Age-associated changes in the shell gland transcriptomics and eggshell quality of broiler breeder hens

The egg production cycle of broiler breeder hens is comparatively shorter than that of laying hens, and as they age, their egg productivity and eggshell quality decline. Eggshell formation occurs in the shell gland, which is influenced by several factors, including aging. The objectives of this study were to 1) determine the age-associated changes in eggshell quality and mineral compositions, and 2) identify differentially expressed genes (DEGs) and biological pathways in the shell glands (young vs aged). The shell gland 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”) at 10–15 h post-ovulation (uncalcified 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 quantitative 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. The strength, thickness, and palisade layer of the eggshells were significantly greater in younger hens, while the mammillary knobs were wider in older hens. Additionally, the levels of sulfur and potassium in the eggshells were higher in the younger hens compared to the older ones. Although the young hens exhibited a higher concentration of calcium in their eggshells, the difference was not statistically significant. RNA-Seq data identified highly up-regulated genes in young hens specific to eggshell biomineralization, such as SPP1 (binds to hydroxyapatite), OTOP2 (maintains high concentration of cytosolic Ca2+), PKD2 (helps in releasing Ca2+), SLC22A15 (transports organic ions), and STAB2 (binds to gram-positive and gram-negative bacteria), which are downregulated in aged hens. The DEGs showed significant enrichment for biological pathways associated with the transport of monoatomic and inorganic cations across the cell membrane (SLC6A6, KCNK7, UCP3, SCNN1A, PKD2, OTOP2), 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 aging downregulates the shell gland transcriptomes involved in eggshell biomineralization, reducing ion transport required for eggshell formation and resulting in poor eggshell quality.