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Effect of Various Dietary Factors and Age on Plasma α-Tocopherol Concentration of Turkeys1
METABOLISM AND NUTRITION Effect of Various Dietary Factors and Age on Plasma a-Tocopherol Concentration of Turkeys1 I. BARTOV Division of Poultry Science, Agricultural Research Organization, The Volcani Center, P.O.B. 6, Bet Dagan 50250, Israel (Received for publication June 24, 1982) ABSTRACT Plasma a-tocopherol concentration of 15- to 22-week-old turkeys was found to be linearly related to dietary all-rac-a-tocopheryl acetate at levels ranging from 10 to 100 mg/kg. However, in 4-week-old poults a plateau was observed at a level of 30 mg a-tocopheryl acetate/kg diet. Increasing soybean oil level in diets containing 10, 50, and 100 mg a-tocopheryl acetate/kg from .5 to 3.0% significantly (P<.05) elevated plasma a-tocopherol. The latter was not affected by the type of dietary fat (soybean vs. tallow). Supplementary cholic acid (.075 - .1%) in diets containing 20 mg a-tocopheryl acetate/kg elevated plasma a-tocopherol slightly but significantly (P<.05); however, higher levels of this bile acid had no effect. Increasing dietary selenium supplementation from .1 to 1.1 ppm did not affect this parameter either. a-Tocopherol and a-tocopheryl acetate, when dissolved in soybean oil or in ethoxyquin at levels equivalent to 40 mg/kg diet, had a similar effect on plasma a-tocopherol concentration. (Key words: turkey, plasma, a-tocopherol, dietary fat, cholic acid) 1983 Poultry Science 62:635-641 INTRODUCTION
Turkeys are known for their poor ability to accumulate vitamin E in their tissues as compared with chicks (Mecchi et al, 1956; Marusich et al, 1975; Sklan et al, 1982). Mecchi et al. (1956) and Marusich et al. (1975) attribute this phenomenon to decreased gastrointestinal absorption. Vitamin E is usually incorporated into the diet as a-tocopheryl acetate, which is a stable form of the vitamin. Bile and pancreatic juice have been reported to be essential for the intestinal absorption of this ester, which is poorly absorbed from the intestine (GalloTorres, 1970). MuUer et al (1976) noted that bile salts increase the activity of pancreatic esterase against tocopheryl acetate. Thompson and Scott (1970) reported that selenium deficiency resulted in an atrophy of the pancreas and impaired vitamin E absorption. Combs (1978) and Combs and Regenstern (1980) observed that selenium supplementation of diets containing low vitamin E level increased plasma tocopherol in chicks. Marusich et al. (1975) did not notice any 1 Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 461-E, 1982 series.
difference between the effects of high levels of free tocopherol or of its acetate ester on liver and muscle tocopherol concentrations in chicks. Combs (1978). however, reported that at dietary concentrations less than (or equal to) 40 mg dl-a-tocopherol equivalent/kg, tocopheryl acetate was utilized by the chick with an efficiency equal to that of free tocopherol, but at higher levels the free form appeared to be better utilized. Baker et al. (1980) also noted higher plasma tocopherol levels in man after the ingestion of free tocopherol than after the ingestion of the esterified vitamin. To the best of our knowledge the effect of dietary fat on the absorption and deposition of vitamin E in turkeys has not been studied. Using rats, Herting et al. (1969) observed that vitamin E is absorbed and utilized irrespective of dietary fat content. Type and amount of dietary fat supplement had no consistent effect on tissue tocopherol in chicks (Bartov and Bornstein, 1977). The efficiency of vitamin E absorption and its deposition in the various tissues is of great practical importance in determining carcass shelf-life, because oxidative rancidity of stored poultry meat is markedly dependent on the vitamin E status of the bird prior to slaughter (Webb et al, 1973; Crawford et al, 1974; Marusich et al, 1975; Bartov and Bornstein,
635
636
BARTOV TABLE 1. Percentage composition of the basal diets Experiment 1 Diet A, 0-4 wk
Ingredients Sorghum grains Soybean meal (45% protein) Soybean oil a DL-Methionine L-Lysine (monohydrochloride) Dicalcium phosphate Limestone Sodium chloride Vitamin and trace mineral mix' 5 Calculated values Crude protein Metabolizable energy (kcal/kg) Assayed a-tocopherol (mg/kg)
44.675 50.0 1.0 .215 .110 2.2 1.2 .25 .35 27.0 2661 9
2,5,7 Diet B, 13-16 wk
2,3 Diet C, 13-16 wk
4,6 Diet D, 17-20 wk
71.108 25.0 .5 .092 .100 2.1 .5 .25 .35
66.133 27.5 3.0 .092 .075 2.1 .5 .25 .35
77.176 19.0 .5 .059 .065 2.1 .5 .25 .35
18.0 2936 10 (23) c
18.7 3048 15 (24)c
15.8 3001 10
Tallow was also used in diet C (Experiment 3). Hurwitz et al. (1978), but without a-tocopheryl acetate. In parentheses, values observed in the basal diets used in Experiment 2. These values were, for unknown reasons, higher than expected.
1977). The purpose of the present study was, therefore, to determine the effect of a few dietary additives that might be involved in the absorption and utilization of vitamin E, on plasma tocopherol concentration, used here as a parameter of the vitamin E status of the turkey. METHODS Birds and Diets. The experiments (except Experiment 1) were carried out with 13- to 21-week-old B.U.T. turkey males kept individually in specially constructed cages with raised wire floors located in a temperaturecontrolled (24 ± 2 C) building. The birds (around 30% more than required for each of the experiments) were raised on commercial rations, which were replaced by the appropriate basal diet (Table 1 ) 1 0 days before the start of the experiment. At that time, birds were weighed, the lightest and heaviest turkeys were rejected, and the remaining birds were distributed into groups of 8 birds, equalizing both mean weight among groups and weight dis2
Koffolk Ltd, Tel Aviv, Israel.
tribution within groups. Each of the dietary treatments was tested in one group of birds. Unless otherwise mentioned, vitamin E was added to the diets as a commercial concentrate containing 27.5 mg all-rac-a-tocopheryl acetate/g. 2 The experimental diets, in mash form, were fed ad libitum for 12 to 17 days. At the termination of the experiments birds were weighed and blood was taken from the brachial vein with a heparinized syringe for tocopherol determination. The amount of feed consumed during the experimental period was also recorded. Experiment 1. The effect of increasing dietary levels of vitamin E on plasma tocopherol concentration of young poults was studied. Day-old birds housed in electrically heated battery brooders were fed for their first week of life on diet A (Table 1). They were then divided into five groups of 8 birds each, which for the next 3 weeks received the experimental diets (based on diet A) supplemented with either 0, 10, 30, 60, or 100 mg a-tocopheryl acetate/kg. At the termination of the experiment the birds were bled by cardiac puncture.
PLASMA TOCOPHEROL CONCENTRATIONS OF TURKEYS
Experiments 2 and 3. The effect of the level and type of dietary fat and the level of vitamin E on plasma tocopherol was studied. Two levels of refined soybean oil (.5 and 3.0%) and three levels of vitamin E (obtained by adding 10, 50, or 100 mg a-tocopheryl acetate to the basal diets) were compared in Experiment 2 in which diets B and C (Table 1) were used. Two fat supplements (soybean oil and tallow) and the three mentioned supplements of vitamin E were compared in Experiment 3 using basal diet C (Table 1). In both trials the experimental diets were fed for 12 days to 13-week-oId turkeys. Experiments 4 and 5. In these trials the effect of added cholic acid 3 in diets containing .5% soybean oil and 25 mg a-tocopheryl acetate/kg was evaluated. These supplements were added to basal diet D (Table 1) fed for 14 days to 20-week-old turkeys (Experiment 4) and to basal diet B fed for the same period to 15-week-old birds (Experiment 5). Experiment 6. The effect of selenium on plasma a-tocopherol was studied. Four levels of selenium (.1, .4, .7, and 1.1 ppm) were added to basal diet D (Table 1), which was also supplemented with 50 mg a-tocopheryl acetate, by means of a commercial concentrate of sodium selenite. 2 These experimental diets were fed for 12 days to 17-week-old birds, 8 turkeys per treatment. Experiment 7. The effect of dietary dl-atocopherol 4 vs. dl-a-tocopheryl acetate 4 (both supplying 40 mg a-tocopherol/kg diet) on plasma tocopherol was determined. The vitamin E sources were dissolved in ethoxyquin or in soybean oil prior to their incorporation into the experimental diets (based on diet B). The latter were fed for 17 days to 14-week-old turkeys. Chemical Methods. Plasma a-tocopherol was determined after cold saponification (Kaufmann, 1958), treatment with aqueous sulfuric acid (85% H2SC»4 by volume) according to Parker and McFarlane (1940) as described by Bartov et al. (1965), and colorimetric reaction with ferric chloride and bathophenanthroline (Erickson and Dunkley, 1964). Dietary atocopherol was determined as previously described (Bartov et al, 1965) with the mentioned colorimetric reaction. Statistics. Means were subjected to analysis of variance (Snedecor and Cochran, 1967) and
3 4
Sigma, St. Louis, MO. Pure materials, Fluka AG, Bucks, Switzerland.
637
TABLE 2. Effect of various levels of dietary a-tocopheryl acetate on plasma a-tocopherol concentration of 28-day-old turkeys (Experiment 1) Added dietary a-tocopheryl acetate
Plasma a-tocopherol 1
(mg/kg)
teg/ml)
0 10 30 60 100
1.1 ± . l c 1.9±.lb 2.7 ± . 2 a 2.8±.la 2.8 ± . 2 a
' ' Values not followed by the same letter differ significantly (P<.05). 1
Mean ± SE of four pooled samples of 2 birds each.
to Duncan's multiple range test (1955). Regression lines were compared by analysis of covariance in which variances due to slope and elevation were separated (Snedecor and Cochran, 1967). RESULTS
Experiment 1. Increasing dietary vitamin E level did not affect weight gain nor feed-to-gain ratio (data not detailed). It caused, however, a significant increase in plasma tocopherol concentrations, which plateaued at 30 mg a-tocopheryl acetate/kg diet (Table 2). Experiment 2. Plasma tocopherol concentrations were significantly elevated due to the increase in dietary vitamin E with both basal diets (Table 3). Within each level of vitamin E supplement, the turkeys fed the diet containing 3% soybean oil had a significantly (P<.05) higher plasma tocopherol concentration than those fed the diet containing .5% oil. The regression equations between dietary and plasma tocopherol (X and Y, respectively) obtained with the diets containing .5 and 3.0% soybean oil were: Y = .25 + .056X, r = .943, and Y = 2.6 + .045X, r = .848, respectively. Analysis of covariance shows that the difference between the elevations of these two regression equations was significant (P<.01), whereas slopes did not differ significantly (P>.05). Weight gain and feed utilization were not markedly affected by dietary treatments and are not detailed.
638
BARTOV TABLE 3. Effect of the concentration of dietary a-tocopheryl acetate and of the level and type of dietary fat supplement on plasma a-tocopherol concentration of 15-week-old turkeys (Experiments 2 and 3) Dietary variable
Experiment no.
Fats upplement
(%)
Type
Added a-tocopheryl acetate
a-tocopherol 1
(mg/kg)
(Mg/ml)
2
Soybean Soybean Soybean Soybean Soybean Soybean
oil oil oil oil oil oil
.5 .5 .5 3.0 3.0 3.0
10 50 100 10 50 100
2.3 ± 4.1 ± 7.3 ± 4.3 ± 6.5 ± 8.6 ±
.2d 3C 3b 3C ob 4a
3
Soybean oil Soybean oil Soybean oil Tallow Tallow Tallow
3.0 3.0 3.0 3.0 3.0 3.0
10 50 100 10 50 100
2.8 ± 4.2 + 7.0 ± 2.3 ± 5.3 ± 7.2 +
2C 3b 3a
a
2C
3b 3*
' ' c Within each experiment, values not followed by the same letter differ significantly (P<.05).
1
Mean + SE of 8 turkeys.
Experiment 3. Plasma tocopherol concentrations were significantly elevated due to the increase in dietary vitamin E levels (Table 3). The response was independent of the type of dietary lipid (unsaturated or saturated). The regression equations between dietary and plasma tocopherol (X and Y, respectively) obtained with diets containing soybean oil and tallow were: Y = 1.5 + .047X, r = .927, and Y = 1.3 + .054X, r = .921, respectively. According to analysis of covariance, neither elevations nor slopes of these two regression equations differed significantly (P>.05). Likewise, dietary treatments did not affect the performance of the turkeys. Experiments 4 and 5. In both experiments the lower levels of dietary cholic acid slightly increased plasma tocopherol concentrations (this effect was significant only in Experiment 5). However, the higher levels of this bile acid did not affect this parameter (Table 4). Weight gains were not consistently or significantly affected by dietary treatments (data not detailed). Experiment 6. Increasing dietary selenium supplementation from .1 to 1.1 ppm did not result in any significant changes in plasma tocopherol concentrations which ranged from 4.8 to 5.5 /L(g/ml. Weight gains were not af-
fected significantly by dietary selenium level, but the highest value was observed in birds fed 1.1 ppm selenium. Experiment 7. Neither form of dietary
TABLE 4. Effect of dietary cholic acid on plasma a-tocopherol concentration of turkeys (Experiments 4 and 5)'
Experiment no.
43
53
Added dietary cholic acid
Plasma a-tocopherol 2
(%)
(Mg/ml)
0 .1 .3
5.0 ± . 3 a b 6.1 ± .6 a 4.6 ± .4°
.075 .15
3.2 + . 3 b 4.2 ± . 3 a 3.2 ± . 2 b
0
a ' bWithin each experiment, values not followed by the same letter differ significantly (P<.05). 1 The diets contained 25 mg a-tocopheryl acetate per kilogram. 2 3
Mean ± SE of 8 turkeys.
Twenty-two- and 17-week-old birds in Experiments 4 and 5, respectively.
PLASMA TOCOPHEROL CONCENTRATIONS OF TURKEYS
vitamin E (free or esterified) nor mode of its incorporation into the diets (dissolved in ethoxyquin or in soybean oil) affected significantly plasma tocopherol concentrations (Table 5). The performance of the turkeys was not affected by dietary treatments. DISCUSSION
There is a good correlation between plasma and liver tocopherol (Pudelkiewicz and Mary, 1969; Bartov and Bornstein, 1977; Sklan et al, 1982), between plasma and carcass fat tocopherol (Bartov and Bornstein, 1977), and between liver and muscle tocopherol (Marusich et al, 1975). Therefore, plasma tocopherol levels can be used to estimate the tocopherol status of the bird. Increasing dietary a-tocopheryl acetate levels resulted in a linear increase in plasma tocopherol (Tables 2 and 3), but 4-week-old turkeys responded differently to increasing dietary vitamin E levels than older birds. In the former, plasma tocopherol reached a plateau at 30 mg a-tocopheryl acetate/kg diet, but no such trend was observed in the older birds. Dror and Bartov (1982) observed that increasing levels of dietary a-tocopheryl acetate beyond 30 mg/kg only slightly elevated liver tocopherol concentration in week-old chicks. It appears, therefore, that high levels of dietary a-tocopheryl acetate are not well utilized by young birds. Increasing the level of soybean oil in diets
TABLE 5. Effect of vitamin E source and the vehicle with which it was incorporated into the diets, on plasma a-tocopherol concentration of 16-week-old turkeys (Experiment 7) Source of vitamin E a and mode of incorporation into the diet
Plasma a-tocopherol D (Mg/ml)
DL-Q-tocopherol in ethoxyquin DL-a- tocopheryl acetate in ethoxyquin DL-a-tocopherol in soybean oil DL-a-tocopheryl acetate in soybean oil
3.8 ± .2 3.6 ± .5 4.1 ± .2 3.5 ± .2
a Both sources of vitamin E supplied 40 mg a.tocopherol per kilogram diet.
Mean ± SE of 8 turkeys. No significant (P>.05) differences were observed among treatment means.
639
containing various levels of a-tocopheryl acetate in Experiment 2 significantly raised plasma tocopherol concentration in turkeys (Table 2). This is in contrast to observations in chicks (Bartov and Bornstein, 1977). Such a species difference may be due to the difference in their vitamin E metabolism (Sklan et al, 1982). Analysis of the regression equations between dietary and plasma tocopherol levels reveals that the increase in plasma tocopherol in birds fed the additional oil is due only to the significant increase in the elevation. The slope, which is an estimate of the utilization of dietary nutrient (Hurwitz, 1964), was not affected by oil level. It might be suggested, therefore, that the additional oil improves only the utilization of the ingredient derived atocopherol. Alternately, the increase content of dietary oil could also change the metabolism of vitamin E in the body and improve thereby its basal status. Type of dietary fat (soybean vs. tallow), however, did not affect plasma tocopherol level (Table 3) in agreement with data observed in chicks (Bartov and Bornstein, 1977). However, it shoud be emphasized that despite the same vitamin E status of birds fed these two fat supplements, the meat of birds fed the more saturated fat supplement (tallow) is more stable during storage (Bartov and Bornstein, 1977). The role of cholic acid supplementation on vitamin E status of the turkey is not clear. Low levels of this bile acid slightly increased plasma tocopherol concentrations in the two experiments (significantly so in one experiment), while higher levels inhibited this effect (Table 4). It seems, therefore, that if cholic acid supplementation has any effect on increasing plasma tocopherol, the effect is relatively small and markedly dependent on the level of this bile acid in the diet and possibly other factors as well. This phenomenon might be explained by the ability of dietary bile acids to inhibit the biosynthesis of bile acids (Danielsson, 1973). This inhibition can partly prevent the expected increase in bile acids concentration in the intestine and also change their natural composition. The latter factor may affect the efficiency of lipid absorption. The addition of selenium to diets containing 50 mg tocopheryl acetate per kilogram did not affect plasma tocopherol concentration. This agrees with the findings of Combs (1978) and
640
BARTOV
Combs and Regenstein ( 1 9 8 0 ) w h e n they used diets s u p p l e m e n t e d with vitamin E. These data are evidence t h a t w h e n diets containing natural ingredients are s u p p l e m e n t e d with vitamin E, t h e utilization of this vitamin is n o t improved b y increasing dietary selenium. Results o b t a i n e d in t h e present study s h o w t h a t free and esterified t o c o p h e r o l at a level of 4 0 mg dl-a-tocopherol equivalent per kilogram feed are equally utilized by t h e t u r k e y (Table 5), in agreement with t h e data of Combs ( 1 9 7 8 ) with chicks. T h u s , it seems t h a t there is n o advantage in feeding free t o c o p h e r o l in t h e m e n t i o n e d , or lower, levels t o t u r k e y s in order t o improve their vitamin E status. T h e m o d e of incorporation of vitamin E into t h e diets (dispersed in e t h o x y q u i n or soybean oil) did n o t affect plasma t o c o p h e r o l concentrations of t u r k e y s fed t h e t w o forms of vitamin E (Table 5). It should be m e n t i o n e d , however, t h a t in a n o t h e r e x p e r i m e n t ( n o t detailed) in which an old sample of e t h o x y q u i n had been used, plasma t o c o p h e r o l concentrations of t u r k e y s fed free t o c o p h e r o l were significantly ( P < . 0 5 ) lower t h a n values observed in birds fed t h e ester (2.7 ± .3 vs. 4.2 ± .4 /ig/ml, respectively). This occurred despite t h e similar levels of t o c o p h e r o l in t h e diets containing these t w o vitamin E sources at t h e end of t h e e x p e r i m e n t ( 6 4 vs. 6 7 mg a-tocopherol/kg, respectively). ACKNOWLEDGMENTS This s t u d y was s u p p o r t e d b y grants from t h e U.S.-Israel Agricultural Research and Development F u n d ( B A R D ) and from the Israel Egg and Poultry Marketing Board. T h e technical assistance of M. Ben-Mosheh is gratefully acknowledged. REFERENCES Baker, H..O. Frank, B. De Angelis, and S. Feingold, 1980. Plasma tocopherol in man at various times after ingesting free or acetylated tocopherol. Nutr. Rep. Int. 21:531-536. Bartov, I., and S. Bornstein, 1977. Stability of abdominal fat and meat of broilers: the interrelationship between the effects of dietary fat and vitamin E supplements. Br. Poultry Sci. 18:47-57. Bartov, I., P. Budowski, and S. Bornstein, 1965. The relation between a-tocopherol content of the breeder diet and that of the newly hatched chick. Poultry Sci. 44:1489-1494. Combs, G. F. Jr., 1978. Studies on the utilization of vitamin E alcohol and esters by the chick. Poul-
try Sci. 57:223-229. Combs, G. F., Jr., and J. M. Regenstern, 1980. Influence of selenium, vitamin E and ethoxyquin on lipid peroxidation in muscle tissues from fowl during low temperature storage. Poultry Sci. 59:347-351. Crawford, L., D. W. Peterson, M. J. Kretsch, A. L. Lilyblade, and H. S. Olcott, 1974. The effects of dietary a-tocopherol and tuna, safflower, and linseed oils on the flavor of turkey. Fish. Bull. 72:1032-1038. Danielsson, H., 1973. Influence of dietary bile acids on formation of bile acids in rat. Steroids 22: 667-676. Dror, Y., and I. Bartov, 1982. Dietary factors affecting experimental models of nutritional encephalomalacia. Poultry Sci. 6 1 : 8 4 - 9 3 . Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1—42. Erickson, D. R., and W. L. Dunkley, 1964. Spectrophotometric determination of tocopherol in milk and milk lipides. Anal. Chem. 36:1055— 1058. Gallo-Torres, H. E., 1970. Obligatory role of bile for the intestinal absorption of vitamin E. Lipids 5:379-384. Herring, D. C , M. I. Ludwig, and E. E. Drury, 1969. Absorption of vitamin E by the rat from a low fat diet. J. Nutr. 99:481-484. Hurwitz, S., 1964. Estimation of net phosphorus utilization by the "slope" method. J. Nutr. 84:83-92. Hurwitz, S., D. Dubrov, U. Eisner, G. Riesenfeld, and A. Bar, 1978. Phosphate absorption and excretion in the young turkey, as influenced by calcium intake. J. Nutr. 108:1329-1335. Kaufmann, H. P., 1958. Page 980 in Analyse der Fette und Fettproducte. Vol. I. Springer-Verlag, Berlin. Marusich, W. L., E. De Ritter, E. F. Ogrinz, J. Keating, M. Mitrovic, and R. H. Bunnell, 1975. Effect of supplemental vitamin E in control of rancidity in poultry meat. Poultry Sci. 54:831-844. Mecchi, E. P., M. F. Pool, G. A. Behman, M. Hamachi, and A. A. Klose, 1956. The role of tocopherol content in the comparative stability of chicken and turkey fat. Poultry Sci. 35:1238-1246. Muller, D.P.R., J. A. Manning, P. M. Mathias, and J. T. Harries, 1976. Studies on the intestinal hydrolysis of tocopheryl esters. Int. J. Vit. Nutr. Res. 46:207-210. Parker, W. E., and W. D. McFarlane, 1940. A proposed modification of Emmeri's iron-dipyridyl method for determining tocopherol content of oils. Can. J. Res. 18B:405-409. Pudelkiewicz, W. J., and N. Mary, 1969. Some relationships between plasma, liver and excreta tocopherol in chicks fed graded levels of alphatocopheryl acetate. J. Nutr. 97:303—306. Sklan, D., I. Bartov, and S. Hurwitz, 1982. Tocopherol absorption and metabolism in the chick and turkey. J. Nutr. 112:1394-1400. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods. 6th ed. Iowa State Univ. Press, Ames, IA. Thompson, J. N., and M. L. Scott, 1970. Impaired lipid and vitamin E absorption related to atrophy
PLASMA TOCOPHEROL CONCENTRATIONS OF TURKEYS of the pancreas in selenium-deficient chicks. J. Nutr. 100:797—809. flavor Webb, J. E., C. C. Brunson, and J. D. Yates, 1973.
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Effects of feeding fish meal and tocopherol on of precooked, frozen turkey meat. Poultry Sci. 52:1029-1034.
Turkeys are known for their poor ability to accumulate vitamin E in their tissues as compared with chicks (Mecchi et al, 1956; Marusich et al, 1975; Sklan et al, 1982). Mecchi et al. (1956) and Marusich et al. (1975) attribute this phenomenon to decreased gastrointestinal absorption. Vitamin E is usually incorporated into the diet as a-tocopheryl acetate, which is a stable form of the vitamin. Bile and pancreatic juice have been reported to be essential for the intestinal absorption of this ester, which is poorly absorbed from the intestine (GalloTorres, 1970). MuUer et al (1976) noted that bile salts increase the activity of pancreatic esterase against tocopheryl acetate. Thompson and Scott (1970) reported that selenium deficiency resulted in an atrophy of the pancreas and impaired vitamin E absorption. Combs (1978) and Combs and Regenstern (1980) observed that selenium supplementation of diets containing low vitamin E level increased plasma tocopherol in chicks. Marusich et al. (1975) did not notice any 1 Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 461-E, 1982 series.
difference between the effects of high levels of free tocopherol or of its acetate ester on liver and muscle tocopherol concentrations in chicks. Combs (1978). however, reported that at dietary concentrations less than (or equal to) 40 mg dl-a-tocopherol equivalent/kg, tocopheryl acetate was utilized by the chick with an efficiency equal to that of free tocopherol, but at higher levels the free form appeared to be better utilized. Baker et al. (1980) also noted higher plasma tocopherol levels in man after the ingestion of free tocopherol than after the ingestion of the esterified vitamin. To the best of our knowledge the effect of dietary fat on the absorption and deposition of vitamin E in turkeys has not been studied. Using rats, Herting et al. (1969) observed that vitamin E is absorbed and utilized irrespective of dietary fat content. Type and amount of dietary fat supplement had no consistent effect on tissue tocopherol in chicks (Bartov and Bornstein, 1977). The efficiency of vitamin E absorption and its deposition in the various tissues is of great practical importance in determining carcass shelf-life, because oxidative rancidity of stored poultry meat is markedly dependent on the vitamin E status of the bird prior to slaughter (Webb et al, 1973; Crawford et al, 1974; Marusich et al, 1975; Bartov and Bornstein,
635
636
BARTOV TABLE 1. Percentage composition of the basal diets Experiment 1 Diet A, 0-4 wk
Ingredients Sorghum grains Soybean meal (45% protein) Soybean oil a DL-Methionine L-Lysine (monohydrochloride) Dicalcium phosphate Limestone Sodium chloride Vitamin and trace mineral mix' 5 Calculated values Crude protein Metabolizable energy (kcal/kg) Assayed a-tocopherol (mg/kg)
44.675 50.0 1.0 .215 .110 2.2 1.2 .25 .35 27.0 2661 9
2,5,7 Diet B, 13-16 wk
2,3 Diet C, 13-16 wk
4,6 Diet D, 17-20 wk
71.108 25.0 .5 .092 .100 2.1 .5 .25 .35
66.133 27.5 3.0 .092 .075 2.1 .5 .25 .35
77.176 19.0 .5 .059 .065 2.1 .5 .25 .35
18.0 2936 10 (23) c
18.7 3048 15 (24)c
15.8 3001 10
Tallow was also used in diet C (Experiment 3). Hurwitz et al. (1978), but without a-tocopheryl acetate. In parentheses, values observed in the basal diets used in Experiment 2. These values were, for unknown reasons, higher than expected.
1977). The purpose of the present study was, therefore, to determine the effect of a few dietary additives that might be involved in the absorption and utilization of vitamin E, on plasma tocopherol concentration, used here as a parameter of the vitamin E status of the turkey. METHODS Birds and Diets. The experiments (except Experiment 1) were carried out with 13- to 21-week-old B.U.T. turkey males kept individually in specially constructed cages with raised wire floors located in a temperaturecontrolled (24 ± 2 C) building. The birds (around 30% more than required for each of the experiments) were raised on commercial rations, which were replaced by the appropriate basal diet (Table 1 ) 1 0 days before the start of the experiment. At that time, birds were weighed, the lightest and heaviest turkeys were rejected, and the remaining birds were distributed into groups of 8 birds, equalizing both mean weight among groups and weight dis2
Koffolk Ltd, Tel Aviv, Israel.
tribution within groups. Each of the dietary treatments was tested in one group of birds. Unless otherwise mentioned, vitamin E was added to the diets as a commercial concentrate containing 27.5 mg all-rac-a-tocopheryl acetate/g. 2 The experimental diets, in mash form, were fed ad libitum for 12 to 17 days. At the termination of the experiments birds were weighed and blood was taken from the brachial vein with a heparinized syringe for tocopherol determination. The amount of feed consumed during the experimental period was also recorded. Experiment 1. The effect of increasing dietary levels of vitamin E on plasma tocopherol concentration of young poults was studied. Day-old birds housed in electrically heated battery brooders were fed for their first week of life on diet A (Table 1). They were then divided into five groups of 8 birds each, which for the next 3 weeks received the experimental diets (based on diet A) supplemented with either 0, 10, 30, 60, or 100 mg a-tocopheryl acetate/kg. At the termination of the experiment the birds were bled by cardiac puncture.
PLASMA TOCOPHEROL CONCENTRATIONS OF TURKEYS
Experiments 2 and 3. The effect of the level and type of dietary fat and the level of vitamin E on plasma tocopherol was studied. Two levels of refined soybean oil (.5 and 3.0%) and three levels of vitamin E (obtained by adding 10, 50, or 100 mg a-tocopheryl acetate to the basal diets) were compared in Experiment 2 in which diets B and C (Table 1) were used. Two fat supplements (soybean oil and tallow) and the three mentioned supplements of vitamin E were compared in Experiment 3 using basal diet C (Table 1). In both trials the experimental diets were fed for 12 days to 13-week-oId turkeys. Experiments 4 and 5. In these trials the effect of added cholic acid 3 in diets containing .5% soybean oil and 25 mg a-tocopheryl acetate/kg was evaluated. These supplements were added to basal diet D (Table 1) fed for 14 days to 20-week-old turkeys (Experiment 4) and to basal diet B fed for the same period to 15-week-old birds (Experiment 5). Experiment 6. The effect of selenium on plasma a-tocopherol was studied. Four levels of selenium (.1, .4, .7, and 1.1 ppm) were added to basal diet D (Table 1), which was also supplemented with 50 mg a-tocopheryl acetate, by means of a commercial concentrate of sodium selenite. 2 These experimental diets were fed for 12 days to 17-week-old birds, 8 turkeys per treatment. Experiment 7. The effect of dietary dl-atocopherol 4 vs. dl-a-tocopheryl acetate 4 (both supplying 40 mg a-tocopherol/kg diet) on plasma tocopherol was determined. The vitamin E sources were dissolved in ethoxyquin or in soybean oil prior to their incorporation into the experimental diets (based on diet B). The latter were fed for 17 days to 14-week-old turkeys. Chemical Methods. Plasma a-tocopherol was determined after cold saponification (Kaufmann, 1958), treatment with aqueous sulfuric acid (85% H2SC»4 by volume) according to Parker and McFarlane (1940) as described by Bartov et al. (1965), and colorimetric reaction with ferric chloride and bathophenanthroline (Erickson and Dunkley, 1964). Dietary atocopherol was determined as previously described (Bartov et al, 1965) with the mentioned colorimetric reaction. Statistics. Means were subjected to analysis of variance (Snedecor and Cochran, 1967) and
3 4
Sigma, St. Louis, MO. Pure materials, Fluka AG, Bucks, Switzerland.
637
TABLE 2. Effect of various levels of dietary a-tocopheryl acetate on plasma a-tocopherol concentration of 28-day-old turkeys (Experiment 1) Added dietary a-tocopheryl acetate
Plasma a-tocopherol 1
(mg/kg)
teg/ml)
0 10 30 60 100
1.1 ± . l c 1.9±.lb 2.7 ± . 2 a 2.8±.la 2.8 ± . 2 a
' ' Values not followed by the same letter differ significantly (P<.05). 1
Mean ± SE of four pooled samples of 2 birds each.
to Duncan's multiple range test (1955). Regression lines were compared by analysis of covariance in which variances due to slope and elevation were separated (Snedecor and Cochran, 1967). RESULTS
Experiment 1. Increasing dietary vitamin E level did not affect weight gain nor feed-to-gain ratio (data not detailed). It caused, however, a significant increase in plasma tocopherol concentrations, which plateaued at 30 mg a-tocopheryl acetate/kg diet (Table 2). Experiment 2. Plasma tocopherol concentrations were significantly elevated due to the increase in dietary vitamin E with both basal diets (Table 3). Within each level of vitamin E supplement, the turkeys fed the diet containing 3% soybean oil had a significantly (P<.05) higher plasma tocopherol concentration than those fed the diet containing .5% oil. The regression equations between dietary and plasma tocopherol (X and Y, respectively) obtained with the diets containing .5 and 3.0% soybean oil were: Y = .25 + .056X, r = .943, and Y = 2.6 + .045X, r = .848, respectively. Analysis of covariance shows that the difference between the elevations of these two regression equations was significant (P<.01), whereas slopes did not differ significantly (P>.05). Weight gain and feed utilization were not markedly affected by dietary treatments and are not detailed.
638
BARTOV TABLE 3. Effect of the concentration of dietary a-tocopheryl acetate and of the level and type of dietary fat supplement on plasma a-tocopherol concentration of 15-week-old turkeys (Experiments 2 and 3) Dietary variable
Experiment no.
Fats upplement
(%)
Type
Added a-tocopheryl acetate
a-tocopherol 1
(mg/kg)
(Mg/ml)
2
Soybean Soybean Soybean Soybean Soybean Soybean
oil oil oil oil oil oil
.5 .5 .5 3.0 3.0 3.0
10 50 100 10 50 100
2.3 ± 4.1 ± 7.3 ± 4.3 ± 6.5 ± 8.6 ±
.2d 3C 3b 3C ob 4a
3
Soybean oil Soybean oil Soybean oil Tallow Tallow Tallow
3.0 3.0 3.0 3.0 3.0 3.0
10 50 100 10 50 100
2.8 ± 4.2 + 7.0 ± 2.3 ± 5.3 ± 7.2 +
2C 3b 3a
a
2C
3b 3*
' ' c Within each experiment, values not followed by the same letter differ significantly (P<.05).
1
Mean + SE of 8 turkeys.
Experiment 3. Plasma tocopherol concentrations were significantly elevated due to the increase in dietary vitamin E levels (Table 3). The response was independent of the type of dietary lipid (unsaturated or saturated). The regression equations between dietary and plasma tocopherol (X and Y, respectively) obtained with diets containing soybean oil and tallow were: Y = 1.5 + .047X, r = .927, and Y = 1.3 + .054X, r = .921, respectively. According to analysis of covariance, neither elevations nor slopes of these two regression equations differed significantly (P>.05). Likewise, dietary treatments did not affect the performance of the turkeys. Experiments 4 and 5. In both experiments the lower levels of dietary cholic acid slightly increased plasma tocopherol concentrations (this effect was significant only in Experiment 5). However, the higher levels of this bile acid did not affect this parameter (Table 4). Weight gains were not consistently or significantly affected by dietary treatments (data not detailed). Experiment 6. Increasing dietary selenium supplementation from .1 to 1.1 ppm did not result in any significant changes in plasma tocopherol concentrations which ranged from 4.8 to 5.5 /L(g/ml. Weight gains were not af-
fected significantly by dietary selenium level, but the highest value was observed in birds fed 1.1 ppm selenium. Experiment 7. Neither form of dietary
TABLE 4. Effect of dietary cholic acid on plasma a-tocopherol concentration of turkeys (Experiments 4 and 5)'
Experiment no.
43
53
Added dietary cholic acid
Plasma a-tocopherol 2
(%)
(Mg/ml)
0 .1 .3
5.0 ± . 3 a b 6.1 ± .6 a 4.6 ± .4°
.075 .15
3.2 + . 3 b 4.2 ± . 3 a 3.2 ± . 2 b
0
a ' bWithin each experiment, values not followed by the same letter differ significantly (P<.05). 1 The diets contained 25 mg a-tocopheryl acetate per kilogram. 2 3
Mean ± SE of 8 turkeys.
Twenty-two- and 17-week-old birds in Experiments 4 and 5, respectively.
PLASMA TOCOPHEROL CONCENTRATIONS OF TURKEYS
vitamin E (free or esterified) nor mode of its incorporation into the diets (dissolved in ethoxyquin or in soybean oil) affected significantly plasma tocopherol concentrations (Table 5). The performance of the turkeys was not affected by dietary treatments. DISCUSSION
There is a good correlation between plasma and liver tocopherol (Pudelkiewicz and Mary, 1969; Bartov and Bornstein, 1977; Sklan et al, 1982), between plasma and carcass fat tocopherol (Bartov and Bornstein, 1977), and between liver and muscle tocopherol (Marusich et al, 1975). Therefore, plasma tocopherol levels can be used to estimate the tocopherol status of the bird. Increasing dietary a-tocopheryl acetate levels resulted in a linear increase in plasma tocopherol (Tables 2 and 3), but 4-week-old turkeys responded differently to increasing dietary vitamin E levels than older birds. In the former, plasma tocopherol reached a plateau at 30 mg a-tocopheryl acetate/kg diet, but no such trend was observed in the older birds. Dror and Bartov (1982) observed that increasing levels of dietary a-tocopheryl acetate beyond 30 mg/kg only slightly elevated liver tocopherol concentration in week-old chicks. It appears, therefore, that high levels of dietary a-tocopheryl acetate are not well utilized by young birds. Increasing the level of soybean oil in diets
TABLE 5. Effect of vitamin E source and the vehicle with which it was incorporated into the diets, on plasma a-tocopherol concentration of 16-week-old turkeys (Experiment 7) Source of vitamin E a and mode of incorporation into the diet
Plasma a-tocopherol D (Mg/ml)
DL-Q-tocopherol in ethoxyquin DL-a- tocopheryl acetate in ethoxyquin DL-a-tocopherol in soybean oil DL-a-tocopheryl acetate in soybean oil
3.8 ± .2 3.6 ± .5 4.1 ± .2 3.5 ± .2
a Both sources of vitamin E supplied 40 mg a.tocopherol per kilogram diet.
Mean ± SE of 8 turkeys. No significant (P>.05) differences were observed among treatment means.
639
containing various levels of a-tocopheryl acetate in Experiment 2 significantly raised plasma tocopherol concentration in turkeys (Table 2). This is in contrast to observations in chicks (Bartov and Bornstein, 1977). Such a species difference may be due to the difference in their vitamin E metabolism (Sklan et al, 1982). Analysis of the regression equations between dietary and plasma tocopherol levels reveals that the increase in plasma tocopherol in birds fed the additional oil is due only to the significant increase in the elevation. The slope, which is an estimate of the utilization of dietary nutrient (Hurwitz, 1964), was not affected by oil level. It might be suggested, therefore, that the additional oil improves only the utilization of the ingredient derived atocopherol. Alternately, the increase content of dietary oil could also change the metabolism of vitamin E in the body and improve thereby its basal status. Type of dietary fat (soybean vs. tallow), however, did not affect plasma tocopherol level (Table 3) in agreement with data observed in chicks (Bartov and Bornstein, 1977). However, it shoud be emphasized that despite the same vitamin E status of birds fed these two fat supplements, the meat of birds fed the more saturated fat supplement (tallow) is more stable during storage (Bartov and Bornstein, 1977). The role of cholic acid supplementation on vitamin E status of the turkey is not clear. Low levels of this bile acid slightly increased plasma tocopherol concentrations in the two experiments (significantly so in one experiment), while higher levels inhibited this effect (Table 4). It seems, therefore, that if cholic acid supplementation has any effect on increasing plasma tocopherol, the effect is relatively small and markedly dependent on the level of this bile acid in the diet and possibly other factors as well. This phenomenon might be explained by the ability of dietary bile acids to inhibit the biosynthesis of bile acids (Danielsson, 1973). This inhibition can partly prevent the expected increase in bile acids concentration in the intestine and also change their natural composition. The latter factor may affect the efficiency of lipid absorption. The addition of selenium to diets containing 50 mg tocopheryl acetate per kilogram did not affect plasma tocopherol concentration. This agrees with the findings of Combs (1978) and
640
BARTOV
Combs and Regenstein ( 1 9 8 0 ) w h e n they used diets s u p p l e m e n t e d with vitamin E. These data are evidence t h a t w h e n diets containing natural ingredients are s u p p l e m e n t e d with vitamin E, t h e utilization of this vitamin is n o t improved b y increasing dietary selenium. Results o b t a i n e d in t h e present study s h o w t h a t free and esterified t o c o p h e r o l at a level of 4 0 mg dl-a-tocopherol equivalent per kilogram feed are equally utilized by t h e t u r k e y (Table 5), in agreement with t h e data of Combs ( 1 9 7 8 ) with chicks. T h u s , it seems t h a t there is n o advantage in feeding free t o c o p h e r o l in t h e m e n t i o n e d , or lower, levels t o t u r k e y s in order t o improve their vitamin E status. T h e m o d e of incorporation of vitamin E into t h e diets (dispersed in e t h o x y q u i n or soybean oil) did n o t affect plasma t o c o p h e r o l concentrations of t u r k e y s fed t h e t w o forms of vitamin E (Table 5). It should be m e n t i o n e d , however, t h a t in a n o t h e r e x p e r i m e n t ( n o t detailed) in which an old sample of e t h o x y q u i n had been used, plasma t o c o p h e r o l concentrations of t u r k e y s fed free t o c o p h e r o l were significantly ( P < . 0 5 ) lower t h a n values observed in birds fed t h e ester (2.7 ± .3 vs. 4.2 ± .4 /ig/ml, respectively). This occurred despite t h e similar levels of t o c o p h e r o l in t h e diets containing these t w o vitamin E sources at t h e end of t h e e x p e r i m e n t ( 6 4 vs. 6 7 mg a-tocopherol/kg, respectively). ACKNOWLEDGMENTS This s t u d y was s u p p o r t e d b y grants from t h e U.S.-Israel Agricultural Research and Development F u n d ( B A R D ) and from the Israel Egg and Poultry Marketing Board. T h e technical assistance of M. Ben-Mosheh is gratefully acknowledged. REFERENCES Baker, H..O. Frank, B. De Angelis, and S. Feingold, 1980. Plasma tocopherol in man at various times after ingesting free or acetylated tocopherol. Nutr. Rep. Int. 21:531-536. Bartov, I., and S. Bornstein, 1977. Stability of abdominal fat and meat of broilers: the interrelationship between the effects of dietary fat and vitamin E supplements. Br. Poultry Sci. 18:47-57. Bartov, I., P. Budowski, and S. Bornstein, 1965. The relation between a-tocopherol content of the breeder diet and that of the newly hatched chick. Poultry Sci. 44:1489-1494. Combs, G. F. Jr., 1978. Studies on the utilization of vitamin E alcohol and esters by the chick. Poul-
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Effects of feeding fish meal and tocopherol on of precooked, frozen turkey meat. Poultry Sci. 52:1029-1034.