2017, Samiullah, Samiullah, Roberts, Julie R, Wu, Shubiao

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.

2016, Samiullah, Samiullah, Roberts, Julie R, Chousalkar, Kapil

The aim of the current study was to assess any effect of wild and vaccine Australian infectious bronchitis virus (IBV) strains on shell colour in brown-shelled eggs. In Experiment 1, eggs were collected from day 1 to day 13 post-inoculation (p.i.) from unvaccinated laying hens challenged with IBV wild strains T and N1/88 and from a negative control group of hens. In Experiment 2, eggs were collected from 2 to 22 days p.i. from unvaccinated and vaccinated laying hens challenged with either a wild or a vaccine strain of IBV. In Experiment 1, there was a significant effect (P < 0.05) of day p.i. and of viral strain on shell reflectivity, L* and protoporphyrin IX (PP IX) in eggshells, with and without cuticle. The mean PP IX/g of shell with and without cuticle was significantly higher on day 1 p.i. compared to day 7, after which PP IX increased with day p.i. In Experiment 2, shell reflectivity and L* increased and PP IX decreased with increased day p.i. until day 12. Shell reflectivity and L* decreased slightly after day 12 and increased again towards day 22. Shell reflectivity, L* and PP IX were not significantly different for eggshells from unvaccinated and vaccinated laying hens in the intact eggshell, but were significantly different in shells from which cuticle had been removed. In conclusion, the IBV strains reduced the intensity of brown shell colour to different extents with a lower amount of PP IX in eggshells.

2016, Samiullah, Samiullah, Omar, Amal Saleh, Roberts, Julie, Chousalkar, Kapil

Egg quality was measured in eggs from different flocks that were reared together and then allocated to different production systems. Eggs were processed for measurements of eggshell and egg internal quality variables, scoring of ultrastructural mammillary layer features, completeness of cuticle cover, and protoporphyrin IX (PP IX) quantification. There was a significant main effect (P < 0.05) of production system on shell reflectivity, egg weight, and egg internal quality and significant effects of flock age on most measurements. The mammillary layer ultrastructural variables showed no clear relationship with production system and flock age. However, there was a significant interaction between production system and flock age for mammillary cap, early and late fusions. Cuticle cover ( ΔE∗ab ), was significantly higher in barn eggs (19.20), followed by free range (17.57), and cage eggs (15.99). Completeness of cuticle cover was significantly higher in eggs from the 44 week old flock than for 64 week and 73 week old flocks. For eggshells with cuticle intact, there was a significant main effect of both production system and flock age, and significant interaction between the two, for shell reflectivity, L*a*b* values and amount of PP IX. For PP IX, when this difference was calculated for the cuticle alone, there were no statistically significant differences. In 1 g of shell with and without cuticle, there was more PP IX in cage eggs (9.49 × 10−8, 7.90 × 10−8 mM) followed by free range (8.24 × 10−8, 6.90 × 10−8 mM), and barn eggs (8.64 × 10−8, 7.28 × 10−8 mM). Similar trends were recorded for the amount of PP IX in 1 g of cuticle, but the difference was not statistically significant. The amount of PP IX decreased significantly with increasing flock age. Comparing the cage and barn production systems at 68 week of flock age, there was no difference for the amount of PP IX in shell with or without cuticle, or in the cuticle alone. Eggs from the cage production system were darker in color and contained more PP IX mainly within the calcareous part of the shell. For the barn production system, the completeness of cuticle cover was higher and egg weight generally lower.

2013, Samiullah, Samiullah, Roberts, Julie R

Protoporphyrin has been identified as the main eggshell pigment in brown-shelled eggs. However, there has been some uncertainty concerning the distribution of the pigment within the shell (and cuticle) in brown-shelled eggs. Most previous studies have suggested that the bulk of the shell pigment is deposited in the cuticle of the shell. The present study measured the levels of protoporphyrin in intact eggshells and in shells from which the cuticle had been removed, using eggs from flocks at 3 different ages. This enabled the calculation of the relative amount of protoporphyrin in the calcareous eggshell and the cuticle layer of the eggshell. The majority of the protoporphyrin pigment was located in the calcareous part of the eggshell (80-87%) with a minority contained within the cuticle (13-20%). These findings suggest that studies focused on maintenance of shell color in brown-shelled eggs need to consider the stage of egg formation at which the reduction in pigment deposition is occurring.

2013, Samiullah, Samiullah, Roberts, Julie R

Eggs were collected from Hy-Line brown flocks aged 33, 50 and 67 wk. Thirty eggs from each flock were analyzed to determine the reliability of MST (MS Technologies, U.K.) cuticle blue stain as an indicator of the presence of cuticle and the effective removal of cuticle by use of an EDTA solution. Another 30 eggs, collected at the same time from each flock, were processed for the quantification of protoporphyrin IX (PP IX) from the eggshell with and without the presence of cuticle. The L*a* components of the colour space system were significantly different among the age groups. There was a high degree of correlation between the extent of MST cuticle blue staining and the amount of cuticle on the eggshell as recorded by scanning electron microscopy. PP IX pigment was quantified by spectrophotometric analysis of digested eggshell solutions.

2016, Samiullah, Samiullah, Omar, A S, Roberts, Julie R, Chousalkar, K

Egg quality parameters were measured in eggs from flocks reared together and then allocated to different production systems. Eggs were processed for measurement of egg quality variables, scoring of ultrastructural mammillary layer features, completeness of cuticle cover and protoporphyrin IX (PPIX) quantification. There was a significant main effect (P<0.05) of production system on shell reflectivity, egg weight and egg internal quality and significant effects of flock age on most measurements. The mammillary layer ultrastructural variables showed no clear relationship with production system and flock age. Cuticle cover was significantly higher in barn eggs, followed by free range and cage eggs. Completeness of cuticle cover was significantly higher in eggs from the 44 week old flock than for 64 week and 73 week old flocks. There was a significant main effect of both production system and flock age, but no significant interaction for shell reflectivity, L* and amount of PPIX. There was no statistically significant difference for cuticle cover. In 1g of shell with and without cuticle, there was more PPIX in cage eggs followed by free range and barn eggs. Similar trends were recorded for the amount of PPIX in 1g of cuticle, but the difference was not statistically significant. The amount of PPIX decreased significantly with increasing flock age.

2013, Roberts, Julie R, Chousalkar, Kapil, Samiullah, Samiullah

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.

2013, Samiullah, Samiullah, Chousalkar, Kapil, Roberts, Julie R, Sexton, M, Day, M, Kiermeier, A

The vast majority of eggs in Australia are washed prior to packing to remove dirt and fecal material and to reduce the microbial contamination of the egg shell. The egg contents can be an ideal growth medium for microorganisms which can result in human illness if eggs are stored improperly and eaten raw or undercooked, and it is estimated that egg-related salmonellosis is costing Australia $44 million per year. Egg shell characteristics such as shell thickness, amount of cuticle present, and thickness of individual egg shell layers can affect the ease with which bacteria can penetrate the egg shell and washing could partially or completely remove the cuticle layer. The current study was conducted to investigate the effects of egg washing on cuticle cover and effects of egg shell quality and cuticle cover on 'Salmonella' Infantis penetration of the egg shell. A higher incidence of unfavorable ultrastructural variables of the mammillary layer such as late fusion, type B bodies, type A bodies, poor cap quality, alignment, depression, erosion and cubics were recorded in 'Salmonella' penetrated areas of egg shells. The influence of egg washing on the ability of 'Salmonella' Infantis on the egg shell surface to enter the egg internal contents was also investigated using culture-based agar egg penetration and real-time qPCR based experiments. The results from the current study indicate that washing affected cuticle cover. There were no significant differences in 'Salmonella' Infantis penetration of washed or unwashed eggs. Egg shell translucency may have effects on 'Salmonella' Infantis penetration of the egg shell. The qPCR assay was more sensitive for detection of 'Salmonella' Infantis from egg shell wash and internal contents than traditional microbiological methods. The agar egg and whole egg inoculation experiments indicated that 'Salmonella' Infantis penetrated the egg shells. Egg washing not only can be highly effective at removing 'Salmonella' Infantis from the egg shell surface, but also allows subsequent trans-shell and trans-membrane penetration into the egg. Consequently, it is important to prevent recontamination of the egg after washing.

2012, Samiullah, Samiullah, Roberts, Juliet R, Chousalkar, Kapil

Eggs were collected from a cage and a free range flock at the ages of 25, 35 and 45 weeks and divided into 4 different groups after candling and scoring for translucency. Thirty eggs were used for the estimation of the amount of cuticle present on the egg shell using a hand held Konica Minolta spectrophotometer before and after MST Cuticle Blue staining. The cage system eggs were visually more uniformly stained than the free range eggs. Significant effects were recorded for production system and age for L*a*b* values, but there was no significant interaction between the two. Under the scanning electron microscope (SEM), unstained patches showed no cuticle whereas stained areas had intact cuticle irrespective of the rearing system. None of the egg shells showed 100% cuticle coverage. Traditional egg quality measurements were carried out using specialized equipment supplied by TSS (UK). A significant effect was recorded for flock age and production system for all the egg quality variables except shell breaking strength, while interaction between the two was significant for translucency score, egg weight, shell weight, shell thickness and yolk score. Shell ultrastructural features of thirty eggs per flock were scored using SEM following plasma etching. Shell ultrastructural variables significantly affected by production system and age were mammillary cap size, early fusion, late fusion and depression, while interaction between production system and flock age was recorded only for confluence, alignment, type A bodies, cuffing, changed membrane, depression and erosion. The remaining 60 eggs per flock were used for microbiological studies, which showed negative results for 'Salmonella' spp.

2014, Samiullah, Samiullah, Roberts, Julie R

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.