Brown eggshell fading with layer ageing: dynamic change in the content of protoporphyrin IX

Brown eggshell fading with layer ageing: dynamic change in the content of protoporphyrin IX ABSTRACT The dynamic change in brown eggshell color as hens age has been observed, but much uncertainty still exists. We aimed to analyze the depth of eggshell color and quantity of protoporphyrin ΙΧ throughout the laying period to explore the reasons for color variation. In this study, 120 Rhode Island Red hens were used, and two eggs were collected from each individual at 26, 34, 42, 50, 60, and 70 wk of age. The eggshell color (L*, a*, b*), egg weight, eggshell dry weight, and protoporphyrin ΙΧ quantity in eggshell were measured for individual eggs. Our results showed that the intensity of brown eggshell color weaken as hens aged from 26 to 60 wk of age (L* gradually increased from 61.43 to 68.07), while eggshell lightness recovered slightly at 70 wk (L* = 64.77). The correlation analysis indicated that the content of protoporphyrin ΙΧ deposited in the eggshell was an important factor for lightness fading with the ageing process (the average r was 0.66, P < 0.01), while the egg weight had little impact on the eggshell color (the average r was 0.07, P > 0.05). The shade of the eggshell color (L* and a*) at the early laying period (26 or 34 wk) had a low correlation with the other age points (42, 50, 60, and 70 wk). However, high correlations between the shell color at 42 wk of age and subsequent ages (50, 60, and 70 wk) were found, suggesting that the intensity of eggshell color is more stable after egg-laying peaks (such as 42 wk of age). In conclusion, the intensity of brown eggshell color varies greatly among the whole laying cycle and breeders can choose the proper age for eggshell color measurements to ensure the degree of shell color in the late laying period. INTRODUCTION Brown eggshell color, as an economic trait, has been a source of concern for a long time. However, the degree of brownness changes over the entire laying cycle, which has decreased interest among retailers. Understanding the immediate causes of variation in coloration is beneficial to guarantee the color intensity of eggs. The chemical basis and mechanisms of color production have been characterized (Kennedy and Vevers, 1973; Ajioka et al., 2006; Zhao et al., 2006; Hanley et al., 2015). Though brown eggs have various intensities of color, several tetrapyrrole compounds, especially protoporphyrin ΙΧ (With, 1974; Gorchein et al., 2009; Igic et al., 2010), are responsible for generating this spectrum of colors via subtle concentration changes (Hanley et al., 2015). These pigments allow eggshells to perform many functions, including improving eggshell quality (Yang et al., 2009), decreasing incubation period (Sanz and García-Navas, 2009), preventing Gram-positive bacterial infections (Ishikawa et al., 2010), and protecting the embryo from solar radiation (Lahti and Ardia, 2016). As hypotheses on the functions of eggshell color have proliferated and the precision of color measurement has improved, many studies have focused on the factors regulating or disrupting eggshell color. In these studies, eggshell color has been correlated with many environmental factors, such as housing systems (Sekeroglu et al., 2010), nutrition (Seo et al., 2010; Yuan et al., 2016), and environmental pollutants (Hanley and Doucet, 2012). Compared with external environmental factors, poultry breeders are more interested in determining the internal factors that produce dark brown eggshells through selective breeding programs. Finally, the studies recognized that hen age can influence eggshell color, especially in the late laying period. Generally, eggshell becomes paler as the laying period progresses (Hunton, 1926; Hall, 1944; Ingram et al., 2008; Tumova and Ledvinka, 2009; Zita et al., 2009). However, it remains unclear how this process happens. As early as ten years ago, Odabasi (Odabasi et al., 2007) reported that older hens laid lighter colored eggs due to an increase in egg size associated with no proportionate change in the quantity of pigment deposited over the shell surface. In contrast, recent research demonstrated that eggshell color was correlated not with egg weight but with eggshell pigmentation (protoporphyrin) (Samiullah et al., 2016), which is the precursor of protoheme (Heinemann et al., 2008). Thus, reduced pigmentation is expected as a hen ages because hematopoiesis functions appear to progressively decline (Brusnahan et al., 2010). Despite relentless efforts that have been made, the reason why eggs fade over time and the way to resolve this problem were still unclear. Analysis of data and of information from scientific experiments is used for evidence-based decision making regarding how to make eggshell color more consistent and how to adapt eggshell color to local and national markets. The main objective of this paper is to describe the dynamic change in brown eggshell color with the increasing age of hens and to provide a breeding strategy in order to improve the color intensity of eggs during the whole egg-laying period. MATERIALS AND METHODS Egg Collection This study was carried out on 120 Rhode Island Red hens, which were selected based on clutch trait, during the whole egg-laying period. The hens were reared in individual cages under the 16L:8D lighting schedule and had ad libitum access to feed and water. At 26, 34, 42, 50, 60, and 70 wk of age, the eggs were collected from each layer on 3 successive days to ensure 2 eggs for each hen. The time interval of measurement was extended from eight weeks to ten weeks because the hens were transferred to other houses at 56 week of age due to the need for production. To minimize the influence of stress, we collected eggs four weeks later when laying performance returned to a normal level. Eggshell Color Measurement Eggshell color was measured with a reflectometer (Konica Minolta, Tokyo, Japan) using three parameters: L* measures lightness (0 is black; 100 is white), a* describes the balance of red and green (< 0 is green; > 0 is red), and b* represents hue as a function of a blue-yellow scale (< 0 is blue; > 0 is yellow). Protoporphyrin ΙΧ Measurement Eggs were cleaned with distilled water, air dried, and weighed by a micro-electronic balance. Egg internal contents and eggshell membrane were removed, and the remaining eggshell was ground into powder. After 24 h of drying in the oven, the total weight of the eggshell power was recorded, and a small amount (0.25 g) was dissolved in 6 mL extraction solution, a mixture of 4 mL methanol and 2 mL concentrated HCl. The solution was allowed to stand in the dark for 48 h until the pigment was completely dissolved. After centrifugation at 1,369.55 × g for 45 min, the absorbance of supernatant solution was measured using a microplate spectrophotometer (Tecan, Port Melbourne, Australia) at a wavelength of 412 nm. The concentration of protoporphyrin ΙΧ in eggshell (nmol g−1, CPES) was calculated by substituting absorbance in the linear regression equation deduced from the standard curve of protoporphyrin ΙΧ (Wang et al., 2009). The total content of protoporphyrin ΙΧ in eggshell (nmol, TPES) was equal to the product of its concentration and dry weight. Statistical Analysis First, the average value of two eggs from the same hen in the same week was calculated and the descriptive analyses for all variables (egg weight, eggshell dry weight, concentration of eggshell protoporphyrin ΙΧ, total content of eggshell protoporphyrin ΙΧ, and eggshell color: L*a*b*) were performed to summarize the characteristics of hens at different weeks of age. Second, a mixed linear model where time was the fixed effect and the hen was the random effect was used. After comparing covariance structures via goodness of fit criteria that were printed by PROC MIXED, we choose the best fit and calculated the least square means for multiple comparisons with the Bonferroni adjustment using SAS 9.2 software (SAS Institute Inc., Cary, NC). The level of statistical significance was set to P-value of <0.01. Third, the correlation coefficient between different traits and different weeks was analyzed with the Pearson correlation using R version 3.0.3, while the missing data were pairwise deleted. RESULTS Comparison of Eggshell Color, Egg Weight, and Content of Protoporphyrin ΙΧ in Eggshell at Different Weeks of Age During the egg-laying period, the average value of L* was gradually increased and reached its peak at 60 wk of age, while a slight but significant decline could be observed at 70 wk (Table 1). The average value of a* started high and experienced a consistent decrease over time, except at 70 wk of age, when an upward trend occurred. These two results showed that the brown eggshell color faded with the ageing of hens from 26 to 60 wk of age, while the shade of color recovered slightly at 70 wk. The value of b* showed a significant increase at 34 wk of age, and after this point, the value levelled off. In the early laying period, egg weight (EW) showed an upward trend, and after 50 wk, its mean value was flat, with no significant shift over time. A similar trend was observed for eggshell dry weight (ESDW). As opposed to the subtle change in eggshell dry weight, eggshell pigmentation showed a surprising variation, as it dropped by 43% between 26 and 34 wk of age for the concentration of CPES and the total content of TPES. The downward trend of CPES continued until 60 wk of age, though the change narrowed to a small extent. In comparison with 60 wk of age, CPES at 70 wk significantly increased and reached the same level as at 42 and 50 wk. Additionally, the variation of TPES over time followed the similar direction as CPES. Table 1. Comparison of eggshell color, content of protoporphyrin ΙΧ in eggshell, and egg weight at different weeks of age. 2Variables Age (wk) 26 34 42 50 60 70 L* 161.43 ± 0.49E 62.19 ± 0.48DE 63.17 ± 0.36CD 63.71 ± 0.33C 68.07 ± 0.35A 64.77 ± 0.37B a* 17.71 ± 0.29A 16.98 ± 0.28A 15.61 ± 0.19B 15.41 ± 0.19B 13.22 ± 0.21D 14.27 ± 0.21C b* 29.14 ± 0.25B 30.36 ± 0.23A 27.74 ± 0.18C 27.47 ± 0.18C 27.24 ± 0.20C 27.55 ± 0.21C EW (g) 55.24 ± 0.54C 57.23 ± 0.56BC 58.65 ± 0.38B 60.70 ± 0.40A 60.12 ± 0.41A 60.01 ± 0.45A ESDW (g) 4.89 ± 0.06D 4.94 ± 0.05CD 5.11 ± 0.04BC 5.44 ± 0.05A 5.35 ± 0.05A 5.27 ± 0.05AB CPES (nmol g−1) 121.49 ± 2.85A 68.87 ± 1.88B 61.46 ± 1.41C 59.67 ± 1.38C 49.82 ± 1.38D 60.21 ± 1.63C TPES (nmol) 591.77 ± 14.00A 339.96 ± 9.82B 311.98 ± 7.24C 323.07 ± 7.13BC 264.21 ± 7.27D 315.09 ± 8.58BC 2Variables Age (wk) 26 34 42 50 60 70 L* 161.43 ± 0.49E 62.19 ± 0.48DE 63.17 ± 0.36CD 63.71 ± 0.33C 68.07 ± 0.35A 64.77 ± 0.37B a* 17.71 ± 0.29A 16.98 ± 0.28A 15.61 ± 0.19B 15.41 ± 0.19B 13.22 ± 0.21D 14.27 ± 0.21C b* 29.14 ± 0.25B 30.36 ± 0.23A 27.74 ± 0.18C 27.47 ± 0.18C 27.24 ± 0.20C 27.55 ± 0.21C EW (g) 55.24 ± 0.54C 57.23 ± 0.56BC 58.65 ± 0.38B 60.70 ± 0.40A 60.12 ± 0.41A 60.01 ± 0.45A ESDW (g) 4.89 ± 0.06D 4.94 ± 0.05CD 5.11 ± 0.04BC 5.44 ± 0.05A 5.35 ± 0.05A 5.27 ± 0.05AB CPES (nmol g−1) 121.49 ± 2.85A 68.87 ± 1.88B 61.46 ± 1.41C 59.67 ± 1.38C 49.82 ± 1.38D 60.21 ± 1.63C TPES (nmol) 591.77 ± 14.00A 339.96 ± 9.82B 311.98 ± 7.24C 323.07 ± 7.13BC 264.21 ± 7.27D 315.09 ± 8.58BC 1Values are presented as the mean ± S.E. Means with different superscripts within each row differ significantly (P < 0.01). 2EW = egg weight, ESDW = eggshell dry weight, CPES = concentration of protoporphyrin ΙΧ in eggshell, TPES = total content of protoporphyrin ΙΧ in eggshell. View Large Table 1. Comparison of eggshell color, content of protoporphyrin ΙΧ in eggshell, and egg weight at different weeks of age. 2Variables Age (wk) 26 34 42 50 60 70 L* 161.43 ± 0.49E 62.19 ± 0.48DE 63.17 ± 0.36CD 63.71 ± 0.33C 68.07 ± 0.35A 64.77 ± 0.37B a* 17.71 ± 0.29A 16.98 ± 0.28A 15.61 ± 0.19B 15.41 ± 0.19B 13.22 ± 0.21D 14.27 ± 0.21C b* 29.14 ± 0.25B 30.36 ± 0.23A 27.74 ± 0.18C 27.47 ± 0.18C 27.24 ± 0.20C 27.55 ± 0.21C EW (g) 55.24 ± 0.54C 57.23 ± 0.56BC 58.65 ± 0.38B 60.70 ± 0.40A 60.12 ± 0.41A 60.01 ± 0.45A ESDW (g) 4.89 ± 0.06D 4.94 ± 0.05CD 5.11 ± 0.04BC 5.44 ± 0.05A 5.35 ± 0.05A 5.27 ± 0.05AB CPES (nmol g−1) 121.49 ± 2.85A 68.87 ± 1.88B 61.46 ± 1.41C 59.67 ± 1.38C 49.82 ± 1.38D 60.21 ± 1.63C TPES (nmol) 591.77 ± 14.00A 339.96 ± 9.82B 311.98 ± 7.24C 323.07 ± 7.13BC 264.21 ± 7.27D 315.09 ± 8.58BC 2Variables Age (wk) 26 34 42 50 60 70 L* 161.43 ± 0.49E 62.19 ± 0.48DE 63.17 ± 0.36CD 63.71 ± 0.33C 68.07 ± 0.35A 64.77 ± 0.37B a* 17.71 ± 0.29A 16.98 ± 0.28A 15.61 ± 0.19B 15.41 ± 0.19B 13.22 ± 0.21D 14.27 ± 0.21C b* 29.14 ± 0.25B 30.36 ± 0.23A 27.74 ± 0.18C 27.47 ± 0.18C 27.24 ± 0.20C 27.55 ± 0.21C EW (g) 55.24 ± 0.54C 57.23 ± 0.56BC 58.65 ± 0.38B 60.70 ± 0.40A 60.12 ± 0.41A 60.01 ± 0.45A ESDW (g) 4.89 ± 0.06D 4.94 ± 0.05CD 5.11 ± 0.04BC 5.44 ± 0.05A 5.35 ± 0.05A 5.27 ± 0.05AB CPES (nmol g−1) 121.49 ± 2.85A 68.87 ± 1.88B 61.46 ± 1.41C 59.67 ± 1.38C 49.82 ± 1.38D 60.21 ± 1.63C TPES (nmol) 591.77 ± 14.00A 339.96 ± 9.82B 311.98 ± 7.24C 323.07 ± 7.13BC 264.21 ± 7.27D 315.09 ± 8.58BC 1Values are presented as the mean ± S.E. Means with different superscripts within each row differ significantly (P < 0.01). 2EW = egg weight, ESDW = eggshell dry weight, CPES = concentration of protoporphyrin ΙΧ in eggshell, TPES = total content of protoporphyrin ΙΧ in eggshell. View Large Correlations between Eggshell Color with Egg Weight and Content of Protoporphyrin ΙΧ in Eggshell at the Same Week of Age Table 2 reflects that the lightness (L*, P < 0.01) and redness (a*, P < 0.01) of eggshell were both highly correlated with main brown eggshell pigment quantity, including the concentration and the total content of protoporphyrin ΙΧ in the eggshell, indicating a strong correlation between eggshell color and its quantity of protoporphyrin ΙΧ. The correlation was significant throughout the laying period, even though the association appeared slightly lower 70 wk of age. Looking at the correlation between yellowness (b*) of the eggshell and quantity of protoporphyrin ΙΧ, we observed a much weaker relationship compared with lightness and redness. In terms of the relationship between eggshell color and egg weight and eggshell dry weight, almost no correlation was found regardless of the time and trait. Overall, brown eggshell color was highly correlated with the content of protoporphyrin ΙΧ rather than egg size. Table 2. Correlations between eggshell color and egg weight and content of protoporphyrin ΙΧ in eggshell at the same week of age. Age (wk) Eggshell Colour 2EW ESDW CPES TPES 26 1L* −0.10 −0.12 −0.81** −0.85** a* 0.11 0.12 0.72** 0.76** b* 0.09 0.00 0.21 0.22 34 L* −0.18 −0.07 −0.66** −0.61** a* 0.24 0.18 0.58** 0.59** b* 0.16 0.16 0.38** 0.42** 42 L* 0.00 0.01 −0.79** −0.80** a* 0.03 0.04 0.79** 0.82** b* −0.06 −0.04 0.50** 0.49** 50 L* −0.07 −0.10 −0.80** −0.85** a* 0.09 0.07 0.77** 0.82** b* 0.02 −0.13 0.44** 0.40** 60 L* −0.06 −0.16 −0.77** −0.83** a* 0.14 0.23 0.72** 0.80** b* −0.04 0.07 0.42** 0.43** 70 L* −0.05 −0.05 −0.51** −0.52** a* 0.16 0.21 0.49** 0.58** b* 0.04 −0.03 0.26 0.22 Age (wk) Eggshell Colour 2EW ESDW CPES TPES 26 1L* −0.10 −0.12 −0.81** −0.85** a* 0.11 0.12 0.72** 0.76** b* 0.09 0.00 0.21 0.22 34 L* −0.18 −0.07 −0.66** −0.61** a* 0.24 0.18 0.58** 0.59** b* 0.16 0.16 0.38** 0.42** 42 L* 0.00 0.01 −0.79** −0.80** a* 0.03 0.04 0.79** 0.82** b* −0.06 −0.04 0.50** 0.49** 50 L* −0.07 −0.10 −0.80** −0.85** a* 0.09 0.07 0.77** 0.82** b* 0.02 −0.13 0.44** 0.40** 60 L* −0.06 −0.16 −0.77** −0.83** a* 0.14 0.23 0.72** 0.80** b* −0.04 0.07 0.42** 0.43** 70 L* −0.05 −0.05 −0.51** −0.52** a* 0.16 0.21 0.49** 0.58** b* 0.04 −0.03 0.26 0.22 1*significant linkage at P < 0.05; **highly significant linkage at P < 0.01. 2EW = egg weight, ESDW = eggshell dry weight, CPES = concentration of protoporphyrin ΙΧ in eggshell, TPES = total content of protoporphyrin ΙΧ in eggshell. View Large Table 2. Correlations between eggshell color and egg weight and content of protoporphyrin ΙΧ in eggshell at the same week of age. Age (wk) Eggshell Colour 2EW ESDW CPES TPES 26 1L* −0.10 −0.12 −0.81** −0.85** a* 0.11 0.12 0.72** 0.76** b* 0.09 0.00 0.21 0.22 34 L* −0.18 −0.07 −0.66** −0.61** a* 0.24 0.18 0.58** 0.59** b* 0.16 0.16 0.38** 0.42** 42 L* 0.00 0.01 −0.79** −0.80** a* 0.03 0.04 0.79** 0.82** b* −0.06 −0.04 0.50** 0.49** 50 L* −0.07 −0.10 −0.80** −0.85** a* 0.09 0.07 0.77** 0.82** b* 0.02 −0.13 0.44** 0.40** 60 L* −0.06 −0.16 −0.77** −0.83** a* 0.14 0.23 0.72** 0.80** b* −0.04 0.07 0.42** 0.43** 70 L* −0.05 −0.05 −0.51** −0.52** a* 0.16 0.21 0.49** 0.58** b* 0.04 −0.03 0.26 0.22 Age (wk) Eggshell Colour 2EW ESDW CPES TPES 26 1L* −0.10 −0.12 −0.81** −0.85** a* 0.11 0.12 0.72** 0.76** b* 0.09 0.00 0.21 0.22 34 L* −0.18 −0.07 −0.66** −0.61** a* 0.24 0.18 0.58** 0.59** b* 0.16 0.16 0.38** 0.42** 42 L* 0.00 0.01 −0.79** −0.80** a* 0.03 0.04 0.79** 0.82** b* −0.06 −0.04 0.50** 0.49** 50 L* −0.07 −0.10 −0.80** −0.85** a* 0.09 0.07 0.77** 0.82** b* 0.02 −0.13 0.44** 0.40** 60 L* −0.06 −0.16 −0.77** −0.83** a* 0.14 0.23 0.72** 0.80** b* −0.04 0.07 0.42** 0.43** 70 L* −0.05 −0.05 −0.51** −0.52** a* 0.16 0.21 0.49** 0.58** b* 0.04 −0.03 0.26 0.22 1*significant linkage at P < 0.05; **highly significant linkage at P < 0.01. 2EW = egg weight, ESDW = eggshell dry weight, CPES = concentration of protoporphyrin ΙΧ in eggshell, TPES = total content of protoporphyrin ΙΧ in eggshell. View Large Correlation of Eggshell Color among Different Weeks of Age The correlation of eggshell color in different weeks is illustrated in Figure 1. Looking at the L* value as an example, we found that the L* value was highly correlated with two adjacent time periods, especially in the similar period when production was divided by the egg-laying peak. To be specific, during the early stage including 26 and 34 wk, the correlation coefficient of L* was as high as 0.85 (P < 0.01), and after the egg-laying peak, the coefficients were 0.80 (P < 0.01), 0.83 (P < 0.01), and 0.76 (P < 0.01) between the two neighboring weeks, respectively, while only a low correlation was found between the two laying periods, as illustrated by 0.30 (P < 0.01) between 34 and 42 wk of age. The highly significant correlation of L* between 42 and 60 wk (r = 0.77, P < 0.01), 42 and 70 wk (r = 0.70, P < 0.01), and 50 and 70 wk (r = 0.77, P < 0.01) were observed, which indicates a constant intensity of eggshell color after the egg-laying peak. Replicating the analysis on a* and b*, similar patterns appeared and are depicted in Figure 1. The redness and yellowness of eggshell at 42 wk was highly correlated with itself in later weeks. Figure 1. View largeDownload slide The correlation matrix plots of L*(A), a*(B), and b*(C) values at different weeks of age. The upper panel shows the correlation matrix circles, and the larger area represents a higher correlation. The lower panel shows the pairwise correlations. Figure 1. View largeDownload slide The correlation matrix plots of L*(A), a*(B), and b*(C) values at different weeks of age. The upper panel shows the correlation matrix circles, and the larger area represents a higher correlation. The lower panel shows the pairwise correlations. Since an obvious distinction occurred at 42 wk of age, we selected this point as the base time and calculated the correlation with other time points. As is shown in Table 3 and Figure 1, a weak and then much stronger correlation appeared on the first two weeks and the next three weeks respectively, no matter which parameters we studied. Take lightness (L*) and redness (a*) of eggshell as an example, the correlation between base time and the earlier time (26 wk, r = 0.22 and 34 wk, r = 0.30) was so weak that we cannot deduce that eggshell color relied on the former record. Instead, the intensity of the color of eggs at the late laying period can be predicted according to the data at 42 wk due to the high correlation. As long as we select layers producing dark brown eggs at 42 wk of age, we would likely be able to obtain darker eggs during the following egg-raising period. Table 3. Correlation of variables at 42 weeks of age with other weeks. Age (wk) EW ESDW CPES TPES 26 10.24** 0.16 0.33** 0.31** 34 0.11 0.16 0.34** 0.31** 50 0.81** 0.76** 0.90** 0.91** 60 0.74** 0.67** 0.87** 0.84** 70 0.71** 0.55** 0.62** 0.56** Age (wk) EW ESDW CPES TPES 26 10.24** 0.16 0.33** 0.31** 34 0.11 0.16 0.34** 0.31** 50 0.81** 0.76** 0.90** 0.91** 60 0.74** 0.67** 0.87** 0.84** 70 0.71** 0.55** 0.62** 0.56** 1*significant linkage at P < 0.05; **highly significant linkage at P < 0.01. View Large Table 3. Correlation of variables at 42 weeks of age with other weeks. Age (wk) EW ESDW CPES TPES 26 10.24** 0.16 0.33** 0.31** 34 0.11 0.16 0.34** 0.31** 50 0.81** 0.76** 0.90** 0.91** 60 0.74** 0.67** 0.87** 0.84** 70 0.71** 0.55** 0.62** 0.56** Age (wk) EW ESDW CPES TPES 26 10.24** 0.16 0.33** 0.31** 34 0.11 0.16 0.34** 0.31** 50 0.81** 0.76** 0.90** 0.91** 60 0.74** 0.67** 0.87** 0.84** 70 0.71** 0.55** 0.62** 0.56** 1*significant linkage at P < 0.05; **highly significant linkage at P < 0.01. View Large To detect whether eggshell color was consistent during the late laying period and whether the selection process could be effective for screening the hens that laid darker eggs during the whole late process, we picked the first sixty hens listed according to the L* value of the eggshell at the last four weeks and found the overlapping individuals (Figure 2A). Only one hen laid eggs with a deeper intensity eggshell color at 42 wk but not at later weeks. There were 35 hens that ranked in the top half during the whole late laying process, and almost 82% of hens emerged in the top half three times. As opposed to setting 42 wk as the base time, the Venn diagram (Figure 2B) showed that 16 hens laid darker eggs just at 26 wk of age and that there were 21 overlapping individuals between 26 wk and the late laying period, which was reduced by 23%. Thus, we believe it is an effective and efficient program to choose the darker egg layers based on the eggshell color measurement after egg-laying peak. Figure 2. View largeDownload slide Venn diagram of hens ranked among the top half according to the L* value of eggshell color at different weeks of age. (A) Overlapping hens appeared at 42, 50, 60, and 70 wk of age. (B) Overlapping hens appeared at 26, 50, 60, and 70 wk of age. Figure 2. View largeDownload slide Venn diagram of hens ranked among the top half according to the L* value of eggshell color at different weeks of age. (A) Overlapping hens appeared at 42, 50, 60, and 70 wk of age. (B) Overlapping hens appeared at 26, 50, 60, and 70 wk of age. DISCUSSION The eggshell color turned pale with age increasing within 60 wk, which was consistent with the findings of many studies, even though eggshell color was measured using different methods (Hunton, 1926; Hall, 1944; Ingram et al., 2008; Tumova and Ledvinka, 2009; Zita et al., 2009). Considering the two assumptions regarding why an eggshell gradually fades over the ageing process (Odabasi et al., 2007; Samiullah et al., 2016), we compared the change in the egg weight and quantity of protoporphyrin ΙΧ in eggshell. There was a tendency for main brown eggshell pigment, including CPES and TPES, to change considerably during the first two weeks and still display a dynamic state during the late laying period, while the egg weight and eggshell dry weight changed slowly in the first four age points and remained comparatively stable after 50 wk. The change tendency, however, did not decrease our confusion; thus, we performed a correlation analysis between eggshell color and egg weight and the content of main brown eggshell pigment. From the results, we found that the shade of eggshell color was highly correlated with the quantity of protoporphyrin ΙΧ in the eggshell. Somewhat unexpectedly, the egg weight and eggshell dry weight, which were reported as the main effectors of eggshell color in a previous study (Odabasi et al., 2007), were found to poorly correlate with eggshell color (L* or a*). The comparison between these two correlation coefficients led us to the conclusion that the content of main brown eggshell pigment had a much greater influence on eggshell color than egg weight and eggshell dry weight. The higher the quantity of protoporphyrin ΙΧ deposited in eggshell, the darker the eggshell color will be. Thus, the fading of eggshell color may be mainly caused by a decrease in available protoporphyrin ΙΧ in the eggshell, which is a result of degradation of the hen's physiological functioning. Eggshell color did not maintain a downward trend during the whole laying process and displayed an increase at 70 wk of age compared with 60 wk. Another important difference is that content of protoporphyrin ΙΧ in eggshell significantly increased, while egg weight and eggshell dry weight were not altered. In 1944, Hall (Hall, 1944) observed this phenomenon in a crossbreed population of White Leghorn and Rhode Red, and he proposed seasonal variation as an explanation. Samiullah et al. (2016) found that the concentration of protoporphyrin ΙΧ in eggshell was recovered to the previous level, which is in line with our research. This phenomenon may be due to decreased egg production and a prolonged oviposition interval. As hens age, their laying performance degrades, which was indicated by the decrease in the egg-laying rate from 95% at 26 wk of age to 86% at 70 wk. The oviposition interval became longer, providing enough time for hens to produce and accumulate protoporphyrin ΙΧ. The large quantity of available protoporphyrin ΙΧ was conducive for hens to produce darker eggshell. Because eggshell color fades with the ageing process, it is imperative for us to select chickens that consistently produce dark eggs. In 2011, Institute de Selection Animal proposed the breeding program“Breeding for 500 eggs in 100 weeks” and aimed to extend the laying cycle (Sambeek, 2011); however, the fact that the intensity of eggshell color fades with ageing set a barrier for brown egg chickens. The statistics indicate that it is economical and effective to select chickens with darker eggshell after the egg-laying peak (such as 42 wk of age) to guarantee the degree of darkness of eggs at the end of the laying period. In conclusion, our study described the dynamic change in brown eggshell color with ageing and revealed the relationship between eggshell color and the quantity of protoporphyrin ΙΧ in eggshell. We also proposed that eggshell color should be measured after the egg-laying peak in breeding programs to ensure the consistent color of eggs can be produced at the late laying period. ACKNOWLEDGMENTS This work was funded in part by the Programs for Changjiang Scholars and Innovative Research in University (IRT_15R62) and by the China Agriculture Research Systems (CARS-41). REFERENCES Ajioka R. S. , Phillips J. D. , Kushner J. P. . 2006 . Biosynthesis of heme in mammals . BBA-Mol. Cell Res. 1763 : 723 – 736 . Brusnahan S. K. , McGuire T. R. , Jackson J. D. , Lane J. T. , Garvin K. L. , O’Kane B. J. , Berger A. M. , Tuljapurkar S. R. , Kessinger M. A. , Sharp J. G. . 2010 . 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The effect of time of oviposition and age on egg weight, egg components weight and eggshell quality . Arch. Geflugelkd . 73 : 110 – 115 . Wang X. T. , Zhao C. J. , Li J. Y. , Xu G. Y. , Lian L. S. , Wu C. X. , Deng X. M. . 2009 . Comparison of the total amount of eggshell pigments in Dongxiang brown-shelled eggs and Dongxiang blue-shelled eggs . Poult. Sci. 88 : 1735 – 1739 . Google Scholar CrossRef Search ADS PubMed With T. K. 1974 . Porphyrins in egg-shells . Biochem. J. 137 : 597-& . Google Scholar CrossRef Search ADS PubMed Yang H. M. , Wang Z. Y. , Lu J. . 2009 . Study on the relationship between eggshell colors and egg quality as well as shell ultrastructure in Yangzhou chicken . Afr. J. Biotechnol. 8 : 2898 – 2902 . Yuan Z. H. , Zhang K. Y. , Ding X. M. , Luo Y. H. , Bai S. P. , Zeng Q. F. , Wang J. P. . 2016 . Effect of tea polyphenols on production performance, egg quality, and hepatic antioxidant status of laying hens in vanadium-containing diets . Poult. Sci. 95 : 1709 – 1717 . Google Scholar CrossRef Search ADS PubMed Zhao R. , Xu G. Y. , Liu Z. Z. , Li J. Y. , Yang N. . 2006 . A study on eggshell pigmentation: Biliverdin in blue-shelled chickens . Poult. Sci. 85 : 546 – 549 . Google Scholar CrossRef Search ADS PubMed Zita L. , Tůmová E. , Štolc L. . 2009 . Effects of Genotype, Age and Their Interaction on Egg Quality in Brown-Egg Laying Hens . Acta. Vet. Brno. 78 : 85 – 91 . Google Scholar CrossRef Search ADS © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Brown eggshell fading with layer ageing: dynamic change in the content of protoporphyrin IX

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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Abstract

ABSTRACT The dynamic change in brown eggshell color as hens age has been observed, but much uncertainty still exists. We aimed to analyze the depth of eggshell color and quantity of protoporphyrin ΙΧ throughout the laying period to explore the reasons for color variation. In this study, 120 Rhode Island Red hens were used, and two eggs were collected from each individual at 26, 34, 42, 50, 60, and 70 wk of age. The eggshell color (L*, a*, b*), egg weight, eggshell dry weight, and protoporphyrin ΙΧ quantity in eggshell were measured for individual eggs. Our results showed that the intensity of brown eggshell color weaken as hens aged from 26 to 60 wk of age (L* gradually increased from 61.43 to 68.07), while eggshell lightness recovered slightly at 70 wk (L* = 64.77). The correlation analysis indicated that the content of protoporphyrin ΙΧ deposited in the eggshell was an important factor for lightness fading with the ageing process (the average r was 0.66, P < 0.01), while the egg weight had little impact on the eggshell color (the average r was 0.07, P > 0.05). The shade of the eggshell color (L* and a*) at the early laying period (26 or 34 wk) had a low correlation with the other age points (42, 50, 60, and 70 wk). However, high correlations between the shell color at 42 wk of age and subsequent ages (50, 60, and 70 wk) were found, suggesting that the intensity of eggshell color is more stable after egg-laying peaks (such as 42 wk of age). In conclusion, the intensity of brown eggshell color varies greatly among the whole laying cycle and breeders can choose the proper age for eggshell color measurements to ensure the degree of shell color in the late laying period. INTRODUCTION Brown eggshell color, as an economic trait, has been a source of concern for a long time. However, the degree of brownness changes over the entire laying cycle, which has decreased interest among retailers. Understanding the immediate causes of variation in coloration is beneficial to guarantee the color intensity of eggs. The chemical basis and mechanisms of color production have been characterized (Kennedy and Vevers, 1973; Ajioka et al., 2006; Zhao et al., 2006; Hanley et al., 2015). Though brown eggs have various intensities of color, several tetrapyrrole compounds, especially protoporphyrin ΙΧ (With, 1974; Gorchein et al., 2009; Igic et al., 2010), are responsible for generating this spectrum of colors via subtle concentration changes (Hanley et al., 2015). These pigments allow eggshells to perform many functions, including improving eggshell quality (Yang et al., 2009), decreasing incubation period (Sanz and García-Navas, 2009), preventing Gram-positive bacterial infections (Ishikawa et al., 2010), and protecting the embryo from solar radiation (Lahti and Ardia, 2016). As hypotheses on the functions of eggshell color have proliferated and the precision of color measurement has improved, many studies have focused on the factors regulating or disrupting eggshell color. In these studies, eggshell color has been correlated with many environmental factors, such as housing systems (Sekeroglu et al., 2010), nutrition (Seo et al., 2010; Yuan et al., 2016), and environmental pollutants (Hanley and Doucet, 2012). Compared with external environmental factors, poultry breeders are more interested in determining the internal factors that produce dark brown eggshells through selective breeding programs. Finally, the studies recognized that hen age can influence eggshell color, especially in the late laying period. Generally, eggshell becomes paler as the laying period progresses (Hunton, 1926; Hall, 1944; Ingram et al., 2008; Tumova and Ledvinka, 2009; Zita et al., 2009). However, it remains unclear how this process happens. As early as ten years ago, Odabasi (Odabasi et al., 2007) reported that older hens laid lighter colored eggs due to an increase in egg size associated with no proportionate change in the quantity of pigment deposited over the shell surface. In contrast, recent research demonstrated that eggshell color was correlated not with egg weight but with eggshell pigmentation (protoporphyrin) (Samiullah et al., 2016), which is the precursor of protoheme (Heinemann et al., 2008). Thus, reduced pigmentation is expected as a hen ages because hematopoiesis functions appear to progressively decline (Brusnahan et al., 2010). Despite relentless efforts that have been made, the reason why eggs fade over time and the way to resolve this problem were still unclear. Analysis of data and of information from scientific experiments is used for evidence-based decision making regarding how to make eggshell color more consistent and how to adapt eggshell color to local and national markets. The main objective of this paper is to describe the dynamic change in brown eggshell color with the increasing age of hens and to provide a breeding strategy in order to improve the color intensity of eggs during the whole egg-laying period. MATERIALS AND METHODS Egg Collection This study was carried out on 120 Rhode Island Red hens, which were selected based on clutch trait, during the whole egg-laying period. The hens were reared in individual cages under the 16L:8D lighting schedule and had ad libitum access to feed and water. At 26, 34, 42, 50, 60, and 70 wk of age, the eggs were collected from each layer on 3 successive days to ensure 2 eggs for each hen. The time interval of measurement was extended from eight weeks to ten weeks because the hens were transferred to other houses at 56 week of age due to the need for production. To minimize the influence of stress, we collected eggs four weeks later when laying performance returned to a normal level. Eggshell Color Measurement Eggshell color was measured with a reflectometer (Konica Minolta, Tokyo, Japan) using three parameters: L* measures lightness (0 is black; 100 is white), a* describes the balance of red and green (< 0 is green; > 0 is red), and b* represents hue as a function of a blue-yellow scale (< 0 is blue; > 0 is yellow). Protoporphyrin ΙΧ Measurement Eggs were cleaned with distilled water, air dried, and weighed by a micro-electronic balance. Egg internal contents and eggshell membrane were removed, and the remaining eggshell was ground into powder. After 24 h of drying in the oven, the total weight of the eggshell power was recorded, and a small amount (0.25 g) was dissolved in 6 mL extraction solution, a mixture of 4 mL methanol and 2 mL concentrated HCl. The solution was allowed to stand in the dark for 48 h until the pigment was completely dissolved. After centrifugation at 1,369.55 × g for 45 min, the absorbance of supernatant solution was measured using a microplate spectrophotometer (Tecan, Port Melbourne, Australia) at a wavelength of 412 nm. The concentration of protoporphyrin ΙΧ in eggshell (nmol g−1, CPES) was calculated by substituting absorbance in the linear regression equation deduced from the standard curve of protoporphyrin ΙΧ (Wang et al., 2009). The total content of protoporphyrin ΙΧ in eggshell (nmol, TPES) was equal to the product of its concentration and dry weight. Statistical Analysis First, the average value of two eggs from the same hen in the same week was calculated and the descriptive analyses for all variables (egg weight, eggshell dry weight, concentration of eggshell protoporphyrin ΙΧ, total content of eggshell protoporphyrin ΙΧ, and eggshell color: L*a*b*) were performed to summarize the characteristics of hens at different weeks of age. Second, a mixed linear model where time was the fixed effect and the hen was the random effect was used. After comparing covariance structures via goodness of fit criteria that were printed by PROC MIXED, we choose the best fit and calculated the least square means for multiple comparisons with the Bonferroni adjustment using SAS 9.2 software (SAS Institute Inc., Cary, NC). The level of statistical significance was set to P-value of <0.01. Third, the correlation coefficient between different traits and different weeks was analyzed with the Pearson correlation using R version 3.0.3, while the missing data were pairwise deleted. RESULTS Comparison of Eggshell Color, Egg Weight, and Content of Protoporphyrin ΙΧ in Eggshell at Different Weeks of Age During the egg-laying period, the average value of L* was gradually increased and reached its peak at 60 wk of age, while a slight but significant decline could be observed at 70 wk (Table 1). The average value of a* started high and experienced a consistent decrease over time, except at 70 wk of age, when an upward trend occurred. These two results showed that the brown eggshell color faded with the ageing of hens from 26 to 60 wk of age, while the shade of color recovered slightly at 70 wk. The value of b* showed a significant increase at 34 wk of age, and after this point, the value levelled off. In the early laying period, egg weight (EW) showed an upward trend, and after 50 wk, its mean value was flat, with no significant shift over time. A similar trend was observed for eggshell dry weight (ESDW). As opposed to the subtle change in eggshell dry weight, eggshell pigmentation showed a surprising variation, as it dropped by 43% between 26 and 34 wk of age for the concentration of CPES and the total content of TPES. The downward trend of CPES continued until 60 wk of age, though the change narrowed to a small extent. In comparison with 60 wk of age, CPES at 70 wk significantly increased and reached the same level as at 42 and 50 wk. Additionally, the variation of TPES over time followed the similar direction as CPES. Table 1. Comparison of eggshell color, content of protoporphyrin ΙΧ in eggshell, and egg weight at different weeks of age. 2Variables Age (wk) 26 34 42 50 60 70 L* 161.43 ± 0.49E 62.19 ± 0.48DE 63.17 ± 0.36CD 63.71 ± 0.33C 68.07 ± 0.35A 64.77 ± 0.37B a* 17.71 ± 0.29A 16.98 ± 0.28A 15.61 ± 0.19B 15.41 ± 0.19B 13.22 ± 0.21D 14.27 ± 0.21C b* 29.14 ± 0.25B 30.36 ± 0.23A 27.74 ± 0.18C 27.47 ± 0.18C 27.24 ± 0.20C 27.55 ± 0.21C EW (g) 55.24 ± 0.54C 57.23 ± 0.56BC 58.65 ± 0.38B 60.70 ± 0.40A 60.12 ± 0.41A 60.01 ± 0.45A ESDW (g) 4.89 ± 0.06D 4.94 ± 0.05CD 5.11 ± 0.04BC 5.44 ± 0.05A 5.35 ± 0.05A 5.27 ± 0.05AB CPES (nmol g−1) 121.49 ± 2.85A 68.87 ± 1.88B 61.46 ± 1.41C 59.67 ± 1.38C 49.82 ± 1.38D 60.21 ± 1.63C TPES (nmol) 591.77 ± 14.00A 339.96 ± 9.82B 311.98 ± 7.24C 323.07 ± 7.13BC 264.21 ± 7.27D 315.09 ± 8.58BC 2Variables Age (wk) 26 34 42 50 60 70 L* 161.43 ± 0.49E 62.19 ± 0.48DE 63.17 ± 0.36CD 63.71 ± 0.33C 68.07 ± 0.35A 64.77 ± 0.37B a* 17.71 ± 0.29A 16.98 ± 0.28A 15.61 ± 0.19B 15.41 ± 0.19B 13.22 ± 0.21D 14.27 ± 0.21C b* 29.14 ± 0.25B 30.36 ± 0.23A 27.74 ± 0.18C 27.47 ± 0.18C 27.24 ± 0.20C 27.55 ± 0.21C EW (g) 55.24 ± 0.54C 57.23 ± 0.56BC 58.65 ± 0.38B 60.70 ± 0.40A 60.12 ± 0.41A 60.01 ± 0.45A ESDW (g) 4.89 ± 0.06D 4.94 ± 0.05CD 5.11 ± 0.04BC 5.44 ± 0.05A 5.35 ± 0.05A 5.27 ± 0.05AB CPES (nmol g−1) 121.49 ± 2.85A 68.87 ± 1.88B 61.46 ± 1.41C 59.67 ± 1.38C 49.82 ± 1.38D 60.21 ± 1.63C TPES (nmol) 591.77 ± 14.00A 339.96 ± 9.82B 311.98 ± 7.24C 323.07 ± 7.13BC 264.21 ± 7.27D 315.09 ± 8.58BC 1Values are presented as the mean ± S.E. Means with different superscripts within each row differ significantly (P < 0.01). 2EW = egg weight, ESDW = eggshell dry weight, CPES = concentration of protoporphyrin ΙΧ in eggshell, TPES = total content of protoporphyrin ΙΧ in eggshell. View Large Table 1. Comparison of eggshell color, content of protoporphyrin ΙΧ in eggshell, and egg weight at different weeks of age. 2Variables Age (wk) 26 34 42 50 60 70 L* 161.43 ± 0.49E 62.19 ± 0.48DE 63.17 ± 0.36CD 63.71 ± 0.33C 68.07 ± 0.35A 64.77 ± 0.37B a* 17.71 ± 0.29A 16.98 ± 0.28A 15.61 ± 0.19B 15.41 ± 0.19B 13.22 ± 0.21D 14.27 ± 0.21C b* 29.14 ± 0.25B 30.36 ± 0.23A 27.74 ± 0.18C 27.47 ± 0.18C 27.24 ± 0.20C 27.55 ± 0.21C EW (g) 55.24 ± 0.54C 57.23 ± 0.56BC 58.65 ± 0.38B 60.70 ± 0.40A 60.12 ± 0.41A 60.01 ± 0.45A ESDW (g) 4.89 ± 0.06D 4.94 ± 0.05CD 5.11 ± 0.04BC 5.44 ± 0.05A 5.35 ± 0.05A 5.27 ± 0.05AB CPES (nmol g−1) 121.49 ± 2.85A 68.87 ± 1.88B 61.46 ± 1.41C 59.67 ± 1.38C 49.82 ± 1.38D 60.21 ± 1.63C TPES (nmol) 591.77 ± 14.00A 339.96 ± 9.82B 311.98 ± 7.24C 323.07 ± 7.13BC 264.21 ± 7.27D 315.09 ± 8.58BC 2Variables Age (wk) 26 34 42 50 60 70 L* 161.43 ± 0.49E 62.19 ± 0.48DE 63.17 ± 0.36CD 63.71 ± 0.33C 68.07 ± 0.35A 64.77 ± 0.37B a* 17.71 ± 0.29A 16.98 ± 0.28A 15.61 ± 0.19B 15.41 ± 0.19B 13.22 ± 0.21D 14.27 ± 0.21C b* 29.14 ± 0.25B 30.36 ± 0.23A 27.74 ± 0.18C 27.47 ± 0.18C 27.24 ± 0.20C 27.55 ± 0.21C EW (g) 55.24 ± 0.54C 57.23 ± 0.56BC 58.65 ± 0.38B 60.70 ± 0.40A 60.12 ± 0.41A 60.01 ± 0.45A ESDW (g) 4.89 ± 0.06D 4.94 ± 0.05CD 5.11 ± 0.04BC 5.44 ± 0.05A 5.35 ± 0.05A 5.27 ± 0.05AB CPES (nmol g−1) 121.49 ± 2.85A 68.87 ± 1.88B 61.46 ± 1.41C 59.67 ± 1.38C 49.82 ± 1.38D 60.21 ± 1.63C TPES (nmol) 591.77 ± 14.00A 339.96 ± 9.82B 311.98 ± 7.24C 323.07 ± 7.13BC 264.21 ± 7.27D 315.09 ± 8.58BC 1Values are presented as the mean ± S.E. Means with different superscripts within each row differ significantly (P < 0.01). 2EW = egg weight, ESDW = eggshell dry weight, CPES = concentration of protoporphyrin ΙΧ in eggshell, TPES = total content of protoporphyrin ΙΧ in eggshell. View Large Correlations between Eggshell Color with Egg Weight and Content of Protoporphyrin ΙΧ in Eggshell at the Same Week of Age Table 2 reflects that the lightness (L*, P < 0.01) and redness (a*, P < 0.01) of eggshell were both highly correlated with main brown eggshell pigment quantity, including the concentration and the total content of protoporphyrin ΙΧ in the eggshell, indicating a strong correlation between eggshell color and its quantity of protoporphyrin ΙΧ. The correlation was significant throughout the laying period, even though the association appeared slightly lower 70 wk of age. Looking at the correlation between yellowness (b*) of the eggshell and quantity of protoporphyrin ΙΧ, we observed a much weaker relationship compared with lightness and redness. In terms of the relationship between eggshell color and egg weight and eggshell dry weight, almost no correlation was found regardless of the time and trait. Overall, brown eggshell color was highly correlated with the content of protoporphyrin ΙΧ rather than egg size. Table 2. Correlations between eggshell color and egg weight and content of protoporphyrin ΙΧ in eggshell at the same week of age. Age (wk) Eggshell Colour 2EW ESDW CPES TPES 26 1L* −0.10 −0.12 −0.81** −0.85** a* 0.11 0.12 0.72** 0.76** b* 0.09 0.00 0.21 0.22 34 L* −0.18 −0.07 −0.66** −0.61** a* 0.24 0.18 0.58** 0.59** b* 0.16 0.16 0.38** 0.42** 42 L* 0.00 0.01 −0.79** −0.80** a* 0.03 0.04 0.79** 0.82** b* −0.06 −0.04 0.50** 0.49** 50 L* −0.07 −0.10 −0.80** −0.85** a* 0.09 0.07 0.77** 0.82** b* 0.02 −0.13 0.44** 0.40** 60 L* −0.06 −0.16 −0.77** −0.83** a* 0.14 0.23 0.72** 0.80** b* −0.04 0.07 0.42** 0.43** 70 L* −0.05 −0.05 −0.51** −0.52** a* 0.16 0.21 0.49** 0.58** b* 0.04 −0.03 0.26 0.22 Age (wk) Eggshell Colour 2EW ESDW CPES TPES 26 1L* −0.10 −0.12 −0.81** −0.85** a* 0.11 0.12 0.72** 0.76** b* 0.09 0.00 0.21 0.22 34 L* −0.18 −0.07 −0.66** −0.61** a* 0.24 0.18 0.58** 0.59** b* 0.16 0.16 0.38** 0.42** 42 L* 0.00 0.01 −0.79** −0.80** a* 0.03 0.04 0.79** 0.82** b* −0.06 −0.04 0.50** 0.49** 50 L* −0.07 −0.10 −0.80** −0.85** a* 0.09 0.07 0.77** 0.82** b* 0.02 −0.13 0.44** 0.40** 60 L* −0.06 −0.16 −0.77** −0.83** a* 0.14 0.23 0.72** 0.80** b* −0.04 0.07 0.42** 0.43** 70 L* −0.05 −0.05 −0.51** −0.52** a* 0.16 0.21 0.49** 0.58** b* 0.04 −0.03 0.26 0.22 1*significant linkage at P < 0.05; **highly significant linkage at P < 0.01. 2EW = egg weight, ESDW = eggshell dry weight, CPES = concentration of protoporphyrin ΙΧ in eggshell, TPES = total content of protoporphyrin ΙΧ in eggshell. View Large Table 2. Correlations between eggshell color and egg weight and content of protoporphyrin ΙΧ in eggshell at the same week of age. Age (wk) Eggshell Colour 2EW ESDW CPES TPES 26 1L* −0.10 −0.12 −0.81** −0.85** a* 0.11 0.12 0.72** 0.76** b* 0.09 0.00 0.21 0.22 34 L* −0.18 −0.07 −0.66** −0.61** a* 0.24 0.18 0.58** 0.59** b* 0.16 0.16 0.38** 0.42** 42 L* 0.00 0.01 −0.79** −0.80** a* 0.03 0.04 0.79** 0.82** b* −0.06 −0.04 0.50** 0.49** 50 L* −0.07 −0.10 −0.80** −0.85** a* 0.09 0.07 0.77** 0.82** b* 0.02 −0.13 0.44** 0.40** 60 L* −0.06 −0.16 −0.77** −0.83** a* 0.14 0.23 0.72** 0.80** b* −0.04 0.07 0.42** 0.43** 70 L* −0.05 −0.05 −0.51** −0.52** a* 0.16 0.21 0.49** 0.58** b* 0.04 −0.03 0.26 0.22 Age (wk) Eggshell Colour 2EW ESDW CPES TPES 26 1L* −0.10 −0.12 −0.81** −0.85** a* 0.11 0.12 0.72** 0.76** b* 0.09 0.00 0.21 0.22 34 L* −0.18 −0.07 −0.66** −0.61** a* 0.24 0.18 0.58** 0.59** b* 0.16 0.16 0.38** 0.42** 42 L* 0.00 0.01 −0.79** −0.80** a* 0.03 0.04 0.79** 0.82** b* −0.06 −0.04 0.50** 0.49** 50 L* −0.07 −0.10 −0.80** −0.85** a* 0.09 0.07 0.77** 0.82** b* 0.02 −0.13 0.44** 0.40** 60 L* −0.06 −0.16 −0.77** −0.83** a* 0.14 0.23 0.72** 0.80** b* −0.04 0.07 0.42** 0.43** 70 L* −0.05 −0.05 −0.51** −0.52** a* 0.16 0.21 0.49** 0.58** b* 0.04 −0.03 0.26 0.22 1*significant linkage at P < 0.05; **highly significant linkage at P < 0.01. 2EW = egg weight, ESDW = eggshell dry weight, CPES = concentration of protoporphyrin ΙΧ in eggshell, TPES = total content of protoporphyrin ΙΧ in eggshell. View Large Correlation of Eggshell Color among Different Weeks of Age The correlation of eggshell color in different weeks is illustrated in Figure 1. Looking at the L* value as an example, we found that the L* value was highly correlated with two adjacent time periods, especially in the similar period when production was divided by the egg-laying peak. To be specific, during the early stage including 26 and 34 wk, the correlation coefficient of L* was as high as 0.85 (P < 0.01), and after the egg-laying peak, the coefficients were 0.80 (P < 0.01), 0.83 (P < 0.01), and 0.76 (P < 0.01) between the two neighboring weeks, respectively, while only a low correlation was found between the two laying periods, as illustrated by 0.30 (P < 0.01) between 34 and 42 wk of age. The highly significant correlation of L* between 42 and 60 wk (r = 0.77, P < 0.01), 42 and 70 wk (r = 0.70, P < 0.01), and 50 and 70 wk (r = 0.77, P < 0.01) were observed, which indicates a constant intensity of eggshell color after the egg-laying peak. Replicating the analysis on a* and b*, similar patterns appeared and are depicted in Figure 1. The redness and yellowness of eggshell at 42 wk was highly correlated with itself in later weeks. Figure 1. View largeDownload slide The correlation matrix plots of L*(A), a*(B), and b*(C) values at different weeks of age. The upper panel shows the correlation matrix circles, and the larger area represents a higher correlation. The lower panel shows the pairwise correlations. Figure 1. View largeDownload slide The correlation matrix plots of L*(A), a*(B), and b*(C) values at different weeks of age. The upper panel shows the correlation matrix circles, and the larger area represents a higher correlation. The lower panel shows the pairwise correlations. Since an obvious distinction occurred at 42 wk of age, we selected this point as the base time and calculated the correlation with other time points. As is shown in Table 3 and Figure 1, a weak and then much stronger correlation appeared on the first two weeks and the next three weeks respectively, no matter which parameters we studied. Take lightness (L*) and redness (a*) of eggshell as an example, the correlation between base time and the earlier time (26 wk, r = 0.22 and 34 wk, r = 0.30) was so weak that we cannot deduce that eggshell color relied on the former record. Instead, the intensity of the color of eggs at the late laying period can be predicted according to the data at 42 wk due to the high correlation. As long as we select layers producing dark brown eggs at 42 wk of age, we would likely be able to obtain darker eggs during the following egg-raising period. Table 3. Correlation of variables at 42 weeks of age with other weeks. Age (wk) EW ESDW CPES TPES 26 10.24** 0.16 0.33** 0.31** 34 0.11 0.16 0.34** 0.31** 50 0.81** 0.76** 0.90** 0.91** 60 0.74** 0.67** 0.87** 0.84** 70 0.71** 0.55** 0.62** 0.56** Age (wk) EW ESDW CPES TPES 26 10.24** 0.16 0.33** 0.31** 34 0.11 0.16 0.34** 0.31** 50 0.81** 0.76** 0.90** 0.91** 60 0.74** 0.67** 0.87** 0.84** 70 0.71** 0.55** 0.62** 0.56** 1*significant linkage at P < 0.05; **highly significant linkage at P < 0.01. View Large Table 3. Correlation of variables at 42 weeks of age with other weeks. Age (wk) EW ESDW CPES TPES 26 10.24** 0.16 0.33** 0.31** 34 0.11 0.16 0.34** 0.31** 50 0.81** 0.76** 0.90** 0.91** 60 0.74** 0.67** 0.87** 0.84** 70 0.71** 0.55** 0.62** 0.56** Age (wk) EW ESDW CPES TPES 26 10.24** 0.16 0.33** 0.31** 34 0.11 0.16 0.34** 0.31** 50 0.81** 0.76** 0.90** 0.91** 60 0.74** 0.67** 0.87** 0.84** 70 0.71** 0.55** 0.62** 0.56** 1*significant linkage at P < 0.05; **highly significant linkage at P < 0.01. View Large To detect whether eggshell color was consistent during the late laying period and whether the selection process could be effective for screening the hens that laid darker eggs during the whole late process, we picked the first sixty hens listed according to the L* value of the eggshell at the last four weeks and found the overlapping individuals (Figure 2A). Only one hen laid eggs with a deeper intensity eggshell color at 42 wk but not at later weeks. There were 35 hens that ranked in the top half during the whole late laying process, and almost 82% of hens emerged in the top half three times. As opposed to setting 42 wk as the base time, the Venn diagram (Figure 2B) showed that 16 hens laid darker eggs just at 26 wk of age and that there were 21 overlapping individuals between 26 wk and the late laying period, which was reduced by 23%. Thus, we believe it is an effective and efficient program to choose the darker egg layers based on the eggshell color measurement after egg-laying peak. Figure 2. View largeDownload slide Venn diagram of hens ranked among the top half according to the L* value of eggshell color at different weeks of age. (A) Overlapping hens appeared at 42, 50, 60, and 70 wk of age. (B) Overlapping hens appeared at 26, 50, 60, and 70 wk of age. Figure 2. View largeDownload slide Venn diagram of hens ranked among the top half according to the L* value of eggshell color at different weeks of age. (A) Overlapping hens appeared at 42, 50, 60, and 70 wk of age. (B) Overlapping hens appeared at 26, 50, 60, and 70 wk of age. DISCUSSION The eggshell color turned pale with age increasing within 60 wk, which was consistent with the findings of many studies, even though eggshell color was measured using different methods (Hunton, 1926; Hall, 1944; Ingram et al., 2008; Tumova and Ledvinka, 2009; Zita et al., 2009). Considering the two assumptions regarding why an eggshell gradually fades over the ageing process (Odabasi et al., 2007; Samiullah et al., 2016), we compared the change in the egg weight and quantity of protoporphyrin ΙΧ in eggshell. There was a tendency for main brown eggshell pigment, including CPES and TPES, to change considerably during the first two weeks and still display a dynamic state during the late laying period, while the egg weight and eggshell dry weight changed slowly in the first four age points and remained comparatively stable after 50 wk. The change tendency, however, did not decrease our confusion; thus, we performed a correlation analysis between eggshell color and egg weight and the content of main brown eggshell pigment. From the results, we found that the shade of eggshell color was highly correlated with the quantity of protoporphyrin ΙΧ in the eggshell. Somewhat unexpectedly, the egg weight and eggshell dry weight, which were reported as the main effectors of eggshell color in a previous study (Odabasi et al., 2007), were found to poorly correlate with eggshell color (L* or a*). The comparison between these two correlation coefficients led us to the conclusion that the content of main brown eggshell pigment had a much greater influence on eggshell color than egg weight and eggshell dry weight. The higher the quantity of protoporphyrin ΙΧ deposited in eggshell, the darker the eggshell color will be. Thus, the fading of eggshell color may be mainly caused by a decrease in available protoporphyrin ΙΧ in the eggshell, which is a result of degradation of the hen's physiological functioning. Eggshell color did not maintain a downward trend during the whole laying process and displayed an increase at 70 wk of age compared with 60 wk. Another important difference is that content of protoporphyrin ΙΧ in eggshell significantly increased, while egg weight and eggshell dry weight were not altered. In 1944, Hall (Hall, 1944) observed this phenomenon in a crossbreed population of White Leghorn and Rhode Red, and he proposed seasonal variation as an explanation. Samiullah et al. (2016) found that the concentration of protoporphyrin ΙΧ in eggshell was recovered to the previous level, which is in line with our research. This phenomenon may be due to decreased egg production and a prolonged oviposition interval. As hens age, their laying performance degrades, which was indicated by the decrease in the egg-laying rate from 95% at 26 wk of age to 86% at 70 wk. The oviposition interval became longer, providing enough time for hens to produce and accumulate protoporphyrin ΙΧ. The large quantity of available protoporphyrin ΙΧ was conducive for hens to produce darker eggshell. Because eggshell color fades with the ageing process, it is imperative for us to select chickens that consistently produce dark eggs. In 2011, Institute de Selection Animal proposed the breeding program“Breeding for 500 eggs in 100 weeks” and aimed to extend the laying cycle (Sambeek, 2011); however, the fact that the intensity of eggshell color fades with ageing set a barrier for brown egg chickens. The statistics indicate that it is economical and effective to select chickens with darker eggshell after the egg-laying peak (such as 42 wk of age) to guarantee the degree of darkness of eggs at the end of the laying period. 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Poultry ScienceOxford University Press

Published: Mar 1, 2018

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