Genetic patterns and genome-wide association analysis of eggshell quality traits of egg-type chicken across an extended laying period.

The industry of egg-type chicken has shown a trend of extending the rearing period, with the goal of breeding chicken breeds capable of producing 500 qualified eggs by 700 d of age. However, the rapid decline in eggshell quality during the late laying period is one of the major challenges. In this study, a total of 3,261 Rhode Island Red chickens were used to measure eggshell quality traits including eggshell strength (ESS), eggshell thickness (EST), eggshell color (ESC) and eggshell gloss (ESG) at seven age points ranging from 36 to 90 wk of age. Phenotypic variations increased with the aging process, especially during the late laying period (> 55 wk), and the heritability during this period decreased by 22.7 to 81.4% compared to the initial and peak laying periods. Then we performed genome-wide association study (GWAS) to identify the genomic variants that associated with eggshell quality, with a custom Illumina 50K BeadChip, named PhenoixChip-I. The results indicated that 2 genomic regions on GGA1(23.24-25.15Mb; 175.95-176.05 Mb) were significantly (P < 4.48E-06) or suggestively (P < 8.97E-05) associated with ESS, which can explain 9.59% and 0.48% of the phenotypic variations of ESS46 and ESS36, respectively. Three genes, FRY, PCNX2, and ENSGALG00000052468, were considered to be the candidate genes for ESS. For other traits, the genome-wide suggestive SNPs were identified at each age point, exhibiting a certain trend with aging process. Additionally, SNP enrichment analysis and functional annotation of cross-tissue regulatory elements to ESS36 revealed a high concentration of enhancer elements specific to shell gland and kidney tissues. This study, deepened our knowledge of eggshells and laying a valued scientific foundation for chicken molecular breeding.

Exploiting the aggregation propensity of beta-lactamases to design inhibitors that induce enzyme misfolding.

There is an arms race between beta-lactam antibiotics development and co-evolving beta-lactamases, which provide resistance by breaking down beta-lactam rings. We have observed that certain beta-lactamases tend to aggregate, which persists throughout their evolution under the selective pressure of antibiotics on their active sites. Interestingly, we find that existing beta-lactamase active site inhibitors can act as molecular chaperones, promoting the proper folding of these resistance factors. Therefore, we have created Pept-Ins, synthetic peptides designed to exploit the structural weaknesses of beta-lactamases by causing them to misfold into intracellular inclusion bodies. This approach restores sensitivity to a wide range of beta-lactam antibiotics in resistant clinical isolates, including those with Extended Spectrum variants that pose significant challenges in medical practice. Our findings suggest that targeted aggregation of resistance factors could offer a strategy for identifying molecules that aid in addressing the global antibiotic resistance crisis.

Enhanced therapeutic window for antimicrobial Pept-ins by investigating their structure-activity relationship.

The overconsumption and inappropriate use of antibiotics is escalating antibiotic resistance development, which is now one of the 10 top threats to global health. Introducing antibiotics with a novel mode of action into clinical use is urgently needed to address this issue. Deliberately inducing aggregation of target proteins and disrupting protein homeostasis in bacteria via amyloidogenic peptides, also called Pept-ins (from peptide interferors), can be lethal to bacteria and shows considerable promise as a novel antibiotic strategy. However, the translation of Pept-ins into the clinic requires further investigation into their mechanism of action and improvement of their therapeutic window. Therefore, we performed systematic structure modifications of 2 previously discovered Pept-ins, resulting in 179 derivatives, and investigated the corresponding impact on antimicrobial potency, cellular accumulation, and ability to induce protein aggregation in bacteria, in vitro aggregation property, and toxicity on mammalian cells. Our results show that both Pept-in accumulation and aggregation of target proteins in bacteria are requisite for Pept-in mediated antimicrobial activity. Improvement of these two parameters can be achieved via increasing the number of arginine residues, increasing Pept-in aggregation propensity, optimizing the aggregate core structure, adopting ß-turn linkers, or forming a disulphide bond. Correspondingly, improvement of these two parameters can enhance Pept-in antimicrobial efficacy against wildtype E. coli BL21 used in the laboratory as well as clinically isolated multidrug-resistant strain E. coli ATCC, A. baumannii, and K. pneumoniae.

Genetic and microbiome analysis of feed efficiency in laying hens.

Improving feed efficiency is an important target for poultry breeding. Feed efficiency is affected by host genetics and the gut microbiota, but many of the mechanisms remain elusive in laying hens, especially in the late laying period. In this study, we measured feed intake, body weight, and egg mass of 714 hens from a pedigreed line from 69 to 72 wk of age and calculated the residual feed intake (RFI) and feed conversion ratio (FCR). In addition, fecal samples were also collected for 16S ribosomal RNA gene sequencing (V4 region). Genetic analysis was then conducted in DMU packages by using AI-REML with animal model. Moderate heritability estimates for FCR (h2 = 0.31) and RFI (h2 = 0.52) were observed, suggesting that proper selection programs can directly improve feed efficiency. Genetically, RFI was less correlated with body weight and egg mass than that of FCR. The phenotypic variance explained by gut microbial variance is defined as the microbiability (m2). The microbiability estimates for FCR (m2 = 0.03) and RFI (m2 = 0.16) suggested the gut microbiota was also involved in the regulation of feed efficiency. In addition, our results showed that the effect of host genetics on fecal microbiota was minor in three aspects: 1) microbial diversity indexes had low heritability estimates, and genera with heritability estimates more than 0.1 accounted for only 1.07% of the tested fecal microbiota; 2) the genetic relationship correlations between host genetics and different microbial distance were very weak, ranging from -0.0057 to -0.0003; 3) the microbial distance between different kinships showed no significant difference. Since the RFI has the highest microbiability, we further screened out three genera, including Anaerosporobacter, Candidatus Stoquefichus, and Fournierella, which were negatively correlated with RFI and played positive roles in improving the feed efficiency. These findings contribute to a great understanding of the genetic background and microbial influences on feed efficiency.

A guide to studying protein aggregation.

Disrupted protein folding or decreased protein stability can lead to the accumulation of (partially) un- or misfolded proteins, which ultimately cause the formation of protein aggregates. Much of the interest in protein aggregation is associated with its involvement in a wide range of human diseases and the challenges it poses for large-scale biopharmaceutical manufacturing and formulation of therapeutic proteins and peptides. On the other hand, protein aggregates can also be functional, as observed in nature, which triggered its use in the development of biomaterials or therapeutics as well as for the improvement of food characteristics. Thus, unmasking the various steps involved in protein aggregation is critical to obtain a better understanding of the underlying mechanism of amyloid formation. This knowledge will allow a more tailored development of diagnostic methods and treatments for amyloid-associated diseases, as well as applications in the fields of new (bio)materials, food technology and therapeutics. However, the complex and dynamic nature of the aggregation process makes the study of protein aggregation challenging. To provide guidance on how to analyse protein aggregation, in this review we summarize the most commonly investigated aspects of protein aggregation with some popular corresponding methods.

Bacterial Protein Homeostasis Disruption as a Therapeutic Intervention.

Cells have evolved a complex molecular network, collectively called the protein homeostasis (proteostasis) network, to produce and maintain proteins in the appropriate conformation, concentration and subcellular localization. Loss of proteostasis leads to a reduction in cell viability, which occurs to some degree during healthy ageing, but is also the root cause of a group of diverse human pathologies. The accumulation of proteins in aberrant conformations and their aggregation into specific beta-rich assemblies are particularly detrimental to cell viability and challenging to the protein homeostasis network. This is especially true for bacteria; it can be argued that the need to adapt to their changing environments and their high protein turnover rates render bacteria particularly vulnerable to the disruption of protein homeostasis in general, as well as protein misfolding and aggregation. Targeting bacterial proteostasis could therefore be an attractive strategy for the development of novel antibacterial therapeutics. This review highlights advances with an antibacterial strategy that is based on deliberately inducing aggregation of target proteins in bacterial cells aiming to induce a lethal collapse of protein homeostasis. The approach exploits the intrinsic aggregation propensity of regions residing in the hydrophobic core regions of the polypeptide sequence of proteins, which are genetically conserved because of their essential role in protein folding and stability. Moreover, the molecules were designed to target multiple proteins, to slow down the build-up of resistance. Although more research is required, results thus far allow the hope that this strategy may one day contribute to the arsenal to combat multidrug-resistant bacterial infections.

Microbiota continuum along the chicken oviduct and its association with host genetics and egg formation.

The microbiota of female reproductive tract have attracted considerable attention in recent years due to their effects on host fitness. However, the microbiota throughout the chicken oviduct and its symbiotic relationships with the host have not been well characterized. Here, we characterized the microbial composition of six segments of the reproductive tract, including the infundibulum, magnum, isthmus, uterus, vagina and cloaca, in pedigreed laying hens with phenotypes of egg quality and quantity. We found that the microbial diversity gradually increased along the reproductive tract from the infundibulum to the cloaca, and the microbial communities were distinct among the cloaca, vagina and four other oviductal segments. The magnum exhibited the lowest diversity, given that the lysozyme and other antimicrobial proteins are secreted at this location. The results of correlation estimated showed that the relationship between host genetic kinship and microbial distance was negligible. Additionally, the genetically related pairwise individuals did not exhibit a more similar microbial community than unrelated pairs. Although the egg might be directly contaminated with potential pathogenic bacteria during egg formation and oviposition, some microorganisms provide long-term benefits to the host. Among these, we observed that increased abundance of vaginal Staphylococcus and Ralstonia was significantly associated with darker eggshells. Meanwhile, vaginal Romboutsia could be used as a predictor for egg number. These findings provide insight into the nature of the chicken reproductive tract microbiota and highlight the effect of oviductal bacteria on the process of egg formation.

Design and evaluation of a custom 50K Infinium SNP array for egg-type chickens.

With the development of molecular genetics and high-throughput sequencing technology, genotyping arrays consisting of large numbers of SNP have raised great interest in animal and plant research. However, the application of commercial chicken 600K SNP arrays has varied in different populations of egg-type chickens. Moreover, their genotyping cost is too high for large-scale population applications. Herein, we independently developed a custom Illumina 50K BeadChip, named PhenoixChip-I, for egg-type chickens based on SNP from 479 sequenced individuals in 7 lines. We filtered and selected SNP with stringent criteria, such as high polymorphism, genome coverage, design score, and priorities. Finally, a total of 43,681 effective SNP successfully genotyped were included on our custom array. Approximately 14K SNP were previously reported to be associated with important economic traits in egg-type chickens. Subsequently, we verified the applicability and efficiency of the PhenoixChip-I SNP array from many aspects, including evaluating its use scientific research (population structure analysis and genome-wide association study) and the poultry breeding industry (genomic selection). The findings in our study will play a crucial role in accelerating the genetic improvement of egg-type chickens.

Investigating the mechanism of action of aggregation-inducing antimicrobial Pept-ins.

Aggregation can be selectively induced by aggregation-prone regions (APRs) contained in the target proteins. Aggregation-inducing antimicrobial peptides (Pept-ins) contain sequences homologous to APRs of target proteins and exert their bactericidal effect by causing aggregation of a large number of proteins. To better understand the mechanism of action of Pept-ins and the resistance mechanisms, we analyzed the phenotypic, lipidomic, and transcriptomic as well as genotypic changes in laboratory-derived Pept-in-resistant E. coli mutator cells. The analysis showed that the Pept-in resistance mechanism is dominated by a decreased Pept-in uptake, in both laboratory-derived mutator cells and clinical isolates. Our data indicate that Pept-in uptake involves an electrostatic attraction between the Pept-in and the bacterial membrane and follows a complex mechanism potentially involving many transporters. Furthermore, it seems more challenging for bacteria to become resistant toward Pept-ins that are less dependent on electrostatic attraction for uptake, suggesting that future Pept-ins should be selected for this property.

Genome-wide association analysis of egg production performance in chickens across the whole laying period.

BACKGROUND: Egg production is the most economically-important trait in layers as it directly influences benefits of the poultry industry. To better understand the genetic architecture of egg production, we measured traits including age at first egg (AFE), weekly egg number (EN) from onset of laying eggs to 80 weeks which was divided into five stage (EN1: from onset of laying eggs to 23 weeks, EN2: from 23 to 37 weeks, EN3: from 37 to 50 weeks, EN4: from 50 to 61 weeks, EN5: from 61 to 80 weeks) based on egg production curve and total egg number across the whole laying period (Total-EN). Then we performed genome-wide association studies (GWAS) in 1078 Rhode Island Red hens using a linear mixed model. RESULTS: Estimates of pedigree and SNP-based genetic parameter showed that AFE and EN1 exhibited high heritability (0.51 ± 0.09, 0.53 ± 0.08), while the h2 for EN in other stages varied from low (0.07 ± 0.04) to moderate (0.24 ± 0.07) magnitude. Subsequently, seven univariate GWAS for AFE and ENs were carried out independently, from which a total of 161 candidate SNPs located on GGA1, GGA2, GGA5, GGA6, GGA9 and GGA24 were identified. Thirteen SNP located on GGA6 were associated with AFE and an interesting gene PRLHR that may affect AFE through regulating oxytocin secretion in chickens. Sixteen genome-wide significant SNPs associated with EN3 were in a strong linkage disequilibrium (LD) region spanning from 117.87 Mb to 118.36 Mb on GGA1 and the most significant SNP (rs315777735) accounted for 3.57% of phenotypic variance. Genes POLA1, PDK3, PRDX4 and APOO identified by annotating sixteen genome-wide significant SNPs can be considered as candidates associated with EN3. Unfortunately, our study did not find any candidate gene for the total egg number. CONCLUSIONS: Findings in our study could provide promising genes and SNP markers to improve egg production performance based on marker-assisted breeding selection, while further functional validation is still needed in other populations.

Differential proteomic analysis revealed crucial egg white proteins for hatchability of chickens.

For healthy development, an avian embryo needs the nutritional and functional molecules maternally deposited in avian eggs. Egg white not only provides nutritional components but also exhibits functional properties, such as defenses against microbial invasion. However, the roles of the more detailed messages in embryo development remain unclear. In this study, a tandem mass tag labeling quantitation approach was used to innovatively identify the differential proteins in the egg whites of fresh eggs produced by hens with divergent high/low hatchability and in the egg whites of embryonated eggs with healthy and dead embryos. A total of 378 proteins were quantified in egg white, which is the most complete proteome identified for egg white to date, and up to 102 differential proteins were identified. GO enrichment, pathway, and hierarchical clustering analysis revealed some of the differential proteins that are the main participants in several biological processes, including blood coagulation, intermediate filament, antibacterial activity, and neurodevelopment. A list of 11 putative protein biomarkers, such as keratin (KRT19, KRT12, KRT15, and KRT6A), which is involved in cell architecture, and fibrinogen (fibrinogen alpha chain, fibrinogen beta chain, and fibrinogen gamma chain), which is related to blood coagulation, were ultimately screened. The current study screened egg white proteins that can predict low hatchability and embryonic death and deciphered the role of these proteins in embryonic development, which is meaningful for the comprehensive understanding of embryonic growth.

The Value of Percutaneous Shoulder Puncture with Contrast-enhanced Ultrasound in Differentiation of Rotator Cuff Tear Subtypes: A Preliminary Prospective Study.

Imaging tests perform relatively well in the detection of rotator cuff tears (RCTs), exhibiting high sensitivity and specificity, mainly among larger full-thickness tears (tear width >1 cm). However, these tests are relatively less accurate in the detection of small full-thickness tears and partial-thickness tears. The purpose of this study was to determine the feasibility of percutaneous ultrasound-guided tendon lesionography (PUTL) using the SonoVue and the value of percutaneous shoulder puncture via contrast-enhanced ultrasound (CEUS)-a combination of percutaneous ultrasound-guided subacromial bursography (PUSB) and PUTL-in the detection of RCT subtypes. Conventional ultrasound (US), CEUS and magnetic resonance imaging (MRI) were performed and prospectively evaluated in 97 patients who had undergone arthroscopy because of suspected RCTs. The rates of detection of the various subtypes of RCTs using CEUS, PUSB, PUTL, US and MRI were evaluated. The RCT subtype detection rate via CEUS was significantly higher than the rates via US and MRI (96.9%, 74.2% and 76.3%, respectively), as were the detection rates for small full-thickness tears combined with partial-thickness tears (98.2%, 60.0% and 61.8%, respectively). The detection rate with PUSB was significantly higher than those with US and MRI in assessing full-thickness tears combined with bursal-side partial-thickness tears (93.9%, 65.3% and 65.3%, respectively). The detection rate with PUTL was significantly higher than those with US and MRI in assessing the corresponding subtypes (100.0%, 69.2% and 76.9%, respectively). On the basis of our findings, we consider PUTL a tolerable and feasible procedure. Percutaneous shoulder puncture using CEUS can be an effective alternative method with better diagnostic performance than US and MRI for the detection of RCT subtypes.

Genetic variations for egg quality of chickens at late laying period revealed by genome-wide association study.

With the extension of the egg-laying cycle, the rapid decline in egg quality at late laying period has aroused great concern in the poultry industry. Herein, we performed a genome-wide association study (GWAS) to identify genomic variations associated with egg quality, employing chicken 600 K high-density SNP arrays in a population of 1078 hens at 72 and 80 weeks of age. The results indicated that a genomic region spanning from 8.95 to 9.31 Mb (~0.36 Mb) on GGA13 was significantly associated with the albumen height (AH) and the haugh unit (HU), and the two most significant SNPs accounted for 3.12 ~ 5.75% of the phenotypic variance. Two promising genes, MSX2 and DRD1, were mapped to the narrow significant region, which was involved in embryonic and ovary development and found to be related to egg production, respectively. Moreover, three interesting genes, RHOA, SDF4 and TNFRSF4, identified from three significant loci, were considered to be candidate genes for egg shell colour. Findings in our study could provide worthy theoretical basis and technological support to improve late-stage egg quality for breeders.

Genome-Wide Association Analysis of Age-Dependent Egg Weights in Chickens.

Egg weight (EW) is an economically-important trait and displays a consecutive increase with the hen's age. Because extremely large eggs cause a range of problems in the poultry industry, we performed a genome-wide association study (GWAS) in order to identify genomic variations that are associated with EW. We utilized the Affymetrix 600 K high density SNP array in a population of 1,078 hens at seven time points from day at first egg to 80 weeks age (EW80). Results reveal that a 90 Kb genomic region (169.42 Mb ~ 169.51 Mb) in GGA1 is significantly associated with EW36 and is also potentially associated with egg weight at 28, 56, and 66 week of age. The leading SNP could account for 3.66% of the phenotypic variation, while two promising genes (DLEU7 and MIR15A) can be mapped to this narrow significant region and may affect EW in a pleiotropic manner. In addition, one gene (CECR2 on GGA1) and two genes (MEIS1 and SPRED2 on GGA3), which involved in the processes of embryogenesis and organogenesis, were also considered to be candidates related to first egg weight (FEW) and EW56, respectively. Findings in our study could provide worthy theoretical basis to generate eggs of ideal size based on marker assisted breeding selection.

Brown eggshell fading with layer ageing: dynamic change in the content of protoporphyrin IX.

The dynamic change in brown eggshell color as hens age has been observed, but much uncertainty still exists. We aimed to analyze the depth of eggshell color and quantity of protoporphyrin ΙΧ throughout the laying period to explore the reasons for color variation. In this study, 120 Rhode Island Red hens were used, and two eggs were collected from each individual at 26, 34, 42, 50, 60, and 70 wk of age. The eggshell color (L*, a*, b*), egg weight, eggshell dry weight, and protoporphyrin ΙΧ quantity in eggshell were measured for individual eggs. Our results showed that the intensity of brown eggshell color weaken as hens aged from 26 to 60 wk of age (L* gradually increased from 61.43 to 68.07), while eggshell lightness recovered slightly at 70 wk (L* = 64.77). The correlation analysis indicated that the content of protoporphyrin ΙΧ deposited in the eggshell was an important factor for lightness fading with the ageing process (the average r was 0.66, P < 0.01), while the egg weight had little impact on the eggshell color (the average r was 0.07, P > 0.05). The shade of the eggshell color (L* and a*) at the early laying period (26 or 34 wk) had a low correlation with the other age points (42, 50, 60, and 70 wk). However, high correlations between the shell color at 42 wk of age and subsequent ages (50, 60, and 70 wk) were found, suggesting that the intensity of eggshell color is more stable after egg-laying peaks (such as 42 wk of age). In conclusion, the intensity of brown eggshell color varies greatly among the whole laying cycle and breeders can choose the proper age for eggshell color measurements to ensure the degree of shell color in the late laying period.

Genomic prediction in a nuclear population of layers using single-step models.

Single-step genomic prediction method has been proposed to improve the accuracy of genomic prediction by incorporating information of both genotyped and ungenotyped animals. The objective of this study is to compare the prediction performance of single-step model with a 2-step models and the pedigree-based models in a nuclear population of layers. A total of 1,344 chickens across 4 generations were genotyped by a 600 K SNP chip. Four traits were analyzed, i.e., body weight at 28 wk (BW28), egg weight at 28 wk (EW28), laying rate at 38 wk (LR38), and Haugh unit at 36 wk (HU36). In predicting offsprings, individuals from generation 1 to 3 were used as training data and females from generation 4 were used as validation set. The accuracies of predicted breeding values by pedigree BLUP (PBLUP), genomic BLUP (GBLUP), SSGBLUP and single-step blending (SSBlending) were compared for both genotyped and ungenotyped individuals. For genotyped females, GBLUP performed no better than PBLUP because of the small size of training data, while the 2 single-step models predicted more accurately than the PBLUP model. The average predictive ability of SSGBLUP and SSBlending were 16.0% and 10.8% higher than the PBLUP model across traits, respectively. Furthermore, the predictive abilities for ungenotyped individuals were also enhanced. The average improvements of prediction abilities were 5.9% and 1.5% for SSGBLUP and SSBlending model, respectively. It was concluded that single-step models, especially the SSGBLUP model, can yield more accurate prediction of genetic merits and are preferable for practical implementation of genomic selection in layers.

Transcriptome sequencing identifies potential regulatory genes involved in chicken eggshell brownness.

Brown eggs are popular in many countries, and consumers regard eggshell brownness as an important indicator of egg quality. Brown eggshell color is controlled by polygene. However, the responsible genes and detailed molecular mechanisms regulating eggshell brownness have not been defined. In the present study, we applied the RNA-seq technology to analyze the transcriptome data of the shell gland epithelium of hens and investigated the candidate genes associated with eggshell brownness. The results indicated that 8461 genes were expressed in the shell gland epithelium, of which 34 genes were differentially expressed in hens laying dark vs. light brown eggs. Functional analysis revealed that two genes, ovotransferrin (TF) and heat-shock protein 70 (HSP70), as well as the oxidative phosphorylation pathway were involved in the synthesis and transport of protoporphyrin Ⅸ, which might influence the formation of eggshell brownness and result in different shades of brown.

Genome-wide association study reveals putative role of gga-miR-15a in controlling feed conversion ratio in layer chickens.

BACKGROUND: Efficient use of feed resources for farm animals is a critical concern in animal husbandry. Numerous genetic and nutritional studies have been conducted to investigate feed efficiency during the regular laying cycle of chickens. However, by prolonging the laying period of layers, the performance of feed utilization in the late-laying period becomes increasingly important. In the present study, we measured daily feed intake (FI), residual feed intake (RFI) and feed conversion ratio (FCR) of 808 hens during 81-82 weeks of age to evaluate genetic properties and then used a genome-wide association study (GWAS) to reveal the genetic determinants. RESULTS: The heritability estimates for the investigated traits were medium and between 0.15 and 0.28 in both pedigree- and genomic-based estimates, whereas the genetic correlations among these traits were high and ranged from 0.49 to 0.90. Three genome-wide significant SNPs located on chromosome 1 (GGA1) were detected for FCR. Linkage disequilibrium (LD) and conditional GWA analysis indicated that these 3 SNPs were highly correlated with one another, located at 13.55-45.16 Kb upstream of gga-miR-15a. Results of quantitative real-time polymerase chain reaction (qRT-PCR) analysis in liver tissue showed that the expression of gga-miR-15a was significantly higher in the high FCR birds than that in the medium or low FCR birds. Bioinformatics analysis further revealed that gga-mir-15a could act on many target genes, such as forkhead box O1 (FOXO1) that is involved in the insulin-signaling pathway, which influences nutrient metabolism in many organisms. Additionally, some suggestively significant variants, located on GGA3 and GGA9, were identified to associate with FI and RFI. CONCLUSIONS: This GWA analysis was conducted on feed intake and efficiency traits for chickens and was innovative for application in the late laying period. Our findings can be used as a reference in the genomic breeding programs for increasing the efficiency performance of old hens and to improve our understanding of the molecular determinants for feed efficiency.

iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness.

Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57) would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 (SLC25A5) and down-regulated translocator protein (TSPO) would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF). In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation.

Polymorphisms in Ion Transport Genes Are Associated with Eggshell Mechanical Property.

Eggshell mechanical property traits such as eggshell breaking strength (ESS), eggshell thickness (EST) and eggshell weight (ESW) are most common and important indexes to evaluate eggshell quality in poultry industry. Uterine ion transporters involve in eggshell formation and might be associated with eggshell mechanical property traits. In this study, 99 SNPs in 15 ion transport genes were selected to genotype 976 pedigreed hens of Rhode Island Red. ESS, EST and ESW were measured for each bird at 55 weeks of age. The association study showed that 14 SNPs in 8 genes were significantly related (p < 0.05) with at least one trait, and their contributions to phenotypic variance ranged from 0.23% to 4.14%. Both ATP2A3 and SLC4A5 had a significant effect on all the three traits. Strong linkage disequilibrium (LD) was detected among SNPs in four genes: ATP2A3, ITPR1, SLC8A3, SCNN1a. The significant effects of those diplotypes on eggshell mechanical property traits were found, and their contributions to phenotypic variance ranged from 0.50% to 0.70%. It was concluded that the identified SNPs and diplotypes in this study were potential markers influencing the eggshell mechanical properties, which could contribute to the genetic improvement of eggshell quality.