Project description:The egg production cycle of broiler breeder hens is comparatively shorter than laying hens, and as they age, their egg production and eggshell quality decline. The eggshell formation occurs in the shell glands, which are influenced by several factors, including aging. The objectives of the study were to 1) identify differentially expressed genes (DEGs) and biological pathways in the shell glands (young vs aged) and 2) determine the age-associated changes in eggshell quality. The shell glands tissues were collected from broiler breeder hens at peak-lay (35 weeks of age; termed as “young”) and late-lay phases (50 weeks of age; termed as “aged”) (n=30/group) at 10-15 hours post-ovulation (unclassified egg present in the shell glands). To delineate the genes and biological pathways associated with eggshell biomineralization, total RNAs extracted from the shell glands of young and aged hens (n=6/group) were analyzed using RNA sequencing and validated using real-time PCR. The ultrastructure quality of eggshells (n=10 eggs/group) was analyzed using a Scanning Electron Microscope (SEM), and the elemental composition of eggshells was measured using SEM-Energy Dispersive Spectrometry, and their variability was confirmed by t-test in RStudio. Eggshell strength, thickness, palisade layer, and mammillary width were significantly higher in the young, while mammillary knobs were wider in aged hens (p<0.05). The sulfur and potassium levels in eggshells were higher in young hens than aged ones. Although the young group had a higher calcium concentration in the eggshells, the difference was statistically insignificant (p>0.05). RNA-Seq data identified highly upregulated genes specific to eggshell biomineralization, such as SPP1 (binds to hydroxyapatite), OTOP2 (maintains high conc. of cytosolic Ca2+), PKD2 (helps in releasing Ca2+), SLC22A15 (transports organic ions), and STAB2 (binds to gram-positive and gram-negative bacteria). The DEGs showed significant enrichment for biological pathways (SLC6A6, KCNK7, UCP3, SCNN1A, PKD2, OTOP2) associated with the transport of monoatomic and inorganic cations across the cell membrane, molecular functions related to the transport of potassium ions and the activity of monoatomic cation channels (KCNK7, PKD2, OTOP2), and the cellular components involved in the luminal side of the endoplasmic reticulum membrane (CALR, PKD2). These findings suggest that the aging process downregulates the transcriptomes of the shell glands, negatively impacting the transportation of ions required for eggshell formation, resulting in poor eggshell quality.

Project description:Purpose: With the advent of Next-generation sequencing (NGS), several novel genes/proteins and cellular pathways in wide variety of tissues has been discovered. The aim of this study are to perform uterine transcriptome profiling (RNA-seq) to determine differently expressed genes in laying and non-laying hens and to further validate the expression of candidate genes using real-time quantitative reverse transcription polymerase chain reaction (qRT–PCR) in laying, non-laying and molting hens. Methods: Uterine mRNA profiles of 35-60 weeks-old laying and non-laying hens, three each, were generated with NextSeq 500 sequencer in single-end mode with a read length of 1x76 bp. Raw sequencing reads were cleaned and trimmmed with Prinseq tool and good reads were aligned against the chicken reference gemone (Galgal 5.0) in Array Studio. Differential gene expression analysis was performed by the DESeq2 algorithm as implemented in Array Studio. The genes with at least two-fold change (FC) and Benjamini and Hochberg q-value < 0.05 were called differentially expressed. Results: Using an optimized data analysis workflow, we mapped about 32 million reads from layers and 28 million reads from non-layers to the chicken genome. A total of 19,152 gene transcripts were annotated from Ensembl alignment which represents 50.24% of the chicken genome assembly. Differential gene expression analysis showed 616 were differentially expressed between layer and non-layer hens. 229 DEGs were significantly up-regulated and 286 were significantly down-regulated in the laying hens when compared to the non-laying hens. Twelve candiate genes, linked to calcium remodeling, were identified by gene function analysis and validated using qPCR. MEPE, CALCB, OTOP2, STC2 and ATP2C2 were confirmed to be highly expressed in laying hens as compared to molting and non-laying hens. RNA-seq and qPCR data for relative gene expression were highly correlated (R2 =0.99). Conclusions: Our study reports the expression of four novel genes that are speculated to transport calcium ions across the uterine epithellium for eggshell mineralization. These genes can be used as quantitative basis of selecting hens with an improved eggshell quality.

Dataset Information

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets