Effect of Dietary Vitamin A on Egg Yolk Retinol and Tocopherol Levels

Transcription

1 2002 Poultry Science Association, Inc. Effect of Dietary Vitamin A on Egg Yolk Retinol and Tocopherol Levels C. X. Mendonça Jr., 1 C. R. M. Almeida, A. V. Mori, and C. Watanabe Departamento de Clínica Médica, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Cidade Universitária Armando de Salles Oliveira, Av. Prof. Orlando Marques de Paiva 87, São Paulo, , Brazil Primary Audience: Nutritionists, Researchers, Poultry Producers, Feed Manufacturers SUMMARY Vitamin A deficiency is a serious public health problem in developing countries, and it causes death or blindness among children in these countries. Fortification of food, such as eggs, could be an important source of vitamins to control deficiency. To evaluate the effect of dietary levels of vitamin A upon reproductive performance, egg quality, and egg yolk retinol and tocopherol concentrations, laying hens were fed a basal diet (commercial ration) supplemented with increased retinyl acetate. It was observed that hen performance was not significantly affected by addition of vitamin A. Progressive increase in the incorporation of retinol into egg yolk was verified when vitamin A was supplemented to the basal diet; the percentage of increment reached 50.6% with 25,000 IU of dietary retinyl acetate/kg. Egg yolk retinol content increased linearly as dietary vitamin A increased. However, significant decreases in the egg yolk tocopherol concentrations showed an adverse effect of vitamin A supplementation. The nutritional value of eggs, related to vitamin A, can be improved by dietary manipulation of hen diets. Key words: egg yolk, hen, retinol, vitamin A, vitamin E, tocopherol 2002 J. Appl. Poult. Res. 11: DESCRIPTION OF PROBLEM A recent report [1] has shown that over 100 million preschool-aged children suffer from vitamin A deficiency. The deficiency is also the most frequent cause of blindness among children in developing countries. Supplementation with vitamin A is estimated to lower a child s risk of dying by approximately 23%. According to this report [1], micronutrient malnutrition cannot be eradicated, but elimination and control of these deficiencies and their health-related consequences as public health problems are currently targets of global programs. Because consumers regard eggs as a highcholesterol dietary product, and attempts to re- duce cholesterol levels in eggs have met with only marginal results, several approaches have been explored to improve its health quality. Modification in the egg composition is attractive to the health-conscious consumer, and this demand has encouraged studies related mainly to changes in egg lipids and vitamins. Levels of fatsoluble vitamins, vitamin B 12, iodine, selenium, and fatty acids in eggs are easily manipulated through dietary modification of these nutrients [2]. Egg vitamin A level is significantly changed by its increase in the diet [2]. Vitamin A supplied at two or three times the NRC values provided the most efficient vitamin transfer (60 to 80%) from the diet [3]. 1 To whom correspondence should be addressed: cxmendon@usp.br.

2 374 In developed countries, fortification of food, such as margarine and milk-derived products, is frequently used as an important source of vitamin A. In developing countries, this supplementation has been included in a program for the remedy of vitamin A deficiency, which is responsible for the annual death of approximately 500,000 children [4]. The vitamin A recommendation for laying hens is 4,000 IU/kg of diet, as reported by NRC [5]. Bárdos [6] showed that dietary retinyl acetate from 12,500 to 300,000 IU caused a peak of vitamin A in plasma after 4 h. The egg yolk stores vitamin A for the embryo development during incubation and also for the first stages of life [7]. According to Joshi et al. [8], the great majority of yolk vitamin A is deposited as retinol and smaller amounts as retinyl esters. Coskun et al. [9] reported no effects on egg production due to supplementing hen diets with increasing levels of vitamin A (0, 4,000, 12,000, and 24,000 IU/kg of diet) for 72 wk. On the other hand, higher levels of vitamin A (210,000 and 410,000 IU/kg) caused a marked decline in egg production and egg weight in research by March et al. [10]. Squires and Naber [11] reported an efficiency of 80% vitamin A transference from rations to eggs when supplementing 8,000 IU/kg of diet, which means 244 IU of vitamin A per egg. Surai et al. [12] found µg of vitamin A/g of yolk when hens were fed 120 µg of retinol/g of diet, whereas in a control group without supplementation only 4.01 µg of vitamin A/ g of yolk was found. Studies have demonstrated a nutritional interaction between vitamin A and other vitamins in a several animal species, and a high intake of vitamin A may interfere negatively in other fat-soluble vitamins, particularly vitamin E [12, 13]. Jiang et al. [13] reported a decrease in α- tocopherol deposition in egg yolk when diets were supplemented with β-carotene. After feeding laying hens for 3 mo with different levels of vitamin A, Surai et al. [12] described a significant decrease of about 20% in yolk vitamin E. Production of eggs with increased levels of vitamin A should improve the reputation of the egg s nutritional value, which has been tainted because of its high cholesterol level. The aim of this study was to evaluate the effect of dietary JAPR: Research Report levels of vitamin A, as retinyl acetate, upon reproductive performance, egg quality, and egg yolk retinol and tocopherol concentrations. MATERIALS AND METHODS A total of 192, 33-wk-old W-36 Hy-Line hens, a commercial white egg strain, was distributed into 24 replicates (four cages per replicate with two birds per cage) and randomly assigned to one of the six experimental diets (four replicates per treatment) for 15 wk. The control group (CON) was composed of hens fed a commercial layer ration with (calculated) 17% CP, 2.0% crude fat, 4.5% calcium, and 0.6% total phosphorus. A vitamin mix in the ration provided 8,800 IU of vitamin A (retinyl acetate) and 7.0 IU of vitamin E/kg of ration. The basal diet was analyzed for vitamin A and E contents and showed values of 9,050 IU and 7.39 IU/kg of ration, respectively. The basal diet met all NRC nutrient requirements for laying hens [5]. This basal diet was supplemented with 5,000 IU (T5), 10,000 IU (T10), 15,000 IU (T15), 20,000 IU (T20), or 25,000 IU (T25) of retinyl acetate/kg. Egg production and egg weight data were recorded daily; feed consumption was recorded weekly. The average egg production, egg weight, feed intake and feed conversion (kilograms of feed consumed per dozen eggs and per kilogram of eggs) were calculated for each replicate group. For evaluation of shell quality, the specific gravity of 16 eggs per treatment (four per replicate) was determined by the saline solutions method [14]. Albumen quality (Haugh units) was evaluated with a S-8400 micrometer (Ames, Waltham, MA). Eggshells were individually weighed, and eggshell thickness was measured with a 25M-5 micrometer (Ames). Shell index (I) was calculated according to Sauver [15] using the following formula: SI = SW/S 100, where SW = shell weight (g), and S = surface (cm 2 ). S is calculated from the egg weight (EW) from the equation S = K EW 2/3, where K = 4.67, 4.68, or 4.69 depending on egg weight (being less than 60 g, between 60 and 70 g, or greater than 70 g, respectively). At the end of the experiment, four eggs were randomly collected from each replicate. Eggs were weighed and albumen and yolk were separated. Yolks were individually weighed and

3 MENDONÇA ET AL.: VITAMIN A IN EGG YOLK 375 TABLE 1. Performance of laying hens (33 to 48 wk of age) fed diets supplemented with retinyl acetate Egg Egg Feed Feed conversion (kg of consumed feed per) weight production consumption Kilogram Treatment A (g) (%) (g/hen/d) Dozen eggs of eggs CON 60.9 ± ± ± 0.9 ab 1.43 ± ± 0.02 T ± ± ± 1.4 ab 1.39 ± ± 0.02 T ± ± ± 1.1 b 1.40 ± ± 0.04 T ± ± ± 1.6 ab 1.41 ± ± 0.02 T ± ± ± 0.9 a 1.42 ± ± 0.03 T ± ± ± 1.2 ab 1.41 ± ± 0.01 a,b Means ± SEM within columns with no common superscript differ significantly (P < 0.05) by Tukey s test. A CON = control (no supplementation); T5 = 5,000 IU, T10 = 10,000 IU, T15 = 15,000 IU, T20 = 20,000 IU, and T25 = 25,000 IU retinyl acetate/kg. were prepared by pooling and blending four yolks per sample. After samples were subjected to extraction and saponification [13, 16], vitamin A and E levels were determined using HPLC [17] as described by Albalá-Hurtado et al. [18]. Statistical analysis was performed using the one-way ANOVA procedure of the SAS Institute [19], and Tukey s test was used to compare treatment means [19]. For egg vitamin data, regression equations and correlation coefficients were calculated according to the vitamin A level of the diets. Significance implies P < RESULTS AND DISCUSSION Hen Performance Diets supplemented with retinyl acetate had no adverse effects on egg weight, egg production, or feed consumption (Table 1). These observations confirm the reports of Squires and Naber [11] and Coskun et al. [9]. March et al. [10] reported a decrease in egg weight and a marked decline in the rate of egg production but only when higher levels of vitamin A were supplemented, from 210,000 to 410,000 IU/kg of ration. Therefore, greater amounts of vitamin A could have some negative effects on egg production due to its indirect action on thyroid activity. Supplemental retinyl acetate did not improve feed conversion, in contrast to the results of Reid et al. [20], who supplemented hen diets with vitamin A. Egg Quality Specific gravity, shell index (SI), and albumen quality of eggs obtained from hens fed supplemental vitamin A did not differ from those (Table 2) laid by hens fed the basal diet (CON). These results agree with Squires and Naber [11], who found no significant differences between albumen quality of eggs obtained from unsup- TABLE 2. Egg quality of laying hens (33 to 48 wk of age) fed diets supplemented with retinyl acetate Shell Haugh Specific Shell thickness unit Treatment A gravity index (SI) B (mm) (%) CON ± ± ± a 88.6 ± 1.2 T ± ± ± b 86.9 ± 1.5 T ± ± ± 0.06 ab 83.3 ± 2.1 T ± ± ± ab 88.5 ± 1.3 T ± ± ± b 83.4 ± 2.6 T ± ± ± ab 84.3 ± 2.3 a,b Means ± SEM within columns with common superscript do not differ significantly (P < 0.05) by Tukey s test. A CON = control (no supplementation); T5 = 5,000 IU, T10 = 10,000 IU, T15 = 15,000 IU, T20 = 20,000 IU, and T25 = 25,000 IU retinyl acetate/kg. B Shell index (SI) = SW/S 100; where SW = shell weight in grams, and S = surface in cm 2.

4 376 JAPR: Research Report TABLE 3. Egg yolk retinol and tocopherol content, and percentage change in comparison to a control group of laying hens (33 to 48 wk of age) fed diets supplemented with retinyl acetate Retinol Change B Tocopherol Change B Treatment A (IU/g) (%) (µg/g) (%) CON ± 0.27 d ± 0.45 a T ± 0.25 c ± 0.55 a 3.2 T ± 0.50 b ± 0.78 a 3.2 T ± 0.47 b ± 2.38 b 20.0 T ± 0.96 a ± 1.23 b 25.3 T ± 0.55 a ± 0.52 c 32.9 a d Means ± SEM within columns with no common superscript differ significantly (P < 0.05) by Tukey s test. A CON = control (no supplementation); T5 = 5,000 IU, T10 = 10,000 IU, T15 = 15,000 IU, T20 = 20,000 IU, and T25 = 25,000 IU retinyl acetate/kg. B Percentage change in comparison to the control group. plemented and supplemented (9,000 IU/kg) diets. The values of eggshell thickness from hens supplemented with 5,000 (T5) or 20,000 (T20) IU of retinyl acetate were statistically lower than the CON (Table 2). Egg Retinol and Tocopherol Contents Significant and progressive incorporation of retinol into egg yolk was achieved by feeding laying hens a basal diet supplemented with retinyl acetate. These findings are in agreement with those of Hill et al. [21] who demonstrated the influence of diet on yolk vitamin A content. In the present study, the percentage increase of yolk retinol (Table 3) ranged from 8.6% (T5) to 50.6% (T25) in comparison to the CON, and the enrichment of vitamin A content was greater in T25 (35.94 IU/g). Our results corroborate the data reported by Squires and Naber [11] and Surai et al. [12]. The former authors, by feeding vitamin A at 16,000 IU/kg, observed an enrich- ment of 24 IU/g of yolk, greater than that reported herein. In another study, Squires and Naber [11] reported an average vitamin A concentration of 8.5 IU/g of yolk when birds were fed 9,000 IU of vitamin A/kg, which is comparable to 8.9 IU/ g found in the present study (Table 3) from hens fed 10,000 IU/kg (T10). There was no significant difference in the yolk retinol content between treatments T10 and T15, and the same was observed between treatments T20 and T25 (Table 3). Supplemental retinyl acetate at 15,000 IU (T15), 20,000 (T20), and 25,000 (T25) IU/kg caused significant reductions in yolk tocopherol content in comparison to CON (Table 3). The percentage of reduction ranged from 3.2% (T5 and T10) to 32.9% (T25). The regression analysis clearly showed a significant positive correlation between dietary retinyl acetate and egg retinol (r = ). The regression equation (Figure 1) was as follows: FIGURE 1. Relationship between retinyl acetate (IU) supplemented in the diet and retinol levels (IU/g) in the egg yolk.

5 MENDONÇA ET AL.: VITAMIN A IN EGG YOLK 377 FIGURE 2. Relationship between retinyl acetate (IU) supplemented in the diet and tocopherol levels (µg/g) in the egg yolk. y = x , where y = egg retinol (IU/g), and x = dietary retinyl acetate (IU/kg), indicating that egg retinol increased linearly as dietary vitamin A increased. This equation allows a prediction of yolk retinol concentration as a function of dietary supplementation. Our results agree with Jiang et al. [13], who reported a linear improvement in egg yolk retinol content in response to increases in dietary β-carotene. Although the authors found a very small absolute increase, this result demonstrated that some β- carotene was converted into retinol and deposited into the ovum. Figure 2 shows the relationship between dietary retinyl acetate and egg tocopherol. The regression analysis was significant (r = ), and the regression equation (Figure 2) was as follows: y = x , where y = egg tocopherol (µg/g), and x = dietary retinyl acetate (IU), indicating that egg tocopherol decreased linearly as dietary retinyl acetate increased. Retinol and tocopherol values found in egg yolk agree closely with that previously reported by Squires and Naber [11], who used similar levels of vitamin A supplementation and reported incorporation of dietary vitamin A into the egg yolk after 13 wk of supplementation. According to the authors, the liver probably plays an important regulatory role in the metabolism of this vitamin. A certain period is necessary for liver concentrations of the vitamins to stabilize and lead to a consistent change in egg yolk concentration by dietary manipulation. Jiang et al. [13] found increased deposition of egg yolk vitamin A after 8 wk of β-carotene supplementation. Tocopherol concentration was also influenced by dietary vitamin A levels (Table 3), reaching a reduction of 32.9% when compared to the CON, corroborating the findings of Surai et al. [12], who demonstrated marked decline in yolk tocopherol levels (24.33 µg/g of yolk) in hens fed diets containing 120 µg retinol/g. These authors demonstrated that high concentrations of dietary vitamin A decreased liver and egg concentrations of vitamin E and showed that the liver is not a good reservoir of vitamin E in laying hens. Therefore, the continuous supplementation of this vitamin is very important, mainly when increased vitamin A is supplemented to the diet. This adverse effect of increased levels of dietary vitamin A on vitamin E absorption agrees with previous findings of Sklan and Donoghue [22], Frigg and Broz [23], and Abawi and Sullivan [24]. Greater intake of vitamin A could promote destruction of vitamin E in the intestine or interfere with its absorption [23]. According to Sklan and Donoghue [22], vitamin A from the diet increases tocopherol oxidation, decreasing its bioavailability. These assertions are in agreement with the results of the present experiment, which showed significant correlation coefficients, indicating that yolk tocopherol tended to decline when retinyl acetate concentration increased in the diet and in the egg.

6 378 JAPR: Research Report CONCLUSIONS AND APPLICATIONS 1. Diets supplemented with retinyl acetate from 5,000 IU/kg (T5) to 25,000 IU/kg (T25) did not affect egg weight, egg production, or feed consumption of laying hens. 2. The results attained herein evidenced great possibilities to improve the nutritional value of eggs through the dietary manipulation of hen diets. Progressive incorporation of retinol into egg yolk was verified when retinyl acetate was supplemented to the diet; the percentage increment ranged from 8.6% (T5) to 50.6% (T25). Egg retinol content increased linearly as dietary retinyl acetate increased. 3. Decreases in the egg yolk tocopherol concentrations in T15 (20.0%), T20 (25.3%), and T25 (32.9%) treatments showed the antagonist effect of dietary retinyl acetate on tocopherol in the egg yolk. Attention must be focused on the inter-relationship between vitamin A and other vitamins in order to enrich eggs. REFERENCES AND NOTES 1. Underwood, B. A Perspectives from micronutrient malnutrition eradication programs. Bull. World Health Org. 76(Suppl. 2): Naber, E. C The effect of nutrition on the composition of eggs. Poult. Sci. 58: Naber, E. C., and M. W. Squires Vitamin profiles of eggs as indicators of nutritional status in the laying hen: Diet to egg transfer and commercial flock survey. Poult. Sci. 72: Murphy, P. A Technology of vitamin A fortification of foods in developing countries. Food Technol. 50: National Research Council Nutrient Requirements of Domestic Animals. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Sci., Washington, DC. 6. Bárdos, L Absorption of retinyl acetate in laying hens. Int. J. Vitamin Nutr. Res. 66: Bárdos, L Plasma vitamin A composition and retinolbinding protein concentration during egg formation in laying hens. Int. J. Vitam. Nutr. Res. 59: Joshi, P. S., S. N. Mathur, S. K. Murthy, and J. Ganguly Vitamin A economy of the developing embryo and of the freshly hatched chick. Biochem. J. 136: Coskun, B., F. Inal, I. Celik, O. Erganis, A. M. Tiftik, F. Kurtoglu, Y. Kuyucuoglu, and U. Ok Effects of dietary levels of vitamin A on the egg yield and immune responses of laying hens. Poult. Sci. 77: March, B. E., V. Coates, and C. Goudie Delayed hatching time of chicks from dams fed excess vitamin A and from eggs injected with vitamin A. Poult. Sci. 51: Squires, M. W., and E. C. Naber Vitamin profiles of eggs as indicators of nutritional status in the laying hen: Vitamin A study. Poult. Sci. 72: Surai, P. F., I. A. Ionov, T. V. Kuklenko; I. A. Kostjuk, A. Macpherson, B. K. Speake, R. C Noble, and N. H. C. Sparks Effect of supplementing the hen s diet with vitamin A on the accumulation of vitamins A and E, ascorbic acid and carotenoids in the egg yolk and in the embryonic liver. Br. Poult. Sci. 39: Jiang, Y. H., R. B. McGeachin, and C. A. Bailey α- Tocopherol, β-carotene, and retinol enrichment of chicken eggs. Poult. Sci. 73: Hamilton, R. M. G Methods and factors that affect the measurement of egg shell quality. Poult. Sci. 61: Sauveur, B Page 385 in Reproduction des Volailles et Production d Oeufs. INRA, Paris. 16. A 1-g sample of pooled yolk was weighed into a 100-mL flask with 30 ml of extraction solvent (hexane:acetone:toluene:ethanol; 10:7:7:6) and stored overnight under nitrogen gas at room temperature in the dark. Saponification was achieved by adding 2 ml of methanolic KOH and storing the solution overnight. This was followed by adding 30 ml of hexane to the total extract and mixing, dilution to 100 ml with 10% aqueous Na 2 SO 4, and then mixing and waiting 1 h before taking 5 ml of the upper organic layer and evaporating it with nitrogen gas. The organic phase was dissolved in methanol. 17. SPD-&AV, Shimadzu, Kyoto, Japan. 18. Albalá-Hurtado, S., S. Novella-Rodriguez, M. T. Veciana- Noguês, and A. Mariné-Font Determination of vitamin A and E in infant formulae by high-performance liquid chromatography. J. Chromatogr. A 778: SAS Institute SAS User Guide: Statistics. SAS Institute Inc., Cary, NC. 20. Reid, B. L., B. W. Heywang, A. A. Kurnick, M. G. Vavich, and J. Hulett Effect of vitamin A and ambient temperature on reproductive performance of White Leghorn pullets. Br. J. Nutr. 14: Hill, F. W., M. L. Scott, L. C. Norris, and G. F. Heuser Reinvestigation of the vitamin A requirements of laying and breeding hens and their progeny. Poult. Sci. 40: Sklan, D., and S. Donoghue Vitamin E response to high dietary vitamin A in the chick. J. Nutr. 112, Frigg, M., and J. Broz Relationships between vitamin A and vitamin E in the chick. Int. J. Vitam. Nutr. Res. 54, Abawi, F. Q., and T. W. Sullivan Interactions of vitamins A, D 3, E, and K in the diet of broiler chicks. Poult. Sci. 68: Acknowledgment The financial support of the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and the supply of retinyl acetate from Roche Nutrição e Saúde Animal are gratefully acknowledged.