Effects of Different Levels of Vitamins A and E on the Utilization of Cholecalciferol by Broiler Chickens 1

Transcription

1 Effects of Different Levels of Vitamins A and E on the Utilization of Cholecalciferol by Broiler Chickens 1 A. ABURTO and W. M. BRITTON2 Department of Poultry Science, University of Georgia, Athens, Georgia ABSTRACT Three experiments were conducted to determine the effects of high dietary levels of vitamins A and E on the utilization of cholecalciferol by broiler chicks. In Experiment 1, chicks were fed six levels of vitamin A (5,000, 10,000, 20,000, 40,000, 80,000, and 160,000 IU/kg). Cholecalciferol (vitamin D 3 ) was not added to the basal diet but all birds were exposed to ultraviolet (UV) fluorescent light. Body weight was decreased only at levels of vitamin A of 80,000 IU/kg or above. In Experiment 2, birds were exposed to UV fluorescent light or no UV light, two levels of dietary vitamin A (1,500 and 45,000 IU/kg) and three levels of dietary vitamin D 3 (0, 500, and 2,500 IU/kg) in a factorial arrangement. The high level of vitamin A reduced (P < 0.001) bone ash but only at a marginal level of vitamin D 3 (500 IU/kg) and when the birds were not exposed to UV light. In Experiment 3, birds were exposed to UV fluorescent light or no UV light, two levels of dietary vitamin E (10 and 10,000 IU/kg) and three levels of dietary vitamin D 3 (0; 500 and 2,500 IU/ kg) in a factorial arrangement. The high level of vitamin E significantly (P < 0.05) reduced body weight, bone ash, plasma calcium, and increased rickets but only at 500 IU/kg of vitamin D 3. Feeding 2,500 IU/kg of vitamin D 3 overcame the effects of the high level of vitamin E, causing a significant (P < 0.05) interaction. Ultraviolet light also prevented the detrimental effects of the high level of vitamin E. The results of these studies indicate that high dietary levels of vitamins A and E negatively affected the utilization of vitamin D 3 only when D 3 was present at a marginal level (500 IU/kg) in the diet but not when it was synthesized in the bird by exposure to UV light or supplemented at 2,500 IU/kg in the diet. (Key words: vitamin A, cholecalciferol, vitamin E, ultraviolet light, broiler) 1998 Poultry Science 77: INTRODUCTION Received for publication June 10, Accepted for publication November 19, 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 High dietary levels of vitamins A and E are believed to interact with vitamin D 3. March et al. (1973) reported that with a calcium-deficient or vitamin D-deficient diet, bone calcification was further 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. Abawi and Sullivan (1989) found that feeding high levels of vitamin A decreased body weight when dietary vitamin D was low; however, increasing dietary vitamin D reversed the effect producing a significant A by D interaction. The administration of single high levels of vitamins A or D 3 has been shown to affect growth and bone metabolism. Davies and Moore (1934), and Moore and Wang (1945) showed that hypervitaminosis A caused bone fragility. Administration of excess vitamin A reduced the effects of hypervitaminosis D in the rat (Clark and Bassett, 1962; Clark and Smith, 1964); whereas extra vitamin D 3 protected the rat (Vedder and Rosenberg, 1938), dog (Frey et al., 1975), and the chick (Taylor et al., 1968; Veltmann and Jensen, 1985; Veltmann et al., 1986, 1987) against vitamin A toxicosis. Although most workers have agreed on the existence of nutritional relationships among fat-soluble vitamins in general and nutritional interactions among vitamins A, D 3, and E in particular, there has been considerable disagreement as to whether it is due essentially to an interaction among the three vitamins in the intestinal tract, prior to or during absorption, or in the tissues of the animals after absorption. Unfortunately, there are not very sensitive methods available to evaluate the status of vitamins A and E of animals. The measurement of vitamins A and E in plasma and liver by HPLC analysis has been used for several years, but the values obtained are not easily related to dietary needs. Abbreviation Key: UV = ultraviolet; vitamin D 3 = cholecalciferol. 570

2 UTILIZATION OF CHOLECALCIFEROL WITH HIGH VITAMIN A AND E 571 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 Calculated composition Crude protein ME, kcal/kg 3, Calcium 0.98 Phosphorus, nonphytate Vitamin B premix provided in milligrams per kilogram 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 in milligrams per kilogram 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, However, bone ash has proved to be a very sensitive measure relative to dietary need for vitamin D in broiler chicks. In preliminary studies, we used HPLC techniques to measure vitamins A and E, and bone parameters (bone ash and rickets) for vitamin D 3 status in broiler chicks, in an attempt to determine the nutritional relationships among these vitamins. We found that the nutritional antagonism occurs, at least in large proportion, at the intestinal absorption level (Aburto and Britton, unpublished observations). Edwards et al. (1994) reported that birds fed no vitamin D 3 that were exposed to ultraviolet light from battery fluorescence tubes required 800 to 1,600 IU/kg of dietary vitamin D 3 to provide maximum response for 16-d body weight, gain:feed ratio, bone ash, and plasma calcium, and reduction of rickets comparable to the UV light values. Ultraviolet light causes a photochemical reaction in the skin, in which 7-dehydrocholesterol is converted to previtamin D and then to vitamin D (Holick, 1981). If the nutritional antagonism among vitamins A, D 3, and E occurs at the intestinal level prior to absorption and vitamin D 3 is supplied by exposure to UV light then the feeding of high levels of dietary vitamins A and E should not effect vitamin D 3 metabolism. The purpose of the experiments reported herein was to elucidate the effects of high dietary levels of vitamins A and E on the utilization of vitamin D 3 by broiler chicks when vitamin D 3 was supplied in the diet or supplied by UV light induction. 3Arm-a-lite, Thermoplastic Processes, Stirling, NJ Hoffmann-La Roche Co., Nutley, NJ MATERIALS AND METHODS Day-old male (Ross Ross) broiler chicks were used in all experiments. Four replicates of 10 chicks each were 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 provided for ad libitum consumption and all experiments were conducted for 16 d. Experiments were approved by the University of Georgia Animal Care Committee. 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 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 covered with plastic sleeves to prevent exposure of the chicks 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. This experiment was conducted to determine the effects of feeding increasing levels of vitamin A on the utilization of vitamin D 3 by broiler chicks and evaluate whether the effect of vitamin A occurs at the absorption level. Six levels of vitamin A (as retinyl acetate4 5,000, 10,000, 20,000, 40,000, 80,000, and 160,000 IU/kg) were added to the basal diet. No vitamin D 3 was supplemented to the basal diet. However, all pens of chicks were exposed to UV light from the fluorescent lights detailed above in the arrangement described by Edwards et al. (1994). The other fat-soluble vitamins were added individually (20 IU/kg of vitamin E as dl-atocopheryl acetate4 and 2 mg/kg of vitamin K as menadione sodium bisulfite4) to the basal diet. The sources of the vitamins used in these experiments were commercial concentrate vitamins. Vitamins A, D 3,4 and E were spray-dried, water-dispersible products. Vitamin A activity was 500,000 IU/g; vitamin D 3 activity was 500,000 IU/g; vitamin E activity was 500 IU/g; and vitamin K had 33% menadione. These concentrated forms of the vitamins were premixed with rice hulls where appropriate for mixing into the feed. Analysis of variance and simple

3 572 ABURTO AND BRITTON TABLE 2. Effects of dietary vitamin A on 16-d body weight, gain:feed ratio, bone ash, and the incidence and severity of rickets in broiler chicks receiving ultraviolet light (UV) and no dietary cholecalciferol, Experiment 1 Treatments 16-d Gain:feed Bone Rickets Vitamin A UV BW 1 ratio 1 ash 1,2 Score 1 Incidence 1 No. 3 Score 1 (IU/kg) (+/ ) (g/chick) (g:g) (%) (%) 5, , , , , , Pooled SEM Probabilities ANOVA Source df Vitamin A Regression analysis Vitamin A Means of four pens per treatment with 10 chicks per pen. 2Percentage of dry fat-free bone. regression analysis for levels of vitamin A were computed (SAS Institute, 1990). Experiment 2. This experiment was conducted to determine the effects of feeding low and high levels of vitamin A on the absorption of vitamin D 3 by broiler chicks. Half of the 48 pens of chicks were exposed to UV light from the fluorescent lights and the other half of the chicks were placed in pens with sleeves covering the fluorescent light to prevent exposure to UV light. Two supplemental levels of vitamin A (1,500 and 45,000 IU/kg) and three levels of vitamin D 3 (0, 500, and 2,500 IU/kg) were added to the basal diet. Vitamins E and K were maintained constant by adding 20 IU/kg and 2 mg/kg, respectively, in the basal diet. The experimental design was a factorial arrangement of treatments and the data were analyzed by analysis of variance with UV light, vitamin A, and vitamin D 3 as main effects. This analysis was performed overall and by category of UV light (with or without) (SAS Institute, 1990). Experiment 3 was conducted to determine the effects of feeding low and very high levels of vitamin E on the absorption of vitamin D 3 by broiler chicks. Half of the 48 pens of chicks were exposed to UV light from the fluorescent lights and the other half of the chicks were placed in pens with sleeves covering the fluorescent light to prevent exposure to UV light. Two supplemental levels of vitamin E (10 and 10,000 IU/kg) and three levels of vitamin D 3 (0; 500; and 2,500 IU/kg) were added to the basal diet. Vitamins A and K were maintained constant by adding 8,000 IU/kg and 2 mg/kg, respectively, to the basal diet. The experimental design was a factorial 5Section N-31, Techincon Autoanalyzer Methodology, (1969) Techincon Corp., Tarrytown, NY Section N-46, Techincon Autoanalyzer Methodology, (1969) Techincon Corp., Tarrytown, NY Sigma Chemical Co., St Louis, MO arrangement of treatments and the data were analyzed by analysis of variance with UV light, vitamin E, and vitamin D 3 as main effects. This analysis was performed overall and by category of UV light (SAS Institute, 1990). Plasma and Tissue Analysis At termination of Experiments 2 and 3, blood samples were obtained from two birds per pen by cardiac puncture, and the plasma was analyzed for total Ca5 and dialyzable P.6 Plasma vitamin A and E concentrations were also determined from the same plasma samples by HPLC analysis. Vitamins A and E were extracted from the plasma 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), except that a spectrophotometric detector was used at 292 nm, which allowed detection of vitamin A and E in the same analysis. All-trans-retinol and a-tocopherol were used as standards.7 Liver samples from the same two birds used to obtain plasma in Experiment 2 were extracted by the procedure of Buttriss and Diplock (1984). Vitamin A was extracted with hexane following saponification. The HPLC analysis was conducted as described above. Experiment 1 RESULTS Regression analysis showed a significant (P < 0.02) decrease in body weight at vitamin A levels exceeding 40,000 IU/kg (Table 2). Gain:feed ratio was significantly decreased (P < 0.04) by the same levels of vitamin A; however, neither bone ash nor rickets were affected by

4 UTILIZATION OF CHOLECALCIFEROL WITH HIGH VITAMIN A AND E 573 TABLE 3. Effects of ultraviolet light (UV) exposure, and different levels of vitamin A and cholecalciferol (D 3 ) on 16-d body weight, gain:feed ratio, bone ash, incidence and severity of rickets, plasma calcium, plasma phosphorus, plasma vitamin A, and liver vitamin A in broiler chicks, Experiment 2 Treatments Rickets Plasma concentrations UV A D 3 16-d BW 1 Gain: feed 1 Bone ash 1,2 Score 1 Inc 1 #3 Score 1 Ca 3 P 3 A 3 (+/ ) (IU/kg) (g/chick)(g:g) (%) (%) (mg/100 ml) (mg/ml) (mg/g) + 1, , ,500 2, , , ,000 2, , , ,500 2, , , ,000 2, Pooled SEM Main effect means UV a a 0.2 b 9 b 3 b b 34.2 b 307 b b 2.3 a 77 a 72 a a 41.7 a A 1, x x 1.2 x 43 x 36 x x 2.6 y 45, x y 1.3 x 44 x 39 x x 73.3 x D f g 1.6 e 56 e 53 e e 40.6 e e f 1.3 f 49 f 41 f f 39.5 e 2, e e 0.7 g 26 g 19 g g 33.8 e ANOVA Source df Probabilities UV 1 < <0.001 <0.001 <0.001 < < A < <0.001 D 3 2 < <0.001 <0.001 <0.001 < UV A UV D <0.001 <0.001 <0.001 <0.001 < A D UV A D UV (+) A <0.001 D < A D UV ( ) A <0.001 D 3 2 < <0.001 <0.001 <0.001 <0.001 < A D a,b;e g;x,ymeans within a variable with no common superscript differ significantly (P < 0.05). 1Means of four pens per treatment with 10 chicks per pen. 2Percentage of dry fat-free bone. 3Means of 8 samples from two chicks per pen per treatment. 4Means of 8 livers from two chicks per pen per treatment. Liver A 4 increasing the dietary level of vitamin A, indicating that the UV light was meeting most of the vitamin D 3 need of the chicks and that vitamin A influence on growth was caused by an effect on something other than vitamin D 3. Experiment 2 Groups of birds exposed to radiation from unfiltered fluorescent light had higher body weight and bone ash and a lower incidence of rickets than birds exposed to filtered fluorescent light (Table 3). Plasma and liver vitamin A concentrations were lower in birds exposed to unfiltered fluorescent light than those exposed to filtered fluorescent light. Gain:feed ratio, plasma calcium, and plasma phosphorus were not influenced by UV light. In chicks fed 0 IU/kg of vitamin D with or without UV light there was no change in body weights when 45,000 IU/kg of vitamin A was consumed. The same was true for chicks fed 2,500 IU/kg of vitamin D 3, but body weight was decreased by 45,000 IU/kg of vitamin A (P < 0.08) in the chicks fed the marginal level of vitamin D 3 (500 IU/kg). The 45,000 IU/kg level of vitamin A significantly (P <

5 ) reduced bone ash, but only at the 500 IU/kg level of vitamin D 3 (500 IU/kg) when the birds were not exposed to UV light. Vitamin D-type rickets, plasma calcium, plasma phosphorus, and plasma vitamin A concentration were not significantly influenced by dietary vitamin A. Liver vitamin A significantly increased (P < 0.001) when 45,000 IU/kg of vitamin A was fed. When dietary vitamin D 3 was changed from 0 to 500 IU/kg, a significant (P < 0.01) increase in body weight, bone ash, and plasma vitamin A concentration, and decreases in rickets score, rickets incidence and number 3 scores were seen. When dietary vitamin D 3 was increased to 2,500 IU/kg, a further increase in bone ash and a decrease in rickets were seen. Plasma vitamin A concentration was significant decreased (P < 0.02) by dietary vitamin D 3 only when the birds were not exposed to UV light. The response to vitamin D 3 for plasma calcium approached significance (P < 0.09) and no effects were observed on gain:feed, plasma phosphorus, and liver vitamin A. Interactions. A significant interaction (P < 0.05) between UV light and vitamin A was observed for bone ash and liver vitamin A concentration. Bone ash was decreased by high levels of vitamin A but only when birds were not exposed to UV light. Liver vitamin A was reduced by exposure to UV light but it increased by increasing vitamin A in the diet. When birds were not exposed to UV light, body weight, bone ash, and plasma calcium all decreased, with a corresponding increase in rickets. The addition of dietary vitamin D 3 prevented these effects (P < 0.05). Experiment 3 Chicks exposed to UV light had significantly higher (P < 0.01) body weight, bone ash, and plasma calcium, and reduced rickets, plasma and liver vitamin A concentrations compared to chicks receiving no UV light (Table 4). High dietary vitamin E significantly (P < 0.05) reduced body weight, bone ash, and plasma calcium, and increased rickets. However, the effect of high dietary vitamin E on body weight, bone ash, and rickets was more severe at the marginal level of vitamin D 3 (500 IU/kg) and when the birds were exposed to filtered fluorescent light. No effect of high level of vitamin E was observed in any of the groups when vitamin D 3 was not supplemented to the basal diet. The response to increasing dietary vitamin D 3 was significant (P < 0.05) for all the criteria measured except plasma phosphorus. Chicks fed 500 IU of D 3 /kg had maximum vitamin E levels in the plasma and plasma vitamin E declined slightly when 2,500 IU of D 3 /kg was fed. Interactions. A significant interaction (P < 0.05) between UV light and vitamin E was observed for plasma vitamin E concentration, and the same interaction approached significance (P < 0.06 and P < 0.09) for the severity of rickets (number 3 scores) and plasma calcium, respectively. The high level of dietary vitamin E greatly ABURTO AND BRITTON increased plasma vitamin E when birds were not exposed to UV light. The interaction between UV light and vitamin D 3 was highly significant (P < 0.001) for body weight, bone ash, rickets score, rickets incidence, number 3 scores, and plasma calcium, and approached significance (P < 0.07) for gain:feed ratio. In the absence of UV light, body weight, bone ash, and plasma calcium increased and the values for the rickets variables decreased when vitamin D 3 was added to the diet; however, this response to D 3 was much smaller when UV light was present. The interaction of vitamins E by D 3 was significant (P < 0.05) for bone ash, rickets score, and severity of rickets (number 3 scores). High dietary vitamin E decreased bone ash and increased rickets that was prevented by vitamin D 3. The three-way interaction among UV light, vitamin E, and vitamin D 3 was significant (P < 0.05) for bone ash, rickets score, rickets incidence, number 3 scores, and plasma calcium. The high dietary vitamin E reduced bone ash and plasma calcium and increased rickets in the absence of UV light and presence of 500 IU/kg vitamin D 3, but these changes were corrected by the addition of 2,500 IU/kg vitamin D 3. DISCUSSION The results of the experiments described above indicate that the nutritional antagonism among vitamins A, D 3, and E after absorption from the intestinal tract is of minor importance. Based on the criteria measured, high dietary levels of vitamins A and E did not interfere with the metabolism of vitamin D 3 when it was synthesized in the skin from UV light exposure. When the birds were exposed to filtered fluorescent light (no UV), high dietary levels of vitamins A and E significantly affected the utilization of dietary vitamin D 3, which reduced body weight, bone ash, and plasma calcium and increased rickets; however, these changes occurred only at marginal dietary vitamin D 3 (500 IU/ kg). In Experiment 1, feeding vitamin A at 80,000 IU/kg of diet caused a small reduction in body weight. This weight reduction would appear to be caused by something other than a vitamin A effect on vitamin D 3, as bone ash and the incidence and severity of rickets were not affected. No vitamin D 3 was supplemented in the diet of these birds, but all groups were exposed to direct fluorescent light that provided about 3.4% of the wattage in the UV range (260 to 400 nm) (Edwards et al., 1994). In experiments from our laboratory (unpublished observations), body weight was reduced when vitamin A was fed at 40,000 IU/kg of diet. In these experiments, a marginal vitamin D 3 (500 IU/kg) level was added to the diet and all birds were exposed to filtered fluorescent light (no UV). These results indicated that 500 IU/ kg of diet of vitamin D 3 appeared not to be enough to produce maximum tibia bone ash and to control rickets when there was no UV light and that increasing dietary vitamin A decreased bone ash and increased rickets. In Experiment 1, it appeared that the UV light alone was

6 UTILIZATION OF CHOLECALCIFEROL WITH HIGH VITAMIN A AND E 575 TABLE 4. Effects of ultraviolet light (UV) exposure, and different levels of vitamin E and cholecalciferol (D 3 ) on 16-d body weight, gain:feed ratio, bone ash, incidence and severity of rickets, plasma calcium, plasma phosphorus, and plasma vitamin E in broiler chicks, Experiment 3 Treatments Rickets Plasma concentrations 16-d Bone UV E D BW 1 ash 1,2 3 Gain:feed 1 Score 1 Inc 1 #3 Score 1 Ca 3 P 3 E 3 D (+/ ) (IU/kg) (g/chick) (g:g) (%) (%) (mg/100 ml) (mg/ml) , , , ,000 2, , , , ,000 2, Pooled SEM Main effect means UV a a 37.7 a 0.3 b 11 b 5 b 9.0 a b 375 b a 32.1 b 1.4 a 48 a 45 a 7.8 b a E x x 35.6 x 0.6 y 24 y 21 y 8.5 x y 10, y y 34.2 y 1.0 x 36 x 30 x 8.2 x x f e 30.0 g 1.7 e 63 e 56 e 7.1 g f e e 36.2 f 0.6 f 24 f 20 f 8.8 f e 2, e f 38.5 e 0.04 g 2 g 1 g 9.2 e e ANOVA Source df Probabilities UV 1 < <0.001 <0.001 <0.001 <0.001 < E 1 < <0.001 <0.001 <0.001 < <0.001 D 3 2 < <0.001 <0.001 <0.001 <0.001 < UV E UV D 3 2 < <0.001 <0.001 <0.001 <0.001 < E D L E D < < UV (+) E <0.001 D < E D UV ( ) E <0.001 <0.001 <0.001 < <0.001 D 3 2 < <0.001 <0.001 <0.001 <0.001 < E D <0.001 <0.001 <0.001 < a,b;e g;x,ymeans within a variable with no common superscript differ significantly (P 0.05). 1Means of four pens per treatment with 10 chicks per pen. 2Percentage of dry fat-free bone. 3Means of 8 samples from two chicks per pen per treatment. not enough to produce maximum bone ash or to control rickets, but these parameters were not affected by dietary vitamin A. There is a possibility that under the conditions of our studies, the birds exposed to fluorescent light were unable to synthesize enough vitamin D 3 by photolysis of UV light. In contrast, Edwards et al. (1994) reported that the exposure to UV light seemed to give maximum response for all of the criteria that they measured, including 16-d body weight, gain:feed ratio, bone ash, plasma calcium, and the incidence of rickets, as compared to values observed for birds receiving 800 or 1,600 IU/kg of vitamin D 3. Birds exposed to UV light in Experiment 2 showed a small positive response in body weight, increased bone ash, and lower incidence of rickets when 500 IU/kg of vitamin D 3 was added to the diet, suggesting that UV light alone was probably not enough to support maximum response of these criteria. This positive response to dietary vitamin D 3 was produced only when vitamin A was added at 1,500 IU/kg of diet, because vitamin A (45,000 IU/kg) interfered with dietary vitamin D 3 (500 IU/kg) in birds exposed to UV light. This effect was more obvious when the birds were not exposed to UV light and all their vitamin D 3 came from

7 576 the diet. Another observation that supports the inhibition of absorption of vitamin D 3 by high dietary levels of vitamin A was when the birds were not exposed to UV light and were not supplemented with vitamin D 3 in the diet. These groups did not show any effect of high dietary vitamin A on body weight (271 vs 272 g), bone ash (24.5 vs 23.6%), incidence and severity of rickets (100 vs 100%), and plasma calcium (5.8 vs 6.2 mg/100 ml). In Experiment 3, adding vitamin D 3 (500 IU/kg) to the diet of birds exposed to UV light gave a slight positive response in body weight, an increase in bone ash, and a small decline in rickets, showing again that UV light without supplementary vitamin D 3 was probably not adequate. This positive effect of dietary vitamin D 3 to increase growth and bone ash was observed at the low level of vitamin E (10 IU/kg) when 500 IU/kg of vitamin D 3 was fed; however, at the very high dietary level of vitamin E (10,000 IU/kg), 500 IU/ kg of dietary vitamin D 3 overcame the bone problems but did not return growth to normal even at 2,500 IU/ kg of diet, which suggests that high dietary vitamin E may affect something other than vitamin D 3, causing the growth depression. Birds exposed to filtered fluorescent light (no UV light) were severely affected by the higher level of vitamin E (10,000 IU/kg) when the level of vitamin D 3 was supplemented at the marginal level (500 IU/kg). The effect was seen in body weight (417 vs 379 g), bone ash (36.0 vs 31.9%), rickets incidence (25 vs 60%), rickets number 3 scores (18 vs 53%), and plasma calcium (9.1 vs 8.0 mg/100 ml). As seen with high vitamin A (45,000 IU/kg) in Experiment 2, birds without UV light and without dietary vitamin D 3 showed no effect of high dietary vitamin E on body weight (305 vs 305 g), bone ash (24.5 vs 23.6%), and incidence and severity of rickets (100 vs 100%). These results again suggest that the most important quantitative effect of high dietary levels of vitamins A and E on the utilization of vitamin D 3 occur at the intestinal level prior to or during the absorption process. The results described above are in agreement with the findings of other investigators that reported that extra vitamin D 3 protected the rat (Vedder and Rosenberg, 1938), dog (Frey et al., 1975), cattle (Payne and Manston, 1967), and poultry (Taylor et al., 1968; Veltmann and Jensen, 1985; Veltmann et al., 1986, 1987) against vitamin A toxicosis. March et al. (1973) and Murphy et al. (1981) reported that excess vitamin E can be toxic for chicks. They could not completely prevent the toxicity with vitamin D 3, but the highest level they fed was 500 IU/kg of diet. Furthermore, when birds were exposed to UV light, we did not find any effect of feeding high dietary levels of vitamins A and E, suggesting that internally produced vitamin D 3 is not affected by high dietary levels of these vitamins. This approach, using UV light to produce vitamin D 3 compared to dietary vitamin D 3, has not been used before in the experimental designs of experiments looking for nutritional antagonisms of vitamins A and E on vitamin D 3. ABURTO AND BRITTON Although the absorption of vitamin D 3 was not measured in these experiments, the evidence points to absorption as the major cause for the adverse effect of high levels of vitamins A and E on vitamin D 3 ; suggestive of an antagonism of vitamin A and E on Vitamin D 3. In the marginal vitamin D 3 diet (500 IU/ kg), 12.5 mg/kg was added to the diet and even with the high vitamin D 3 diet (2,500 IU/kg) only 62.5 mg/kg was added to the diet. In contrast, vitamin A was added at 450 mg/kg (1,500 IU/kg) or 13.5 mg/kg (45,000 IU/kg) and vitamin E was added at 10 mg/kg (10 IU/kg) or 10,000 mg/kg (10,000 IU/kg). If the vitamins share any common mechanism of absorption, then by mass action it is apparent that vitamin E and vitamin A would be favored over vitamin D 3. For example, if 500 IU/kg of dietary vitamin D 3 is expressed on a molar basis compared to 45,000 IU/kg of vitamin A or 10,000 IU/kg of vitamin E, the molar concentrations are micromolar for vitamin D 3, millimolar for vitamin A and millimolar for vitamin E (dl-atocopherol). Although there are large differences in molar concentrations of vitamin A and vitamin E from vitamin D 3, increasing the molar concentration of vitamin D 3 to micromolar (2,500 IU/kg of diet) overcame the problems caused by the high levels of vitamin A and vitamin E. It would appear that studies to work out the proper molar ratios among these vitamins are needed. ACKNOWLEDGMENT We wish to thank the Hoffmann-LaRoche Company for supplying the vitamins for these 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: Clark, I., and C.A.L. Bassett, The amelioration of hypervitaminosis D in the rat with vitamin A. Exptl. Med. 115: Clark, I., and M. R. Smith, Effects of hypervitaminosis A and D on skeletal metabolism. J. Biol. Chem. 239: Davies, A. W., and T. Moore, Vitamin A and carotene. XI. The distribution of vitamin A in the organs of the normal and hypervitaminotic rat. Biochem. J. 28: 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: Frey, R. A., M. M. Guffy, and H. W. Leipold, Hypervitaminosis A in the dog. Am. J. Vet. Res. 36:

8 UTILIZATION OF CHOLECALCIFEROL WITH HIGH VITAMIN A AND E 577 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: Holick, M. F., The cutaneous photosynthesis of previtamin D 3 : A unique photoendocrine system. J. Invest. Dermatol. 76: 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: Moore, T., and Y. L. Wang, Hypervitaminosis A. Biochem. J. 39: 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: Payne, J. M., and R. Manston, The safety of massive doses of vitamin D 3 in the prevention of milk fever. Vet. Rec. 81: SAS Institute, SAS/STAT User s Guide. Vol. I and II. Version 6. 4th ed. SAS Institute Inc., Cary, NC. 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: Vedder, E. B., and C. Rosenberg, Concerning the toxicity of vitamin A. J. Nutr. 16: 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: