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R. E. CLEGG AND S. K.
REFERENCES Block, R. J., E. L. Durrum and G. Zweig, 1955.
A Manual of Paper Chromatography and Paper Electrophoresis. Academic Press, Inc., New . York. Clegg, R. E., and R. E. Hein, 1953. P-32 distribution in the serum proteins of the chicken. Science, 117: 714-715. Levy, A. L., and D. Chung, 1953. Two dimensional chromatography of amino acids on buffered papers. Anal. Chem. 25: 396-399. Misra, U. K., 1959. Fractionation of high phosphorus containing proteins of chicken blood serum. Ph.D. thesis, Kansas State University. Nelson, G. F., and N. K. Freeman, 1959. Serum phospholipide analysis by chromatography and infrared spectrophotometry. J. Biol. Chem. 234: 1375-1380. Perlman, G. E., 1955. The nature of phosphorus linkages in phosphoproteins. Adv. in Prot. Chem. X. Academic Press, Inc., New York. Shore, B., and V. Shore, 1960. NH 2 -terminal amino acids of the serum lipoproteins of normal and hypercholesterolemic rabbits. J. Lipid Research, 1: 321-325.
Effect of Dietary Fat on the Fatty Acid Composition of Eggs and Tissues of the Hen L. J. MACHLIN AND R. S. GORDON Monsanto, Chemical Co., St. Louis, Missouri, AND
J. MARR AND C. W. POPE Ralston Purina Co., St. Louis, Missouri (Received for publication January 23, 1962)
T
HE proportion of polyunsaturated fatty acids (PUFA) in egg fat can be readily increased by increasing the level of PUFA in the diet (Cruickshank, 1934; Fisher and Leveille, 1957; Feigenbaum and Fisher, 1959; Wheeler et al., 1959; Murty and Reiser, 1961). Feigenbaum and Fisher (1959) reported that the egg fat was not influenced by dietary saturated fat whereas the body fat was influenced by ingestion of either unsaturated or saturated fat. In these studies of Feigenbaum and Fisher (1959) the alkaline isomerization method
was used. Since this method measures saturated fatty acids indirectly, it was impossible to detect any changes in the relative quantities of the saturated fatty acids such as might occur when short chain ( < C-14) fatty acids are fed. In the following study, gas chromatography was employed in order to determine more definitively whether egg yolk saturated fatty acids could be influenced by dietary saturated acids. In addition, the influence of the fatty acid composition of the diet on the fatty acid composition non-adipose tissue was studied.
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In our investigation, threonine was not evident as a NH2-terminal amino acid in the nondelipidized Compound-6. The appearance of threonine as NH2-terminal amino acid in the case of the delipidized Component-6 indicated that this amino may link the protein and the lipid of the blood serum. This is another indication that the hydroxyamino acids (threonine and serine) may serve as a link between the protein moiety and the lipid portion of lipo-proteins. Since Component-6 is found in the sera of laying hens as well as in the sera of diethylstilbestrol-treated cockerels, the threonine may be implicated in lipid transport during egg formation.
MALIK
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FATTY ACID COMPOSITION OF EGGS AND TISSUES
RESULTS AND DISCUSSION To our knowledge this is the first report of a study on the effect of diet on the fatty acid composition of non-adipose tissues in the hen. In general the pattern of change in the fatty acid composition of the liver and heart (Table 2) parallels those observed with the young, rapidly growing chicken (Machlin and Gordon, 1961) and reflects the composition of the fat consumed in the diets. However, in the chick it was observed that feeding of linoleic acid (18:2) 2 increased the proportion of arachidonic acid (20:4) in all non-adipose tissues, whereas in the hen 20:4 was not affected by feeding of a diet high or low in 18:2. There was also no increase in 20:3 3 when the hens were fed a diet devoid of essential fatty acids (18:2 and 20:4) for 12 weeks. This suggests that arachidonic acid is contained in lipids which have extremely slow turnover rates in contrast to shorter chain acids ( < C-20) which presumably are concentrated in lipids with appreciable turnover rates. The stability of tissue arachidonic acid and the absence of 20:3 from tissues of 1 These hens were fed a commercial laying ration (Purina Layena) until they were fed the experimental diets. 2 First number is the number of carbon atoms, the second is the number double bonds. 3 The presence of this acid is usually assumed to be an index of essential fatty acid deficiency.
TABLE 1.—Experimental diets Ingredients
Hen diet S-25
% Isolated soybean protein Safflower oil or hydrogenated coconut oil1 Na 2 Se0 3 -5H 2 0 Hen salt mixture 2 * Limestone Methionine hydroxy analogue 3 Cellulose Vitamin mixture 4 Choline chloride Vitamin A (10,000 I.U./g.) Vitamin D 3 (7,500 I.C.U./g.) Santoquin6 Vitamin E (20,000 I.U./lb.) Glucose (Cerelose)
25.00 15.00 0.0001 5.00 2.80 0.40 8.00 0.60 0.25 0.20 0.05 0.075 0.50 q.s. 100
1 Hydrol-Durkee Food Company, Chicago, Illinois. 2 This supplied as % of the diet; Ca 3 (P0 4 ) 2 , 2.8; K 2 HP0 4 , 1.0; MgSCv7H 2 0, 0.25; Fe(C 6 H 5 0 7 ) • 6H 2 0,0.14; ZnC0 3 ,0.015; KI, 0.004; CuSCv 5H 2 0, 0.002; H 3 B0 3 , 0.009; CoS0 4 -7H 2 0, 0.0001; MnS0 4 , 0.065; NaCl, 0.70. 3 Registered trademark of the Monsanto Chemical Company for calcium DL-2-hydroxy-4-methylthiobutyrate. 4 This will supply in mg./kilo of finished diet: vitamin Bi 2 ,0.03; biotin 0.30; menadione, 1.0; pyridoxine HC1 8.0; folic acid, 4.0; riboflavin, 16.0; calcium pantothenate, 20.0; thiamine HC1, 24.0; nicotinic acid, 100.0. This mixture, Alphacel, and salts "A" can all be obtained from the Nutritional Biochemicals Corp., Cleveland, Ohio. 6 Registered trademark of the Monsanto Chemical Company for 6-ethoxy, 1,2-dihydro, 2,2,4-trimethylquinoline (an antioxidant).
hens fed EFA deficient diets also suggests that these hens did not develop an EFA deficiency. Reiser (1950) and Reiser et al. (1951) reported that EFA are not required by the laying hen for maintenance of egg production although the hen cannot synthesize linoleic acid from acetate (Murty et al:, 1960). However, as shown in Table 3, the depot fat of hens fed linoleic-free diets for 12 weeks still contained a large reserve of linoleic acid and appreciable quantities of 20:4 and 18:2 were present in eggs from these same hens. Therefore, it may take many months before the EFA content of eggs is depleted sufficiently to affect egg production or hatchability.
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EXPERIMENTAL White Leghorn hens in their second year of egg production1 were fed the experimental diets given in Table 1 for 12 weeks. The hens were then fasted overnight, sacrificed and the abdominal fat, liver, heart and oviduct removed for analysis. All tissues were kept at — 15°C. until analyzed. Fatty acid analyses were conducted using procedures previously described (Marco et al., 1961).
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L. J. MACHLIN, R. S. GORDON, J. MARR AND C. W. POPE TABLE 2.—Fatty acid analysis of dietary fat, heart, and liver of adult hens1 after 12 weeks on experimental diets Dietary • fats
Fatty acid2
HCO (Hydrogenated coconut oil)
%
SAF (SafHower oil)
Dietary fat source SAF (3)4
HCO (4)
SAF (3)
HCO (3)
%
%
%
%
0.2 + 0.2 0.1+0.1 0.0 0.9 + 0.1 0.0 1.4 + 0.2 16.1 + 1.9 2.5 + 0.8 0.0 16.3 + 2.9 19.8 + 2.9 31.0 + 6.0 0.0 0.0 10.3±3.2
0.6 + 0.5 14.9 + 4.8** 0.0 4.4+1.7* 0.0 1.2 + 1.4 13.8 + 3.1 2.3 + 0.9 0.0 18.8 + 4.6 20.0 + 4.9 14.6 + 3.0** 0.0 0.0 9.2 + 3.2
0.0 0.3+0.3 0.0 0.7+0.6 0.0 0.2 + 0.03 20.0 + 1.5 1.6 + 0.8 0.0 16.4 + 8.4 24.0 + 2.6 30.1+8.5 0.0 0.0 5.4 + 2.9
6
. %
8.4 51.8 0.0 19.9 0.0 0.0 9.7 0.0 0.0 8.3 1.9 0.0 0.0
0.1 0.1 0.0 0.4 0.0 0.0 14.4 0.0 0.0 1.1 11.6 71.7 0.8
0.0
0.0
3
1.7 + 1.4**6 1.4 + 0.2** 0.0 3.1+0.4** 0.6±0.1 23.8 + 1.7* 3.0 + 0.8 0.2±0.2 15.8 + 0.8 32.7 + 2.6** 13.3 + 0.9** 0.4 + 0.4 0.0 5.3±0.9
1
All hens were fed Purina layena until beginning of experiment. Carbon chain length: number of double bonds. 15% of the fat was added to an otherwise "fat-free" diet. 4 Number of replicates. 5 Expressed as percent by weight of total fatty acids + standard deviation. 6 Two asterisks indicate difference between treatments is significant at the .01 probability level, one asterisk at the .05 probability level. 2 s
TABLE 3.—Fatly acid analysis of egg yolk and depot fat from adult hens after 12 weeks on experimental diets Depot fat
Egg yolk Dietary fat source Fatty Acid
SAF 1 3 (6)
4
% 10 or less 12:0 12:1 14:0 14:1 16:0 16:1 17:0 or 16:2 18:0 18:1 18:2 18:3 20:1-20:3 20:4 1 2 3 4
1.7 0.3+0.2 0.2 + 0.2 0.5 + 0.4 0.0 18.8 + 1.7 0.6 + 1.3 1.1 + 1.0 10.0 + 1.7 25.6 + 1.7 37.1 + 1.9 0.3 + 0.3 0.5 + 0.4 2.5 + 0.5
HCO (5)
2
% 4.9 1.8 + 0.5** 0.0 8.6 + 2.0** 3.4 + 0.4** 19.9 + 3.3 8.0 + 0.8** 0.0 8.2+0.2 36.1+4.6** 6.9 + 0.3** 0.7 + 0.1 0.0 1.6 + 0.7*
SAF . (4)
% 2.0 0.2 + 0.2 0.3 + 0.2 0.5 + 0.1 0.0 12.8 + 1.9 2.6 + 0.5 0.7 + 0.1 3.8 + 1.4 30.4 + 5.1 45.0 + 4.7 1.0 + 0.5 0.0 0.0
Diet contains 15% safflower oil. Diet contains 15% hydrogenated coconut oil. Number of replicates. Expressed as percent by weight of total fatty acids + standard deviation.
HCO (4)
% 3.3 28.4 + 5.6** 0.0 13.6 + 0.8** 1.6 + 0.5* 12.5 + 1.4 3.3 + 1.0 0.0 6.4 + 0.9** 22.7 + 2.6* 6.9 + 1.5** 1.0 + 0.6 0.0 0.0
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10:0 or less 12:0 12:1 14:0 14:1 15:0 or 14:2 16:0 16:1 17:0 or 16:2 18:0 18:1 18:2 18:3 20:1-20:3 20:4
Liver
Heart
FATTY ACID COMPOSITION OF EGGS AND TISSUES SUMMARY
REFERENCES Cruickshank, E. M., 1934. Studies in fat metabolism in the fowl. 1. The composition of the
egg fat and depot fat of the fowl as affected by ingestion of large amounts of different fats. Biochem. J. 28: 967-977. Feigenbaum, A. S., and H. Fisher, 1959. The influence of dietary fat on the incorporation of fatty acids into body and egg fat of the hen. Arch. Biochem. Biophys. 79: 302-306. Fisher, H., and G. A. Leveille, 1957. Observations on the cholesterol, linoleic and linolenic acid content of eggs as infiunced by dietary fats. J. Nutrition, 63: 119-124. Machlin, L. J., and R. S. Gordon, 1961. Effect of dietary fatty acids and cholesterol on growth and fatty acid composition of the chicken. J. Nutrition, 75: 157-164. Marco, G. J., L. J. Machlin, E. Emery and R. S. Gordon, 1961. Dietary effects of fats upon fatty acid composition of the mitochondria. Arch. Biochem. Biophys. 94: 115-120. Murty, N. L., M. C. Williams and R. Reiser, 1960. Non-synthesis of linoleic acid from acetate-l-C' 4 by the laying hen. J. Nutrition, 72: 451-454. Murty, N. L., and R. Reiser, 1961. Influence of graded levels of dietary linoleic and linolenic acids on the fatty acid composition of hen's eggs. J. Nutrition, 75: 261-294. Reiser, R., 1950. Fatty acid changes in egg yolk of hens on a fat-free and cottonseed oil ration. J. Nutrition, 40: 429-440. Reiser, R., B. Gibson, J. J. Carr and B. G. Lamp, 1951. The synthes:s and interconversions of polyunsaturated fatty acids by the laying hen. J. Nutrition, 44: 159-176. Wheeler, P., D. W. Peterson and G. D. Michaels, 1959. Fatty acid distribution in egg yolk as influenced by type and level of dietary fat. J. Nutrition, 69: 253-260.
NEWS AND NOTES (Continued from page 1317) ARIZONA NOTES Vicar, Oklahoma State University, representing the American Association of Land Grant Colleges and At the annual meeting of the Arizona Poultry Federation held in Tucson, a special plaque was State Universities; N. L. Jacobson, Iowa State University, representing the American Dairy Science presented to B. W. Heywang, Superintendent of the Southwest Poultry Experiment Station at GlenAssociation; T. J. Cunha, University of Florida, representing the American Society of Animal Prodale for his long service to the poultry industry. He is a joint employee of the U.S. Department of Agriduction; and W. M. Insko, Jr., University of Kentucky, representing the Poultry Science Association. culture and the University of Arizona. (Continued
on page 1347)
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One and one-half-year-old hens were fed purified diets containing either 15% hydrogenated coconut oil or 15% safflower oil as the sole fat source for 12 weeks. Hens fed a diet containing hydrogenated coconut oil layed eggs containing considerable quantities of lauric, myristic, and myristoleic acids and significantly less arachidonic acid than hens fed safflower oil. The depot fat, liver and heart of hens fed hydrogenated coconut oil contained significantly more lauric and myristic acid and significantly less linoleic than tissues from hens fed safflower oil. The proportion of arachidonic acid or C-20 triene in tissues was not influenced by diet. It was concluded that: 1. The fatty acid composition of nonadipose as well as adipose tissues and eggs will reflect the fatty acid composition of the diet. 2. Saturated as well as unsaturated dietary fats can influence egg and tissue fat. 3. Adult hens are not sensitive to deficiencies of essential fatty acids as a result of large reserves of linoleic acid in adipose tissue and a slow rate of depletion of arachidonic acid.
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