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Effect of Antibiotics on Growth and Nutrient Absorption of Chicks
Effect of Antibiotics on Growth and Nutrient Absorption of Chicks H. EYSSEN AND P. D E SOMER Department of Microbiology, Rega Institute for Medical Research, University of Louvain, Louvain, (Received for publication March 11, 1963)
INTRODUCTION
The present investigations were performed to obtain additional information on the correlation between the growthpromoting action of various antibiotics such as penicillin, virginiamycin, chloramphenicol and chlortetracycline, and their effect on intestinal absorption of fats. MATERIALS AND METHODS
The composition of the basal diet is shown in Table 1. This diet corresponds closely with the diet no. 3 described by Stokstad et al. (1953). The regular diet
contains 3 % corn oil. Higher levels of corn oil were added at the expense of carbohydrates. In the low-fat diet, corn oil was omitted and replaced by 3 % carbohydrate. Antibiotics were added to a concentration of 100 p.p.m. Feces used for fatty acid analyses were collected on glass plates placed underneath the wire floors. Plates were removed daily and the feces were scraped off and dried under reduced pressure over phosphorpentoxide. Fatty acids were determined by the method of Van de Kamer etal. (1949). TABLE 1.—Composition of diet Grams Casein (crude) Gelatin Carbohydrate" Calcium triphosphate Calcium gluconate Salt mixture b Vitamins in glucose0 Corn oil and vitamin A, D, E d DL-methionine L-cystine Choline chloride
20 8 58.5 2 5 2 1 3 0.15 0.15 0.2
a Sucrose, cornstarch, glucose, or a mixture of equal parts of glucose and fructose. b 600 mg. sodium chloride; 600 mg. dipotassium phosphate; 450 mg. monopotassium phosphate; 250 mg. magnesium sulfate monohydrate; 40 mg. manganese sulfate; 50 mg. ferric citrate; 2 mg. cupric sulfate; 0.6 mg. potassium iodide; 0.8 mg. potassium bromide; 1.4 mg. zinc acetate; 1.6 mg. aluminium sulfate; 0.4 mg. cobalt acetate tetrahydrate; 0.2 mg. nickel sulfate; 0.05 mg. sodium molybdate. Water to dissolve trace minerals up to 2 g. 0 1 mg. thiamine hydrochloride; 1 mg. riboflavin; 5 mg. niacinamide; 1 mg. pyridoxine; 5 mg. calcium pantothenate; 0.5 mg. pteroyl-glutamic acid; 0.5 mg. vitamin K; 0.02 mg. biotin; 5 meg. vitamin B12; 100 mg. inositol; glucose up to 1 g. d 1,500 U. vitamin A acetate; 200 U. vitamin D 3 ; 10 mg. vitamin E acetate; corn oil up to 3 g.
1373
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I
N A recent study from this laboratory, on the mode of action of antibiotics as growth stimulants for young animals, a transitory period of growth depression was observed around the 8th day of life in chicks fed an antibiotic-free, caseinsucrose diet (Eyssen and De Somer, 1963). The inhibition of weight gain was found to be associated with decreased efficiency of feed utilization and malabsorption of fats and carbohydrates, and the syndrome was most pronounced when sucrose was fed as the sole source of dietary carbohydrate. Addition to the feed of 20 p.p.m. of virginiamycin, an antibiotic active only against Gram-positive microorganisms, resulted in improved intestinal absorption and stimulated growth. As a result of these studies, the hypothesis was proposed that the growth-stimulating effect of antibiotics is most likely due to suppression of intestinal Gram-positive microorganisms which interfere with the absorption of nutrients.
1374
H. EYSSEN AND P. D E SOMER
mainly to Gram-positive microorganisms. In this respect it is similar to virginiao mycin. Chloramphenicol is a broad spec9 -o Chlortetracycline trum antibiotic which generally fails to o o Penicillin elicit a positive growth response, whereas • o Virginiamycin chlortetracycline is a widely used broad spectrum growth stimulant. In previous experiments, chlortetracycline regularly gave a growth increase of 15 to 25% but lost its activity after it was in use for 6 months. This is in agreement with other reports that continuous feeding of an antibiotic results in a decrease in the growth response (Waibel et al., 1954; Libby and Schaible, 1955). L, 6 8 10 12 Figure 1 illustrates the evolution of the Age of chicks in days daily weight increase, and Table 2 sumFIG. 1. Effect of 100 p.p.m. of various antibiotics marizes the body weight data. In the on daily weight gain of chicks fed a casein-sucrose control animals a growth depression is obdiet. served after the 5th day. Weight gain reAll experiments were carried out in mains low for 4 days, and thereafter tends thermostatically controlled conventional to become normal. Procaine penicillin and battery brooders with raised wire screen virginiamycin stimulate growth during floors under continuous illumination, feed this critical period while chloramphenicol and water being supplied ad libitum. and chlortetracycline are ineffective. Dropping trays were removed daily. All An example of the evolution of the fecal animals were one-day-old broiler chicks of fat excretion is presented in Figure 2. mixed sexes obtained from a commercial During the critical period from the 6th hatchery. Each experimental group con- to the 9th day of the experiment, chicks sisted of 2 replicates of 10 to 12 chicks, 120 and the experimental diets were fed from • — • Control = no antibiotics o—© Chloramphenicol the first day on. Only short term experio—« Chlortetracycline „ioo o—o Penicillin ments over a 2 week period were perVirginiamycin formed. " C o n t r o l : no antibiotics o Chloramphenicol
1. Effect of antibiotics on growth and on fat absorption. To establish whether the growth-promoting effect of various antibiotics is correlated with their influence on fat absorption, a first series of experiments with a casein-sucrose diet compared the effect of virginiamycin with that of penicillin, chloramphenicol, and chlortetracycline. Penicillin was chosen because it is a potent growth-stimulating agent with an antibacterial spectrum restricted
-80
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FIG. 2. Effect of 100 p.p.m. of various antibiotics on the fecal fat excretion of chicks fed a caseinsucrose diet.
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RESULTS
1375
EFFECT OF ANTIBIOTICS
TABLE 2.—Growth-promoting effect of various antibiotics on chicks fed a purified casein-sucrose diet
Diet Basal diet +Virginiamycin lOOp.p.m. +Chlortetracycline lOOp.p.m. +Proc. penicillin 100 p.p.m. +Chloramphenicol 100 p.p.m. :
No. of chicks 24 24 23 23 22
Average body weight (gm.) Start
2 Weeks
% Weight increase over basal
34 34 34 34 34
130 150 133 147 130
+20.8 + 3.5* + 18.3 + 0
Not significantly different at 0.05 level of probability (p>0.05).
2. Effect of various dietary carbohydrates. Stokstad el al. (1953) reported that an antibiotic-free sucrose diet did not support as rapid growth as an antibiotic-free starch diet, although the sucrose diet supplemented with chlortetracycline resulted in better growth than did the starch diet containing the antibiotic. Chloretracycline was virtually inactive when added to a starch diet. The results of our studies on the growth-promoting effect of virginiamycin were in agreement with these data (Eyssen et al., 1962). Since Borgstrom el al. (1959) found that the intestinal flora of the rat inactivates invertase but not amylase, several experiments were performed to establish whether
a similar mechanism could explain the observed differences between the two carbohydrates in the chick. Therefore, glucose and fructose—the hydrolytic products of sucrose—were substituted for the intact disaccharide. Table 3 shows that the growth rate on the diets without antibiotics was not improved when sucrose was replaced by the glucose-fructose mixture, but that glucose alone led to significantly better growth. Consequently, in the absence of antibiotics the fructose moiety of the sucrose molecule seems to be less well utilized by the chick, perhaps because it is more slowly resorbed from the intestine (Bogner, 1961) thus remaining available for bacterial fermentation. Although the basal glucose diet supported growth almost comparable to that obtained with a starch diet, it was still further improved by virginiamycin, whereas a starch diet was not. The evolution of the fecal fat excretion throughout these experiments is illustrated in Figure 3. Glucose, sucrose, and glucose-fructose provoke a peak of malabsorption of fats from the 5th to the 9th day of the experiment, and this period of steatorrhea is suppressed by 100 p.p.m. of virginiamycin. These experiments provide additional evidence for a close correlation between the activity of antibiotics as growth stimulants and their effect on intestinal absorption of nutrients. Dietary composi-
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receiving a sucrose diet without antibiotics lost 70 to 90 mg. fatty acids per gram dried feces. Comparison of Figures 1 and 2 shows that only the two growthpromoting antibiotics penicillin and virginiamycin reduced the fecal fat excretion to about 40 mg. per gram. Chloramphenicol and chlortetracycline did not reduce fecal fat loss and, concomitantly, they were ineffective in stimulating growth. Although it still remains to be elucidated why the malabsorption does not develop until the 5th or the 6th day and why spontaneous recovery occurs after several days, our observations suggest that antibiotics stimulate growth of chicks particularly for a few days around the 8th day of life.
1376
H. EYSSEN AND P. D E SOMER TABLE 3.—KJJect of the type of dietary carbohydrate on growth of chicks and on growth-stimulating effect of virginiamycin No. of chicks
Type of diet
Start
2 weeks
% Weight increase over basal
Basal sucrose +virginiamycin 100 p.p.m.
24 24
37 37
133 156
+23.9
Basal glucose-fructose +virginiamycin 100 p.p.m.
22 22
37 37
126 148
+24.7
Basal glucose +virginiamycin 100 p.p.m.
24 24
37 37
148 163
+ 13.5
Basal starch +virginiamycin 100 p.p.m.
24 24
37 37
152 155
+ 2.6*
Not significantly different at 0.05 level of probability (p>0.05).
tions containing low molecular carbohydrates result in high fecal fat excretion and are significantly improved by virginiamycin. Little or no malabsorption is observed when a starch diet is fed, and in this case the antibiotic fails to stimulate growth significantly. 3. Effect of different fat levels. The diets used in the foregoing experiments contained 3 % corn oil as the sole source of fat. Since in birds fed the antibiotic-free
sucrose diet from 30 to 50% of the dietary fat is lost during the period of malabsorption and since the chick is known for its high linoleic acid requirements (Machlin and Gordon, 1961), it seemed by no means impossible that virginiamycin stimulates growth not only by improving total nutrient absorption, but also by alleviating specific linoleic acid deficiency symptoms. In this case, however, one should expect a decreased activity of the antibiotic when higher levels of corn oil are fed.
- • Sucrose diet o--o Glucose diet ®~© Glucose - levulose • - • • Starch diet
100p.p.m.Virginiamycin added
2
4
6
8
10 12 tt 2 4 Age of chicks in days
6
8
10
FIG. 3. Effect of type of dietary carbohydrate on fecal fat excretion in chicks.
12
u
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:
Average body weight (gm.)
1377
EFFECT OF ANTIBIOTICS TABLE 4.—Influence of increasing dietary fat levels on chick growth and response to 100 p.p.m. of virginiamycin
Type of diet
Averages body weight (gm.)
No. of chicks
Start
2 weeks
Gain due to Gain due to antibiotic fat supplement (gm.) (gm-)
Basal sucrose +virginiamycin
0 0
24 24
36 36
108 127
+ 19
Basal sucrose +virginiamycin
1 1
24 24
36 36
120 139
+ 19
+ 12 + 12
Basal sucrose +virginiamycin
3 3
24 24
36 36
136 151
+ 15
+28 +24
Basal sucrose +virginiamycin
6 6
24 23
36 36
135 154
+ 19
+27 +27
Basal starch +virginiamycin
0 0
24 24
34 34
134 133
-
Basal starch +virginiamycin
1 1
24 24
34 34
136 141
+ 5*
+ 2* + 8*
Basal starch +virginiamycin
3 3
23 24
34 34
143 147
+ 4*
+ 9 + 14
Basal starch +virginiamycin
6 6
24 24
34 34
145 136
9*
,
1*
+ 11 + 3*
Not significantly different at 0.05 level of probability (p>0.05).
Whereas growth on the basal sucrose diet was significantly improved by increasing the corn oil from 0 to 3%, no additional weight gain resulted from the 6% corn oil ration (Table 4) and in every case the response to virginiamycin was virtually unaffected by the dietary fat level. The effect of increasing fat levels was less pronounced on a starch diet, although a small weight increase was noted. Table 4 also shows that chicks grew as fast on a fat-free starch diet as on an antibiotic-free 6% corn oil sucrose diet. This would indicate that, at least during the first 2 weeks of the chicks life, the growthpromoting effect of antibiotics is not due to a therapeutic effect on an essential fatty acid deficiency. DISCUSSION Numerous workers have reported that the growth-promoting effect of antibiotics
is most often associated with an increased efficiency of feed utilization and that antibiotics under certain dietary conditions exert a sparing action on vitamins, minerals, and proteins. These findings might be explained by an antimicrobial effect of the antibiotics upon Gram-positive intestinal bacteria. In animals given an antibiotic-free diet these microorganisms could provoke a polymalabsorption syndrome including not only vitamins and minerals but also proteins, fats and carbohydrates. Our previous work with the antibiotic virginiamycin supported this hypothesis (Eyssen and De Somer, 1963). Further evidence is provided by the present investigations in which a close correlation has been observed between the activity of antibiotics as growthpromoting agents and their influence on fat absorption. Growth rate was increased by penicillin and virginiamycin, and these
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:
Corn oil %
1378
H. E Y S S E N AND P . D E SOMER
T h e activity of penicillin and virginiamycin, which are active only against Gram-positive microorganisms proves t h a t the causative microorganism is a Gram-positive microbe. T h e bacterial flora in the proximal intestinal segments of the young chick consists mainly of lactobacilli and enterococci. Nevertheless, preliminary unpublished experiments on monocontaminated birds indicate t h a t neither a pure flora of lactobacilli nor a monoflora of enterococci reduces chick growth, and t h a t those floras do not provoke malabsorption of fats. F u r t h e r studies on chicks monocontaminated with other intestinal microorganisms are in progress. Although a growth response was obtained to 1 % and 3 % corn oil, no further increase in growth was observed a t the 6 % corn oil level either on a sucrose or on a starch diet, and in every case the growth response to antibiotics was independent of the dietary fat level and was influenced only by the type of carbohydrate. Nevertheless D a m et al. (1959) observed improved chick growth when the dietary fat was increased from 3 % to 1 0 % . E m p h a sis must be placed, however, on the statement b y Donaldson et al. (1957), t h a t for satisfactory use of high fat levels the ration must be properly balanced in respect to other nutrients. Perhaps minor differences in the dietary composition
might explain the varying responses to fat supplements. A striking feature is the excellent fat tolerance on a starch diet, whereas animals receiving a sucrose, glucose or glucosefructose diet, lost considerable quantities of fat. These experiments clearly prove t h a t fat absorption studies m a y be profoundly influenced b y the age of the test animals, the type of dietary carbohydrate, and the presence or absence of antibiotics. This is in agreement with the studies of Supplee (1960) and Donaldson (1962), who felt t h a t the microflora status possibly influences studies of dietary fat effects in poults and chicks. T h e importance of intestinal bacteria in fat absorption is also known from h u m a n cases of steatorrhea and has been further illustrated in work from this laboratory with blind-loop rats (Hoet et al., 1962). These studies showed t h a t rats with an artificial cecum on the proximal jejunum excreted twice as much dietary fat as did their controls. In these experiments, too, the carbohydrate composition of the diet influenced fat absorption, and the fecal fat loss could be brought down to controlvalues by the administration of virginiamycin b u t not b y chlortetracycline.
SUMMARY A close correlation between the activity of antibiotics as growth promotants and the influence of the antibiotics on intestinal fat absorption has been observed in chicks fed purified diets. Only those antibiotics which reduced fecal fat excretion improved growth. Basal diets containing sucrose, glucose, or glucose-fructose as the sole source of carbohydrate resulted in high fecal fat excretion and could be improved by antibiotics. With a corn starch diet, however, little or no malabsorption was observed and this diet
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two antibiotics also reduced fecal fat excretion. Chlortetracycline and chloramphenicol were without effect on fecal fat and failed to stimulate growth. On the other hand, the basal diets containing low molecular carbohydrates resulted in high fecal fat excretion and were significantly improved by the addition of virginiamycin. Little or no malabsorption was observed when a corn starch diet was fed, and in this case virginiamycin did not stimulate growth.
EFFECT OF ANTIBIOTICS
was not improved by the addition of antibiotics. These investigations provide further evidence that the growth-stimulating effect of antibiotics is most likely due to suppression of Gram-positive intestinal bacteria which interfere with the absorption of nutrients.
REFERENCES Bogner, P. H., 1961. Alimentary absorption of reducing sugars by embryos and young chicks. Proc. Soc. Exp. Biol. Med. 107: 263-265. Borgstrom, B., A. Dahlqvist, B. E. Gustafsson, G. Lundh and J. Malmquist, 1959. Trypsin, invertase and amylase content of feces of germfree rats. Proc. Soc. Exp. Biol. Med. 102: 154-155. Dam, R., R. M. Leach, T. S. Nelson, L. C. Norris and F. W. Hill, 1959. Studies on the effect of quantity and type of fat on chick growth. J. Nutrition, 68: 615-632. Donaldson, W. E., G. F. Combs, G. L. Romoser and W. C. Supplee, 1957. Studies on energy levels in poultry rations. 2. Tolerance of growing chicks to dietary fat. Poultry Sci. 36: 807-815. Donaldson, W. E., 1962. The response of chicks to dietary animal fat in new and old environments.
Poultry Sci. 41: 1106-1108. Eyssen, H., V. De Prins and P. De Somer, 1962. The growth-promoting action of virginiamycin and its influence on the crop flora in chickens. Poultry Sci. 41: 227-233. Eyssen, H., and P. De Somer, 1963. The mode of action of antibiotics in stimulating growth of chicks. J. Exp. Med. 117:127-138. Hoet, P. P., J. V. Joossens, E. Evrard, H. Eyssen and P. De Somer, 1962. Intestinal bacteria and fecal fat. Proc. Internat. Conference on the Biochemical Problems of Lipids, held at Birmingham, U. K., July 23-27. Elsevier Publishing Company, Amsterdam. In press. Libby, D. A., and P. S. Schaible, 1955. Observations on growth responses to antibiotics and arsonic acids. Science, 121: 733-734. Machlin, L. S., and R. S. Gordon, 1961. Effects of dietary fatty acids and cholesterol on growth and fatty acid composition of the chicken. J. Nutrition, 75: 157-164. Stokstad, E. L. R., T. H. Jukes and W. L. Williams, 1953. The growth-promoting effect of Aureomycin on various types of diet. Poultry Sci. 32:10541058. Supplee, W. C , 1960. The effect of antibiotic supplementation on the response of poults to dietary corn oil. Poultry Sci. 39: 227-229. Van de Kamer, J. H., H. Ten Bokkel Huinink and H. A. Weyers, 1949. A rapid method for the determinination of fat in feces. J. Biol. Chem. 212: 347-355. Waibel, P. E., O. J. Abbott, C. A. Baumann and H. R. Bird, 1954. Disappearance of the growth response of chicks to dietary antibiotics in an "old" environment. Poultry Sci. 33:1141-1146.
NEWS AND NOTES (Continued from page 1372) served as a member of the Committee. Dr. J. C. Huttar, Trumansburg, New York, Head of the Grange League Federation's Farm Management Department, was presented with the Industry Service Award. The Award is for service to the industry above and beyond self-gain. He has spent all his adult life in various phases of the poultry industry. The "extra curricular" projects in which he has been a leading figure in the poultry industry through the years are legion in number and high in importance. The industry was proud and pleased to see him receive this highly deserved recognition.
L. Campbell has been appointed Washington Representative for the Institute. This branch office opened about June 15. Joanne Alwes, Bloomington, Illinois, was named the winner of the annual Story Award contest of the Junior Poultry and Egg Fact Finding Conference. She was the choice of the Conference sponsors for best reporting the personal value of her trip to Kansas City where she participated with 72 other delegates from 22 states in the Annual Career Guidance program. Second place was accorded Larry Hock, Minden, Louisiana, with third place going to Nancy Coufal, Seward, Nebraska.
(Continued on page 1392)
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ACKNOWLEDGMENT This work was partly supported by a grant from the "Instituut tot aanmoediging van het wetenschappelijk onderzoek in nijverheid en landbouw" (I.W.O.N.L.).
1379
INTRODUCTION
The present investigations were performed to obtain additional information on the correlation between the growthpromoting action of various antibiotics such as penicillin, virginiamycin, chloramphenicol and chlortetracycline, and their effect on intestinal absorption of fats. MATERIALS AND METHODS
The composition of the basal diet is shown in Table 1. This diet corresponds closely with the diet no. 3 described by Stokstad et al. (1953). The regular diet
contains 3 % corn oil. Higher levels of corn oil were added at the expense of carbohydrates. In the low-fat diet, corn oil was omitted and replaced by 3 % carbohydrate. Antibiotics were added to a concentration of 100 p.p.m. Feces used for fatty acid analyses were collected on glass plates placed underneath the wire floors. Plates were removed daily and the feces were scraped off and dried under reduced pressure over phosphorpentoxide. Fatty acids were determined by the method of Van de Kamer etal. (1949). TABLE 1.—Composition of diet Grams Casein (crude) Gelatin Carbohydrate" Calcium triphosphate Calcium gluconate Salt mixture b Vitamins in glucose0 Corn oil and vitamin A, D, E d DL-methionine L-cystine Choline chloride
20 8 58.5 2 5 2 1 3 0.15 0.15 0.2
a Sucrose, cornstarch, glucose, or a mixture of equal parts of glucose and fructose. b 600 mg. sodium chloride; 600 mg. dipotassium phosphate; 450 mg. monopotassium phosphate; 250 mg. magnesium sulfate monohydrate; 40 mg. manganese sulfate; 50 mg. ferric citrate; 2 mg. cupric sulfate; 0.6 mg. potassium iodide; 0.8 mg. potassium bromide; 1.4 mg. zinc acetate; 1.6 mg. aluminium sulfate; 0.4 mg. cobalt acetate tetrahydrate; 0.2 mg. nickel sulfate; 0.05 mg. sodium molybdate. Water to dissolve trace minerals up to 2 g. 0 1 mg. thiamine hydrochloride; 1 mg. riboflavin; 5 mg. niacinamide; 1 mg. pyridoxine; 5 mg. calcium pantothenate; 0.5 mg. pteroyl-glutamic acid; 0.5 mg. vitamin K; 0.02 mg. biotin; 5 meg. vitamin B12; 100 mg. inositol; glucose up to 1 g. d 1,500 U. vitamin A acetate; 200 U. vitamin D 3 ; 10 mg. vitamin E acetate; corn oil up to 3 g.
1373
Downloaded from http://ps.oxfordjournals.org/ at Wayne State University on April 13, 2015
I
N A recent study from this laboratory, on the mode of action of antibiotics as growth stimulants for young animals, a transitory period of growth depression was observed around the 8th day of life in chicks fed an antibiotic-free, caseinsucrose diet (Eyssen and De Somer, 1963). The inhibition of weight gain was found to be associated with decreased efficiency of feed utilization and malabsorption of fats and carbohydrates, and the syndrome was most pronounced when sucrose was fed as the sole source of dietary carbohydrate. Addition to the feed of 20 p.p.m. of virginiamycin, an antibiotic active only against Gram-positive microorganisms, resulted in improved intestinal absorption and stimulated growth. As a result of these studies, the hypothesis was proposed that the growth-stimulating effect of antibiotics is most likely due to suppression of intestinal Gram-positive microorganisms which interfere with the absorption of nutrients.
1374
H. EYSSEN AND P. D E SOMER
mainly to Gram-positive microorganisms. In this respect it is similar to virginiao mycin. Chloramphenicol is a broad spec9 -o Chlortetracycline trum antibiotic which generally fails to o o Penicillin elicit a positive growth response, whereas • o Virginiamycin chlortetracycline is a widely used broad spectrum growth stimulant. In previous experiments, chlortetracycline regularly gave a growth increase of 15 to 25% but lost its activity after it was in use for 6 months. This is in agreement with other reports that continuous feeding of an antibiotic results in a decrease in the growth response (Waibel et al., 1954; Libby and Schaible, 1955). L, 6 8 10 12 Figure 1 illustrates the evolution of the Age of chicks in days daily weight increase, and Table 2 sumFIG. 1. Effect of 100 p.p.m. of various antibiotics marizes the body weight data. In the on daily weight gain of chicks fed a casein-sucrose control animals a growth depression is obdiet. served after the 5th day. Weight gain reAll experiments were carried out in mains low for 4 days, and thereafter tends thermostatically controlled conventional to become normal. Procaine penicillin and battery brooders with raised wire screen virginiamycin stimulate growth during floors under continuous illumination, feed this critical period while chloramphenicol and water being supplied ad libitum. and chlortetracycline are ineffective. Dropping trays were removed daily. All An example of the evolution of the fecal animals were one-day-old broiler chicks of fat excretion is presented in Figure 2. mixed sexes obtained from a commercial During the critical period from the 6th hatchery. Each experimental group con- to the 9th day of the experiment, chicks sisted of 2 replicates of 10 to 12 chicks, 120 and the experimental diets were fed from • — • Control = no antibiotics o—© Chloramphenicol the first day on. Only short term experio—« Chlortetracycline „ioo o—o Penicillin ments over a 2 week period were perVirginiamycin formed. " C o n t r o l : no antibiotics o Chloramphenicol
1. Effect of antibiotics on growth and on fat absorption. To establish whether the growth-promoting effect of various antibiotics is correlated with their influence on fat absorption, a first series of experiments with a casein-sucrose diet compared the effect of virginiamycin with that of penicillin, chloramphenicol, and chlortetracycline. Penicillin was chosen because it is a potent growth-stimulating agent with an antibacterial spectrum restricted
-80
"60
-•2 4 0 -
n20
Age
4 of
6 chicks
8 in
10 days
K
FIG. 2. Effect of 100 p.p.m. of various antibiotics on the fecal fat excretion of chicks fed a caseinsucrose diet.
Downloaded from http://ps.oxfordjournals.org/ at Wayne State University on April 13, 2015
RESULTS
1375
EFFECT OF ANTIBIOTICS
TABLE 2.—Growth-promoting effect of various antibiotics on chicks fed a purified casein-sucrose diet
Diet Basal diet +Virginiamycin lOOp.p.m. +Chlortetracycline lOOp.p.m. +Proc. penicillin 100 p.p.m. +Chloramphenicol 100 p.p.m. :
No. of chicks 24 24 23 23 22
Average body weight (gm.) Start
2 Weeks
% Weight increase over basal
34 34 34 34 34
130 150 133 147 130
+20.8 + 3.5* + 18.3 + 0
Not significantly different at 0.05 level of probability (p>0.05).
2. Effect of various dietary carbohydrates. Stokstad el al. (1953) reported that an antibiotic-free sucrose diet did not support as rapid growth as an antibiotic-free starch diet, although the sucrose diet supplemented with chlortetracycline resulted in better growth than did the starch diet containing the antibiotic. Chloretracycline was virtually inactive when added to a starch diet. The results of our studies on the growth-promoting effect of virginiamycin were in agreement with these data (Eyssen et al., 1962). Since Borgstrom el al. (1959) found that the intestinal flora of the rat inactivates invertase but not amylase, several experiments were performed to establish whether
a similar mechanism could explain the observed differences between the two carbohydrates in the chick. Therefore, glucose and fructose—the hydrolytic products of sucrose—were substituted for the intact disaccharide. Table 3 shows that the growth rate on the diets without antibiotics was not improved when sucrose was replaced by the glucose-fructose mixture, but that glucose alone led to significantly better growth. Consequently, in the absence of antibiotics the fructose moiety of the sucrose molecule seems to be less well utilized by the chick, perhaps because it is more slowly resorbed from the intestine (Bogner, 1961) thus remaining available for bacterial fermentation. Although the basal glucose diet supported growth almost comparable to that obtained with a starch diet, it was still further improved by virginiamycin, whereas a starch diet was not. The evolution of the fecal fat excretion throughout these experiments is illustrated in Figure 3. Glucose, sucrose, and glucose-fructose provoke a peak of malabsorption of fats from the 5th to the 9th day of the experiment, and this period of steatorrhea is suppressed by 100 p.p.m. of virginiamycin. These experiments provide additional evidence for a close correlation between the activity of antibiotics as growth stimulants and their effect on intestinal absorption of nutrients. Dietary composi-
Downloaded from http://ps.oxfordjournals.org/ at Wayne State University on April 13, 2015
receiving a sucrose diet without antibiotics lost 70 to 90 mg. fatty acids per gram dried feces. Comparison of Figures 1 and 2 shows that only the two growthpromoting antibiotics penicillin and virginiamycin reduced the fecal fat excretion to about 40 mg. per gram. Chloramphenicol and chlortetracycline did not reduce fecal fat loss and, concomitantly, they were ineffective in stimulating growth. Although it still remains to be elucidated why the malabsorption does not develop until the 5th or the 6th day and why spontaneous recovery occurs after several days, our observations suggest that antibiotics stimulate growth of chicks particularly for a few days around the 8th day of life.
1376
H. EYSSEN AND P. D E SOMER TABLE 3.—KJJect of the type of dietary carbohydrate on growth of chicks and on growth-stimulating effect of virginiamycin No. of chicks
Type of diet
Start
2 weeks
% Weight increase over basal
Basal sucrose +virginiamycin 100 p.p.m.
24 24
37 37
133 156
+23.9
Basal glucose-fructose +virginiamycin 100 p.p.m.
22 22
37 37
126 148
+24.7
Basal glucose +virginiamycin 100 p.p.m.
24 24
37 37
148 163
+ 13.5
Basal starch +virginiamycin 100 p.p.m.
24 24
37 37
152 155
+ 2.6*
Not significantly different at 0.05 level of probability (p>0.05).
tions containing low molecular carbohydrates result in high fecal fat excretion and are significantly improved by virginiamycin. Little or no malabsorption is observed when a starch diet is fed, and in this case the antibiotic fails to stimulate growth significantly. 3. Effect of different fat levels. The diets used in the foregoing experiments contained 3 % corn oil as the sole source of fat. Since in birds fed the antibiotic-free
sucrose diet from 30 to 50% of the dietary fat is lost during the period of malabsorption and since the chick is known for its high linoleic acid requirements (Machlin and Gordon, 1961), it seemed by no means impossible that virginiamycin stimulates growth not only by improving total nutrient absorption, but also by alleviating specific linoleic acid deficiency symptoms. In this case, however, one should expect a decreased activity of the antibiotic when higher levels of corn oil are fed.
- • Sucrose diet o--o Glucose diet ®~© Glucose - levulose • - • • Starch diet
100p.p.m.Virginiamycin added
2
4
6
8
10 12 tt 2 4 Age of chicks in days
6
8
10
FIG. 3. Effect of type of dietary carbohydrate on fecal fat excretion in chicks.
12
u
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:
Average body weight (gm.)
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EFFECT OF ANTIBIOTICS TABLE 4.—Influence of increasing dietary fat levels on chick growth and response to 100 p.p.m. of virginiamycin
Type of diet
Averages body weight (gm.)
No. of chicks
Start
2 weeks
Gain due to Gain due to antibiotic fat supplement (gm.) (gm-)
Basal sucrose +virginiamycin
0 0
24 24
36 36
108 127
+ 19
Basal sucrose +virginiamycin
1 1
24 24
36 36
120 139
+ 19
+ 12 + 12
Basal sucrose +virginiamycin
3 3
24 24
36 36
136 151
+ 15
+28 +24
Basal sucrose +virginiamycin
6 6
24 23
36 36
135 154
+ 19
+27 +27
Basal starch +virginiamycin
0 0
24 24
34 34
134 133
-
Basal starch +virginiamycin
1 1
24 24
34 34
136 141
+ 5*
+ 2* + 8*
Basal starch +virginiamycin
3 3
23 24
34 34
143 147
+ 4*
+ 9 + 14
Basal starch +virginiamycin
6 6
24 24
34 34
145 136
9*
,
1*
+ 11 + 3*
Not significantly different at 0.05 level of probability (p>0.05).
Whereas growth on the basal sucrose diet was significantly improved by increasing the corn oil from 0 to 3%, no additional weight gain resulted from the 6% corn oil ration (Table 4) and in every case the response to virginiamycin was virtually unaffected by the dietary fat level. The effect of increasing fat levels was less pronounced on a starch diet, although a small weight increase was noted. Table 4 also shows that chicks grew as fast on a fat-free starch diet as on an antibiotic-free 6% corn oil sucrose diet. This would indicate that, at least during the first 2 weeks of the chicks life, the growthpromoting effect of antibiotics is not due to a therapeutic effect on an essential fatty acid deficiency. DISCUSSION Numerous workers have reported that the growth-promoting effect of antibiotics
is most often associated with an increased efficiency of feed utilization and that antibiotics under certain dietary conditions exert a sparing action on vitamins, minerals, and proteins. These findings might be explained by an antimicrobial effect of the antibiotics upon Gram-positive intestinal bacteria. In animals given an antibiotic-free diet these microorganisms could provoke a polymalabsorption syndrome including not only vitamins and minerals but also proteins, fats and carbohydrates. Our previous work with the antibiotic virginiamycin supported this hypothesis (Eyssen and De Somer, 1963). Further evidence is provided by the present investigations in which a close correlation has been observed between the activity of antibiotics as growthpromoting agents and their influence on fat absorption. Growth rate was increased by penicillin and virginiamycin, and these
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:
Corn oil %
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H. E Y S S E N AND P . D E SOMER
T h e activity of penicillin and virginiamycin, which are active only against Gram-positive microorganisms proves t h a t the causative microorganism is a Gram-positive microbe. T h e bacterial flora in the proximal intestinal segments of the young chick consists mainly of lactobacilli and enterococci. Nevertheless, preliminary unpublished experiments on monocontaminated birds indicate t h a t neither a pure flora of lactobacilli nor a monoflora of enterococci reduces chick growth, and t h a t those floras do not provoke malabsorption of fats. F u r t h e r studies on chicks monocontaminated with other intestinal microorganisms are in progress. Although a growth response was obtained to 1 % and 3 % corn oil, no further increase in growth was observed a t the 6 % corn oil level either on a sucrose or on a starch diet, and in every case the growth response to antibiotics was independent of the dietary fat level and was influenced only by the type of carbohydrate. Nevertheless D a m et al. (1959) observed improved chick growth when the dietary fat was increased from 3 % to 1 0 % . E m p h a sis must be placed, however, on the statement b y Donaldson et al. (1957), t h a t for satisfactory use of high fat levels the ration must be properly balanced in respect to other nutrients. Perhaps minor differences in the dietary composition
might explain the varying responses to fat supplements. A striking feature is the excellent fat tolerance on a starch diet, whereas animals receiving a sucrose, glucose or glucosefructose diet, lost considerable quantities of fat. These experiments clearly prove t h a t fat absorption studies m a y be profoundly influenced b y the age of the test animals, the type of dietary carbohydrate, and the presence or absence of antibiotics. This is in agreement with the studies of Supplee (1960) and Donaldson (1962), who felt t h a t the microflora status possibly influences studies of dietary fat effects in poults and chicks. T h e importance of intestinal bacteria in fat absorption is also known from h u m a n cases of steatorrhea and has been further illustrated in work from this laboratory with blind-loop rats (Hoet et al., 1962). These studies showed t h a t rats with an artificial cecum on the proximal jejunum excreted twice as much dietary fat as did their controls. In these experiments, too, the carbohydrate composition of the diet influenced fat absorption, and the fecal fat loss could be brought down to controlvalues by the administration of virginiamycin b u t not b y chlortetracycline.
SUMMARY A close correlation between the activity of antibiotics as growth promotants and the influence of the antibiotics on intestinal fat absorption has been observed in chicks fed purified diets. Only those antibiotics which reduced fecal fat excretion improved growth. Basal diets containing sucrose, glucose, or glucose-fructose as the sole source of carbohydrate resulted in high fecal fat excretion and could be improved by antibiotics. With a corn starch diet, however, little or no malabsorption was observed and this diet
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two antibiotics also reduced fecal fat excretion. Chlortetracycline and chloramphenicol were without effect on fecal fat and failed to stimulate growth. On the other hand, the basal diets containing low molecular carbohydrates resulted in high fecal fat excretion and were significantly improved by the addition of virginiamycin. Little or no malabsorption was observed when a corn starch diet was fed, and in this case virginiamycin did not stimulate growth.
EFFECT OF ANTIBIOTICS
was not improved by the addition of antibiotics. These investigations provide further evidence that the growth-stimulating effect of antibiotics is most likely due to suppression of Gram-positive intestinal bacteria which interfere with the absorption of nutrients.
REFERENCES Bogner, P. H., 1961. Alimentary absorption of reducing sugars by embryos and young chicks. Proc. Soc. Exp. Biol. Med. 107: 263-265. Borgstrom, B., A. Dahlqvist, B. E. Gustafsson, G. Lundh and J. Malmquist, 1959. Trypsin, invertase and amylase content of feces of germfree rats. Proc. Soc. Exp. Biol. Med. 102: 154-155. Dam, R., R. M. Leach, T. S. Nelson, L. C. Norris and F. W. Hill, 1959. Studies on the effect of quantity and type of fat on chick growth. J. Nutrition, 68: 615-632. Donaldson, W. E., G. F. Combs, G. L. Romoser and W. C. Supplee, 1957. Studies on energy levels in poultry rations. 2. Tolerance of growing chicks to dietary fat. Poultry Sci. 36: 807-815. Donaldson, W. E., 1962. The response of chicks to dietary animal fat in new and old environments.
Poultry Sci. 41: 1106-1108. Eyssen, H., V. De Prins and P. De Somer, 1962. The growth-promoting action of virginiamycin and its influence on the crop flora in chickens. Poultry Sci. 41: 227-233. Eyssen, H., and P. De Somer, 1963. The mode of action of antibiotics in stimulating growth of chicks. J. Exp. Med. 117:127-138. Hoet, P. P., J. V. Joossens, E. Evrard, H. Eyssen and P. De Somer, 1962. Intestinal bacteria and fecal fat. Proc. Internat. Conference on the Biochemical Problems of Lipids, held at Birmingham, U. K., July 23-27. Elsevier Publishing Company, Amsterdam. In press. Libby, D. A., and P. S. Schaible, 1955. Observations on growth responses to antibiotics and arsonic acids. Science, 121: 733-734. Machlin, L. S., and R. S. Gordon, 1961. Effects of dietary fatty acids and cholesterol on growth and fatty acid composition of the chicken. J. Nutrition, 75: 157-164. Stokstad, E. L. R., T. H. Jukes and W. L. Williams, 1953. The growth-promoting effect of Aureomycin on various types of diet. Poultry Sci. 32:10541058. Supplee, W. C , 1960. The effect of antibiotic supplementation on the response of poults to dietary corn oil. Poultry Sci. 39: 227-229. Van de Kamer, J. H., H. Ten Bokkel Huinink and H. A. Weyers, 1949. A rapid method for the determinination of fat in feces. J. Biol. Chem. 212: 347-355. Waibel, P. E., O. J. Abbott, C. A. Baumann and H. R. Bird, 1954. Disappearance of the growth response of chicks to dietary antibiotics in an "old" environment. Poultry Sci. 33:1141-1146.
NEWS AND NOTES (Continued from page 1372) served as a member of the Committee. Dr. J. C. Huttar, Trumansburg, New York, Head of the Grange League Federation's Farm Management Department, was presented with the Industry Service Award. The Award is for service to the industry above and beyond self-gain. He has spent all his adult life in various phases of the poultry industry. The "extra curricular" projects in which he has been a leading figure in the poultry industry through the years are legion in number and high in importance. The industry was proud and pleased to see him receive this highly deserved recognition.
L. Campbell has been appointed Washington Representative for the Institute. This branch office opened about June 15. Joanne Alwes, Bloomington, Illinois, was named the winner of the annual Story Award contest of the Junior Poultry and Egg Fact Finding Conference. She was the choice of the Conference sponsors for best reporting the personal value of her trip to Kansas City where she participated with 72 other delegates from 22 states in the Annual Career Guidance program. Second place was accorded Larry Hock, Minden, Louisiana, with third place going to Nancy Coufal, Seward, Nebraska.
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ACKNOWLEDGMENT This work was partly supported by a grant from the "Instituut tot aanmoediging van het wetenschappelijk onderzoek in nijverheid en landbouw" (I.W.O.N.L.).
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