METABOLISM AND NUTRITION. Effects and Interactions of Dietary Levels of Vitamins A and E and Cholecalciferol in Broiler Chickens 1

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1 METABOLISM AND NUTRITION Effects and Interactions of Dietary Levels of Vitamins A and E and Cholecalciferol in Broiler Chickens 1 A. ABURTO and W. M. BRITTON2 Department of Poultry Science, University of Georgia, Athens, Georgia ABSTRACT Four experiments were conducted to determine the effects and interactions of feeding different levels of vitamins A, cholecalciferol (vitamin D 3 ), and E on broiler chicks. In Experiment 1, chicks were fed marginal vitamin D 3 (500 IU/kg) and increasing dietary levels of vitamin A (5,000, 10,000, 20,000, 40,000, 80,000, and 160,000 IU/kg). Bone ash was reduced by 10,000 IU/kg of vitamin A in the diet and at vitamin A levels above 20,000 IU/kg of diet body weight was reduced. In Experiment 2, two levels of vitamin A (1,500 and 15,000 IU/kg) and six levels of vitamin E (10, 500, 1,000, 2,500, 5,000, and 10,000 IU/kg) were added to the basal diet. High levels of vitamins A and E significantly (P < 0.001) reduced bone ash. The vitamin A E interaction was significant (P 0.05) for rickets. In Experiment 3, the same two levels of vitamin A as Experiment 2 and six levels of vitamin D 3 (500, 1,000, 1,500, 2,000, 2,500, and 3,000 IU/kg) were added to the basal diet that contained 10,000 IU/kg of vitamin E. Body weight and bone ash were increased by increasing vitamin D 3 with a corresponding reduction (P 0.05) in rickets. In Experiment 4, three levels of vitamin A (1,500, 15,000, and 45,000 IU/kg), three levels of vitamin D 3 (500, 1,500, and 2,500 IU/kg), and three levels of vitamin E (10, 5,000, and 10,000 IU/kg) were added to the basal diet. Significant negative responses (P 0.05) to increasing dietary vitamin A were observed for bone ash, rickets, and plasma and liver vitamin E. A significant (P < 0.001) increase in bone ash and plasma calcium with a corresponding reduction in rickets was observed by increasing vitamin D 3. Increasing dietary vitamin E adversely affected (P 0.01) bone ash, plasma calcium, and plasma and liver vitamin A concentrations. These results indicate the need for making feed with the proper ratios of vitamins A, D 3, and E. (Key words: vitamin A, cholecalciferol, vitamin E, bone ash, broiler) 1998 Poultry Science 77: INTRODUCTION Researchers originally suggested in the 1930s that nutritional relationships exist among vitamins A, D 3 (cholecalciferol), and E in humans and animals. All of these vitamins are lipid-soluble and occur in nature with lipids. Certain interactions occur among these fat-soluble vitamins through their common physical characteristic of lipid solubility in lipid systems. Studies have demonstrated that the feeding of high dietary levels of vitamin A leads to decreased tissue and plasma tocopherol concentrations in chicks (Pudelkiewicz et al., 1964; Combs and Scott, 1974; Combs, 1976; Sklan and Donoghue, 1982; Frigg and Broz, 1984; Abawi and Sullivan, 1989). Conversely, high dietary tocopherol alleviated hypervitaminosis A in chicks (McCuaig and Motzok, 1970; Sklan and Donoghue, 1982). Received for publication May 23, Accepted for publication December 15, Supported by state and Hatch funds allocated to the Georgia Agricultural Stations of the University of Georgia. 2 To whom correspondence should be addressed: wmbritt@uga.cc.uga.edu March et al. (1973) reported that with calcium- or vitamin D-deficient diets, bone calcification was depressed when chicks were given excess vitamin E. Similarly, Murphy et al. (1981) observed reduced bone ash and plasma calcium and phosphorus when chicks were given large doses of vitamin E. A significant threeway interaction among vitamins A, D 3, and E was noted for plasma vitamin E concentrations in broiler chickens (Abawi and Sullivan, 1989). Extra vitamin D 3 has been shown to protect the chick (Taylor et al., 1968; Veltmann and Jensen, 1985; Veltmann et al., 1986, 1987) against vitamin A toxicosis. Most workers agree on the existence of the nutritional relationship of dietary vitamins A, E, and D 3. A better understanding of this relationship is especially important as it relates to feed mixing. Feed mixing mistakes in the addition or deletion of the proper amounts of the fat-soluble vitamins could create a dietary condition of Abbreviation Key: MSBC = menadione sodium bisulfite; UV = ultraviolet; vitamin D 3 = cholecalciferol. 666

2 DIETARY VITAMINS A, D 3, AND E 667 TABLE 1. Composition of the basal diet Ingredients Amount (%) Ground yellow corn Soybean meal (Dehulled) Poultry fat 5.00 Dicalcium phosphate 1.86 Limestone 1.28 Iodized sodium chloride 0.45 DL-methionine 0.20 Vitamin B premix Mineral premix Fat-soluble vitamin premix Calculated composition Crude protein ME, kcal/kg 3, Calcium 0.98 Phosphorus, nonphytate Vitamin B premix provided in milligrams per kilogram of diet (except as noted): riboflavin, 4.4; calcium pantothenate, 12; nicotinic acid, 44; choline Cl, 220; vitamin B 12,9mg; vitamin B 6, 3; thiamin (as thiamin mononitrate), 2.2; folic acid, 3; biotin, 0.3; and ethoxyquin, Trace mineral premix provided (milligrams per kilogram of diet): MnO 2, 222; ZnO, 150; FeSO 4 7H 2 O, 200; FeCO 3, 83; CuSO 4 5H 2 O, 29; and Ca(IO 3 ) 2, 15, Na 2 SeO 3, Fat soluble vitamin premix provided 2 mg/kg of diet of vitamin K as menadione sodium bisulfite complex, and vitamin A (all-transretinol), cholecalciferol, and vitamin E, and was added according to treatment need using rice hulls as a carrier. vitamin imbalance. Unfortunately, the methods available to determine the vitamin A and E status of animals are difficult to do and harder to interpret. The measurement of plasma and liver vitamin A and E concentrations has been used. An HPLC analysis for plasma and liver vitamins A and E has been determined to be a good method but it is a chemical analysis attempting to measure biological response. Bone ash and vitamin D- type rickets have proven to be very sensitive measurements of vitamin D status in broiler chicks. The purpose of the investigation reported herein was to study the effects and interactions of dietary vitamins A, D 3, and E to better understand the dietary ratio of these vitamins needed for performance of broiler chicks. The effects of vitamin A and vitamin E on vitamin D 3 will be the major thrust of the investigation using the response of the bone parameters to vitamin D 3 as the major measure of interaction of the vitamins. Data generated could be important in understanding feed mixing errors of these three vitamins. MATERIALS AND METHODS Day-old male (Ross Ross) broiler chicks were used in all experiments. Four replicates of 10 chicks each were 3Arm-a-Lite, Thermoplastic Processes, Stirling, NJ Rovimix A-500W, Rovimix D S.D., Rovimix E-50% S. D., Roche Menadione Sodium Bisulfate Complex (MSBC). Hoffmann- LaRoche Inc., Nutley, NJ fed each dietary treatment. Chicks were wing-banded and housed in electrically heated battery brooders with wire mesh floors. The temperature of the room was maintained at 22 C. Feed and water were always available and all experiments were conducted for 16 d. The basal diet, shown in Table 1, was used in all experiments. Sunlight was excluded from the room by taping black plastic over the windows. The overhead fluorescent lights in the room were fitted with Arm-a- Lite 3 sleeves, FR312W-T-12, to prevent emission of ultraviolet light (UV) into the room. The fluorescent lights used in the batteries were General Electric, F15T8- CW, providing 3.4% of the wattage in the ultraviolet range (260 to 400 nm). These lights were also covered with plastic sleeves to prevent exposure to UV light. A diagram of the configuration of the pens and lights in relation to chicks is described by Edwards et al. (1994). At the termination of the experiments, birds were weighed by pen and their feed consumption recorded. They were then killed by carbon dioxide asphyxiation and examined for vitamin D-type rickets without knowledge of treatment. The birds were diagnosed as having rickets when the subepiphyseal growth-plate band was lengthened (Long et al., 1984). The degree was scored on a 0 to 3 basis, with 0 being no rickets and 3 very severe rickets. The left tibia was removed for bone ash determination on a dry fat-free basis (AOAC, 1995). Experimental Design Experiment 1 was conducted to determine the effects of feeding high levels of vitamin A on the performance and some bone parameters in broiler chicks. Six levels of vitamin A (5,000, 10,000, 20,000, 40,000, 80,000, and 160,000 IU/kg) were added to the basal diet. The other fatsoluble vitamins were added individually. Vitamin D 3 was added at 500 IU/kg of diet and this was considered to be a marginal level of this vitamin (Edwards et al., 1994). Vitamin E (20 IU/kg) and vitamin K (2 mg/kg) were considered to be adequate to meet the requirements of the chick. The source of the vitamins used in this and subsequent experiments were commercial concentrate vitamins.4 Vitamins A, D 3, and E were spray-dried, waterdispersible products. Vitamin A activity was 500,000 IU/g or 150 mg/g. Vitamin D 3 activity was 500,000 IU/g or 12.5 mg/g. Vitamin E activity was 500 IU/g or 500 mg/g. Vitamin K was supplied as the menadione sodium bisulfite (MSBC), which has 33% menadione. These concentrated forms of the vitamins were premixed with rice hulls where appropriate for mixing into the feed. Analysis of variance and regression analysis for levels of vitamin A were computed on the data in Experiment 1 (SAS Institute, 1990). The pen was considered the experimental unit in this and all subsequent analyses. Experiment 2 was conducted to determine the effects and interactions of feeding two levels of vitamin A (1,500 and 15,000 IU/kg of diet) and six levels of vitamin E (10, 500, 1,000, 2,500, 5,000, and 10,000 IU/kg of diet) on the

3 668 ABURTO AND BRITTON TABLE 2. Effects of different levels of vitamin A on 16-d body weight, gain:feed ratio, bone ash, and the incidence and severity of rickets in broiler chicks, Experiment 1 1 Treatments Rickets 16-d Gain:feed Bone Vitamin A Cholecalciferol BW ratio ash Score Incidence No. 3 score (IU/kg) (g) (g:g) (%) (%) 5, a a 36.1 a 1.8 b 79 b 39 bc 10, a a 33.7 b 1.7 b 79 b 25 c 20, a a 33.4 b 1.9 ab 81 b 35 bc 40, ab a 31.6 c 2.2 ab 79 b 48 ab 80, ab a 32.0 c 2.4 a 85 ab 56 a 160, b a 30.3 d 2.4 a 90 a 47 ab Pooled SEM ANOVA Source df Probabilities Vitamin A < Regression analysis Vitamin A < a dmeans with no common superscript differ significantly (P 0.05). 1Means of four pens per treatment with 10 chicks per pen. performance and some bone parameters in broiler chicks. Vitamins D 3 (500 IU/kg) and K (2 mg/kg) were fed as in Experiment 1. The experiment was a 2 6 factorial arrangement of treatments and the data were analyzed by analysis of variance with vitamin A and vitamin E as main effects (SAS Institute, 1990). Experiment 3 was conducted to determine the effects and interactions of feeding two levels of vitamin A (1,500 and 15,000 IU/kg of diet) and six levels of vitamin D 3 (500, 1,000, 1,500, 2,000, 2,500, and 3,000 IU/kg of diet) on the performance and some bone parameters of broiler chicks fed excess vitamin E. The vitamin E excess was 10,000 IU/ kg of diet. Vitamin K was again added at 2 mg/kg of diet. The experiment was a 2 6 factorial arrangement of treatments and the data were analyzed by analysis of variance with vitamin A and vitamin D 3 as main effects. Linear and quadratic regression analysis for levels of vitamin D 3 were performed by level of vitamin A (SAS Institute, 1990). Experiment 4 was conducted to determine the effects and interactions of feeding three levels of vitamin A (1,500, 15,000, and 45,000 IU/kg), three levels of vitamin D 3 (500, 1,500, and 2,500 IU/kg), and three levels of vitamin E (10, 5,000, and 10,000 IU/kg) to broiler chicks. Vitamin K was maintained constant by adding 2 mg/kg to the basal diet. The experiment was a factorial arrangement of treatments and the data was analyzed by analysis of variance with vitamin A, D 3, and E as main effects (SAS Institute, 1990). 5Section N-31, Technicon Autoanalyzer Methodology, Technicon Corp., Tarrytown, NY Section N-46, Technicon Autoanalyzer Methodology, Technicon Corp., Tarrytown, NY Sigma Chemical Co., St. Louis, MO Plasma and Tissue Analysis At termination of Experiment 4, blood samples were obtained from two randomly selected birds per pen by cardiac puncture, and the plasma was analyzed for total Ca5 and dialyzable P.6 Plasma vitamin A and E were extracted using the method of Jansson et al. (1981). The plasma was extracted with ethanol and hexane, the top layer removed, dried, and resuspended in ethanol for HPLC injection. The HPLC method was the method described by Hatam and Kayden (1979), but a spectrophotometric detector was used at 292 nm, which allowed for detection of vitamin A and E. All-trans-retinol7 and a-tocopherol were used as standards. Liver samples from the same two birds used to obtain plasma were extracted by the procedure of Buttriss and Diplock (1984). Vitamin A and E were extracted with hexane following saponification. The HPLC analysis was conducted as described above. Experiment 1 RESULTS Regression analysis of body weight was significant (P 0.005) for vitamin A level (Table 2). The reduction in weight was seen only at levels of vitamin A of 40,000 IU/ kg of diet or above. Bone ash was reduced and rickets measurements were increased by the increase in vitamin A in the diet as shown by the significant linear regression for all these parameters. Bone ash appeared to be the most sensitive measure of the effect of vitamin A with only 10,000 IU/kg causing a reduction in bone ash. Experiment 2 A highly significant reduction in body weight (P < 0.001) was observed when very high levels of vitamin E

4 DIETARY VITAMINS A, D 3, AND E 669 TABLE 3. Effects of different levels of vitamins A and E on 16-d body weight, gain:feed ratio, bone ash, and the incidence and severity of rickets in broiler chicks, Experiment 2 1 Treatments Rickets 16-d Gain:feed Bone Vitamin A Vitamin E BW ratio ash Score Incidence No. 3 score (IU/kg) (g) (g:g) (%) (%) 1, , ,500 1, ,500 2, ,500 5, ,500 10, , , ,000 1, ,000 2, ,000 5, ,000 10, Pooled SEM Main effect means 1, , , , , , ANOVA Source df Probabilities Vitamin A <0.001 <0.001 <0.001 Vitamin E 5 < <0.001 <0.001 <0.001 <0.001 Vitamin A vitamin E Means of four pens per treatment with 10 chicks per pen. were fed and this body weight depression was more severe at the lower level of vitamin A (Table 3). However, the interaction between vitamin A and E for body weight was not significant. Gain:feed ratio was not significantly influenced by treatment. Both vitamins A and E significantly affected bone ash and their interaction approached significance (P 0.06). Rickets score, rickets incidence, and number three scores were all significantly increased by raising the dietary levels of both vitamins. The vitamin A by E interaction was significant for all three. It was obvious that the effect of increasing levels of vitamin E on rickets score, rickets incidence, and number three scores was more severe at the higher level of vitamin A. Experiment 3 In this experiment, in which all chicks were fed excess vitamin E (10,000 IU/kg), the chicks fed high levels of vitamin A (15,000 IU/kg) had greater body weights at all levels of vitamin D 3 with linear and quadratic effects being significant (P 0.05) (Table 4). The body weight response was much greater at the higher level of vitamin A. Experimental treatments did not significantly affect gain:feed ratio except for the interaction of vitamins A and D 3 (P 0.04), reflecting the effect of vitamin D 3 on this parameter at the higher level of vitamin A. Increasing vitamin A significantly decreased bone ash (P < 0.001) with a corresponding increase in rickets score, rickets incidence, and number three scores. Bone ash increased and rickets decreased significantly in response to increasing dietary vitamin D 3. The interaction between vitamins A and D 3 was significant only for bone ash. Bone ash was reduced to a greater extent by low levels of vitamin D 3 when the higher level of vitamin A was fed. Significant interactions were approached for rickets score, rickets incidence, and number three scores with probabilities of 0.07, 0.10, and 0.08, respectively for the same reason as for bone ash. Linear and quadratic vitamin D 3 effects were significant for most of the parameters measured when analyzed by level of vitamin A. Experiment 4 Feeding the high levels of vitamin E (5,000 or 10,000 IU/kg) reduced body weight and gain:feed ratio, but increasing vitamins A and D 3 improved body weights and gain:feed ratio in chicks fed high levels of vitamin E (Table 5). Bone ash, rickets score, and plasma and liver vitamin E concentrations significantly decreased as dietary vitamin A increased; whereas significant increases in plasma

5 670 dialyzable phosphorus, and plasma and liver vitamin A concentrations were produced by increasing dietary levels of vitamin A. A significant (P < 0.001) increase in bone ash and plasma calcium with a corresponding reduction in rickets score, rickets incidence, and number three scores were observed by increasing the dietary levels of vitamin D 3. As dietary vitamin D 3 was increased, plasma vitamin A declined. This value approached significance (P 0.06). Conversely, increasing levels of vitamin E decreased (P < 0.01) bone ash, plasma calcium, and plasma and liver vitamin A concentrations, with significant increases (P < 0.001) in rickets score, rickets incidence, number three scores, and plasma and liver vitamin E concentrations. Interactions. A highly significant A D 3 (P < 0.001) interaction was observed for bone ash, with bone ash being depressed by high levels of vitamin A especially at low levels of vitamin D 3. The interaction of A E was significant (P 0.05) for rickets score, rickets incidence, and plasma vitamin A, and was highly significant for ABURTO AND BRITTON plasma and liver vitamin E concentrations. Bone quality was decreased by vitamin A in the presence of low dietary vitamin E but had little effect when the vitamin E level of the diet was high. Plasma and liver vitamin E concentrations were increased by feeding high dietary levels of vitamin E but addition of more vitamin A to the diet reduced the vitamin E concentrations in the plasma and liver. The interaction of vitamin D 3 E was highly significant for body weight, bone ash, and rickets number three scores and they were significant (P 0.05) for gain: feed ratio and rickets score. High dietary levels of vitamin E depressed growth at low dietary levels of vitamin D 3 but as vitamin D 3 was increased the growth depression was less. Bone ash and rickets followed a similar pattern. A pattern for gain:feed ratio was not clear. Plasma calcium approached significance (P 0.08) for this interaction. There were no significant three-way interactions for any of the criteria in this experiment. TABLE 4. Effects of different levels of vitamins A and D 3 (cholecalciferol) on 16-d body weight, gain:feed ratio, bone ash, and the incidence and severity of rickets in broiler chicks fed 10,000 IU/kg of vitamin E, Experiment 3 1 Treatments Rickets 16-d Gain:feed Bone Vitamin A Vitamin D 3 BW ratio ash Score Incidence No. 3 score (IU/kg) (g) (g:g) (%) (%) 1, ,500 1, ,500 1, ,500 2, ,500 2, ,500 3, , ,000 1, ,000 1, ,000 2, ,000 2, ,000 3, Pooled SEM Main effect means 1, , , , , , , ANOVA Source df Probabilities Vitamin A 1 < <0.001 <0.001 < Vitamin D 3 5 < <0.001 <0.001 <0.001 <0.001 Vitamin A vitamin D < Regression analysis 2 Low vitamin A Linear 1 < <0.001 <0.001 <0.001 Quadratic <0.001 <0.001 < High vitamin A Linear 1 < <0.001 <0.001 <0.001 <0.001 Quadratic 1 < < Means of four pens with 10 chicks per pen. 2Probability values shown for most appropriate model as determined by R 2.

6 DISCUSSION Results of these experiments show that very high dietary levels of vitamins A and E negatively affect broiler chick performance and bone metabolism especially at marginal dietary levels (500 IU/kg) of vitamin DIETARY VITAMINS A, D 3, AND E 671 D 3. If we examine the actual amounts of each of these vitamins in the diet they suggest that vitamin A and E might antagonize vitamin D 3. In the marginal vitamin D 3 diet (500 IU/kg) the actual amount of the vitamin added to the diet was 12.5 mg/kg and even in the diets with the largest amount of vitamin D 3 (3,000 IU/kg) TABLE 5. Effects of different levels of vitamins A, D 3, and E on 16-d body weight, gain:feed ratio, bone ash, incidence and severity of rickets, plasma calcium, plasma phosphorus, plasma vitamin A, plasma vitamin E, liver vitamin A, and liver vitamin E in broiler chicks, Experiment 4 Dietary treatments Gain: Rickets Plasma concentrations Liver concentration A D 3 E 16-d BW 1 feed ratio 1 Bone ash 1 Score 1 Inc 1 No. 3 score 1 Ca 2 P 2 A 2 E 2 A 2 E 2 (IU/kg) (g) (g:g) (%) (%) (mg/ 100 ml) (mg/ml) (mg/g) 1, , , , , ,500 1, ,500 1,500 5, ,500 1,500 10, ,500 2, ,500 2,500 5, ,500 2,500 10, , , , , , ,000 1, ,000 1,500 5, ,000 1,500 10, ,000 2, ,000 2,500 5, ,000 2,500 10, , , , , , ,000 1, ,000 1,500 5, ,000 1,500 10, ,000 2, ,000 2,500 5, ,000 2,500 10, Pooled SEM Main effect means 1, , , , , , , ANOVA Source df Probabilities Vitamin A < <0.001 <0.001 <0.001 <0.001 Vitamin D 3 2 < <0.001 <0.001 <0.001 <0.001 < Vitamin E 2 < <0.001 <0.001 <0.001 < <0.001 <0.001 <0.001 <0.001 Vitamin A vitamin D < Vitamin A vitamin E < <0.001 Vitamin D 3 vitamin E 4 < < < Vitamin A vitamin D 3 vitamin E Means of four pens per treatment with 10 chicks per pen. 2Means of two samples per pen per treatment.

7 672 ABURTO AND BRITTON only 75 mg/kg were added. In contrast, the smallest amount of vitamin A added to the diet was 450 mg/kg (1,500 IU/kg) and the largest was 48 mg/kg (160,000 IU/kg). Vitamin E was added to the diet from a low of 10 mg/kg (10 IU/kg) to a high of 10,000 mg/kg (10,000 IU/kg). If these vitamins share any common mechanisms in their biological activity then by mass action it is apparent that vitamin A and vitamin E would be favored over vitamin D 3. For example, a 500 IU/kg diet of vitamin D 3 is micromolar for vitamin D 3, 45,000 IU/kg of diet of vitamin A is millimolar for vitamin A, and 10,000 IU/kg of dietary vitamin E is millimolar for vitamin E. The data from these experiments indicate that a mixing error that would add a marginal level of dietary vitamin D 3 to the diet of broiler chicks would have effects, especially on bone quality and that adding excess vitamin A or vitamin E would further antagonize the problem. The work of Edwards et al. (1994) under conditions similar to these studies indicated a vitamin D 3 requirement for maximum response to be 275 IU/kg for growth, 503 IU/kg for bone ash, 552 IU/kg for plasma calcium, and 904 IU/kg for rickets prevention. Their studies were conducted with 5,500 IU/kg of dietary vitamin A and 11 IU/kg of vitamin E. It is apparent from the studies reported here that increasing the dietary vitamin A to levels of 10,000 IU/ kg or 3 mg/kg (Experiment 1) and 15,000 IU/kg or 4.5 mg/kg or greater (Experiments 2, 3, and 4) increased the need for vitamin D 3 in the chicks fed 500 IU/kg or 12.5 mg/kg of vitamin D 3 for bone ash and rickets. These dietary levels of vitamin A would not be considered much in excess by the industry, as the industry average in a survey by Ward (1993) was 8,822 IU/kg or 2.6 mg/ kg. Dietary vitamin E also depressed bone ash and increased rickets in marginal vitamin D 3 diets. The actual amounts of vitamin E required (2,500 IU/kg or 2,500 mg/kg) to cause a problem were much greater than those required using vitamin A, but the effect on vitamin D 3 was greater if the high levels of vitamin A and vitamin E were fed together. The addition of vitamin E at the levels of 2,500 to 10,000 IU/kg of the diet would probably only occur due to a serious feed mixing error. In Experiment 2, there is some indication that lower levels may have some effect on bone quality especially at higher levels of vitamin A. It is apparent that high levels of vitamin A and E depress bone ash and increase rickets if the vitamin D 3 level of the diet is marginal. The level of vitamin D 3 considered marginal appears to increasing as the vitamin A and E level of the diet increases. It appears, however, these negative effects of feeding high levels of vitamin A were overcome by feeding levels of vitamin D 3 (Experiments 3 and 4) that would be considered to be practical by the industry (2,000 to 3,000 IU/kg) (Ward, 1993). March et al. (1973) reported that excess vitamin E administered orally or parenterally depressed calcification when the diet was deficient either in calcium or vitamin D 3. They suggested that excess vitamin E, like other fat-soluble vitamins, must be considered potentially toxic. In Experiments 3 and 4, calcification problems were not produced by excess vitamin E when the dietary level of vitamin D 3 was adequate. Therefore, it appears from our work that the potential vitamin E toxic effects reported by March et al. (1973) and Murphy et al. (1981) can be prevented by feeding practical levels of vitamin D 3. It also could be suggested that increasing the dietary levels of vitamin A and E in order to improve immune response (Friedman and Sklan, 1997) of the chicken could create a condition in which these vitamins could increase the dietary need for vitamin D 3. Any situation 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. The data in this paper clearly show that this relationship is true for vitamins A, E, and D 3. This work indicates the need to understand the proper ratios of the fat-soluble vitamins for the best performance of chickens. One of the most noteworthy properties of vitamin A is the extent to which it can be accumulated in the liver. In Experiment 4, we found that the concentration of vitamin A in the liver significantly (P < 0.001) increased as the level of the vitamin was increased in the diet. Plasma vitamin A concentrations showed a quadratic response to increasing dietary vitamin A but the concentration of vitamin A in the liver was linear, confirming the high capacity of the liver to accumulate vitamin A. The amount of vitamin A found in the liver was several times higher than its plasma levels when feeding the same levels of vitamin A, confirming that the liver is the first choice for vitamin A storage in the body. Yet, excess levels of vitamin E (5,000 and 10,000 IU/kg of diet), significantly (P < 0.001) reduced the concentration of vitamin A in plasma and liver. In Experiment 4, we also studied the relationship between high dietary levels of vitamin A and plasma and liver vitamin E concentrations. Plasma and liver vitamin E concentrations were increased by increasing the dietary level of vitamin E (P < 0.001) in the absence of excess vitamin A. Nockels et al. (1976) found that vitamin E increased in plasma and liver of broiler chickens fed even higher levels of vitamin E than those used here. In our work there was a highly significant (P < 0.001) reduction in plasma and liver vitamin E concentrations by feeding high levels of vitamin A (15,000 or 45,000 IU/kg). Although it was not a major objective of these studies to look at the dietary interactions of vitamin A and vitamin E it was clear from the blood levels in Experiment 4 that this relationship does exist. Feeding excess vitamin A decreases the blood and liver levels of vitamin E and feeding excess vitamin E decreases the blood and liver levels of vitamin A. The mechanism of this relationship needs further study.

8 DIETARY VITAMINS A, D 3, AND E 673 Perhaps the most consistent and important finding throughout all the experiments reported herein was the fact that the nutritional antagonism from feeding high levels of vitamins A or E on performance and bone metabolism in broiler chicks was observed when marginal levels of vitamin D 3 were fed. However, the supplementation of practical levels of vitamin D 3 (2,000 to 3,000 IU/kg of diet) seems to have prevented much of the body weight depression, produced maximum bone ash, and controlled the incidence and severity of rickets. ACKNOWLEDGMENT We wish to thank the Hoffmann-La Roche Company for supplying the vitamins for these and other experiments. REFERENCES Abawi, F. G., and T. W. Sullivan, Interactions of vitamins A, D 3, E, and K in the diet of broiler chicks. Poultry Sci. 68: Association of Official Agricultural Chemists, Official Methods of Analysis. 16th ed. Association of Official Agricultural Chemists, Washington, DC. Buttriss, J. L., and A. T. Diplock, High-performance liquid chromatography methods for vitamin E in tissues. Methods Enzymol. 105: Combs, G. F., Jr., Differential effects of high dietary levels of vitamin A on the vitamin E-selenium nutrition of young and adult chickens. J. Nutr. 106: Combs, G. F., Jr., and M. L. Scott, Dietary requirements for vitamin E and selenium measured at the cellular level in the chick. J. Nutr. 104: Edwards, H. M., Jr., M. A. Elliot, S. Sooncharernying, and W. M. Britton, Quantitative requirement of cholecalciferol in the absence of ultraviolet light. Poultry Sci. 73: Friedman, A., and D. Sklan, Effects of retinoids on immune responses in birds. World s Poult. Sci. J. 53: Frigg, M., and J. Broz, Relationships between vitamin A and vitamin E in the chick. Int. J. Vit. Nutr. Res. 54: Hatam, L. J., and H. J. Kayden, A high-performance liquid chromatographic method for the determination of tocopherol in plasma and cellular elements of the blood. J. Lipid Res. 20: Jansson, L., B. Akesson, and L. Holmberg, Vitamin E and fatty acid composition of human milk. Am. J. Clin. Nutr. 34:8 13. Long, P. H., S. R. Lee, G. N. Rowland, and W. M. Britton, Experimental rickets in broilers: Gross, microscopic, and radiographic lesions. II. Calcium deficiency. Avian Dis. 28: March, B. E., E. Wong, S. J. Sim, and J. Biely, Hypervitaminosis E in the chick. J. Nutr. 103: McCuaig, L. W., and I. Motzok, Excessive dietary vitamin E: Its alleviation of hypervitaminosis A and lack of toxicity. Poultry Sci. 49: Murphy, T. P., K. E. Wright, and W. J. Pudelkiewicz, An apparent rachitogenic effect of excessive vitamin E intakes in the chick. Poultry Sci. 60: Nockels, C. F., D. L. Menge, and E. W. Kienholz, Effect of excessive dietary vitamin E on the chick. Poultry Sci. 55: Pudelkiewicz, W. J., L. Webster, and L. D. Matterson, Effects of high levels of dietary vitamin A acetate on tissue tocopherol and some related analytical observations. J. Nutr. 84: SAS Institute, SAS/STAT User s Guide. Vol. I and II. Version 6. 4th ed. SAS Institute Inc., Cary, NC. Sklan, D., and S. Donoghue, Vitamin E response to high dietary vitamin A in the chick. J. Nutr. 112: Taylor, T. G., K.M.L. Morris, and J. Kirkely, Effects of dietary excesses of vitamins A and D on some constituents of the blood of the chicks. Br. J. Nutr. 22: Veltmann, J. R., Jr., and L. S. Jensen, Interaction between high dietary vitamin A and low dietary vitamin D 3 in the chick and turkey poult. Nutr. Rep. Int. 31: Veltmann, J. R., Jr., L. S. Jensen, and G. N. Rowland, Excess dietary vitamin A in the growing chick: Effect of fat source and vitamin D. Poultry Sci. 65: Veltmann, J. R., Jr., L. S. Jensen, and G. N. Rowland, Partial amelioration of vitamin A toxicosis in the chick and turkey poult by extra dietary vitamin D 3. Nutr. Rep. Int. 35: Ward, N. E., Vitamin supplementation rates for U. S. commercial broilers, turkeys, and layers. J. Appl. Poult. Res. 2: