Functional inhibition of deep brain non-visual opsins facilitates acute long day induction of reproductive recrudescence in male Japanese quail.

For nearly a century, we have known that brain photoreceptors regulate avian seasonal biology. Two photopigments, vertebrate ancient opsin (VA) and neuropsin (OPN5), provide possible molecular substrates for these photoreceptor pathways. VA fulfills many criteria for providing light input to the reproductive response, but a functional link has yet to be demonstrated. This study examined the role of VA and OPN5 in the avian photoperiodic response of Japanese quail (Coturnix japonica). Non-breeding male quail were housed under short days (6L:18D) and received an intracerebroventricular infusion of adeno-associated viral vectors with shRNAi that selectively inhibited either VA or OPN5. An empty viral vector acted as a control. Quail were then photostimulated (16L:8D) to stimulate gonadal growth. Two long days significantly increased pituitary thyrotrophin-stimulating hormone ß-subunit (TSHß) and luteinizing hormone ß-subunit (LHß) mRNA of VA shRNAi treated quail compared to controls. Furthermore, at one week there was a significant increase, compared to controls, in both hypothalamic gonadotrophin releasing hormone-I (GnRH-I) mRNA and paired testicular mass in VA shRNAi birds. Opn5 shRNAi facilitated the photoinduced increase in TSHß mRNA at 2 days, but no other differences were identified compared to controls. Contrary to our expectations, the silencing of deep brain photoreceptors enhanced the response of the reproductive axis to photostimulation rather than preventing it. In addition, we show that VA opsin plays a dominant role in the light-dependent neuroendocrine control of seasonal reproduction in birds. Together our findings suggest the photoperiodic response involves at least two photoreceptor types and populations working together with VA opsin playing a dominant role.

Genetic markers associated with bone composition in Rhode Island Red laying hens.

BACKGROUND: Bone damage has welfare and economic impacts on modern commercial poultry and is known as one of the major challenges in the poultry industry. Bone damage is particularly common in laying hens and is probably due to the physiological link between bone and the egg laying process. Previous studies identified and validated quantitative trait loci (QTL) for bone strength in White Leghorn laying hens based on several measurements, including bone composition measurements on the cortex and medulla of the tibia bone. In a previous pedigree-based analysis, bone composition measurements showed heritabilities ranging from 0.18 to 0.41 and moderate to strong genetic correlations with tibia strength and density. Bone composition was measured using infrared spectroscopy and thermogravimetry. The aim of this study was to combine these bone composition measurements with genotyping data via a genome-wide association study (GWAS) to investigate genetic markers that contribute to genetic variance in bone composition in Rhode Island Red laying hens. In addition, we investigated the genetic correlations between bone composition and bone strength. RESULTS: We found novel genetic markers that are significantly associated with cortical lipid, cortical mineral scattering, medullary organic matter, and medullary mineralization. Composition of the bone organic matter showed more significant associations than bone mineral composition. We also found interesting overlaps between the GWAS results for tibia composition traits, particularly for cortical lipid and tibia strength. Bone composition measurements by infrared spectroscopy showed more significant associations than thermogravimetry measurements. Based on the results of infrared spectroscopy, cortical lipid showed the highest genetic correlations with tibia density, which was negative (- 0.20 ± 0.04), followed by cortical CO3/PO4 (0.18 ± 0.04). Based on the results of thermogravimetry, medullary organic matter% and mineral% showed the highest genetic correlations with tibia density (- 0.25 ± 0.04 and 0.25 ± 0.04, respectively). CONCLUSIONS: This study detected novel genetic associations for bone composition traits, particularly those involving organic matter, that could be used as a basis for further molecular genetic investigations. Tibia cortical lipids displayed the strongest genetic associations of all the composition measurements, including a significantly high genetic correlation with tibia density and strength. Our results also highlighted that cortical lipid may be a key measurement for further avian bone studies.

Comparative analyses of dynamic transcriptome profiles highlight key response genes and dominant isoforms for muscle development and growth in chicken.

BACKGROUND: Modern breeding strategies have resulted in significant differences in muscle mass between indigenous chicken and specialized broiler. However, the molecular regulatory mechanisms that underlie these differences remain elusive. The aim of this study was to identify key genes and regulatory mechanisms underlying differences in breast muscle development between indigenous chicken and specialized broiler. RESULTS: Two time-series RNA-sequencing profiles of breast muscles were generated from commercial Arbor Acres (AA) broiler (fast-growing) and Chinese indigenous Lushi blue-shelled-egg (LS) chicken (slow-growing) at embryonic days 10, 14, and 18, and post-hatching day 1 and weeks 1, 3, and 5. Principal component analysis of the transcriptome profiles showed that the top four principal components accounted for more than 80% of the total variance in each breed. The developmental axes between the AA and LS chicken overlapped at the embryonic stages but gradually separated at the adult stages. Integrative investigation of differentially-expressed transcripts contained in the top four principal components identified 44 genes that formed a molecular network associated with differences in breast muscle mass between the two breeds. In addition, alternative splicing analysis revealed that genes with multiple isoforms always had one dominant transcript that exhibited a significantly higher expression level than the others. Among the 44 genes, the TNFRSF6B gene, a mediator of signal transduction pathways and cell proliferation, harbored two alternative splicing isoforms, TNFRSF6B-X1 and TNFRSF6B-X2. TNFRSF6B-X1 was the dominant isoform in both breeds before the age of one week. A switching event of the dominant isoform occurred at one week of age, resulting in TNFRSF6B-X2 being the dominant isoform in AA broiler, whereas TNFRSF6B-X1 remained the dominant isoform in LS chicken. Gain-of-function assays demonstrated that both isoforms promoted the proliferation of chicken primary myoblasts, but only TNFRSF6B-X2 augmented the differentiation and intracellular protein content of chicken primary myoblasts. CONCLUSIONS: For the first time, we identified several key genes and dominant isoforms that may be responsible for differences in muscle mass between slow-growing indigenous chicken and fast-growing commercial broiler. These findings provide new insights into the regulatory mechanisms underlying breast muscle development in chicken.

The expression of the gastrin/cholecystokinin (GAST/CCK) family and their receptors (CCKAR/CCKBR) in the chicken changes in response to quantitative restriction and reveals a functional role of CCK in the crop.

Gastrin and cholecystokinin peptides bind a common G-protein coupled receptor, cholecystokinin receptor B (CCKBR) whilst cholecystokinin receptor A (CCKAR) is preferentially bound by CCK. Gastrin and cholecystokinin mediate signalling from the gastrointestinal tract to regulate appetite and digestive function. In this study, expression of the cholecystokinin/gastrin family and distribution of their receptors expression was measured to understand the target organs for the peptides and how expression responds to changes in food intake. We confirmed the restricted expression of gastrin in the antrum and the abundant expression of cholecystokinin in the hypothalamus. The expression of gastrin in the antrum was significantly elevated in broiler breeders when released from feed restriction. CCKBR was most abundant in the hypothalamus and proventriculus. CCKAR was most abundant in the pancreas and crop, more than tenfold greater than the gastrointestinal tract. Cholecystokinin expression in the pancreas increased after removal of food restriction. CCKAR in the gastrointestinal tract peaks around the distal ileum, distal to the peak of cholecystokinin expression. There was virtually no cholecystokinin expression in the caecum but CCKAR expression was high. The CCKAR expression in the crop was unexpected, supporting a role of cholecystokinin in mediating crop emptying which was supported by the observation of in-vitro contraction after cholecystokinin administration. The response to changes in food intake and the expression pattern of the cholecystokinin/gastrin family and their receptors will stimulate and inform new hypotheses on their role in growth in poultry.

No evidence that selection for egg production persistency causes loss of bone quality in laying hens.

BACKGROUND: The physiological adaptations that have evolved for egg laying make hens susceptible to bone fractures and keel bone damage. In modern laying hen breeds, longer periods of egg laying could result in a greater risk of poor bone quality, and selection for increased egg production has frequently been stated to be a cause. However, the existing literature does not support this hypothesis. To test the hypothesis that egg production is associated with quality, breaking strength and density of bone, genetic correlations between these traits were estimated in White Leghorn and Rhode Island Red breeds. Genetic correlations of cortical and medullary bone material chemical properties with bone quality were also estimated, in order to identify methods to improve bone quality with appropriately targeted measurement of key traits. RESULTS: Estimates of heritability for bone quality traits were moderate (0.19-0.59) for both White Leghorn and Rhode Island Red breeds, except for the keel bone trait, which had a heritability estimate equal to zero. There was no evidence for genetic or phenotypic relationships between post-peak egg production and bone quality. In the White Leghorn breed, the estimate of the genetic correlation between pre-peak production/age at first egg and bone quality was significant and negative (- 0.7 to - 0.4). Estimates of heritability of thermogravimetric measurements of tibial medullary bone mineralisation were significant (0.18-0.41), as were estimates of their genetic correlations with tibia breaking strength and density (0.6-0.9). CONCLUSIONS: The low genetic correlation of post-peak egg production with bone quality suggests that selection for increased persistency of egg production may not adversely affect bone quality. Onset of puberty and mineralisation of the medullary bone, which is a specialised adaptation for egg laying, were identified as important factors associated with the quality of the skeleton later during egg production. These are traits for which genetic, as well as environmental and management factors can positively impact the overall quality of the skeleton of laying hens.

A genome-wide association analysis for body weight at 35 days measured on 137,343 broiler chickens.

BACKGROUND: Body weight (BW) is an economically important trait in the broiler (meat-type chickens) industry. Under the assumption of polygenicity, a "large" number of genes with "small" effects is expected to control BW. To detect such effects, a large sample size is required in genome-wide association studies (GWAS). Our objective was to conduct a GWAS for BW measured at 35 days of age with a large sample size. METHODS: The GWAS included 137,343 broilers spanning 15 pedigree generations and 392,295 imputed single nucleotide polymorphisms (SNPs). A false discovery rate of 1% was adopted to account for multiple testing when declaring significant SNPs. A Bayesian ridge regression model was implemented, using AlphaBayes, to estimate the contribution to the total genetic variance of each region harbouring significant SNPs (1 Mb up/downstream) and the combined regions harbouring non-significant SNPs. RESULTS: GWAS revealed 25 genomic regions harbouring 96 significant SNPs on 13 Gallus gallus autosomes (GGA1 to 4, 8, 10 to 15, 19 and 27), with the strongest associations on GGA4 at 65.67-66.31 Mb (Galgal4 assembly). The association of these regions points to several strong candidate genes including: (i) growth factors (GGA1, 4, 8, 13 and 14); (ii) leptin receptor overlapping transcript (LEPROT)/leptin receptor (LEPR) locus (GGA8), and the STAT3/STAT5B locus (GGA27), in connection with the JAK/STAT signalling pathway; (iii) T-box gene (TBX3/TBX5) on GGA15 and CHST11 (GGA1), which are both related to heart/skeleton development); and (iv) PLAG1 (GGA2). Combined together, these 25 genomic regions explained ~ 30% of the total genetic variance. The region harbouring significant SNPs that explained the largest portion of the total genetic variance (4.37%) was on GGA4 (~ 65.67-66.31 Mb). CONCLUSIONS: To the best of our knowledge, this is the largest GWAS that has been conducted for BW in chicken to date. In spite of the identified regions, which showed a strong association with BW, the high proportion of genetic variance attributed to regions harbouring non-significant SNPs supports the hypothesis that the genetic architecture of BW35 is polygenic and complex. Our results also suggest that a large sample size will be required for future GWAS of BW35.

Transcriptome analysis of the uterus of hens laying eggs differing in cuticle deposition.

BACKGROUND: Avian eggs have a proteinaceous cuticle. The quantity of cuticle varies and the deposition of a good cuticle in the uterus (Shell-gland) prevents transmission of bacteria to the egg contents. RESULTS: To understand cuticle deposition, uterus transcriptomes were compared between hens with i) naturally good and poor cuticle and, ii) where manipulation of the hypothalamo-pituitary-gonadal-oviduct axis produced eggs with or without cuticle. The highest expressed genes encoded eggshell matrix and cuticle proteins, e.g. MEPE (OC-116), BPIFB3 (OVX-36), RARRES1 (OVX-32), WAP (OVX-25), and genes for mitochondrial oxidative phosphorylation, active transport and energy metabolism. Expression of a number of these genes differed between hens laying eggs with or without cuticle. There was also a high expression of clock genes. PER2, CRY2, CRY1, CLOCK and BMAL1 were differentially expressed when cuticle deposition was prevented, and they also changed throughout the egg formation cycle. This suggests an endogenous clock in the uterus may be a component of cuticle deposition control. Cuticle proteins are glycosylated and glycosaminoglycan binding genes had a lower expression when cuticle proteins were deposited on the egg. The immediate early genes, JUN and FOS, were expressed less when the cuticle had not been deposited and changed over the egg formation cycle, suggesting they are important in oviposition and cuticle deposition. The uterus transcriptome of hens with good and poor cuticle deposition did not differ. CONCLUSIONS: We have gained insights into the factors that can affect the production of the cuticle especially clock genes and immediate early genes. We have demonstrated that these genes change their expression over the period of eggshell formation supporting their importance. The lack of differences in expression between the uterus of hens laying eggs with the best and worse cuticle suggest the genetic basis of the trait may lie outside the oviduct.

An eQTL in the cystathionine beta synthase gene is linked to osteoporosis in laying hens.

BACKGROUND: Skeletal damage is a challenge for laying hens because the physiological adaptations required for egg laying make them susceptible to osteoporosis. Previously, we showed that genetic factors explain 40% of the variation in end of lay bone quality and we detected a quantitative trait locus (QTL) of large effect on chicken chromosome 1. The aim of this study was to combine data from the commercial founder White Leghorn population and the F2 mapping population to fine-map this QTL and understand its function in terms of gene expression and physiology. RESULTS: Several single nucleotide polymorphisms on chromosome 1 between 104 and 110 Mb (galGal6) had highly significant associations with tibial breaking strength. The alternative genotypes of markers of large effect that flanked the region had tibial breaking strengths of 200.4 vs. 218.1 Newton (P < 0.002) and, in a subsequent founder generation, the higher breaking strength genotype was again associated with higher breaking strength. In a subsequent generation, cortical bone density and volume were increased in individuals with the better bone genotype but with significantly reduced medullary bone quality. The effects on cortical bone density were confirmed in a further generation and was accompanied by increased mineral maturity of the cortical bone as measured by infrared spectrometry and there was evidence of better collagen cross-linking in the cortical bone. Comparing the transcriptome of the tibia from individuals with good or poor bone quality genotypes indicated four differentially-expressed genes at the locus, one gene, cystathionine beta synthase (CBS), having a nine-fold higher expression in the genotype for low bone quality. The mechanism was cis-acting and although there was an amino-acid difference in the CBS protein between the genotypes, there was no difference in the activity of the enzyme. Plasma homocysteine concentration, the substrate of CBS, was higher in the poor bone quality genotype. CONCLUSIONS: Validated markers that predict bone strength have been defined for selective breeding and a gene was identified that may suggest alternative ways to improve bone health in addition to genetic selection. The identification of how genetic variants affect different aspects of bone turnover shows potential for translational medicine.

Genetic variation and potential for genetic improvement of cuticle deposition on chicken eggs.

BACKGROUND: The cuticle is an invisible glycosylated protein layer that covers the outside of the eggshell and forms a barrier to the transmission of microorganisms. Cuticle-specific staining and in situ absorbance measurements have been used to quantify cuticle deposition in several pure breeds of chicken. For brown eggs, a pre-stain and a post-stain absorbance measurement is required to correct for intrinsic absorption by the natural pigment. For white eggs, a post-stain absorbance measurement alone is sufficient to estimate cuticle deposition. The objective of the research was to estimate genetic parameters and provide data to promote adoption of the technique to increase cuticle deposition and reduce vertical transmission of microorganisms. RESULTS: For all pure breeds examined here, i.e. Rhode Island Red, two White Leghorns, White Rock and a broiler breed, the estimate of heritability for cuticle deposition from a meta-analysis was moderately high (0.38 ± 0.04). In the Rhode Island Red breed, the estimate of the genetic correlation between measurements recorded at early and late times during the egg-laying period was ~ 1. There was no negative genetic correlation between cuticle deposition and production traits. Estimates of the genetic correlation of cuticle deposition with shell color ranged from negative values or 0 in brown-egg layers to positive values in white- or tinted-egg layers. Using the intrinsic fluorescence of tryptophan in the cuticle proteins to quantify the amount of cuticle deposition failed because of complex quenching processes. Tryptophan fluorescence intensity at 330 nm was moderately heritable, but there was no evidence of a non-zero genetic correlation with cuticle deposition. This was complicated furthermore by a negative genetic correlation of fluorescence with color in brown eggs, due to the quenching of tryptophan fluorescence by energy transfer to protoporphyrin pigment. We also confirmed that removal of the cuticle increased reflection of ultraviolet wavelengths from the egg. CONCLUSIONS: These results provide additional evidence for the need to incorporate cuticle deposition into breeding programs of egg- and meat-type birds in order to reduce vertical and horizontal transmission of potentially pathogenic organisms and to help improve biosecurity in poultry.

Gastrointestinal distribution of chicken gastrin-cholecystokinin family transcript expression and response to short-term nutritive state.

The related peptide hormones cholecystokinin (CCK) and gastrin are conserved throughout vertebrate clades and implicated in energy homeostasis. CCK is generally accepted as a satiety hormone in poultry, but the role of gastrin remains poorly studied. Functional dissection of these ligands is required to characterise the molecular control of growth & satiety in the domestic chicken, for which there is an increasingly pressing mandate. There are limited descriptions of physiological distributions for the two genes in birds, and these are mostly reliant on immunohistochemistry which can prove problematic due to the shared structure of the targets. Therefore, we have defined the tissue distributions of CCK and gastrin in the chicken, focussing on the gastrointestinal tract, by using transcript-dependent techniques to improve reliability by increasing specificity. Though considerably more highly expressed in the brain, gastrointestinal CCK transcripts were dispersed throughout the small intestine and particularly around the proximal ileum. Gastrin expression was strictly limited to the gastric antrum region of the intestinal tract, albeit very highly expressed. We demonstrate that CCK mRNA expression does not respond as expected for a short-term satiety hormone, and that the short-term response of gastrin expression is paradoxical compared to its role in mammals. These results partially corroborate previous peptide distribution studies and initiate exploration of the nutrient-responsive roles of these hormones in avian energy balance.

Understanding avian egg cuticle formation in the oviduct: a study of its origin and deposition.

The cuticle is a unique invisible oviduct secretion that protects avian eggs from bacterial penetration through gas exchange pores. Despite its importance, experimental evidence is lacking for where, when, and what is responsible for its deposition. By using knowledge about the ovulatory cycle and oviposition, we have manipulated cuticle deposition to obtain evidence on these key points. Cuticle deposition was measured using staining and spectrophotometry. Experimental evidence supports the location of cuticle deposition to be the shell gland pouch (uterus), not the vagina, and the time of deposition to be within the final hour before oviposition. Oviposition induced by arginine vasotocin or prostaglandin, the penultimate and ultimate factors for the induction of oviposition, produces an egg with no cuticle; therefore, these factors are not responsible for cuticle secretion. Conversely, oviposition induced by GNRH, which mimics the normal events of ovulation and oviposition, results in a normal cuticle. There is no evidence that cuticle deposition differs at the end of a clutch and, therefore, there is no evidence that the ovulatory surge of progesterone affects cuticle deposition. Overall, the results demonstrate that the cuticle is a specific secretion and is not merely an extension of the organic matrix of the shell. Cuticle deposition was found to be reduced by an environmental stressor, and there is no codependence of the deposition of pigment and cuticle. Defining the basic facts surrounding cuticle deposition will help reduce contamination of hen's eggs and increase understanding of the strategies birds use to protect their eggs.

Pancreatic PYY but not PPY expression is responsive to short-term nutritional state and the pancreas constitutes the major site of PYY mRNA expression in chickens.

PP-fold peptides such as peptide YY (PYY) and pancreatic polypeptide (PPY) are known to play key roles in vertebrate energy homeostasis. Until recently, no gene sequence was available for avian PYY and therefore a gap in knowledge of regulation of its expression exists in avian species. Here we further evidence the mRNA sequence for chicken PYY and show that the pancreas is the major site of its mRNA expression, with a secondary peak of expression around the distal jejunum, in contrast to mammals where the large intestine is the major site of PYY expression. We also demonstrate that pancreatic PYY expression is responsive to short-term and long-term nutritional state, increasing within hours of feeding, in contrast to intestinal PYY which does not fluctuate to the same extent, and pancreatic PPY which appears to be primarily determined by long-term energy state. Both pancreatic PYY and PPY expression were found to exhibit ontogeny, being evenly distributed throughout the pancreas in young (2wk) chicks but having a decreasing splenic to duodenal gradient by adolescence (12wk).

Direct functional consequences of ZRS enhancer mutation combine with secondary long range SHH signalling effects to cause preaxial polydactyly.

Sonic hedgehog (SHH) plays a central role in patterning numerous embryonic tissues including, classically, the developing limb bud where it controls digit number and identity. This study utilises the polydactylous Silkie (Slk) chicken breed, which carries a mutation in the long range limb-specific regulatory element of SHH, the ZRS. Using allele specific SHH expression analysis combined with quantitative protein analysis, we measure allele specific changes in SHH mRNA and concentration of SHH protein over time. This confirms that the Slk ZRS enhancer mutation causes increased SHH expression in the posterior leg mesenchyme. Secondary consequences of this increased SHH signalling include increased FGF pathway signalling and growth as predicted by the SHH/GREM1/FGF feedback loop and the Growth/Morphogen models. Manipulation of Hedgehog, FGF signalling and growth demonstrate that anterior-ectopic expression of SHH and induction of preaxial polydactyly is induced secondary to increased SHH signalling and Hedgehog-dependent growth directed from the posterior limb. We predict that increased long range SHH signalling acts in combination with changes in activation of SHH transcription from the Slk ZRS allele. Through analysis of the temporal dynamics of anterior SHH induction we predict a gene regulatory network which may contribute to activation of anterior SHH expression from the Slk ZRS.

Ovodefensins, an Oviduct-Specific Antimicrobial Gene Family, Have Evolved in Birds and Reptiles to Protect the Egg by Both Sequence and Intra-Six-Cysteine Sequence Motif Spacing.

Ovodefensins are a novel beta defensin-related family of antimicrobial peptides containing conserved glycine and six cysteine residues. Originally thought to be restricted to the albumen-producing region of the avian oviduct, expression was found in chicken, turkey, duck, and zebra finch in large quantities in many parts of the oviduct, but this varied between species and between gene forms in the same species. Using new search strategies, the ovodefensin family now has 35 members, including reptiles, but no representatives outside birds and reptiles have been found. Analysis of their evolution shows that ovodefensins divide into six groups based on the intra-cysteine amino acid spacing, representing a unique mechanism alongside traditional evolution of sequence. The groups have been used to base a nomenclature for the family. Antimicrobial activity for three ovodefensins from chicken and duck was confirmed against Escherichia coli and a pathogenic E. coli strain as well as a Gram-positive organism, Staphylococcus aureus, for the first time. However, activity varied greatly between peptides, with Gallus gallus OvoDA1 being the most potent, suggesting a link with the different structures. Expression of Gallus gallus OvoDA1 (gallin) in the oviduct was increased by estrogen and progesterone and in the reproductive state. Overall, the results support the hypothesis that ovodefensins evolved to protect the egg, but they are not necessarily restricted to the egg white. Therefore, divergent motif structure and sequence present an interesting area of research for antimicrobial peptide design and understanding protection of the cleidoic egg.

Genetic loci inherited from hens lacking maternal behaviour both inhibit and paradoxically promote this behaviour.

BACKGROUND: A major step towards the success of chickens as a domesticated species was the separation between maternal care and reproduction. Artificial incubation replaced the natural maternal behaviour of incubation and, thus, in certain breeds, it became possible to breed chickens with persistent egg production and no incubation behaviour; a typical example is the White Leghorn strain. Conversely, some strains, such as the Silkie breed, are prized for their maternal behaviour and their willingness to incubate eggs. This is often colloquially known as broodiness. RESULTS: Using an F2 linkage mapping approach and a cross between White Leghorn and Silkie chicken breeds, we have mapped, for the first time, genetic loci that affect maternal behaviour on chromosomes 1, 5, 8, 13, 18 and 19 and linkage group E22C19W28. Paradoxically, heterozygous and White Leghorn homozygous genotypes were associated with an increased incidence of incubation behaviour, which exceeded that of the Silkie homozygotes for most loci. In such cases, it is likely that the loci involved are associated with increased egg production. Increased egg production increases the probability of incubation behaviour occurring because egg laying must precede incubation. For the loci on chromosomes 8 and 1, alleles from the Silkie breed promote incubation behaviour and influence maternal behaviour (these explain 12 and 26% of the phenotypic difference between the two founder breeds, respectively). CONCLUSIONS: The over-dominant locus on chromosome 5 coincides with the strongest selective sweep reported in chickens and together with the loci on chromosomes 1 and 8, they include genes of the thyrotrophic axis. This suggests that thyroid hormones may play a critical role in the loss of incubation behaviour and the improved egg laying behaviour of the White Leghorn breed. Our findings support the view that loss of maternal incubation behaviour in the White Leghorn breed is the result of selection for fertility and egg laying persistency and against maternal incubation behaviour.

Regulation of the avian central melanocortin system and the role of leptin.

The avian central melanocortin system is well conserved between birds and mammals in terms of the component genes, the localisation of their expression in the hypothalamic arcuate nucleus, the effects on feeding behaviour of their encoded peptides and the sensitivity of agouti-related protein (AGRP) and pro-opiomelanocortin (POMC) gene expression to changes in energy status. Our recent research has demonstrated that AGRP gene expression precisely differentiates between broiler breeder hens with different histories of chronic food restriction and refeeding. We have also shown that the sensitivity of AGRP gene expression to loss of energy stores is maintained even when food intake has been voluntarily reduced in chickens during incubation and in response to a stressor. However, the similarity between birds and mammals does not appear to extend to the way AGRP and POMC gene expression are regulated. In particular, the preliminary evidence from the discovery of the first avian leptin (LEP) genes suggests that LEP is more pleiotropic in birds and may not even be involved in regulating energy balance. Similarly, ghrelin exerts inhibitory, rather than stimulatory, effects on food intake. The fact that the importance of these prominent long-term regulators of AGRP and POMC expression in mammals appears diminished in birds suggests that the balance of regulatory inputs in birds may have shifted to more short-term influences such as the tone of cholecystokinin (CCK) signalling. This is likely to be related to the different metabolic fuelling required to support flight.

Cold atmospheric pressure air plasma jet disinfection of table eggs: Inactivation of Salmonella enterica, cuticle integrity and egg quality.

Eggshell cuticles are first lines of defense against egg-associated pathogens, such as Salmonella enterica serovar Enteritidis (SE). Infections from eggs contaminated with this strain remain a significant risk. In addition, changes in the cuticle are closely related to changes in egg safety. The emerging non-thermal atmospheric pressure plasma technology enables a high rate of microbial inactivation at near-ambient temperatures, making it ideal for food safety applications. This study examines the effects of a cold atmospheric pressure air plasma jet (CAAP-J) on eggshell cuticle and egg quality whilst inactivating SE. Shell eggs inoculated with SE (7 log10 cfu/egg) were used as the samples to test the decontamination performance of the device. The tests were conducted using an industrial CAAP-J with different power levels (600-800 W), exposure times (60-120 s), at a fixeddistance of 20 mm from the plasma jet and an air flow rate of 3600 L/h. It was found that the best results were obtained after 120 s at maximum plasma power (800 W). Subsequent to the implementation of this plasma procedure, it was determined that no viable cells could be detected. After CAAP-J treatment, the temperature remains below 50.5 °C, thereby minimizing the risk of altering egg quality. All specific measurements (egg white pH, yolk pH, yolk color, HU, and eggshell breaking strength) have shown that CAAP-J treatment has no negative effect on egg quality. No changes in eggshell cuticle quality after CAAP-J treatment was confirmed through scanning electron microscope (SEM).

Decreased expression of the satiety signal receptor CCKAR is responsible for increased growth and body weight during the domestication of chickens.

Animal domestication has resulted in changes in growth and size. It has been suggested that this may have involved selection for differences in appetite. Divergent growth between chickens selected for egg laying or meat production is one such example. The neurons expressing AGRP and POMC in the basal hypothalamus are important components of appetite regulation, as are the satiety feedback pathways that carry information from the intestine, including CCK and its receptor CCKAR (CCK1 receptor). Using 16 generations of a cross between a fast and a relatively slow growing strain of chicken has identified a region on chromosome 4 downstream of the CCKAR gene, which is responsible for up to a 19% difference in body weight at 12 wk of age. Animals possessing the high-growth haplotype at the locus have lower expression of mRNA and immunoreactive CCKAR in the brain, intestine, and exocrine organs, which is correlated with increased levels of orexigenic AGRP in the hypothalamus. Animals with the high-growth haplotype are resistant to the anorectic effect of exogenously administered CCK, suggesting that their satiety set point has been altered. Comparison with traditional breeds shows that the high-growth haplotype has been present in the founders of modern meat-type strains and may have been selected early in domestication. This is the first dissection of the physiological consequences of a genetic locus for a quantitative trait that alters appetite and gives us an insight into the domestication of animals. This will allow elucidation of how differences in appetite occur in birds and also mammals.

Genome wide association analysis of cuticle deposition in laying hens.

The cuticle is an invisible barrier that protects the internal egg contents from microorganisms entering through gas exchange pores. Eggs which have a good cuticle are least likely to be penetrated by microorganisms and improved cuticle cover should reduce vertical transmission of microorganisms and improve biosecurity. The aim was to carry out a genome wide association study for cuticle deposition in 3 independent populations of laying hens using tartrazine and lissamine green staining. Eggs from ∼8,000 hens represented 2 White Leghorn and 1 Rhode Island Red breed. Estimates of heritability using pedigree or genomic relationship matrices were in the 0.2 to 0.3 range. The results were breed specific. Across the populations, genomic regions on chromosomes 1, 2, 4, 5, and 8 were identified as significantly associated with cuticle deposition. No single loci had a large effect. A comparison was made with genes differentially expressed in the shell gland when cuticle deposition was manipulated, however none were obvious candidates for cuticle deposition. The results support the polygenic nature of the trait and the information will help in the future to understand the genetic variance and what might control cuticle deposition and the microbiological safety of the egg.

Genetics of tibia bone properties of crossbred commercial laying hens in different housing systems.

Osteoporosis and bone fractures are a severe problem for the welfare of laying hens, with genetics and environment, such as housing system, each making substantial contributions to bone strength. In this work, we performed genetic analyses of bone strength, bone mineral density, and bone composition, as well as body weight, in 860 commercial crossbred laying hens from 2 different companies, kept in either furnished cages or floor pens. We compared bone traits between housing systems and crossbreds and performed a genome-wide association study of bone properties and body weight. As expected, the 2 housing systems produced a large difference in bone strength, with layers housed in floor pens having stronger bones. These differences were accompanied by differences in bone geometry, mineralization, and chemical composition. Genome scans either combining or independently analyzing the 2 housing systems revealed no genome-wide significant loci for bone breaking strength. We detected 3 loci for body weight that were shared between the housing systems on chromosomes 4, 6, and 27 (either genome-wide significant or suggestive) and these coincide with associations for bone length. In summary, we found substantial differences in bone strength, content, and composition between hens kept in floor pens and furnished cages that could be attributed to greater physical activity in pen housing. We found little evidence for large-effect loci for bone strength in commercial crossbred hens, consistent with a highly polygenic architecture for bone strength in the production environment. The lack of consistent genetic associations between housing systems in combination with the differences in bone phenotypes could be due to gene-by-environment interactions with housing system or a lack of power to detect shared associations for bone strength.