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Dietary Lipid Effect on Atherogenesis and Plasma Lipids in Cockerels1
Dietary Lipid Effect on Atherogenesis and Plasma Lipids in Cockerels1 R. R. EPLEY AND S. L. BALLOUN Iowa State University of Science and Technology, Ames, Iowa 50010 (Received for publication June 24, 1970)
A
1
Journal paper No. J-6613 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 1720.
and thereby decreases their reabsorption from the intestine. This decreased recirculation stimulates cholesterol oxidation to bile acids to replace acids lost by excretion. Our series of experiments was initiated to study several effects of feeding cholestyramine, cholesterol, coconut oil, dried whole egg and soy oil on plasma free cholesterol, cholesterol esters, glycerides, lipoproteins and severity of atherosclerosis in cockerels. MATERIALS AND METHODS Blood samples were obtained from a pricked brachial vein after cockerels were fasted 12 hr. In Experiment 1, blood was allowed to clot at room temperature for 24 hr. to express serum. In Experiments 2 and 3, blood was collected in oxalated test tubes. Serum and plasma were stored in glass vials at — 17.8°C. Aortas were stored in formalin solution (200 ml. formaldehyde, 37.5%, 1800 ml. distilled water, 80 gm.NaH 2 P0 4 and 130 gm.NaHPO*), (Ball, 1968). Aortas were opened with a scalpel longitudinally on the dorsal side and scrutinized by one judge and scored either as 1, having one or more plaques, or 0, having no plaques. Apparent ether-extract digestibility was determined by using chromic oxide as a marker and by using the apparent digestibility equation described by Maynard and Loosli (1962). Serum total cholesterol was assayed with methodology described by Technicon Instruments Corp. (1964) using a Technicon Autoanalyzer.
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THEROGENESIS and plasma lipids • levels may be affected by dietary constituents. Christensen (1964) and Banerjee et al. (1965) reported increased uptake of cholesterol by the aorta of cockerels fed cholesterol. Pick et al. (1967) reported a positive relationship between the degree of hypercholesterolemia and coronary atherosclerosis in cholesterol-fed cockerels. Dayton and Hashimoto (1966) demonstrated that lipoprotein is necessary in the surrounding medium if labeled cholesterol is to be transported into the intima. Orma (1957) and Leveille et al. (1960) separated lipoproteins by paper electrophoresis and noted, in the hypercholesterolemic condition, an increased cholesterol content of the a-lipoprotein and increased lipid content of the ^-lipoprotein. Leveille et al. (1960) reported that chicks fed a diet containing 10% coconut oil had higher plasma cholesterol levels than those receiving a similar diet supplemented with corn oil. Gosling et al. (1969) and Fisher et al. (1963) reported increased abdominal atherosclerosis when cockerels were fed diets containing whole, dried eggs. Tennent et al. (1961) demonstrated that feeding cholestyramine, an anion-exchange resin, to cockerels with a normal plasma cholesterol level results in a decreased plasma cholesterol concentration. Van Itallie and Hashim (1963) postulated that cholestyramine binds bile acids in the gut
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R. R. EPLEY AND S. L. BALLOUN
methionine 0.075% and Santoquin 0.0125%. Cholesterol and cholestyramine were substituted for yellow corn on an equal weight basis. When the cockerels were 10 weeks old, dietary protein was reduced from 22% to 15% by increased corn and decreased sybean meal. Experiment 2 was designed to study several effects of feeding dried whole egg or crystalline cholesterol, plus soy oil with and without 1% cholestyramine. Diets were formulated to be isocaloric and isonitrogenous and to provide the same concentrations of fat and cholesterol. Nine Hubbard broiler cockerels (2 months oil) were allotted at random to each of 4 dietary treatments arranged in a 2 X 2 factorial. During the 4-month experimental period, cockerels were housed in 30 X 50 X 40 cm. cages in a 22°C. environmental chamber. The composition of diets fed in Experiment 2 are listed in Table 1. Samples of initial and final blood plasma were obtained and aortas were removed from all cockerels at the end of the experiment. Experiment 3 was designed to study several effects of feeding 10% coconut oil or soy oil with and without 1% cholestyramine. Thirty-six Hubbard broiler cockerels (2.5 months of age) were used in Experiment 3. Otherwise, the experimental design and procedure used for Experiment 2 and 3 were identical. The composition of diets fed in Experiment 3 are listed in Table 1. RESULTS Experiment 1. Neither weight gains nor feed consumption of chicks were significantly affected by feeding cholestyramine or cholesterol. Apparent ether-extract digestibility was significantly decreased by feeding cholestyramine but was not affected by feeding cholesterol (Table 2). When cockerels were fed diets containing 1% added cholesterol, adding cholestyramine to the diet reduced serum cholesterol
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Plasma glycerides, free cholesterol and cholesterol esters were assayed by extracting plasma with chloroform-methanol (2: 1). The ratio of solvent to plasma was 20: 1. This extract was then analyzed according to Galanos et al. (1964). Total cholesterol was calculated by summing free and esterified cholesterol. Percentages of al, al and ^-lipoproteins were determined according to Gebott (1968). This technique uses cellulose polyacetate strips and the Schiff's reagent staining procedure that stains the carboncarbon double bonds present in each lipoprotein. Experiment 1 was designed to study several effects of feeding cholesterol with or without 1% dietary cholestyramine. Hubbard broiler males were randomly allotted at one day of age to each of 8 pens with 12 chicks per pen. Four of the pens of chicks received the basal diet and four were fed the basal diet plus 1% cholesterol for 45 days. After 45 days, diets with 1% cholestyramine were fed for 17 days to 2 groups that received the basal diet and to 2 groups that received 1% cholesterol during the first 45-day period. Serum samples were collected on day 45, before feeding cholestyramine, and again when the experiment ended. The apparent ether-extract digestibility was determined during the last week of the experiment. Diets fed in Experiment 1 contained the following ingredients, expressed as percentages: yellow corn 51, soybean meal (48% protein) 28, soy oil 10, fish meal (70% protein) 5, dehydrated alfalfa meal 2, dicalcium phosphate 1, oyster shell 0.7, salt 0.3 and premix concentrate 1. The premix concentrate provided per kilogram of diet: riboflavin 5 mg., niacin 25 mg., calcium panthothenate 10 mg., choline 450 mg., vitamin B12 10 meg., vitamin K. 1 mg., vitamin E 10 I.U., vitamin A 7000 I.U., vitamin D 1000 I.C.U., penicillin 6.6. mg., streptomycin 33 mg. and also
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DIET LIPIDS AND PLASMA LIPIDS TABLE 1.—Diets fed in experiments Z and 3 Experiment 2
Experiment 3
Ingredients 1
2
3
4
1
2
3
4
75.4 10.6
74.4 10.6
70.17 21.34
69.17 21.34
69.5 12.0
69.5 12.0
68.5 12.0
68.5 12.0
—
—
—
—
5.0
5.0
5.0
5.0
1.4 1.6 0.5 0.5
1.4 1.6 0.5 0.5
1.52 1.50 0.5 0.50
1.52 1.50 0.5 0.50
1.0 1.5 0.5 0.5
—
—
1.0 1.5 0.5 0.5 10.0
1.0 1.5 0.5 0.5
— — —
1.0 1.5 0.5 0.5 10.0
— — — —
10.0
—
10.0 1.0
10.0
—
100.0
— 1.0 10.0 — 100.0
4.22 — — 0.253 100.003
—
4.22 1.00
—
0.253 100.003
100.0
—
—
1.0
— — —
— —
100.0
— —
100.0
100.0
a Mineralized salt mixture provided (per kilogram): 4.49 gm. NaCl, 60 mg. Mn, 75 mg. Zn, 55 mg. Fe, 9 mg. Cu, 1.1 mg. I 2 , and 0.55 mg. Co. b Vitamin concentrate provided (per kilogram): 7500 I.U. vitamin A, 1000 I.TJ. vitamin D3, 10 I.U. vitamin E, 1 mg. menadione sodium bisulfite, 10 meg. vitamin B12, 5 mg. riboflavin, 450 mg. choline, 10 mg. pantothenic acid, 25 mg. niacin, 1000 mg. methionine equivalent, 100 mg. Ethoxyquin, 8.25 mg. penicillin and 41 mg. streptomycin.
concentration significantly (P < 0.01). When diets contained no added cholesterol, serum cholesterol levels were reduced by feeding cholestyramine, but the reduction was not statistically significant. Chicks fed cholesterol but no cholestyramine had significantly higher serum cholesterol levels than chicks fed any of the other three diets (Table 2). Experiment 2. Cockerels fed diets containing 10% dried whole egg gained slightly more weight (1.8 ± 0.1 kg.) than cockerels fed diets with cholesterol and soy oil (1.6 ± 0.1 kg.). Mean weight gain was 1.7 kg. in the groups fed no cholestyramine and the same in groups fed diets containing 1% cholestyramine. Feed consumption was not affected by treatments. None of the treatments significantly affected the presence of plaques in the aorta. The significant main effects of feeding cholestyramine were elevated plasma glyceride and lowered cholesterol ester and total cholesterol concentrations (Table 3). Free plasma cholesterol level and the al + a2)/^-lipoprotein
ratio were not significantly affected by feeding cholestyramine (Table 3). Plasma glycerides were increased by feeding dried whole egg plus cholestyramine and unchanged by feeding dried whole eggs plus no cholestyramine (Table 4). The significant main effects of feeding dried whole egg were lowered plasma cholesterol ester and total cholesterol levels compared with those of cockerels fed cholesterol plus soy oil. TABLE 2.—Percentage apparent ether-extract digestibility and mean change of serum cholesterol levels in experiment P-
Dietary treatment No cholesterol+ cholestyramine Cholesterol-|cholestyramine Cholesterol+ no cholestyramine No cholesterol-rno cholestyramine
Percentage digestibility
Serum cholesterol
(%)
(mg./lOOml.)
70.5 + 1.8*
-
77.4+1.8°
-342b
85.9 + 1.8b 90.0+1.8b
4f>
462° -
14.0"
1 Means followed by the same superscript are not significantly different (P<0.05).
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Yellow corn Soybean meal (50% protein) Fish meal (70% protein) Dical. phosphate Calcium carbonate Mineralized salt" Vitamin etc. b Coconut oil Soy oil Cholestyramine Dried whole egg Cholesterol Total
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R. R. EPLEY AND S. L. BALLOUN TABLE 3.—Mean net changes of plasma lipids in experiment 2, main effects (mg./lOO ml.) Diet Variables
Glycerides Cholesterol ester Free cholesterol Total cholesterol Lipoprotein ratio 2
Whole egg
Cholesterol+soy oil
Csm3
No Csms
+ 3 2 . 8 + 14.6 1 - 2 9 . 4 + 8.4 b - 8 . 5 + 5.2 -35.0±10.9d -0.30±0.34d
+ 3 2 . 2 + 14.6 - 1 2 . 4 + 8.4 b -5.4±5.2 - 1 7 . 7 + 10.9 d - 1 . 0 0 + 0.34"
+ 4 5 . 0 + 14.6° - 3 2 . 7 + 8.4" - 5 . 3 + 5.2 - 3 8 . 0 + 10.9 b - 0 . 6 4 + 0.34
+ 2 0 . 0 + 14.6° -18.3+8.4" - 5 . 6 + 5.2 - 1 4 . 7 + 10.9 b - 0 . 6 0 + 0.34
1 Means with the same superscripts are significantly different. Superscripts a, b, c, and d indicate significant differences at the 1, 5, 10 and 25% level of probability, respectively, here and in Table 4. 2 Mean changes of the (al+a2)/(3-lipoprotein ratio. 3 Csm is an abbreviation for cholestyramine in this and following tables.
TABLE 4.—Mean net changes of plasma lipids in experiment 2 (mg./lOO ml.) Whole dried egg Lipid component Glycerides Cholesterol ester' Free cholesterol Total cholesterol1 1 Lipoprotein ration
Csm 64.9 -25.0 - 2.4 -24.5 - 0.16
i Interaction was significant.
No Csm 0.8 -33.9 - 8.6 -42.5 - 0.45
Cholesterol+Soy oil Csm
No Csm
25.0 39.3 -40.4 15.6 - 8.2 - 2.5 -48.5 13.1 - 1.13 - 0.87
cients between the decrease in (al +
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Cockerels fed dried whole egg and no cholestyramine had lower plasma cholesterol ester and total cholesterol levels than those fed dried whole egg plus cholestyramine (Table 4). Cholestyramine more effectively reduced plasma cholesterol ester and total cholesterol when cockerels were fed cholesterol plus soy oil than when fed dried whole egg. Plasma cholesterol ester and total cholesterol were decreased by feeding dried whole egg with no cholestyramine, but increased by feeding cholesterol and soy oil with no cholestyramine (Table 4). The (al + «2)/^-lipoprotein ratio decreased when cockerels were fed cholesterol plus soy oil. This decreased ratio was associated with an increased number of carboncarbon double bonds in the al-lipoprotein in plasma of cockerels fed diets containing whole dried egg and a concomitant increase in the number of double bonds present in ^-lipoprotein of cockerels fed diets with cholesterol plus soy oil. Correlation coeffi-
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DIET LIPIDS AND PLASMA LIPIDS TABXE 5.—Mean net changes of plasma lipids in experiment 3, main effects (mg./lOO ml.) Diet variables Lipid components Coconut oil Glycerides Cholesterol esters Free cholesterol Total cholesterol Lipoprotein 1
+ 12.6 + -5.4 + -1.0 + -4.8 + -8.8 +
12.7 7.9" 2.8 9.5" 2.3 b
Soy oil
Csm +3.0 + -28.1 + -2.0 + -30.4 + -7.4 +
-3.6+12.7 - 4 0 . 0 + 7.9" - 4 . 0 + 2.8 -44.1+9.5" - 2 . 8 + 2.3 b
No Csm + 6 . 0 + 12.7 - 1 7 . 3 + 7.9 — 1.2 + 2.8 - 1 8 . 5 + 9.5 -4.1±2.3d
12.7 7.9 2.8 9.5 2.3 d
• Mean change of percent al-lipoprotein.
coefficients are not significant (Snedecor, 1956), but they can aid in the interpretation of the correlation between (al + a 2 ) / /3-lipoprotein ratio and cholesterol ester levels. The decrease in (al + a2)//3-lipoprotein ratio was due to decreased a2-lipoprotein relative to increased al- and /3-lipoproteins. These relative changes occurred when plasma cholesterol ester levels decreased. Conversely, as the cholesterol ester level increased, there was an increased number of double bonds in the a2-lipoprotein and a decreased number of double bonds in the al- and /3-lipoprotein. This decrease in double bonds indicates an increased amount of cholesterol ester carried by the al- and ^-lipoproteins. DISCUSSION Apparent ether-extract digestibility was decreased by feeding diets containing cholestyramine with or without added dietary cholesterol. The decreased digestibility may be related to the bile acid-binding property TABLE
6.-
-Mean net changes of plasma lipids in experiment 3 (mg./lOO ml.) Coconut oil
Soy oil
Lipid component Csm Glycerides Cholesterol esters Free cholesterol Total cholesterol Lipoprotein a l 1 1
17.9 -13.1 1.2 -12.5 -13.5
Interaction was significant.
N o Csm
-
7.9 2.4 0.7 3.0 4.1
Csm
N o Csm
-11.3 -43.1 - 5.1 -48.2 - 1.4
4.0 -37.0 - 3.0 -30.0 - 4.1
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cockerels fed coconut oil compared with plasma from cockerels fed soy oil. Plasma cholesterol ester and total cholesterol, however, were significantly decreased by feeding soy oil. The al-lipoprotein was decreased by feeding coconut oil plus cholestyramine (Table 6). This decrease was associated with increased plasma glycerides. The increased plasma glycerides should have contained saturated fatty acids from coconut oil. Saturated fatty acids in lipoproteins from coconut oil would not be detected with the Schiff's stain. From this relationship, we conclude that al-lipoprotein was carrying more saturated fatty acids when coconut oil plus cholestyramine was added to the diet. Correlation coefficients between the decrease in (al + a2)//3-lipoprotein ratio and decreased free cholesterol, cholesterol ester and glycerides were 0.0, 0.4 and —0.2, respectively. Since the correlation coefficients of the lipoprotein ratio to cholesterol and glycerides are not significant (Snedecor, 1956), there is no reason to believe that a relationship exists between the ratio and these plasma lipid components. However, the correlation coefficient between the decreased (al + «2)//3-lipoprotein ratio and decrease in plasma cholesterol ester is significant. Correlation coefficients between decreased cholesterol ester and decreased al-, al- and /3-lipoprotein were —0.2, 0.3 and —0.2, respectively. These correlation
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R. R. EPLEY AND S. L. BALLOUN
terol levels were elevated and weight gains were decreased by feeding coconut oil. Lofland (1965) reported that feeding coconut oil as 10% of the diet was not atherogenic. Results of our experiment agree with those of Leveille et al. (1960) and Lofland (1965). Cockerels fed 10% coconut oil gained less weight and had higher plasma cholesterol ester and total cholesterol levels than cockerels fed diets containing 10% soy oil. The presence of soy oil decreased these cholesterol components more than coconut oil increased them. Cockerels fed coconut oil or soy oil had equally severe atherosclerosis and similar free cholesterol and glyceride concentrations in the plasma. Coconut oil fed as 10% of the diet did not appear to be atherogenic, although it did alter levels of plasma lipid components. When cockerels were fed coconut oil, there was a significant correlation between (al + «2) ^-lipoprotein ratio and plasma cholesterol ester level. This indicated an increased concentration of cholesterol ester carried by the al- and /3-lipoproteins. Orma (1957) noted an increased concentration of cholesterol in the a-lipoprotein band which had been separated from the ^-lipoprotein band by paper electrophoresis. Leveille et al. (1960) noted that the amount of lipid bound to /^-lipoprotein was significantly increased by feeding cholesterol. SUMMARY The effect of feeding cholestyramine on feed consumption and weight gains was negligible. Apparent ether-extract digestibility was decreased by feeding cholestyramine. This effect was consistent regardless of whether the diet contained added cholesterol. Total plasma cholesterol and cholesterol ester concentrations were decreased by feeding cholestyramine when diets contained added crystalline cholesterol ( 1 % or 0.252%). Cholestyramine more effectively
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of cholestyramine. Van Itallie and Hashim (1963) reviewed bile acid-binding by cholestyramine and Senior (1964) reviewed the essentiality of bile acids for micelle formation and subsequent lipid absorption. Cholestyramine binds bile acids in the gut and decreases lipid absorption by interferring with micelle formation. The overall effect of feeding cholestyramine, therefore, is a decrease in apparent ether-extract digestibility. Pick et al. (1965) and Tennent et al. (1961) demonstrated that feeding cholestyramine to cockerels resulted in decreased plasma cholesterol concentration. Van Itallie and Hashim (1963) related the decreased absorption of bile acids caused by feeding cholestyramine to increased oxidation of cholesterol to bile acids. In this series of experiments total cholesterol and cholesterol ester levels were decreased by feeding cholestyramine except in the presence of dried whole egg. Since free plasma cholesterol concentration was not decreased by feeding cholestyramine, the effect of cholestyramine on total plasma cholesterol was caused by a decrease in cholesterol ester and not by decreased free cholesterol. Even though total cholesterol and cholesterol ester concentrations were lowered by feeding cholestyramine, the severity of atherosclerosis was not altered by feeding cholestyramine. Diets containing whole egg have been considered atherogenic (Weiss et al., 1966; Gosling et al., 1969; Fisher et al., 1963). Cockerels fed dried whole egg or cholesterol plus soy oil had equally severe atherosclerosis in our studies. The presence of whole egg lipid or soy oil with dietary cholesterol did not affect the atherogenic effect of dietary cholesterol. Leveille et al. (1960) compared plasma cholesterol levels and body weights of chicks fed diets containing either 10% coconut oil or 10% corn oil. Plasma choles-
DIET LIPIDS AND PLASMA LIPIDS
REFERENCES Ball, R. A., 1968. Veterinary Medical Research Institute, Ames, Iowa. Personal communication. Banerjee, S., P. N. Rao and S. K. Ghosh, 1965. Biochemical and histochemical changes in aorta of chicks fed vegetable oils and cholesterol. Proc. Soc. Exptl. Biol. Med. 119: 1081-1086. Christensen, S., 1964. Transfer of labelled cholesterol across the aortic intimal surface of normal and cholesterol-fed cockerels. J. Atherosclerosis Res. 4 : 151-160. Dayton, S., and S. Hashimoto, 1966. Movement of labeled cholesterol between plasma lipoprotein and normal arterial wall across the intimal surface. Circulation Res. 19: 1041-1049. Fisher, H., H. S. Weiss and P. Griminger, 1963. Effect of prolonged dietary treatment on atherosclerosis in the mature fowl. J. Atherosclerosis Res. 3 : 57-62. Galanos, D. S., G. A. M. Aivazis and V. M. Kapoulas, 1964. A simple method for the determination of serum glycerides, free cholesterol, and cholesterol esters using a binary solvent system. J. Lipid Res. 5: 242-244. Gebott, M. C , 1968. New, easier procedures for lipoprotein electrophoresis. Current Laboratory Practice 1 ( 1 ) : 25-28. The Gelman Instrument Company, 690 South Wagner Road, Ann Arbor, Michigan. Gosling, R. G., J. A. Hayes and W. SegreMackay, 1969. Induction of atheroma in cockerels as a model for studying alterations in blood flow. J. Atherosclerosis Res. 9: 47-56. Leveille, G. A., A. S. Feigenbaum and H. Fisher, 1960. The effect of dietary protein, fat and cholesterol on plasma cholesterol and serum protein components of the growing chick. Arch. Biochem. Biophys. 86: 67-70. Lofiand, H. B., Jr., 1965. Experimental atherosclerosis in pigeons. Comparative Atherosclerosis, Eds. J. C. Roberts and R. Strauss, Harper and Row. Maynard, L. A., and J. K. Loosli, 1962. Animal Nutrition, McGraw-Hill Book Company, Inc., N.Y. Orma, E. J. 1957. Effect of physical activity on atherogenesis. An experimental study in cockerels. Acta Physiol. Scand. 41, Suppl. 142: 1-75. Pick, R., S. Jain and L. N. Katz, 1965. Effect of cholestyramine and pectin in induction and regression of atherosclerosis in cholesterol-fed cockerels. Circulation, 32: Supl. 11—26. Pick, R., C. Kakita and P. Johnson. 1967. Relationship between plasma cholesterol level and
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reduced plasma cholesterol ester and total cholesterol when cockerels were fed cholesterol plus soy oil than when fed dried whole egg. When the diet contained 10% coconut oil or soy oil, total cholesterol and cholesterol ester levels were decreased by feeding cholestyramine, but the decrease was not significant in all experiments. When the diet contained no added fat or cholesterol, cholestyramine decreased serum cholesterol, but not significantly. Free plasma cholesterol level was not significantly affected by feeding cholestyramine. Plasma glyceride concentration was increased by feeding cholestyramine plus dried whole egg, but was unaffected by other dietary modifications. The effect of feeding cholestyramine on plasma lipoproteins and severity of atherosclerosis was negligible. Cockerels fed diets containing 10% dried whole egg gained slightly more weight, but consumed the same amount of feed as cockerels fed cholesterol plus soy oil. Cockerels fed diets containing dried whole egg had equally severe atherosclerosis as those fed crystalline cholesterol plus soy oil and had similar plasma free cholesterol levels. Feeding dried whole egg decreased plasma cholesterol ester and total cholesterol levels. The ratio of (al + «2)/^-lipoproteins was decreased by feeding crystalline cholesterol. Cockerels fed diets with 10% coconut oil consumed more feed and gained less weight than cockerels fed 10% soy oil, but the type of oil did not affect the severity of atherosclerosis and plasma free cholesterol and glyceride concentrations. Cockerels fed coconut oil had higher plasma cholesterol ester and total cholesterol levels than cockerels fed soy oil. Plasma al-lipoprotein was significantly decreased by feeding coconut oil plus cholestyramine and there was a consistent relationship between the (al + a2)/;8-lipoprotein ratio and cholesterol ester level.
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coronary atherosclerosis in cholesterol-fed cockerels. Fed. Proc. 26:490. Senior, J. R., 1964. Intestinal absorption fats. J. Lipid Res. 5: 495-521. Snedecor, G. W., 1956. Statistical Methods, The Iowa State University Press, Ames, Iowa. Technicon Instruments Corp., 1964. Total cholesterol. Technicon Auto Analyzer MethodologyMethod File N-24. Technicon Instruments Corporation, Chauncey, N.Y. Tennent, D. M., G. W. Kuron, M. E. Zanetti and W. H. Ott, 1961. Plasma cholesterol concentra-
tion in cockerels and dogs treated with bile acid binding polymer and cholesterol synthesis inhibitors. Proc. Soc. Exptl. Biol. Med. 108214-216. van Itallie, T. B., and S. A. Hashim, 1963. Clinical and experimental aspects of bile acid metabolism. Medical Clinics of North America, 47: 629-648. Weiss, H. S., F. D. Brown, P. Griminger and H. Fisher, 1966. Physical activity and atherosclerosis in the adult chicken. J. Atherosclerosis Res. 6: 407-414.
A. W. ADAMS AND M. E. JACKSON Department of Dairy and Poultry Science, Kansas State University, Manhattan, Kansas 66502 (Received for publication June 27, 1970)
T
HE trend toward housing egg-producing strains of chickens at high densities results from rising costs of buildings, equipment and labor. Cages are used more with birds housed at high densities. Logan (1965) reported if birds are to be housed at high densities cages should be used, while floor housing is advantageous for low densities. Effects of cage size, number of birds per cage, and bird density on performance of egg-producing birds have been widely studied (Shupe and Quisenberry, 1961; Lowe and Heywang, 1964; Moore et al., 1965; Cook and Dembnicki, 1966; Elmslie et al., 1966; Wilson et al., 1967; and Champion and Zindel, 1968). In general, egg production declined and mortality increased as bird population or bird density increased. Several researchers found that responses of layer strains differ significantly with various cage-size and bird-density environ1 Contribution No. 766, Department of Dairy and Poultry Science, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas 66502.
ments (Gowe, 1956; Francis, 1957a; Cook and Dembnicki, 1966; Elmslie et al., 1966; Wilson et al., 1967; and Craig, 1969). Lowe and Heywang (1964) and Wilson et al. (1967) have reported cannibalism as a major cause of increased mortality from housing layers in multiple-bird cages or at high densities. However, several studies suggest increased mortality may be caused by general stress that results from high bird densities and flock sizes common to multiple-bird cage systems. Siegel (1960) produced characteristics of adaptation normally associated with physiological stress by subjecting pullets and cockerels to high population densities in floor pens. Incidences of cage-layer fatigue differed significantly among seven strains of White Leghorns (Francis, 1957b). Elmslie et al. (1966) reported that a hysterical and featherless condition developed among birds housed 12-14 per 41 X 123 cm. cage, but not among birds housed 3 per 41 X 41 cm. cage. Reported here are effects of cage size and bird density on performance of six commercial strains of layers.
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Effect of Cage Size and Bird Density on Performance of Six Commercial Strains of Layers1
A
1
Journal paper No. J-6613 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 1720.
and thereby decreases their reabsorption from the intestine. This decreased recirculation stimulates cholesterol oxidation to bile acids to replace acids lost by excretion. Our series of experiments was initiated to study several effects of feeding cholestyramine, cholesterol, coconut oil, dried whole egg and soy oil on plasma free cholesterol, cholesterol esters, glycerides, lipoproteins and severity of atherosclerosis in cockerels. MATERIALS AND METHODS Blood samples were obtained from a pricked brachial vein after cockerels were fasted 12 hr. In Experiment 1, blood was allowed to clot at room temperature for 24 hr. to express serum. In Experiments 2 and 3, blood was collected in oxalated test tubes. Serum and plasma were stored in glass vials at — 17.8°C. Aortas were stored in formalin solution (200 ml. formaldehyde, 37.5%, 1800 ml. distilled water, 80 gm.NaH 2 P0 4 and 130 gm.NaHPO*), (Ball, 1968). Aortas were opened with a scalpel longitudinally on the dorsal side and scrutinized by one judge and scored either as 1, having one or more plaques, or 0, having no plaques. Apparent ether-extract digestibility was determined by using chromic oxide as a marker and by using the apparent digestibility equation described by Maynard and Loosli (1962). Serum total cholesterol was assayed with methodology described by Technicon Instruments Corp. (1964) using a Technicon Autoanalyzer.
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THEROGENESIS and plasma lipids • levels may be affected by dietary constituents. Christensen (1964) and Banerjee et al. (1965) reported increased uptake of cholesterol by the aorta of cockerels fed cholesterol. Pick et al. (1967) reported a positive relationship between the degree of hypercholesterolemia and coronary atherosclerosis in cholesterol-fed cockerels. Dayton and Hashimoto (1966) demonstrated that lipoprotein is necessary in the surrounding medium if labeled cholesterol is to be transported into the intima. Orma (1957) and Leveille et al. (1960) separated lipoproteins by paper electrophoresis and noted, in the hypercholesterolemic condition, an increased cholesterol content of the a-lipoprotein and increased lipid content of the ^-lipoprotein. Leveille et al. (1960) reported that chicks fed a diet containing 10% coconut oil had higher plasma cholesterol levels than those receiving a similar diet supplemented with corn oil. Gosling et al. (1969) and Fisher et al. (1963) reported increased abdominal atherosclerosis when cockerels were fed diets containing whole, dried eggs. Tennent et al. (1961) demonstrated that feeding cholestyramine, an anion-exchange resin, to cockerels with a normal plasma cholesterol level results in a decreased plasma cholesterol concentration. Van Itallie and Hashim (1963) postulated that cholestyramine binds bile acids in the gut
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R. R. EPLEY AND S. L. BALLOUN
methionine 0.075% and Santoquin 0.0125%. Cholesterol and cholestyramine were substituted for yellow corn on an equal weight basis. When the cockerels were 10 weeks old, dietary protein was reduced from 22% to 15% by increased corn and decreased sybean meal. Experiment 2 was designed to study several effects of feeding dried whole egg or crystalline cholesterol, plus soy oil with and without 1% cholestyramine. Diets were formulated to be isocaloric and isonitrogenous and to provide the same concentrations of fat and cholesterol. Nine Hubbard broiler cockerels (2 months oil) were allotted at random to each of 4 dietary treatments arranged in a 2 X 2 factorial. During the 4-month experimental period, cockerels were housed in 30 X 50 X 40 cm. cages in a 22°C. environmental chamber. The composition of diets fed in Experiment 2 are listed in Table 1. Samples of initial and final blood plasma were obtained and aortas were removed from all cockerels at the end of the experiment. Experiment 3 was designed to study several effects of feeding 10% coconut oil or soy oil with and without 1% cholestyramine. Thirty-six Hubbard broiler cockerels (2.5 months of age) were used in Experiment 3. Otherwise, the experimental design and procedure used for Experiment 2 and 3 were identical. The composition of diets fed in Experiment 3 are listed in Table 1. RESULTS Experiment 1. Neither weight gains nor feed consumption of chicks were significantly affected by feeding cholestyramine or cholesterol. Apparent ether-extract digestibility was significantly decreased by feeding cholestyramine but was not affected by feeding cholesterol (Table 2). When cockerels were fed diets containing 1% added cholesterol, adding cholestyramine to the diet reduced serum cholesterol
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Plasma glycerides, free cholesterol and cholesterol esters were assayed by extracting plasma with chloroform-methanol (2: 1). The ratio of solvent to plasma was 20: 1. This extract was then analyzed according to Galanos et al. (1964). Total cholesterol was calculated by summing free and esterified cholesterol. Percentages of al, al and ^-lipoproteins were determined according to Gebott (1968). This technique uses cellulose polyacetate strips and the Schiff's reagent staining procedure that stains the carboncarbon double bonds present in each lipoprotein. Experiment 1 was designed to study several effects of feeding cholesterol with or without 1% dietary cholestyramine. Hubbard broiler males were randomly allotted at one day of age to each of 8 pens with 12 chicks per pen. Four of the pens of chicks received the basal diet and four were fed the basal diet plus 1% cholesterol for 45 days. After 45 days, diets with 1% cholestyramine were fed for 17 days to 2 groups that received the basal diet and to 2 groups that received 1% cholesterol during the first 45-day period. Serum samples were collected on day 45, before feeding cholestyramine, and again when the experiment ended. The apparent ether-extract digestibility was determined during the last week of the experiment. Diets fed in Experiment 1 contained the following ingredients, expressed as percentages: yellow corn 51, soybean meal (48% protein) 28, soy oil 10, fish meal (70% protein) 5, dehydrated alfalfa meal 2, dicalcium phosphate 1, oyster shell 0.7, salt 0.3 and premix concentrate 1. The premix concentrate provided per kilogram of diet: riboflavin 5 mg., niacin 25 mg., calcium panthothenate 10 mg., choline 450 mg., vitamin B12 10 meg., vitamin K. 1 mg., vitamin E 10 I.U., vitamin A 7000 I.U., vitamin D 1000 I.C.U., penicillin 6.6. mg., streptomycin 33 mg. and also
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DIET LIPIDS AND PLASMA LIPIDS TABLE 1.—Diets fed in experiments Z and 3 Experiment 2
Experiment 3
Ingredients 1
2
3
4
1
2
3
4
75.4 10.6
74.4 10.6
70.17 21.34
69.17 21.34
69.5 12.0
69.5 12.0
68.5 12.0
68.5 12.0
—
—
—
—
5.0
5.0
5.0
5.0
1.4 1.6 0.5 0.5
1.4 1.6 0.5 0.5
1.52 1.50 0.5 0.50
1.52 1.50 0.5 0.50
1.0 1.5 0.5 0.5
—
—
1.0 1.5 0.5 0.5 10.0
1.0 1.5 0.5 0.5
— — —
1.0 1.5 0.5 0.5 10.0
— — — —
10.0
—
10.0 1.0
10.0
—
100.0
— 1.0 10.0 — 100.0
4.22 — — 0.253 100.003
—
4.22 1.00
—
0.253 100.003
100.0
—
—
1.0
— — —
— —
100.0
— —
100.0
100.0
a Mineralized salt mixture provided (per kilogram): 4.49 gm. NaCl, 60 mg. Mn, 75 mg. Zn, 55 mg. Fe, 9 mg. Cu, 1.1 mg. I 2 , and 0.55 mg. Co. b Vitamin concentrate provided (per kilogram): 7500 I.U. vitamin A, 1000 I.TJ. vitamin D3, 10 I.U. vitamin E, 1 mg. menadione sodium bisulfite, 10 meg. vitamin B12, 5 mg. riboflavin, 450 mg. choline, 10 mg. pantothenic acid, 25 mg. niacin, 1000 mg. methionine equivalent, 100 mg. Ethoxyquin, 8.25 mg. penicillin and 41 mg. streptomycin.
concentration significantly (P < 0.01). When diets contained no added cholesterol, serum cholesterol levels were reduced by feeding cholestyramine, but the reduction was not statistically significant. Chicks fed cholesterol but no cholestyramine had significantly higher serum cholesterol levels than chicks fed any of the other three diets (Table 2). Experiment 2. Cockerels fed diets containing 10% dried whole egg gained slightly more weight (1.8 ± 0.1 kg.) than cockerels fed diets with cholesterol and soy oil (1.6 ± 0.1 kg.). Mean weight gain was 1.7 kg. in the groups fed no cholestyramine and the same in groups fed diets containing 1% cholestyramine. Feed consumption was not affected by treatments. None of the treatments significantly affected the presence of plaques in the aorta. The significant main effects of feeding cholestyramine were elevated plasma glyceride and lowered cholesterol ester and total cholesterol concentrations (Table 3). Free plasma cholesterol level and the al + a2)/^-lipoprotein
ratio were not significantly affected by feeding cholestyramine (Table 3). Plasma glycerides were increased by feeding dried whole egg plus cholestyramine and unchanged by feeding dried whole eggs plus no cholestyramine (Table 4). The significant main effects of feeding dried whole egg were lowered plasma cholesterol ester and total cholesterol levels compared with those of cockerels fed cholesterol plus soy oil. TABLE 2.—Percentage apparent ether-extract digestibility and mean change of serum cholesterol levels in experiment P-
Dietary treatment No cholesterol+ cholestyramine Cholesterol-|cholestyramine Cholesterol+ no cholestyramine No cholesterol-rno cholestyramine
Percentage digestibility
Serum cholesterol
(%)
(mg./lOOml.)
70.5 + 1.8*
-
77.4+1.8°
-342b
85.9 + 1.8b 90.0+1.8b
4f>
462° -
14.0"
1 Means followed by the same superscript are not significantly different (P<0.05).
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Yellow corn Soybean meal (50% protein) Fish meal (70% protein) Dical. phosphate Calcium carbonate Mineralized salt" Vitamin etc. b Coconut oil Soy oil Cholestyramine Dried whole egg Cholesterol Total
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R. R. EPLEY AND S. L. BALLOUN TABLE 3.—Mean net changes of plasma lipids in experiment 2, main effects (mg./lOO ml.) Diet Variables
Glycerides Cholesterol ester Free cholesterol Total cholesterol Lipoprotein ratio 2
Whole egg
Cholesterol+soy oil
Csm3
No Csms
+ 3 2 . 8 + 14.6 1 - 2 9 . 4 + 8.4 b - 8 . 5 + 5.2 -35.0±10.9d -0.30±0.34d
+ 3 2 . 2 + 14.6 - 1 2 . 4 + 8.4 b -5.4±5.2 - 1 7 . 7 + 10.9 d - 1 . 0 0 + 0.34"
+ 4 5 . 0 + 14.6° - 3 2 . 7 + 8.4" - 5 . 3 + 5.2 - 3 8 . 0 + 10.9 b - 0 . 6 4 + 0.34
+ 2 0 . 0 + 14.6° -18.3+8.4" - 5 . 6 + 5.2 - 1 4 . 7 + 10.9 b - 0 . 6 0 + 0.34
1 Means with the same superscripts are significantly different. Superscripts a, b, c, and d indicate significant differences at the 1, 5, 10 and 25% level of probability, respectively, here and in Table 4. 2 Mean changes of the (al+a2)/(3-lipoprotein ratio. 3 Csm is an abbreviation for cholestyramine in this and following tables.
TABLE 4.—Mean net changes of plasma lipids in experiment 2 (mg./lOO ml.) Whole dried egg Lipid component Glycerides Cholesterol ester' Free cholesterol Total cholesterol1 1 Lipoprotein ration
Csm 64.9 -25.0 - 2.4 -24.5 - 0.16
i Interaction was significant.
No Csm 0.8 -33.9 - 8.6 -42.5 - 0.45
Cholesterol+Soy oil Csm
No Csm
25.0 39.3 -40.4 15.6 - 8.2 - 2.5 -48.5 13.1 - 1.13 - 0.87
cients between the decrease in (al +
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Cockerels fed dried whole egg and no cholestyramine had lower plasma cholesterol ester and total cholesterol levels than those fed dried whole egg plus cholestyramine (Table 4). Cholestyramine more effectively reduced plasma cholesterol ester and total cholesterol when cockerels were fed cholesterol plus soy oil than when fed dried whole egg. Plasma cholesterol ester and total cholesterol were decreased by feeding dried whole egg with no cholestyramine, but increased by feeding cholesterol and soy oil with no cholestyramine (Table 4). The (al + «2)/^-lipoprotein ratio decreased when cockerels were fed cholesterol plus soy oil. This decreased ratio was associated with an increased number of carboncarbon double bonds in the al-lipoprotein in plasma of cockerels fed diets containing whole dried egg and a concomitant increase in the number of double bonds present in ^-lipoprotein of cockerels fed diets with cholesterol plus soy oil. Correlation coeffi-
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DIET LIPIDS AND PLASMA LIPIDS TABXE 5.—Mean net changes of plasma lipids in experiment 3, main effects (mg./lOO ml.) Diet variables Lipid components Coconut oil Glycerides Cholesterol esters Free cholesterol Total cholesterol Lipoprotein 1
+ 12.6 + -5.4 + -1.0 + -4.8 + -8.8 +
12.7 7.9" 2.8 9.5" 2.3 b
Soy oil
Csm +3.0 + -28.1 + -2.0 + -30.4 + -7.4 +
-3.6+12.7 - 4 0 . 0 + 7.9" - 4 . 0 + 2.8 -44.1+9.5" - 2 . 8 + 2.3 b
No Csm + 6 . 0 + 12.7 - 1 7 . 3 + 7.9 — 1.2 + 2.8 - 1 8 . 5 + 9.5 -4.1±2.3d
12.7 7.9 2.8 9.5 2.3 d
• Mean change of percent al-lipoprotein.
coefficients are not significant (Snedecor, 1956), but they can aid in the interpretation of the correlation between (al + a 2 ) / /3-lipoprotein ratio and cholesterol ester levels. The decrease in (al + a2)//3-lipoprotein ratio was due to decreased a2-lipoprotein relative to increased al- and /3-lipoproteins. These relative changes occurred when plasma cholesterol ester levels decreased. Conversely, as the cholesterol ester level increased, there was an increased number of double bonds in the a2-lipoprotein and a decreased number of double bonds in the al- and /3-lipoprotein. This decrease in double bonds indicates an increased amount of cholesterol ester carried by the al- and ^-lipoproteins. DISCUSSION Apparent ether-extract digestibility was decreased by feeding diets containing cholestyramine with or without added dietary cholesterol. The decreased digestibility may be related to the bile acid-binding property TABLE
6.-
-Mean net changes of plasma lipids in experiment 3 (mg./lOO ml.) Coconut oil
Soy oil
Lipid component Csm Glycerides Cholesterol esters Free cholesterol Total cholesterol Lipoprotein a l 1 1
17.9 -13.1 1.2 -12.5 -13.5
Interaction was significant.
N o Csm
-
7.9 2.4 0.7 3.0 4.1
Csm
N o Csm
-11.3 -43.1 - 5.1 -48.2 - 1.4
4.0 -37.0 - 3.0 -30.0 - 4.1
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cockerels fed coconut oil compared with plasma from cockerels fed soy oil. Plasma cholesterol ester and total cholesterol, however, were significantly decreased by feeding soy oil. The al-lipoprotein was decreased by feeding coconut oil plus cholestyramine (Table 6). This decrease was associated with increased plasma glycerides. The increased plasma glycerides should have contained saturated fatty acids from coconut oil. Saturated fatty acids in lipoproteins from coconut oil would not be detected with the Schiff's stain. From this relationship, we conclude that al-lipoprotein was carrying more saturated fatty acids when coconut oil plus cholestyramine was added to the diet. Correlation coefficients between the decrease in (al + a2)//3-lipoprotein ratio and decreased free cholesterol, cholesterol ester and glycerides were 0.0, 0.4 and —0.2, respectively. Since the correlation coefficients of the lipoprotein ratio to cholesterol and glycerides are not significant (Snedecor, 1956), there is no reason to believe that a relationship exists between the ratio and these plasma lipid components. However, the correlation coefficient between the decreased (al + «2)//3-lipoprotein ratio and decrease in plasma cholesterol ester is significant. Correlation coefficients between decreased cholesterol ester and decreased al-, al- and /3-lipoprotein were —0.2, 0.3 and —0.2, respectively. These correlation
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R. R. EPLEY AND S. L. BALLOUN
terol levels were elevated and weight gains were decreased by feeding coconut oil. Lofland (1965) reported that feeding coconut oil as 10% of the diet was not atherogenic. Results of our experiment agree with those of Leveille et al. (1960) and Lofland (1965). Cockerels fed 10% coconut oil gained less weight and had higher plasma cholesterol ester and total cholesterol levels than cockerels fed diets containing 10% soy oil. The presence of soy oil decreased these cholesterol components more than coconut oil increased them. Cockerels fed coconut oil or soy oil had equally severe atherosclerosis and similar free cholesterol and glyceride concentrations in the plasma. Coconut oil fed as 10% of the diet did not appear to be atherogenic, although it did alter levels of plasma lipid components. When cockerels were fed coconut oil, there was a significant correlation between (al + «2) ^-lipoprotein ratio and plasma cholesterol ester level. This indicated an increased concentration of cholesterol ester carried by the al- and /3-lipoproteins. Orma (1957) noted an increased concentration of cholesterol in the a-lipoprotein band which had been separated from the ^-lipoprotein band by paper electrophoresis. Leveille et al. (1960) noted that the amount of lipid bound to /^-lipoprotein was significantly increased by feeding cholesterol. SUMMARY The effect of feeding cholestyramine on feed consumption and weight gains was negligible. Apparent ether-extract digestibility was decreased by feeding cholestyramine. This effect was consistent regardless of whether the diet contained added cholesterol. Total plasma cholesterol and cholesterol ester concentrations were decreased by feeding cholestyramine when diets contained added crystalline cholesterol ( 1 % or 0.252%). Cholestyramine more effectively
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of cholestyramine. Van Itallie and Hashim (1963) reviewed bile acid-binding by cholestyramine and Senior (1964) reviewed the essentiality of bile acids for micelle formation and subsequent lipid absorption. Cholestyramine binds bile acids in the gut and decreases lipid absorption by interferring with micelle formation. The overall effect of feeding cholestyramine, therefore, is a decrease in apparent ether-extract digestibility. Pick et al. (1965) and Tennent et al. (1961) demonstrated that feeding cholestyramine to cockerels resulted in decreased plasma cholesterol concentration. Van Itallie and Hashim (1963) related the decreased absorption of bile acids caused by feeding cholestyramine to increased oxidation of cholesterol to bile acids. In this series of experiments total cholesterol and cholesterol ester levels were decreased by feeding cholestyramine except in the presence of dried whole egg. Since free plasma cholesterol concentration was not decreased by feeding cholestyramine, the effect of cholestyramine on total plasma cholesterol was caused by a decrease in cholesterol ester and not by decreased free cholesterol. Even though total cholesterol and cholesterol ester concentrations were lowered by feeding cholestyramine, the severity of atherosclerosis was not altered by feeding cholestyramine. Diets containing whole egg have been considered atherogenic (Weiss et al., 1966; Gosling et al., 1969; Fisher et al., 1963). Cockerels fed dried whole egg or cholesterol plus soy oil had equally severe atherosclerosis in our studies. The presence of whole egg lipid or soy oil with dietary cholesterol did not affect the atherogenic effect of dietary cholesterol. Leveille et al. (1960) compared plasma cholesterol levels and body weights of chicks fed diets containing either 10% coconut oil or 10% corn oil. Plasma choles-
DIET LIPIDS AND PLASMA LIPIDS
REFERENCES Ball, R. A., 1968. Veterinary Medical Research Institute, Ames, Iowa. Personal communication. Banerjee, S., P. N. Rao and S. K. Ghosh, 1965. Biochemical and histochemical changes in aorta of chicks fed vegetable oils and cholesterol. Proc. Soc. Exptl. Biol. Med. 119: 1081-1086. Christensen, S., 1964. Transfer of labelled cholesterol across the aortic intimal surface of normal and cholesterol-fed cockerels. J. Atherosclerosis Res. 4 : 151-160. Dayton, S., and S. Hashimoto, 1966. Movement of labeled cholesterol between plasma lipoprotein and normal arterial wall across the intimal surface. Circulation Res. 19: 1041-1049. Fisher, H., H. S. Weiss and P. Griminger, 1963. Effect of prolonged dietary treatment on atherosclerosis in the mature fowl. J. Atherosclerosis Res. 3 : 57-62. Galanos, D. S., G. A. M. Aivazis and V. M. Kapoulas, 1964. A simple method for the determination of serum glycerides, free cholesterol, and cholesterol esters using a binary solvent system. J. Lipid Res. 5: 242-244. Gebott, M. C , 1968. New, easier procedures for lipoprotein electrophoresis. Current Laboratory Practice 1 ( 1 ) : 25-28. The Gelman Instrument Company, 690 South Wagner Road, Ann Arbor, Michigan. Gosling, R. G., J. A. Hayes and W. SegreMackay, 1969. Induction of atheroma in cockerels as a model for studying alterations in blood flow. J. Atherosclerosis Res. 9: 47-56. Leveille, G. A., A. S. Feigenbaum and H. Fisher, 1960. The effect of dietary protein, fat and cholesterol on plasma cholesterol and serum protein components of the growing chick. Arch. Biochem. Biophys. 86: 67-70. Lofiand, H. B., Jr., 1965. Experimental atherosclerosis in pigeons. Comparative Atherosclerosis, Eds. J. C. Roberts and R. Strauss, Harper and Row. Maynard, L. A., and J. K. Loosli, 1962. Animal Nutrition, McGraw-Hill Book Company, Inc., N.Y. Orma, E. J. 1957. Effect of physical activity on atherogenesis. An experimental study in cockerels. Acta Physiol. Scand. 41, Suppl. 142: 1-75. Pick, R., S. Jain and L. N. Katz, 1965. Effect of cholestyramine and pectin in induction and regression of atherosclerosis in cholesterol-fed cockerels. Circulation, 32: Supl. 11—26. Pick, R., C. Kakita and P. Johnson. 1967. Relationship between plasma cholesterol level and
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reduced plasma cholesterol ester and total cholesterol when cockerels were fed cholesterol plus soy oil than when fed dried whole egg. When the diet contained 10% coconut oil or soy oil, total cholesterol and cholesterol ester levels were decreased by feeding cholestyramine, but the decrease was not significant in all experiments. When the diet contained no added fat or cholesterol, cholestyramine decreased serum cholesterol, but not significantly. Free plasma cholesterol level was not significantly affected by feeding cholestyramine. Plasma glyceride concentration was increased by feeding cholestyramine plus dried whole egg, but was unaffected by other dietary modifications. The effect of feeding cholestyramine on plasma lipoproteins and severity of atherosclerosis was negligible. Cockerels fed diets containing 10% dried whole egg gained slightly more weight, but consumed the same amount of feed as cockerels fed cholesterol plus soy oil. Cockerels fed diets containing dried whole egg had equally severe atherosclerosis as those fed crystalline cholesterol plus soy oil and had similar plasma free cholesterol levels. Feeding dried whole egg decreased plasma cholesterol ester and total cholesterol levels. The ratio of (al + «2)/^-lipoproteins was decreased by feeding crystalline cholesterol. Cockerels fed diets with 10% coconut oil consumed more feed and gained less weight than cockerels fed 10% soy oil, but the type of oil did not affect the severity of atherosclerosis and plasma free cholesterol and glyceride concentrations. Cockerels fed coconut oil had higher plasma cholesterol ester and total cholesterol levels than cockerels fed soy oil. Plasma al-lipoprotein was significantly decreased by feeding coconut oil plus cholestyramine and there was a consistent relationship between the (al + a2)/;8-lipoprotein ratio and cholesterol ester level.
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R. R. EPLEY AND S. L. BALLOUN
coronary atherosclerosis in cholesterol-fed cockerels. Fed. Proc. 26:490. Senior, J. R., 1964. Intestinal absorption fats. J. Lipid Res. 5: 495-521. Snedecor, G. W., 1956. Statistical Methods, The Iowa State University Press, Ames, Iowa. Technicon Instruments Corp., 1964. Total cholesterol. Technicon Auto Analyzer MethodologyMethod File N-24. Technicon Instruments Corporation, Chauncey, N.Y. Tennent, D. M., G. W. Kuron, M. E. Zanetti and W. H. Ott, 1961. Plasma cholesterol concentra-
tion in cockerels and dogs treated with bile acid binding polymer and cholesterol synthesis inhibitors. Proc. Soc. Exptl. Biol. Med. 108214-216. van Itallie, T. B., and S. A. Hashim, 1963. Clinical and experimental aspects of bile acid metabolism. Medical Clinics of North America, 47: 629-648. Weiss, H. S., F. D. Brown, P. Griminger and H. Fisher, 1966. Physical activity and atherosclerosis in the adult chicken. J. Atherosclerosis Res. 6: 407-414.
A. W. ADAMS AND M. E. JACKSON Department of Dairy and Poultry Science, Kansas State University, Manhattan, Kansas 66502 (Received for publication June 27, 1970)
T
HE trend toward housing egg-producing strains of chickens at high densities results from rising costs of buildings, equipment and labor. Cages are used more with birds housed at high densities. Logan (1965) reported if birds are to be housed at high densities cages should be used, while floor housing is advantageous for low densities. Effects of cage size, number of birds per cage, and bird density on performance of egg-producing birds have been widely studied (Shupe and Quisenberry, 1961; Lowe and Heywang, 1964; Moore et al., 1965; Cook and Dembnicki, 1966; Elmslie et al., 1966; Wilson et al., 1967; and Champion and Zindel, 1968). In general, egg production declined and mortality increased as bird population or bird density increased. Several researchers found that responses of layer strains differ significantly with various cage-size and bird-density environ1 Contribution No. 766, Department of Dairy and Poultry Science, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas 66502.
ments (Gowe, 1956; Francis, 1957a; Cook and Dembnicki, 1966; Elmslie et al., 1966; Wilson et al., 1967; and Craig, 1969). Lowe and Heywang (1964) and Wilson et al. (1967) have reported cannibalism as a major cause of increased mortality from housing layers in multiple-bird cages or at high densities. However, several studies suggest increased mortality may be caused by general stress that results from high bird densities and flock sizes common to multiple-bird cage systems. Siegel (1960) produced characteristics of adaptation normally associated with physiological stress by subjecting pullets and cockerels to high population densities in floor pens. Incidences of cage-layer fatigue differed significantly among seven strains of White Leghorns (Francis, 1957b). Elmslie et al. (1966) reported that a hysterical and featherless condition developed among birds housed 12-14 per 41 X 123 cm. cage, but not among birds housed 3 per 41 X 41 cm. cage. Reported here are effects of cage size and bird density on performance of six commercial strains of layers.
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Effect of Cage Size and Bird Density on Performance of Six Commercial Strains of Layers1