J APPL POULT RES 2006. 15:425-432
© 2006 Poultry Science Association
Vitamin C Overcomes the Detrimental Effect of Vanadium on Brown Eggshell Pigmentation
A. Z. Odaba
i*,
R. D. Miles
,1,
M. O. Balaban*,
K. M. Portier
and
V. Sampath
* Food Science and Human Nutrition Department, Department of Animal Sciences, and Department of Statistics, University of Florida, Gainesville 32611
1 Corresponding author: rdmiles{at}ufl.edu
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SUMMARY
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Phosphorus supplementation from poor quality feed-grade sources may introduce excessive levels of vanadium (V) into the diets of laying hens. Vanadium in the diet of chickens has been shown to be detrimental to egg production, albumen height, BW, and feed consumption. This study showed that dietary V also has a negative effect on the color of brown eggshells from commercial-type layers. Commercial-type brown egg layers were fed a cornsoybean meal basal diet supplemented with 0, 50, or 100 ppm of V as NH4VO3 to determine the effect on shell pigmentation. Hens fed V at both dietary concentrations laid lighter colored eggs (less redness) after only 2 d. Another experimental group of the same type of hens that were fed lower V concentrations (0, 15, or 30 ppm) also had less shell pigmentation. After the detrimental "bleaching" effect on shell color was observed, these diets were supplemented with 1 of the following: no supplement, 100 ppm of vitamin C, 100 IU of vitamin E, or 100 ppm of ß-carotene. Only vitamin C restored the eggshell color of eggs from hens fed both levels of V. When these same antioxidants were added as preventive agents to diets supplemented with 15 ppm of V before the effects of V were established, brown layers benefited again from 100 ppm of vitamin C but not from vitamin E or ß-carotene.
Key Words: vanadium • shell color • egg-type layer • vitamin • brown egg
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DESCRIPTION OF PROBLEM
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Phosphorus sources are known to be the cause of excessive V in various types of bird diets [1, 2]. Sullivan et al. [3] reported V concentrations ranging from 36 to 185 ppm in commercially available feed phosphates of both domestic and foreign origin (Russian Federation, Poland, Japan). In another study [4] comparable means (134 vs. 139 ppm) and ranges (2 to 195 ppm) of V were reported in commercial feed-grade phosphate sources from Brazil. The NRC [5] recommends the presence of very low levels of V in poultry diets, with the maximum tolerance level established as 10 ppm [6].
Vanadium in the diet of chickens has been shown to be detrimental to egg production, interior quality of eggs (albumen height), BW, and feed consumption [7, 8, 9]. The exact mechanism of V toxicosis in birds has not yet been elucidated. However, V appears to exert its toxic effect through inhibition of enzymes and cell damage from lysis [10]. The first report in the literature documenting the negative (depigmenting) effect that dietary V has on brown eggshell color was that of Sutly et al. [11]. Recently, Miles and Henry [12] reported the negative effect of storage time and conditions on albumen quality of eggs from hens fed V. The negative influence of V on performance and immune responses of commercial egg-type hens has been documented [13]. However, no studies have been reported that were designed to investigate the effects of V on shell pigmentation of eggs obtained from commercial-type brown egg layers.
Bone, kidney, liver, and oviduct (magnum) were found to be the primary retention sites for V administered orally to laying hens [14]. The magnum is the albumen-secreting portion of the hen’s oviduct. It was suggested that the decrease in magnum weight observed in hens fed diets containing 30 ppm of V was due to muscle atrophy, which was in turn caused by decreased motility [7]. Hens laying eggs with poor albumen quality (lower Haugh units) have also been reported to have magnum folds with less height, which has been attributed to either genetics or consumption of V [15].
Several dietary supplements have been investigated with regards to their ability to counteract the detrimental effects of V, including vitamin C [16, 17, 18], chromium [16, 17], and EDTA [17, 19]. Interference with V absorption [11, 19], interactions with other mineral elements, increased body elimination, or reducing the toxicity of V in tissues [16] are suggested mechanisms in counteracting the effects of V. Feed-grade phosphate sources containing high concentrations of V can originate from any part of the world where rock phosphate deposits are mined. Normally, poultry diets contain supplemental phosphate supplied from good quality feed-grade sources. However, when worldwide feed phosphate prices increase, the cheaper, inferior sources will often find their way into the feed ingredient market. In such cases, when diets of laying hens contain higher than desired concentrations of V and detrimental effects on performance are expected, knowledge of how to prevent and overcome these negative effects would be beneficial.
Although shell color is not an indication of internal quality of eggs, consumers in some markets throughout the world prefer brown eggs to white eggs (United Kingdom, Italy, Portugal, Ireland, Southeast Asia, Australia, New Zealand). Shell color intensity within each country is dictated by consumer preference. For example, in the Japanese egg market there are standards for a uniform dark shell color, whereas other markets prize a uniform light brown tint to the shell, which requires crossing of white and brown egg laying lines [20]. When selecting egg-laying lines with specific dark and light brown shell tints for such markets, capability of objective color measurement becomes critical. For this purpose, companies that produce these genetic lines of birds commonly use a colorimeter that measures the eggs’ lightness (L*) and hue [as a function of a red-green (a*) and a yellow-blue (b*) scale]. We recommend the use of a color machine vision (CMV) system to measure the color units more accurately. A colorimeter measures the color of only a small portion of the shell’s surface area, whereas with CMV the color of the entire surface facing the camera is determined. The advanced CMV systems in use today have numerous advantages and uses. For example, it has recently been reported that the CMV system can successfully be used to detect different dirt stains on brown eggs [21].
The first objective of this study was to determine if V has a negative effect on shell pigmentation of eggs obtained from commercial-type brown egg layers. Having established such an effect, the second objective was to characterize the changes in eggshell color as a result of feeding concentrations of 15, 30, 50, and 100 ppm of V in the diet and to test different antioxidants (ß-carotene, vitamin C, vitamin E) for their ability to reverse the negative changes in eggshell pigmentation.
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MATERIALS AND METHODS
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Experiment 1
This experiment was conducted to determine if V affected eggshell color. From a flock of 500 sixty-week-old Hy-Line Brown egg layers [22], 3 eggs laid in sequence were collected from each bird, and their eggshell pigmentation was determined. The images of the eggs were acquired using a CMV system, which consisted of a rectangular light box (42.4 x 61 x 68.6 cm) and an analog color video camera, which was linked to a personal computer with Color Expert software [23]. A complete description of the CMV system is described by Odabai [24].
Based on the pigmentation of the shells, hens were selected, and 2 groups of birds were established from these hens, which laid eggs having distinctly different "shades" of shell color (dark or light brown). These 2 shade groups constituted the hens used in Experiment 1. Each group, consisting of 60, hens received 3 different experimental diets formulated from the corn or soybean meal basal diet (Table 1
) that otherwise met all the dietary requirements for layers in production [5]. The treatments were the addition of 0, 50, or 100 ppm of V as NH4VO3 to the basal diet. Each treatment was fed to 4 pens of 5 individually caged hens. The hens consuming the basal diet containing 0 ppm of V served as the control. Eggshell color of individual eggs was measured on the first 3 eggs laid by each hen after the onset of V feeding and every 3 d thereafter (d 1, 2, 3, 6, 9, 12, 15).
Experiment 2
This experiment was conducted to determine if the "bleaching" effect of V on eggshell pigmentation would be observed at lower dietary V concentrations and if the loss of pigmentation could be restored by feeding the antioxidants vitamin C, vitamin E, and ß-carotene. Sixty individually caged, 38-wk-old Hy-Line Brown hens received 1 of 3 different experimental diets: a corn or soybean meal basal diet or basal diet supplemented with NH4VO3 to provide concentrations of 0, 15, or 30 ppm of V. These levels of dietary V were used because we felt that even if diets were supplemented with high-V phosphate sources, they would supply no more than 30 ppm of V, the highest dietary concentration used in this experiment. The basal diet was similar to that used in Experiment 1 and contained all the dietary requirements for layers of this production age [5]. Each experimental diet was fed to 4 pens containing 5 individually caged hens. Eggs collected on d 3, 6, and 9 after the initiation of feeding were used in shell color analyses. This part of the study will be called Experiment 2a from this point on. After the eggs were collected on the ninth day, and because the bleaching effect was observed from d 3, the diets were changed so that each pen of 5 individually caged hens initially fed the V-containing diets was supplemented with 1 of the following anti-oxidants: none, 100 ppm of vitamin C, 100 IU of vitamin E, or 100 ppm of ß-carotene. Eggs collected on d 3, 6, 9, 12, and 15 following the diet change were used for shell color analyses. This portion of the study, from this point on, will be called Experiment 2b. The hens had ad libitum access to their experimental diets and water in Experiments 1, 2a, and 2b.
Experiment 3
This experiment was designed to investigate whether the previously observed negative effect of V on eggshell pigmentation could be prevented from occurring when V was added to layer diets already containing the antioxidants, supplemented at different dietary concentrations. The 6 dietary treatments were as follows: 1) basal diet; 2) basal +15 ppm of V; 3) basal +15 ppm of V and 100 ppm of ß-carotene; 4) basal +15 ppm of V and 100 ppm of vitamin C; 5) basal +15 ppm of V and 100 IU of vitamin E; 6) basal +15 ppm of V plus a mixture of 100 ppm of ß-carotene, 100 ppm of vitamin C, and 100 IU of vitamin E.
Each of the 6 experimental diets was fed to 5 randomly assigned, individually caged 42-wk-old Hy-Line Brown hens. Hens had ad libitum access to experimental diets and water for 10 d. To obtain a sufficient number of eggs from the hens, the eggs were collected from individual hens on d 8, 9, and 10, and their eggshell color was measured as described previously.
Statistical Analysis
A linear mixed model with first-order autoregressive covariance structure was used for eggshell color data collected from Experiments 1, 2a, and 2b. Shade (dark brown, light brown) was used as a block effect in Experiment 1. Multiple comparisons of the least squares means were carried out with adjustments such as the Bonferroni adjustment to prevent the inflation of the experiment-wise type I error. All statistical analyses were done using the SAS System for Windows, Release 8.01 [25].
The shell color data collected in Experiment 3 were analyzed using ANOVA. Due to the small sample size for each treatment, data from d 8, 9, and 10 were combined to test for differences among the treatment means. The least squares means of each group were compared with that of the control group, which consisted of hens fed the basal diet.
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RESULTS AND DISCUSSION
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Experiment 1: 0, 50, or 100 ppm of V
Feeding the hens diets supplemented with V resulted in a negative, bleaching effect on eggshell color. The effects of V, shade, and time on the eggshell color were significant (P 
0.01). The shade of the eggshell (dark brown, light brown) did not influence the effect of V. When V was added to the diets of hens laying dark or light brown shells, the magnitude of decline in pigmentation was essentially the same. Correspondingly, the effect of V on L*, a*, or b* did not change if the eggs were from a dark brown or a light brown group. Along the same lines, it did not matter if a hen typically laid an egg with a dark or a light shell (shade); the effect of V was the same. The interaction data for shade and V are not shown.
Because the effects of dietary V level on L* were not significantly different in light or dark shade groups (nonsignificant V by shade interaction), the data from dark and light groups were combined. The effect of both dietary levels of V was significant on d 3 (Table 2). Supplementing V to the diet at 100 ppm did not cause any further increase in bleaching of the eggshells compared with that observed with the diet containing 50 ppm of V.
The term "brown egg" is used universally. However, the effect of V on eggshell color is essentially an increase in L*. With the machine vision system, the actual cause of the bleaching effect could be elucidated.
Vanadium causes a reduction in the a* value of eggshells (Table 3). That is, significantly less red color is present in the eggshells when V is supplemented to the diet for longer than 2 d. Compared with 50 ppm of V, supplementing 100 ppm of V in the diet did not cause any further decline in the amount of a* of the shell. Because eggshell color is composed of a yellow component along with a red component, the amount of b* in the shell was also determined (Table 4). Feeding 50 or 100 ppm of V had no significant effect on the amount of yellow color deposited on the eggshell during the first 3 d. However, by d 6, a significant decrease in the b* value was observed at the 100-ppm level of V. This significant decrease in yellow color remained at least to d 12; the b* value increased to a point in which it was not significantly different from the other treatments, whereas the a* value (Table 3) did not return by d 15. It should be pointed out at this time that in the laboratory, a visual observation of the eggs by the investigators indicated that the bleaching effect seemed to be brought about by more yellow being observed as a* declined.
Experiment 2a: 0, 15, or 30 ppm of V
In Experiment 1 (Table 2
), feeding 50 or 100 ppm of V resulted in a significant decrease in eggshell pigmentation, as reflected in the L* values on d 3. Similarly, in Experiment 2a, as indicated by the L* values in Table 5, decreasing the level to 30 ppm, but not 15 ppm, also resulted in a significant decline in eggshell pigmentation by d 3, which continued through d 9.
It may be noted that the overall L* values for the eggshells from hens used in Experiment 1 (Table 2) are higher than the values in Experiment 2 (Table 5). This was because the hens used in Experiment 1 were 60 wk of age, compared with 38-wk-old hens in Experiment 2. The eggs from hens in Experiment 1 were larger than those collected from younger hens in Experiment 2; therefore, the amount of pigment in the shell of eggs in Experiment 2 was dispersed over a smaller area and thus resulted in a lower overall L* value.
The a* value for eggshells from hens fed 15 ppm of V did not differ significantly from the control at any period during the study (Table 6). However, feeding 30 ppm of V resulted in a similar significant decline in the amount of a* in the shell, as previously observed for 50 and 100 ppm of V (Table 3). There were no significant differences in the amount of b* in the shell due to feeding either 15 or 30 ppm of V (Table 7). These data would indicate that there seems to be a dietary threshold somewhere from 15 to 30 ppm of V that significantly affects the amount of a* deposited in the eggshell. Similarly, considering the b* data presented in Tables 4 and 7, V levels below 50 ppm do not seem to influence the amount of b* imparted to the shell.
Whether V is directly involved in enzyme or cofactor inhibition within the tubular gland cells of the shell gland, which results in deposition of less pigment in the shell, or it modifies the pigment molecules structurally at other sites before being delivered to the shell gland for deposition is not known. Further research to clarify the mechanism(s) of the change in shell color due to dietary V is needed.
Experiment 2b: Lower Levels of V Supplemented with Vitamins
The first 9 d of Experiment 2 (part 2a) served to establish the effects of V before the vitamins were introduced to the diets. As previously found in Experiment 2a, adding V at 15 and 30 ppm affected the L* value, and adding 30 ppm of V affected the amount of a* in the eggshells (P 0.01), but the amount of b* was not affected (Table 8).
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Table 8. Changes in lightness (L*), redness (a*), and yellowness (b*) of eggshells as influenced by V in experiment 2b1
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When ß-carotene, vitamin C, and vitamin E were supplemented to the diets containing either 15 or 30 ppm of V, only vitamin C restored eggshell pigmentation (P 0.05) to the level observed in the control eggshells (Table 9). Further analysis of the pigmentation data revealed that the a* component was totally restored, when compared with the control, by the addition of vitamin C (data not shown). Vitamin supplementation had no effect on the pigmentation of eggshells from hens fed the control diet, which contained no V.
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Table 9. Changes in lightness (L*) of eggshells from eggs from hens fed diets supplemented with antioxidants to counteract the previously established negative effect of V on pigmentation (Experiment 2b)1
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These results imply that, in a practical field situation, the bleaching effect on eggshells caused by hens consuming a diet containing 30 ppm of V or less can be overcome, and complete restoration of normal eggshell pigmentation would be expected when vitamin C is supplemented to the diet at 100 ppm.
The exact mechanism of restoration of pigmentation by vitamin C is not known. However, in this study, it was the only supplemental anti-oxidant that was water soluble, and this may have been a contributing factor, because it was more easily available for enzyme-catalyzed pigmentation reactions occurring in the water phase of the uterine (shell gland) cells. Similarly, the exact mechanism of action involved in the ability of vitamin C and other dietary components to restore egg albumen quality is also not known but has been speculated by several investigators [14, 16]. For example, Benabdeljelil and Jensen [16] were able to reduce the negative effects of 10 ppm of V on albumen quality by supplementing 100 ppm of vitamin C to the diet. If a crisis situation occurs that necessitates the use of poor-quality, high-V phosphate sources, the detrimental effect of V on brown eggshell pigmentation would be minimal if a diet contained at least 100 ppm of supplemental vitamin C.
Experiment 3: Simultaneous Supplementation of the Diets with 15 ppm of V and Antioxidants
In Experiment 2b, vitamin C was able to restore the eggshell pigmentation after the negative, bleaching effect of V had already been established. This experiment investigated whether 1 or more of the antioxidants would be able to prevent the negative bleaching effect of V when supplemented simultaneously with V at the initiation of the feeding period. When the pigmentation data (Table 10) for each treatment were compared with the control, vitamin C was the only antioxidant that prevented the negative effect that V had on pigmentation, as indicated by L* values that did not differ significantly from control. Neither vitamin E nor ß-carotene was able to prevent the negative effect of V on pigmentation when they were fed alone in the diet. A combination of all 3 antioxidants in the diet prevented the negative effect that V had on eggshell pigmentation, thus indicating that vitamin C indeed can prevent a decline in pigmentation from occurring. The pigmentation data (Table 10) indicated that vitamin C prevents the decline in a* value associated with V. The b* component was not affected by V at 15 ppm (Table 7). Research is needed to determine the minimum concentration of vitamin C in the diet that will successfully overcome the negative bleaching effect of V on brown eggshell pigmentation.
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Table 10. Changes in lightness (L*), redness (a*), and yellowness (b*) of eggshells from eggs from hens fed diets supplemented with V and antioxidants simultaneously (Experiment 3)1
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CONCLUSIONS AND APPLICATIONS
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- Vanadium, when fed in the diet at 15 ppm or above, has a detrimental effect on eggshell pigmentation of eggs from commercial-type brown egg layers.
- Supplementing 100 ppm of vitamin C to diets already containing up to 30 ppm of V will completely restore eggshell pigmentation.
- When up to 15 ppm of V is present in the diet, supplementing 100 ppm of vitamin C before or at the same time V is introduced to the diet will "prevent" a decline in eggshell pigmentation.
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REFERENCES AND NOTES
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- Berg, L. R. 1963. Evidence of vanadium toxicity resulting from the use of certain commercial phosphorus supplements in chick rations. Poult. Sci. 42:766–769.
- Romoser, G. L., L. Loveless, L. J. Machlin, and R. S. Gordon. 1960. Toxicity of vanadium and chromium for the growing chicken. Poult. Sci. 39:1288. (Abstr.)
- Sullivan, T. W., J. H. Douglas, and N. J. Gonzalez. 1994. Levels of various elements of concern in feed phosphates of domestic and foreign origin. Poult. Sci. 73:520–528.[Web of Science][Medline]
- Lima, F. R., C. X. Mendonça, J. C. Alvarez, G. Ratti, S. L. R. Lenharo, H. Kahn, and J. M. F. Garzillo. 1995. Chemical and physical evaluations of commercial dicalcium phosphates as sources of phosphorus in animal nutrition. Poult. Sci. 74:1659–1670.[Web of Science][Medline]
- National Research Council. 1994. Nutrient Requirements of Poultry. 9th ed. Natl Acad. Sci. Washington, DC.
- Henry, P. R., and R. D. Miles. 2001. Heavy metals—vanadium in poultry. Ciênc. Anim. Bras. 2:11–26.
- Eyal, A., and J. R. Moran. 1984. Egg changes associated with reduced interior quality because of dietary vanadium toxicity in the hen. Poult. Sci. 63:1378–1385.
- Sell, J. L., J. A. Arthur, and I. L. Williams. 1982. Adverse effect of dietary vanadium, contributed by dicalcium phosphate, on albumen quality. Poult. Sci. 61:2112–2116.[Web of Science][Medline]
- Bressman, R. B., R. D. Miles, C. W. Comer, H. R. Wilson, and G. D. Butcher. 2002. Effect of dietary supplementation of vanadium in commercial egg-type laying hens. J. Appl. Poult. Res. 11:46–53.[Abstract/Free Full Text]
- Waters, M. D., D. E. Gardner, C. Aranyi, and D. L. Coffin. 1975. Metal toxicity for rabbit alveolar macrophages in vitro. Environ. Res. 9:32–47.[Medline]
- Sutly, J. P., R. D. Miles, C. W. Comer, and M. O. Balaban. 2001. The influence of vanadium on pigmentation of brown-shelled eggs. Poult. Sci. 80(Suppl. 1):1039. (Abstr.)
- Miles, R. D., and P. R. Henry. 2004. Effect of time and storage conditions on albumen quality of eggs from hens fed vanadium. J. Appl. Poult. Res. 13:619–627.[Abstract/Free Full Text]
- Davis, E. G., R. D. Miles, G. D. Butcher, and C. W. Comer. 2002. Effect of vanadium on performance and immune responses of commercial egg-type laying hens. J. Appl. Anim. Res. 22:113–124.
- Sell, J. L., C. Y. Davis, and S. E. Scheideler. 1986. Influence of cottonseed meal on vanadium toxicity and 48vanadium distribution in body tissues of laying hens. Poult. Sci. 65:138–146.[Web of Science][Medline]
- Toussant, M. J., D. E. Swayne, and J. D. Latshaw. 1995. Morphological characteristics of oviducts from hens producing eggs of different Haugh units caused by genetics and by feeding vanadium as determined with computer software-integrated digitizing technology. Poult. Sci. 74:1671–1676.[Web of Science][Medline]
- Benabdeljelil, K., and L. S. Jensen. 1990. Effectiveness of ascorbic acid and chromium in counteracting the negative effects of dietary vanadium on interior egg quality. Poult. Sci. 69:781–786.[Web of Science][Medline]
- Ousterhout, L. E., and L. R. Berg. 1981. Effects of diet composition on vanadium toxicity in laying hens. Poult. Sci. 60:1152–1159.[Web of Science][Medline]
- Toussant, M. L., and J. D. Latshaw. 1994. Evidence of multiple metabolic routes in vanadium’s effects on layers. Ascorbic acid differential effects on prepeak egg production parameters following prolonged vanadium feeding. Poult. Sci. 73:1572–1580.[Web of Science][Medline]
- Hathcock, J. N., C. H. Hill, and G. Matrone. 1964. Vanadium toxicity and distribution in chicks and rats. J. Nutr. 82:106–110.[Abstract/Free Full Text]
- Arthur, J. A., and N. O’Sullivan. 2005. Breeding chickens to meet egg quality needs. Int. Hatchery Pract. 19:7–9.
- Mertens, K., B. De Ketelaere, B. Kamers, F. R. Bamelis, B. J. Kemps, E. M. Verhoelst, J. G. De Baerdemaeker, and E. M. Decuypere. 2005. Dirt detection on brown eggs by means of color computer vision. Poult. Sci. 84:1653–1659.[Abstract/Free Full Text]
- Hy-Line Int., West Des Moines, IA.
- The software has been developed by M.O. Balaban, who can be contacted about the color machine vision system at mobalaban{at}i-fas.ufl.edu. The color of each individual egg was calculated from the computer images and reported as the L*, a*, and b* values of the eggshell surface facing the camera. The L* value represents lightness and ranges from 0 to 100, with 0 corresponding to black and 100 to white. The chromatic attributes, redness-greenness and yellowness-blueness are measured by a* and b*, respectively. Positive values of a* represent the amount of redness of the shell color, whereas negative values of a* indicate the amount of greenness in the shell color. Similarly, the yellow and blue components in any color are represented by positive and negative values of b*, respectively.
- Odabai, A. Z. 2003. Shell color and other quality attributes of brown eggs as affected by the hens’ age and vanadium in their diet. PhD Thesis. Univ. Florida. Gainesville.
- SAS Inst. Inc. Cary, NC.
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DESCRIPTION OF PROBLEM
