IntroductionKeel bone damage is a severe animal welfare problem in laying hens. Although it is influenced by husbandry and diet, the selection for laying performance seems to play a key role.Materials and MethodsIn order to learn more about the pathogenesis of keel bone damage, this study aims to characterize and compare keel bone health and potentially related traits in Lohmann Selected Leghorn (LSL, n = 12) and two low-performing genotypes that have not intensively been selected for laying performance: Junglefowl phenotype (JF, n = 14) and Sumatra (Su, n = 12). X-ray imaging, blood sampling, and ultrasonography were conducted at five different time points between the 16th and 72nd week of age. The X-ray images were evaluated for fractures, deviated keel bone area, radiographic density, length, and ossification of the keel bone. Blood samples were used to determine blood ionized calcium, as well as plasma total calcium, phosphate, and 17-β-estradiol. Laying activity and eggshell quality were assessed at group level. Ultrasonography was used to detect visible follicles and assess pectoral muscle thickness.ResultsKeel bone fractures were detected in five out of twelve LSL hens, but in none of the two low-performing genotypes. The first egg was laid distinctly earlier in LSL than in JF and Su (18th vs. 24th and 31st week of age, respectively). Keel bone ossification was completed significantly later in Su than in LSL and JF, but there was no significant difference between the latter two. Visible follicles at the ovary were associated with significantly higher plasma calcium and 17-β-estradiol levels.DiscussionThis study provides a deeper insight into keel bone health and related traits in LSL and low-performing chicken genotypes. Our findings indicate that the earlier onset of lay in LSL does not correspond with earlier keel bone maturation, which could increase later susceptibility for keel bone fractures.

BACKGROUND:Understanding the genetic architecture of economically important traits in poultry is critical for improving breeding strategies. In this study, we investigated a backcrossing scheme between a White Layer line and Araucana chickens. Genome-wide association studies (GWAS) were performed using array-genotyped and imputed data. We also explored the use of correlated traits as covariates, which helps to distinguish between pleiotropic and trait-specific associations. We applied two Bayesian fine-mapping methods to refine GWAS-identified QTLs and pinpoint candidate variants and genes associated with egg number (EN, from 20 to 71 weeks), egg weight (EW, from 30 to 70 weeks), and body weight (BW, at 32 weeks): the forward selection approach and functional annotation enrichment implemented in BFMAP, and the shotgun stochastic search algorithm implemented in FINEMAP. RESULTS:GWAS identified multiple significant loci associated with BW and EW, with high heritability estimated for these traits. EN showed a more polygenic architecture with lower heritability across most periods. Including correlated traits as covariates in GWAS revealed pleiotropic loci, particularly on chromosomes 1 and 4, that influenced both BW and EW, as well as loci specific to individual traits. Both fine-mapping methods successfully pinpointed candidate genes such as NCAPG, LCORL, and IGF2BP1, which are well known for their roles in growth and body size across species. Several novel candidate genes were also highlighted for EN. Notably, some fine-mapped results reflected patterns consistent with the covariate-adjusted GWAS results. CONCLUSIONS:This study demonstrates the power of combining GWAS with imputation and fine-mapping methods in chickens to uncover the genetic basis of economically important traits. Furthermore, incorporating correlated traits as covariates in GWAS provided valuable insights, enabling the distinction between pleiotropic and trait-specific loci. Together, these approaches refine GWAS signals and deepened our understanding of the genetic architecture underlying complex traits.

Avian influenza viruses pose a threat to both wild and domestic bird populations worldwide. In particular, chickens kept outdoors have contact with wild birds and thus an increased risk of infection with influenza viruses. Increasing use of high-performance chicken breeds in growing poultry production reduces genetic diversity whereas local breeds may offer valuable traits such as disease resilience important for sustainable agriculture. This study investigates the immunological performance of three local chicken breeds (Altsteirer, Ramelsloher, and Bielefelder) in Germany to assess their potential resilience to infectious diseases such as avian influenza. Viral resilience was assessed by inoculating 6-week-old chickens with a highly pathogenic avian influenza virus H7N1 and the moderately pathogenic recombinant avian influenza virus TG05-HAR65. Differences in mortality, immune response, transmission, virus shedding, and viral load in certain organs were observed. The Ramelsloher chickens had the lowest clinical score and the highest survival rate. Chicken-to-chicken transmission was only observed in the Altsteirer breed. Lung infiltration by T cells was detected in the Bielefelder and Ramelsloher breeds. In addition, 35-week-old laying hens at peak laying performance were infected with TG05-HAR65, to assess the impact of laying activity on viral resilience and immunocompetence. Compared with juvenile chickens, clinical signs, virus shedding, and seroconversion were detected at later time points. At the end of the experiment, most hens showed egg yolk peritonitis, likely caused by the influenza virus infection. This study indicates differences in the immunocompetence and viral resilience of local chicken breeds and provides breeding and biosafety management recommendations for organic farming.

Animal welfare monitoring is rapidly becoming a critical concern, with an ongoing search for biomarkers. DNA methylation has the potential to serve as such a biomarker, as environment and genotype can interact to shape DNA methylation. We tested the hypothesis, whether genetically distinct laying hens display divergent methylation patterns at selected genes relevant for behavior in response to housing conditions. The aims of the present study were therefore to determine the effect of age (two-day-old chicks versus one-year-old laying hens), genetics (brown versus white laying hens), laying intensity (high versus low), and the effect of housing conditions (layer cage versus small group housing) on the methylation levels of selected genes implicated in behavior in chicken. DNA methylation at the OXT, OXTR, ESR1, HTR1A, HTR2A, and HTR3A gene were determined using direct bisulfite-PCR sequencing, with OXTR being the only gene displaying consistent effects. The greatest effect of age on methylation was evident for OXTR, with adult animals displaying significantly higher methylation than 2-day old chicks. While methylation levels for OXTR did not differ between chicks, significant differences in methylation levels were detected for OXTR when comparing samples from adult animals. For the majority of CpG sites, an interactive effect of laying intensity x housing and genetics x housing on OXTR DNA methylation was evident. When evaluating OXTR methylation status separated by housing condition, a clear difference between layer lines was revealed. While BLA (brown, high laying intensity) animals exhibited consistent methylation levels independent of the housing condition, WLA (white, high laying intensity), G11 (white, low laying intensity) and L68 (brown, low laying intensity) animals displayed differences in OXTR methylation depending on the housing condition. In summary, age, genetics, laying intensity, and housing conditions are associated with differences in DNA methylation of the oxytocin receptor in female chicken, suggesting that OXTR methylation may represent a candidate epigenetic biomarker linking genetics, environment, and laying performance. Future studies will have to determine its correlation with animal welfare status.

Chickens are one of the world’s most important farm animals. With an increasing demand for poultry meat and eggs in recent years, chickens play an essential role in global nutrition and agriculture. However, a severe loss of genetic diversity in livestock has been caused by the focus on high-performance lines, with many traditional and local breeds being threatened with extinction. Although it is assumed that traditional local breeds are more resilient to disease and less susceptible to external influences, little is known about their immunocompetence. This study focuses on the immunological performance of three local chicken breeds (Altsteirer, Bielefelder, and Ramelsloher) in Germany. To evaluate general immunocompetence, blood samples from naïve chicken throughout their lifespan were analyzed by flow cytometry. In adult chickens, minor breed differences were detected regarding the composition of T cell subtypes. However, in day-old chicks the presence of these T cells differs greatly between breeds. To assess the immunological memory after Newcastle Disease Virus vaccination, cellular and humoral immune responses were analyzed. Both, in vivo and in vitro experiments revealed that the duration of immunity depends on the genetic background. Breed-specific proliferation phenotypes were observed up to tolerance induction in one breed after viral re-stimulation. The immune differences among local chicken breeds can explain their differential response to Newcastle Disease vaccination, providing immune markers for breed selection in organic farming. The results of the present study represent the genetic diversity of chicken breeds and show differences in the immunocompetence of local breeds. Thus, this study provides valuable insights into the genetic variation of immunological traits beyond commercial hybrids.

Bone damages in laying hens are of great concern in poultry farming. Besides various risk factors like housing systems or nutrient supply during egg production, it has often been hypothesized that genetically high-performing laying hens may be more prone to bone damages. The relevance of dietary support during the rearing period of pullets for optimal bone development has been little addressed so far. In the present study, an increasing dietary vitamin D3 content within EU legislation was tested during the first 12 weeks of life in two high and two moderate-performing pullet lines (white and brown layer lines). For this purpose, a total of 940 chickens of both sexes were housed at the Institute of Animal Welfare and Animal Husbandry (Friedrich-Loeffler-Institut, Germany). The three experimental diets differed only regarding the added vitamin D3 amount (300/1000/3000 IU Cholecalciferol/kg diet). After every 4 weeks, randomly chosen animals per genotype and dietary treatment were slaughtered for dissection. Serum 25(OH)-Vitamin D3 concentrations reflected the dietary treatment. Body weight differed regarding genotype. No effect of dietary vitamin D3 content as a single influence factor on bone parameters like breaking strength, bone dimensions or mineral content could be shown, but age, sex and genotype had impacts and influenced traits in an interactive manner. Therefore, during the first 12 weeks of the rearing period of layer pullets, the different dietary vitamin D3 contents did not influence performance or bone parameters in the four genetically diverse purebred layer lines. Adjusted dietary vitamin D3 recommendations for pullets depending on genetically predetermined egg-laying performance do not appear to be necessary if dietary vitamin D3 contents are within EU legislation.

ABSTRACT 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.

it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed.The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge.Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals. Frontiers journal seriesThe Frontiers journal series is a multi-tier and interdisciplinary set of openaccess, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing.All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community.At the same time, the Frontiers journal series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too. Dedication to qualityEach Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians.Research must be certified by peers before entering a stream of knowledge that may eventually reach the public -and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view.By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

Einleitung In einer Leistungsstudie der Hühnerrassen „Dresdner” und „Zwerg- Dresdner” fielen zahlreiche Tiere mit tastbaren Wirbelsäulendeformationen auf. Untersuchungen zur näheren Beschreibung der Deformationen und zur Ermittlung möglicher Ursachen wurden durchgeführt.

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.

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.

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.

Gene banks are a component of a national strategy for the preservation of genetic diversity. Gene bank managers need to have a global and comparable picture of the diversity in their collections in order to rationalize them. Facing a diversity of molecular tools is a difficulty. The IMAGE H2020 project aimed at developing a low cost 60k SNP array to facilitate the mapping of diversity in gene banks. The first test of this array was performed for chicken with 204 samples from 18 local breeds and nine experimental lines provided by Germany, France and Spain. The MAF across population was 0.34, showing that this tool is useful over a range of populations. The principal component analysis and the Neighbor-joining tree showed that local breeds did not cluster according to country and were generally homogenous. Comparison with on-farm populations remains to be done to assess the value of the gene bank collections.

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.

Structural variants (SV) have gained increasing interest within the last years. However, calling SVs from array genotypes or short-read data is known to suffer from high false-positive rates and low specificity at the same time. In contrast, long-read technologies promise to derive accurate and sensitive SV callsets. We hereby present the first study that uses PacBio and Nanopore long-reads to derive quantitative estimates of the amount of SVs in chicken, based on four trios. We estimated the mean size of the reference genome affected by at least heterozygous SV calls to range from 0.2% for insertions to 0.7% for deletions. We further compared the results to a short-read based calling approach. This revealed more than 2/3 of short-read based initial SV calls not being backed by long-read based calls, while more than 50% of the long-read based calls would have been missed by the short-read approach.

Poultry production systems are currently dealing with some important issues like animal welfare, environmental impacts of soy imports from overseas, and the decline of genetic diversity. Therefore, an alternative approach to poultry production was tested in this study using six dierent chicken genotypes in terms of fattening performance of males and laying performance of females. Two traditional chicken breeds, Vorwerkhuhn (VH) and Bresse Gauloise (BG), and a commercial layer genotype, White Rock (WR) as well as crossbreds thereof were fed diets containing either 20% vicin-rich or vicin-poor faba beans. The genotype BG and the crossing genotypes with BG exhibited the best growth performance. WR and BG × WR cross showed the best performance regarding the egg production and egg quality. Taken together, the dietary effect revealed only small negative impacts of vicin. In terms of dual-purpose usage, crossing BG with WR seems to be the most promising variant studied here.

Genetic rescue using external breeders or cryo-preserved semen is a reasonable strategy to restore diversity in small breeds. It is also known from natural populations that animals from large populations can convey substantial genetic load when they migrate into small, highly inbred populations, which may compromise the recipient population. We used stochastic simulation to evaluate different scenarios of genetic rescue of two conservation stocks of different sizes and found that introgression of males from larger stocks had a more detrimental effect on the viability of offspring than from smaller stocks, even though both strategies partially restored diversity. Using samples from gene-banks had a comparably negative effect on offspring viability, if the population was substantially larger at the time of cryo-conservation. Our results suggest that careful assessment of genetic load and its consequences is required whenever external animals should be transferred into endangered stocks. As larger breeds have higher genetic load due to less efficient purging, animals from small breeds seem beneficial for genetic rescue.

Several PCR-based methods are known for sexing of chickens and other birds (Çakmak et al., 2017; Eiras et al., 2018; Gruszczyńska & Grzegrzółka, 2021; Morinha et al., 2012). While some methods for bird sexing are suitable for large-scale analyses (Chen et al., 2012; Clinton et al., 2016; He et al., 2019; Margulis & Danielli, 2019; Morinha et al., 2013; Rosenthal et al., 2010), research is still ongoing because most of these methods are costly and time consuming. Here we report a newly developed, easy to use competitive allele-specific PCR (KASP) assay that is suitable for large-scale sexing in chickens and other birds. The KASP assay is based on an A/G difference in exon 17 between the W- and Z-chromosomal variants of the conserved chromodomain helicase DNA binding protein 1 (CHD1) in exon 17 (Figure S1). Sex-specific primers were designed up- and downstream of this variant in CHD1 genes to amplify a product of 46 bp (Figure 1; Table S1, Figure S1). The amplicon overlaps with the PCR product for sex genotyping of the method of Fridolfsson and Ellegren (1999) (Figure S1). Furthermore, similar sequences were obtained from NCBI databases using blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for duck, goose, quail and turkey and were aligned with the chicken sequence (Figure 1). In total, 734 chicken samples with known sex were analysed (Table S2). In addition, 55 samples of other species of the orders Galliformes and Anseriformes were analysed (Table S2). If the sex of non-chicken samples was unknown it was verified by multiplex PCR modified from Fridolfsson and Ellegren (1999) (Table S1, Figure S1). Samples were mainly taken from an extensive DNA collection, which was set up within the framework of the projects AVIANDIV (Lyimo et al., 2014) and SYNBREED (www.synbreed.tum.de). Each KASP reaction contained 20–50 ng of template DNA, KASP v. 4.0 2× Master mix standard ROX and the KASP-by-Design assay mix (LGC Genomics). The standard KASP thermal cycling conditions according to LGC protocols were performed in an Eppendorf Mastercycler (Eppendorf). After amplification, microplates were analysed with FLUOstar Omega (BMG Labtech) using excitation and emission values of 485/520 nm for the FAM-labelled-FRET cassette, 530/560 nm for the HEX-labelled-FRET cassette and 584/620 nm for the ROX standard. All chickens, ducks, geese, quails and turkeys were correctly assigned to their sex using the newly developed KASP assay (Table S2). This newly developed KASP assay is well suited to chicken, duck, goose, quail and turkey for efficient sex determination on a larger scale. The research leading to these results has received funding from the European Union's Horizon 2020 Research and Innovation Programme under the grant agreement no. 677353 IMAGE. The authors thank Alina Kirchhoff, Kim Köhler, Christin Moelders and Katrin Zimmermann for technical assistance. Open access funding was enabled and organised by ProjektDEAL. Open access funding enabled and organized by ProjektDEAL. The authors declare no potential conflicts of interest. The data supporting the findings of the present study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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.