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.

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.

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.

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.

Organic Turkey Flocks: A Reservoir of Streptococcus gallolyticus subspecies gallolyticus.

Streptococcus gallolyticus subspecies gallolyticus (S. gallolyticus) can colonise the gastrointestinal tract of humans and animals and is known to cause similar infections in both humans and animals. Data about the spread or prevalence in farm animals are missing. In this study, Trypton Soya Agar was modified to a selective medium enabling the isolation and quantification of S. gallolyticus from faecal samples. The bacterium was observed in 82 out of 91 faecal samples obtained from 18 different organic turkey flocks. The prevalence of shedding birds was estimated by the number of positive fresh droppings and reached up to 100% on most farms. Furthermore, for the first time S. gallolyticus was quantified in faeces from poultry flocks. The median of colony forming units (CFU) per gramme faeces was 3.6 x 10(5) CFU/g. Typing of one isolate from each positive faecal sample by multilocus sequence typing delivered 24 sequence types (STs). Most of the isolates belonged to the clonal complex CC58. The same STs of this complex were detected in up to six different flocks. Partly, these flocks were located in various regions and stocked with varying breeding lines. Regarding the biochemical profiles of the same STs from different farms, the results did not contradict a spread of specific STs in the organic turkey production. Moreover, checking the pubMLST database revealed that STs found in this study were also found in other animal species and in humans. The high detection rate and the number of S. gallolyticus in turkey faeces indicate that this bacterium probably belongs to the common microbiota of the gastrointestinal tract of turkeys from organic flocks. Furthermore, the findings of this study support the suggestion of a possible interspecies transmission.

A replication study for genome-wide gene expression levels in two layer lines elucidates differentially expressed genes of pathways involved in bone remodeling and immune responsiveness.

The current replication study confirmed significant differences in gene expression profiles of the cerebrum among the two commercial layer lines Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB). Microarray analyses were performed for 30 LSL and another 30 LB laying hens kept in the small group housing system Eurovent German. A total of 14,103 microarray probe sets using customized Affymetrix ChiGene-1_0-st Arrays with 20,399 probe sets were differentially expressed among the two layer lines LSL and LB (FDR adjusted P-value <0.05). An at least 2-fold change in expression levels could be observed for 388 of these probe sets. In LSL, 214 of the 388 probe sets were down- and 174 were up-regulated and vice versa for the LB layer line. Among the 174 up-regulated probe sets in LSL, we identified 51 significantly enriched Gene ontology (GO) terms of the biological process category. A total of 63 enriched GO-terms could be identified for the 214 down-regulated probe sets of the layer line LSL. We identified nine genes significantly differentially expressed between the two layer lines in both microarray experiments. These genes play a crucial role in protection of neuronal cells from oxidative stress, bone mineral density and immune response among the two layer lines LSL and LB. Thus, the different regulation of these genes may significantly contribute to phenotypic trait differences among these layer lines. In conclusion, these novel findings provide a basis for further research to improve animal welfare in laying hens and these layer lines may be of general interest as an animal model.

Analysis of heterophil to lymphocyte ratios in laying hens kept in a small group housing system.

The objective of the present investigation was to assess the level of stress imposed on two different layer lines kept in a small group housing system Eurovent German with two group sizes and three tiers. A total of 615 Lohmann Selected Leghorn (LSL) and 633 Lohmann Brown (LB) hens were examined in four consecutive trials. Based on differential white blood cell counts, the heterophil to lymphocyte ratio (H/L-ratio) was calculated as an indicator of stress. The H/L-ratios significantly differed among the two layer lines, with 2.5-fold higher H/L-ratios in LB than in LSL. No significant differences across and within layer lines could be found between the different group sizes. A significant 0.7-fold decrease of the H/L-ratio could be shown in LSL layers when the space per hen was increased from 828 to 920 cm2.

Differential gene expression from genome-wide microarray analyses distinguishes Lohmann Selected Leghorn and Lohmann Brown layers.

The Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB) layer lines have been selected for high egg production since more than 50 years and belong to the worldwide leading commercial layer lines. The objectives of the present study were to characterize the molecular processes that are different among these two layer lines using whole genome RNA expression profiles. The hens were kept in the newly developed small group housing system Eurovent German with two different group sizes. Differential expression was observed for 6,276 microarray probes (FDR adjusted P-value <0.05) among the two layer lines LSL and LB. A 2-fold or greater change in gene expression was identified on 151 probe sets. In LSL, 72 of the 151 probe sets were up- and 79 of them were down-regulated. Gene ontology (GO) enrichment analysis accounting for biological processes evinced 18 GO-terms for the 72 probe sets with higher expression in LSL, especially those taking part in immune system processes and membrane organization. A total of 32 enriched GO-terms were determined among the 79 down-regulated probe sets of LSL. Particularly, these terms included phosphorus metabolic processes and signaling pathways. In conclusion, the phenotypic differences among the two layer lines LSL and LB are clearly reflected in their gene expression profiles of the cerebrum. These novel findings provide clues for genes involved in economically important line characteristics of commercial laying hens.