Juliet Roberts

Egg formation takes place in the oviduct of laying hens over a 24 h period. Infectious bronchitis virus (IBV) causes pathological lesions in the chicken oviduct. In the current study, mitochondrial counts were determined in three different segments of the oviduct during egg formation in laying chickens challenged with IBV T strain. Nuclear DNA encoded genes that are involved in mitochondrial biogenesis, fission and function were studied in the shell gland of the oviduct undergoing virus multiplication. In the shell gland, the mitochondrial count was significantly lower (P < 0.05) in the challenged group, compared with the control group. However, it did not vary in response to IBV challenge in the isthmus and magnum regions of the oviduct. The gene succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA) was down-regulated in the shell gland by IBV challenge (P < 0.05), while other genes being studied did not show responses to the challenge (P > 0.05). Differential expression of the genes was observed at different time-points of egg-shell formation. The expression levels of citrate synthase (CS), cytochrome C, somatic (CYC, S) and sodium-potassium adenosine triphosphatase (Na+-K+ATPase) genes were significantly higher, while those of SDHA and dynamin related protein 1 (Drp1) genes were significantly lower, at 15 h compared with 5 h following oviposition of the previous egg. The expression level of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) did not show significant change at different time-points. It was concluded that IBV T strain infection in laying hens reduced mitochondrial counts only in the shell gland region of the oviduct. The genes involved in mitochondrial biogenesis or function may not show synchronised responses to that of mitochondria in the shell gland of chickens under T strain of IBV challenge.

Eggshell 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. Gene 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. The 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.

The cuticle is the outermost layer, deposited on the palisade layer of the eggshell during the last 1.5-2 hr of eggshell formation in utero. It is a non-calcified, thin, water-insoluble layer composed mainly of glycoproteins with some carbohydrate and fat constituents. The cuticle functions as a protective layer which regulates gaseous exchange across the shell, acts as a first line of defence against microbial penetration across the eggshell and is associated with termination of calcite crystal growth during shell formation. The extent of cuticle deposition is influenced by hen age and strain. The physiological and microbiological characteristics of the hen eggshell cuticle are reviewed in relation to its protective functions.

Eggs were collected from commercial caged layer flocks in early, mid, late and very late lay. Eggs were candled and scored for translucency. Cuticle cover was estimated using MST Cuticle Stain and a Konica Minolta hand-held spectrophotometer. Traditional measures of egg quality were determined using specialised equipment. Shell ultrastructural features were scored following plasma ashing of shell samples and viewing under a benchtop scanning electron microscope. Translucency score was significantly higher in late lay than for all other age groups. Shell quality declined with increasing flock age. However, the extent of cuticle cover on the egg shell was not significantly different among flock age groups. The incidence of shell ultrastructural features associated with good quality shells was lower for older flocks and incidence of ultrastructural features associated with poorer quality shells was higher for older flocks. Translucency score had a low correlation with the ultrastructural features of the mammillary layer.