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Cuticle deposition duration in the uterus is correlated with eggshell cuticle quality in White Leghorn laying hens

ABSTRACT: The cuticle formed in the uterus is the outermost layer as the first defense line of eggshell against microbial invasions in most avian species, and analyzing its genetic regulation and influencing factors are of great importance to egg biosecurity in poultry production worldwide. The current study compared the uterine transcriptome and proteome of laying hens producing eggs with good and poor cuticle quality (GC and PC, the top and tail of the cuticle quality distribution), and identified several genes involved with eggshell cuticle quality (ESCQ). Overall, transcriptomic analysis identified 53 differentially expressed genes (DEGs) between PC versus GC group hens, among which 25 were up-regulated and 28 were down-regulated. No differences were found in the uterine proteome. Several DEGs, including PTGDS, PLCG2, ADM and PRLR related to uterine functions and reproductive hormones, were validated by qPCR analysis. Egg quality measurements between GC and PC hens showed GC hens had longer laying interval between two consecutive ovipositions (25.64 ± 1.23 vs 24.94 ± 1.12 h) and thicker eggshell thickness (352.01 ± 23.04 vs 316.20 ± 30.58 μm) (P < 0.05). Apart from eggshell traits, other egg quality traits didn’t differ. The result demonstrated eggshell and cuticle deposition duration in the uterus is one of the major factors affecting ESCQ in laying hens. PTGDS, PLCG2, ADM and PRLR genes were discovered and might play crucial roles in cuticle deposition by regulating the uterine muscular activities and secretion function. The findings in the present study provide new insights into the genetic regulation of cuticle deposition in laying hens and establish a foundation for further investigations.

SUBMITTER: Chen X

PROVIDER: S-EPMC8586345 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Avian eggs have a proteinaceous cuticle. The quantity of cuticle varies and the deposition of a good cuticle in the uterus (Shell-gland) prevents transmission of bacteria to the egg contents. RESULTS:To understand cuticle deposition, uterus transcriptomes were compared between hens with i) naturally good and poor cuticle and, ii) where manipulation of the hypothalamo-pituitary-gonadal-oviduct axis produced eggs with or without cuticle. The highest expressed genes encoded eggshell matrix and cuticle proteins, e.g. MEPE (OC-116), BPIFB3 (OVX-36), RARRES1 (OVX-32), WAP (OVX-25), and genes for mitochondrial oxidative phosphorylation, active transport and energy metabolism. Expression of a number of these genes differed between hens laying eggs with or without cuticle. There was also a high expression of clock genes. PER2, CRY2, CRY1, CLOCK and BMAL1 were differentially expressed when cuticle deposition was prevented, and they also changed throughout the egg formation cycle. This suggests an endogenous clock in the uterus may be a component of cuticle deposition control. Cuticle proteins are glycosylated and glycosaminoglycan binding genes had a lower expression when cuticle proteins were deposited on the egg. The immediate early genes, JUN and FOS, were expressed less when the cuticle had not been deposited and changed over the egg formation cycle, suggesting they are important in oviposition and cuticle deposition. The uterus transcriptome of hens with good and poor cuticle deposition did not differ. CONCLUSIONS:We have gained insights into the factors that can affect the production of the cuticle especially clock genes and immediate early genes. We have demonstrated that these genes change their expression over the period of eggshell formation supporting their importance. The lack of differences in expression between the uterus of hens laying eggs with the best and worse cuticle suggest the genetic basis of the trait may lie outside the oviduct.

Project description:The decline in eggshell quality resulting from aging hens poses a threat to the financial benefits of the egg industry. The deterioration of eggshell quality with age can be attributed to changes in its ultrastructure and chemical composition. Specific matrix proteins in eggshells have a role in controlling crystal growth and regulating structural organization. However, the variations in ultrastructure and organic matrix of eggshells in aging hens remain poorly understood. This study assessed the physical traits, mechanical quality, chemical content, as well as the microstructural and nanostructural properties of eggs from Jing Tint 6 hens at 38, 58, 78, and 108 wk of age. Subsequently, a quantitative proteomic analysis was conducted to identify differences in protein abundance in eggshells between the ages of 38 and 108 wk. The results indicated a notable decline in shell thickness, breaking strength, index, fracture toughness, and stiffness in the 108-wk-age group compared to the other groups (P < 0.05). The ultrastructure variations primarily involved an increased ratio of the mammillary layer and a reduced thickness of the effective layer of eggshell in the 108-wk-age group (P < 0.05). However, no significant differences in eggshell compositions were observed among the various age groups (P > 0.05). Proteomic analysis revealed the identification of 76 differentially expressed proteins (DEPs) in the eggshells of the 38-wk-age group and 108-wk-age group, which comprised proteins associated with biomineralization, calcium ion binding, immunity, as well as protein synthesis and folding. The downregulation of ovocleidin-116, osteopontin, and calcium-ion-related proteins, together with the upregulation of ovalbumin, lysozyme C, and antimicrobial proteins, has the potential to influence the structural organization of the eggshell. Therefore, the deterioration of eggshell quality with age may be attributed to the alterations in ultrastructure and the abundance of matrix proteins.

Project description:Calcium (Ca) and phosphorus (P) transporters are responsible for their absorption and transport in small intestine and kidney, contributing to eggshell formation. The light-dark cycle is a primary cue in the reproduction of laying hen. In this study, we investigated the effect of different light-dark programs on eggshell quality and the expression of genes related to Ca and P transportation in laying hens. Seventy-two 56-week-old laying hens were randomly divided into two groups and reared at 16-h light and 8-h dark (control) or 9-h light and 15-h dark regime (long dark phase, LDP). The expressions of calcium transporter calbindin-D28k (CaBP-D28k), plasma membrane Ca ATPase 1b (PMCA1b), and phosphorus transporter NaPi-IIb (NPt2b) and NaPi-IIa (NPt2a) were measured in the small intestine, kidney, and eggshell gland. The results showed that feed intake (P < 0.001) and egg weight (P = 0.05) were decreased by LDP treatment. Compared with control, the eggshell hardness was increased (P = 0.011) by LDP treatment, but the eggshell thickness and the percentage of eggshell were not changed. The Ca and P contents in eggshell were increased by LDP treatment. During the scotophase, LDP-hens showed higher serum Ca (P = 0.0056) and P levels (P = 0.079) but lower alkaline phosphatase (ALP) activity than that of control hens. In the duodenum, the relative higher expression of CaBP-D28k and PMCA1b in scotophase compared to photophase was masked by LDP treatment. The expression of CaBP-D28k and osteopontin (OPN) in the eggshell gland were increased by LDP treatment, compared to control hens. In the jejunum, the protein expression levels of CaBP-D28k and PMCA1b decreased during photophase in LDP-hens. The result indicates that the increased blood Ca and P concentration during scotophase by LPD treatment is beneficial to the deposition of Ca and P in the eggshell. The result offers an alternative strategy for managing laying hens with poor eggshell quality.

Project description:Insufficient calcium supply during the dark period is an important reason for deteriorated eggshell quality in laying hens. In the present study, the feeding time of hens was altered in order to investigate whether the changes in feeding time and feed consumption could influence the laying performance and eggshell quality of hens. A total of 192, 60-wk-old Hy-line Brown hens with similar body weight and laying rate were obtained. The hens were randomly divided into 4 groups and subjected to the following feeding strategies: feeding 3 times a day (control group, CON), or feeding once a day in the morning at 08:00 (MF), in the noon at 12:00 (NF), or in the afternoon at 16:00 (AF), respectively. The feeding strategies had no significant effect (P > 0.05) on laying rate, egg weight, and egg mass. Although the feed intake did not differ among treatments, the time phase of feed consumption was changed. From 15:00 to 21:00 h, hens consumed 49.7%, 42.4%, 49.1%, and 70.8% of daily feed intake in the CON, MF, NF, and AF groups, respectively. Feeding strategy had no detectable influence (P > 0.05) on egg shape index, eggshell strength, and eggshell percentage. Compared to CON, AF hens tended to have a higher eggshell thickness (P = 0.053). In MF and NF treatments, plasma calcium (Ca), phosphorus (P) levels, and alkaline phosphatase (ALP) activity did not differ (P > 0.05) compared with CON. In contrast, AF-hens had lower Ca and P levels, but a higher ALP activity than CON (P < 0.01). The AF hens had higher uterine fluid Ca than MF and NF hens (P < 0.05). Compared to CON, the expression level of CaBP-D28K was increased in the shell gland mucosa of MF-hens. Also, MF-, NF-, and AF-hens had higher Osteopontin (OPN) expression level (P < 0.05), whereas NF had a higher expression of OC-116 (P < 0.01). In conclusion, the results indicated that feeding in the afternoon changed the pattern of feed consumption and exerted a positive influence on eggshell thickness.

Project description:BackgroundIn this study, we explored the characteristics and causes of freckle formation. We collected 15 normal and freckled eggs each for eggshell index testing and hypothesized that the structure and function of the uterus would have a direct effect on freckled egg production given that eggshells are formed in the uterus. To test this hypothesis, we collected uterine tissue from laying hens (418 days of age) that laid normal (Group C, n = 13) and freckled (Group T, n = 16) eggs for 7 consecutive days.ResultsWhen we examined the eggshell quality, we found that the L value was significantly lower (P < 0.05) in the freckled site group of freckled eggs compared to the normal egg group during the detection of blunt pole, equator, and sharp pole of the eggshell color. The a-values of the three positions were significantly higher (P < 0.05) in the freckled site group of freckled eggs, and the a-values of the blunt pole were significantly lower (P < 0.05) in the background site group of freckled eggs, compared to the normal egg group. The b-values were significantly higher (P < 0.05) at three locations in the freckled site group of freckled eggs compared to the normal egg group. During the detection of eggshell thickness, the blunt pole was significantly higher (P < 0.05) in the freckled egg site group of freckled eggs compared to the normal egg group, and there was no significant difference between the other groups (P > 0.05). There was no significant difference (P > 0.05) between the transverse and longitudinal diameters of the eggs in each group.We then performed histopathology and transcriptome analyses on the collected tissue. When compared with group C, uterine junctional epithelial cells in group T showed significant defects and cilia loss, and epithelial tissue was poorly intact. From transcriptomics, genes that met (|log2FC|) ≥ 1 and P < 0.05 criteria were screened as differentially expressed genes (DEGs). We identified a total of 136 DEGs, with 101 up- and 35 down-regulated genes from our RNA-seq data. DEGs identified by enrichment analyses, which were potentially associated with freckled egg production were: IFI6, CCL19, AvBD10, AvBD11, S100A12, POMC, and UCN3. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that pathways were associated with immunoreaction and stress stimulation, e.g., complement activation, interleukin-1 cell reactions, viral responses, cell reactions stimulated by corticotropin releasing hormone, steroid hormone mediated signaling pathways, staphylococcal infections, B cell receptor signaling pathways, and natural killer cell mediated cytotoxicity.ConclusionsFrom these data, freckled areas deepen freckled eggshell color, but background areas are not affected. At the same time,we reasoned that freckle eggs may result from abnormal immune responses and impaired uterine functions induced by stress. Therefore, the uterus of laying hens in a state of stress and abnormal immune function can cause the appearance of freckled eggs.

Project description:Eggshell is composed of a very ordered and mineralized structure and is important for egg quality. Eggshell strength is particularly important because of its direct association with economic outcomes and egg safety. Various factors related to laying hens and their environment affects eggshell strength. However, the molecular mechanisms of liver functions related to decreased eggshell strength of aged laying hens are largely unknown. Therefore, this study aimed to identify potential factors affecting eggshell strength in aged laying hens at the hepatic transcriptomic level. A total of five hundred 92-wk-old Hy-line Brown laying hens were screened to select those exhibiting the greatest variation in eggshell strength. Based on the final eggshell strength, 12 hens producing eggs with strong eggshell strength (SES) and weak eggshell strength (WES) were finally selected (n = 6) for liver tissue sampling. The RNA-sequencing was performed to identify differentially expressed genes (DEGs) between the 2 groups. We identified a total of 2,084 DEGs, of which 1,358 genes were upregulated and 726 genes were downregulated in the WES group compared with SES group. According to the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, the DEGs indicated the mammalian target of rapamycin signaling pathway, the Janus kinase-signal transducer and activator of transcription pathway, the mitogen‑activated protein kinase signaling pathway, and the insulin resistance pathways. Genes related to fatty liver disease were upregulated in WES group compared with SES group. In addition, expression of several genes associated with oxidative stress and bone resorption activity was altered in aged laying hens with different eggshell strength. Overall, these findings contribute to the identification of genes involved in different intensity of eggshell strength, enabling more understanding of the hepatic molecular mechanism underlying in decreased eggshell strength of aged laying hens.

Project description:BackgroundFloor eggs, which are defined as eggs that hens lay off-nest, are a major issue in cage-free layer poultry systems. They create additional work for farmers because they must be collected by hand. They are also usually soiled or broken, which results in economic losses. Nonetheless, knowledge about the genetics of nesting behavior is limited. The aim of this study was to estimate genetic parameters for traits related to nest preference for laying and to time spent in the nests used for laying (laying duration).MethodsTwo pure lines of laying hens were studied: 927 Rhode Island Red and 980 White Leghorn. Electronic nests were used to record the nesting behavior of these hens in floor pens from 24 to 64 weeks of age. Nest preference was studied based on the mean distance between nests used for laying and the percentage of nests used for laying. Laying duration was studied based on mean laying duration, mean duration in the nest before laying, and mean duration in the nest after laying. Genetic parameters were estimated for each line using a restricted maximum-likelihood method applied to a pedigree-based multi-trait animal model.ResultsEstimates of genetic parameters were similar for the two lines. Estimates of heritability ranged from 0.18 to 0.37 for nest preference traits and from 0.54 to 0.70 for laying duration traits. Estimates of genetic correlations of these traits with clutch number or mean oviposition time were favorable. Positive genetic correlations were estimated between nest preference and laying rate in the nests or nest acceptance for laying (+ 0.06 to + 0.37).ConclusionsThese results show that genetics influences traits related to nest preference and laying duration. Selecting hens that have no preference for particular nests and spend little time laying in the nests could help optimize nest use, reduce their occupation rate, and thus decrease the incidence of floor eggs in cage-free systems. Genetic correlations of these traits with other traits of interest related to hen welfare and egg quality have yet to be estimated.

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Cuticle deposition duration in the uterus is correlated with eggshell cuticle quality in White Leghorn laying hens

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