Physicochemical Index Analyses of the Egg White in Blue-Shelled Eggs and Commercial Brown-Shelled Eggs during Storage.

To compare the physical and chemical changes in egg whites during storage, assisting in the evaluation of differences in egg freshness between various chicken breeds, we chose 240 blue-shelled eggs (Blue group) and 240 commercial brown-shelled eggs (Brown group) that 28-week-old hens had laid. In this study, all eggs were stored at 25 °C. The egg weight, egg components' weight and proportion, Haugh Unit value and the contents of S-ovalbumin, ovomucin and lysozyme in the thick albumen (KA) and thin albumen (NA) were measured at eight time points every 3 days until the 21st day of storage. The eggshell, yolk and KA proportions in the Brown group were significantly lower, whereas the NA proportion was significantly higher than that in the Blue group (p < 0.001). The Haugh Unit value and S-ovalbumin in the Brown group were significantly higher, whereas KA ovomucin and NA lysozyme were significantly lower than those in the Blue group (p < 0.001). There existed significant negative correlations between the KA and NA, irrespective of weight or proportion. The Haugh Unit value was significantly positively correlated with lysozyme and ovomucin, but significantly negatively correlated with S-ovalbumin. During storage, the KA weight (proportion), Haugh Unit value, lysozyme and ovomucin decreased, whereas the NA weight (proportion) and S-ovalbumin increased. At each time point, the NA lysozyme in the Brown group was lower than that in the Blue group (p < 0.05). After storage for 6 days, the KA ovomucin in the Brown group began to be lower than that in the Blue group (p < 0.05). The study showed that the weight (proportion) differences in egg components between blue-shelled eggs and commercial brown-shelled eggs are mainly due to the NA. The Haugh Unit value and albumin protein indexes of blue-shelled eggs were better than those of brown-shelled eggs, and showed mild changes during storage, indicating the better storage performance of blue-shelled eggs.

Pigment concentrations in eggshell and their related gene expressions in uterus of Changshun blue eggshell chickens.

1. The goal of this study was to investigate the colour diversity of egg shells and expression of related genes in the uterus of chickens that produce eggs of different colours.2. Four colour types of Changshun blue-shell chickens, producing dark or light blue, greenish-brown and brown shelled eggs, were selected. The eggshell pigment concentration and colour values in each group were examined. The relative gene expression of solute carrier organic anion transporter family member 1C1 (SLCO1C1), ferrochelatase (FECH), haem oxygenase 1 (HO-1), ovotransferrin (OF) and biliverdin reductase A (BLVRA) in eggshell gland were measured.3. The Δb, ΔE and protoporphyrin in brown and greenish-brown groups were significantly higher in the blue egg group (P < 0.01), whereas ΔL was significantly lower than that in the blue eggs (P < 0.01). There was no significant difference in biliverdin concentration between the brown and blue groups.4. The Δa values, in descending order, were 8.27 ± 2.76 in the brown, -3.79 ± 2.39 in the greenish-brown and -7.29 ± 2.27 in the blue groups, respectively. The relative expression of HO-1 in the greenish-brown and light blue groups was significantly higher than in the dark blue and brown groups. The relative expression of FECH in the light blue group was significantly lower than that in the dark blue, greenish-brown or brown group (P < 0.01). The relative expression of HO-1 and BLVRA genes in the dark blue group was significantly higher than that in the light blue, greenish-brown and the brown group (P < 0.01).5. The Δa might provide a better index than protoporphyrin and biliverdin contents for eggshell colour breeding. Overall, HO-1 as well as BLVRA were important candidate genes for the selection of dark blue eggs.

An EAV-HP insertion in the 5' flanking region of SLCO1B3 is associated with its tissue-expression profile in blue-eggshell Yimeng chickens (Gallus gallus).

We previously reported that blue eggshell color in chickens is associated with a partial endogenous retroviral (EAV-HP) insertion in the promoter region of the solute carrier organic anion transporter family member 1B3 (SLCO1B3) gene. The EAV-HP sequence includes numerous regulatory elements, which may modulate the expression of adjacent genes. To determine whether this insertion influences the expression of neighboring genes, we screened the expression of solute carrier organic anion transporter family members 1C1, 1B1 (SLCO1C1, SLCO1B1), and SLCO1B3 in 13 and 10 tissues from female and male Yimeng chickens, respectively. We observed that the insertion only significantly modulated the expression of SLCO1B3 and did not majorly affect that of SLCO1C1 and SLCO1B1. High expression of SLCO1B3 was detected in the shell gland, magnum, isthmus, and vagina of the oviduct in female blue-eggshell chickens. We also observed ectopic expression of SLCO1B3 in the testes of male chickens. SLCO1B3 is typically highly expressed in the liver; however, the EAV-HP insertion significantly reduces SLCO1B3 expression. As a liver-specific transporter, a reduction in the expression of SLCO1B3 may affect liver metabolism, particularly that of bile acids. We also detected higher ectopic expression of SLCO1B3 in the lungs of birds heterozygous for the EAV-HP insertion than in homozygous genotypes. In conclusion, we confirmed that the EAV-HP insertion modifies SLCO1B3 expression, and showed, for the first time, similar expression profile of this gene in all parts of the oviduct in females and testis in males. We also observed different levels of SLCO1B3 expression in the liver, which were associated with the EAV-HP insertion, and significantly higher expression in the lungs of birds with heterozygous genotype. The effects of these changes in the SLCO1B3 expression pattern on the function of the tissues warrant further investigation.

Transcriptional responses of LSm14A after infection of blue eggshell layers with Newcastle disease viruses.

LSm14A is a key innate immunity component of processing body (P-body) that mediates interferon-ß (IFN-ß) signaling by viral RNA. This is the first study to report chicken LSm14A (cLSm14A) cloning from blue eggshell layer, high tibia and frizzle chickens. The cLSm14A gene, encoding 461 amino acids, is highly homologous in the three types of chickens. The cLSm14A was extensively expressed in several tissues. The transcriptional level of cLSm14A was significantly increased in various stages of Newcastle disease virus (NDV) infection. In HEK293 cells, full length cLSm14A from blue eggshell layer was localized in the cytoplasm as dots. The results of this study indicated that cLSm14A is an important sensor that mediates innate immunity in chicken against NDV infections.

A selection method of chickens with blue-eggshell and dwarf traits by molecular marker-assisted selection.

The blue-eggshell and dwarf traits have an important economic value in poultry production. Using a genetic aggregation-based strategy, the molecular marker-assisted selection technology was jointly used to provide a rapid breeding method for pure strain chickens simultaneously with hens exhibiting the blue-eggshell and dwarf traits. Overall, 80 male dwarf chickens and 1,000 hybrid blue-eggshell hens (F0) were used for the hybridization experiment. Subsequently, the crossing of F1 or F2 chicks was performed in succession. The F1 and F2 chicks were respectively detected by the joint molecular markers of the solute carrier organic anion transporter family, namely, 1B3 (SLCO1B3) and the growth hormone receptor (GHR) genes, which relate to blue-eggshell and dwarf traits. Meanwhile, the selection of blue-eggshell and dwarf phenotypes was used to validate the data obtained by the molecular markers. The results showed that F1 chicks included the heterozygous and wild-type of SLCO1B3, as well as the homozygous (hens) and heterozygous (roosters) of GHR. However, F2 chicks included 3 different genotypes of both SLCO1B3 and GHR. Ultimately, 196 F1 roosters (concurrently with heterozygous genotype of SLCO1B3 and GHR) and 1,073 F1 hens (concurrently with heterozygous genotype of SLCO1B3 and homozygous genotype of GHR) were obtained from the initial 10,040 F1 chicks. Further, 27 F2 roosters and 345 F2 hens, which simultaneously carried the homozygous genotype of SLCO1B3 and GHR, were screened from the initial 6,000 F2 chicks. Data obtained on the blue-eggshell and dwarf phenotypes were consistent with the results by molecular markers. Similarly, the purity verification of the strain obtained through 2 crossing experiments (F0♂ × F2♀ and F2♂ × F2♀) revealed that all chickens had the blue-eggshell and dwarf traits, supporting that the obtained F2 strain was pure. In summary, for the first time, we successfully bred a pure strain chicken with blue-eggshell and dwarf traits by jointly using the molecular markers of the SLCO1B3 and GHR genes. Our study provides a new method for the rapid cultivation of new chicken strains.

Association Between the Methylation Statuses at CpG Sites in the Promoter Region of the SLCO1B3, RNA Expression and Color Change in Blue Eggshells in Lushi Chickens.

The formation mechanism underlying the blue eggshell characteristic has been discovered in birds, and SLCO1B3 is the key gene that regulates the blue eggshell color. Insertion of an endogenous retrovirus, EAV-HP, in the SLCO1B3 5' flanking region promotes SLCO1B3 expression in the chicken shell gland, and this expression causes bile salts to enter the shell gland, where biliverdin is secreted into the eggshell, forming a blue shell. However, at different laying stages of the same group of chickens, the color of the eggshell can vary widely, and the molecular mechanism underlying the eggshell color change remains unknown. Therefore, to reveal the molecular mechanism of the blue eggshell color variations, we analyzed the change in the eggshell color during the laying period. The results indicated that the eggshell color in Lushi chickens can be divided into three stages: 20-25 weeks for dark blue, 26-45 weeks for medium blue, and 46-60 weeks for light blue. We further investigated the expression and methylation levels of the SLCO1B3 gene at eight different weeks, finding that the relative expression of SLCO1B3 was significantly higher at 25 and 30 weeks than at other laying weeks. Furthermore, the overall methylation rate of the SLCO1B3 gene in Lushi chickens increased gradually with increasing weeks of egg production, as shown by bisulfite sequencing PCR. Pearson correlation analysis showed that methylation of the promoter region of SLCO1B3 was significantly negatively correlated with both SLCO1B3 expression in the shell gland tissue and eggshell color. In addition, we predicted that CpG5 and CpG8 may be key sites for regulating SLCO1B3 gene transcription. Our findings show that as the level of methylation increases, methylation of the CpG5 and CpG8 sites hinders the binding of transcription factors to the promoter, reducing SLCO1B3 expression during the late period and resulting in a lighter eggshell color.