informative single nucleotide polymorphisms (SNP) were identified using Random Forests classification. We then searched for genes known to be related to bone stability in close proximity to the SNPs and identified 16 potential candidates. Some of them had human orthologues. Based on our findings, we can support the assumption that multiple genes determine bone strength, with each of them having a rather small effect, as illustrated by our SNP effect estimates. Furthermore, the enrichment analysis showed that some of these candidates are involved in metabolic pathways critical for bone integrity. In conclusion, the identified candidates represent genes that may play a role in the bone integrity of chickens. Although further studies are needed to determine causality, the genes reported here are promising in terms of alleviating bone disorders in laying hens.

Cultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens.

The chicken (Gallus gallus) is one of the most common and widespread domestic species, with an estimated total population of 25 billion birds worldwide. The vast majority of chickens in agriculture originate from hybrid breeding programs and is concentrated on few commercially used high performance lines, whereas numerous local and indigenous breeds are at risk to become extinct. To preserve the genomic resources of rare and endangered chicken breeds innovative methods are necessary. Here, we established a solid workflow for the derivation and biobanking of chicken primordial germ cells (PGCs) from blue layer hybrids. To achieve this, embryos of a cross of heterozygous blue egg layers were sampled to obtain blood derived and gonadal male as well as female PGCs of different genotypes (homozygous, heterozygous and nullizygous blue-allele bearing). The total efficiency of established PGC lines was 45% (47/104) within an average of 49 days until they reached sufficient numbers of cells for cryopreservation. The stem-cell character of the cultivated PGCs was confirmed by SSEA-1 immunostaining, and RT-PCR amplification of the pluripotency- and PGC-specific genes cPOUV, cNANOG, cDAZL and CVH. The Sleeping Beauty transposon system allowed to generate a stable integration of a Venus fluorophore reporter into the chicken genome. Finally, we demonstrated that, after re-transfer into chicken embryos, Venus-positive PGCs migrated and colonized the forming gonads. Semen samples of 13 raised cell chimeric roosters were analyzed by flow cytometry for the efficiency of germline colonization by the transferred PGCs carrying the Venus reporter and their proper differentiation into vital spermatids. Thus, we provide a proof-of-concept study for the potential use of PGCs for the cryobanking of rare breeds or rare alleles.

Keel Bone Damage in Laying Hens-Its Relation to Bone Mineral Density, Body Growth Rate and Laying Performance.

Keel bone damage is an important animal welfare problem in laying hens. Two generations of four layer lines, differing in phylogenetic background and performance level and kept in single cages or floor pens were weighed and scored for keel bone deformities (KBD) during the laying period. KBD, keel bone fractures (KBF) and the bone mineral density (BMD) of the keels were assessed post mortem. For BMD, relationships to laying performance and body growth were estimated. Caged hens showed more deformities, but fewer fractures and a lower BMD of the keel bone than floor-housed hens. White-egg layers had a lower BMD (0.140-0.165 g/cm2) and more KBD than brown-egg layers (0.179-0.184 g/cm2). KBF occurred more often in the high-performing lines than the moderate-performing ones. However, in the high-performing lines, BMD was positively related to total egg number from 18 to 29 weeks of age. The adult body weight derived from fitted growth curves (Gompertz function) had a significant effect (p < 0.001) on keels' BMD. The study contributes to the understanding of predisposing factors for keel bone damage in laying hens. It showed that the growth rate has a rather subordinate effect on keels' BMD, while the BMD itself greatly affects KBD.

Influence of Age and Phylogenetic Background on Blood Parameters Associated With Bone Metabolism in Laying Hens.

The high laying performance of today's laying hens places enormous demands on their mineral metabolism. While up-to-date data are rare, the present study aimed to describe blood parameters associated with egg laying and bone metabolism during the pre-laying period, in the course of the laying period and the daily egg laying cycle. Ten to 15 laying hens of two high-performing, phylogenetically divergent lines (BLA: brown-egg layer; WLA: white-egg layer), kept in single cages were blood sampled at 17, 25, 29, 49, and 69 weeks of age. Sampling was made at 6 a.m., 10 a.m., 2 p.m. and, with the exception of week 17, 6 p.m. Blood samples were analyzed for concentrations of total and ionized calcium, inorganic phosphate (PO4), markers of bone formation (osteocalcin) and resorption [carboxyterminal crosslinked telopeptide of type I collagen (CTX-I)], 25-hydroxycholecalciferol (25(OH)D3) and estradiol-17ß. In the pre-laying period (17 week), the estradiol-17ß level calculated for WLA was more than twice as high as the level calculated for BLA, while no significant difference could be observed in the laying period (25 to 69 weeks). BLA hens had significantly higher total calcium concentrations at 49 weeks of age as well as up to twice as high levels of osteocalcin and 25(OH)D3 than WLA at any time of the day from 25 to 69 weeks of age. While osteocalcin, CTX-I and 25(OH)D3 concentrations were significantly higher before the onset of lay, total calcium and estradiol-17ß levels significantly increased from 17 to 69 weeks of age. In contrast, PO4 values varied only slightly during the experimental period and ionized calcium was highest at 17 and 49 weeks of age and lowest around peak production (29 week). In the course of the daily egg laying cycle blood concentrations clearly reflected the stage of egg formation. Our results provide up-to-date data of bone- and egg laying-associated blood parameters of two contemporary purebred layer lines over the course of the pre- and egg-laying period and the daily egg laying cycle. Differences between brown- and white-egg layers raise questions, whether phylogenetic background determines their efficiency to cope with high calcium demands relating to egg production.

How imputation can mitigate SNP ascertainment Bias.

BACKGROUND: Population genetic studies based on genotyped single nucleotide polymorphisms (SNPs) are influenced by a non-random selection of the SNPs included in the used genotyping arrays. The resulting bias in the estimation of allele frequency spectra and population genetics parameters like heterozygosity and genetic distances relative to whole genome sequencing (WGS) data is known as SNP ascertainment bias. Full correction for this bias requires detailed knowledge of the array design process, which is often not available in practice. This study suggests an alternative approach to mitigate ascertainment bias of a large set of genotyped individuals by using information of a small set of sequenced individuals via imputation without the need for prior knowledge on the array design. RESULTS: The strategy was first tested by simulating additional ascertainment bias with a set of 1566 chickens from 74 populations that were genotyped for the positions of the Affymetrix Axiom™ 580 k Genome-Wide Chicken Array. Imputation accuracy was shown to be consistently higher for populations used for SNP discovery during the simulated array design process. Reference sets of at least one individual per population in the study set led to a strong correction of ascertainment bias for estimates of expected and observed heterozygosity, Wright's Fixation Index and Nei's Standard Genetic Distance. In contrast, unbalanced reference sets (overrepresentation of populations compared to the study set) introduced a new bias towards the reference populations. Finally, the array genotypes were imputed to WGS by utilization of reference sets of 74 individuals (one per population) to 98 individuals (additional commercial chickens) and compared with a mixture of individually and pooled sequenced populations. The imputation reduced the slope between heterozygosity estimates of array data and WGS data from 1.94 to 1.26 when using the smaller balanced reference panel and to 1.44 when using the larger but unbalanced reference panel. This generally supported the results from simulation but was less favorable, advocating for a larger reference panel when imputing to WGS. CONCLUSIONS: The results highlight the potential of using imputation for mitigation of SNP ascertainment bias but also underline the need for unbiased reference sets.

Genetic diversity in global chicken breeds in relation to their genetic distances to wild populations.

BACKGROUND: Migration of a population from its founder population is expected to cause a reduction of its genetic diversity and facilitates differentiation between the population and its founder population, as predicted by the theory of genetic isolation by distance. Consistent with that theory, a model of expansion from a single founder predicts that patterns of genetic diversity in populations can be explained well by their geographic expansion from their founders, which is correlated with genetic differentiation. METHODS: To investigate this in chicken, we estimated the relationship between the genetic diversity of 160 domesticated chicken populations and their genetic distances to wild chicken populations. RESULTS: Our results show a strong inverse relationship, i.e. 88.6% of the variation in the overall genetic diversity of domesticated chicken populations was explained by their genetic distance to the wild populations. We also investigated whether the patterns of genetic diversity of different types of single nucleotide polymorphisms (SNPs) and genes are similar to that of the overall genome. Among the SNP classes, the non-synonymous SNPs deviated most from the overall genome. However, genetic distance to the wild chicken still explained more variation in domesticated chicken diversity across all SNP classes, which ranged from 83.0 to 89.3%. CONCLUSIONS: Genetic distance between domesticated chicken populations and their wild relatives can predict the genetic diversity of the domesticated populations. On the one hand, genes with little genetic variation across populations, regardless of the genetic distance to the wild population, are associated with major functions such as brain development. Changes in such genes may be detrimental to the species. On the other hand, genetic diversity seems to change at a faster rate within genes that are associated with e.g. protein transport and protein and lipid metabolic processes. In general, such genes may be flexible to changes according to the populations' needs. These results contribute to the knowledge of the evolutionary patterns of different functional genomic regions in the chicken.

How array design creates SNP ascertainment bias.

Single nucleotide polymorphisms (SNPs), genotyped with arrays, have become a widely used marker type in population genetic analyses over the last 10 years. However, compared to whole genome re-sequencing data, arrays are known to lack a substantial proportion of globally rare variants and tend to be biased towards variants present in populations involved in the development process of the respective array. This affects population genetic estimators and is known as SNP ascertainment bias. We investigated factors contributing to ascertainment bias in array development by redesigning the Axiom™ Genome-Wide Chicken Array in silico and evaluating changes in allele frequency spectra and heterozygosity estimates in a stepwise manner. A sequential reduction of rare alleles during the development process was shown. This was mainly caused by the identification of SNPs in a limited set of populations and a within-population selection of common SNPs when aiming for equidistant spacing. These effects were shown to be less severe with a larger discovery panel. Additionally, a generally massive overestimation of expected heterozygosity for the ascertained SNP sets was shown. This overestimation was 24% higher for populations involved in the discovery process than not involved populations in case of the original array. The same was observed after the SNP discovery step in the redesign. However, an unequal contribution of populations during the SNP selection can mask this effect but also adds uncertainty. Finally, we make suggestions for the design of specialized arrays for large scale projects where whole genome re-sequencing techniques are still too expensive.

Correction: Jansen, S., et al. Relationship between Bone Stability and Egg Production in Genetically Divergent Chicken Layer Lines. Animals 2020, 10, 850.

The authors wish to make the following corrections to this paper [...].

Bone Health or Performance? Adaptation Response of Genetically Divergent Chicken Layer Lines to a Nutritive Calcium Depletion.

In modern laying hybrids, calcium (Ca) homeostasis is immensely challenged by daily eggshell calcification. However, excessive mobilization of Ca from bones may lead to osteoporosis, which then manifests in a high incidence of poor bone quality. The aim of this study was to characterize the hens' adaptation response to an alternating dietary Ca restriction. The animal model consisted of four purebred layer lines, differing in laying performance (high vs. moderately performing lines) and phylogenetic origin (white- vs. brown-egg lines). According to the resource allocation theory, hens selected for high egg production were assumed to show a different response pattern to cope with this nutritive challenge compared to moderately performing lines. Data collected included egg number, egg quality traits, body weight and bone characteristics. The Ca depletion led to a temporary drop in egg production and shell quality and a loss of bone stability due to Ca mobilization. The white-egg lines response was more pronounced, whereas the brown-egg lines were less sensitive towards reduced Ca supply. Our study shows that the hens' responsiveness to coping with a nutritive Ca depletion is not ultimately linked to genetic selection for increased egg production but rather to phylogenetic origin.

Meat Quality Parameters and Sensory Properties of One High-Performing and Two Local Chicken Breeds Fed with Vicia faba.

The current practices of the poultry industry have raised concerns among consumers. Among these is the culling of day-old male chicks of laying hybrids; a suitable alternative for this could be the use of dual-purpose breeds where both sexes are used. Another practice that causes concern is the import of large quantities of soybeans for feedstuff production. Substitutes for these soybean-based products are regional protein crops, such as faba beans (Vicia faba L.; FBs). The objective of this study was to test the suitability of FB as a locally produced soybean meal replacement for two local dual-purpose chicken breeds and one high-performing layer line. The breast and leg meat of male Bresse Gauloise (BG), Vorwerkhuhn (VH), and White Rock (WR) animals was evaluated for different meat quality parameters: pH, color, water holding capacity, and tenderness. Sensory properties of the samples were evaluated by a trained panel with a conventional descriptive analysis. Results show different effects of FB diets on meat quality parameters in the different breeds. The attributes mostly affected by the diet are related to aroma, flavor, and texture, particularly in VH and WR. Overall, faba beans appear to be an acceptable dietary protein source for rearing these breeds for meat production.

Egg Production and Bone Stability of Local Chicken Breeds and Their Crosses Fed with Faba Beans.

Poultry production is raising concerns within the public regarding the practice of culling day-old chicks and the importation of soy from overseas for feedstuff. Therefore, an alternative approach to poultry production was tested. In two consecutive experiments, two traditional chicken breeds, Vorwerkhuhn and Bresse Gauloise, and White Rock as a commercial layer genotype as well as crossbreds thereof were fed diets containing either 20% vicin-rich or vicin-poor faba beans, though addressing both subjects of debate. Hen performance traits and bone stability were recorded. All parameters were considerably influenced by the genotype with White Rock showing the significantly highest (p < 0.05) laying performance (99.4% peak production) and mean egg weights (56.6 g) of the purebreds, but the lowest bone breaking strength (tibiotarsus 197.2 N, humerus 230.2 N). Regarding crossbreds, the Bresse Gauloise × White Rock cross performed best (peak production 98.1%, mean egg weight 58.0 g). However, only limited dietary effects were found as only the feeding of 20% vicin-rich faba beans led to a significant reduction of egg weights of at most 1.1 g (p < 0.05) and to a significant reduction of the shell stability in the crossbred genotypes. In terms of dual-purpose usage, crossing of Bresse Gauloise with White Rock seems to be the most promising variant studied here.

Relationship between Bone Stability and Egg Production in Genetically Divergent Chicken Layer Lines.

Impaired animal welfare due to skeletal disorders is likely one of the greatest issues currently facing the egg production industry. Reduced bone stability in laying hens is frequently attributed to long-term selection for increased egg production. The present study sought to analyse the relationship between bone stability traits and egg production. The study comprised four purebred layer lines, differing in their phylogenetic origin and performance level, providing extended insight into the phenotypic variability in bone characteristics in laying hens. Data collection included basic production parameters, bone morphometry, bone mineral density (BMD) and bone breaking strength (BBS) of the tibiotarsus and humerus. Using a multifactorial model and regression analyses, BMD proved to be of outstanding importance for bone stability. Only for the tibiotarsus were morphometric parameters and the bone weight associated with BBS. Within the chicken lines, no effect of total eggshell production on BBS or BMD could be detected, suggesting that a high egg yield itself is not necessarily a risk for poor bone health. Considering the complexity of osteoporosis, the estimated genetic parameters confirmed the importance of genetics in addressing the challenge of improving bone strength in layers.