Dataset Information

Reference gene selection for the shell gland of laying hens in response to time-points of eggshell formation and nicarbazin.

ABSTRACT: Ten reference genes were investigated for normalization of gene expression data in the shell gland of laying hens. Analyses performed with geNorm revealed that hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS) were the two most stable reference genes in response to post-oviposition time alone (POT) or with nicarbazin treatment (POT+N) of laying hens. NormFinder analyses showed that the two most stable reference genes in response to POT and POT+N were 18S ribosomal RNA (18S rRNA), ribosomal protein L4 (RPL4) and HMBS, RPL4, respectively. BestKeeper analyses showed that 18S rRNA, RPL4 and HPRT1, HMBS were the two most stable reference genes for POT, and POT+N, respectively. Of the ten reference genes, all except B2M showed geNorm M <0.5, suggesting that they were stably expressed in the shell gland tissue. Consensus from these three programs suggested HPRT1 and HMBS could be used as the two most stable reference genes in the present study. Expression analyses of four candidate target genes with the two most and the two least stable genes showed that a combination of stable reference genes leads to more discriminable quantification of expression levels of target genes, while the least stable genes failed to do so. Therefore, HMBS and HPRT1 are recommended as the two most stable reference genes for the normalization of gene expression data at different stages of eggshell formation in brown-egg laying hens. Available statistical programs for reference gene ranking should include more robust analysis capability to analyse the gene expression data generated from factorial design experiments.

SUBMITTER: Samiullah S

PROVIDER: S-EPMC5495395 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Reference gene selection for the shell gland of laying hens in response to time-points of eggshell formation and nicarbazin.

Samiullah Sami S Roberts Juliet J Wu Shu-Biao SB

PloS one 20170703 7

Ten reference genes were investigated for normalization of gene expression data in the shell gland of laying hens. Analyses performed with geNorm revealed that hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS) were the two most stable reference genes in response to post-oviposition time alone (POT) or with nicarbazin treatment (POT+N) of laying hens. NormFinder analyses showed that the two most stable reference genes in response to POT and POT+N were 18S ri ...[more]

PMID: 28671969

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Project description:In this study, we identified cadmium (Cd) as a potential endocrine disruptor that impairs laying performance, egg quality, and eggshell deposition and induces oxidative stress and inflammation in the eggshell glands of laying hens. A total of 480 38-wk-old laying hens were randomly assigned into 5 groups that were fed a basal diet (control) or a basal diet supplemented with Cd (provided as CdCl2·2.5 H2O) at 7.5, 15, 30, and 60 mg Cd per kg feed for 9 wk. The results showed that, when compared with the control group, a low dose of dietary Cd (7.5 mg/kg) had positive effects on egg quality by improving albumen height, Haugh unit, yolk color, and shell thickness at the third or ninth week. However, with the increase in the dose and duration of Cd exposure, the laying performance, egg quality, and activities of eggshell gland antioxidant enzymes (catalase [CAT], glutathione peroxide [GSH-Px]), and ATPase (Na+/K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase) deteriorated, and the activity of total nitric oxide synthase (T-NOS) and the level of malondialdehyde (MDA) increased significantly (P < 0.05). The histopathology and real-time quantitative PCR results showed that Cd induced endometrial epithelial cell proliferation accompanied by upregulation of the mRNA levels of progesterone receptor (PgR) and epidermal growth factor receptor (EGFR), downregulation of the mRNA levels of estrogen receptor α (ERα) and interleukin 6 (IL6), and inflammation of the eggshell gland accompanied by significantly increased expression of complement C3 and pro-inﬂammatory cytokine tumor necrosis factor α (TNFα) (P < 0.05). In addition, the ultrastructure of the eggshell showed that dietary supplementation with 7.5 mg/kg Cd increased the palisade layer and total thickness of the shell, but with the increase in dietary Cd supplementation (30 and 60 mg/kg) the thickness of the palisade layer and mammillary layer decreased significantly (P < 0.05), and the outer surface of the eggshell became rougher. Correspondingly, the expression of calbindin 1 (CALB1), ovocalyxin-32 (OCX-32), ovocalyxin-36 (OCX-36), osteopontin (SPP1), and ovocledidin-17 (OC-17) decreased significantly (P < 0.05) with increasing dietary Cd supplementation. Conclusively, the present study demonstrates that dietary supplementation with Cd negatively affects laying performance, egg quality, and eggshell deposition by disturbing the metabolism of eggshell glands in laying hens but has a positive effect on egg quality at low doses.

Project description:BackgroundEggshell formation takes place in the shell gland of the oviduct of laying hens. The eggshell is rich in calcium and various glycoproteins synthesised in the shell gland. Although studies have identified genes involved in eggshell formation, little is known about the regulation of genes in the shell gland particularly in a temporal manner. The current study investigated the global gene expression profile of the shell gland of laying hens at different time-points of eggshell formation using RNA-Sequencing (RNA-Seq) analysis.ResultsGene expression profiles of the shell gland tissue at 5 and 15 h time-points were clearly distinct from each other. Out of the 14,334 genes assessed for differential expression in the shell gland tissue, 278 genes were significantly down-regulated (log2 fold change > 1.5; FDR < 0.05) and 413 genes were significantly up-regulated at 15 h relative to the 5 h time-point of eggshell formation. The down-regulated genes annotated to Gene Ontology (GO) terms showed anion transport, synaptic vesicle localisation, organic anion transport, secretion and signal release as the five most enriched terms. The up-regulated gene annotation showed regulation of phospholipase activities, alanine transport, transmembrane receptor protein tyrosine kinase signalling pathway, regulation of blood vessels diameter and 3, 5-cyclic nucleotide phosphodiesterase activity as the five most enriched GO terms. The putative functions of genes identified ranged from calcium binding to receptor activity. Validation of RNA-Seq results through qPCR showed a positive correlation.ConclusionsThe down-regulated genes at 15 h relative to the 5 h time-point were most likely involved in the transport of molecules and synthesis activities, initiating the formation of the eggshell. The up-regulated genes were most likely involved in calcium transportation, as well as synthesis and secretory activities of ions and molecules, reflecting the peak stage of eggshell formation. The findings in the current study improve our understanding of eggshell formation at the molecular level and provide a foundation for further studies of mRNA and possibly microRNA regulation involved in eggshell formation in the shell gland of laying hens.

Project description:Chicken eggshell (ES) waste is a rich source of calcium carbonate (CaCO3); however, the potential of ES as dietary calcium (Ca) in old laying hens has not been explored. This study compared the effects of feeding limestone, cockle shell, oyster shell, fine ES, and coarse ES as the sole Ca source on production performance, egg quality, blood biochemical constituents, and tibia characteristics in old laying hens. A total of 450 ISA-Brown laying hens at 73 wk of age with similar egg production rate (EPR) were randomly assigned to 5 treatment groups (90 hens/group, 9 hens/replicate) for 7 wk. Dietary treatment groups comprised a corn-soybean meal based diet containing different Ca sources: (i) limestone (LS; < 2 mm and 2-4 mm mixed in the ratio of 3:7) as control, (ii) cockle shell (CS; 1-4 mm), (iii) oyster shell (OS; 3-16 mm), (iv) ES fine particles (ESF; < 1 mm), and (v) ES coarse particles (ESC; 3-5 mm). Results indicated that dietary inclusion of coarse ES particles significantly increased average egg weight (P < 0.001) and daily egg mass (P < 0.05), and decreased feed conversion ratio (P < 0.001) as compared with the other treatments. However, no significant differences in EPR, feed intake, cracked egg proportion, and mortality were observed among the dietary treatments (P > 0.05). Notably, the use of ESF led to a lower proportion of cracked eggs than ESC (P < 0.05). ESC fed hens produced the heaviest eggs whereas CS fed hens produced the lightest (P < 0.001); the particle size of ES also affected the egg weight (P < 0.05). The eggs from OS and ESC fed hens showed a greater albumen height in comparison to eggs from CS group (P < 0.05); but no significant difference was observed among the LS, OS, ESF, and ESC groups (P > 0.05). The yolk color was darker in the eggs of group ESF as compared with other dietary groups (P < 0.01). However, no significant effects on Haugh units and shell properties were observed among the treatments (P > 0.05). The blood biochemistry results were not affected by the dietary Ca (P > 0.05) except for lower levels of high-density lipoprotein percentage (HDL %) in OS and ESC fed hens (P < 0.05). The tibia characteristics including weight, length, width, and breaking strength did not differ among the dietary groups (P > 0.05). However, the ESC and OS fed hens showed higher tibia bone mineral density (BMD) than the other groups (P < 0.001). In conclusion, coarse ES as a sole Ca source had beneficial effects on the production performance, egg quality, and tibia BMD in old laying hens.

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:This study evaluated the changes in eggshell mechanical properties, ultrastructure, calcium metabolism-related serum indices, and gene expression in eggshell gland during eggshell formation between laying ducks in the peak (young duck) and late phase (aged duck) of production. A total of 84 healthy young (31 wk of age) and 84 healthy aged (65 wk of age) Longyan laying ducks were each divided into 6 replicates of 14 birds, and caged individually. All the ducks were fed in one house with the same corn-soybean meal-based diet for 5 wk. The eggshell mechanical properties (shell proportion, thickness, breaking strength, and fracture toughness) and chemical components (matrix proteins, calcium, phosphorus, and magnesium) decreased in aged laying ducks (P < 0.05). Shell structural indices: total thickness, effective thickness and its proportion decreased, whereas mammillary knob width and its proportion increased (P < 0.05). The regulation values of early fusion, cuffing, caps, and total score of mammillary knobs were higher in aged laying ducks relative to the young ducks (P < 0.05). During the initial, growth and terminal stages of eggshell formation, shell thickness and breaking strength (terminal), shell weight, and its proportion (terminal) decreased in aged laying ducks (P < 0.05). Ultrastructural changes during shell formation indicated that the mammillary-knob density and effective thickness decreased (P < 0.05). Decreases occurred in serum content of phosphorus (growth), and estradiol and calcium contents (terminal) (P < 0.05). Relative expression of Ca2+ transporter and HCO3- exchanger, and matrix proteins genes decreased in aged laying ducks (P < 0.05) at all stages of eggshell formation. Collectively, the decreased incidence of early fusion and caps, increased thickness and width of mammillary knobs, and decreased effective thickness are the crucial differences leading to the compromised mechanical properties of eggshell in the late laying period. A disturbed regulation of calcium metabolism and uterine expression of ion transporters, especially for HCO3- exchange of aged laying ducks likely contribute to age-induced ultrastructural deterioration of the eggshell.

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.

Dataset Information

Reference gene selection for the shell gland of laying hens in response to time-points of eggshell formation and nicarbazin.

Publications

Reference gene selection for the shell gland of laying hens in response to time-points of eggshell formation and nicarbazin.

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