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The cuticle is the outmost layer of the eggshell and may affect the hatchability by modulating eggshell conductance. Three different solutions using acetic acid (AA), vinegar (V), and citric acid (CA) for cuticle removal by egg washing were developed, and the effects of cuticle removal on hatching performance of quail hatching eggs were evaluated. A total of 5,238 fresh quail hatching eggs were randomly divided into 9 treatments as follows: unwashed control, nondipped (CND); washed control, water dipped (CWD); standard control, 0.13% sodium hyperchlorite (CSH); 2% AA (AA2); 4% AA (AA4); 44.4% V (V2); 88.8% V (V4); 2% CA (CA2); and 4% CA (CA4). Overall, AA4, V4, and CA4 treatments significantly improved the hatchability of fertile eggs (95.42%, 94.16% and 95.66%, respectively) (P < 0.05) and the hatchability of CND, CWD, CSH, AA2, V2 and CA2 treatments were 90.98%, 93.00%, 92.27%, 79.44%, 90.37%, and 90.59%, respectively. The eggshell thickness and cuticle quality results showed that all AA, V, and CA solutions can effectively remove the quail eggshell cuticle, and AA4, V4 and CA4 significantly decreased eggshell thickness (P 0.05). There were no significant differences between treatments for chick livability and live weight at the first 21 D of life. Results of the present study indicate that cuticle removal with AA4, V4, or CA4 could effectively decrease the microbial activity on the eggshell surface during the incubation period and improve hatchability of quail hatching eggs without negative effects on hatch time and performance of quail chicks.

The monitoring of eggshell quality is important mainly in terms of production economy. Eggshell appearance is one of the most characteristics, influencing the purchasing behavior of consumers. Besides numerous eggshell appearance quality (color, shape, etc.), gloss is an important trait to reflect the eggshell appearance. In this study, 2 experiments were conducted to investigate the effect of breed and age on the gloss of eggshells. In experiment 1, we compared the eggshell gloss of 7 chicken breeds. In experiment 2, 105 Wanan (WA) chickens were raised, and 1 egg was collected from each individual at 26, 32, 40, and 50 wks of age. Eggshell gloss, color (L*, a*, b*), cuticle coverage (ΔE*ab), and thickness were measured. The results of experiment 1 showed that the average gloss values were highly variable among different breeds, and the highest was found in WA (gloss unit [GU] = 8.12), almost 2.5 folds as many as the lowest in Rhode Island Red (GU = 3.23). Also, the eggshell gloss of the local chicken breeds was significantly higher than the highly selected lines of egg-type chicken breeds (P < 0.001). In experiment 2, the results showed that gloss ranged from 9.08 GU to 12.12 GU with a variation of 28.38 to 39.71%. It fluctuated with the increasing age of hens and had the peak value at 26 wk. But, the correlation analysis between eggshell gloss and other eggshell quality traits were very low (−0.07 to 0.25). This study laid a foundation for improving the uniformity and intensity of eggshell gloss for breeders.

The nanostructure greatly contributes to eggshell formation, the mechanical properties of eggshells, and mineral dissolution during incubation. In this study, to investigate the effect of the nanostructure on the gloss of eggs, the gloss and eggshell quality (cuticle coverage, color, and thickness) of 105 eggs were measured. According to the order of the gloss, the surface roughness of 30 high-gloss and 30 low-gloss eggs was compared. The gloss had no significant correlation with the eggshell color and thickness (P > 0.05) and a significant relationship with the cuticle coverage (r = 0.19, P < 0.01). The surface roughness significantly differed between the high- and low-gloss eggs (P < 0.001), and the gloss was negatively correlated with the surface roughness (r(high-group) = -0.61, r(low-group) = -0.56, P < 0.01). The shell gloss of 30 oiled eggs with mineral oil and 30 normal eggs from commercial brown-egg layers was also compared. The oil coating increased the eggshell gloss, but the roughness was unchanged. This is the first report to establish the contribution of nanostructure for the gloss of chicken eggshell. The surface roughness can be used as an indicator of the gloss, which could be helpful for selective breeding to improve the eggshell brightness. Our research also provides the foundation for further investigation of the effect of non-pigmentary contributors on the chicken eggshell appearance.

Poultry eggs from different species varied significantly, due to their divergent process of evolution. However, the information on egg characteristics has been limited mostly to chicken. The current study compared the egg quality, albumen physical and nutritional property for domesticated chicken, duck, goose, turkey, quail, and pigeon. Egg quality traits among different species differed significantly, such as egg weight (from 11 to 139 g), egg shape (from 1.28 to 1.44), proportion of yolk (from 19.3 to 37.9%), and breaking strength (from 0.91 to 8.04 kg/cm2). For the physical property of albumen gel, pigeon egg was the most special one. The albumen gel of pigeon egg had a transparent appearance (A = 1.23), and it had the highest hardness (121.7 g) and water-holding capacity (96%) but a medium level of total cutting work (440 g⋅s). Hence, it was easy for deformation but was hard to cut off when external force was applied. For nutritional compositions of egg albumen, goose egg had the highest moisture (89.21%) and lowest crude protein (8.5%) contents. Specific to amino acid, glutamic acid was found the most abundant in albumen of all six species. Egg albumen of turkey had the highest quantity of essential amino acids (EAA) and total amino acids (TAA), while duck and goose had relatively higher EAA/TAA ratios. Both PCA and cluster analysis revealed the high similarity of amino acid composition between duck and goose, and among quail, turkey, and chicken. The comparative data will improve the understanding of egg qualities of major poultry species and can be helpful in technological application of egg albumen.

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.

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, P0.01), while the egg weight had little impact on the eggshell color (the average r was 0.07, P0.05). 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.

As a member of the suppressor of cytokine signaling (SOCS) family, the SOCS2 gene has been associated with negative regulation of the inflammatory immune response and a positive effect on growth when it is overexpressed. In our previous study, using next-generation sequencing, it was found that the Luxi Game (LX, a Chinese indigenous gamecock) had an increased copy number (CN) of the SOCS2 gene. To verify the copy number increase of the SOCS2 gene in gamecocks, we quantified the copy number value in 8 chicken breeds by quantitative PCR. The results showed that the average CN values of the SOCS2 gene were highly variable among the 8 breeds, and the highest was found in LX (CN = 5.0), almost 3.5 folds as many as the lowest in Rhode Island Red (CN = 1.43). All 4 gamecock breeds possessed significantly higher CN values than others, including broilers and commercial breeds. Thus, it is suggested that most Chinese gamecocks possess an increased copy number in the SOCS2 gene, indicating the plausible functions of the SOCS2 gene in inflammation control and increased body size.

Campylobacter jejuni (C. jejuni) is one of major foodborne pathogen that cause human diarrhea by consuming C. jejuni contaminated chicken products. MicroRNAs play an integral role in many different biological processes including bacteria and virus inoculation in chickens. In this study, we identified chicken miRNAs responding to C. jejuni inoculation through Solexa sequencing in the cecum. As a result, four miRNAs were significantly differentially expressed between inoculated and non-inoculated groups. There were 1,114 putative target genes regulated by those differentially expressed miRNAs predicted by miRanda, TargetScan, and miRTarget softwares. Functional analysis of those target genes showed that 113 gene ontology biological process terms and 14 pathways were significantly enriched. Hedgehog signaling pathway may contribute to chicken C. jejuni inoculation. MiR-155 played vital role in the C. jejuni inoculation. The result herein will lay the foundation for the further study of regulatory mechanism of chicken miRNAs in the response to C. jejuni inoculation.

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.

Environmental stimuli resulting from immunological stress can induce transgenerational phenotypic inheritance, but few similar studies are found in avian. Here, we challenged F0 hens with polyinosinic: polycytidylic acid [Poly(I: C)] and lipopolysaccharide (LPS) at 53 wk of age, and then investigated the ethology of the challenged hens. In the unchallenged F1 descendants, the egg quality at 23 wk of age and laying rate (LR) at different stages were measured. Mortality rate (MR) and the days of population LR reaching 50% (D50%LR) at 33 wk of age were also tested in F1 hens. Pearson correlation analysis was subsequently calculated between F1 peripheral blood lymphocytes transcriptome and LR (in L vs. C) and EW (in P vs. C), respectively. The results showed that the ethology and egg-laying variations of stimuli-challenged hens and their descendants could be affected by the 2 kinds of immune stimuli. Poly(I: C) was likely to increase LR, especially in the early laying period and advance the D50%LR in F1 hens. It also reduced the MR, albumen height, and Haugh units of the unchallenged offspring. Whereas LPS could induce a sickness behavior of the challenged F0 hens, it also reduced the LR of F1 hens throughout the study, prolonged the D50%LR, and faded the eggshell color. Correlation analysis showed that Poly(I: C) mainly affected EW, while LPS mainly influenced LR of F1 offspring. All findings in the present study were the first time to be revealed in laying chickens, suggesting the different effects of Poly(I: C) and LPS on chickens and their descendants, and laying the foundation for the study of the influence of maternal experience on offspring in avian.

Marek's disease (MD), caused by Marek's disease virus (MDV), is a lymphoproliferative neoplastic disease of chickens and is characterized by MD lymphoma in multiple visceral organs of chicken. It causes great damage to poultry health. Recently, miRNA has been reported to be involved in Marek's disease lymphomagenesis. Our previous study showed that gga-miR-181a was downregulated in MDV-induced lymphoma, and its target gene, v-myb myeloblastosis viral oncogene homolog-like 1 (MYBL1), was predicted. In this study, the interaction between gga-miR-181a and MYBL1 was further verified by detecting protein expression levels of MYBL1 after transfecting miR-181a mimic into MD lymphoma cell line, MSB1. The result showed that protein level of MYBL1 was lower in gga-miR-181a mimic transfecting group than that in the negative control group at 96 h post transfection, which indicated that MYBL1 was a target gene of gga-miR-181a. Additionally, we found that the expression of MYBL1 was higher in MDV-infected samples than that in non-infected controls, which agreed with the proposition that miRNA showed a negatively correlated expression pattern with its target gene. We observed the inhibitory effect of gga-miR-181a on MSB1 cell proliferation. Collectively, the aberrant expression of gga-miR-181a and MYBL1 in MD lymphoma suggested that they might be involved in MD tumor transformation and played important roles.

Marek's disease (MD) is lymphoproliferative neoplastic disease in chickens, which is caused by Marek's disease virus (MDV). Our previous study profiled microRNA (miRNA) transcriptome in MD lymphoma, and found that gga-miR-199-3p, gga-miR-140-3p, and gga-miR-221-5p were down-regulated in MD lymphoma. In this study, we further investigated their differential expression between MDV-infected spleens and noninfected spleens at 4, 7, 14, 21, and 28 d postinfection (dpi) to elucidate whether deregulation of them was specific to late tumor transformation phase or not. The results showed that gga-miR-199-3p was down-regulated at 14 and 28 dpi, and the expression of gga-miR-140-3p was decreased at 14 dpi, which indicated that deregulation of these miRNAs appeared since early stage of MD tumor transformation. Additionally, the inhibitory effects of gga-miR-199-3p, gga-miR-140-3p, and gga-miR-221-5p on MDV-transformed lymphoid cell line (MSB1) cells proliferation were observed, which suggested that these miRNAs acted as MD tumor suppressors. Their aberrant expression at early tumor transformation phase and suppressive role in cell proliferation indicated that they were involved in MD lymphoma transformation, and might play crucial roles in MD tumorigenesis.

This study was conducted to investigate the feeding behaviors of slow-growing yellow broilers and the relationships of feeding behaviors with performance traits. With the help of automatic recording systems in floor houses, feeding events from a pure line of slow-growing yellow broilers were recorded from 57 to 77 d of age. After data quality control, a total of 116,477 feeding records from 319 birds were used for analyses. Feeding behaviors including number of visits per day (18.74), feeding duration per day (71.17 min/d), feeding duration per visit (262.00 s/visit), feeding rate (2.19 g/min), and feed intake per visit (8.52 g/visit) were calculated according to feeding records. Correlation analyses and comparisons between divergent efficiency groups were performed to examine the relationships between feed efficiency and feeding behaviors. Absolute correlations between residual feed intake (RFI) and feeding behaviors (except for feed intake per visit) were significant but weak (r = 0.18 to 0.34, P0.05), whereas feed conversion ratio (FCR) was not significantly correlated with any feeding behaviors. All of the weight-associated traits were positively correlated with feeding rate and feed intake per visit (r = 0.19 to 0.25, P0.05). Compared with the inefficient birds with the 20% highest RFI or FCR (HRFI or HFCR), the efficient ones with the 20% lowest RFI or FCR (LRFI or LFCR) ate faster (P0.05), spent shorter eating duration (P0.05) and had similar feed intakes per visit (P0.05). However, number of visits per day and the feeding duration per day were lower in the LRFI group than in the HRFI group (P0.05) but were not significantly different between the LFCR and HFCR groups (P0.05). In summary, this study shows the feeding behaviors of group-housed slow-growing yellow broilers and observed that RFI has closer relationships with feeding behaviors than FCR does, and the selection for birds with improved RFI may result in fewer visits, shorter duration and faster feeding rate.

Feed consumption accounts for the major cost of broiler production. Improving the efficiency of feed utilization is a primary goal in breeding strategies, although few studies have focused on slower growing broilers. Here, we recorded the feed intake (FI) during the fast-growing period (d 56 to 76) and measured the live weight, body measurements, carcass characteristics, and intramuscular fat (IMF) content of Chinese yellow broilers. Then, the residual feed intake (RFI) and feed conversion ratio (FCR) were calculated for each individual. Pair-wise phenotypic correlations were subsequently calculated between feed efficiency traits and others. Finally, we separately selected the more efficient individuals based on RFI and FCR values to evaluate the impacts on the traits of FI, growth, carcass characteristics, and meat quality. The results showed higher correlations between FCR and production traits than with RFI, while RFI showed a moderate and positive phenotypic correlation with abdominal fat. FCR was weakly correlated with FI and slightly positively correlated with IMF content. The correlation coefficient between RFI and FI was 0.62, and that between RFI and IMF content was close to zero. Without increasing FI, decreasing FCR could effectively enhance the growth rate and market weight with no adverse effect on meat quality. In contrast, by improving RFI, FI and abdominal fat mass were significantly reduced and thus increased the yield with no unfavorable effects on meat quality. In consideration of consumer preference and overall economical benefits, RFI is a more suitable index to improve feed efficiency in slower growing broilers.

Marek's disease (MD) is a viral neoplastic disease of chickens caused by Marek's disease virus (MDV), which is serious threat to worldwide poultry industry. Our previous studies showed that the CD30 gene was hypomethylated in MD lymphoma. In this study, we further analyzed differential expression patterns and methylation levels of the CD30 gene between MDV-infected and noninfected spleens at 4, 7, 14, 21, and 28 d postinfection (dpi). The results showed that the expression of CD30 in MDV-infected spleens was significantly lower than that in noninfected spleens at 4 dpi. The expression of CD30 did not present significant difference between MDV-infected and noninfected spleens at 7 and 14 dpi. However, an increased expression of CD30 was presented in MDV-infected spleens at both 21 and 28 dpi. Simultaneously, CD30 showed a lower DNA methylation level in MDV-infected spleens at 14, 21, and 28 dpi. The results indicated that CD30 gene was involved in the whole process of MD tumorigenesis and upregulated expression of CD30 in MDV-infected spleens might be attributed to the hypomethylation of promoter of CD30 gene.

Laying records on 1,534 F2 hens, derived from a reciprocal cross between White Leghorns and Dongxiang blue-shelled chickens, were used to estimate genetic parameters for residual feed intake (RFI), feed conversion ratio (FCR), daily feed intake (FI), metabolic BW (MBW), BW gain (BWG), and daily egg mass (EM) at 37 to 40 (T1) and 57 to 60 wk age (T2), respectively. Genetic analysis was subsequently conducted with the AI-REML method using an animal model. Estimates for heritability of RFI, FCR, and FI were 0.21, 0.19, and 0.20 in T1, and 0.29, 0.13, and 0.26 in T2, respectively. In T1 and T2, RFI showed high and positive genetic correlations with FCR (0.51, 0.43) and FI (0.72, 0.84), whereas the genetic correlation between FI and FCR was very low (-0.09, 0.11). Genetically, negative correlations were found between RFI and its component traits (-0.01 to -0.47). In addition, high genetic correlations, from 0.76 to 0.94, were observed between T1 and T2 for RFI, FCR, and FI, suggesting that feed efficiency traits in the 2 stages had a similar genetic background. The results indicate that selection for low RFI could reduce FI without significant changes in EM, while selection on FCR will increase EM. The present study lays the foundation for genetic improvement of feed efficiency during the laying period of chickens.

Shank color of domestic chickens varies from black to blue, green, yellow, or white, which is controlled by the combination of melanin and xanthophylls in dermis and epidermis. Dermal shank pigmentation of chickens is determined by sex-linked inhibitor of dermal melanin (Id), which is located on the distal end of the long arm of Z chromosome, through controlling dermal melanin pigmentation. Although previous studies have focused on the identification of Id and the linear relationship with barring and recessive white skin, no causal mutations have yet been identified in relation to the mutant dermal pigment inhibiting allele at the Id locus. In this study, we first used the 600K Affymetrix Axiom HD genotyping array, which includes ~580,961 SNP of which 26,642 SNP were on the Z chromosome to perform a genome-wide association study on pure lines of 19 Tibetan hens with dermal pigmentation shank and 21 Tibetan hens with yellow shank to refine the Id location. Association analysis was conducted by the PLINK software using the standard chi-squared test, and then Bonferroni correction was used to adjust multiple testing. The genome-wide study revealed that 3 SNP located at 78.5 to 79.2 Mb on the Z chromosome in the current assembly of chicken genome (galGal4) were significantly associated with dermal shank pigmentation of chickens, but none of them were located in known genes. The interval we refined was partly converged with previous results, suggesting that the Id gene is in or near our refined genome region. However, the genomic context of this region was complex. There were only 15 SNP markers developed by the genotyping array within the interval region, in which only 1 SNP marker passed quality control. Additionally, there were about 5.8-Mb gaps on both sides of the refined interval. The follow-up replication studies may be needed to further confirm the functional significance for these newly identified SNP.

Many genetic factors influence the growth and feed intake of birds. In the current study, we evaluated the association of 8 previously reported SNP in the chicken leptin receptor (LEPR) gene with BW, BW gain (BWG), feed intake (FI), and feed conversion ratio (FCR). Four SNP with a very low minor allele frequency were removed by genotype quality control. The experimental population consisted of 796 pedigreed males from 2 genetically unrelated yellow meat-type chicken strains, 335 chickens from N202, and 461 chickens from N301. The BW at 49 (BW49) and 70 d of age (BW70) and FI (from 49 to 70 d of age) were measured individually. The BWG and FCR were calculated based on BW and FI in the interval between 49 to 70 d. The results indicated that rs14657336, rs13684613, rs13684615, and rs13684616 were found in strong linkage disequilibrium. This linkage disequilibrium block was significantly associated with BW49 (P < 0.05), BW70 (P < 0.05), and FI (P < 0.05) in the N202 strain, and FCR (P < 0.01) in the N301 strain, respectively. In addition, the GTACGTAC diplotype had the highest BWG and FI in both strains. The association revealed in this study suggests the need for further functional study on the role of LEPR gene in regulating feed intake and FCR of chickens.

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.

Translucent eggshells negatively affect the appearance of eggs and decrease their economic value. Translocation and accumulation of water from the contents to the shells of eggs are frequent occurrences. Causes of translucent eggshell formation have been investigated, but the primary reason is uncertain. In previous studies, scientists have found that the thickness of the eggshell membrane was significantly different between translucent and opaque eggs. However, there are some conflicts among studies. We performed 2 experiments with 3 breeding flocks of chickens to target the reasons for egg translucence. In experiment 1, eggs of 1,024 Brown-Egg Dwarf Layers (DWL) were used. Approximately 1,600 eggs were collected over 2 consecutive days. They were stored for 3 days, and then 120 translucent and 120 opaque eggs were selected for measurement of egg quality traits and weight loss over several weeks. In experiment 2, we used DWL and White Leghorn pure line (WLL) for assessment of eggshell ultrastructure and membrane traits. We chose 120 translucent and 120 opaque eggs from 3,500 DWL eggs and 125 translucent and 125 opaque eggs from 5,028 WLL eggs. The results are as follows: (1) translucent eggs had greater eggshell strength and lower ultimate failure stress of shell membrane than opaque eggs in both DWL and WLL groups, (2) translucent eggs had thicker shells and thinner shell membranes than opaque eggs in DWL, (3) no significant differences were found in either gas pore or bubble pore traits between translucent and opaque eggs in either line, and (4) no significant differences were detected in internal egg quality or weight loss between translucent and opaque eggs in either line. In summary, the present study suggests that variations in both eggshells and shell membrane structures are implicated in the formation of translucent eggs.

Toll-like receptors (TLR) are trans-membrane sensors recognizing invading microbes. Toll-like receptors play a central role in initiating immune responses against several pathogens. In this study, we investigated the response of TLR and downstream genes to Marek's disease virus (MDV) infection. Forty 1-d-old chicks were randomly divided into 2 groups, with 20 chicks infected with MDV and 20 chicks mock-infected. Four chickens were euthanized respectively from infected and age-matched noninfected groups at 4, 7, 14, 21, and 28 d postinfection (dpi). Bursas, spleens, and thymuses were removed. The differential expression of TLR genes, including TLR3, TLR5, TLR7, TLR15, and TLR21, and downstream genes of TLR7, including MyD88, TRAF3, TRAF6, IFNA, IFNB, and IL6, in lymphoid tissues of MDV-infected and noninfected chickens was determined by real-time PCR. The results showed that the change of TLR genes was different in 3 lymphoid tissues. Expression of TLR7 and MyD88 was upregulated at 14 dpi and downregulated at 28 dpi in MDV-infected compared with noninfected spleens. The TRAF6 and IFNB were upregulated, and TRAF3, IFNA, and IL6 genes showed increasing trends in MDV-infected compared with noninfected spleens at 14 dpi. The expression of TLR3 and TLR15 genes was downregulated in MDV-infected compared with noninfected spleens at 28 dpi. The results indicated that TLR7 and its downstream genes were a response to MDV infection at 14 dpi. However, the function of TLR was impaired when the infection entered the tumor transformation phase. In bursas, TLR3 and TLR15 genes were upregulated at 7 and 4 dpi, respectively. It indicated that TLR3 and TLR15 might be involved in response to MDV infection in bursa at early phases. However, no differential expression of TLR genes was observed between MDV-infected and noninfected thymuses, which indicated that the thymus had little response to MDV infection mediated by TLR.

Damaged eggshells result in losses of eggs. Numerous efforts have been carried out to improve eggshell quality, which may lead to increased eggshell thickness. The conventional way of enhancing eggshell strength with thicker eggshell on average may be replaced by a new strategy to improve eggshell uniformity without increasing eggshell thickness. To achieve this, it is necessary to investigate global variation of eggshell thickness. In this study, we used 100 fresh eggs from 52-wk-old layers of a commercial brown-egg variety. To determine the global variation of eggshell thickness, 42 points for each egg along both longitudinal and latitudinal axes were selected to measure thickness using an eggshell thickness gauge. The eggshell thickness from blunt to sharp end varied significantly (P < 0.05). The area surrounding the blunt end was the thinnest (0.341 ± 0.025 mm), whereas the area surrounding the sharp end was the thickest (0.367 ± 0.023 mm). It was found that the thickness of the sharp end was the closest to the average thickness of the whole eggshell and could be considered as a valid measurement of eggshell thickness. A new parameter, eggshell thickness uniformity, was defined as the reciprocal of the coefficient of variation (1/CV) of eggshell thickness from 42 points of each egg and can be used to evaluate the eggshell quality. Eggshell thickness uniformity was positively correlated with breaking strength (r = 0.341; P < 0.01), suggesting that the parameter may be used as a potential selection criterion in breeding program to improve eggshell quality without increasing eggshell thickness.

Chicken is considered to be an excellent model for genetic studies of phenotypic and genomic evolution, with large effective population size, specialized commercial lines, and strong human-driven selection. High-density chicken SNP chips can help to achieve a better understanding of the selection mechanisms in artificially selected populations. We performed the genome-wide tests for the selection signature in 385 White Leghorn hens and mapped positively selected regions to the genome annotations. Ten QTL related to egg production, egg quality, growth, and disease resistance traits were selected for extended haplotype homozygosity tests to give a brief overview of recent selection signatures in chicken QTL. We also reported 185 candidate genes/CDSs showing top P-values and slower decay of haplotype homozygosities. Some of these genes seemed to have significant effects on important economical traits, and most of them have not been reported in chickens. The current study provides a genome-wide map of linkage disequilibrium extents and distributions and selection footprints in the chicken genome. A panel of genes, including PRL, NCKX1, NRF1, LHX2, and SFRP1 associated with egg production, metabolism traits, and response to illumination were identified. In addition, there were more genes identified that have not yet been reported in chickens, and our results provide new clues for further study.

Investigations on regulatory genes of feed intake will provide a rational scientific basis to improve future selection indices for more efficient chickens. In the present study, we investigated the association of 13 previously reported SNPs in the chicken adenosine monophosphate activated protein kinase (AMPK) subunits PRKAB1, PRKAG2, and PRKAG3 genes with body weight (BW), body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) in two distinct yellow meat-type strains. Six SNPs with a very low minor allele frequency were removed by genotype quality control and data filtering. The experimental population comprised 796 pedigreed males from two strains with different genetic backgrounds, 335 chickens from N202 and 461 chickens from N301. BW at 49 (BW49) and 70 days of age (BW70) and FI (from 49 to 70 days of age) were determined individually. BWG and FCR were computed based on BW and FI in the interval between 49 to 70 days. The results indicated that PRKAB1 SNPs rs14094358 and rs14094362 were significantly associated with BW70, BWG, and FI in the N202 strain, and rs14094361 and rs14094363 were significantly associated with FI and FCR in the N301 strain (P < 0.05). In addition, the PRKAG2 SNP rs14133282 showed significant association with FI in N202, and rs13535812 was significantly associated with BW70 in N202 (P < 0.05). Moreover, the PRKAG3 SNP rs13595570 was significantly associated with BW in N202 (P < 0.05), and significantly associated with FI and FCR in N301 (P < 0.05). Additionally, a two-SNP haplotype comprising rs14094361 and rs14094362 in PRKAB1 was significantly associated with BWG in N202 (P < 0.05). Meanwhile, haplotypes based on two SNPs, rs14133282, and rs13535812, showed significant effects on FI in N202 (P < 0.05). Our findings therefore provide important evidence for association of AMPK subunits polymorphisms with body weight, feed intake, and feed efficiency that may be applied in meat-type chicken breeding programs.

Epigenetic modification is widely known to be involved in embryo development, aging, tumorigenesis, and many complex diseases. Both hypermethylation of CpG islands at the gene promoters and global hypomethylation are involved in the initiation and progression of carcinogenesis. However, only a small portion of hypomethylation occurs at gene promoters and leads to the overexpression of certain oncogenes. To determine whether DNA methylation plays a role in tumorigenesis of Marek's disease, we selected one putative oncogene and 8 tumor suppressor genes from the gene expression profile for the analysis of DNA methylation variation. Four normal spleen tissues and 4 Marek's disease virus-infected tumor spleen tissues were collected, and the methylation level of the promoter region of each gene was analyzed using MassARRAY. As a result, the promoter region of CD30 was hypomethylated and displayed a significantly higher expression in Marek's disease virus-infected tumor spleen tissues compared with normal ones (P0.05). In neoplastic cells, CD30 was known to promote the survival and proliferation of T-cell lymphomas. This result suggests that activation of CD30 is possibly associated with the tumorigenesis of Marek's disease.

Avian leukosis virus (ALV) subgroup E (ALVE) is an endogenous retrovirus in the chicken genome. The chickens carrying ALVE locus 3 (ALVE3), 6 (ALVE6), 9 (ALVE9), and 21 (ALVE21) have been proved to be susceptible to ALV. Tumor virus locus B (TVB) encodes the cellular receptor for ALV subgroups B, D, and E. The insertions of the 4 ALVE loci and the genotypes of TVB have not been demonstrated in Chinese indigenous chicken breeds. In the present study, the existence of ALVE3, ALVE6, ALVE9, and ALVE21 were detected in 10 native breeds of Chinese chickens and an introduced breed, the White Leghorn (2 populations in this study, WL1 and WL2), by locus-specific PCR. The PCR products of ALVE were further confirmed by sequencing assay. We also surveyed the status of genotypes of TVB in Silkie, Beijing You, and White Leghorn (WL1 and WL2) chickens with pyrosequencing assays. The results showed that the carrier frequency of ALVE3 was 1.3% in the Chinese chicken population, and was 10.3 in WL1 and 49.2% in WL2. The carrier frequency of ALVE6 was 5.4% in native breeds of Chinese birds, in contrast with 0% in WL1 and 6.8% in WL2. The carrier frequency of ALVE9 was 0.1% in the Chinese indigenous population, and was 16.0% in WL1 and 11.9% in WL2. The carrier frequency of ALVE21 was 10.4% in Chinese chickens, whereas ALVE21 was detected with a frequency of 0% in WL1 and 50% in WL2. The frequency of the TVB resistance allele (TVB*R and TVB*R') was 0.4% in Beijing You chickens, whereas it was 70.5% in WL1 and 54.5% in WL2. No carriers of ALVE3, ALVE9, and ALVE21 were detected in Silkie fowl, a famous Chinese native breed that has been used as a source for alternative medicine. These results present molecular evidence of ALVE3, ALVE6, ALVE9, and ALVE21 insertions and TVB genotypes in Chinese indigenous chickens and could provide potential molecular insights into anti-ALV breeding in chickens.

The objective of the present study was to evaluate the role of myofiber characteristics and the thickness of 2 major muscle membranes, perimysium and endomysium, in determining the breast meat tenderness of chickens. Birds from 2 breeds (White Leghorn and a line of broiler) were chosen. Chicks were sexed and wing-banded at hatch and were grown in separate cages in a single house. Sixty broilers and 60 White Leghorns were harvested at 6 wk of age, respectively, whereas another 60 White Leghorns were slaughtered at 18 wk of age. An equal number of males and females was maintained for each group. Body weight, breast muscle weight, pH, drip loss, cooking loss, Warner-Bratzler shear force value (SFV), total energy of shear force, fiber diameter, sarcomere length, myofiber density, and the thickness of endomysium and perimysium of the breast were determined for each bird. At 6 wk of age, histological examination indicated that the size of myofiber and thickness of endomysium and perimysium of broilers were larger than that those of White Leghorns (P < 0.01), whereas the SFV, drip loss, and cooking loss of broilers were smaller (P < 0.01). A comparison between the White Leghorns at 18 wk and the broilers at 6 wk, which were at similar BW but different ages, showed that the breast muscle weight of broilers was larger (P < 0.01) than that of White Leghorns. For breast muscle, the endomysium of broilers at 6 wk was thicker than that of White Leghorns at 18 wk (P < 0.01), whereas the perimysium was thinner (P < 0.01). The SFV, drip loss, and the cooking loss of broilers were smaller than those of White Leghorns at similar BW (P < 0.01). Meat tenderness was negatively correlated with myofiber density (-0.27) and the thickness of endomysium (-0.29) and positively correlated with the thickness of perimysium (0.20). It is suggested that muscle membrane should be considered in evaluating meat tenderness of the chicken.

Blue-shelled eggs are gaining popularity as the consumption demand diversifies in some countries. This study was carried out to investigate the laying performance and egg quality of the blue-shelled egg layers as well as the effects of different housing systems on egg production and quality traits. One thousand pullets from Dongxiang blue-shelled layers were divided into 2 even groups and kept in different housing systems (outdoor vs. cage). Daily laying performance was recorded from 20 to 60 wk of age. External and internal egg quality traits were examined at 26, 34, 42, and 50 wk. Yolk cholesterol concentration and whole egg cholesterol content were measured at 40 wk of age. Average laying rate from 20 to 60 wk for the cage (54.7%) was significantly higher than that of outdoor layers (39.3%). Among all of the egg quality traits, only eggshell color was affected by housing system. Interaction between housing system and layer age was found in egg weight, eggshell color, eggshell ratio, yolk color, and yolk weight. Meanwhile, cholesterol concentration in yolk was 8.64 +/- 0.40 mg/g in the outdoor eggs, which was significantly lower than that of eggs from the cage birds (10.32 +/- 0.48 mg/g; P < 0.05). Whole egg cholesterol content in the outdoor eggs (125.23 +/- 6.32 mg/egg) was also significantly lower than that of eggs from the caged layers (158.01 +/- 8.62 mg/egg). The results demonstrated that blue-shelled layers have lower productivity in the outdoor system than in the cage system. Blue-shelled layers have lower egg weight, larger yolk proportion, and lower cholesterol content compared with commercial layers. In a proper marketing system, lower productivity could be balanced by a higher price for the better quality of blue-shelled eggs.

This study was conducted to estimate genetic parameters of meat quality-related traits by a MTDFREML procedure, using 1,069 purebred Beijing-You full-sib male chickens derived from the first 2 generations of divergent selection for the percentage of intramuscular fat (IMF) and selection for increased inosine-5'-monophosphate content (IMP) in breast meat. The results show that estimated heritability of IMP was moderate (0.23), and heritability of IMF was low (0.11). Other traits with high heritabilities, ranging between 0.56 and 0.79, were BW, abdominal fat weight (AFW), breast meat yield, ratio of breast meat yield to evisceration weight (BMP), leg muscle yield, comb weight, and ratio of comb weight to BW (CWP). Moderate heritabilities for the ratio of AFW to BW (AFP) and leg muscle yield to evisceration weight were estimated, 0.24 and 0.32, respectively. Lower significant phenotypic correlations of IMP with BMP and ratio of leg muscle weight to evisceration weight were discovered (P < 0.05), whereas IMF exhibited slightly positive, though significant, phenotypic correlations with BW (0.11) and AFP (0.27). Genetic correlations of IMP with BW and CWP were negative (-0.38 and -0.62, respectively), whereas a high positive genetic correlation was found between IMP and BMP (0.57). It was found that IMF had high genetic correlations with BW (0.75) and AFW (0.66) and moderate correlations with AFP (0.32) and CWP (0.40). A low positive genetic correlation was estimated between IMP and IMF (0.27). In conclusion, both IMP and IMF contents in chicken meat have the potential to be increased through genetic selection with little or no positive effect on BW. Furthermore, close managerial control of growth rate (and BW) will be needed to assure high quality of chicken meat so that increased IMP and IMF can be obtained with less abdominal fat deposited.

Sex-linked dwarf chickens caused by the mutation of the growth hormone receptor gene are characterized by normal growth hormone (GH), very low insulin-like growth factor I (IGF-I) level in the blood, and reduced growth. It has been demonstrated that the sex-linked dwarfing gene has negative effects on female reproduction. In the current study, dwarf cocks and their phenotypic normal siblings were used to investigate the effects of dwarf gene on male reproduction. Dwarf cocks grew slower than the normal cocks did, and at 20 wk of age, their BW were 36.4% smaller. However, all parameters for semen quality, including volume, sperm concentration, viability, mobility, pH, and percentage of abnormal sperms, examined at 30 wk of age showed no significant difference between normal and dwarf cocks. The fertility of dwarf cocks was 95.2%, and the normal was 92.4%. The concentrations of GH and IGF-I in serum and seminal plasma were measured with RIA and ELISA, respectively. The serum GH in the dwarf cocks was significantly higher than their normal siblings (P < 0.05), whereas the serum IGF-I in the dwarf cocks was very low. However, the concentration of seminal IGF-I in dwarf cocks was similar to that of their normal siblings, indicating that IGF-I might be produced and acted independently in testis. In conclusion, the deficiency in GH receptor did not affect the male reproduction in dwarf chickens, and the fertility of dwarf cocks could be satisfactory for production when artificial insemination was adopted.

Campylobacter jejuni (C. jejuni) is a leading cause of human bacterial gastroenteritis worldwide. Previous research has shown that circadian rhythm plays a critical role in host response to C. jejuni colonization. The CLOCK gene is one of the core genes regulating circadian rhythms and shows significant expression on 7 d post-C. jejuni inoculation. The objective of this study was to investigate temporal and spatial expression of chicken CLOCK gene post-C. jejuni inoculation. Cecal and splenic RNA were isolated from 2 distinct chicken breeds and used to compare the mRNA expression of CLOCK gene between inoculated and noninoculated chickens within each breed and between breeds within each of inoculated and noninoculated groups. Our results showed that the CLOCK gene was significantly down-regulated at 20 h postinoculation (hpi) in cecum and spleen in Jiningbairi chicken. CLOCK gene was significantly down-regulated at 4 and 16 hpi and up-regulated at 8 hpi in cecum and spleen in specific pathogen free white leghorn noninoculated chicken. The findings suggested that expression of CLOCK gene was significantly changed post C. jejuin inoculation. This change was affected by genetic background, tissue, and time points postinoculation.

The peroxisome proliferators-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was investigated as a candidate gene for growth and fatness traits in chicken because of its prominent role in muscle fiber specialization and adipogenesis. A single nucleotide polymorphism (SNP) from G to A at position 646 of the open reading frame of chicken PGC-1alpha gene causing an Asp216Asn amino acid substitution was identified. The frequencies of alleles and genotypes were significantly different among 6 chicken breeds (P < 0.01). The White Plymouth Rock had the highest frequency (0.67) of allele G, whereas the White Leghorn had the lowest (0.18). The associations of the SNP with the growth and fatness traits were evaluated in 332 F(2) birds from an experimental cross of White Plymouth Rock x Silkies. No association was found between the SNP and growth-related traits. However, abdominal fat weight at 12 wk of age for birds with genotype GG was 34.26 and 28.71% higher than those with genotypes AA and AG, respectively (P < 0.01), indicating that the Asp216Asn polymorphism of the PGC-1alpha gene could be used as a novel potential molecular marker for selection against abdominal fatness without interfering in regular breeding for growth rate of chickens.

Thyroid hormone responsive Spot 14 (THRSP) is suggested as a transcription factor involved in the regulation of adipogenic enzymes by 3 thyroid response elements in the promoter region. In the chicken genome, THRSP gene was identified to duplicate into 2 paralogs, THRSPalpha and THRSPbeta. In the current study, cDNA sequences of the duplicated duck THRSP genes were cloned by real-time PCR and rapid amplification of cDNA ends. Duck THRSPalpha and THRSPbeta were predicted to encode peptides with 133 amino acids, which had 74 and 68% sequence identity at cDNA level, 78 and 74% identity at amino acid level to the chicken counterparts, respectively. A high percentage (73.1%) of G and C nucleotides were found in the 3' untranslated region of duck THRSPbeta cDNA. Although a low similarity of peptide composition was shared between ducks and mammals, and a moderate similarity was shared between ducks and chickens, many predicted properties of THRSP, including the pI, subcellular localization and functional domains seemed to be highly conserved. The present study demonstrated that the duck THRSP gene duplicates into the 2 paralogs as in chickens. Phylogenetic analysis indicated that the duplication for THRSP paralogs appeared to have taken place preceding the chicken-duck split, and the diverging rate between THRSP paralogs seemed faster in the chicken genome than that in the duck genome. Expression analysis by real-time quantitative PCR showed that THRSP paralogs in ducks were more actively transcribed in fat tissues (i.e., s.c. fat and abdominal fat) than in liver, and the mRNA concentrations of THRSPbeta were higher than that of THRSPalpha in liver and s.c. fat.