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The Comparative Effects of Various Antibiotics and an Arsenical upon the Growth, Health, and Certain Blood Constituents of Dairy Calves1,2
T I I E C O M P A R A T I V E E F F E C T S OF V A R I O U S A N T I B I O T I C S A N D AN ARSENICAL UPON THE GROWTH, HEALTH, AND CERTAIN B L O O D C O N S T I T U E N T S O F D A I R Y C A L V E S 1'2 F. G. OWEN, I-I. H. VOELKER, N. L. JACOBSON, AI~DR. S. ALLEN
Depart.merits of Animal Husbandry and Chemistry Iowa State College, Ames
The effects of antibiotic feeding to d a i r y animals have been summarized in recent articles (15, 22); therefore, extensive review seems unnecessary. Numerous investigators have observed t h a t aureomycin has a growth-stimulating effect on d a i r y calves, and several reports have a t t r i b u t e d a similar effect to t e r r a m y c i n . A slight growth stimulation f r o m bacitracin supplementation of calf rations also has been suggested (10, 27). Studies with p-amino phenyl arsonic acid indicated an i m p r o v e m e n t of hair coat and general a p p e a r a n c e of calves but growth effects were inconsistent (3, 11). Penicillin a p p e a r s to be ineffective in p r o m o t i n g calf growth (16, 21, 27). The occurrence of macrocytie anemia and leucopenia following the feeding of either penicillin or aureomycin to calves in amounts of 50 and 100 rag. per day, respectively, has been indicated in a recent r e p o r t f r o m D e n m a r k (18). However, no serious hematological reactions to antibiotic feeding have been observed in studies conducted in the United States with various species of animals. Therapeutic administration of chloromycetin has been suggested as an etiological agent in the development of certain blood dyscrasias in h u m a n beings (17). No report on the feeding of chloromycetin to r u m i n a n t s has been encountered; however, inhibition of in vitro cellulose digestion b y r m n e n microorganisms has been demonstrated (12, 28). The mechanism of antibiotic and arsenical action in growth stimulation is u n k n o w n ; nevertheless, the p r e p o n d e r a n c e of evidence suggests t h a t control of " d e b i l i t a t i n g i n f e c t i o n " m a y be involved (14). I n view of this hypothesis and the established relationship between blood morphology a n d infectious conditions. it was anticipated t h a t blood cell counts might be of value in the clarification of the mode of action of antibiotics and arsenicals. Since the state of c u r r e n t information r e g a r d i n g the physiological effects of antibiotics and arsenicals administered orally to calves is incomplete and inconclusive, the present s t u d y was u n d e r t a k e n to compare the effects of several of these substances on growth, feed utilization, health, blood cell numbers, hemoglobin, plasma f a t , and fecal p H . Received for publication February 7, 195.5. Journal Paper No. J-2695, Iowa Agricultural Experiment Station, Ames, Iowa. Project No. 814. " Supported in part through funds provided by Lederle .Laboratories Division, American Cyanamid Co., Pearl River, N. Y. 891
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F . G . OWEN E T A L
EXPERIMENTAL PROCEDURE
The studies reported herein were conducted in two experiments with 62 Holstein calves. Aureomycin, terramycin, and penicillin were evaluated in the first experiment and t e r r a m y c i n , bacitracin, chloromycetin, and 3-nitro-4-hydroxy phenyl arsonic acid were studied in the second2 I n E x p e r i m e n t I each group consisted of eight calves (four males, four females), and in E x p e r i m e n t I I each g r o u p w a s composed of six calves (three males, three females). The calves were p e r m i t t e d to remain with their dams for 3 days p o s t p a r t u m and subsequently were t r a n s f e r r e d to individual pens and were allotted r a n d o m l y to the respective groups. T h e r e a f t e r the q u a n t i t y of liquid fed was based on body weights at 4 days of age. Whole milk averaging 3.0% ( E x p e r i m e n t I ) a n d 2.8% (Experim e n t I I ) f a t was fed at the daily rates of 5.0 a n d 3.6 lb. per 100 lb. body weight d u r i n g the first and second weeks, respectively, of the experiments. A milk replacement, ~ containing p r i m a r i l y dried whey p r o d u c t reconstituted with water (14% milk replacement, 86% water), was fed at the following daily rates per 100 lb. body weight f r o m the first through the 7th weeks, respectively: 3.0, 5.4, 10, 8, 8, 6, and 4 lb. One half of the calves in E x p e r i m e n t I were fed milk replacement in two feedings daily (6 A.M., 4 P.M.) ; the other half received the same level in four feedings daily (6 A.~., 11 A.M., 4 P.~., 9 P.~.). I n E x p e r i m e n t I I all calves were fed twice daily (6 A.~., 4 P.~.). S t a r t e r 5 consumption was limited to a m a x i m u m of 2 lb. per day during the first 7 weeks a n d to a m a x i m u m of 4 lb. the 8th t h r o u g h the 12th week, when experimental feeding was concluded. Medium quality alfalfa h a y was provided ad libitum. The antibiotic supplements were fed at the rate of 40 rag. daily per calf in the milk replacement d u r i n g the initial 7 weeks ; t h e r e a f t e r 80 rag. daily were fed in the calf starter. The arsenical was administered in the fluid portion of the diet at the daily rate of 30 rag. through the 6th week of the experiment and in the starter t h r o u g h o u t the 12 weeks of the experiment at the rate of 30 rag. per pound. The intake of arsenical was a p p r o x i m a t e l y 57 p.p.m, total d r y m a t t e r in the ration. B o d y weights and measurements were t a k e n at the beginning of the experimental period. B o d y weights also were taken each week and other body measurements (height at withers, chest circumference, and barrel circumference) every 4 weeks d u r i n g the study. Additional weights were obtained at 4 and 8 weeks a f t e r t r e a t m e n t termination. Incidence and severity of diarrhea were estimated b y daily observations of fecal samples obtained b y manual stimulation of defecation. Rectal t e m p e r a t u r e s were taken daily. Samples of venous blood were drawn at weekly intervals for hematological observations, which included erythrocyte, 3 Aureomycin is the trade name for the antibiotic chlortetracycline, fed as Aurorae 2A and D; terramycin is the trade name for oxytetracyeline, fed as Bi-Con-TiY[-5; and chloromycetin is the trade name for ch]oramphenico], fed as the mycelial meal. Supplied through the courtesy of Western Condensing Co., Appleton, Wise. 5 Composed of 40% ground corn, 30% ground oats, 28% soybean oil meal, 1% steamed bone meal, and 1% iodized salt.
893
P H Y S I O L O G I C A L E F F E C T S OF A N T I B I O T I C S ON C A L V E S
t o t a l l e u c o c y t e , a n d d i f f e r e n t i a l l e u c o c y t e c o u n t s ( l y m p h o c y t e , n e u t r o p h i l , monocyte, eosinophi!, a n d b a s o p h i l ) ( 2 5 ) . I n E x p e r i m e n t I h e m o g l o b i n ( 2 5 ) , b l o o d p l a s m a f a t ( 1 ) a n d f e c a l p H also w e r e d e t e r m i n e d e a c h week. D u r i n g d i a r r h e a p H v a l u e s a n d d i f f e r e n t i a l leucocyte counts were obtained daily. RESULTS A s u m m a r y of t h e g r o w t h d a t a f o r t h e two e x p e r i m e n t s is p r e s e n t e d i n T a b l e 1. T h e m e a n b o d y w e i g h t g a i n s of t h e a u r e o m y c i n a n d t e r r a m y c i n g r o u p s in E x p e r i m e n t I e x c e e d e d those of the c o n t r o l s a t 12 weeks b y 51 a n d 5 5 % , r e s p e c t i v e l y . A g r o w t h s t i m u l a t i o n f r o m a u r e o m y c i n a n d t e r r a m y c i n was a p p a r e n t as TABLE 1 Effect of antibiotic and arsenical supplementation on weight gains and certain body measurements Weight gains to : Experimental Dietary Initial group" weight
Expt. I
C A T P
Expt. II
C B C1 AA T
Increasesin body meas-
Post-treatment
urements--O-12weeks
4 wk.
8 wk.
12 wk.
16 wk.
20 wk.
Wither height
Chest circ.
Barrel circ.
(lb.) 93 91 91 88
(lb.) 5 14 14 7
(lb.) 33 51 51 31
(lb.) 69 104 b 107 b 59
(lb.) 117 129 139 97
(lb.) 164 177 180 151
(in.) 2.9 4.8 5.0 3.6
(in.) 6.9 7.8 7.8 6.4
(in.) 15.6 16.6 16.7 14.8
80 92 88 92 88
8 12 12 15 11
32 38 39 41 41
65 82 72 83 88
101 112 106 125 118
156 162 144 167 160
3.6 3.7 3.3 4.2 4.4
5.9 7.1 7.8 7.9 7.5
14.7 15.8 15.1 16.2 15.3
a C = control, A ----aureomycin, T ----terramycin, P ----penicillin, B ----bacitracln, C1 ----chloromycetin, AA = 3-nltro-4-hydroxy phenyl arsonic acid. b Statistically significant (P -- 0.01) compared with the control and penicillin-fed groups in Expt. I. e a r l y as 28 d a y s of age. A n a l y s i s of v a r i a n c e of b o d y w e i g h t g a i n s to 88 d a y s of age i n d i c a t e d t h a t t h e calves f e d a u r e o m y c i n a n d t e r r a m y c i n h a d g a i n e d sign i f i c a n t l y more than other groups (P = 0.01). Gains of the penicillin-fed calves
averaged 14% less than those of the control group, a reduction of growth which approached statistical significance at P = 0.05. Weight gains of calves fed milk replacement four times per day were similar to those of calves fed twice daily. Mean body weight gains during the 12-week period for the chloromyeetin, b a e i t r a e i n , a r s o n i e acid, a n d t e r r a m y c i n g r o u p s of E x p e r i m e n t I I e x c e e d e d those of t h e c o n t r o l g r o u p b y 1 1 % , 2 6 % , 2 8 % , a n d 3 5 % , r e s p e c t i v e l y . D i f f e r e n c e s i n weight gains for the controls versus antibiotic- and arsenical-treated animals at 88 d a y s of age w e r e f o u n d to a p p r o a c h s t a t i s t i c a l s i g n i f i c a n c e ( P -----0.07). A f t e r d i s c o n t i n u a n c e of a n t i b i o t i c s u p p l e m e n t a t i o n t h e r e l a t i v e g r o w t h adv a n t a g e f o r a u r e o m y c i n a n d t e r r a m y c i n g r o u p s t e n d e d to d e c r e a s e i n r e l a t i o n to t h e o t h e r g r o u p s , t h e r e b y r e d u c i n g t h e e a r l i e r w e i g h t a d v a n t a g e ( T a b l e 1 ) . l~ates of g a i n f o r t h e p e n i c i l l i n a n d c o n t r o l g r o u p s e x c e e d e d those of o t h e r g r o u p s f o r t h e 8-week p o s t - t r e a t m e n t p e r i o d .
894
F.G.
O W E N E T AL
TABLE 2 Mean feed consumption and efficiency of feed utilization :Milk replacement b
Dietary group a
Whole milk
Expt. I
C A T P
54 54 55 53
(mean per calf, lb.) 280 289 284 274
Expt. II
C B C1 AA T
47 54 54 55 50
234 264 242 268 260
Hay
TDN (total all feeds)
TDN per lb. gain
]47 179 174 119
72 70 88 68
183 204 212 160
2.7 2.0 2.0 2.8
144 162 158 161 161
61 69 62 72 70
171 194 185 195 192
2.9 2.4 2.6 2.4 2.2
Starter
a C -- control, A = aureomycin, T = terramycin, P ----penicillin, B = bacitracin, C1 = chloromycetin, AA = 3-nitro-4-hydroxy phenyl arsonic acid. b Liquid milk replacement, 14% solids. Feed efficiency (calculated T D N (20) per p o u n d weight gain) was superior and starter consumption greatest in the groups which gained weight most rapidly (Table 2). Efficiency of feed utilization was improved significantly (P ----0.01) in Experiment I b y aureomycin and terramycin as compared with the penicillin and control groups. Comparison of all treated groups in E x p e r i m e n t I I with the controls also suggested (P = 0.09) an improvement in the efficiency of feed utilization. The arsenical-, terramycin-, and aureomycin-fed calves appeared to be more t h r i f t y and their hair coats were somewhat smoother than those of other calves at the conclusion of the supplemental feeding period. The appearance and vigor of the penicillin and chloromycetin calves seemed to be inferior to those of other treated groups. No treatment effects on blood constituents were found (Table 3). Since the trends were relatively consistent among groups and between experiments, the values for all groups were combined to establish normal curves for the conditions imposed in this s t u d y (Figures 1 and 2). A sudden drop in erythrocyte, leucocyte, and neutrophil counts d u r i n g the period from 2 to 5 weeks was typical of all groups. These values gradually increased d u r i n g the period from 6 to 12 weeks. Hemoglobin also exhibited age trends, the mean changes tending to parallel those for erythrocytes (Figure 1). Blood plasma lipid levels, as measured b y the method of Allen (1), also appeared unaffected by treatment (Experiment I). The values were relatively high at 4 days, decreased to minimum values at about 5 weeks, and subsequently rose gradually (Figure 1). Diarrhea appeared to be primarily of the noninfectious type, with greatest incidence and severity during the period of the 3rd through the 5th weeks (Figure 3). None of the experimental treatments employed were effective in relieving the laxative symptoms, but relief was sometimes obtained by t e m p o r a r y reduction of milk a n d / o r milk replacement intake.
---
--
---
C B
C1
AA T
Expt.
II
-~ ---
Total
9.2 9.5 9.2 8.9 9.3
8.9 9.0 8.7 9.0 8.4 8.3 8.2 8.6 8.4
9.4 8.6 9.5 9.3
(millions/e,mm.) (thousands~cram.)
Erythrocytes
70 70 68 75 72
69 70 67 72
(%)
Lymphoc yt e s
28 27 30 24 25
29 27 29 27
(%)
Neutrophils
Leucocytes
2 2 2 1 2
2 2 2 1
(%)
Monoe yt e s
~1 ~1 ~1 ~1 ~1
~1 ~1 ,~1 ~1
(%)
Eosino~ phils
~1 ~1 ~1 ~1 ~1
~1 ~1 ~1 ~1
(%)
Basophils
" C = control, A = a u r e o m y c i n , T = t e r r a m y c i n , P = p e n i c i l l i n , B = b a c i t r a e i n , C1 = c h l o r o m y e e t i n , A A = 3-nitro-4-hydroxy p h e n y l arsonic acid.
Expt. I
11.9 11.4 11.9 11.8
(g/lO0 ml.)
(mg/lO0 rot.)
89 84 86 80
Hemoglobin
Plasma (Allen) f a t levels
C A T P
Dietary group a
TABLE 3
Hematological data (mean of wecI~ly samples during 12 w~. period)
896
F.G. OWEN ET Ah
0 o
n,.
I
9.0
8.5
r~ z o -1I.-
_i
"7
8.0'
~
7.1" 12C
0 Z I00 ",J
6"
80
60 40 4
I 18
I 3Z
I, I 46 60 AGE, DAYS
I 74
I 88
FIG. 1. Mean weekly values for certain blood constituents (hemoglobin and blood fat, 32 calves; erythrocytes and leucocytes, 62 calves).
There was no relation between fecal pH and antibiotic feeding or severity of diarrhea (Experiment I). The mean fecal pH values for the respective groups were : control, 6.6 ; aureomycin, 6.5 ; terramycin, 6.6 ; penicillin, 6.8. DISCUSSION
The observed growth promotion from aureomycin and terramycin is in accord with previous work. Because of differences in experimental procedure it is difficult to compare the results from bacitracin supplementation in this study with those of other experiments. Practically identical gains for bacitracin and control groups were reported by Rusoff and Davis (23), whereas results of other studies (10, 27) indicate a tendency toward growth promotion by this antibiotic, as was observed in the present study. The observed effect of 3-nitro-4-hydroxy phenyl arsonic acid on calf growth corresponds to the results in swine (7, 26) and poultry (2, 4, 19) and is in agreement with the preliminary findings of Graf and Holdaway (11) using p-aminophenyl arsonic acid. The ineffectiveness of chloromycetin corroborates, in general, observations with other species (6, 9, 13, 26).
897
PHYSIOLOGICAL EFFECTS O~ ANTIBIOTICS ON CALVES
80
7¢
(D
w t..>-
6C
0
oiJ 3 w "-= 3,( J
NEU ~ D P H I L E S o
/
/
2O ~,,,,,
MONOCYTES
,., 2.0 (,,) I,i,I
EOSINOPHILES
~- 1.0 O.O l'O
BASOPHILES
0.0 4
I 18
I 52
I 46
I 60
I 74
I 88
AGE, DAYS FZG. 2. M e a n t r e n d s in differential leucocyte c o u n t s (62 calves).
x tu o
15
o
I0
~o o
5
z z
0
7.0 ~6.5 6.0 FECAL 5.5 4
I 18
pH I 32
I 46
I 60
I 74
I 88
A G E . DAYS
FIG. 3. M e a n t r e n d s i n diurrheu (62 calves) a n d f e c a l p H (32 calves).
898
F . G . O W E N ET AL
The failure of penicillin to stimulate growth substantiates earlier reports (16, 21, 27). The effectiveness of aureomycin, terramycin, baeitracin, and arsonic acid in improving feed efficiency appears to be the result of increased feed consumption with a concurrent increase in rate of gain and consequent lowered maintenance per pound of gain. A temporary reduction in rate of gain following discontinuance of the feeding of some of the antibiotics also has been observed by others (8, 10). This phenomenon may have resulted from an alteration in gastrointestinal microflora to which these animals could not adjust immediately. This alteration in growth rate would appear to add support to the hypothesis that an alteration of microflora is responsible for at least a part of the growth-stimulating effects of antibiotics. The absence of a reduced growth rate in the arsenical group following discontinuance of supplementation might indicate a different mode of action and/or a residual effect. The contrasting results of the hematological studies conducted in Denmark (18) and those of the present investigation are difficult to reconcile. If there are deleterious hematological effects from antibiotic supplementation, there apparently are important conditional influences. According to a popular theory of antibiotic action, benefits result from repression of low-grade infections. If such infections are reflected to a detectable extent by elevated white blood cell counts, an inverse relationship between response to antibiotics and white cell counts might be expected. In the present study, although growth responses to antibiotics were observed, there were no appreciable differences among groups in white blood cell counts. Any attempt to explain the characteristic trends of blood cell counts certainly would be tenuous. It seems possible, however, that the peculiar segment of the leucocyte curve noted between the 2nd and 6th weeks might be associated with the amount of diarrhea prevailing during this period. Since increased numbers of lymphocytes in the intestinal wall would be anticipated, large fluid losses through these tissues may possibly produce excessive losses of these cells (29). The age trends in hemoglobin levels noted in the present study also have been observed by Thomas et al. (24); however, the minimum values reported by them were much lower. A number of workers have noted a reduced incidence and/or severity of diarrhea of antibiotic-fed calves. Since none of the supplements used in this study controlled the diarrhea incurred, it might be proposed that this laxation was of noninfectious origin and that the antibiotics and arsenical employed are ineffective in controlling this type of scours. Blaxter et al. (5) reported pH values in normal calves of 6.8 as compared with 6.2 in " l o o s e " calves and 6.0 in calves with severe diarrhea. These findings were not supported by data of the present study (Figure 3). The apparent superiority of the 3-nitro-4-hydroxy phenyl arsonic acid in maintaining an increased rate of gain during the post-treatment period would indicate a need for additional investigations to (a) supplement the data herein reported regarding its growth-promoting potential, (b) determine if this arseni-
PtfYSIOLOGICAL EFFECTS OF ANTIBIOTICS ON CALVES
899
eal will interact with antibiotics in calf growth stimulation, (c) determine whether a mechanism different from that of antibiotics is involved in response to arsenicals, and (d) ascertain whether there is any residual effect after removal of the al-senical from the diet. Since antibiotic-fed calves tend to lose much of their growth advantage sub:sequent to cessation of antibiotic feeding, the practicality of supplementing calf rations might be questioned. However, the rapid early growth, increased feed consumption, and improved feed efficiency may be of real value in reducing unthriftiness, "debilitating infection," and even the high mortality that is often encountered in raising dairy calves. SUM/~ARY
The comparative effects of feeding aureomycin, terramycin, and penicillin (Experiment I) and terramycin, bacitracin, ehloromycetin, and 3-nitro-4-hydroxy phenyl arsonic acid (]~xperiment II) to dairy calves were evaluated. Mean body weight gains over a 12-week period as per cent of the mean gains of the controls were: Experiment I, terramycin, 155%; aureomycin, 151%; and penicillin, 86% ; Experiment II, terramycin, 135% ; arsonic acid, 1.28% ; bacitraein, 126% ; and chloromyeetin, 111%. The treatments that stimulated growth also improved efficiency of feed utilization. The most rapidly gaining groups also attained greater height at withers and appeared somewhat superior in condition and hair coat at completion of the experiment; however, these differences were not marked. No pronounced ration effects were noted oll incidence and severity of diarrhea, although both were relatively high during weeks 3 to 5, inclusive. Blood cell counts (erythrocytes, leucocytes, and differential leucocytes), hemoglobin, plasnm fat, and fecal pH appeared to be unaffected by antibiotic or arsenical supplementation. Also, there was no apparent effect of diarrhea on fecal pI~. Rather marked age trends in the blood constituents were noted. ACK~qOWLEDGMENTS The ~uthors wish to express their appreciation to R. F. Elliott of Lederle Laboratories Division, American Cyanamia Co., Pearl l¢iver, N. Y., for the Aurofac 2A and D; to H. G. Luther of Chas. Pfizer and Co., Inc., Brooklyn, N. Y., for the Bi-Con-TM-5; to L. Michaud, o f Merck and Co., Rahway, N. J., for the procaine penicillin G; to F. E. Eads, Parke Davis and Co., Detroit, Mich., for the chloromycetin mycelial meal; to K. E. Passoth, S. B. Penick and Co., New York, N. ¥., for the bacitracin; and to T. W. Zbornik, Dr. Salsbury's Laboratories, Charles City, Iowa, for the 3-nitro-4-hydroxy pheny] arsonic acid. REFERENCES (1) ALLEN, N. N. A Simple Volumetric ~ e t h o d for Determination of F a t in Blood Plasma. Proc. Soe. Expt. Biol. Med., 31" 991. 1934. (2) A!XTDEaSON,G. W., CU~INGHA~, J. D., AND SLINGEr, S. ,]-. Effect of Terramycin and Certain Phenylarsonie Acid Derivatives on the Growth and Intestinal Flora of Turkey Poults. J. Nutrition, 48: 539. 2952. (3) BARTLEY, E. E. Personal communication. Kansas State College, Manhattan. 1954. (4) BIRD, H. R., GaOSCHI
900
F.G. OWEN ET AL
(6) ]3RANION, H. D., AND HILL, D. C. The Comparative Effect of Antibiotics on the Growth of PouRs. Poultry Sci., 30: 793. 1951. (7) CARPENTER, L. E. The Effect of 3-Nitro-4-Hydroxy Phenyl Arsonic Acid on the Growth of Swine. Arch. Biochem. and Biophys., 32: 181. 1951. (8) EDO~LY, C. G. M. Antibiotics for Dairy Calves. The Ayrshire Digest, 39: 853. 1953. (9) ELAI~, J. F., JACOBS, R. L., FOWLER, JEAN, HALE, F., AND COUCH, J. R. Effect of Feedi n g Chloromycetin Mycelial Meal upon the Growth and Fecal Microflora of Swine. J. Animal Sci., 12: 819. 1953. (10) ELLSW0aTH, S. A., ItUFF~AN, C. F., S~I~H, C. K., AND RALSTON, N. P. Effect of Feeding Antibiotics to Dairy Calves: I. Aureomycin and ]3acitracin Feed Supplements. Mich. Agr. Expt. Sta., Quart. Bull. 36: 60. 1953. (11) GRakF, G. C., AND HOLDAWAY, C. W. The Value of Arsonic Acid Derivatives as a Growth Stimulant when Fed to Calves. (Abs.) J. Dairy Sci., 35: 492. 1952. (12) HAI~DIE, W. E., DOWNING, H. E., REYNOLDS, W. M., AND LUTttEI~, H. G. Antibiotics in Ruminants. I n Antibiotics-Annual, 1953-1954. p. 372. Medical Encyclopedia, Inc., New York. 1953. (13) HEUSEE, G. F., AND Noaais, L. C. Some Results of Feeding Antibiotics to Chickens. Poultry Sci., 31: 857. 1952. (14) JuK~S, T. H., AND WILLIAMS, W. L. Nutritional Effects of Antibiotics. Pharm. ]¢ev., 5: 381. 1953. (15) KNOb% C. ]3. Antibiotics in the Growth of Ruminant Animals. Antibiotics and Chemotherapy, 3: 442. 1953. (16) KNO~)T, C. B., AND ]3LOOM, S. The Value of Vitamin BI:, DL-Methionine and PotassiumPenicillin in Milk Replacement Formulas for Dairy Calves. J. Dairy Sci., 35: 675. 1952. (17) LEWIS, C. N., PUTNAM, L. E., ~IENDtCICKS, F. D., KERLAN, I., AND WELCH, H. Chloramphenicol (Chloromycetin) in Relation to Blood Dyscrasias with Observations on other Drugs. Antibiotics and Chemotherapy, 2: 601. 1952. (18) MOLLGAARD,H. The New F o o d s - - A Warning from Abroad. Farmer and Stock-Breeder, 67: 43. 1953. (19) M0~EHO~SE, N. F. Accelerated Growth in Chickens and Turkeys Produced by 3-Nitro-4Hydroxyphenylarsonic Acid. Poultry Sci., 28: 375. 1949. (20) MOEEISON, F. B. Feeds and Feeding. 21st ed. The Morrison Publ. Co., Ithaca, N. Y. 1948. Appendix Table I. p. 1086. (21) MomxISON, S. H., DEAL, J. F., ANn DALTON, It. L. Effect of Feeding Penicillin to Dairy Calves. Georgia Agr. Expt. Sta., Mimeo. Series No. 2. 1954. (22) REID, J. T., WAI~NER, R. G., AND LOOSLI, J. K. Antibiotics in the 1N~utrition of Ruminants. Agr. and Food Chem., 2: 186. 1954. (23) RUSOFF, L. L., AND DAVIS, A. V. Antibiotic Studies with Dairy Calves--Tyrothricin and ]3acitracin. (Abs.) Paper presented at Assoc. of Southern Agr. Workers Meeting, Atlanta, Ga. 1952. (24) THOMAS, J. W., OKA~OT0, M., JACOBSON, W. C., AND M00I~E, L. A. A Study of Hemoglobin Levels in the Blood of Young Dairy Calves and the Alleviation of Anemia by Iron. J. Dairy Sci., 37: 805. 1954. (25) TODD, J. C., AND SANF0aD, A. It. Clinical D~agnosis by Laboratory Methods. 11th ed. W. B. Saunders Co., Philadelphia. 1948. (26) W~LLACE, H. D., NEY, W. A., AND CUNHA, T. J. Various Antibiotics and 3-Nitro-4Hydroxyphenyl Arsonic Acid in Corn-Peanut Meal Rations for Swine. Proc. Soc. J~xptl. Biol. Med., 78: 807. 1951. (27) WARNS, R. G. Recent Findings on Antibiotics and Surfactants for Ruminants. Proc. Corneal Nutrition Conference. Ithaca, N. Y. p. 126. 1952. (28) WASSEt~]V[AN,R. H., DUNCAN, C. W., CHURCHILL, E. S., AND HUI~F~AN, C. ~. The Effect of Antibiotics on in vitro Cellulose Digestion by Rumen Microorganisms. J. Dairy Sci., 35: 571. 1952. (29) WINTROBE, M. M. Clinical Hematology. 2nd ed. Lea and Febiger. Philadelphia. 1946.
Depart.merits of Animal Husbandry and Chemistry Iowa State College, Ames
The effects of antibiotic feeding to d a i r y animals have been summarized in recent articles (15, 22); therefore, extensive review seems unnecessary. Numerous investigators have observed t h a t aureomycin has a growth-stimulating effect on d a i r y calves, and several reports have a t t r i b u t e d a similar effect to t e r r a m y c i n . A slight growth stimulation f r o m bacitracin supplementation of calf rations also has been suggested (10, 27). Studies with p-amino phenyl arsonic acid indicated an i m p r o v e m e n t of hair coat and general a p p e a r a n c e of calves but growth effects were inconsistent (3, 11). Penicillin a p p e a r s to be ineffective in p r o m o t i n g calf growth (16, 21, 27). The occurrence of macrocytie anemia and leucopenia following the feeding of either penicillin or aureomycin to calves in amounts of 50 and 100 rag. per day, respectively, has been indicated in a recent r e p o r t f r o m D e n m a r k (18). However, no serious hematological reactions to antibiotic feeding have been observed in studies conducted in the United States with various species of animals. Therapeutic administration of chloromycetin has been suggested as an etiological agent in the development of certain blood dyscrasias in h u m a n beings (17). No report on the feeding of chloromycetin to r u m i n a n t s has been encountered; however, inhibition of in vitro cellulose digestion b y r m n e n microorganisms has been demonstrated (12, 28). The mechanism of antibiotic and arsenical action in growth stimulation is u n k n o w n ; nevertheless, the p r e p o n d e r a n c e of evidence suggests t h a t control of " d e b i l i t a t i n g i n f e c t i o n " m a y be involved (14). I n view of this hypothesis and the established relationship between blood morphology a n d infectious conditions. it was anticipated t h a t blood cell counts might be of value in the clarification of the mode of action of antibiotics and arsenicals. Since the state of c u r r e n t information r e g a r d i n g the physiological effects of antibiotics and arsenicals administered orally to calves is incomplete and inconclusive, the present s t u d y was u n d e r t a k e n to compare the effects of several of these substances on growth, feed utilization, health, blood cell numbers, hemoglobin, plasma f a t , and fecal p H . Received for publication February 7, 195.5. Journal Paper No. J-2695, Iowa Agricultural Experiment Station, Ames, Iowa. Project No. 814. " Supported in part through funds provided by Lederle .Laboratories Division, American Cyanamid Co., Pearl River, N. Y. 891
892
F . G . OWEN E T A L
EXPERIMENTAL PROCEDURE
The studies reported herein were conducted in two experiments with 62 Holstein calves. Aureomycin, terramycin, and penicillin were evaluated in the first experiment and t e r r a m y c i n , bacitracin, chloromycetin, and 3-nitro-4-hydroxy phenyl arsonic acid were studied in the second2 I n E x p e r i m e n t I each group consisted of eight calves (four males, four females), and in E x p e r i m e n t I I each g r o u p w a s composed of six calves (three males, three females). The calves were p e r m i t t e d to remain with their dams for 3 days p o s t p a r t u m and subsequently were t r a n s f e r r e d to individual pens and were allotted r a n d o m l y to the respective groups. T h e r e a f t e r the q u a n t i t y of liquid fed was based on body weights at 4 days of age. Whole milk averaging 3.0% ( E x p e r i m e n t I ) a n d 2.8% (Experim e n t I I ) f a t was fed at the daily rates of 5.0 a n d 3.6 lb. per 100 lb. body weight d u r i n g the first and second weeks, respectively, of the experiments. A milk replacement, ~ containing p r i m a r i l y dried whey p r o d u c t reconstituted with water (14% milk replacement, 86% water), was fed at the following daily rates per 100 lb. body weight f r o m the first through the 7th weeks, respectively: 3.0, 5.4, 10, 8, 8, 6, and 4 lb. One half of the calves in E x p e r i m e n t I were fed milk replacement in two feedings daily (6 A.M., 4 P.M.) ; the other half received the same level in four feedings daily (6 A.~., 11 A.M., 4 P.~., 9 P.~.). I n E x p e r i m e n t I I all calves were fed twice daily (6 A.~., 4 P.~.). S t a r t e r 5 consumption was limited to a m a x i m u m of 2 lb. per day during the first 7 weeks a n d to a m a x i m u m of 4 lb. the 8th t h r o u g h the 12th week, when experimental feeding was concluded. Medium quality alfalfa h a y was provided ad libitum. The antibiotic supplements were fed at the rate of 40 rag. daily per calf in the milk replacement d u r i n g the initial 7 weeks ; t h e r e a f t e r 80 rag. daily were fed in the calf starter. The arsenical was administered in the fluid portion of the diet at the daily rate of 30 rag. through the 6th week of the experiment and in the starter t h r o u g h o u t the 12 weeks of the experiment at the rate of 30 rag. per pound. The intake of arsenical was a p p r o x i m a t e l y 57 p.p.m, total d r y m a t t e r in the ration. B o d y weights and measurements were t a k e n at the beginning of the experimental period. B o d y weights also were taken each week and other body measurements (height at withers, chest circumference, and barrel circumference) every 4 weeks d u r i n g the study. Additional weights were obtained at 4 and 8 weeks a f t e r t r e a t m e n t termination. Incidence and severity of diarrhea were estimated b y daily observations of fecal samples obtained b y manual stimulation of defecation. Rectal t e m p e r a t u r e s were taken daily. Samples of venous blood were drawn at weekly intervals for hematological observations, which included erythrocyte, 3 Aureomycin is the trade name for the antibiotic chlortetracycline, fed as Aurorae 2A and D; terramycin is the trade name for oxytetracyeline, fed as Bi-Con-TiY[-5; and chloromycetin is the trade name for ch]oramphenico], fed as the mycelial meal. Supplied through the courtesy of Western Condensing Co., Appleton, Wise. 5 Composed of 40% ground corn, 30% ground oats, 28% soybean oil meal, 1% steamed bone meal, and 1% iodized salt.
893
P H Y S I O L O G I C A L E F F E C T S OF A N T I B I O T I C S ON C A L V E S
t o t a l l e u c o c y t e , a n d d i f f e r e n t i a l l e u c o c y t e c o u n t s ( l y m p h o c y t e , n e u t r o p h i l , monocyte, eosinophi!, a n d b a s o p h i l ) ( 2 5 ) . I n E x p e r i m e n t I h e m o g l o b i n ( 2 5 ) , b l o o d p l a s m a f a t ( 1 ) a n d f e c a l p H also w e r e d e t e r m i n e d e a c h week. D u r i n g d i a r r h e a p H v a l u e s a n d d i f f e r e n t i a l leucocyte counts were obtained daily. RESULTS A s u m m a r y of t h e g r o w t h d a t a f o r t h e two e x p e r i m e n t s is p r e s e n t e d i n T a b l e 1. T h e m e a n b o d y w e i g h t g a i n s of t h e a u r e o m y c i n a n d t e r r a m y c i n g r o u p s in E x p e r i m e n t I e x c e e d e d those of the c o n t r o l s a t 12 weeks b y 51 a n d 5 5 % , r e s p e c t i v e l y . A g r o w t h s t i m u l a t i o n f r o m a u r e o m y c i n a n d t e r r a m y c i n was a p p a r e n t as TABLE 1 Effect of antibiotic and arsenical supplementation on weight gains and certain body measurements Weight gains to : Experimental Dietary Initial group" weight
Expt. I
C A T P
Expt. II
C B C1 AA T
Increasesin body meas-
Post-treatment
urements--O-12weeks
4 wk.
8 wk.
12 wk.
16 wk.
20 wk.
Wither height
Chest circ.
Barrel circ.
(lb.) 93 91 91 88
(lb.) 5 14 14 7
(lb.) 33 51 51 31
(lb.) 69 104 b 107 b 59
(lb.) 117 129 139 97
(lb.) 164 177 180 151
(in.) 2.9 4.8 5.0 3.6
(in.) 6.9 7.8 7.8 6.4
(in.) 15.6 16.6 16.7 14.8
80 92 88 92 88
8 12 12 15 11
32 38 39 41 41
65 82 72 83 88
101 112 106 125 118
156 162 144 167 160
3.6 3.7 3.3 4.2 4.4
5.9 7.1 7.8 7.9 7.5
14.7 15.8 15.1 16.2 15.3
a C = control, A ----aureomycin, T ----terramycin, P ----penicillin, B ----bacitracln, C1 ----chloromycetin, AA = 3-nltro-4-hydroxy phenyl arsonic acid. b Statistically significant (P -- 0.01) compared with the control and penicillin-fed groups in Expt. I. e a r l y as 28 d a y s of age. A n a l y s i s of v a r i a n c e of b o d y w e i g h t g a i n s to 88 d a y s of age i n d i c a t e d t h a t t h e calves f e d a u r e o m y c i n a n d t e r r a m y c i n h a d g a i n e d sign i f i c a n t l y more than other groups (P = 0.01). Gains of the penicillin-fed calves
averaged 14% less than those of the control group, a reduction of growth which approached statistical significance at P = 0.05. Weight gains of calves fed milk replacement four times per day were similar to those of calves fed twice daily. Mean body weight gains during the 12-week period for the chloromyeetin, b a e i t r a e i n , a r s o n i e acid, a n d t e r r a m y c i n g r o u p s of E x p e r i m e n t I I e x c e e d e d those of t h e c o n t r o l g r o u p b y 1 1 % , 2 6 % , 2 8 % , a n d 3 5 % , r e s p e c t i v e l y . D i f f e r e n c e s i n weight gains for the controls versus antibiotic- and arsenical-treated animals at 88 d a y s of age w e r e f o u n d to a p p r o a c h s t a t i s t i c a l s i g n i f i c a n c e ( P -----0.07). A f t e r d i s c o n t i n u a n c e of a n t i b i o t i c s u p p l e m e n t a t i o n t h e r e l a t i v e g r o w t h adv a n t a g e f o r a u r e o m y c i n a n d t e r r a m y c i n g r o u p s t e n d e d to d e c r e a s e i n r e l a t i o n to t h e o t h e r g r o u p s , t h e r e b y r e d u c i n g t h e e a r l i e r w e i g h t a d v a n t a g e ( T a b l e 1 ) . l~ates of g a i n f o r t h e p e n i c i l l i n a n d c o n t r o l g r o u p s e x c e e d e d those of o t h e r g r o u p s f o r t h e 8-week p o s t - t r e a t m e n t p e r i o d .
894
F.G.
O W E N E T AL
TABLE 2 Mean feed consumption and efficiency of feed utilization :Milk replacement b
Dietary group a
Whole milk
Expt. I
C A T P
54 54 55 53
(mean per calf, lb.) 280 289 284 274
Expt. II
C B C1 AA T
47 54 54 55 50
234 264 242 268 260
Hay
TDN (total all feeds)
TDN per lb. gain
]47 179 174 119
72 70 88 68
183 204 212 160
2.7 2.0 2.0 2.8
144 162 158 161 161
61 69 62 72 70
171 194 185 195 192
2.9 2.4 2.6 2.4 2.2
Starter
a C -- control, A = aureomycin, T = terramycin, P ----penicillin, B = bacitracin, C1 = chloromycetin, AA = 3-nitro-4-hydroxy phenyl arsonic acid. b Liquid milk replacement, 14% solids. Feed efficiency (calculated T D N (20) per p o u n d weight gain) was superior and starter consumption greatest in the groups which gained weight most rapidly (Table 2). Efficiency of feed utilization was improved significantly (P ----0.01) in Experiment I b y aureomycin and terramycin as compared with the penicillin and control groups. Comparison of all treated groups in E x p e r i m e n t I I with the controls also suggested (P = 0.09) an improvement in the efficiency of feed utilization. The arsenical-, terramycin-, and aureomycin-fed calves appeared to be more t h r i f t y and their hair coats were somewhat smoother than those of other calves at the conclusion of the supplemental feeding period. The appearance and vigor of the penicillin and chloromycetin calves seemed to be inferior to those of other treated groups. No treatment effects on blood constituents were found (Table 3). Since the trends were relatively consistent among groups and between experiments, the values for all groups were combined to establish normal curves for the conditions imposed in this s t u d y (Figures 1 and 2). A sudden drop in erythrocyte, leucocyte, and neutrophil counts d u r i n g the period from 2 to 5 weeks was typical of all groups. These values gradually increased d u r i n g the period from 6 to 12 weeks. Hemoglobin also exhibited age trends, the mean changes tending to parallel those for erythrocytes (Figure 1). Blood plasma lipid levels, as measured b y the method of Allen (1), also appeared unaffected by treatment (Experiment I). The values were relatively high at 4 days, decreased to minimum values at about 5 weeks, and subsequently rose gradually (Figure 1). Diarrhea appeared to be primarily of the noninfectious type, with greatest incidence and severity during the period of the 3rd through the 5th weeks (Figure 3). None of the experimental treatments employed were effective in relieving the laxative symptoms, but relief was sometimes obtained by t e m p o r a r y reduction of milk a n d / o r milk replacement intake.
---
--
---
C B
C1
AA T
Expt.
II
-~ ---
Total
9.2 9.5 9.2 8.9 9.3
8.9 9.0 8.7 9.0 8.4 8.3 8.2 8.6 8.4
9.4 8.6 9.5 9.3
(millions/e,mm.) (thousands~cram.)
Erythrocytes
70 70 68 75 72
69 70 67 72
(%)
Lymphoc yt e s
28 27 30 24 25
29 27 29 27
(%)
Neutrophils
Leucocytes
2 2 2 1 2
2 2 2 1
(%)
Monoe yt e s
~1 ~1 ~1 ~1 ~1
~1 ~1 ,~1 ~1
(%)
Eosino~ phils
~1 ~1 ~1 ~1 ~1
~1 ~1 ~1 ~1
(%)
Basophils
" C = control, A = a u r e o m y c i n , T = t e r r a m y c i n , P = p e n i c i l l i n , B = b a c i t r a e i n , C1 = c h l o r o m y e e t i n , A A = 3-nitro-4-hydroxy p h e n y l arsonic acid.
Expt. I
11.9 11.4 11.9 11.8
(g/lO0 ml.)
(mg/lO0 rot.)
89 84 86 80
Hemoglobin
Plasma (Allen) f a t levels
C A T P
Dietary group a
TABLE 3
Hematological data (mean of wecI~ly samples during 12 w~. period)
896
F.G. OWEN ET Ah
0 o
n,.
I
9.0
8.5
r~ z o -1I.-
_i
"7
8.0'
~
7.1" 12C
0 Z I00 ",J
6"
80
60 40 4
I 18
I 3Z
I, I 46 60 AGE, DAYS
I 74
I 88
FIG. 1. Mean weekly values for certain blood constituents (hemoglobin and blood fat, 32 calves; erythrocytes and leucocytes, 62 calves).
There was no relation between fecal pH and antibiotic feeding or severity of diarrhea (Experiment I). The mean fecal pH values for the respective groups were : control, 6.6 ; aureomycin, 6.5 ; terramycin, 6.6 ; penicillin, 6.8. DISCUSSION
The observed growth promotion from aureomycin and terramycin is in accord with previous work. Because of differences in experimental procedure it is difficult to compare the results from bacitracin supplementation in this study with those of other experiments. Practically identical gains for bacitracin and control groups were reported by Rusoff and Davis (23), whereas results of other studies (10, 27) indicate a tendency toward growth promotion by this antibiotic, as was observed in the present study. The observed effect of 3-nitro-4-hydroxy phenyl arsonic acid on calf growth corresponds to the results in swine (7, 26) and poultry (2, 4, 19) and is in agreement with the preliminary findings of Graf and Holdaway (11) using p-aminophenyl arsonic acid. The ineffectiveness of chloromycetin corroborates, in general, observations with other species (6, 9, 13, 26).
897
PHYSIOLOGICAL EFFECTS O~ ANTIBIOTICS ON CALVES
80
7¢
(D
w t..>-
6C
0
oiJ 3 w "-= 3,( J
NEU ~ D P H I L E S o
/
/
2O ~,,,,,
MONOCYTES
,., 2.0 (,,) I,i,I
EOSINOPHILES
~- 1.0 O.O l'O
BASOPHILES
0.0 4
I 18
I 52
I 46
I 60
I 74
I 88
AGE, DAYS FZG. 2. M e a n t r e n d s in differential leucocyte c o u n t s (62 calves).
x tu o
15
o
I0
~o o
5
z z
0
7.0 ~6.5 6.0 FECAL 5.5 4
I 18
pH I 32
I 46
I 60
I 74
I 88
A G E . DAYS
FIG. 3. M e a n t r e n d s i n diurrheu (62 calves) a n d f e c a l p H (32 calves).
898
F . G . O W E N ET AL
The failure of penicillin to stimulate growth substantiates earlier reports (16, 21, 27). The effectiveness of aureomycin, terramycin, baeitracin, and arsonic acid in improving feed efficiency appears to be the result of increased feed consumption with a concurrent increase in rate of gain and consequent lowered maintenance per pound of gain. A temporary reduction in rate of gain following discontinuance of the feeding of some of the antibiotics also has been observed by others (8, 10). This phenomenon may have resulted from an alteration in gastrointestinal microflora to which these animals could not adjust immediately. This alteration in growth rate would appear to add support to the hypothesis that an alteration of microflora is responsible for at least a part of the growth-stimulating effects of antibiotics. The absence of a reduced growth rate in the arsenical group following discontinuance of supplementation might indicate a different mode of action and/or a residual effect. The contrasting results of the hematological studies conducted in Denmark (18) and those of the present investigation are difficult to reconcile. If there are deleterious hematological effects from antibiotic supplementation, there apparently are important conditional influences. According to a popular theory of antibiotic action, benefits result from repression of low-grade infections. If such infections are reflected to a detectable extent by elevated white blood cell counts, an inverse relationship between response to antibiotics and white cell counts might be expected. In the present study, although growth responses to antibiotics were observed, there were no appreciable differences among groups in white blood cell counts. Any attempt to explain the characteristic trends of blood cell counts certainly would be tenuous. It seems possible, however, that the peculiar segment of the leucocyte curve noted between the 2nd and 6th weeks might be associated with the amount of diarrhea prevailing during this period. Since increased numbers of lymphocytes in the intestinal wall would be anticipated, large fluid losses through these tissues may possibly produce excessive losses of these cells (29). The age trends in hemoglobin levels noted in the present study also have been observed by Thomas et al. (24); however, the minimum values reported by them were much lower. A number of workers have noted a reduced incidence and/or severity of diarrhea of antibiotic-fed calves. Since none of the supplements used in this study controlled the diarrhea incurred, it might be proposed that this laxation was of noninfectious origin and that the antibiotics and arsenical employed are ineffective in controlling this type of scours. Blaxter et al. (5) reported pH values in normal calves of 6.8 as compared with 6.2 in " l o o s e " calves and 6.0 in calves with severe diarrhea. These findings were not supported by data of the present study (Figure 3). The apparent superiority of the 3-nitro-4-hydroxy phenyl arsonic acid in maintaining an increased rate of gain during the post-treatment period would indicate a need for additional investigations to (a) supplement the data herein reported regarding its growth-promoting potential, (b) determine if this arseni-
PtfYSIOLOGICAL EFFECTS OF ANTIBIOTICS ON CALVES
899
eal will interact with antibiotics in calf growth stimulation, (c) determine whether a mechanism different from that of antibiotics is involved in response to arsenicals, and (d) ascertain whether there is any residual effect after removal of the al-senical from the diet. Since antibiotic-fed calves tend to lose much of their growth advantage sub:sequent to cessation of antibiotic feeding, the practicality of supplementing calf rations might be questioned. However, the rapid early growth, increased feed consumption, and improved feed efficiency may be of real value in reducing unthriftiness, "debilitating infection," and even the high mortality that is often encountered in raising dairy calves. SUM/~ARY
The comparative effects of feeding aureomycin, terramycin, and penicillin (Experiment I) and terramycin, bacitracin, ehloromycetin, and 3-nitro-4-hydroxy phenyl arsonic acid (]~xperiment II) to dairy calves were evaluated. Mean body weight gains over a 12-week period as per cent of the mean gains of the controls were: Experiment I, terramycin, 155%; aureomycin, 151%; and penicillin, 86% ; Experiment II, terramycin, 135% ; arsonic acid, 1.28% ; bacitraein, 126% ; and chloromyeetin, 111%. The treatments that stimulated growth also improved efficiency of feed utilization. The most rapidly gaining groups also attained greater height at withers and appeared somewhat superior in condition and hair coat at completion of the experiment; however, these differences were not marked. No pronounced ration effects were noted oll incidence and severity of diarrhea, although both were relatively high during weeks 3 to 5, inclusive. Blood cell counts (erythrocytes, leucocytes, and differential leucocytes), hemoglobin, plasnm fat, and fecal pH appeared to be unaffected by antibiotic or arsenical supplementation. Also, there was no apparent effect of diarrhea on fecal pI~. Rather marked age trends in the blood constituents were noted. ACK~qOWLEDGMENTS The ~uthors wish to express their appreciation to R. F. Elliott of Lederle Laboratories Division, American Cyanamia Co., Pearl l¢iver, N. Y., for the Aurofac 2A and D; to H. G. Luther of Chas. Pfizer and Co., Inc., Brooklyn, N. Y., for the Bi-Con-TM-5; to L. Michaud, o f Merck and Co., Rahway, N. J., for the procaine penicillin G; to F. E. Eads, Parke Davis and Co., Detroit, Mich., for the chloromycetin mycelial meal; to K. E. Passoth, S. B. Penick and Co., New York, N. ¥., for the bacitracin; and to T. W. Zbornik, Dr. Salsbury's Laboratories, Charles City, Iowa, for the 3-nitro-4-hydroxy pheny] arsonic acid. REFERENCES (1) ALLEN, N. N. A Simple Volumetric ~ e t h o d for Determination of F a t in Blood Plasma. Proc. Soe. Expt. Biol. Med., 31" 991. 1934. (2) A!XTDEaSON,G. W., CU~INGHA~, J. D., AND SLINGEr, S. ,]-. Effect of Terramycin and Certain Phenylarsonie Acid Derivatives on the Growth and Intestinal Flora of Turkey Poults. J. Nutrition, 48: 539. 2952. (3) BARTLEY, E. E. Personal communication. Kansas State College, Manhattan. 1954. (4) BIRD, H. R., GaOSCHI
900
F.G. OWEN ET AL
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