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Influence of Fats and Choline on Liver and Serum Cholesterol in the Chick1
1712
E. L. WISMAN, W. L. BEANE, W. R. LUCKHAM AND E. 0. ESSAEY
correlations for six feathering characteristics. Poultry Sci. 36: 621-628. Siegel, P. B., and R. H. Coles, 1958. Effects of floor space on broiler performance. Poultry Sci. 37: 1243. Smith, R. C , 1956. Kind of litter and breast blisters on broilers. Poultry Sci. 35: 593-595. Smith, R. C , 1959. Age and weight of marketing affects broiler profits. Delaware Agr. Exp. Sta.
Tech. Bui. 328. Smith, R. M., N. R. Gyles and J. C. Gilbreath, 1954. The influence of producing sexes separately on growth, feed utilization, and dressed grade of broilers. Poultry Sci. 33: 1082. Winget, C. M., A. H. Smith and C. F. Kelly, 1958. The effect of body weight on joint and skin disorders of domestic birds. Poultry Sci. 37: 1253.
N. J. DAGHIR AND S. L. BALLOUN Department of Poultry Husbandry, Iowa State University, Ames (Received for publication February 27. 1961)
D
URING the past decade, there has been a great deal of interest in investigating the effects of different dietary components on serum cholesterol levels in a large number of species. Previous papers have shown that dietary fats and choline are among the various components that affect serum cholesterol levels in the chick. It is well established that dietary fat may either increase or decrease blood cholesterol depending upon the type of fat and the level at which it is fed. When feeding S to 10 percent of added animal fat to laying chickens, Weiss and Fisher (1957) observed high plasma cholesterol concentrations, and friable and fatty liver development which suggested that high animal fat diets result in a derangement in lipid metabolism in the chicken. Leveille and Fisher (1958) reported no significant increases in plasma cholesterol when 10 percent tallow was fed to Single Comb White Leghorn hens as compared to hens fed diets containing 10 percent of corn oil or the basal diet containing no added fat. The data presented, 1 Journal Paper No. J-4067 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project No. 1062.
however, show a definite trend toward higher plasma cholesterol levels in the groups fed animal fats. March and Biely (1959) fed chicks various fats in diets containing 20 and 26 percent protein. With both protein levels, 8 percent of Crisco, butter, lard or chicken fat increased serum cholesterol. Herring oil did not increase serum cholesterol with either diet, and corn oil did so only in the case of the chicks fed a 20 percent protein diet. This indicates that fats high in saturated fatty acids tend to raise serum cholesterol levels in the chick. Similar results were obtained by Hegsted et al. (1960), who clearly demonstrated that fats high in saturated fatty acids promote hypercholesterolemia and that this effect is counteracted by unsaturated fatty acids. Mone et al. (1959), on the other hand, concluded that serum cholesterol was not affected in their experiments by increasing the fat from 4 to 10 percent, or by the degree of unsaturation of the fat. Since several natural oils, high in polyunsaturated fatty acids, have been shown to lower serum cholesterol in several species, Quackenbush and Pawlowski (1960) stud-
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 9, 2015
Influence of Fats and Choline on Liver and Serum Cholesterol in the Chick 1
1713
CHOLESTEROL LEVELS
sclerosis in the rabbit. They showed that the above amount of choline, fed with one gram of cholesterol daily, did not affect the development of atherosclerosis or the cholesterol content of the blood. Svacha et al. (1958), on the other hand, reported that the feeding of 0.25 percent choline chloride with 2 percent cholesterol resulted in a decrease in serum cholesterol in chicks in the presence or absence of fat. The effects of fats and choline on cholesterol metabolism are not well elucidated as the previous brief review indicates. Hence, more work on the effects of these two dietary components and their interactions is considered in order. The following experiment was designed to study the effect of dietary fats of two types, each fed with three different levels of choline, on weight gains, feed efficiency, liver lipids, and liver and serum cholesterol of growing chicks. EXPERIMENTAL PROCEDURE
Two hundred fifty two day-old cross-bred sexed chicks (Vantress $ X Arbor Acre 9 ) were kept on diet 1 (Table 1) for one
TABLE 1.—Experimental diets Diet composition (%) 1
2
3
4
5
6
7
8
9
49 48.75 48.50 48.75 48.50 49 58.75 59 Ground yellow corn 58.50 10 10 10 10 10 10 Oat hulls — — — 1 8 8 8 Animal fat — — — — — — 8 8 8 Soybean oil — — — — — — 8 8 Dextrose 8 0.25 0.50 0.25 0.50 0.50 0.25 Choline chloride (25%) — — — 33 33 33 33 35 33 33 33 33 Basal components 2 Calculated composition 20.6 20.6 20.6 20.6 20.6 20.0 20.0 20.6 20.0 Protein, % Productive energy, 1,036 1 ,036 1,036 1 ,036 1 ,036 1,005 1 ,005 1 ,036 Cal./lb. 1 ,005 536 820 1 ,104 802 1 ,104 536 536 820 1 ,104 Choline, mg./lb. 1 White grease—moisture 0.34%, free fatty acids 1.38%, unsaponifiable matter 0.52%, titer 39.1°C, stability (A.O.M.) over 60 hours. 2 The basal components portion of the diet was composed of the following: (values are expressed as percentages of the complete diet or amounts per lb. of diet). Soybean oil meal (50% protein) 22%, meat scraps (50% protein) 5%, fish meal (65% protein) 1.5%, alfalfa leaf meal (20% protein) 2%, steamed bone meal 1%, NaCl 0.4%, manganese 55 mg., iodine 0.25 mg., iron 18 mg., copper 1.36 mg., zinc 1.30 mg., cobalt 0.23 mg., sulfur 7.7 mg., riboflavin 2 mg., niacin 17 mg., Ca. pantothenate 4 mg., vit. B12 7.5 meg., menadione 1 mg., vit. E. 5 I.U., vit. A. 3,000 I. U. and vit. D, 500 I.C.U.
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 9, 2015
ied the effects of individual fatty acids on serum cholesterol in the rat. Linoleic acid ester was found to lower markedly the plasma cholesterol level of hypercholesterolemic cholesterol-fed rats. It also lowered liver cholesterol of the rats fed a low-fat diet but not those receiving a high-fat diet. Sex differences were reported by Stamler et al. (1954), who indicated that twice the amount of dietary cholesterol was required to cause the same degree of plasma cholesterol increase in females as in males. Reports in the literature related to the action of choline on serum cholesterol are not in complete agreement. Weiss et al. (1952) observed that the feeding of 1 percent choline, plus inositol, produced a marked hypercholesterolemia in rats fed a 2 percent cholesterol diet. In rats fed a lowcholesterol diet, however, plasma cholesterol was not significantly changed. A series of three experiments were conducted by Duff and Meissner (1954) to determine the effect of oral administration of 1 to 3 grams of choline chloride daily on the development of experimental athero-
1714
N. J. DAGHIR AND S. L. BALLOUN
T A B L E 2.—Effect
Diet additions Choline F a t (%) (nig./ lb.)
Ani. Ani. Ani. Soy. Soy. Soy. 1
8 8 8 8 8 8
0 284 568 0 284 568 0 284 568
fat and choline on weight gains, feed efficiency, cholesterol of growing chicks1
Wt. Gains in Pounds 2 Males
Females
2.54 2.42 2.32 2.40 2.38 2.52 2.40 2.48 2.52
2.06 1.92 2.06 2.11 1.89 2.00 1.82 2.03 2.22
Feed/ Gain Mixed 2.26 2.27 2.26 2.20 2.16 2.10 2.22 2.06 2.22
Liver Lipids (% of dry tissue)
liver
lipids,
Liver cholesterol (mg./g. of dry tissue)
'• liver i
Serum cholesterol (mg. % )
Males
Females
Males
Females
Males
Females
16.8+0.64 15.8+0.99 17.0+0.60 17.3 + 0.75 15.8+0.41 16.4+0.67 16.5 + 0.31 15.7 + 0.26 16.4+0.19
18.8+0.63 17.1 + 0.54 16.1 + 0.42 15.6+0.56 17.3 + 0.78 17.2 + 0.87 15.7 + 0.29 16.4+0.87 16.4 + 0.39
26.7+1.16 24.4+0.99 25.3+0.67 25.5+0.74 2 4 . 8 + 1.23 25.7+0.88 24.8+0.72 25.4+0.92 26.7 + 0.56
27.1 + 0.88 26.1 + 0.10 25.0+1.48 25.0+0.33 27.0+1.10 24.9+1.90 24.3+1.47 25.4+0.92 26.7+0.56
152+ 4 . 9 162+ 7.9 156+ 6.4 172+ 6.4 182+ 9.2 172+10.1 159+ 8.6 158+10.4 156+ 6 . 9
146+ 7.7 154+ 9 . 5 148+ 6.7 154+ 2 . 8 165+ 6.6 170+ 6 . 6 144+ 5 . 1 145+10.3 149+ 3 . 5
All values represent means or means + their standard errors. Values represent weight gains over a period of 6 weeks (from 1 to 7 weeks of agi
week after which they were randomly assigned to 18 pens, each pen containing 7 birds of each sex. Two pens were allotted to each of the 9 experimental diets (Table 1) under a restricted randomized block design. Groups were chosen to be approximately of equal weights. The birds were kept in thermostatically-controlled batteries for the first 4 weeks after which they were transferred to non-heated batteries for the remainder of the experimental period. Feed and water were supplied ad libitum throughout the test period, and both birds and feed were weighed at weekly intervals. The diets were made approximately isocaloric and isonitrogenous by substituting oat hulls in the added-fat diets for part of the corn, and dextrose in the no-added-fat diets for the 8 percent fat in the remaining diets. Three different levels of choline were used, 536, 820, and 1104 mg. of total choline per pound of diet. Choline was added in the form of choline chloride (25 percent). At both 5 and 7 weeks of age, individual blood samples were collected by heart puncture from two male and two female birds chosen at random from each pen. The birds were then sacrificed by dislocation of the spinal cord, and livers were immediately removed, washed of adhering blood and frozen. Total cholesterol in serum was de-
termined by the method described by Zlatkis et al. (1953). For lipid and cholesterol determinations, the livers from two chicks of each sex from each pen were first individually homogenized with 15 ml. of distilled water in a Waring blendor and then dried in an oven at 100°C. for 12 hours. The dry homogenate was then ground in a Servall OmniMixer. Duplicate, 2 gram samples of each chick liver homogenate were extracted with the following solvent mixture for 10 hours: 5 parts ethyl ether, 5 parts Skellysolve B and 2 parts ethanol. Percent lipids was calculated from the weight loss. Extracts were diluted to 200 ml. with the solvent mixture and a 1 ml. sample was evaporated to dryness for total cholesterol determination as outlined in the method of Zlatkis et al. (1953). The analyses of variance were made according to Snedecor (1957). RESULTS AND DISCUSSION The data presented in Table 2 indicate that the various fat and choline treatments had no marked effect on weight gains of birds to 7 weeks of age. The fat treatments, however, regardless of the level of choline, resulted in a significant (P < 0.01) improvement in feed efficiency. These results are in agreement with previous papers. Aitken et al. (1954) obtained no significant
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1
0 0 0 Fat Fat Fat Oil Oil Oil
ofdietary
1715
CHOLESTEROL LEVELS TABLE 3.—Analysis of variance of serum cholesterol of chicks1 rce of variation
d.f.
Mean squares
Blocks Fat (F) Choline (C) Sex (S) FXC FXS CXS FXCXS Error
1 2 2 1 4 2 2 4 17
612.0 1,056.0** 116.0 1,071.0** 44.0 34.5 45.5 25.3 92.1
Total
35
1 Combined data of 5 and 7 week determinations. ** Significant at P < 0 . 0 1 .
Analysis of variance of serum cholesterol data is presented in Table 3. Feeding 8 percent of animal fat resulted in significantly higher serum cholesterol values in both male and female birds, while 8 percent of soybean oil did not elevate serum cholesterol. An evaluation of the role of fat upon serum cholesterol levels must always relate both the amount and kind of fat used. Since, in these studies, the amount of fat fed was constant, the difference in effect on serum cholesterol was mainly due to the kind of fat used. The results confirm reports in the literature that the feeding of a highly-saturated fat to chicks increases serum cholesterol and that a highly-unsaturated fat counteracts this effect (March and Biely, 1959; Hegsted et al., 1960). The results are also similar to those obtained by Ahrens et al. (1957) in humans fed diets of the same composition and a constant amount of fat but different in the kind of fats fed. The question of posible effects of dietary choline on the cholesterol concentration of serum was studied and the results indicate that choline supplementation in the form of choline chloride (25 percent) to make a total concentration of 820 mg. or 1104 mg. of choline per pound of diet did not influence serum cholesterol. This was also previously observed at this laboratory
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 9, 2015
increase in growth and only a slight improvement in feed efficiency when 10 percent of beef tallow was included in a 22 percent protein diet. Donaldson et al. (1954) reported improved feed utilization but no improvement in weight by the addition of 2.5 to 15 percent of stabilized animal fat to diets containing 785 mg. of choline per pound of diet. They indicated that added fat above 5 percent did not improve feed efficiency when the ration contained 685 mg. of choline per pound. Added choline improved feed efficiency only in diets containing animal fat. The fact that the choline requirement tends to increase in the presence of an excess amount of dietary fat was first shown by Kummerow et al. (1949) who fed diets containing 25 percent linseed oil to chicks to 10 weeks of age. Previous trials in this laboratory with laying hens (Balloun, 1956; Daghir et al. 1960) have shown that choline additions above those normally considered as adequate seem to be beneficial in high animal fat diets. The various fat and choline treatments had no significant effect on either liver lipids or liver cholesterol (Table 2). Since all diets were approximately isocaloric and isonitrogenous, it can be assumed that the only dietary influences that might affect liver lipids, or liver cholesterol, are the type of fat used and the level of choline. Liver lipid values show a slight tendency toward higher values in the case of the animal fat-fed groups than in the soybean oil-fed groups. Benton et al. (1956) showed elevated liver fat values in rats fed 20 percent of butter or lard, and decreased values in those fed 20 percent corn oil. Donovan and Balloun (1955) showed that chicks consuming a high-fat diet (10 percent) accumulated no more fat in their livers than those consuming a low-fat diet (1 percent), indicating that dietary fat per se is not deposited in the chick's liver to any great extent.
1716
X. J. DAGHIR AND S. L.
| 82
_J80 5* 2 '*•*
172
'
BALLOUN
I NO ADDED CHOLINE |284 MG/LB ADDED CHOLINE |568 MG/LB ADDED CHOLINE
•••',!
_l O
*Id 160 — ltf> UJ _j
o X °I40 S
-
Z) £C UJ
en
!00 *
r
uumua
i •
MALES FEMALES 8 % ANIMAL FAT
MALES FEMALES 8 % SOYBEAN OIL
MALES FEMALES NO ADDED FAT
FIG. 1. Effect of fat and choline on serum cholesterol of chicks (average of determinations made at 5 and 7 weeks of age).
in work with laying hens (Daghir, 1960). Duff and Meissner (19S4) showed that the oral administration of 1 to 3 gms. of choline chloride daily to rabbits did not affect serum cholesterol. Similar results were reported by Rawls and Tichner (1956) in human patients. Figure 1 illustrates a tendency toward higher serum cholesterol levels as the choline level of the diet is increased in birds fed 8 percent animal fat. Moyer et al. (1956) and later Cuthbertson et al. (1959) reported similar observations in rats fed high-fat diets. Considering all treatments, males had significantly higher serum cholesterol concentrations than did females. Sutton et al. (1958) observed that changes in serum cholesterol are influenced by both cholesterol concentration in the diet and by the sex of the chick. A significant difference between sexes in chick serum cholesterol concentration at five and seven weeks of age was also reported by Bumgardner and Shaffner (1955).
SUMMARY
Xine diets, approximately equal in energy and protein content, were fed to chicks to study the effect of feeding 8 percent of animal fat (white grease) and 8 percent of soybean oil with three different levels of choline (536, 820, 1104 mg./lb. total choline) on weight gains, feed efficiency, liver lipids and liver and serum cholesterol. Different fat and choline treatments had no marked effect on weight gains up to 7 weeks of age, but a significant (P < 0.01) improvement in feed efficiency resulted from feeding 8 percent of either fat. Choline additions were effective in improving feed efficiency in the animal fat-fed groups, but had no significant effect on those not receiving added fat or those receiving soybean oil. Groups receiving 8 percent of animal fat had significantly higher serum cholesterol values than those not receiving added fat or those receiving 8 percent of soybean oil. When all treatments were considered, concentrations of serum cholesterol were sig-
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 9, 2015
120
CHOLESTEROL LEVELS
nificantly higher in males than in females. Dietary variables did not significantly affect either liver lipids or liver cholesterol. ACKNOWLEDGMENT
Soybean oil for this study was generously supplied by the Spencer-Kellogg Company, Des Moines, Iowa, and the animal fat by Armour and Company, Chicago, Illinois.
Aitken, J. R., G. S. Lindblad and W. G. Hunsaker, 1954. Beef tallow as a source of energy in broiler rations. Poultry Sci. 33: 1038. Balloun, S. L., 1956. Choline and tallow in breeder hen diets. Poultry Sci. 35: 737-738. Benton, D. A., A. E. Harper and C. A. Elvehjem, 1956. The effect of different fats on liver fat deposition. J. Biol. Chem. 218: 693-700. Bumgardner, H. L., and C. S. Shaffner, 1955. Studies on serum cholesterol levels of chickens. Poultry Sci. 34: 1183. Cuthbertson, W. F. J., P. V. Bicoate, D. M. Ireland, D. Mills and P. Shearley, 1959. Serumcholesterol levels and atherosclerosis: effects of composition of diet and triiodothyronine on the rat. British J. Nutrition, 13: 227-242. Daghir, N. J., 1960. Influence of animal and vegetable fats on egg producton, egg quality and serum cholesterol of laying hens. Unpublished M. S. thesis, Iowa State University Library, Ames, Iowa. Daghir, N. J., W. Marion and S. L. Balloun, 1960. Influence of dietary fat and choline on serum and egg yolk cholesterol in the laying chicken. Poultry Sci. 39: 1459-1466. Donaldson, W. E., G. F. Combs and G. L. Romoser, 1954. Results obtained with added fat in chick rations. Poultry Sci. 33: 1053. Donovan, G. A., and S. L. Balloun, 1955. Effect of dietary protein and energy on chick liver fat accumulation. Proc. Soc. Exp. Biol. Med. 90: 692-694. Duff, G. L., and G. F. Meissner, 1954. Effect of choline on the development and regression of cholesterol atherosclerosis in rabbits. Arch. Pathol. 57: 329. Hegsted, D. M., A. Gotsis and F. J. Stare, 1960. The influence of dietary fats on serum choles-
terol levels in cholesterol-fed chicks. J. Nutrition, 70: 119-126. Kummerow, F. A., R. Weaver and H. Honstead, 1949. The choline replacement value of ethanolamine in chickens kept on a high-fat ration. Poultry Sci. 28: 475-478. Leveille, G. A., and H. Fisher, 1958. Observation on lipid utilization in hens fed vegetable and animal fat supplemented diets. Poultry Sci. 37: 658-664. March, B. E., and J. Biely, 1959. Dietary modification of serum cholesterol in the chick. J. Nutrition, 69: 105-110. Mone, P. E., W. D. Warner, C. E. Poling and E. E. Rice, 1959. Influence of dietary fat and protein on serum cholesterol of cholesterol-fed chicks. J. Amer. Oil Chem. Soc, 36: 141-142. Moyer, A. W., D. Kritchevsky, J. Blogan and H. R. Cox, 1956. Dietary protein and serum cholesterol in rats. Proc. Soc. Exp. Biol. Med. 92: 736-737. Quackenbush, F. W., and M. D. Powlowski, 1960. Effects of purified linoleic ester on cholesterol in the rat. J. Nutrition, 72: 196-202. Rawls, W. B., and J. B. Tichner, 1956. Lipid metabolism and the effect of a combined lipotropic agent. J. Amer. Geriatrics Soc. 4 : 89-105. Snedecor, G. W., 1957. Statistical Methods Applied to Experiments in Agriculture and Biology. 5th Ed. The Iowa State College Press, Ames, Iowa. Stamler, J., R. Pick and L. Katz, 1954. Inhibition of cholesterol induced coronary atherogenesis in the egg producing hen. Circulation, 10: 251254. Sutton, J. B., A. A. Kurnick, M. W. Pasvogel and M. G. Vavich, 1958. The effects of graded dietary cholesterol levels on the growth, serum cholesterol and body composition of the chick. Poultry Sci. 37: 1246. Svacha, R. L., T. M. Ferguson, B. L. Reid and J. R. Couch, 1958. Effect of cholesterol, type of fat and lipotropic agents in chick diets. Poultry Sci. 37: 1246. Weiss, H. S., and H. Fisher, 1957. Plasma lipid and organ changes associated with the feeding of animal fat to laying chickens. J. Nutrition, 61: 267-280. Weiss, H. S., L. Marx and W. Marx, 1952. Effects of thyroid and of choline and inositol on cholesterol distribution in rats fed a high cholesterol diet. Endocrinology, 50: 192-198. Zlatkis, A., B. Zak and A. J. Boyle, 1953. A new method for the direct determination of serum cholesterol. J. Lab. Clin. Med. 4 1 : 486-492.
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 9, 2015
REFERENCES Ahrens, E. H., W. Insull, R. Blomstrand, J. Hirsch, T. T. Tsaltas and M. L. Peterson, 1957. The influence of dietary fats on serum-lipid levels in man. Lancet, 272: 943-953.
1717
E. L. WISMAN, W. L. BEANE, W. R. LUCKHAM AND E. 0. ESSAEY
correlations for six feathering characteristics. Poultry Sci. 36: 621-628. Siegel, P. B., and R. H. Coles, 1958. Effects of floor space on broiler performance. Poultry Sci. 37: 1243. Smith, R. C , 1956. Kind of litter and breast blisters on broilers. Poultry Sci. 35: 593-595. Smith, R. C , 1959. Age and weight of marketing affects broiler profits. Delaware Agr. Exp. Sta.
Tech. Bui. 328. Smith, R. M., N. R. Gyles and J. C. Gilbreath, 1954. The influence of producing sexes separately on growth, feed utilization, and dressed grade of broilers. Poultry Sci. 33: 1082. Winget, C. M., A. H. Smith and C. F. Kelly, 1958. The effect of body weight on joint and skin disorders of domestic birds. Poultry Sci. 37: 1253.
N. J. DAGHIR AND S. L. BALLOUN Department of Poultry Husbandry, Iowa State University, Ames (Received for publication February 27. 1961)
D
URING the past decade, there has been a great deal of interest in investigating the effects of different dietary components on serum cholesterol levels in a large number of species. Previous papers have shown that dietary fats and choline are among the various components that affect serum cholesterol levels in the chick. It is well established that dietary fat may either increase or decrease blood cholesterol depending upon the type of fat and the level at which it is fed. When feeding S to 10 percent of added animal fat to laying chickens, Weiss and Fisher (1957) observed high plasma cholesterol concentrations, and friable and fatty liver development which suggested that high animal fat diets result in a derangement in lipid metabolism in the chicken. Leveille and Fisher (1958) reported no significant increases in plasma cholesterol when 10 percent tallow was fed to Single Comb White Leghorn hens as compared to hens fed diets containing 10 percent of corn oil or the basal diet containing no added fat. The data presented, 1 Journal Paper No. J-4067 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project No. 1062.
however, show a definite trend toward higher plasma cholesterol levels in the groups fed animal fats. March and Biely (1959) fed chicks various fats in diets containing 20 and 26 percent protein. With both protein levels, 8 percent of Crisco, butter, lard or chicken fat increased serum cholesterol. Herring oil did not increase serum cholesterol with either diet, and corn oil did so only in the case of the chicks fed a 20 percent protein diet. This indicates that fats high in saturated fatty acids tend to raise serum cholesterol levels in the chick. Similar results were obtained by Hegsted et al. (1960), who clearly demonstrated that fats high in saturated fatty acids promote hypercholesterolemia and that this effect is counteracted by unsaturated fatty acids. Mone et al. (1959), on the other hand, concluded that serum cholesterol was not affected in their experiments by increasing the fat from 4 to 10 percent, or by the degree of unsaturation of the fat. Since several natural oils, high in polyunsaturated fatty acids, have been shown to lower serum cholesterol in several species, Quackenbush and Pawlowski (1960) stud-
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 9, 2015
Influence of Fats and Choline on Liver and Serum Cholesterol in the Chick 1
1713
CHOLESTEROL LEVELS
sclerosis in the rabbit. They showed that the above amount of choline, fed with one gram of cholesterol daily, did not affect the development of atherosclerosis or the cholesterol content of the blood. Svacha et al. (1958), on the other hand, reported that the feeding of 0.25 percent choline chloride with 2 percent cholesterol resulted in a decrease in serum cholesterol in chicks in the presence or absence of fat. The effects of fats and choline on cholesterol metabolism are not well elucidated as the previous brief review indicates. Hence, more work on the effects of these two dietary components and their interactions is considered in order. The following experiment was designed to study the effect of dietary fats of two types, each fed with three different levels of choline, on weight gains, feed efficiency, liver lipids, and liver and serum cholesterol of growing chicks. EXPERIMENTAL PROCEDURE
Two hundred fifty two day-old cross-bred sexed chicks (Vantress $ X Arbor Acre 9 ) were kept on diet 1 (Table 1) for one
TABLE 1.—Experimental diets Diet composition (%) 1
2
3
4
5
6
7
8
9
49 48.75 48.50 48.75 48.50 49 58.75 59 Ground yellow corn 58.50 10 10 10 10 10 10 Oat hulls — — — 1 8 8 8 Animal fat — — — — — — 8 8 8 Soybean oil — — — — — — 8 8 Dextrose 8 0.25 0.50 0.25 0.50 0.50 0.25 Choline chloride (25%) — — — 33 33 33 33 35 33 33 33 33 Basal components 2 Calculated composition 20.6 20.6 20.6 20.6 20.6 20.0 20.0 20.6 20.0 Protein, % Productive energy, 1,036 1 ,036 1,036 1 ,036 1 ,036 1,005 1 ,005 1 ,036 Cal./lb. 1 ,005 536 820 1 ,104 802 1 ,104 536 536 820 1 ,104 Choline, mg./lb. 1 White grease—moisture 0.34%, free fatty acids 1.38%, unsaponifiable matter 0.52%, titer 39.1°C, stability (A.O.M.) over 60 hours. 2 The basal components portion of the diet was composed of the following: (values are expressed as percentages of the complete diet or amounts per lb. of diet). Soybean oil meal (50% protein) 22%, meat scraps (50% protein) 5%, fish meal (65% protein) 1.5%, alfalfa leaf meal (20% protein) 2%, steamed bone meal 1%, NaCl 0.4%, manganese 55 mg., iodine 0.25 mg., iron 18 mg., copper 1.36 mg., zinc 1.30 mg., cobalt 0.23 mg., sulfur 7.7 mg., riboflavin 2 mg., niacin 17 mg., Ca. pantothenate 4 mg., vit. B12 7.5 meg., menadione 1 mg., vit. E. 5 I.U., vit. A. 3,000 I. U. and vit. D, 500 I.C.U.
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on June 9, 2015
ied the effects of individual fatty acids on serum cholesterol in the rat. Linoleic acid ester was found to lower markedly the plasma cholesterol level of hypercholesterolemic cholesterol-fed rats. It also lowered liver cholesterol of the rats fed a low-fat diet but not those receiving a high-fat diet. Sex differences were reported by Stamler et al. (1954), who indicated that twice the amount of dietary cholesterol was required to cause the same degree of plasma cholesterol increase in females as in males. Reports in the literature related to the action of choline on serum cholesterol are not in complete agreement. Weiss et al. (1952) observed that the feeding of 1 percent choline, plus inositol, produced a marked hypercholesterolemia in rats fed a 2 percent cholesterol diet. In rats fed a lowcholesterol diet, however, plasma cholesterol was not significantly changed. A series of three experiments were conducted by Duff and Meissner (1954) to determine the effect of oral administration of 1 to 3 grams of choline chloride daily on the development of experimental athero-
1714
N. J. DAGHIR AND S. L. BALLOUN
T A B L E 2.—Effect
Diet additions Choline F a t (%) (nig./ lb.)
Ani. Ani. Ani. Soy. Soy. Soy. 1
8 8 8 8 8 8
0 284 568 0 284 568 0 284 568
fat and choline on weight gains, feed efficiency, cholesterol of growing chicks1
Wt. Gains in Pounds 2 Males
Females
2.54 2.42 2.32 2.40 2.38 2.52 2.40 2.48 2.52
2.06 1.92 2.06 2.11 1.89 2.00 1.82 2.03 2.22
Feed/ Gain Mixed 2.26 2.27 2.26 2.20 2.16 2.10 2.22 2.06 2.22
Liver Lipids (% of dry tissue)
liver
lipids,
Liver cholesterol (mg./g. of dry tissue)
'• liver i
Serum cholesterol (mg. % )
Males
Females
Males
Females
Males
Females
16.8+0.64 15.8+0.99 17.0+0.60 17.3 + 0.75 15.8+0.41 16.4+0.67 16.5 + 0.31 15.7 + 0.26 16.4+0.19
18.8+0.63 17.1 + 0.54 16.1 + 0.42 15.6+0.56 17.3 + 0.78 17.2 + 0.87 15.7 + 0.29 16.4+0.87 16.4 + 0.39
26.7+1.16 24.4+0.99 25.3+0.67 25.5+0.74 2 4 . 8 + 1.23 25.7+0.88 24.8+0.72 25.4+0.92 26.7 + 0.56
27.1 + 0.88 26.1 + 0.10 25.0+1.48 25.0+0.33 27.0+1.10 24.9+1.90 24.3+1.47 25.4+0.92 26.7+0.56
152+ 4 . 9 162+ 7.9 156+ 6.4 172+ 6.4 182+ 9.2 172+10.1 159+ 8.6 158+10.4 156+ 6 . 9
146+ 7.7 154+ 9 . 5 148+ 6.7 154+ 2 . 8 165+ 6.6 170+ 6 . 6 144+ 5 . 1 145+10.3 149+ 3 . 5
All values represent means or means + their standard errors. Values represent weight gains over a period of 6 weeks (from 1 to 7 weeks of agi
week after which they were randomly assigned to 18 pens, each pen containing 7 birds of each sex. Two pens were allotted to each of the 9 experimental diets (Table 1) under a restricted randomized block design. Groups were chosen to be approximately of equal weights. The birds were kept in thermostatically-controlled batteries for the first 4 weeks after which they were transferred to non-heated batteries for the remainder of the experimental period. Feed and water were supplied ad libitum throughout the test period, and both birds and feed were weighed at weekly intervals. The diets were made approximately isocaloric and isonitrogenous by substituting oat hulls in the added-fat diets for part of the corn, and dextrose in the no-added-fat diets for the 8 percent fat in the remaining diets. Three different levels of choline were used, 536, 820, and 1104 mg. of total choline per pound of diet. Choline was added in the form of choline chloride (25 percent). At both 5 and 7 weeks of age, individual blood samples were collected by heart puncture from two male and two female birds chosen at random from each pen. The birds were then sacrificed by dislocation of the spinal cord, and livers were immediately removed, washed of adhering blood and frozen. Total cholesterol in serum was de-
termined by the method described by Zlatkis et al. (1953). For lipid and cholesterol determinations, the livers from two chicks of each sex from each pen were first individually homogenized with 15 ml. of distilled water in a Waring blendor and then dried in an oven at 100°C. for 12 hours. The dry homogenate was then ground in a Servall OmniMixer. Duplicate, 2 gram samples of each chick liver homogenate were extracted with the following solvent mixture for 10 hours: 5 parts ethyl ether, 5 parts Skellysolve B and 2 parts ethanol. Percent lipids was calculated from the weight loss. Extracts were diluted to 200 ml. with the solvent mixture and a 1 ml. sample was evaporated to dryness for total cholesterol determination as outlined in the method of Zlatkis et al. (1953). The analyses of variance were made according to Snedecor (1957). RESULTS AND DISCUSSION The data presented in Table 2 indicate that the various fat and choline treatments had no marked effect on weight gains of birds to 7 weeks of age. The fat treatments, however, regardless of the level of choline, resulted in a significant (P < 0.01) improvement in feed efficiency. These results are in agreement with previous papers. Aitken et al. (1954) obtained no significant
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1
0 0 0 Fat Fat Fat Oil Oil Oil
ofdietary
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CHOLESTEROL LEVELS TABLE 3.—Analysis of variance of serum cholesterol of chicks1 rce of variation
d.f.
Mean squares
Blocks Fat (F) Choline (C) Sex (S) FXC FXS CXS FXCXS Error
1 2 2 1 4 2 2 4 17
612.0 1,056.0** 116.0 1,071.0** 44.0 34.5 45.5 25.3 92.1
Total
35
1 Combined data of 5 and 7 week determinations. ** Significant at P < 0 . 0 1 .
Analysis of variance of serum cholesterol data is presented in Table 3. Feeding 8 percent of animal fat resulted in significantly higher serum cholesterol values in both male and female birds, while 8 percent of soybean oil did not elevate serum cholesterol. An evaluation of the role of fat upon serum cholesterol levels must always relate both the amount and kind of fat used. Since, in these studies, the amount of fat fed was constant, the difference in effect on serum cholesterol was mainly due to the kind of fat used. The results confirm reports in the literature that the feeding of a highly-saturated fat to chicks increases serum cholesterol and that a highly-unsaturated fat counteracts this effect (March and Biely, 1959; Hegsted et al., 1960). The results are also similar to those obtained by Ahrens et al. (1957) in humans fed diets of the same composition and a constant amount of fat but different in the kind of fats fed. The question of posible effects of dietary choline on the cholesterol concentration of serum was studied and the results indicate that choline supplementation in the form of choline chloride (25 percent) to make a total concentration of 820 mg. or 1104 mg. of choline per pound of diet did not influence serum cholesterol. This was also previously observed at this laboratory
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increase in growth and only a slight improvement in feed efficiency when 10 percent of beef tallow was included in a 22 percent protein diet. Donaldson et al. (1954) reported improved feed utilization but no improvement in weight by the addition of 2.5 to 15 percent of stabilized animal fat to diets containing 785 mg. of choline per pound of diet. They indicated that added fat above 5 percent did not improve feed efficiency when the ration contained 685 mg. of choline per pound. Added choline improved feed efficiency only in diets containing animal fat. The fact that the choline requirement tends to increase in the presence of an excess amount of dietary fat was first shown by Kummerow et al. (1949) who fed diets containing 25 percent linseed oil to chicks to 10 weeks of age. Previous trials in this laboratory with laying hens (Balloun, 1956; Daghir et al. 1960) have shown that choline additions above those normally considered as adequate seem to be beneficial in high animal fat diets. The various fat and choline treatments had no significant effect on either liver lipids or liver cholesterol (Table 2). Since all diets were approximately isocaloric and isonitrogenous, it can be assumed that the only dietary influences that might affect liver lipids, or liver cholesterol, are the type of fat used and the level of choline. Liver lipid values show a slight tendency toward higher values in the case of the animal fat-fed groups than in the soybean oil-fed groups. Benton et al. (1956) showed elevated liver fat values in rats fed 20 percent of butter or lard, and decreased values in those fed 20 percent corn oil. Donovan and Balloun (1955) showed that chicks consuming a high-fat diet (10 percent) accumulated no more fat in their livers than those consuming a low-fat diet (1 percent), indicating that dietary fat per se is not deposited in the chick's liver to any great extent.
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X. J. DAGHIR AND S. L.
| 82
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'
BALLOUN
I NO ADDED CHOLINE |284 MG/LB ADDED CHOLINE |568 MG/LB ADDED CHOLINE
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MALES FEMALES 8 % ANIMAL FAT
MALES FEMALES 8 % SOYBEAN OIL
MALES FEMALES NO ADDED FAT
FIG. 1. Effect of fat and choline on serum cholesterol of chicks (average of determinations made at 5 and 7 weeks of age).
in work with laying hens (Daghir, 1960). Duff and Meissner (19S4) showed that the oral administration of 1 to 3 gms. of choline chloride daily to rabbits did not affect serum cholesterol. Similar results were reported by Rawls and Tichner (1956) in human patients. Figure 1 illustrates a tendency toward higher serum cholesterol levels as the choline level of the diet is increased in birds fed 8 percent animal fat. Moyer et al. (1956) and later Cuthbertson et al. (1959) reported similar observations in rats fed high-fat diets. Considering all treatments, males had significantly higher serum cholesterol concentrations than did females. Sutton et al. (1958) observed that changes in serum cholesterol are influenced by both cholesterol concentration in the diet and by the sex of the chick. A significant difference between sexes in chick serum cholesterol concentration at five and seven weeks of age was also reported by Bumgardner and Shaffner (1955).
SUMMARY
Xine diets, approximately equal in energy and protein content, were fed to chicks to study the effect of feeding 8 percent of animal fat (white grease) and 8 percent of soybean oil with three different levels of choline (536, 820, 1104 mg./lb. total choline) on weight gains, feed efficiency, liver lipids and liver and serum cholesterol. Different fat and choline treatments had no marked effect on weight gains up to 7 weeks of age, but a significant (P < 0.01) improvement in feed efficiency resulted from feeding 8 percent of either fat. Choline additions were effective in improving feed efficiency in the animal fat-fed groups, but had no significant effect on those not receiving added fat or those receiving soybean oil. Groups receiving 8 percent of animal fat had significantly higher serum cholesterol values than those not receiving added fat or those receiving 8 percent of soybean oil. When all treatments were considered, concentrations of serum cholesterol were sig-
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120
CHOLESTEROL LEVELS
nificantly higher in males than in females. Dietary variables did not significantly affect either liver lipids or liver cholesterol. ACKNOWLEDGMENT
Soybean oil for this study was generously supplied by the Spencer-Kellogg Company, Des Moines, Iowa, and the animal fat by Armour and Company, Chicago, Illinois.
Aitken, J. R., G. S. Lindblad and W. G. Hunsaker, 1954. Beef tallow as a source of energy in broiler rations. Poultry Sci. 33: 1038. Balloun, S. L., 1956. Choline and tallow in breeder hen diets. Poultry Sci. 35: 737-738. Benton, D. A., A. E. Harper and C. A. Elvehjem, 1956. The effect of different fats on liver fat deposition. J. Biol. Chem. 218: 693-700. Bumgardner, H. L., and C. S. Shaffner, 1955. Studies on serum cholesterol levels of chickens. Poultry Sci. 34: 1183. Cuthbertson, W. F. J., P. V. Bicoate, D. M. Ireland, D. Mills and P. Shearley, 1959. Serumcholesterol levels and atherosclerosis: effects of composition of diet and triiodothyronine on the rat. British J. Nutrition, 13: 227-242. Daghir, N. J., 1960. Influence of animal and vegetable fats on egg producton, egg quality and serum cholesterol of laying hens. Unpublished M. S. thesis, Iowa State University Library, Ames, Iowa. Daghir, N. J., W. Marion and S. L. Balloun, 1960. Influence of dietary fat and choline on serum and egg yolk cholesterol in the laying chicken. Poultry Sci. 39: 1459-1466. Donaldson, W. E., G. F. Combs and G. L. Romoser, 1954. Results obtained with added fat in chick rations. Poultry Sci. 33: 1053. Donovan, G. A., and S. L. Balloun, 1955. Effect of dietary protein and energy on chick liver fat accumulation. Proc. Soc. Exp. Biol. Med. 90: 692-694. Duff, G. L., and G. F. Meissner, 1954. Effect of choline on the development and regression of cholesterol atherosclerosis in rabbits. Arch. Pathol. 57: 329. Hegsted, D. M., A. Gotsis and F. J. Stare, 1960. The influence of dietary fats on serum choles-
terol levels in cholesterol-fed chicks. J. Nutrition, 70: 119-126. Kummerow, F. A., R. Weaver and H. Honstead, 1949. The choline replacement value of ethanolamine in chickens kept on a high-fat ration. Poultry Sci. 28: 475-478. Leveille, G. A., and H. Fisher, 1958. Observation on lipid utilization in hens fed vegetable and animal fat supplemented diets. Poultry Sci. 37: 658-664. March, B. E., and J. Biely, 1959. Dietary modification of serum cholesterol in the chick. J. Nutrition, 69: 105-110. Mone, P. E., W. D. Warner, C. E. Poling and E. E. Rice, 1959. Influence of dietary fat and protein on serum cholesterol of cholesterol-fed chicks. J. Amer. Oil Chem. Soc, 36: 141-142. Moyer, A. W., D. Kritchevsky, J. Blogan and H. R. Cox, 1956. Dietary protein and serum cholesterol in rats. Proc. Soc. Exp. Biol. Med. 92: 736-737. Quackenbush, F. W., and M. D. Powlowski, 1960. Effects of purified linoleic ester on cholesterol in the rat. J. Nutrition, 72: 196-202. Rawls, W. B., and J. B. Tichner, 1956. Lipid metabolism and the effect of a combined lipotropic agent. J. Amer. Geriatrics Soc. 4 : 89-105. Snedecor, G. W., 1957. Statistical Methods Applied to Experiments in Agriculture and Biology. 5th Ed. The Iowa State College Press, Ames, Iowa. Stamler, J., R. Pick and L. Katz, 1954. Inhibition of cholesterol induced coronary atherogenesis in the egg producing hen. Circulation, 10: 251254. Sutton, J. B., A. A. Kurnick, M. W. Pasvogel and M. G. Vavich, 1958. The effects of graded dietary cholesterol levels on the growth, serum cholesterol and body composition of the chick. Poultry Sci. 37: 1246. Svacha, R. L., T. M. Ferguson, B. L. Reid and J. R. Couch, 1958. Effect of cholesterol, type of fat and lipotropic agents in chick diets. Poultry Sci. 37: 1246. Weiss, H. S., and H. Fisher, 1957. Plasma lipid and organ changes associated with the feeding of animal fat to laying chickens. J. Nutrition, 61: 267-280. Weiss, H. S., L. Marx and W. Marx, 1952. Effects of thyroid and of choline and inositol on cholesterol distribution in rats fed a high cholesterol diet. Endocrinology, 50: 192-198. Zlatkis, A., B. Zak and A. J. Boyle, 1953. A new method for the direct determination of serum cholesterol. J. Lab. Clin. Med. 4 1 : 486-492.
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REFERENCES Ahrens, E. H., W. Insull, R. Blomstrand, J. Hirsch, T. T. Tsaltas and M. L. Peterson, 1957. The influence of dietary fats on serum-lipid levels in man. Lancet, 272: 943-953.
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