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Biochemical Changes in the Penicillin-Fed Chick1
Biochemical Changes in the Penicillin-Fed Chick 1 R. C. BURGESS, M. GLUCK AND D. H. LAUGHLAND
Department of Agriculture, Science Service, Ottawa, Canada (Received for publication September 24, 1952)
I
The above evidence suggests that dietary penicillin and possibly other antibiotics stimulate the intestinal absorption of several vitamins. This phenomenon may be associated with the increased growth of penicillin-fed chicks. The work presented in this paper is an extension of the study of the effect of 1 Contribution No. 229 of the Division of Chemistry, Science Service, Ottawa.
dietary antibiotics on biochemical constituents of the chick. EXPERIMENTAL
The dietary treatment, management and analytical techniques were similar in the two experiments reported herein. Day-old New Hampshire by Broad Breasted White chicks were randomized into two groups of forty-eight chicks each and housed in two compartments of an electrically-heated battery brooder with raised wire floors. One group received the high-energy basal ration shown in Table 1 and the other group received this ration supplemented with 30 milligrams procaine penicillin (Merck) per kilogram. Feed and water were provided ad libitum. The body weight and feed consumption were recorded semi-weekly but only the final weights and cumulative feed consumption are recorded in Table 2. All birds were starved twelve hours before killing. In the first experiment half the birds were killed at fifteen days of age and the remainder at thirty days of age. In the second experiment all birds were killed at thirty days of age. Birds receiving each treatment, in Experiment 1 (fifteen days), Experiment 1 (thirty days) and Experiment 2 (thirty days) were divided into pooled samples of 8, 4 and 4 birds respectively. The birds in each sample were bled from the jugular vein into a single heparinized centrifuge tube. The livers from the birds in each sample were individually weighed, placed in a common jar and frozen in dry ice. At the time of killing a pooled sample of
444
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T HAS been repeatedly demonstrated that dietary antibiotic can stimulate the growth of chicks but little is known concerning the mode of action. Waibel et al. (1952) and Scott (1951) suggest that an alteration in the microflora of the gut improves the nutritional status or health of the host. However, the work of Anderson et al. (1952), March and Biely (1952) and Rosenberg et al. (1952) has not revealed any consistent influence of dietary antibiotic on the intestinal microflora. Some attention has been directed toward the biochemical status of the antibiotic-fed bird. Common et al. (1950) found that aureomycin enhanced the elevation of serum calcium and riboflavin caused by injected estrogen in the immature pullet. Migicovsky et al. (1951) have observed that dietary penicillin increases the absorption of calcium. Burgess et al. (1951) found that dietary penicillin increased the liver storage of vitamin A and the blood level of carotenoid pigments in the chick. Waibel et al. (1952) found that penicillin increased the deposition of biotin and folic acid in the hen's egg.
445
DIETARY PENICILLIN AND BIOCHEMICAL CHANGES TABLE 1.—Composition of the basal diet Ingredient
54.0 17.5 5.0 5.0 6.0 4.0 5.0 1.5 1.4 0.5 0.1 0.025 lb./lOO lb. 0.05 lb./lOO lb.
liver was placed in alcoholic KOH for subsequent vitamin A analysis. When all birds in a given pooled sample had been killed, the heparinized blood was thoroughly mixed and a 0.5 ml. aliquot removed for haemoglobin de-
Lipids were extracted from plasma with Bloor's mixture (Peters and VanSlyke, 1946) and phospholipid phosphorus was
TABLE 2.—Biochemical changes in the liver of the penicillin-fed chick Experiment 2
Experiment 1 Penicillin mg./kg. diet Days on test No. pooled samples No. birds per pool Total no. birds Final body weight gms. Weight gain gms. Feed consumed gms. Weight gain/feed X100
nil 15 3 8 24
30 15 3 8 24
132 102 208 49.0
138 108 205 52.7
nil 30 6 4 24 411 381
30 30 6 4 24
nil 30 12 4 48
30 30 12 4 48
436 406
344 304 683 44.5
416f 376f 808J 46.5
—
— —
•
—
•
Liver weight gms. Liver gms./kg. body wt.
3.39 24.3
3.51 24.4
7.61 18.5
8.14 18.7
7.07 20.7
7.89f 19.0*
Liver Analyses Dry matter % fresh liver Dry matter gms./kg. body wt. Protein % dry liver Protein gms./kg. body wt.
29.1 7.07 68.7 4.85
29.1 7.10 69.1 4.99
29.5 5.46 75.2 4.11
29.7 5.55 75.4 4.18
27.5 5.69 78.5 4.47
28.5* 5.41 77.9 4.21
10.9
10.7
9.8
9.7
10.9
9.9
0.77 0.93 65.7 109 773
0.76 0.89 63.0 123 868
0.53 0.95 51.9 90 492
0.54 1.04 57.7 104 579
0.62 1.31 74.5 124 703
0.54 1.23 66.4 188f
Ether-extractable fraction % dry liver Ether-extractable fraction gms./kg. body wt. Cholesterol % dry liver Cholesterol mg./kg. body wt. Vit. A I.U./gm. dry liver Vit. A I.U./kg. body wt.
* Significantly different from basal at P= .05. t Significantly different from basal at P— .01. j No test of significance possible due to the use of single groups.
l,016f
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Ground yellow corn Soybean oil meal (solvent) Wheat middlings Alfalfa meal (15%) Fish meal (65%) Meat meal (55%) Brewer's yeast Limestone Tri-calcium phosphate Sodium chloride Vadol (2,400 1. units A, 400 A.O.A.C. units D3) Manganese sulphate Choline chloride
Percent
termination by the Wong (1928) method. The remaining blood was chilled in the refrigerator, centrifuged at 2,500 r.p.m. and the packed cell volume recorded. The supernatant plasma was used for the preparation of a protein-free filtrate by the Folin-Wu (1919) method. Nonprotein nitrogen was determined by the nesslerization procedure of Folin-Wu (1919) and creatinine by the picric acid method of Bonsnes and Taussky (1945). In the first experiment plasma glucose was estimated in the tungstic acid filtrate by the micro-method of Folin and Malmros (1929) but in the second experiment the Somogyi-Shaffer-Hartman (1930) procedure was employed.
446
R. C. BURGESS, M. GLUCK AND D. H. LAUGHLAND
determined in this extract by the Fiske and Subbarow (1925) method after digestion of organic matter with sulphuric acid and hydrogen peroxide. Total fatty acid plus cholesterol was estimated by Bloor's (1928) chromate oxidation technique. Total cholesterol was estimated in an aliquot of the Skelly B extract of saponified fat prepared in the determination of total fatty acids plus cholesterol. The aliquot was evaporated to dryness, taken up in chloroform and the colour developed with the acetic anhydridesulphuric acid mixture of Sperry and Brand (1943). Plasma carotenoid pigments and ascorbic acid were determined by the procedures of Sobel and Snow (1947) and Lowry et al. (1945) respectively.
Vitamin A was extracted from the liver samples with diethyl ether after saponification by means of alcoholic KOH. An aliquot of this extract was evaporated to dryness, taken up in chloroform and the vitamin A estimated by the Carr-Price (1926) reaction. The significance of differences between mean values for control and penicillinfed chicks was determined by "Student's" t-test (Snedecor, 1946).
Liver dry matter was obtained by drying a sample to constant weight at 105 degrees Centrigrade. Total nitrogen was determined by the macro-kjeldahl procedure. Liver lipids were extracted from a trichloracetic acid precipitate of liver with Bloor's mixture (Johnson and Dutch, 1951) and total cholesterol was estimated in this extract by the procedure used for plasma.
The lack of a significant growth response to dietary penicillin by the chicks in Experiment 1 (Table 2) is unusual since this antibiotic had consistently increased the growth of New Hampshire by Broad Bjreasted White chicks in previous experiments. In Experiment 2 the antibiotic significantly enhanced growth and markedly increased feed consumption. It was observed in Experiment 2 that the enhanced growth caused by penicillin was accompanied by a significant decrease in liver weight per kilogram body weight. However, there was a compensating increase in the percentage dry matter of the liver. Thus there was no significant
TABLE 3.—Biochemical changes in the blood of the penicillin-fed chick Experiment 2
Experiment 1 Penicillin mg./kg. diet Days on test
nil 15
30 15
nil 30
30 30
nil 30
30 30
Blood Packed cell volume % whole blood Haemoglobin %
30.3 9.2
32.0 9.3
33.7 9.2
33.1 8.9
30.6 9.1
32.2 9.2
22.0 0.95 234 363 135 8.80 416 2.38
22.2 1.07 231 392 138 . 9.44* 591* 2.82
22.3 0.81 245 399 120 8.60 570
22.3 0.81 230 400 112 10.16| 653
21.2 0.71 192
20.8 0.72 193
— —
— —
Plasma Non-protein nitrogen mg. % Creatinine mg. % Glucose mg. % Total lipid mg. % Total cholesterol mg. % Phospholipid P mg. % Carotenoid pigments meg. % Ascorbic acid mg. %
* Significantly different from basal at P= .05. t Significantly different from basal at P = .01.
—
•
—
7.56 786 2.04
8.52f l,272f 2.48f
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RESULTS
DIETARY PENICILLIN AND BIOCHEMICAL CHANGES
DISCUSSION Coates et al. (1952 b) have reported that the penicillin supplementation of a good practical ration did not enhance the growth of chicks which were reared in a laboratory not previously employed for chick experiments. It was suggested that a "growth depression" in the normal bird was due to a transmissible "infection" which could be counteracted by penicillin. The contrast in growth response of chicks to dietary penicillin in Experiments 1 and 2 may be due to such a factor which preconditions the growth response to penicil^ lin. The fact that the increase in liver vitamin A concentration is most striking when the chicks show a growth response to
penicillin has also been noted by Coates et al. (1952 a) and considered by them to be secondary to the more rapid growth. It is possible, however, that the "infection" which preconditions the growth response to penicillin is also necessary for the increase in liver vitamin A concentration. The effect of dietary penicillin on calcium absorption (Migicovsky et al., 1951), on the concentration of serum carotenoid pigments, and liver vitamin A (Burgess et al., 1951) and on the concentration of yolk biotin and folic acid (Waibel et al., 1952) suggests that the antibiotic increases (a) the amount of these nutrients available to the host or (b) the absorptive capacity of the gut wall. Either of these hypotheses explains the "vitamin-sparing" activity of antibiotics when included in diets which contain suboptimal amounts of certain vitamins (Sunde et al., 1951, Coates et al., 1951) and accounts for the observed increase in feed efficiency when antibiotics are added to simplified diets (Slinger et al., 1952; Branion and Hill, 1951). The rise in the plasma phospholipid phosphorus level after penicillin administration is of considerable interest. According to Walker et al. (1951) the plasma phospholipid concentration is unaffected by increases in the dietary fat content. Fishier et al. (1943) have demonstrated that plasma phospholipids are synthesized exclusively in the liver of the dog. Ranney et al. (1949) believe that fowl plasma lecithins and cephalins are synthesized in the liver but do not speculate on the site of plasma sphingomyelin synthesis. In the present work no attempt was made to fractionate the plasma phospholipids. I t should be emphasized that the increase in this serum component was evident both in the presence and absence of a growth response attributable to penicillin and
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change in the protein, ether-extractable and total cholesterol fractions of the liver either when expressed as a percentage of liver dry matter or per kilogram body weight. In contrast to the aforementioned fractions, an increase in the concentration of vitamin A in the liver was observed under conditions of the enhanced growth produced by penicillin. In the absence of a growth response the increase in the concentration of liver vitamin A was not statistically significant at P = .05 (Experiment 1). The results in Table 3 indicate that there was no significant change in the packed cell volume or haemoglobin concentration of the blood, nor in the nonprotein nitrogen, creatinine, glucose, total lipid or cholesterol level of the plasma. However, dietary penicillin significantly increased the plasma phospholipid phosphorus concentration, both in the presence and absence of a growth response. Plasma ascorbic acid and carotenoid pigment concentrations were also increased, particularly in Experiment 2 where growth was enhanced by the antibiotic.
447
448
R. C. BUEGESS, M. GLUCK AND D. H. LAUGHLAND
may be a specific effect of the antibiotic which is not related to its growth promoting property. SUMMARY
In neither experiment did the antibiotic affect packed cell volume, haemoglobin, plasma non-protein nitrogen, creatinine, glucose, total fatty acid or cholesterol levels. It did elevate plasma carotenoid and ascorbic acid levels and caused a small but highly significant rise in plasma phospholipid phosphorus concentration which was independent of a growth response to the antibiotic. ACKNOWLEDGMENT The authors gratefully acknowledge the technical assistance of Miss M. W. Dowler, Mr. P. M. Richard and Mr. L. J. Carter.
Anderson, G. W., J. D . Cunningham and S. J. Slinger, 1952. Effect of protein level and penicillin on growth and intestinal flora of chickens. J. Nutrition, 47:175-190. Bloor, W. R., 1928. The determination of small amounts of lipid in blood plasma. J. Biol. Chem. 77: 53-62. Bonsnes, R. W., and H. H. Taussky, 1945. On the colorimetric determination of creatinine by the Jaffe reaction. J. Biol. Chem. 158: 581-591. Branion, H. D., and D. C. Hill, 1951. The comparative effect of antibiotics on the growth of poults. Poultry Sci. 30: 793-798. Burgess, R. C , M. Gluck, G. Brisson and D. H. Laughland, 1951. Effect of dietary penicillin on liver vitamin A and serum carotenoids in the chick. Arch. Biochem. Biophys. 33: 339-340. Carr, F. H., and E. A. Price, 1926. Colour reactions attributed to vitamin A. Biochem. J. 20: 497501. Coates, M. E., C. D. Dickinson, G. F. Harrison and S. K. Kon, 1951. The effect of antibiotics on the growth of chicks deprived of vitamins of the Bcomplex. Biochem. J. 49: lxviii. Coates, M. E., G. F. Harrison, S. K. Kon, J. W. G. Porter and S. Y. Thompson, 1952a. Antibiotics in chick nutrition and vitamin A metabolism. Chem. Ind., Issue No. 7: 149-150. Coates, M. E., S. K. Kon, C. D. Dickinson, G. F. Harrison, J. W. G. Porter, S. H. Cummins and W. F. J. Cuthbertson, 1952b. A mode of action of antibiotics in chick nutrition. J. Sci. Food Agr. 3:43-48. Common, R. H., T. J. Keefe, R. C. Burgess and W. A. Maw, 1950. Modification of the biochemical responses of the immature pullet to estrogen by means of dietary aureomycin. Nature, 166: 992-993. Fishier, M. C , C. Entenmann, M. L. Montgomery and I. L. Chaikoff, 1943. The formation of phospholipid by the hepatectomized dog as measured with radioactive phosphorus. I. The site of formation of plasma phospholipids. J. Biol. Chem. 150:47-55. Fiske, C. H., and Y. Subbarow, 1925. The colonmetric determination of phosphorus. J. Biol. Chem. 66:375-384. Folin, O., and H. Malmros, 1929. An improved form of Folin's micromethod for blood sugar determinations. J. Biol. Chem. 83: 115-120. Folin, O., and H. Wu, 1919. A system of blood analysis. J. Biol. Chem. 38: 81-105. Johnson, R. M., and D . H . Dutch, 1951. Use of tri-
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 22, 2015
In two experiments with New Hampshire by Broad Breasted White chicks, dietary penicillin had an inconsistent effect on growth; in the first experiment no growth increase was observed, while in the second experiment birds receiving the antibiotic consumed more feed and gained significantly more weight by four weeks of age than did control birds. Although liver weight per kilogram body weight was less and dry matter greater in penicillin-fed birds of Experiment 2, there was no significant difference in the protein, ether-extractable or cholesterol fractions of the liver of control and penicillin-fed chicks on either a percentage dry liver or per kilogram body weight basis. In Experiment 2 penicillin significantly increased the liver vitamin A concentration per gram dry liver and per kilogram body weight. No significant change in the concentration of any of these constituents was observed in Experiment 1 where penicillin also failed to enhance growth.
REFERENCES
FAT LEVELS, CHOLINE, ANTIBIOTIC AND BI 2
Interaction between penicillin and grass juice concentrate in turkeys. Poultry Sci. 31: 187-188. Snedecor, G. W., 1946. Statistical Methods, Fourth Ed., Iowa State College Press, Ames, Iowa. Sobel, A. E., and S. O. Snow, 1947. The estimation of serum vitamin A with activated glycerol dichlorohydrin. J. Biol. Chem. 171: 617-632. Somogyi, M., 1930. A method for the preparation of blood filtrates for the determination of sugar. J. Biol. Chem. 86: 655-663. Sperry, W. M., and F. C. Brand, 1943. Colorimetric determination of total cholesterol. J. Biol. Chem. 150:315-324. Sunde, M. L., P. E. Waibel, W. W. Cravens and C. A. Elvehjem, 1951. A relationship between antibiotics, vitamin B]2, choline and methionine in chick growth. Poultry Sci. 30:668-671. Waibel, P. E., M. L. Sunde and W. W. Cravens, 1952. Effect of addition of penicillin to the hen's ration on biotin and folic acid content of eggs. Poultry Sci. 31: 621-324. Walker, H. A., M. W. Taylor and W. C. Russell, 1951. The level and interrelationship of the plasma lipids of the laying hen. Poultry Sci. 30: 525530. Wong, S. Y., 1928. Colorimetric determination of iron and haemoglobin in blood. J. Biol. Chem. 77: 409-419.
Effect of Feeding Graded Levels of Fat With and Without Choline and Antibiotic + B12 Supplements to Chicks* A . J . SlEDLER AND B . S. SCHWEIGERT Division of Biochemistry and Nutrition, American Meat Institute Foundation and Department of Biochemistry, University of Chicago, Chicago, Illinois (Received for publication September 27, 1952)
T
HE trend to high energy rations in the production of broilers has led to increased interest in the possibility of
* Journal paper No. 62, American Meat Institute Foundation. A report of work done under contract with the U. S. Department of Agriculture and authorized by the Research and Marketing Act. The contract is being supervised by the Eastern Regional Research Laboratory of the Bureau of Agricultural and Industrial Chemistry. We are indebted to the Lederle Laboratories Di-
adding fat to this type ration. Reiser and Pearson (1949) used lard and hydrogenated vegetable oils in chick starter rations and the addition of these fats showed no deleterious effects on the growth of chicks. Buckner et al. (1947) have also indicated that the addition of 17% corn oil to ravision, American Cyanamid Company for supplying the Aurofac and to the A. E. Staley Manufacturing Company for supplying the soybean grits used in the first experiment.
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 22, 2015
chloracetic acid in purification of lipids. Proc. Soc. Biol. Med. 78: 662-664. Lowry, O. H., J. A. Lopez and O. A. Bessey, 1945. The determination of ascorbic acid in small amounts of blood serum. J. Biol. Chem. 160: 609-615. March, B., and J. Biely, 1952. The effect of feeding aureomycin on the bacterial content of chick feces. Poultry Sci. 31:177-178. Migicovsky, B. B., A. M. Nielson, M. Gluck and R. C. Burgess, 1951. Penicillin and calcium absorption. Arch. Biochem. Biophys. 34: 479-480. Peters, J. P., and D. D. VanSlyke, 1946. Quantitative Clinical Chemistry, Vol. II, Second Ed., Williams and Wilkins Co., Baltimore, Md. Ranney, R. E., C. Entenman and I. L. Chaikoff, 1949. The lecithin, cephalin and sphingomyelin contents of plasma and liver of the fowl; their metabolic interrelationships as shown by the administration of diethylstilbestrol. J. Biol. Chem. 180: 307-313. Rosenberg, M. M., W. Morikawa and 0 . A. Bushnell, 1952. The effect of increasing concentrations of terramycin on the growth and intestinal microflora of chicks. Poultry Sci. 31: 708-714. Scott, M. L., 1951. Unidentified vitamins in turkey nutrition. Proc. Cornell Nutrition Conference: pp. 73-77. Slinger, S. J., W. F. Pepper and D. C. Hill, 1952.
449
Department of Agriculture, Science Service, Ottawa, Canada (Received for publication September 24, 1952)
I
The above evidence suggests that dietary penicillin and possibly other antibiotics stimulate the intestinal absorption of several vitamins. This phenomenon may be associated with the increased growth of penicillin-fed chicks. The work presented in this paper is an extension of the study of the effect of 1 Contribution No. 229 of the Division of Chemistry, Science Service, Ottawa.
dietary antibiotics on biochemical constituents of the chick. EXPERIMENTAL
The dietary treatment, management and analytical techniques were similar in the two experiments reported herein. Day-old New Hampshire by Broad Breasted White chicks were randomized into two groups of forty-eight chicks each and housed in two compartments of an electrically-heated battery brooder with raised wire floors. One group received the high-energy basal ration shown in Table 1 and the other group received this ration supplemented with 30 milligrams procaine penicillin (Merck) per kilogram. Feed and water were provided ad libitum. The body weight and feed consumption were recorded semi-weekly but only the final weights and cumulative feed consumption are recorded in Table 2. All birds were starved twelve hours before killing. In the first experiment half the birds were killed at fifteen days of age and the remainder at thirty days of age. In the second experiment all birds were killed at thirty days of age. Birds receiving each treatment, in Experiment 1 (fifteen days), Experiment 1 (thirty days) and Experiment 2 (thirty days) were divided into pooled samples of 8, 4 and 4 birds respectively. The birds in each sample were bled from the jugular vein into a single heparinized centrifuge tube. The livers from the birds in each sample were individually weighed, placed in a common jar and frozen in dry ice. At the time of killing a pooled sample of
444
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 22, 2015
T HAS been repeatedly demonstrated that dietary antibiotic can stimulate the growth of chicks but little is known concerning the mode of action. Waibel et al. (1952) and Scott (1951) suggest that an alteration in the microflora of the gut improves the nutritional status or health of the host. However, the work of Anderson et al. (1952), March and Biely (1952) and Rosenberg et al. (1952) has not revealed any consistent influence of dietary antibiotic on the intestinal microflora. Some attention has been directed toward the biochemical status of the antibiotic-fed bird. Common et al. (1950) found that aureomycin enhanced the elevation of serum calcium and riboflavin caused by injected estrogen in the immature pullet. Migicovsky et al. (1951) have observed that dietary penicillin increases the absorption of calcium. Burgess et al. (1951) found that dietary penicillin increased the liver storage of vitamin A and the blood level of carotenoid pigments in the chick. Waibel et al. (1952) found that penicillin increased the deposition of biotin and folic acid in the hen's egg.
445
DIETARY PENICILLIN AND BIOCHEMICAL CHANGES TABLE 1.—Composition of the basal diet Ingredient
54.0 17.5 5.0 5.0 6.0 4.0 5.0 1.5 1.4 0.5 0.1 0.025 lb./lOO lb. 0.05 lb./lOO lb.
liver was placed in alcoholic KOH for subsequent vitamin A analysis. When all birds in a given pooled sample had been killed, the heparinized blood was thoroughly mixed and a 0.5 ml. aliquot removed for haemoglobin de-
Lipids were extracted from plasma with Bloor's mixture (Peters and VanSlyke, 1946) and phospholipid phosphorus was
TABLE 2.—Biochemical changes in the liver of the penicillin-fed chick Experiment 2
Experiment 1 Penicillin mg./kg. diet Days on test No. pooled samples No. birds per pool Total no. birds Final body weight gms. Weight gain gms. Feed consumed gms. Weight gain/feed X100
nil 15 3 8 24
30 15 3 8 24
132 102 208 49.0
138 108 205 52.7
nil 30 6 4 24 411 381
30 30 6 4 24
nil 30 12 4 48
30 30 12 4 48
436 406
344 304 683 44.5
416f 376f 808J 46.5
—
— —
•
—
•
Liver weight gms. Liver gms./kg. body wt.
3.39 24.3
3.51 24.4
7.61 18.5
8.14 18.7
7.07 20.7
7.89f 19.0*
Liver Analyses Dry matter % fresh liver Dry matter gms./kg. body wt. Protein % dry liver Protein gms./kg. body wt.
29.1 7.07 68.7 4.85
29.1 7.10 69.1 4.99
29.5 5.46 75.2 4.11
29.7 5.55 75.4 4.18
27.5 5.69 78.5 4.47
28.5* 5.41 77.9 4.21
10.9
10.7
9.8
9.7
10.9
9.9
0.77 0.93 65.7 109 773
0.76 0.89 63.0 123 868
0.53 0.95 51.9 90 492
0.54 1.04 57.7 104 579
0.62 1.31 74.5 124 703
0.54 1.23 66.4 188f
Ether-extractable fraction % dry liver Ether-extractable fraction gms./kg. body wt. Cholesterol % dry liver Cholesterol mg./kg. body wt. Vit. A I.U./gm. dry liver Vit. A I.U./kg. body wt.
* Significantly different from basal at P= .05. t Significantly different from basal at P— .01. j No test of significance possible due to the use of single groups.
l,016f
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 22, 2015
Ground yellow corn Soybean oil meal (solvent) Wheat middlings Alfalfa meal (15%) Fish meal (65%) Meat meal (55%) Brewer's yeast Limestone Tri-calcium phosphate Sodium chloride Vadol (2,400 1. units A, 400 A.O.A.C. units D3) Manganese sulphate Choline chloride
Percent
termination by the Wong (1928) method. The remaining blood was chilled in the refrigerator, centrifuged at 2,500 r.p.m. and the packed cell volume recorded. The supernatant plasma was used for the preparation of a protein-free filtrate by the Folin-Wu (1919) method. Nonprotein nitrogen was determined by the nesslerization procedure of Folin-Wu (1919) and creatinine by the picric acid method of Bonsnes and Taussky (1945). In the first experiment plasma glucose was estimated in the tungstic acid filtrate by the micro-method of Folin and Malmros (1929) but in the second experiment the Somogyi-Shaffer-Hartman (1930) procedure was employed.
446
R. C. BURGESS, M. GLUCK AND D. H. LAUGHLAND
determined in this extract by the Fiske and Subbarow (1925) method after digestion of organic matter with sulphuric acid and hydrogen peroxide. Total fatty acid plus cholesterol was estimated by Bloor's (1928) chromate oxidation technique. Total cholesterol was estimated in an aliquot of the Skelly B extract of saponified fat prepared in the determination of total fatty acids plus cholesterol. The aliquot was evaporated to dryness, taken up in chloroform and the colour developed with the acetic anhydridesulphuric acid mixture of Sperry and Brand (1943). Plasma carotenoid pigments and ascorbic acid were determined by the procedures of Sobel and Snow (1947) and Lowry et al. (1945) respectively.
Vitamin A was extracted from the liver samples with diethyl ether after saponification by means of alcoholic KOH. An aliquot of this extract was evaporated to dryness, taken up in chloroform and the vitamin A estimated by the Carr-Price (1926) reaction. The significance of differences between mean values for control and penicillinfed chicks was determined by "Student's" t-test (Snedecor, 1946).
Liver dry matter was obtained by drying a sample to constant weight at 105 degrees Centrigrade. Total nitrogen was determined by the macro-kjeldahl procedure. Liver lipids were extracted from a trichloracetic acid precipitate of liver with Bloor's mixture (Johnson and Dutch, 1951) and total cholesterol was estimated in this extract by the procedure used for plasma.
The lack of a significant growth response to dietary penicillin by the chicks in Experiment 1 (Table 2) is unusual since this antibiotic had consistently increased the growth of New Hampshire by Broad Bjreasted White chicks in previous experiments. In Experiment 2 the antibiotic significantly enhanced growth and markedly increased feed consumption. It was observed in Experiment 2 that the enhanced growth caused by penicillin was accompanied by a significant decrease in liver weight per kilogram body weight. However, there was a compensating increase in the percentage dry matter of the liver. Thus there was no significant
TABLE 3.—Biochemical changes in the blood of the penicillin-fed chick Experiment 2
Experiment 1 Penicillin mg./kg. diet Days on test
nil 15
30 15
nil 30
30 30
nil 30
30 30
Blood Packed cell volume % whole blood Haemoglobin %
30.3 9.2
32.0 9.3
33.7 9.2
33.1 8.9
30.6 9.1
32.2 9.2
22.0 0.95 234 363 135 8.80 416 2.38
22.2 1.07 231 392 138 . 9.44* 591* 2.82
22.3 0.81 245 399 120 8.60 570
22.3 0.81 230 400 112 10.16| 653
21.2 0.71 192
20.8 0.72 193
— —
— —
Plasma Non-protein nitrogen mg. % Creatinine mg. % Glucose mg. % Total lipid mg. % Total cholesterol mg. % Phospholipid P mg. % Carotenoid pigments meg. % Ascorbic acid mg. %
* Significantly different from basal at P= .05. t Significantly different from basal at P = .01.
—
•
—
7.56 786 2.04
8.52f l,272f 2.48f
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 22, 2015
RESULTS
DIETARY PENICILLIN AND BIOCHEMICAL CHANGES
DISCUSSION Coates et al. (1952 b) have reported that the penicillin supplementation of a good practical ration did not enhance the growth of chicks which were reared in a laboratory not previously employed for chick experiments. It was suggested that a "growth depression" in the normal bird was due to a transmissible "infection" which could be counteracted by penicillin. The contrast in growth response of chicks to dietary penicillin in Experiments 1 and 2 may be due to such a factor which preconditions the growth response to penicil^ lin. The fact that the increase in liver vitamin A concentration is most striking when the chicks show a growth response to
penicillin has also been noted by Coates et al. (1952 a) and considered by them to be secondary to the more rapid growth. It is possible, however, that the "infection" which preconditions the growth response to penicillin is also necessary for the increase in liver vitamin A concentration. The effect of dietary penicillin on calcium absorption (Migicovsky et al., 1951), on the concentration of serum carotenoid pigments, and liver vitamin A (Burgess et al., 1951) and on the concentration of yolk biotin and folic acid (Waibel et al., 1952) suggests that the antibiotic increases (a) the amount of these nutrients available to the host or (b) the absorptive capacity of the gut wall. Either of these hypotheses explains the "vitamin-sparing" activity of antibiotics when included in diets which contain suboptimal amounts of certain vitamins (Sunde et al., 1951, Coates et al., 1951) and accounts for the observed increase in feed efficiency when antibiotics are added to simplified diets (Slinger et al., 1952; Branion and Hill, 1951). The rise in the plasma phospholipid phosphorus level after penicillin administration is of considerable interest. According to Walker et al. (1951) the plasma phospholipid concentration is unaffected by increases in the dietary fat content. Fishier et al. (1943) have demonstrated that plasma phospholipids are synthesized exclusively in the liver of the dog. Ranney et al. (1949) believe that fowl plasma lecithins and cephalins are synthesized in the liver but do not speculate on the site of plasma sphingomyelin synthesis. In the present work no attempt was made to fractionate the plasma phospholipids. I t should be emphasized that the increase in this serum component was evident both in the presence and absence of a growth response attributable to penicillin and
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change in the protein, ether-extractable and total cholesterol fractions of the liver either when expressed as a percentage of liver dry matter or per kilogram body weight. In contrast to the aforementioned fractions, an increase in the concentration of vitamin A in the liver was observed under conditions of the enhanced growth produced by penicillin. In the absence of a growth response the increase in the concentration of liver vitamin A was not statistically significant at P = .05 (Experiment 1). The results in Table 3 indicate that there was no significant change in the packed cell volume or haemoglobin concentration of the blood, nor in the nonprotein nitrogen, creatinine, glucose, total lipid or cholesterol level of the plasma. However, dietary penicillin significantly increased the plasma phospholipid phosphorus concentration, both in the presence and absence of a growth response. Plasma ascorbic acid and carotenoid pigment concentrations were also increased, particularly in Experiment 2 where growth was enhanced by the antibiotic.
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R. C. BUEGESS, M. GLUCK AND D. H. LAUGHLAND
may be a specific effect of the antibiotic which is not related to its growth promoting property. SUMMARY
In neither experiment did the antibiotic affect packed cell volume, haemoglobin, plasma non-protein nitrogen, creatinine, glucose, total fatty acid or cholesterol levels. It did elevate plasma carotenoid and ascorbic acid levels and caused a small but highly significant rise in plasma phospholipid phosphorus concentration which was independent of a growth response to the antibiotic. ACKNOWLEDGMENT The authors gratefully acknowledge the technical assistance of Miss M. W. Dowler, Mr. P. M. Richard and Mr. L. J. Carter.
Anderson, G. W., J. D . Cunningham and S. J. Slinger, 1952. Effect of protein level and penicillin on growth and intestinal flora of chickens. J. Nutrition, 47:175-190. Bloor, W. R., 1928. The determination of small amounts of lipid in blood plasma. J. Biol. Chem. 77: 53-62. Bonsnes, R. W., and H. H. Taussky, 1945. On the colorimetric determination of creatinine by the Jaffe reaction. J. Biol. Chem. 158: 581-591. Branion, H. D., and D. C. Hill, 1951. The comparative effect of antibiotics on the growth of poults. Poultry Sci. 30: 793-798. Burgess, R. C , M. Gluck, G. Brisson and D. H. Laughland, 1951. Effect of dietary penicillin on liver vitamin A and serum carotenoids in the chick. Arch. Biochem. Biophys. 33: 339-340. Carr, F. H., and E. A. Price, 1926. Colour reactions attributed to vitamin A. Biochem. J. 20: 497501. Coates, M. E., C. D. Dickinson, G. F. Harrison and S. K. Kon, 1951. The effect of antibiotics on the growth of chicks deprived of vitamins of the Bcomplex. Biochem. J. 49: lxviii. Coates, M. E., G. F. Harrison, S. K. Kon, J. W. G. Porter and S. Y. Thompson, 1952a. Antibiotics in chick nutrition and vitamin A metabolism. Chem. Ind., Issue No. 7: 149-150. Coates, M. E., S. K. Kon, C. D. Dickinson, G. F. Harrison, J. W. G. Porter, S. H. Cummins and W. F. J. Cuthbertson, 1952b. A mode of action of antibiotics in chick nutrition. J. Sci. Food Agr. 3:43-48. Common, R. H., T. J. Keefe, R. C. Burgess and W. A. Maw, 1950. Modification of the biochemical responses of the immature pullet to estrogen by means of dietary aureomycin. Nature, 166: 992-993. Fishier, M. C , C. Entenmann, M. L. Montgomery and I. L. Chaikoff, 1943. The formation of phospholipid by the hepatectomized dog as measured with radioactive phosphorus. I. The site of formation of plasma phospholipids. J. Biol. Chem. 150:47-55. Fiske, C. H., and Y. Subbarow, 1925. The colonmetric determination of phosphorus. J. Biol. Chem. 66:375-384. Folin, O., and H. Malmros, 1929. An improved form of Folin's micromethod for blood sugar determinations. J. Biol. Chem. 83: 115-120. Folin, O., and H. Wu, 1919. A system of blood analysis. J. Biol. Chem. 38: 81-105. Johnson, R. M., and D . H . Dutch, 1951. Use of tri-
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In two experiments with New Hampshire by Broad Breasted White chicks, dietary penicillin had an inconsistent effect on growth; in the first experiment no growth increase was observed, while in the second experiment birds receiving the antibiotic consumed more feed and gained significantly more weight by four weeks of age than did control birds. Although liver weight per kilogram body weight was less and dry matter greater in penicillin-fed birds of Experiment 2, there was no significant difference in the protein, ether-extractable or cholesterol fractions of the liver of control and penicillin-fed chicks on either a percentage dry liver or per kilogram body weight basis. In Experiment 2 penicillin significantly increased the liver vitamin A concentration per gram dry liver and per kilogram body weight. No significant change in the concentration of any of these constituents was observed in Experiment 1 where penicillin also failed to enhance growth.
REFERENCES
FAT LEVELS, CHOLINE, ANTIBIOTIC AND BI 2
Interaction between penicillin and grass juice concentrate in turkeys. Poultry Sci. 31: 187-188. Snedecor, G. W., 1946. Statistical Methods, Fourth Ed., Iowa State College Press, Ames, Iowa. Sobel, A. E., and S. O. Snow, 1947. The estimation of serum vitamin A with activated glycerol dichlorohydrin. J. Biol. Chem. 171: 617-632. Somogyi, M., 1930. A method for the preparation of blood filtrates for the determination of sugar. J. Biol. Chem. 86: 655-663. Sperry, W. M., and F. C. Brand, 1943. Colorimetric determination of total cholesterol. J. Biol. Chem. 150:315-324. Sunde, M. L., P. E. Waibel, W. W. Cravens and C. A. Elvehjem, 1951. A relationship between antibiotics, vitamin B]2, choline and methionine in chick growth. Poultry Sci. 30:668-671. Waibel, P. E., M. L. Sunde and W. W. Cravens, 1952. Effect of addition of penicillin to the hen's ration on biotin and folic acid content of eggs. Poultry Sci. 31: 621-324. Walker, H. A., M. W. Taylor and W. C. Russell, 1951. The level and interrelationship of the plasma lipids of the laying hen. Poultry Sci. 30: 525530. Wong, S. Y., 1928. Colorimetric determination of iron and haemoglobin in blood. J. Biol. Chem. 77: 409-419.
Effect of Feeding Graded Levels of Fat With and Without Choline and Antibiotic + B12 Supplements to Chicks* A . J . SlEDLER AND B . S. SCHWEIGERT Division of Biochemistry and Nutrition, American Meat Institute Foundation and Department of Biochemistry, University of Chicago, Chicago, Illinois (Received for publication September 27, 1952)
T
HE trend to high energy rations in the production of broilers has led to increased interest in the possibility of
* Journal paper No. 62, American Meat Institute Foundation. A report of work done under contract with the U. S. Department of Agriculture and authorized by the Research and Marketing Act. The contract is being supervised by the Eastern Regional Research Laboratory of the Bureau of Agricultural and Industrial Chemistry. We are indebted to the Lederle Laboratories Di-
adding fat to this type ration. Reiser and Pearson (1949) used lard and hydrogenated vegetable oils in chick starter rations and the addition of these fats showed no deleterious effects on the growth of chicks. Buckner et al. (1947) have also indicated that the addition of 17% corn oil to ravision, American Cyanamid Company for supplying the Aurofac and to the A. E. Staley Manufacturing Company for supplying the soybean grits used in the first experiment.
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chloracetic acid in purification of lipids. Proc. Soc. Biol. Med. 78: 662-664. Lowry, O. H., J. A. Lopez and O. A. Bessey, 1945. The determination of ascorbic acid in small amounts of blood serum. J. Biol. Chem. 160: 609-615. March, B., and J. Biely, 1952. The effect of feeding aureomycin on the bacterial content of chick feces. Poultry Sci. 31:177-178. Migicovsky, B. B., A. M. Nielson, M. Gluck and R. C. Burgess, 1951. Penicillin and calcium absorption. Arch. Biochem. Biophys. 34: 479-480. Peters, J. P., and D. D. VanSlyke, 1946. Quantitative Clinical Chemistry, Vol. II, Second Ed., Williams and Wilkins Co., Baltimore, Md. Ranney, R. E., C. Entenman and I. L. Chaikoff, 1949. The lecithin, cephalin and sphingomyelin contents of plasma and liver of the fowl; their metabolic interrelationships as shown by the administration of diethylstilbestrol. J. Biol. Chem. 180: 307-313. Rosenberg, M. M., W. Morikawa and 0 . A. Bushnell, 1952. The effect of increasing concentrations of terramycin on the growth and intestinal microflora of chicks. Poultry Sci. 31: 708-714. Scott, M. L., 1951. Unidentified vitamins in turkey nutrition. Proc. Cornell Nutrition Conference: pp. 73-77. Slinger, S. J., W. F. Pepper and D. C. Hill, 1952.
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