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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.

ABSTRACT: Eggshell translucency is a widespread issue in the field of egg quality. Previous research has established that the heritability of eggshell translucency is relatively low or moderate. Scientists have also successfully identified SNP loci related to eggshell translucency on different chromosomes by using gene chips and single-variant GWAS. However, the specific impact of single or multiple genes on the trait of eggshell translucency remains unknown. In an effort to investigate this, we examined 170 SNPs associated with eggshell translucency obtained by our research group. We selected 966 half-sibling laying hens from 2 generations in 3 pure lines: Dwarf Layer-White, Rhode Island Red-White Strain, and Rhode Island Red. Eggs were collected from each hen over a period of 5 consecutive days, and eggshell translucency was measured using a grading method in which the hens were divided into 2 groups: an opaque group and a translucent group. We collected blood samples from the laying hens and extracted DNA. Time of flight mass spectrometry (TOF-MS) was used for genotyping to identify SNP loci that influence the trait of eggshell translucency. The results of our analysis revealed that using TOF-MS in 3 chicken strains, we were able to eliminate loci with low gene polymorphism, genetic effect contribution less than 1%, and deviation from Hardy-Weinberg equilibrium. Ultimately, 5 SNPs (Affx-50362599, rs15050262, rs312943734, rs316121113, and rs317389181) were identified on chromosomes 1, 5, and 19. Additionally, nine candidate genes (DCN, BTG1, ZFP92, POU2F1, NUCB2, FTL, GGNBP2, ACACA, and TADA2A) were found to be associated with these SNPs. No linkage disequilibrium relationship was observed between the 2 pairs of SNP loci on chromosomes 1 and 19. Based on previous studies on the formation mechanism of eggshell translucency, we hypothesize that NUCB2, FTL, and ACACA genes may be affecting the eggshell structure through different mechanisms, such as increase the water permeability or make thin of eggshell membrane, which promote moisture or part of other egg contents and ultimately lead to the formation of eggshell translucency. These findings validate and identify five SNP loci that regulate the translucency trait, and provide molecular markers for breeding non-translucent populations. Furthermore, this study serves as a reference for further investigation of the genetic regulatory mechanisms underlying eggshell translucency.

ABSTRACT: Granular cell apoptosis is a key factor leading to follicular atresia and decreased laying rate in aged laying hens. Endoplasmic reticulum stress (ERS) induced cell apoptosis is a new type of apoptosis pathway. Previous studies have shown that the ERS pathway is involved in the regulation of follicular development and atresia, and can be regulated by mTOR. Melatonin (MEL) can protect the normal development of follicles, but the precise mechanism by which MEL regulates follicular development is not yet clear. So, we investigated the potential relationship between MEL and ERS and mTOR signaling pathway in vivo through intraperitoneal injection of MEL in aged laying hens. The results show that the laying rate, ovarian follicle number, plasma MEL, E2, LH, FSH concentrations, as well as the mRNA expression of mTOR signaling-associated genes TSC1, TSC2, mTOR, 4E-BP1, and S6K in old later-period chicken control (Old-CN) group was significantly decreased (P < 0.01). In contrast, the ERS-related of plasma and granular cell layer mRNA expression of Grp78, CHOP, and Caspase-3 was significantly increased (P < 0.01). While both of the effects were reversed by MEL. Then, aging granulosa cells were treated with MEL in vitro, followed by RNA seq analysis, and it was found that 259 and 322 genes were upregulated and downregulated. After performing GO enrichment analysis, it was found that DEGs significantly contribute to the biological processes including cell growth and apoptosis. Using pathway enrichment analysis, we found significant overrepresentation of cellular processes related to mTOR signaling and endoplasmic reticulum (ER) stress, involving genes such as GRB10, SGK1, PRKCA, RPS6KA2, RAF1, PIK3R3, FOXO1, DERL3, HMOX1, TLR7, VAMP7 and INSIG2. The obtained results of RT-PCR showed consistency with the RNA-Seq data. In summary, the underlined results revealed that MEL has significantly contributed to follicular development via activating the mTOR signaling pathway-related genes and alleviating ERS-related genes in laying hens. The current study provides a theoretical background for enhancing the egg-laying capability of hens and also providing a basis for elucidating the molecular mechanism of follicular selection.

ABSTRACT: Follicular atresia in chickens reduces the number of follicles that can further develop, leading to decrease egg laying. Endoplasmic reticulum stress (ERS) can initiate a unique pathway inducing the apoptosis of follicular granulosa cells, thus reducing egg laying. Melatonin (MEL) is involved in the regulation of follicle development, ovulation, and oocyte maturation, and is closely related to follicle fate. Mammalian target of Rapamycin (mTOR) signaling pathway plays an important role in cell growth regulation, and that there is a possible crosstalk between melatonin and mTOR activity in granular cells maturation and ovulation. This study aimed to investigate whether MEL inhibits ERS and follicular granulosa cell apoptosis by regulating ATF4 to activate mTOR signaling pathway in chickens. Frist, we established an in vitro ERS cell model using tunicamycin (TM). The results showed that different concentrations of TM exhibited dose-dependent inhibition of cell activity and induction of granulosa cells (P

The decrease in eggshell quality seriously affects production efficiency. Guinea fowl (GF) eggs possess strong eggshells because of their unique crystal structure, and few systematic studies have compared laying hen and GF eggs. Sixty eggs were collected from both 40-week-old Dwarf Layer-White (DWL-White) laying hens and GF, and the eggshell quality, ultrastructure, bubble pores, and composition were measured. The results showed that the DWL-White eggs had a higher egg weight and a lower eggshell strength, strength per unit weight, thickness, and ratio than the GF eggs (p < 0.01). There were differences in the mammillary layer thickness ratio, the effective layer thickness ratio, the quantity of bubble pores (QBPs), the ratio of the sum of the area of bubble pores to the area of the eggshell in each image (ARBE), and the average area of bubble pores (AABPs) between the DWL-White and GF eggs (p < 0.01). The composition analysis demonstrated that there were differences in the organic matter, inorganic matter, calcium, and phosphorus between the DWL-White and GF eggs (p < 0.01). There were positive associations between the mammillary knob number in the image and the QBPs and ARBE and a negative correlation with the AABPs in the DWL-White eggs (p < 0.01). This study observed distinctions that offer new insights into enhancing eggshell quality.

ABSTRACT: Oxidative stress can trigger follicular atresia, and decrease follicles quantity in each development stage, thereby alleviating reproductive activity. The induction of oxidative stress in chickens through intraperitoneal injection of dexamethasone is a reliable and stable method. Melatonin has been shown to mitigate oxidative stress in this model, but the underlying mechanism remains unclear. Therefore, this study aimed to investigate whether melatonin can recover aberrant antioxidant status induced by dexamethasone and the specific mechanism behind melatonin-dependent protection. A total of 150 healthy 40-wk-old Dawu Jinfeng laying hens with similar body weights and laying rates were randomly divided into three groups, with five replicates per group and 10 hens per replicate. The hens in the control group (NS) received intraperitoneal injections of normal saline for 30 d, the dexamethasone group (Dex+NS) received 20 mg/kg dose of dexamethasone for the first 15 d, followed by the 15 d of normal saline treatment. While in the melatonin group (Dex+Mel), dexamethasone (20 mg/kg dose) was injected intraperitoneally in the first 15 d, and melatonin (20 mg/kg/d) was injected in the last 15 d. The results showed that dexamethasone treatment significantly enhanced oxidative stress (P < 0.05), while melatonin not only inhibited the oxidative stress but also notably enhanced the antioxidant enzymes superoxide dismutase (SOD), catalase activity (CAT), glutathione peroxidase (GSH-Px), and antioxidant genes CAT, superoxide dismutase 1 (SOD1), glutathione peroxidase 3 (GPX3), and recombinant peroxiredoxin 3 (PRDX3) expression (P < 0.05). Melatonin treatment also markedly reduced 8-hydroxy deoxyguanosine (8-OHdG), malondialdehyde (MDA), and reactive oxygen species (ROS) levels (P < 0.05) and apoptotic genes Caspase-3, Bim, and Bax in the follicle. In the Dex+Mel group, the Bcl-2 and SOD1 protein levels were also increased (P < 0.05). Melatonin inhibited the forkhead Box Protein O1 (FOXO1) gene and its protein expression (P < 0.05). In general, this investigation revealed that melatonin might decrease oxidative stress and ROS by enhancing antioxidant enzymes and genes, activating the antiapoptotic genes, and inhibiting the FOXO1 pathway in laying hens.

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.

ABSTRACT: The translucency of eggshells is a ubiquitous appearance problem caused by moisture translocation and the accumulation of egg contents into the eggshell ultrastructure. Previous studies have mainly investigated the causes of eggshell translucency from nutritional and environmental perspectives. However, little is known of the effect of genetics the causes of eggshell translucency on hen production performance. To evaluate the genetic parameters of eggshell translucency and other production performance indicators, we performed an experiment on 3 pure hen lines: 624 Dwarf Layer-White, 1,612 Rhode Island Red, and 813 Rhode Island Red-White. We collected eggs from each hen over 5 d and measured eggshell translucent level (TL) using the grading method. Additionally we measured indicators of each hen during the laying period, including age at laying of the first egg (AFE), body weight at laying of the first egg (BWFE), weight of the first egg (FEW), body weight at 40 wk (BW40), egg weight at 40 wk (EW40), egg production up to 40 wk of age (EN), and calculated the genetic parameters among the indicators. The results showed that the estimated heritability of TL in the 3 genotypes were 0.30, 0.24, and 0.20, respectively, suggesting a low or moderate level of heritability. We found a positive correlation between TL and AFE, with genetic correlation coefficients 0.19 to 0.41, and negative genetic correlation between TL and EN, with correlation coefficient −0.36 to −0.19. Additionally, we observed positive correlation exists between AFE and FEW, BWFE and FEW, and BW40 and EW40; and negative correlation between AFE and EN in the 3 pure lines. These results enriched the research on heritability of eggshell translucency in different hen breeds and demonstrated moderate or low heritability of the indicator. Furthermore, eggshell translucency was negatively affected by AFE and EN. Our results provide a valuable reference for predicting selection response of eggshell translucency and production performance in brood hens, and locating the genes regulating eggshell translucency.

The molecular mechanisms underlying reproductive aging in avian species are poorly understood. Previous studies have shown the importance of mechanistic target of rapamycin (mTOR) signaling pathway in aging. In this study, we investigated the relationship between the mTOR signaling pathway and ovarian aging in the peak phase and late phase of egg production in laying hens. The egg production rate and egg quality were tracked for 5 consecutive weeks in 30-week-old and 70-week-old Dawu Jinfeng hens (N = 30/group). During the peak phase (week 35) and late phase (week 75), antioxidant and immune indicators were detected by enzyme-linked immunosorbent assay, and mTOR signaling-related genes (CLIP-170, GRB10, LIPIN-1, ATG1, 4E-BP1, S6K, PKC, RHO, and SGK1) were detected in the follicles by quantitative reverse transcription-PCR technology. The protein expression of key genes (mTOR, PKC, 4EBP1) was evaluated by Western blot analysis. The egg production rate and the antioxidant indexes superoxide dismutase and glutathione peroxidase and the levels of total antioxidant capacity and immunoglobulins (IgM and IgG) were significantly higher at week 35 than those at week 75 (P

Melatonin is a key regulator of follicle granular cell maturation and ovulation. The mammalian target of rapamycin (mTOR) pathway plays an important role in cell growth regulation. Therefore, our aim was to investigate whether the mTOR signaling pathway is involved in the regulation of melatonin-mediated proliferation and apoptotic mechanisms in granulosa cells. Chicken follicle granular cells were cultured with melatonin (0, 2, 20, or 200 μmol/L) for 48 h. The results showed that melatonin treatment enhanced proliferation and suppressed apoptosis in granular cells at 20 μmol/L and 200 μmol/L (P < 0.05) by upregulation of cyclin D1 (P < 0.01) and Bcl-2 (P < 0.01) and downregulation of P21, caspase-3, Beclin1, and LC3-II (P < 0.01). The effects resulted in the activation of the mTOR signaling pathway by increasing the expression of avTOR, PKC, 4E-BP1, S6K (P < 0.05), p-mTOR, and p-S6K. We added an mTOR activator and inhibitor to the cells and identified the optimal dose (10 μmol/L MHY1485 and 100 nmol/L rapamycin) for subsequent experiments. The combination of 20 μmol/L melatonin and 10 μmol/L MHY1485 significantly enhanced granulosa cell proliferation (P < 0.05), while 100 nmol/L rapamycin significantly inhibited proliferation and enhanced apoptosis (P < 0.05), but this action was reversed in the 20-μmol/L melatonin and 100-nmol/L rapamycin cotreatment groups (P < 0.05). This was confirmed by mRNA and protein expression that was associated with proliferation, apoptosis, and autophagy (P < 0.05). The combination of 20 μmol/L melatonin and 10 μmol/L MHY1485 also activated the mTOR pathway upstream genes PI3K, AKT1, and AKT2 and downstream genes PKC, 4E-BP1, and S6K (P < 0.05), as well as protein expression of p-mTOR and p-S6K. Rapamycin significantly inhibited the mTOR pathway–related genes mRNA levels (P < 0.05). In addition, activation of the mTOR pathway increased melatonin receptor mRNA levels (P < 0.05). In conclusion, these findings demonstrate that melatonin regulates chicken granulosa cell proliferation and apoptosis by activating the mTOR signaling pathway via its receptor.

The signal pathway of target of rapamycin (TOR) plays an important role in regulating cell growth and proliferation, follicular development, and ovulation. Melatonin (N-acetyl-5-methoxytryptamine) (MT) is involved in the regulation of many physiological functions in animals. Recent studies have shown that MT affects the number and the degree of maturation of follicles in the ovary, but there are few studies concerning its mechanism. Therefore, the aim of this study was to investigate the mechanism of TOR signal pathway in the regulation of ovarian function by MT in aging laying hens. In the present study, a total of 60 hens (70-week-old) were randomly divided into 2 groups: control group and melatonin group (M). Melatonin was administered intraperitoneally at a dose of 20 mg/kg/D for 28 D in the M group. The results showed that MT significantly increased the levels of the antioxidant enzymes superoxide dismutase and total antioxidant capacity (P < 0.01) as well as levels of immunoglobulin (IgA, IgG, and IgM) (P < 0.05) and the reproductive hormones estradiol and luteinizing hormone (P < 0.01) in the plasma and also increased the numbers of middle white follicles and small white follicles (P < 0.05) and decreased the level of reactive oxygen species in plasma (P < 0.01) in laying hens. There were higher expression levels in MT receptor A (P < 0.05), melatonin receptor B (P < 0.01), and tuberous sclerosis complex 2 (P < 0.01). Activation of TOR, 4E binding protein-l (4E-BP1), and ribosomal protein 6 kinase (P

ABSTRACT: Eggshell translucency is a ubiquitous external eggshell quality problem caused by variations of eggshell ultrastructure or shell membrane. In previous studies, researchers have widely investigated this phenomenon with nutritional, environmental, and genetic perspectives in many breeds. However, most studies referring to phenotypic measurement of shell translucency have been performed using a relatively subjective two-, three-, or four-grading methods, which made it impossible to compare distribution of shell translucency among different breeds. In this study, we aimed to explore variations of translucent eggshell spots in different breeds and their distribution in blunt, middle, and sharp ends of eggshell using a relatively objective grayscale recognition method. We selected 45 eggs from each flock of pure lines, commercial strains, and Chinese local breeds (10 flocks, aged 60 to 70 wk), and stored them in a constant environment for 5 d. Then measured eggshell translucency using grayscale recognition method. Indicators of shell translucency included sum of spot areas on the whole eggshell (SUSA), sum area of the whole eggshell (SUSHA), RSS (ratio of SUSA to SUSHA), quantity of spots (QS), average spot area in eggshell (AAES), and diameter of spots in eggshell (DS). As results, in Hy-Line Brown, Brown-Egg Dwarf Layer, and Taihang (pink-shelled) breeds, phenotypic intensity of eggshell translucency was slight; in Rhode Island Red, Jingfen-1, and Dongxiang breeds, phenotypic intensity of eggshell translucency was relatively extensive; and in Jinghong-1, Hy-Line Sonia, White Leghorn, and Taihang (blue-shelled), phenotypic intensity of eggshell translucency was at an intermediate level. In general, the larger the RSS, the larger the QS, AAES, and DS. Of 3 ends for most breeds, eggshell translucency of blunt and sharp ends was usually greater than that of middle ends, and blunt ends seemed to have the most extensive eggshell translucency. Findings from this study could provide a reference for population selection to locate genes regulating shell translucency and to explore the physical structure mechanism for eggshell translucency formation.

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. [ABSTRACT FROM AUTHOR]

Objective. Chronic hepatitis B liver fibrosis is an important intermediate link in the development of liver cirrhosis. A retrospective cohort study was conducted in Longhua Hospital affiliated to the Shanghai University of Traditional Chinese Medicine in order to prove whether integrated traditional Chinese and Western medicine could improve the incidence of CHB complications and clinical prognosis. There are 130 patients with hepatitis B liver fibrosis (being treated from 2011–2021) included in the study, and the patients were divided into 64 TCM users (NAs combined with TCM) and 66 TCM nonusers (NAs antiviral therapy). The serum noninvasive diagnostic model (APRI, FIB-4) and LSM value were used to classify the stages of fibrosis. The results showed that the LSM value was decreased significantly in TCM users compared with TCM nonusers (40.63% versus 28.79%). Indicators of FIB-4 and APRI of TCM users have improved significantly compared with that of TCM nonusers (32.81% versus 10.61% and 35.94% versus 24.24%). The AST, TBIL, and HBsAg levels in TCM users were lower than those in TCM nonusers, and the HBsAg level was inversely correlated with the CD3+, CD4+, and CD8+ in TCM users. The PLT and spleen thickness of TCM users also were improved considerably. The incidence rate of end-point events (decompensated cirrhosis/liver cancer) in TCM nonusers was higher than that of TCM users (16.67% versus 1.56%). The long course of the disease and a family history of hepatitis B were the risk factors for disease progression, and long-term oral administration of TCM was the protective factor. As a result, the serum noninvasive fibrosis index and imaging parameters in TCM users were lower than those of TCM nonusers. Patients in the treatment of NAs combined with TCM had better prognoses such as a lower HBsAg level, a more stable lymphocyte function, and a lower incidence of end-point events. The present findings suggest the effect of TCM combined with NAs in the treatment of chronic hepatitis B liver fibrosis is better than that of single drug treatment. [ABSTRACT FROM AUTHOR]

Eggshell damages lead to economic losses in the egg production industry and are a threat to human health. We examined 49-wk-old Rhode Island White hens (Gallus gallus) that laid eggs having shells with significantly different strengths and thicknesses. We used HiSeq 2000 (Illumina) sequencing to characterize the chicken transcriptome and whole genome to identify the key genes and genetic mutations associated with eggshell calcification. We identified a total of 14,234 genes expressed in the chicken uterus, representing 89% of all annotated chicken genes. A total of 889 differentially expressed genes were identified by comparing low eggshell strength (LES) and normal eggshell strength (NES) genomes. The DEGs are enriched in calcification-related processes, including calcium ion transport and calcium signaling pathways as revealed by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Some important matrix proteins, such as OC-116, LTF and SPP1, were also expressed differentially between two groups. A total of 3,671,919 single-nucleotide polymorphisms (SNPs) and 508,035 Indels were detected in protein coding genes by whole-genome re-sequencing, including 1775 non-synonymous variations and 19 frame-shift Indels in DEGs. SNPs and Indels found in this study could be further investigated for eggshell traits. This is the first report to integrate the transcriptome and genome re-sequencing to target the genetic variations which decreased the eggshell qualities. These findings further advance our understanding of eggshell calcification in the chicken uterus.