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Eggshell pores can be subdivided into micrometer-scale gas pores, submicroscopic bubble pores, and nanoscale mesopores. All are important indicators of eggshell quality ensuring gas exchange between the inside and outside of the eggshell and preventing invasion by external bacteria. Although previous studies on eggshell pores focused on gas pores, recent studies have shown that bubble pores may play an more important role in regulating gas exchange. In order to investigated the relationship between gas and bubble pores and the mechanisms of genetic regulation. In this study, 40-week-old Brown-Egg Dwarf Layers (DWL) eggs were selected, and the quantity of gas pores (QGP), quantity of mammillary (QM), and quantity of bubble pores (QBP), area sum of bubble pores (ASBP), and other bubble pore-related indexes were determined. The correlation between each index was calculated, and genome-wide association analysis (GWAS) was performed based on whole genome resequencing (WGR). The results showed that the CVs of QGP and QM were 15.69% and 15.49%, respectively, and the CVs of the related pore indices, such as QBP and ASBP, were 29.22%-44.82%. The correlation coefficient between QGP and QM was 0.59 (P< 0.01), and there was no correlation between QGP, QM, and the bubble pore-related indicators (P > 0.05). These results above suggest that the gas and bubble pores may be two independent pore systems. A total of 32 single nucleotide polymorphisms (SNPs) associated with the suggestively significant level of bubble pore correlation indexes were detected in GWAS, and the corresponding genes were ANXA10, CDH10, AADAT, RXFP1, FNIP2, DDX60, PCDH10, RAPGEF2, FSTL5 and SPOCK3. KEGG enrichment analysis showed that these genes were mainly expressed in the calcium ion binding pathway, indicating that the genes and pathways may play a regulatory role in forming bubble pores during eggshell calcification. This study provides a basis for revealing the genetic regulatory mechanism of eggshell pores and a reference and direction for further improvement in eggshell quality.

Eggshell translucency severely affects external egg quality, and variations in the eggshell or eggshell membrane are considered the structural basis of the trait. Research has shown that 1.85% additional mixed fatty acids in the diet would greatly decrease the occurrence of eggshell translucency. Only a few studies have examined the phenotypic regularity of eggshell translucency with the increasing age of hens. Therefore, two strains, 1139 Rhode Island Red-White (RIR-White) and 836 Dwarf Layer-White (DWL-White), were used, and from each strain, 30 hens each that consecutively laid translucent or opaque eggs at 67 wks of age were selected. Subsequently, eggshell translucency, internal quality and external quality of eggs, and total cholesterol, albumin, calcium binding protein and other physiological indicators related to lipid, lipoprotein, and calcium metabolisms at the 75th, 79th, and 83rd wks of age in the late phase of the laying cycle were determined. Results: (1) In terms of flocks, for both strains, the translucency scores of the translucent groups were significantly higher than those of the opaque groups (P < 0.05); in terms of individuals, 81.1% RIR-White and 82.8% DWL-White hens consecutively laid eggs of the same or similar translucency, indicating the stability of the trait with increasing hen age; (2) In RIR-White, the eggshell strength of the translucent group at 75 weeks was significantly higher than that of the opaque group (P < 0.05); in DWL-White, the eggshell membrane thickness of the translucent group at the 75th and 83rd weeks was significantly lower than that of the opaque group (P < 0.05); (3) Compared to the opaque groups, the translucent groups had lower total cholesterol content in both RIR-White and DWL-White, lower albumin content in DWL-White at the 79th weeks (P < 0.05), and higher calcium-binding protein (CALB1) in RIR-White at the 83rd weeks (P < 0.05). In summary, this study illustrates the stability of eggshell translucency in late-phase laying hens and provides a reference of physiological indicators for exploring the formation of translucent eggs.