Supplementing Hen Diets with Vitamins A and E Affects Egg Yolk Retinol and α-tocopherol Levels

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1 2003 Poultry Science Association, Inc. Supplementing Hen Diets with Vitamins A and E Affects Egg Yolk Retinol and α-tocopherol Levels A. V. Mori, C. X. Mendonça Jr., 1 C. R. M. Almeida, and M. C. G. Pita Departamento de Clínica Médica, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Cidade Universitária Armando de Salles Oliveira, Av. Orlando Marques de Paiva 87, São Paulo, , Brazil Primary Audience: Nutritionists, Researchers, Poultry Producers, Feed Manufacturers SUMMARY Laying hens were fed a basal diet supplemented with increasing levels of retinyl acetate and α-tocopheryl acetate to investigate the effects of vitamin A and E supplementation upon egg yolk retinol and tocopherol concentrations. The high concentration of added vitamin E caused a decline in egg production and poor feed conversion. Egg quality was not affected by vitamin A and E levels. Yolk retinol concentration was enhanced by added vitamin A, from 24.6 IU/g for eggs from the control group, to 33.6 and 37.7 IU/g of yolk when hens were fed 15,000 and 30,000 IU/kg of diet. Yolk α-tocopherol was significantly increased by dietary tocopherol supplementation, ranging from 10.9 µg/g (control group) to 160.6, 264.1, and µg/g of yolk, respectively, when 200, 400 and 600 mg/kg of ration were added, respectively. Yolk α-tocopherol was increased by 24.9 and 44.0% with increasing vitamin A supplementation at 15,000 and 30,000 IU/kg of diet, respectively. When correlation coefficients and regression equations were calculated, it was found that yolk α- tocopherol decreased (P < 0.05) as supplemental vitamin A increased, indicating the adverse effect of dietary vitamin A on yolk tocopherol deposition. The nutritional value of eggs, related to retinol and tocopherol, can be improved by dietary manipulation of hens diet, but attention must be focused on their inter-relationship. Key words: egg yolk, hen, retinol, vitamin A, vitamin E, α-tocopherol, laying performance 2003 J. Appl. Poult. Res. 12: DESCRIPTION OF PROBLEM Today, consumers are much more concerned about the influence of diets on health than in previous periods, which has encouraged studies related mainly to changes in egg lipids and vitamins. Dietary vitamin A and E affect egg yolk levels of retinol and tocopherol. Squires and Naber [1] reported that yolk vitamin A content from hens fed 16,000 IU/kg was 24.0 IU/g, more than twice the value of eggs from hens fed diets containing the levels recommended by NRC [2]. Jiang et al. [3] achieved significant and progressive incorporation of α-tocopherol in egg yolk by feeding diets containing 50, 100, 200, or 400 mg α-tocopheryl acetate/kg, without impairing egg production. Besides the increase in yolk vitamin E concentration, the incorporation of tocopherols in hen diets prevents lipid oxidative deterioration of the ration [4, 5]. Studies have demonstrated that a high intake of vitamin A may interfere negatively with other 1 To whom correspondence should be addressed: cxmendon@usp.br.

2 MORI ET AL.: VITAMIN A AND E IN EGG YOLK 107 fat-soluble vitamins, particularly vitamin E. Frigg and Broz [6] supplemented chick diets with two levels of vitamin A (10,000 and 50,000 IU/kg) combined with addition of 0, 50, 100, or 150 mg vitamin E/kg and reported a decrease in plasma tocopherol concentration in birds fed the higher vitamin A level. In laying hens, Jiang et al. [3] showed a decrease in yolk α-tocopherol deposition when diets were supplemented with β-carotene. Attempts to reduce yolk cholesterol have not succeeded to meet the demands of health-conscious consumers, resulting in lower per capita egg consumption in recent years [7, 8, 9]. Fortification of eggs with vitamins A and E, however, has been shown [3, 5] to be a desirable alternative to increased egg consumption. The aim of this study was to evaluate the effect of dietary levels of vitamin A and E upon laying performance, egg quality, and egg yolk retinol and α- tocopherol concentrations. MATERIALS AND METHODS A total of 288, 33-wk-old Hy-Line hens was distributed into 36 replicates (four cages per replicate with two birds per cage) and randomly assigned to each of the 12 experimental diets (three replicates per treatment) for an 11-wk experiment. Egg production, egg weight, and feed consumption were recorded weekly. Average egg production, egg weight, feed intake, and feed conversion (kg of feed consumed per dozen eggs and per kg of eggs) were calculated by replicate group. Egg quality measurements were made during the last experimental week. The control group was composed of hens fed a commercial layer ration (17.0% CP, 4.5% calcium, and 0.6% phosphorus), with assayed levels of vitamin A and E of 9,050 and 7.39 IU/ kg of diet, respectively. This basal diet met all nutrient requirements for laying hens [2] and was formulated to provide three levels of vitamin A (as retinyl acetate 0, 5,000, or 30,000 IU/kg) in combination with four levels of tocopherol (as α-tocopheryl acetate 0, 200, 400, or 600 mg/kg). For the evaluation of shell quality, the specific gravity of 16 eggs per treatment (four per replicate) was determined by the saline solutions method [10]. Albumen quality (Haugh units) was evaluated by a S-8400 micrometer (Ames, Waltham, MA). Egg shells were individually weighed, and egg shell thickness was measured by a 25M-5 micrometer (Ames). Shell index (SI) was calculated according to Sauver [11] using the formula SI = SW/S 100 where SW is the shell weight (g) and S the surface area (cm 2 ). S is calculated from the egg weight (EW) from the equation S = K EW 2/3 where K is 4.67, 4.68, or 4.69, depending on egg weight, for eggs less than 60 g, between 60 and 70 g, or greater than 70 g, respectively. At the end of the experimental period, four eggs were randomly collected from each replicate and weighed, and albumen and yolk were separated. Yolks were individually weighed and were prepared by pooling and blending four yolks per sample. After samples were subjected to extraction and saponification [3, 12], levels of vitamins A and E were determined in triplicate using HPLC [13] as described by Albalá-Hurtado et al. [14]. Statistical analysis was performed using the two-way ANOVA procedure (levels of vitamin A and vitamin E) of the SAS Institute [15], and Tukey s test was used to compare treatment means [15]. For egg vitamin data, regression equations, and correlation coefficients were calculated according to the vitamin level of the diets. Significance implies P < RESULTS AND DISCUSSION Hen Performance Dietary vitamin A supplementation did not affect egg weight (Table 1). Egg production was enhanced by dietary vitamin A, reaching average values of 80.4 and 79.5%, respectively, at 15,000 and 30,000 IU/kg, compared to the unsupplemented vitamin A group (76.7%). However, this effect on average egg production was statistically significant only for the group fed 15,000 IU of vitamin A/kg (Table 1). Feed consumption was significantly increased when 30,000 IU of vitamin A/kg was fed, whereas feed conversion was not affected by addition of vitamin A to

3 108 JAPR: Research Report TABLE 1. Performance of laying hens (44 wk old) fed diets supplemented with retinyl acetate in combination with α-tocopheryl acetate Treatments A Feed conversion (kg of feed per) Vitamin A Vitamin E Egg Egg Feed supplementation supplementation weight production consumption Kilogram (IU/kg of diet) (mg/kg of diet) (g) (%) (g/hen/d) Dozen eggs of eggs ± 0.6 a 78.2 ± 0.5 ab ± 2.0 ab 1.72 ± 0.03 a 2.09 ± 0.03 a ± 0.2 a 79.6 ± 1.0 ab ± 0.9 ab 1.68 ± 0.01 a 2.06 ± 0.01 a ± 0.3 a 76.1 ± 2.4 ab ± 1.7 a 1.72 ± 0.03 a 2.13 ± 0.05 a ± 1.1 a 72.9 ± 4.0 a ± 0.9 ab 1.87 ± 0.11 a 2.27 ± 0.17 a 15, ± 0.6 a 81.5 ± 1.5 ab ± 0.6 ab 1.67 ± 0.04 a 2.05 ± 0.03 a 15, ± 0.4 a 82.1 ± 0.4 b ± 0.1 ab 1.68 ± 0.01 a 2.02 ± 0.01 a 15, ± 0.8 a 80.1 ± 1.8 ab ± 1.9 ab 1.70 ± 0.07 a 2.06 ± 0.06 a 15, ± 0.7 a 77.8 ± 0.9 ab ± 0.7 a 1.72 ± 0.03 a 2.06 ± 0.02 a 30, ± 0.1 a 77.8 ± 1.8 ab ± 1.5 ab 1.72 ± 0.02 a 2.09 ± 0.03 a 30, ± 1.0 a 79.5 ± 0.4 ab ± 1.7 ab 1.72 ± 0.03 a 2.06 ± 0.02 a 30, ± 0.3 a 82.6 ± 1.4 b ± 2.6 ab 1.67 ± 0.03 a 2.01 ± 0.02 a 30, ± 1.4 a 78.2 ± 0.5 a ± 0.9 b 1.79 ± 0.00 a 2.20 ± 0.04 a Vitamin A supplementation (IU/kg of diet) y 76.7 y y 1.75 y 2.14 y 15, y 80.4 z yz 1.69 y 2.04 y 30, y 79.5 yz z 1.73 y 2.09 y Vitamin E supplementation (mg/kg of diet) a 79.2 ab a 1.70 ab 2.07 ab a 80.4 b a 1.69 a 2.05 a a 79.6 ab a 1.70 ab 2.06 ab a 76.3 a a 1.79 b 2.18 b a,b; x,z Means ± SEM within columns with no common superscript differ significantly (P < 0.05) by Tukey s Test. A Assayed vitamin A and E contents of the basal diet were 9,050 and 7.39 IU/kg of diet, respectively. hen diets (Table 1). According to Coskun et al. [16], addition of 24,000 IU vitamin A/kg of ration did not influence feed consumption, egg production, egg weight, or feed efficiency, in comparison to birds fed diets with 4,000 IU vitamin A/kg, as recommended by NRC [2]. On the other hand, March et al. [17] reported egg weight reduction when excessive vitamin A (410,000 IU/kg) was supplied to hens. The maximum tolerable dosage of vitamin A for laying hens is approximately 40,000 IU, and there is evidence in the literature showing toxigenic effects when greater quantities are used [18]. Egg weight and feed consumption were not significantly affected by dietary levels of vitamin E. The present work showed that the highest level of vitamin E (600 mg/kg) produced the poorest egg production and feed conversion, regardless of supplemental vitamin A levels in the experimental rations (Table 1). However, Jiang et al. [3] reported that supplementation with 400 mg α-tocopheryl acetate/kg resulted in a decline in feed consumption, without impairing egg production. Previous reports [19, 20, 21] show an absence of effect on hen performance parameters by the supplementation of various levels of vitamin E. The results attained herein suggested that dietary supplementation with 15,000 IU vitamin A and 200 mg tocopherol/kg among the experimental diets produced better hen performance. Egg Quality Specific gravity, shell index, shell thickness, and albumen quality of eggs from hens fed diets with supplemental vitamins A and E did not differ from those laid by hens fed the basal diet (Table 2). These findings are in agreement with Qi and Sim [20], who supplied hens with 800 mg vitamin E/kg of diet and did not observe changes in the internal quality of eggs. On the

4 MORI ET AL.: VITAMIN A AND E IN EGG YOLK 109 TABLE 2. Egg quality of laying hens (44 wk old) fed diets supplemented with retinyl acetate in combination with α-tocopheryl acetate A Treatments B Vitamin A Vitamin E Shell supplementation supplementation Specific Shell thickness Haugh (IU/kg of diet) (mg/kg of diet) gravity index C (mm) units ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± , ± ± ± ± , ± ± ± ± , ± ± ± ± , ± ± ± ± , ± ± ± ± , ± ± ± ± , ± ± ± ± , ± ± ± ± 2.8 Vitamin A supplementation (IU/kg of diet) , , Vitamin E supplementation (mg/kg of diet) A Means ± SEM within columns do not differ significantly (P < 0.05) by Tukey s Test. B Assayed vitamin A and E contents of the basal diet were 9,050 and 7.39 IU/kg of diet, respectively. C Shell Index = SW/S 100; where SW = shell weight in grams, and S = surface in cm 2. other hand, Squires and Naber [1] found thinner shells around eggs from hens fed 16,000 IU vitamin A/kg of diet compared to eggs from hens fed on diets containing 4,000 or 8,000 IU vitamin A/kg. Egg Yolk Retinol and α-tocopherol Content Egg yolk retinol concentrations were significantly increased by dietary vitamin A, ranging from 24.6 IU/g in the unsupplemented group to 33.6 and 37.7 IU/g of yolk (36.6% and 53.3% increase) for hens fed 15,000 and 30,000 IU/kg of ration, respectively (Table 3 and Figure 1). Regression analysis clearly showed a significant positive correlation between dietary retinyl acetate and egg retinol (r = ). The regression equation was y = x , where y is egg retinol (IU/g), and x is dietary retinyl acetate (IU/kg), indicating that egg retinol increased linearly as dietary vitamin A rose. This equation allows a prediction of yolk retinol concentration as a function of dietary supplementation. These data are in agreement with previous reports indicating the influence of dietary vitamin A on its concentration in the egg yolk [1, 20]. The addition of increasing levels of dietary vitamin E showed a trend to reduce the yolk retinol content, decreasing in 2.7, 3.9, and 5.4%, respectively for 200, 400, and 600 mg of supplemental vitamin E /kg of diet, in comparison with the control group (Table 3 and Figure 2). These results, however, proved to be not significant. Supplementation with vitamin A at 15,000 IU/ kg of ration resulted in a retinol incorporation of 35.0 and 32.2 IU/g of yolk when 0 and 600 mg of vitamin E, respectively, were added to the ration, causing a reduction of 8.9%, which was not significant. Hens fed 30,000 IU of supplemental vitamin A/kg of diet had 38.3, 38.4,

5 110 JAPR: Research Report TABLE 3. Egg yolk retinol (IU/g of yolk), percentage of change, and correlation coefficients (44-wk-old hens) Treatments A Vitamin A Vitamin E Correlation supplementation supplementation Yolk retinol Yolk retinol coefficient (IU/kg of diet) (mg/kg of diet) (IU/g of yolk) (% change) (r) ± 1.61 a ± 1.32 a 6.2 B DNS ± 1.17 a 5.4 B ± 1.50 a 4.7 B 15, ± 0.48 bc 15, ± 1.40 bc 3.4 B DNS 15, ± 0.59 bc 4.6 B 15, ± 1.24 b 8.9 B 30, ± 0.20 c 30, ± 0.72 c +0.3 B * D 30, ± 0.44 bc 2.6 B 30, ± 0.31 bc 3.9 B Vitamin A supplementation (IU/kg of diet) x 15, y C * E 30, z C Vitamin E supplementation (mg/kg of diet) a a 2.7 B a 3.9 B a 5.4 B a c; x z Means ± SEM within columns with no common superscript differ significantly (P < 0.05) by Tukey s test. A Assayed vitamin A and E contents of the basal diet were 9,050 and 7.39 IU/kg of diet, respectively. B Percentage of yolk retinol in comparison with no Vitamin E supplementation. C Percentage of yolk retinol in comparison with no Vitamin A supplementation. D Correlation coefficient between yolk retinol and dietary Vitamin E supplementation at the same level of supplemental Vitamin A. E Correlation coefficient between yolk retinol and dietary Vitamin A supplementation, whole data, regardless of levels of supplemental Vitamin E. NS Not significant. *P < 0.05). 37.3, and 36.8 IU retinol/g of yolk when 0, 200, 400, and 600 mg of vitamin E, respectively, were supplied, showing a trend (P < 0.05) of reduction (3.9%) in yolk retinol. The increase of dietary vitamin E for the groups with zero and 15,000 IU vitamin A supplementation per kilogram of diet did not affect yolk retinol deposition. The correlation coefficient was statistically significant only at the highest level of vitamin A (r = ), indicating that at this level, the increasing vitamin E supplementation resulted in linear decrease in yolk retinol deposition (Table 3). Grobas et al. [22] did not observe an effect of dietary vitamin E on yolk retinol deposition by feeding hens with vitamin E from 0 to 1,280 mg/kg and vitamin A from 4,000 to 40,000 IU/kg. Egg yolk α-tocopherol content was significantly influenced by levels of dietary vitamin E (Table 4). The yolk values ranged from 10.9 µg/ g for eggs not supplemented to 160.6, 264.1, and µg/g of yolk (increases of 1,373, 2,323, and 3,416% in comparison to the control group) for hens fed 200, 400, and 600 mg/kg of ration, respectively (Table 4 and Figure 2). Significant and progressive incorporation of α-tocopherol into the egg yolk was achieved by feeding laying hens a basal diet supplemented with increasing levels of α-tocopheryl acetate, regardless of supplemental vitamin A. The re-

6 MORI ET AL.: VITAMIN A AND E IN EGG YOLK 111 FIGURE 1. Effect of dietary retinyl acetate on egg yolk retinol, according to level of vitamin E supplementation. gression analysis was significant (r = ), and the regression equation was y = x , where y is egg α-tocopherol (µg/g), and x is dietary α-tocopheryl acetate (mg/kg), indicating that egg α-tocopherol increased linearly as dietary vitamin E increased. This elevation FIGURE 2. Effect of dietary retinyl acetate on egg yolk tocopherol, according to level vitamin E supplementation.

7 112 JAPR: Research Report TABLE 4. Egg yolk α-tocopherol (µg/g of yolk), percentage of change, and correlation coefficients (44-wk-old hens) Treatments A Vitamin E Vitamin A Yolk Yolk Correlation supplementation supplementation α-tocopherol α-tocopherol coefficient (mg/kg diet) (IU/kg of diet) (µg/g of yolk) (% change) (r) ± 0.47 a 0 15, ± 0.38 a 0 B DNS 0 30, ± 0.80 a 0 B ± 6.11 cde , ± bc 28.6 B * D , ± 5.73 b 44.3 B ± ef , ± de 26.7 B * D , ± 7.16 bcd 47.0 B ± h , ± g 22.5 B * D , ± ef 42.7 B Vitamin A supplementation (IU/kg of diet) z 15, y 24.9 B 30, x 44.9 B Vitamin E supplementation (mg/kg of diet) a b +1,373 C * E c +2,322 C d +3,415 C a g; x z Means ± SEM within columns with no common superscript differ significantly (P < 0.05) by Tukey s test. A Assayed vitamin A and E contents of the basal diet were 9,050 and 7.39 IU/kg of diet, respectively. B Percentage of yolk α-tocopherol in comparison with no Vitamin A supplementation. C Percentage of yolk α-tocopherol in comparison with no Vitamin E supplementation. D Correlation coefficient between yolk α-tocopherol and dietary Vitamin A supplementation, at the same level of supplemental Vitamin E. E Correlation coefficient between yolk α-tocopherol and dietary Vitamin E supplementation, whole data, regardless of levels of supplemental Vitamin A. *P < in yolk α-tocopherol in response to dietary supplementation confirms previous observations and can be explained by the direct transport of tocopherols accumulated in the liver to egg yolk as compounds of plasma Very-low-density lipoprotein (VLDL) [5]. Because VLDL does not undergo any appreciable lipolysis in the route from the liver to the oocytes [23], transfer of tocopherol from diet to egg yolk is highly efficient. Supplementation with increasing amounts of dietary vitamin A produced significant reductions in average egg yolk α-tocopherol concentrations of 24.9 and 44.9%, respectively, for eggs from hens fed 15,000 and 30,000 IU of supplemental vitamin A/kg (Table 4 and Figure 1). Dietary vitamin E supplementation in combination with 15,000 IU vitamin A/kg caused a decline in yolk α-tocopherol deposition from 22.5 to 28.6% in comparison to the vitamin A group not supplemented. When hens were fed 30,000 IU vitamin A/kg, the reduction of egg α-tocopherol was greater, ranging from 42.7 to 47.0%, in comparison to the vitamin A group not supplemented. For the group with zero vitamin E supplementation, increasing dietary vitamin A did not affect yolk α-tocopherol deposition. Correlation coefficients between levels of supplemental vitamin A and yolk α-tocopherol were statistically significant (Table 4) showing values of , , and for 200, 400, and 600 mg of vitamin E, respectively. This result

8 MORI ET AL.: VITAMIN A AND E IN EGG YOLK 113 indicated that at the same level of vitamin E supplementation, yolk α-tocopherol deposition decreased when dietary supplemental vitamin A increased. These data are in agreement with Grobas et al. [22] and Surai et al. [24], who observed lower yolk α-tocopherol deposition due to high quantities of dietary vitamin A. The interaction between these vitamins was also reported by Aburto and Britton [25], who noted a reduction in plasma and liver vitamin E concentrations of broilers fed high levels of vitamin A (15,000 or 45,000 IU/kg). High quantities of dietary vitamin E decrease vitamin A utilization only when both vitamins are administered simultaneously and by the same route, indicating a mechanism of competition between these nutrients [26]. According to Frigg and Broz [6], the negative effects of high levels of vitamin A upon the vitamin E status are manifested in the intestinal tract. A high vitamin A intake may antagonize vitamin E by promoting its destruction in the gut or by interfering with its absorption. Sklan and Donoughue [27] showed that vitamin A increases oxidation of tocopherol before reaching the absorption sites, reducing its bioavailability and intestinal absorption. Situations in which one fat-soluble vitamin may be fed to birds at something other than normal levels could be a possible problem depending on the dietary level of the other fatsoluble vitamins [25]. Effects of vitamin A and E supplementation are both linear and readily incorporated into egg yolk when provided from the hen diet. However, the concomitant supplementation of these vitamins in the ration could influence the yolk α- tocopherol deposition. The high level (30,000 IU/kg) of vitamin A resulted in a major reduction (44.9%) in the yolk α-tocopherol accumulation, and at this level the yolk retinol was also affected by dietary vitamin E. Considering an average yolk weight of 17 g, a normal egg supplies approximately 449 IU of retinol and 0.2 mg of α- tocopherol. Fortified eggs obtained from supplementation with 15,000 and 30,000 IU vitamin A/kg in hen diets would provide 571 and 641 IU of retinol, respectively, significant amounts, taking into consideration the recommendations for an intake of µg retinol equivalents (3,333 IU of vitamin A) for human adults [28]. Supplementation of hen diets with 200, 400, or 600 mg/kg of vitamin E would provide 2.7, 4.5, or 6.5 mg of α-tocopherol per egg, respectively. These values could also supply a relevant amount of the vitamin E recommendation for human adults, of 15 IU (10 mg of α-tocopherol), but studies have suggested a 10-fold increase (150 IU) of this recommendation [29]. When the aim is the enrichment of eggs with vitamins A and E, the interactions and costs of these vitamins must be considered. CONCLUSIONS AND APPLICATIONS 1. A high level of vitamin E (600 mg/kg) resulted in decreases in egg production and poor feed conversion, regardless of supplemental vitamin A levels in the experimental rations. Levels of 15,000 IU of vitamin A and 200 mg of vitamin E/kg of diet gave better hen performance and may be justified when hens are supplied high quantities of these vitamins in order to enrich eggs. 2. The percentage of increment in yolk retinol ranged from 36.6 to 53.3% at 15,000 and 30,000 IU of vitamin A/kg, respectively. The increase in yolk α-tocopherol reached 1,373, 2,322, and 3,415% at 200, 400, and 600 mg of supplemental vitamin E/kg of diet, respectively. 3. Decreases in egg yolk α-tocopherol concentrations showed the antagonist effect of dietary vitamin A on vitamin E deposition in the egg yolk. Yolk tocopherol was reduced by the increasing rates of vitamin A supplementation, decreasing 24.9 and 44.0%, respectively, for 15,000 and 30,000 IU of supplemental vitamin A/kg of diet. At this highest level (30,000 IU/ kg), the yolk retinol was also affected by dietary vitamin E. Attention must be focused on the inter-relationship between vitamin A and E when they are supplied together in diets in order to enrich eggs.

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