The presence of extreme feather peckers in groups of laying hens

Feather pecking is a serious economic and welfare problem in laying hens. Feather damage occurs mainly through severe feather pecking (SFP). Selection experiments have proved that this behavior is heritable and lines have been divergently selected for high (HFP) and low feather pecking (LFP). The number of bouts of SFP per hen follows a Poisson distribution with a maximum nearby 0. A few studies indicate that the distribution within flocks is not homogenous but contains sub-groups of birds showing extremely high levels of feather pecking (EFP). It was the aim of the current study to re-analyze data on SFP of lines selected for HFP/LFP and their F2 cross so as to uncover hidden sub-populations of EFP birds. Data of seven selection generations of HFP and LFP selection lines as well as their F2 cross have been used. We fitted a two-component mixture of Poisson distributions in order to separate the sub-group of EFP from the remaining birds. HFP and LFP lines differed mainly in mean bouts per bird. The proportion of EFP was only marginal in the LFP as compared with the HFP and the F2 population. Selection for LFP did not result in total elimination of EFP. The presence of even small proportions of EFP may play an important role in initiating outbreaks of feather pecking in large flocks. Further studies on feather pecking should pay special attention to the occurrence of EFP sub-groups.     

Heart rate variability in domestic chicken lines genetically selected on feather pecking behavior

Domestic chicken lines of the White Leghorn type differing in their level of feather pecking were developed by divergent genetic selection specifically on feather pecking behavior. We determined parameters of heart rate variability to elucidate the relative activation of the sympathetic and parasympathetic nervous systems during rest and stressful situations. A total of 48 hens were tested in 8 batches. Segments of 2 min were extracted from electrocardiograms recorded by radio-transmitter implants, before (basal undisturbed conditions) and during physical restraint and a social test. Under basal conditions mean distance between R-waves were shorter in the low and high lines compared to the control. During physical restraint, stress reactions [reduced root of the mean squares of successive differences (RMSSD), reduced high frequency (HF), high low frequency (LF/HF) and low vagal-sympathetic effect (VSE) compared to basal levels] were significant in all lines. During the physical restraint the high feather pecking (HFP) line reacted significantly stronger than control (CON) and low feather pecking (LFP) line. During social test the LFP line reacted different than the other two lines. Seemingly birds from LFP conceived the social test as less stressful than birds from the CON and HFP lines. From this it follows that (1) physical restraint generally induced higher stress reactions than the social test and (2) genetic selection for higher levels of feather pecking increased the autonomic nervous system reaction to physical restraint whereas selection against feather pecking has reduced the response to increased social contact.     

Evidence for epistasis between <Emphasis Type="Italic">SLC6A4</Emphasis> and <Emphasis Type="Italic">ITGB3</Emphasis> in autism etiology and in the determination of platelet serotonin levels

Autism is a neurodevelopmental disorder of unclear etiology. The consistent finding of platelet hyperserotonemia in a proportion of patients and its heritability within affected families suggest that genes involved in the serotonin system play a role in this disorder. The role in autism etiology of seven candidate genes in the serotonin metabolic and neurotransmission pathways and mapping to autism linkage regions (SLC6A4, HTR1A, HTR1D, HTR2A, HTR5A, TPH1 and ITGB3) was analyzed in a sample of 186 nuclear families. The impact of interactions among these genes in autism was assessed using the multifactor-dimensionality reduction (MDR) method in 186 patients and 181 controls. We further evaluated whether the effect of specific gene variants or gene interactions associated with autism etiology might be mediated by their influence on serotonin levels, using the quantitative transmission disequilibrium test (QTDT) and the restricted partition method (RPM), in a sample of 109 autistic children. We report a significant main effect of the HTR5A gene in autism (P = 0.0088), and a significant three-locus model comprising a synergistic interaction between the ITGB3 and SLC6A4 genes with an additive effect of HTR5A (P < 0.0010). In addition to the previously reported contribution of SLC6A4, we found significant associations of ITGB3 haplotypes with serotonin level distribution (P = 0.0163). The most significant models contributing to serotonin distribution were found for interactions between TPH1 rs4537731 and SLC6A4 haplotypes (P = 0.002) and between HTR1D rs6300 and SLC6A4 haplotypes (P = 0.013). In addition to the significant independent effects, evidence for interaction between SLC6A4 and ITGB3 markers was also found. The overall results implicate SLC6A4 and ITGB3 gene interactions in autism etiology and in serotonin level determination, providing evidence for a common underlying genetic mechanism and a molecular explanation for the association of platelet hyperserotonemia with autism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Loss of Function of ATXN1 Increases Amyloid ��-Protein Levels by Potentiating ��-Secretase Processing of ��-Amyloid Precursor Protein

Alzheimer disease (AD) is a devastating neurodegenerative disease with complex and strong genetic inheritance. Four genes have been established to either cause familial early onset AD (APP, PSEN1, and PSEN2) or to increase susceptibility for late onset AD (APOE). To date ∼80% of the late onset AD genetic variance remains elusive. Recently our genome-wide association screen identified four novel late onset AD candidate genes. Ataxin 1 (ATXN1) is one of these four AD candidate genes and has been indicated to be the disease gene for spinocerebellar ataxia type 1, which is also a neurodegenerative disease. Mounting evidence suggests that the excessive accumulation of Aβ, the proteolytic product of β-amyloid precursor protein (APP), is the primary AD pathological event. In this study, we ask whether ATXN1 may lead to AD pathogenesis by affecting Aβ and APP processing utilizing RNA interference in a human neuronal cell model and mouse primary cortical neurons. We show that knock-down of ATXN1 significantly increases the levels of both Aβ40 and Aβ42. This effect could be rescued with concurrent overexpression of ATXN1. Moreover, overexpression of ATXN1 decreased Aβ levels. Regarding the underlying molecular mechanism, we show that the effect of ATXN1 expression on Aβ levels is modulated via β-secretase cleavage of APP. Taken together, ATXN1 functions as a genetic risk modifier that contributes to AD pathogenesis through a loss-of-function mechanism by regulating β-secretase cleavage of APP and Aβ levels.     

Increased DNA methylation of neuropsychiatric genes occurs in borderline personality disorder

Borderline personality disorder (BPD) is a complex psychiatric disease of increasing importance. Epigenetic alterations are hallmarks for altered gene expression and could be involved in the etiology of BPD. In our study we analyzed DNA methylation patterns of 14 neuropsychiatric genes (COMT, DAT1, GABRA1, GNB3, GRIN2B, HTR1B, HTR2A, 5-HTT, MAOA, MAOB, NOS1, NR3C1, TPH1 and TH). DNA methylation was analyzed by bisulfite restriction analysis and pyrosequencing in whole blood samples of patients diagnosed with DSM-IV BPD and in controls. Aberrant methylation was not detectable using bisulfite restriction analysis, but a significantly increased methylation of HTR2A, NR3C1, MAOA, MAOB and soluble COMT (S-COMT) was revealed for BPD patients using pyrosequencing. For HTR2A the average methylation of four CpG sites was 0.8% higher in BPD patients compared to controls (p = 0.002). The average methylation of NR3C1 was 1.8% increased in BPD patients compared to controls (p = 0.0003) and was higher at 2 out of 8 CpGs (p ≤ 0.04). In females, an increased average methylation (1.5%) of MAOA was observed in BPD patients compared to controls (p = 0.046). A similar trend (1.4% higher methylation) was observed for MAOB in female BPD patients and increased methylation was significant for 1 out of 6 CpG sites. For S-COMT, a higher methylation of 2 out of 4 CpG sites was revealed in BPD patients (p ≤ 0.02). In summary, methylation signatures of several promoter regions were established and a significant increased average methylation (1.7%) occurred in blood samples of BPD patients (p < 0.0001). Our data suggest that aberrant epigenetic regulation of neuropsychiatric genes may contribute to the pathogenesis of BPD.     

An approach based on a genome-wide association study reveals candidate loci for narcolepsy

Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness, cataplexy, and a pathological manifestation of rapid eye movement during sleep. Narcoleptic pathogenesis is triggered by both genetic and environmental factors. Recently, development of genome-wide association studies (GWAS) has identified new genetic factors, with many more susceptibility genes yet to be elucidated. Using a new approach that consists of a combination of GWAS and an extensive database search for candidate genes, we picked up 202 candidate genes and performed a replication study in 222 narcoleptic patients and 380 controls. Statistical analysis indicated that six genes, NFATC2, SCP2, CACNA1C, TCRA, POLE, and FAM3D, were associated with narcolepsy (P < 0.001). Some of these associations were further supported by gene expression analyses and an association study in essential hypersomnia (EHS), CNS hypersonia similar to narcolepsy. This novel approach will be applicable to other GWAS in the search of disease-related susceptibility genes.     

Genome‐wide association study confirms that the chromosome Z harbours a region responsible for rumplessness in Hongshan chickens

Rumplessness in Hongshan chickens has been reported as a novel sex‐linked characteristic. Re‐sequencing data suggest that a pseudogene on the Z chromosome, LOC431648, is affiliated with this phenotype. In this study, we chose 23 rumpless and 25 normal Hongshan chickens to localize the potential variation by means of a genome‐wide association study using a high density microarray. A region on the Z chromosome was found to be closely associated with rumplessness in Hongshan chickens. The region, located in gene LINGO2, was approximately 3 Mb away from pseudogene LOC431648. The function of this gene has not yet been studied in birds. Differential expression of the candidate genes in the tail feather follicles was not detected by q‐PCR, which suggests that the rumplessness trait could be attributed to other genetic mechanisms.     

Selection method and early-life history affect behavioural development, feather pecking and cannibalism in laying hens: A review

The aim of this review is to discuss the effects of selection method and early-life history on the behavioural development of laying hens. Especially in larger groups, laying hens often develop damaging behaviours, such as feather pecking and cannibalism, leading to impaired animal welfare. We hypothesise that the propensity to develop feather pecking and cannibalism is affected by a bird's genetic background and by its early-life history. The genetic background can be influenced by genetic selection. Laying hens are traditionally selected on individual performance, which may lead to co-selection of feather pecking and cannibalism. For hens kept in small groups, it has recently been demonstrated that a novel group selection method, focusing on group performance, can help to reduce cannibalism. However, the biological background behind the success of group selection is unknown. It is also not known whether these results from small groups can be translated to larger groups of laying hens. Regarding early-life history, laying, brooding and rearing conditions have been shown to have major effects on behavioural development and on feather pecking and cannibalism. The presence of a hen during rearing has been shown to improve foraging- and social behaviour, to decrease feather pecking and to decrease fearfulness in chicks. Applying group selection and rearing laying hens in a more natural environment may be key factors in solving the problems caused by feather pecking and cannibalism, especially if the promising results of group selection from small groups in experimental settings can be translated to large-group housing systems.     

Identification of molecular markers for oocyte competence in bovine cumulus cells

Cumulus cells (CCs) have an important role during oocyte growth, competence acquisition, maturation, ovulation and fertilization. In an attempt to isolate potential biomarkers for bovine in vitro fertilization, we identified genes differentially expressed in bovine CCs from oocytes with different competence statuses, through microarray analysis. The model of follicle size, in which competent cumulus–oocyte complexes (COCs) were recovered from bigger follicles (≥8.0 mm in diameter) and less competent ones from smaller follicles (1–3 mm), was used. We identified 4178 genes that were differentially expressed (P < 0.05) in the two categories of CCs. The list was further enriched, through the use of a 2.5‐fold change in gene expression as a cutoff value, to include 143 up‐regulated and 80 down‐regulated genes in CCs of competent COCs compared to incompetent COCs. These genes were screened according to their cellular roles, most of which were related to cell cycle, DNA repair, energy metabolism, metabolism of amino acids, cell signaling, meiosis, ovulation and inflammation. Three candidate genes up‐regulated (FGF11, IGFBP4, SPRY1) and three down‐regulated (ARHGAP22, COL18A1 and GPC4) in CCs from COCs of big follicles (≥8.1 mm) were selected for qPCR analysis. The selected genes showed the same expression patterns by qPCR and microarray analysis. These genes may be potential genetic markers that predict oocyte competence in in vitro fertilization routines.     

Three‐dimensional genetic networks among seed oil‐related traits,metabolites and genes reveal the genetic foundations of oil synthesis in soybean

Although the biochemical and genetic basis of lipid metabolism is clear in Arabidopsis, there is limited information concerning the relevant genes in Glycine max (soybean). To address this issue, we constructed three‐dimensional genetic networks using six seed oil‐related traits, 52 lipid metabolism‐related metabolites and 54 294 SNPs in 286 soybean accessions in total. As a result, 284 and 279 candidate genes were found to be significantly associated with seed oil‐related traits and metabolites by phenotypic and metabolic genome‐wide association studies and multi‐omics analyses, respectively. Using minimax concave penalty (MCP) and smoothly clipped absolute deviation (SCAD) analyses, six seed oil‐related traits were found to be significantly related to 31 metabolites. Among the above candidate genes, 36 genes were found to be associated with oil synthesis (27 genes), amino acid synthesis (four genes) and the tricarboxylic acid (TCA) cycle (five genes), and four genes (GmFATB1a, GmPDAT, GmPLDα1 and GmDAGAT1) are already known to be related to oil synthesis. Using this information, 133 three‐dimensional genetic networks were constructed, 24 of which are known, e.g. pyruvate–GmPDAT–GmFATA2–oil content. Using these networks, GmPDAT, GmAGT and GmACP4 reveal the genetic relationships between pyruvate and the three major nutrients, and GmPDAT, GmZF351 and GmPgs1 reveal the genetic relationships between amino acids and seed oil content. In addition, GmCds1, along with average temperature in July and the rainfall from June to September, influence seed oil content across years. This study provides a new approach for the construction of three‐dimensional genetic networks and reveals new information for soybean seed oil improvement and the identification of gene function.     

Candidate gene studies of ADHD: a meta-analytic review

Quantitative genetic studies (i.e., twin and adoption studies) suggest that genetic influences contribute substantially to the development of attention deficit hyperactivity disorder (ADHD). Over the past 15 years, considerable efforts have been made to identify genes involved in the etiology of this disorder resulting in a large and often conflicting literature of candidate gene associations for ADHD. The first aim of the present study was to conduct a comprehensive meta-analytic review of this literature to determine which candidate genes show consistent evidence of association with childhood ADHD across studies. The second aim was to test for heterogeneity across studies in the effect sizes for each candidate gene as its presence might suggest moderating variables that could explain inconsistent results. Significant associations were identified for several candidate genes including DAT1, DRD4, DRD5, 5HTT, HTR1B, and SNAP25. Further, significant heterogeneity was observed for the associations between ADHD and DAT1, DRD4, DRD5, DBH, ADRA2A, 5HTT, TPH2, MAOA, and SNAP25, suggesting that future studies should explore potential moderators of these associations (e.g., ADHD subtype diagnoses, gender, exposure to environmental risk factors). We conclude with a discussion of these findings in relation to emerging themes relevant to future studies of the genetics of ADHD.     

Feather eating and its associations with plumage damage and feathers on the floor in commercial farms of laying hens

Feather eating has been associated with feather pecking, which continues to pose economic and welfare problems in egg production. Knowledge on feather eating is limited and studies of feather eating in commercial flocks of laying hens have not been performed previously. Therefore, the main objective was to investigate feather eating and its association with plumage damage and floor feather characteristics in commercial flocks of layers in barn and organic production systems. The study was performed in 13 flocks of barn layers and 17 flocks of organic layers. Each flock was visited at around 32 and 62 weeks of age. During both visits, the plumage condition was assessed and the density of floor feathers recorded. In week 62, droppings and floor feathers were collected. Droppings were examined for presence of feather content, whereas length, downiness and pecking damage were recorded for each floor feather. In week 62, a higher prevalence of hens with poor plumage condition was found in barn (22.2%) compared with organic production systems (7.4%; P<0.001), but the prevalence of droppings with feather content did not differ between the two production systems (8.5% in barn v. 4.3% in organic; P=0.99). Our hypothesis about a positive correlation between feather eating and plumage damage was not supported as no correlation was found between the prevalence of poor plumage condition and the prevalence of droppings with feather content. However, the prevalence of pecking damaged floor feathers was positively correlated both with prevalence of droppings with feather content (P<0.05) and poor plumage condition (P<0.01), indicating a possible association between feather eating and feather pecking. In conclusion, it was confirmed that feather eating occurs on-farm, but feather eating was only found to be positively correlated to the number of floor feathers with pecking damage and not as expected to the prevalence of plumage damage. More research is needed into the sources from where feathers are selected for ingestion, that is, whether they are picked from the floor litter, plucked directly from other hens or dislodged during preening of own feathers.     

Identification of candidate genes encoding an LDL-C QTL in baboons

Cardiovascular disease (CVD) is the leading cause of death in developed countries, and dyslipidemia is a major risk factor for CVD. We previously identified a cluster of quantitative trait loci (QTL) on baboon chromosome 11 for multiple, related quantitative traits for serum LDL-cholesterol (LDL-C). Here we report differentially regulated hepatic genes encoding an LDL-C QTL that influences LDL-C levels in baboons. We performed hepatic whole-genome expression profiling for LDL-C-discordant baboons fed a high-cholesterol, high-fat (HCHF) diet for seven weeks. We detected expression of 117 genes within the QTL 2-LOD support interval. Three genes were differentially expressed in low LDL-C responders and 8 in high LDL-C responders in response to a HCHF diet. Seven genes (ACVR1B, CALCOCO1, DGKA, ERBB3, KRT73, MYL6B, TENC1) showed discordant expression between low and high LDL-C responders. To prioritize candidate genes, we integrated miRNA and mRNA expression profiles using network tools and found that four candidates (ACVR1B, DGKA, ERBB3, TENC1) were miRNA targets and that the miRNAs were inversely expressed to the target genes. Candidate gene expression was validated using QRT-PCR and Western blotting. This study reveals candidate genes that influence variation in LDL-C in baboons and potential genetic mechanisms for further investigation.     

Assessment of the effect of housing on feather damage in laying hens using IR thermography

Plumage damage represents one of the animal-based measures of laying hens welfare. Damage occurs predominantly due to age, environment and damaging pecking. IR thermography, due to its non-invasiveness, objectivity and repeatability is a promising alternative to feather damage scoring systems such as the system included in the Welfare Quality^R assessment protocol for poultry. The aim of this study was to apply IR thermography for the assessment of feather damage in laying hens kept in two housing systems and to compare the results with feather scoring. At the start of the experiment, 16-week-old laying hens (n=30) were divided into two treatments such as deep litter pen and enriched cage. During 4 months, feather damage was assessed regularly in 2-week intervals. One more single assessment was done nine and a half months after the start of the experiment. The feather damage on four body regions was assessed by scoring and IR thermography: head and neck, back and rump, belly, and underneck and breast. Two variables obtained by IR thermography were used: the difference between the body surface temperature and ambient temperature (ΔT_B) and the proportion of featherless areas, which were defined as areas with a temperature >33.5°C. Data were analyzed using a GLM model. The effects of housing, time, region and their interactions on feather damage, measured by the feather scoring and by both IR thermography measures, were all significant (P<0.001). The ΔT_B in all assessed regions correlated positively with the feather score. Feather scoring revealed higher damage in enriched cages compared with deep litter pens starting from week 6 of the experiment on the belly and back and rump regions, whereas ΔT_B from week 6 in the belly and from week 8 on the back and rump region. The proportion of featherless areas in the belly region differed significantly between the housings from week 8 of the experiment and on the back and rump region from week 12. The IR thermography assessment of the feather damage revealed differences between hens kept in different housing systems in agreement with the feather scoring. In conclusion, it was demonstrated that the IR thermography is a useful tool for the assessment of poultry feather cover quality that is not biased by the subjective component and provides higher precision than feather damage scoring.     

Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer

The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2′-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.     

Quantitative mRNA Analysis of Eight Bovine 5‐HT Receptor Subtypes in Brain,Abomasum, and Intestine by Real‐Time RT‐PCR

Abstract

Serotoninergic pathways are involved in economically important bovine gastrointestinal (GI) motility disorders such as displaced abomasum and cecal dilatation/dislocation. The existing research tools to investigate the role of serotoninergic pathways in such disorders in ruminants comprise functional pharmacological methods, e.g., in vitro contractility studies in tissue baths, and electromyographical recordings in vivo. However, no tools for quantification of bovine serotonin receptor [5‐hydroxytryptamine receptor (5‐HTR)] expression were available so far. This study aimed to develop real‐time RT‐PCR assays for quantitative mRNA analysis of bovine 5‐HTR subtypes. Because the bovine 5‐HTR coding sequences (CDSs) were completely unknown, multiple species (human, mouse, and rat) alignment of complete CDS was used for primer design in highly homologous regions. LightCycler real‐time RT‐PCR assays (partial CDS) for the following bovine 5‐HTR subtypes were developed and validated: 5‐HTR_1A, 5‐HTR_1B, 5‐HTR_1D, 5‐HTR_1F, 5‐HTR_2A, 5‐HTR_2B, 5‐HTR_2C, and 5‐HTR₄. Intra‐ and inter‐assay coefficients of variation (CV) for the eight established assays were small, ranging from 0.49% to 2.46%. As a first physiological application, 5‐HTR mRNA expression levels were measured in brain, abomasum, and intestine of 10 healthy, lactating dairy cows. The 5‐HTR expression was quantified by normalization to the housekeeping gene glyceraldehyde‐phosphate‐dehydrogenase (GAPDH). The 5‐HTR subtype expression levels ranged from 0.001% (5‐HTR_2C in intestine) to 1% 5‐HTR/GAPDH (5‐HTR_1B and 5‐HTR₄ in intestine). There were high variations of 5‐HTR subtype mRNA expression within tissues across receptor subtypes and within receptor subtypes across tissues. In conclusion, accurate real‐time RT‐PCR assays for quantitative analysis of bovine 5‐HTR subtype gene expression were developed and validated.