Parental methyl-enhanced diet and in ovo corticosterone affect first generation Japanese quail (Coturnix japonica) development, behaviour and stress response.

The role of maternal investment in avian offspring has considerable life history implications on production traits and therefore potential for the poultry industry. A first generation (G1) of Japanese quail (Coturnix japonica) were bred from a 2 × 2 factorial design. Parents were fed either a control or methyl-enhanced (HiBET) diet, and their eggs were treated with a vehicle or corticosterone injection during day 5 of incubation. A subset of G1 birds were subjected to an open field trial (OFT) and capture-restraint stress protocol. Significant effects of HiBET diet were found on parental egg and liver weights, G1 hatch, liver and female reproductive tract weights, egg productivity, latency to leave the OFT central zone, male baseline 11-dehydrocorticosterone, and female androstenedione plasma concentrations. In ovo treatment significantly affected latency to return to the OFT, male baseline testosterone and androstenedione, and change in androstenedione plasma concentration. Diet by treatment interactions were significant for G1 liver weight and male baseline plasma concentrations of corticosterone. These novel findings suggest significant positive effects on reproduction, growth, precociousness, and hypothalamic-pituitary-adrenal axis function from enhanced methyl diets, and are important in understanding how in ovo stressors (representing maternal stress), affect the first offspring generation.

Analysis of the Progeny of Sibling Matings Reveals Regulatory Variation Impacting the Transcriptome of Immune Cells in Commercial Chickens.

There is increasing recognition that the underlying genetic variation contributing to complex traits influences transcriptional regulation and can be detected at a population level as expression quantitative trait loci. At the level of an individual, allelic variation in transcriptional regulation of individual genes can be detected by measuring allele-specific expression in RNAseq data. We reasoned that extreme variants in gene expression could be identified by analysis of inbred progeny with shared grandparents. Commercial chickens have been intensively selected for production traits. Selection is associated with large blocks of linkage disequilibrium with considerable potential for co-selection of closely linked "hitch-hiker alleles" affecting traits unrelated to the feature being selected, such as immune function, with potential impact on the productivity and welfare of the animals. To test this hypothesis that there is extreme allelic variation in immune-associated genes we sequenced a founder population of commercial broiler and layer birds. These birds clearly segregated genetically based upon breed type. Each genome contained numerous candidate null mutations, protein-coding variants predicted to be deleterious and extensive non-coding polymorphism. We mated selected broiler-layer pairs then generated cohorts of F2 birds by sibling mating of the F1 generation. Despite the predicted prevalence of deleterious coding variation in the genomic sequence of the founders, clear detrimental impacts of inbreeding on survival and post-hatch development were detected in only one F2 sibship of 15. There was no effect on circulating leukocyte populations in hatchlings. In selected F2 sibships we performed RNAseq analysis of the spleen and isolated bone marrow-derived macrophages (with and without lipopolysaccharide stimulation). The results confirm the predicted emergence of very large differences in expression of individual genes and sets of genes. Network analysis of the results identified clusters of co-expressed genes that vary between individuals and suggested the existence of trans-acting variation in the expression in macrophages of the interferon response factor family that distinguishes the parental broiler and layer birds and influences the global response to lipopolysaccharide. This study shows that the impact of inbreeding on immune cell gene expression can be substantial at the transcriptional level, and potentially opens a route to accelerate selection using specific alleles known to be associated with desirable expression levels.

Dissecting the Genomic Architecture of Resistance to Eimeria maxima Parasitism in the Chicken.

Coccidiosis in poultry, caused by protozoan parasites of the genus Eimeria, is an intestinal disease with substantial economic impact. With the use of anticoccidial drugs under public and political pressure, and the comparatively higher cost of live-attenuated vaccines, an attractive complementary strategy for control is to breed chickens with increased resistance to Eimeria parasitism. Prior infection with Eimeria maxima leads to complete immunity against challenge with homologous strains, but only partial resistance to challenge with antigenically diverse heterologous strains. We investigate the genetic architecture of avian resistance to E. maxima primary infection and heterologous strain secondary challenge using White Leghorn populations of derived inbred lines, C.B12 and 15I, known to differ in susceptibility to the parasite. An intercross population was infected with E. maxima Houghton (H) strain, followed 3 weeks later by E. maxima Weybridge (W) strain challenge, while a backcross population received a single E. maxima W infection. The phenotypes measured were parasite replication (counting fecal oocyst output or qPCR for parasite numbers in intestinal tissue), intestinal lesion score (gross pathology, scale 0-4), and for the backcross only, serum interleukin-10 (IL-10) levels. Birds were genotyped using a high density genome-wide DNA array (600K, Affymetrix). Genome-wide association study located associations on chromosomes 1, 2, 3, and 5 following primary infection in the backcross population, and a suggestive association on chromosome 1 following heterologous E. maxima W challenge in the intercross population. This mapped several megabases away from the quantitative trait locus (QTL) linked to the backcross primary W strain infection, suggesting different underlying mechanisms for the primary- and heterologous secondary- responses. Underlying pathways for those genes located in the respective QTL for resistance to primary infection and protection against heterologous challenge were related mainly to immune response, with IL-10 signaling in the backcross primary infection being the most significant. Additionally, the identified markers associated with IL-10 levels exhibited significant additive genetic variance. We suggest this is a phenotype of interest to the outcome of challenge, being scalable in live birds and negating the requirement for single-bird cages, fecal oocyst counts, or slaughter for sampling (qPCR).

Phenotypic and genetic variation in the response of chickens to Eimeria tenella induced coccidiosis.

BACKGROUND: Coccidiosis is a major contributor to losses in poultry production. With emerging constraints on the use of in-feed prophylactic anticoccidial drugs and the relatively high costs of effective vaccines, there are commercial incentives to breed chickens with greater resistance to this important production disease. To identify phenotypic biomarkers that are associated with the production impacts of coccidiosis, and to assess their covariance and heritability, 942 Cobb500 commercial broilers were subjected to a defined challenge with Eimeria tenella (Houghton). Three traits were measured: weight gain (WG) during the period of infection, caecal lesion score (CLS) post mortem, and the level of a serum biomarker of intestinal inflammation, i.e. circulating interleukin 10 (IL-10), measured at the height of the infection. RESULTS: Phenotypic analysis of the challenged chicken cohort revealed a significant positive correlation between CLS and IL-10, with significant negative correlations of both these traits with WG. Eigenanalysis of phenotypic covariances between measured traits revealed three distinct eigenvectors. Trait weightings of the first eigenvector, (EV1, eigenvalue = 59%), were biologically interpreted as representing a response of birds that were susceptible to infection, with low WG, high CLS and high IL-10. Similarly, the second eigenvector represented infection resilience/resistance (EV2, 22%; high WG, low CLS and high IL-10), and the third eigenvector tolerance (EV3, 19%; high WG, high CLS and low IL-10), respectively. Genome-wide association studies (GWAS) identified two SNPs that were associated with WG at the suggestive level. CONCLUSIONS: Eigenanalysis separated the phenotypic impact of a defined challenge with E. tenella on WG, caecal inflammation/pathology, and production of IL-10 into three major eigenvectors, indicating that the susceptibility-resistance axis is not a single continuous quantitative trait. The SNPs identified by the GWAS for body weight were located in close proximity to two genes that are involved in innate immunity (FAM96B and RRAD).

Phenotypic and genetic integration of personality and growth under competition in the sheepshead swordtail, Xiphophorus birchmanni.

Competition for resources including food, physical space, and potential mates is a fundamental ecological process shaping variation in individual phenotype and fitness. The evolution of competitive ability, in particular social dominance, depends on genetic (co)variation among traits causal (e.g., behavior) or consequent (e.g., growth) to competitive outcomes. If dominance is heritable, it will generate both direct and indirect genetic effects (IGE) on resource-dependent traits. The latter are expected to impose evolutionary constraint because winners necessarily gain resources at the expense of losers. We varied competition in a population of sheepshead swordtails, Xiphophorus birchmanni, to investigate effects on behavior, size, growth, and survival. We then applied quantitative genetic analyses to determine (i) whether competition leads to phenotypic and/or genetic integration of behavior with life history and (ii) the potential for IGE to constrain life history evolution. Size, growth, and survival were reduced at high competition. Male dominance was repeatable and dominant individuals show higher growth and survival. Additive genetic contributions to phenotypic covariance were significant, with the G matrix largely recapitulating phenotypic relationships. Social dominance has a low but significant heritability and is strongly genetically correlated with size and growth. Assuming causal dependence of growth on dominance, hidden IGE will therefore reduce evolutionary potential.

Effects of Eimeria tenella infection on chicken caecal microbiome diversity, exploring variation associated with severity of pathology.

Eimeria species cause the intestinal disease coccidiosis, most notably in poultry. While the direct impact of coccidiosis on animal health and welfare is clear, its influence on the enteric microbiota and by-stander effects on chicken health and production remains largely unknown, with the possible exception of Clostridium perfringens (necrotic enteritis). This study evaluated the composition and structure of the caecal microbiome in the presence or absence of a defined Eimeria tenella challenge infection in Cobb500 broiler chickens using 16S rRNA amplicon sequencing. The severity of clinical coccidiosis in individual chickens was quantified by caecal lesion scoring and microbial changes associated with different lesion scores identified. Following E. tenella infection the diversity of taxa within the caecal microbiome remained largely stable. However, infection induced significant changes in the abundance of some microbial taxa. The greatest changes were detected in birds displaying severe caecal pathology; taxa belonging to the order Enterobacteriaceae were increased, while taxa from Bacillales and Lactobacillales were decreased with the changes correlated with lesion severity. Significantly different profiles were also detected in infected birds which remained asymptomatic (lesion score 0), with taxa belonging to the genera Bacteroides decreased and Lactobacillus increased. Many differential taxa from the order Clostridiales were identified, with some increasing and others decreasing in abundance in Eimeria-infected animals. The results support the view that caecal microbiome dysbiosis associated with Eimeria infection contributes to disease pathology, and could be a target for intervention to mitigate the impact of coccidiosis on poultry productivity and welfare. This work highlights that E. tenella infection has a significant impact on the abundance of some caecal bacteria with notable differences detected between lesion score categories emphasising the importance of accounting for differences in caecal lesions when investigating the relationship between E. tenella and the poultry intestinal microbiome.

Analysis of the function of IL-10 in chickens using specific neutralising antibodies and a sensitive capture ELISA.

In mammals, the inducible cytokine interleukin 10 is a feedback negative regulator of inflammation. To determine the extent to which this function is conserved in birds, recombinant chicken IL-10 was expressed as a secreted human Ig Fc fusion protein (chIL-10-Fc) and used to immunise mice. Five monoclonal antibodies (mAb) which specifically recognise chicken IL-10 were generated and characterised. Two capture ELISA assays were developed which detected native chIL-10 secreted from chicken bone marrow-derived macrophages (chBMMs) stimulated with lipopolysaccharide (LPS). Three of the mAbs detected intracellular IL-10. This was detected in only a subset of the same LPS-stimulated chBMMs. The ELISA assay also detected massive increases in circulating IL-10 in chickens challenged with the coccidial parasite, Eimeria tenella. The same mAbs neutralised the bioactivity of recombinant chIL-10. The role of IL-10 in feedback control was tested in vitro. The neutralising antibodies prevented IL-10-induced inhibition of IFN-γ synthesis by mitogen-activated lymphocytes and increased nitric oxide production in LPS-stimulated chBMMs. The results confirm that IL-10 is an inducible feedback regulator of immune response in chickens, and could be the target for improved vaccine efficacy or breeding strategies.

How integrated are behavioral and endocrine stress response traits? A repeated measures approach to testing the stress-coping style model.

It is widely expected that physiological and behavioral stress responses will be integrated within divergent stress-coping styles (SCS) and that these may represent opposite ends of a continuously varying reactive-proactive axis. If such a model is valid, then stress response traits should be repeatable and physiological and behavioral responses should also change in an integrated manner along a major axis of among-individual variation. While there is some evidence of association between endocrine and behavioral stress response traits, few studies incorporate repeated observations of both. To test this model, we use a multivariate, repeated measures approach in a captive-bred population of Xiphophorus birchmanni. We quantify among-individual variation in behavioral stress response to an open field trial (OFT) with simulated predator attack (SPA) and measure waterborne steroid hormone levels (cortisol, 11-ketotestosterone) before and after exposure. Under the mild stress stimulus (OFT), (multivariate) behavioral variation among individuals was consistent with a strong axis of personality (shy-bold) or coping style (reactive-proactive) variation. However, behavioral responses to a moderate stressor (SPA) were less repeatable, and robust statistical support for repeatable endocrine state over the full sampling period was limited to 11-ketotestosterone. Although post hoc analysis suggested cortisol expression was repeatable over short time periods, qualitative relationships between behavior and glucocorticoid levels were counter to our a priori expectations. Thus, while our results clearly show among-individual differences in behavioral and endocrine traits associated with stress response, the correlation structure between these is not consistent with a simple proactive-reactive axis of integrated stress-coping style. Additionally, the low repeatability of cortisol suggests caution is warranted if single observations (or indeed repeat measures over short sampling periods) of glucocorticoid traits are used in ecological or evolutionary studies focussed at the individual level.

Associations between lamb survival and prion protein genotype: analysis of data for ten sheep breeds in Great Britain.

BACKGROUND: Selective breeding programmes, based on prion protein (PrP) genotype, have been introduced throughout the European Union to reduce the risk of sheep transmissible spongiform encephalopathies (TSEs). These programmes could have negative consequences on other important traits, such as fitness and production traits, if the PrP gene has pleiotropic effects or is in linkage disequilibrium with genes affecting these traits. This paper presents the results of an investigation into associations between lamb survival and PrP genotype in ten mainstream sheep breeds in Great Britain (GB). In addition, the reasons for lamb deaths were examined in order to identify any associations between these and PrP genotype. RESULTS: Survival times from birth to weaning were analysed for over 38000 lambs (2427 dead and 36096 live lambs) from 128 flocks using Cox proportional hazard models for each breed, including additive animal genetic effects. No significant associations between PrP genotype and lamb survival were identified, except in the Charollais breed for which there was a higher risk of mortality in lambs of the ARR/VRQ genotype compared with those of the ARR/ARR genotype. Significant effects of birth weight, litter size, sex, age of dam and year of birth on survival were also identified. For all breeds the reasons for death changed significantly with age; however, no significant associations between reason for death and PrP genotype were found for any of the breeds. CONCLUSION: This study found no evidence to suggest that a selective breeding programme based on PrP genotype will have a detrimental effect on lamb survival. The only significant effect of PrP genotype identified was likely to be of little consequence because an increased risk of mortality was associated with a genotype that is selected against in current breeding strategies.

Predicting the consequences of selecting on PrP genotypes on PrP frequencies, performance and inbreeding in commercial meat sheep populations.

Selection programmes based on prion protein (PrP) genotypes are being implemented for increasing resistance to scrapie. Commercial meat sheep populations participating in sire-referencing schemes were simulated to investigate the effect of selection on PrP genotypes on ARR and VRQ allele frequencies, inbreeding and genetic gain in a performance trait under selection. PrP selection strategies modelled included selection against the VRQ allele and in favour of the ARR allele. Assuming realistic initial PrP frequencies, selection against the VRQ allele had a minimal impact on performance and inbreeding. However, when selection was also in favour of the ARR allele and the frequency of this allele was relatively low, there was a loss of up to three to four years of genetic gain over the 15 years of selection. Most loss in gain occurred during the first five years. In general, the rate of inbreeding was reduced when applying PrP selection. Since animals were first selected on their PrP genotype before being selected on the performance trait, the intensity of selection on performance was weaker under PrP selection (compared with no PrP selection). Eradication of the VRQ allele or fixation of the ARR allele within 15 years of selection was possible only with PrP selection targeting all breeding animals.