Nine experiments were performed to investigate how the synthesis and/or deposition of protoporphyrin IX (PP IX) into eggshell is influenced in brown-egg laying hens. The findings obtained in the first experiment (Chapter 2) showed that flock age and production system affected overall eggshell and egg quality. Egg weight was significantly higher in cage eggs, while albumen height was significantly higher in barn eggs. The mammillary layer ultrastructural variables showed no clear relationship with production system and flock age. Cuticle cover (ΔE*ab) was significantly higher in barn eggs compared with free range and cage eggs and was significantly higher in eggs from the 44 week old flock than for 64 and 73 week old flocks. In 1 gram of eggshell with and without cuticle, there was more PP IX in cage eggs followed by free range and barn eggs. The findings in experiment 2 (Chapter 3) indicate that eggs laid earlier in the day had deeper brown eggshell colour compared with the eggs laid later in the day. Egg position in a clutch had a clearer effect on eggshell quality in long clutches, as compared with medium and short clutches. The findings of the third experiment (Chapter 4) showed that different infectious bronchitis virus (IBV) strains affected the level of PP IX in eggshell differently. In unvaccinated laying hens, the mean PP IX per gram of shell was significantly higher on day 1 post-infection (p.i.) compared to day 7, after which PP IX increased with day p.i. In unvaccinated and vaccinated laying hens, PP IX decreased with increased day p.i. until day 12. The effect on loss of shell colour was more marked in the T strain infected group followed by N1/88, Vic S and A3 strains. Experiment four (Chapter 5) investigated reference gene stability in the shell gland in relation to time-points (time post-oviposition) of eggshell formation and nicarbazin treatment. The two most stable reference genes selected, HPRT1 and HMBS, were used for the normalisation of gene expression data obtained in experiment five (Chapter 6). The findings in experiment five showed that mitochondria per cell did not vary significantly with different time-points of eggshell formation and nicarbazin treatment. Genes involved in the synthesis of PP IX were regulated differentially in relation to time-points of eggshell formation. Feeding nicarbazin caused down-regulation of the ALAS1 gene that resulted in lower production of PP IX appearing in the shell gland tissue and eggshell. Experiment six (Chapter 7) investigated reference gene stability in the shell gland and spleen of infectious bronchitis virus (IBV) infected hens. The two most stable reference genes selected, TBP and YWHAZ, were used for the normalisation of gene expression data obtained in experiments seven and eight (Chapters 8 and 9). The RNA-sequencing findings in experiment seven showed that there were no differentially expressed genes (DEGs) involved in the mucosal immune system and eggshell formation in the shell gland between IBV challenged and control laying hens. However, there were 1608 and 1806 DEGs at 5 and 15 hrs time-points of eggshell formation, respectively. The Gene Ontology (GO) terms and functional gene analysis showed that the DEGs at 5 hr post-oviposition were mainly involved in ion transport and synthetic activities, while the DEGs at 15 hr were involved in energy metabolism and secretory activities, reflecting the peak stage of eggshell formation. The findings in experiment eight (Chapter 9) showed that IBV T infection significantly lowered mitochondrial count per cell in the shell gland region of the oviduct but not in the magnum or isthmus. The expression levels of nuclear DNA encoded genes involved in mitochondrial biogenesis and fission showed no clear correlation with mitochondrial count and were not significantly different between the control and challenged samples. The expression levels of all the genes except for PGC-1α were significantly affected by the time-points of eggshell formation. Experiment nine (Chapter 10) attempted to localize ALAS1, ALAD and FECH enzymes in shell gland tissue collected at different time-points (time post-oviposition) and in response to nicarbazin treatment. The findings showed that these antibodies did not recognise their respective proteins, possibly due to their amino acid sequences being derived from humans. PP IX fluorescence was not detected in Zenker Formol fixed tissue sections, stained or unstained, taken at different time-points and with or without nicarbazin treatment. Future work is suggested using chicken specific antibodies to localise cells involved in PP IX synthesis. Taken together, the findings in the current study broaden the understanding of factors affecting the synthesis and deposition of PP IX into eggshells.