The location of protoporphyrin in the eggshell of brown-shelled eggs

Transcription

1 The location of protoporphyrin in the eggshell of brown-shelled eggs S. Samiullah and J. R. Roberts 1 Animal Science, School of Environmental and Rural Science, Woolshed Building (W49), University of New England, Armidale, New South Wales, 2351, Australia ABSTRACT 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 INTRODUCTION The color of the eggs of commercial laying hens (brown, white, or tinted) is determined primarily by the genotype of the hen. However, eggs of many colors are laid by different avian species, and considerable research attention has been paid to the eggshells of the domestic quail (Soh and Koga, 1999). Brownshelled eggs dominate commercial markets in many countries including Australia and western Europe, so shell color of brown eggs is a quality aspect for consumers (Mertens et al., 2010). Eggshell pigment is used as an assessment tool for stress and disease conditions in commercial laying hens because these conditions may result in lighter colored eggshells (Walker and Hughes, 1998; Moreno and Osorno, 2003; Martinez-de la Puente et al., 2007; Mertens et al., 2010). Eggshell pigment has also been shown to have antimicrobial properties, particularly against gram-positive bacteria (Ishikawa et al., 2010). Eggshell color has been linked to egg quality parameters (Curtis et al., 1985; Jones et al., 2010) and may be related to shell ultrastructure (Richards and Deeming, 2001). Protoporphyrin has been identified as the main eggshell pigment found in the eggs of laying hens, in addition to other pigments such as biliverdin (With, 1973; 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. Key words: laying hen, eggshell, eggshell pigment, shell color, protoporphyrin 2013 Poultry Science 92 : /ps Poultry Science Association Inc. Received January 20, Accepted June 12, Corresponding author: jrobert2@une.edu.au Kennedy and Vevers, 1973, 1976; Miksik et al., 1996; Mertens et al., 2010). Previous research has suggested that most of the protoporphyrin is located in the cuticle (Schwartz et al., 1975; Nys et al., 1991; Sparks, 1994; Miksik et al., 2007; Nys and Guyot, 2011). However, it is important to note that the relative proportions of pigment in the shell cuticle and the calcareous part of the eggshell have not been measured directly in previous studies. Therefore, the purpose of the present study was to quantify the amount of protoporphyrin IX (PP IX) in the cuticle and calcareous shell of brown eggshells. MATERIALS AND METHODS Sixty eggs per flock were collected from conventional cage flocks (HyLine Brown egg laying strain), from the same commercial facility, when flocks were 33, 50, and 67 wk of age. The 67-wk flock had been subjected to an induced molt at the age of 62 wk. Eggs were divided into 2 groups of 30 eggs per flock, one for each of experiments 1 and 2. Experiment 1 was conducted to verify the use of MST cuticle blue dye (MST Technologies, Europe Ltd., Kettering, UK) as an indicator of the presence of cuticle and also to verify the technique used for removal of cuticle without eroding the calcareous shell, in preparation for experiment 2. Experiment 1 used 30 eggs from each flock (33, 50, and 67 wk). Eggs selected were free from extraneous calcareous material. Shell color was measured by the L*a*b* color system using a Konica Minolta spectro- 2783

2 2784 Samiullah and Roberts photometer model CM-2600d (Konica Minolta Sensing, Singapore). The L* has a maximum of 100 (white) and a minimum of 0 (black). For a*, green is toward the negative end of the scale and red toward the positive end. For b*, blue is toward the negative end and yellow toward the positive end of the scale. Shell reflectivity (%) was also measured with a shell reflectivity meter (Technical Services and Supply, Dunnington, UK). Eggs were then soaked in MST cuticle blue dye for 1 min, rinsed in distilled water to remove excess stain, and allowed to dry in air. Shell color (L*a*b*) and shell reflectivity were measured on the stained eggs. The removal of the cuticle followed the method described by Leleu et al. (2011). Each egg, individually, was soaked in an EDTA solution (0.34 M, ph 7.5) for 5 min, and the cuticle was carefully removed in running tap water using a small soft brush. Eggs were then allowed to dry, after which shell color (L*a*b*) and shell reflectivity were measured on the same eggs without cuticle. Eggs were restained as described earlier, and shell color (L*a*b*) and shell reflectivity were again measured. Egg internal contents were removed by making a small hole at the blunt end of the egg using a rotary tool (Dremel High Speed rotary tool, 300 series, Robert Bosch Tool Corporation, Gerlingen, Germany). A small piece of shell from around the equator of the shell was cut out, mounted on an aluminum stub using silver paint (1005 aqueous conductive silver liquid-sem adhesive, ProSciTech, Townsville, QLD, Australia), photographed under a light microscope at 12 magnification, sputter coated with gold for 5 min in a Neocoater (Nikon, Tokyo, Japan), and viewed under a JCM-5000 Neoscope benchtop scanning electron microscope. In experiment 2, the remaining 30 eggs from each of the same 33-, 50-, and 67-wk flocks were analyzed for PP IX in the cuticle and whole eggshell (including the cuticle). All eggs analyzed were free from any extraneous calcareous material. The method described by Poole (1965) and used by Ito et al. (1993) and Wang et al. (2007, 2009) was used with some modification. Egg contents were removed as described above and the inner shell walls rinsed with tap water to remove the adherent albumen. A thin stick was passed across the long axis of the eggshell in such a way as to immerse one longitudinal half of the shell in a glass bowl containing 0.34 M EDTA (ph 7.5) for 5 min, at the same time as maintaining the other half of the shell outside the solution. The cuticle of the soaked longitudinal half side of the eggshell was washed away in running tap water by rubbing with a small soft brush. After removal of the cuticle on one side, the eggshell was cut longitudinally into 2 equal halves, one having cuticle and one without cuticle. From both halves, shell membranes were carefully peeled away and the shells were allowed to dry. After the shells had dried completely, g from the equator of each shell was weighed into a clean 10- ml plastic centrifuge tube into which 4 ml of methanol-concentrated HCl (2:1) solvent was added. For each flock, 30 tubes were prepared with whole eggshell and 30 tubes from the shell without cuticle a total of 180 tubes for the 3 flocks. All tubes were wrapped in aluminum foil to prevent exposure to light and placed in a refrigerator for 12 h. The samples were centrifuged at 800 g at 4 C for 1 h. After centrifugation, the supernatant solution was decanted into disposable plastic 4-mL cuvettes and the absorbance of the supernatant read at 412 nm in a spectrophotometer (UV-1201, Shimadzu, Kyoto, Japan). To confirm that the shell had dissolved completely, the sediment remaining in the bottom of the centrifuge tubes was viewed under a light microscope at various magnifications. A standard solution was prepared by dissolving g of powder PP IX disodium salt (Sigma Aldrich, Castle Hill, NSW, Australia) in 30 ml of methanolconcentrated HCl (2:1) solvent. Serial dilutions were prepared until a 1:128 dilution was reached. A standard curve was constructed using the absorbance at 412 nm of concentrations of protoporphyrin standards from 0 mm to mm. The absorbance values from the analyzed samples were converted into concentration of PP IX in 1 g of eggshell (with and without cuticle present). For determination of the amount of PP IX in the cuticle, the values of the eggshell samples without cuticle were subtracted from the values of the eggshell samples with the cuticle still present. Data were analyzed by one-way ANOVA using Statview Software (version , SAS Institute Inc., Cary, NC). Differences between means were assessed using Fisher s protected least significant difference test. Significance was assumed at P < RESULTS In experiment 1, there were no significant differences among the 3 flocks for egg weight, which was 66.2, 64.4, and 63.1 g for the 33, 50-, and 67-wk flocks, respectively. In addition, there was no significant effect of flock age on shell reflectivity (%) when the cuticle was present (with and without MST cuticle blue staining) (Table 1). However, after the cuticle had been removed with EDTA, there were significant differences among flocks, with and without cuticle blue staining, with the 67-wk flock having the lowest reflectivity. There was a significant main effect of cuticle treatment on shell reflectivity (P < ). Shell reflectivity decreased following staining of eggs with cuticle intact. Shell reflectivity increased after the cuticle was removed from the eggs and was not significantly different following cuticle blue staining of shells with cuticle removed, except for the 33-wk flock in which reflectivity decreased slightly. The L* component of the L*a*b* space system was not significantly affected by flock age when the cuticle was intact (Table 2). However, following the removal of the cuticle, there were differences among flocks with values being highest for the 50-wk flock both before

3 LOCATION OF PIGMENT IN BROWN-SHELLED EGGS Table 1. Shell reflectivity (%) of eggshells from different aged flocks and different cuticle treatment 1, ± 0.75 c ± 0.85 d ± 0.77 a,xy ± 0.78 b,x < ± 0.69 b ± 0.82 c ± 0.63 a,x ± 0.57 a,x < ± 0.55 b ± 0.63 c ± 0.55 a,y ± 0.57 a,y < age comparison x,y Means within a column lacking a common superscript differ (P < 0.05). 1 : eggshell with intact cuticle; : eggshell with cuticle removed; : eggshells stained with MST cuticle blue dye (MST Technologies, Europe Ltd., Kettering, UK); : unstained eggshells. and after the second cuticle staining. There was a significant effect of cuticle treatment on mean values of L* among each age group. Staining of eggs with intact cuticle resulted in lower L* values, whereas cuticle removal increased these values. There was a significant effect of flock age on the a* reading for all cuticle treatments with the 67-wk flock having higher values than the 33- and 50-wk flocks, which were not significantly different from each other (Table 3). There was also a significant effect of cuticle treatment on values for a* for all flocks. The a* reading decreased following staining of eggs with intact cuticle. Removal of the cuticle with EDTA resulted in lower readings than for unstained shells with intact cuticle. Staining with cuticle blue dye of eggs with cuticle removed resulted in a further reduction in the a* value for all flocks. There was a significant effect of flock age on b* values with the 67-wk flock having the highest values (Table 4). There was also a significant effect of cuticle treatment for all flocks. Staining of eggs with intact cuticle resulted in higher b* readings than the unstained eggs with cuticle intact, whereas removal of cuticle resulted in lower readings with or without cuticle blue staining. A high correlation was observed between the presence of cuticle blue stain on eggshells and the amount of cuticle as viewed under the scanning electron microscope. Eggs with good quality intact cuticle stained well; eggs with patchy cuticle acquired patchy stain, whereas in the absence of the cuticle, the eggs did not stain at all. The scanning electron microscope observations of the restained eggshell after the cuticle was removed by EDTA confirmed that, in the absence of the cuticle, eggshells did not stain with cuticle blue dye. However, in some cases, small amounts of cuticle remained in crevices and pores so that there was some staining with cuticle blue dye. In experiment 2, the thickness of the eggshell, with shell membrane removed, was thicker for eggs from the 67-wk flock than for the other 2 ages, with and without the cuticle present (Table 5). However, the difference between the 2 thicknesses, which represents the thickness of the cuticle alone, was not different among the flocks. For a 1-g piece of eggshell, there was more PP IX in the shell with the cuticle intact compared with a piece of shell from the same egg with the cuticle removed. When the difference between the 2 was calculated, there was more protoporphyrin present in the true (calcareous) shell than in the cuticle from the same amount of shell, as shown in Table 5. The total amount of PP IX in 1 g of shell with cuticle intact was not significantly different among the flocks (P = ), although it tended to be lowest at 50 wk. The total amount of PP IX in 1 g of shell without cuticle was not significantly different among the 3 flocks. However, when the amount of PP IX in the cuticle alone of 1 g of total eggshell was calculated, it was significantly higher (P < 0.02) in the 50- and 67-wk flocks compared with the 33-wk flock. For a given weight of whole eggshell, the percentage of total PP IX found in the cuticle Table 2. Spectrophotometric L* values of eggshells from different aged flocks and different cuticle treatment 1, ± 0.63 b ± 0.83 c ± 0.59 a,y ± 0.60 a,y < ± 0.62 c ± 0.85 d ± 0.49 a,x ± 0.46 b,x < ± 0.48 b ± 0.65 c ± 0.43 a,y ± 0.42 a,y < age comparison x,y Means within a column lacking a common superscript differ (P < 0.05). 1 : eggshell with intact cuticle; : eggshell with cuticle removed; : eggshells stained with MST cuticle blue dye (MST Technologies, Europe Ltd., Kettering, UK); : unstained eggshells.

4 2786 Samiullah and Roberts Table 3. Spectrophotometric a* values of eggshells from different aged flocks and different cuticle treatment 1, ± 0.38 a,y 0.21 ± 0.77 d,y ± 0.41 b,y ± 0.42 c,y < ± 0.35 a,y 1.37 ± 0.93 d,y ± 0.31 b,y ± 0.37 c,y < ± 0.28 a,x 6.67 ± 0.77 d,x ± 0.26 b,x ± 0.28 c,x < age comparison < < x,y Means within a column lacking a common superscript differ (P < 0.05). 1 : eggshell with intact cuticle; : eggshell with cuticle removed; : eggshells stained with MST cuticle blue dye (MST Technologies, Europe Ltd., Kettering, UK); : unstained eggshells. was 13, 20, and 18% in 33-, 50-, and 67-wk flock eggs, respectively. Microscopic observations of the digested shell precipitates showed only shell membranes which confirmed that all shells had been dissolved in the solvent. DISCUSSION In experiment 1, as expected, a significant change in shell reflectivity (%) and L*a*b* components of the color space system with the various cuticle treatments confirmed the effectiveness of the procedure used for cuticle removal. For unstained eggs with cuticle intact, there was no difference among the flocks for either shell reflectivity or L* values. Following cuticle removal (but before staining), reflectivity and L* values were highest for the 50-wk flock. From the current experiments, it is concluded that shell reflectivity and L* component of the L*a*b* color space system provide similar, but not always identical, information about overall shell color. The a* value of eggshells stained with MST cuticle blue dye is the most important indicator of the amount of cuticle present on an eggshell. The higher a* values for intact cuticle, stained eggs in the 67-wk flock, compared with the 33- and 50-wk flocks, indicated the presence of less cuticle. When cuticle blue stain was applied to shells with cuticle removed, all flocks showed slight reductions in a* values, reflecting the visual observation that a small amount of staining was occurring where cuticle was present in crevices and pores. The data for the b* component of the L*a*b* color space system are more difficult to interpret. Despite the MST cuticle blue stain being blue when it is dissolved in distilled water, it stains the cuticle of eggshells a green color that is detected by the a* component, as discussed above. Experiment 1 also verified that the MST cuticle blue dye is a reliable indicator of the presence of cuticle on eggshells. The scanning electron microscope observation of shells with cuticle removed confirmed that the EDTA treatment reliably removed the cuticle without eroding the calcium carbonate of the eggshell. Application of the cuticle staining technology in the commercial industry needs to take into consideration that egg washing has the potential to remove cuticle from eggshells and that this extent of cuticle removal will depend on the washing technique used, as well as the washing and sanitizing chemicals employed. The results of experiment 2 showed more pigment (PP IX) in the calcareous components of the eggshell than in the cuticle of commercial brown eggs. These results are in contrast to previous research that reports more pigment in the cuticle than in the calcareous layers of the eggshell (Baird et al., 1975; Schwartz et al., 1975; Miksik et al., 2007; Wang et al., 2007, 2009; Nys and Guyot, 2011). In the present study, the amount of PP IX in the eggshell with the cuticle removed, and in the shell with the cuticle intact, was not significantly different among the 3 age groups. This finding supports the suggestion of Odabasi et al. (2007) that there is a constant rate of secretion of PP IX in the shell gland throughout the production cycle of laying hens Table 4. Spectrophotometric b* values of eggshells from different aged flocks and different cuticle treatment 1, ± 0.34 b,y ± 0.42 a,y ± 0.36 c,y ± 0.34 c,x < ± 0.34 b,y ± 0.24 a,y ± 0.38 c,z ± 0.36 c,y < ± 0.24 b,x ± 0.24 a,x ± 0.29 c,x ± 0.30 d,x < age comparison x z Means within a column lacking a common superscript differ (P < 0.05). 1 : eggshell with intact cuticle; : eggshell with cuticle removed; : eggshells stained with MST cuticle blue dye (MST Technologies, Europe Ltd., Kettering, UK); : unstained eggshells.

5 LOCATION OF PIGMENT IN BROWN-SHELLED EGGS Table 5. Values of protoporphyrin IX (PP IX) in cuticle and calcareous eggshell of HyLine brown eggs Variable P-value PP IX in cuticle of 1 g of eggshell (mm) b a a PP IX in 1 g of shell without cuticle (mm) Shell thickness with cuticle (membrane removed) ± 5.3 b ± 4.8 b ± 3.5 a Shell thickness without cuticle (membrane removed) ± 5.6 b ± 4.7 b ± 3.4 a a,b Means within a row lacking a common superscript differ (P < 0.05). 1 Values are means ± SE. and that shell color may therefore be related to egg size. However, there was a statistically significant difference among the 3 flocks in the amount of PP IX in the cuticle alone. There was some discrepancy between the amount of PP IX measured in the cuticle of the shell and the results of the a* component of the stained intact cuticle. The 33-wk flock eggs had a lower amount of PP IX in the cuticle compared with the 50- and 67-wk eggs, but the a* color of stained eggs with cuticle intact suggested that the 33- and 50-wk eggs had more cuticle than the 67-wk eggs. Campo et al. (2007) have linked the variation in shell color within a brown egg laying breed as timedependent, stating that eggs laid in the afternoon were lighter in color as compared with eggs laid in the morning. Several factors affecting the amount of pigment in eggshells have been described (Lang and Wells, 1987; Charlton et al., 2005). Our finding that there is a greater percentage of total pigment in the calcareous part of the shell than in the cuticle raises questions about the stages of eggshell formation and the times at which protoporphyrin deposition is maximal. Many authors have suggested that most pigment is secreted in the last hour of oviposition and deposited in the cuticle (Poole, 1965; Schwartz et al., 1975; Kennedy and Vevers, 1976). However, the findings of the present study indicate that the amount of pigment in the cuticle is lower than that contained within the outer calcareous layer of the shell. In summary, staining with MST cuticle blue dye is, in general, a reliable indicator of the presence of cuticle. More of the eggshell pigment (PP IX) in brown-shelled eggs is in the calcareous components of the eggshell than in the cuticle. 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. ACKNOWLEDGMENTS This study was supported by funding from Australian Egg Corporation Limited, Sydney, Australia. REFERENCES Baird, T., S. E. Solomon, and D. R. Tedstone Localization and characterization of egg shell porphyrins in several avian species. Br. Poult. Sci. 16: Campo, J. L., M. G. Gil, and S. G. Davila Differences among white, tinted and brown egg laying hens for incidence of eggs laid on the floor and for oviposition time. Arch. Geflugelkd. 71: Charlton, B. R., A. K. Tiwary, A. A. Bickford, and M. Filigenzi Acute depigmentation of fertile brown eggs in a commercial layer operation. J. Vet. Diagn. Invest. 17: Curtis, P. A., F. A. Gardner, and D. B. Mellor A comparison of selected quality and compositional characteristics of brown and white shell eggs. II. Interior quality. Poult. Sci. 64: Ishikawa, S., K. Suzuki, E. Fukuda, K. Arihara, Y. Yamamoto, T. Mukai, and M. Itoh Photodynamic antimicrobial activity of avian eggshell pigments. FEBS Lett. 584: dx.doi.org/ /j.febslet Ito, S., M. Tsudzuki, M. Komori, and M. Mizutani Celadon: An eggshell color mutation in Japanese quail. J. Hered. 84: Jones, D. R., M. T. Musgrove, K. E. Anderson, and H. S. Thesmar Physical quality and composition of retail shell eggs. Poult. Sci. 89: Kennedy, G. Y., and H. G. Vevers Eggshell pigments of the Araucano fowl. Comp. Biochem. Physiol. B 44: Kennedy, G. Y., and H. G. Vevers A survey of avian egg shell pigments. Comp. Biochem. Physiol. 55: Lang, M. R., and J. W. Wells A review of eggshell pigmentation. World s Poult. Sci. J. 43: Leleu, S., M. Bain, L. Herman, M. Heyndrickx, J. De Baerdemaeker, C. W. Michiels, C. Perianu, and W. Messens The effect of micro cracks and the presence of the cuticle on trans-shell penetration of table eggs by Salmonella Enteritidis. Proceedings of Egg Meat Symposia Leipzig, Germany, September 4 8, Martinez-de la Puente, J., S. Merino, J. Moreno, G. Tomas, J. Morales, E. Lobato, S. Garcia-fraile, and J. Martinez Are eggshell spottiness and color indicators of health and condition in blue tits Cyanistes caeruleus? J. Avian Biol. 38: Mertens, K., I. Vaesen, J. Loffel, B. Kemps, B. Kamers, C. Perianu, J. Zoons, P. Darius, E. Decuypere, J. De Baerdemaeker, and B. De Ketelaere The transmission color value: A novel egg quality measure for recording shell color used for monitoring the stress and health status of a brown layer flock. Poult. Sci. 89: Miksik, I., A. Eckhardt, P. Sedlakova, and K. Mikulikova Proteins of insoluble matrix of avian (Gallus gallus) eggshell. Connect. Tissue Res. 48:1 8. Miksik, I., V. Holan, and Z. Deyl Avian eggshell pigments and their variability. Comp. Biochem. Physiol. 113B: Moreno, J., and J. L. Osorno Avian egg color and sexual selection: Does eggshell pigmentation reflect female condition and genetic quality? Ecol. Lett. 6: Nys, Y., and N. Guyot Egg formation and chemistry. Pages in Improving the Safety and Quality of Eggs and Egg Product. Y. Nys, M. Bain, and F. Van Immerseel, ed. Woodhead Publishing, Cambridge, UK. Nys, Y., J. Zawadzki, J. Gautron, and A. D. Mills Whitening of brown-shelled eggs: Mineral composition of uterine fluid and rate of protoporphyrin deposition. Poult. Sci. 70: Odabasi, A. Z., R. D. Miles, M. O. Balaban, and K. M. Porier Changes in brown eggshell color as the hen ages. Poult. Sci. 86:

6 2788 Samiullah and Roberts Poole, H. K Spectrophotometric identification of eggshell pigments and timing of superficial pigment deposition in the Japanese quail. Proc. Soc. Exp. Biol. Med. 119: Richards, P. D. G., and D. C. Deeming Correlation between shell color and ultra structure pheasant eggs. Br. Poult. Sci. 42: Schwartz, S., B. D. Stephenson, D. H. Sarkar, and M. R. Bracho Red, white and blue eggs as models of porphyrin and heme metabolism. Ann. N. Y. Acad. Sci. 244: Soh, T., and O. Koga Effects of phosphate, prostaglandins, arachidonic acid and arginine vasotocin on oviposition and pigment secretion from the shell gland of the Japanese quail. Br. Poult. Sci. 40: Sparks, N. H. C Shell accessory materials: Structure and function. Pages in Microbiology of the Avian Egg. R. G. Board and R. Fuller, ed. Chapman & Hall, London, UK. Walker, A. W., and B. O. Hughes Egg shell color is affected by laying cage design. Br. Poult. Sci. 39: Wang, X. L., J. X. Zheng, Z. H. Ning, L. J. Qu, G. Y. Xu, and N. Yang Laying hen performance and egg quality of blueshelled layers as affected by different housing systems. Poult. Sci. 88: Wang, X. T., X. M. Deng, C. J. Zhao, J. Y. Li, G. Y. Xu, L. S. Lian, and C. X. Wu Study of the deposition process of eggshell pigments using an improved dissolution method. Poult. Sci. 86: With, T. K Porphyrin in eggshell. Biochem. J. 137: