- Email: [email protected]
The Influence of EDTA on Blood Antibiotic Levels Following Oral Administration of Tetracycline Compounds1
1500
F. G. PEOUDFOOT
Becker, W. A., 1964. The storage of White Leghorn hatching eggs in plastic bags. Poultry Sci. 4 3 : 1109-1112. Bohren, B. B., L. B. Crittenden and R. T. King, 1961. Hatching time and hatchability in the fowl. Poultry Sci. 40: 620-633. Kosin, I. L., 1964. Recent research trends in hatchability—related problems of the domestic fowl. World's Poultry Sci. J. 20: 254-268. Merritt, E. S., 1964. Pre-incubation storage effects on subsequent performance of chickens. Brit. Poultry Sci. 5: 67-73.
Proudfoot, F. G., 1964. The effects of plastic packaging and other treatments on hatching eggs. Can. J. Animal Sci. 44: 87-95. Proudfoot, F. G., 1966a. The use of sealed Cryovac and polyethylene case liners with and without nitrogen gas for the preservation of hatching eggs. Poultry Sci. 4 5 : 105-108. Proudfoot, F. G., 1966b. Hatchability of stored chicken eggs as affected by daily turning during storage and pre-warming and vacuuming eggs enclosed in plastic with nitrogen. Can. J. Animal Sci. 46:47-50.
D. E. GREENE, 2 K. E. RINEHART 2 AND E. L. STEPHENSON Department of Animal Sciences, University of Arkansas, FayeUevitte, Arkansas 72701 (Received for publication February 15, 1968) TABLE 1.—Composition of Basal Ration
C
ONSIDERABLE effort has been devoted to increasing the concentration of tetracycline compounds in the blood when a given level of the antibiotic is administered orally. Techniques used to date have not proven entirely satisfactory and thus the search has continued for a practical means of potentiating broad spectrum antibiotics in poultry rations. Price et al. (1959) demonstrated that the calcium ion inhibited absorption of orally administered tetracycline compounds. Reduction of the dietary calcium level from 1.18 to 0.18% resulted in more than a two-fold increase in serum levels of oxytetracycline (OTC) or chlortetracychne (CTC). Harms and Waldroup (1961) reported maximum blood OTC levels when
Ingredient Fine Ground Corn Soybean Meal (50% protein) Alfalfa Meal (17% protein) Fish Meal (65% protein) Animal Fat Sodium Chloride Trace Mineral Mix1 Dicalcium Phosphate Calcium Carbonate Vitamin Mix2 DL-Methionine
Gm./Kg. 552.0 323.0 20.0 50.0 30.0 4.0 0.5 10.0 5.0 5.0 0.5 1000.0
1
Supplies (in ppm), Mn, 50; Fe, 50: Zn, 50: Cu, 5; I, 1.37: Co, 0.5. 2 Suppliers per Kg. of feed: 4400 I.U. vit. A; 1320 I.C.U. vit. D 3 ; 4.4 mg. riboflavin; 22 mg. niacin; 8.8 mg. d-calcium pantothenate; 440 mg. choline chloride; 11 meg. vit. Bi 2 ; and 100 mg. ethoxyquin. Calculated Analyses: Protein (%), 24.6; Calcium (%), 0.82; Phosphorus (%), 0.69; Metabolizable Energy (kcal./kg.), 3113.
1
A portion of the data used herein was taken from a thesis submitted by K. E. Rinehart to the Graduate School, University of Arkansas, in partial fulfillment of the requirements for the degree of Master of Science. 2 Present address, Ralston Purina Company, St. Louis, Missouri.
the diet contained a suboptimum level of calcium. Peterson (1958) reported that the addition of terephthalic acid to a poultry mash containing CTC resulted in a four-fold in-
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The Influence of E D T A on Blood Antibiotic Levels Following Oral Administration of Tetracycline Compounds1
EDTA
AND BLOOD ANTIBIOTIC LEVELS
crease in serum antibiotic levels. A report by Eisner et al. (1953) indicated that oral administration of certain organic acids enhanced absorption of tetracyclines from the gastrointestinal tract of rats, guinea pigs, and dogs. This group included sequestrene or ethylenediamine tetraacetic acid (EDTA). The purpose of the investigations reported in this manuscript was to determine the efficacy of EDTA as a potentiator for tetracycline compounds in the chick.
Antibiotic levels in blood serum and whole blood were determined by the Charles Pfizer Company, Terre Haute, Indiana, and The American Cyanamid Company, Princeton, New Jersey. Birds used for growth studies were fed a common diet from 0-6 days of age and assigned to treatment groups according to body weight in such a manner that the average starting weight for all groups was essentially the same. When both sexes were used the males and females were assigned separately with an equal number of each sex in all pens. Statements of probability in this report are based on the analysis of variance as outlined by Snedecor (1957) with significant differences between treatment means determined by the multiple range test of Duncan (1955). RESULTS AND DISCUSSION
Preliminary studies indicated EDTA to be more effective than some of its related salts in supporting maximum antibiotic concentrations in either blood serum or whole blood. Relatively high levels of EDTA were used in these preliminary evaluations, however, and optimal concentrations were not determined. Experiment 1 employed a wide range of levels of EDTA. Serum and whole blood OTC concentrations were significantly increased (Table 2) when EDTA was added to the diet. The 1.25% level of EDTA appeared to give maximum response. The pattern of OTC levels in serum and whole blood was similar. A narrower range of EDTA levels was used in experiment 2 with diets containing either OTC or CTC. Results (Table 3) indicate no significant response in serum CTC levels beyond the 0.25% level of EDTA. The 0.50% level of EDTA supported maximum whole blood CTC levels. Both serum and whole blood OTC values
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EXPERIMENTAL
Broiler type chicks of both sexes were employed in all experiments except 2 and 7 where all females were used. Birds were reared in electrically heated battery brooders within a temperature controlled environment. In the initial experiment, birds were offered a standard cornsoybean meal ration (Table 1) plus tap water ad libitum from hatch to 28 days of age at which time they were given experimental rations containing EDTA and tetraclines ad libitum for three days. Three birds were selected at random from each experimental group of ten at the end of this three day period and blood samples were taken by cardiac puncture. Three milliliter samples from each of the three birds were pooled for analysis. In subsequent experiments, in which blood samples were taken, birds were fasted for four hours at the end of the three day feeding period and then re-fed for three hours to enhance uniformity of antibiotic intake just prior to bleeding. In experiments with both sexes two males and one female were bled for each pooled sample. Whole blood samples were heparinized, frozen immediately and held for antibiotic analysis. Pooled blood samples for serum analysis were allowed to stand for 12 hours at 5°C. (40°F.), centrifuged, serum collected and frozen until analyzed.
1501
1502
D. E. GREENE, K. E. RINEHART AND E. L. STEPHENSON
TABLE 2.—Effect of EDTA on blood OTC levels1 Experiment 1 Ration
Serum
2
Basal As"l"+0.625%EDTA A s " l " + 1.2S0%EDTA As " 1 " + 1 . 8 7 5 % EDTA As'T*+2.500%EDTA As"l"+3.125%EDTA
1. 2. 3. 4. 5. 6.
0.69 1.35 1.97 1.75 1.91 1.91
b a, b a a a a
Whole Blood 0.93 1.67 1.89 1.74 1.84 1.95
b a, b a a, b a, b a
1 2
Average of 3 replicates reported as /ug./ml. Contained 440 mg. OTC/kg. Values followed by the same superscript are not significantly different (P<0.01).
TABLE 3.—Effect of EDTA and NaSOt on blood antibiotic levels1 Experiment 2
Basal (CTC) 2 As 1 + 0 . 2 5 % As 1 + 0 . 5 0 % As 1 + 1.00% As 1 + 1.40% Basal (OTC)2 As 6 + 0 . 2 5 % As 6 + 0 . 5 0 % As 6 + 1 . 0 0 % As 6 + 1 . 4 0 %
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1
EDTA EDTA EDTA NaS0 4 EDTA EDTA EDTA NaSCu
Serum
Whole Blood
.585 d .790 b .872 b .860 b .782 b .378 e .679 c .011 a .973 a .558 d
.710 d .963 b .033 a ,020 a .785 c .335 g .450 f .610 e .653 e .470 f
Average of 4 replicates reported as ^g./ml. Contained 440 mg. OTC or CTC/kg. Values followed by the same superscript are not significantly different (p<0.01). 2
% Calcium - u ^ x n y.0 2
0 0.25 0.50 1.00 1 2
1.2
0.9
0.6 .353 .532 .672 .932
.302 .453 .552 .717
.225 .390 .430 .503
Each ration contained 440 mg. OTC/kg. Average of 4 replicates reported as /jg./ml.
rangement of treatments with three levels of calcium and four levels of EDTA. The data (Table 4) show a suppression in serum OTC levels as the calcium level of the diet increased within each level of EDTA. Serum OTC levels increased with each increment of EDTA up to 1.0% of the diet. Statistical analyses indicated significant (P < 0.01) effects of EDTA level and of calcium level with the interaction being nonsignificant. These results are not surprising if it is postulated that the mechanism of action of EDTA is that of binding calcium to prevent its interference with absorption of the tetracycline compound. Experiment 4 was an attempt to more closely define the optimal level of EDTA needed for potentiation of oxytetracycline (Table 5). A level of 0.2% EDTA signifiTABLE 5.—Effect of EDTA and NaSOi on OTC levels1 Experiment 4 Ration 1. 2. 3. 4. 5. 6. 7. 8. 1
Serum OTC
2
Basal As " l " + 0 . 2 0 % As " l " + 0 . 3 0 % As " l " + 0 . 4 0 % As " l " + 0 . 5 0 % As " 1 " + 1 . 0 0 % As " 1 " + 1.40% As "5*'+"7"
EDTA EDTA EDTA EDTA EDTA NaS0 4
.353 .530 .591 .651 .672 .868 .470 .685
e c, d b, c, d b, c b a d, e b
Average of 4 replicates reported as jug./ml. Contained 440 mg. OTC/kg. Values followed by same superscript are not significantly different (P<0.01). 2
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 9, 2015
were maximum when the diet contained 0.50% EDTA. The addition of 1.4% NaS0 4 significantly increased both serum and whole blood antibiotic levels but the response was somewhat less than obtained with 0.5% EDTA. Although absolute values differed slightly between OTC and CTC, the pattern of response to EDTA was essentially the same. The magnitude of the potentiating effect was greatest in serum OTC values which indicated greater sensitivity in this measurement than in other values. Therefore, subsequent studies employed OTC and only serum antibiotic levels were determined. Experiment 3 involved a factorial ar-
Ration
TABLE A.—Effect of dietary EDTA and calcium on serum OTC levels1 Experiment 3
EDTA
AND BLOOD ANTIBIOTIC LEVELS
1503
different (P<0.01).
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TABLE 7.—Effect of EDTA, zinc, and manganese cantly increased serum OTC level. The reon chick performance1 sponse appeared to plateau between 0.4 Experiment 6 and 0.5% EDTA; however, the 1.00% 7-28 Day 7-28 Day level gave significantly higher serum OTC Ration Gain (gm.) F/G levels than the 0.5% level of EDTA. It 532 a 1.80 a cannot be determined whether maximum 1. Basal 2. A s " l " + 0 . 5 0 % E D T A 454 d 2.09 b 2 potentiation was reached in this experi- 3. As " l " + 0 . 5 0 % EDTA 498 b 1.88 a ment. Sodium sulfate did not significantly 4. As " l " + 0 . 5 0 % E D T A + 100 p.p.m. Zinc 456 c, d 2 . 1 2 b increase blood OTC levels. The combina- 5. A s " l " + 0 . 5 0 % E D T A - | 100 p.p.m. Manganese 464 c, d 2.07 b tion of EDTA and NaS0 4 did not indicate 6. A s " l " + 0 . 5 0 % E D T A + an additive effect. 100 p.p.m. Zinc+lOO 100 p.p.m. Manganese 488 b, c 1.95 a, b With indications that 0.5% EDTA 1 would be an effective level for OTC potenAverage of 4 replicates of 10 birds each. 2 Fed 7-14 days; unsupplemented basal 14-28 tiation, experiment 5 was designed to deterdays. mine the effect of this compound on growth Values followed by the same superscript are not and feed consumption. It is interesting to significantly different (P<0.01). note (Table 6) that weight gain from 6 to 26 days of age decreased with each increment of EDTA. This was true in the pres- statistically significant differences among ence or absence of OTC. When OTC levels birds fed the various levels of EDTA. were combined for analysis, the effect of Birds were removed from the experimental each increment of EDTA was statistically rations in this test at 26 days of age and significant. Feed intake was also reduced fed a practical broiler ration with no with each increment of EDTA although EDTA or OTC until 47 days of age. All this did not appear to be sufficient to ac- birds appeared to gain at a similar rate folcount for the total effect on growth. Oxytet- lowing withdrawal of EDTA. Those that racycline did not significantly influence encountered suppression in weight before weight gain or feed per unit of gain. Bone 26 days of age were unable to make comash analyses on tibias from ten birds per pensatory gains during the 26-47 day petreatment at 26 days of age did not show riod. It was postulated that the effect of EDTA on growth might be that of binding certain trace minerals rendering them unTABLE 6.—Effect of EDTA on chick performance1 Experiment 5 available to the bird. Experiment 6 was designed to determine the effect of adding «T26 Fd. Bone «T472 supplemental manganese and/or zinc to Ration g& Intake F/G Ash § £ (gm % (gm.) -> (gm.) diets containing EDTA. Growth rate (Table 994 443 a 799 a, b 1.81 4 0 . 6 8 1. Basal 7) was significantly (P < 0.01) reduced 2. A s " l " + 0 . 2 5 % EDTA 417 a, h 790 a, b 1.89 38.56 993 when birds were fed diets containing 3. A s " l " + 0 . 5 0 % EDTA 379 b, c. 785 a, b, c 2 . 0 8 39.80 952 0.50% EDTA for 21 days or when fed this 4. A s " l " + 1 . 0 0 % ration for a 7-day period followed by the EDTA 317 d 723 c, d 2.15 39.24 919 5. A s " l " + O T O 425 a 816 a 1.94 4 0 . 0 8 1049 953 control ration from 12-28 days of age. ft. A s " 2 " + 0 T C 3 407 a, b 786 a, b, c 1.94 39.12 748 b, c, d 2 . 0 3 38.64 941 7. A s " 3 " + 0 T C 3 368 c Supplementing the diet with 100 ppm zinc 703 d 2.32 39.76 835 8 . A s " 4 " + O T O > 303 d or manganese alone had no effect on weight 1 Average of 5 replicates of 5 males and 5 females per pen. 2 gain, however, gains were significantly (P All birds fed diet 1 from 26-47 days of age. 3 Contained 440 mg./kg. of diet. < 0.01) increased when the two were Values followed by same superscript are not significantly
1504
D. E. GREENE, K. E. RINEHART AND E. L. STEPHENSON
TABLE 8.—Effect of selected mineral elements on performance of birds fed EDTA1 Experiment 7 Ration 1. 2. 3. 4. 5. 6. 7. 8.
Basal As"l"+1.0%EDTA As "2" 2 As "2" crumbled As "2"+100 p.p.m. Zinc As "5"+100 p.p.m. Manganese As "6"+100 p.p.m. Magnesium As " 7 " + 5 0 p.p.m. Copper As " 8 " + 1 . 0 % CaCOs As " 8 " + 1.5% Dicalcium Phosphate
366 317 337 334 257
a b a, b a, b c
2.04 2.10 2.10 1.86 2.24
a, b a, b a, b b a
341a, b
1.99 a, b
348 a, b
2.03 a, b
336 a, b 340 a, b
1.93 b 1.89 b
272 c
2.07 a, b
1
Average of 5 replicates of 7 females each. As " 2 " for 6-13 days and as " 1 " for 14-26 days. Values followed by the same superscript are not significantly different (P<0.05). 2
added in combination. Following the observation that the addition of zinc and manganese improved performance of birds fed EDTA, an additional experiment was designed to test these in combination with copper, magnesium, calcium, and phosphorus. Results (Table 8) confirmed that a combination of zinc and manganese was effective in partially alleviating the adverse effect of EDTA on growth and feed per unit of gain. No additional improvement in gain was noted when magnesium, copper, calcium, and phosphorus were added. Feeding the ration containing EDTA in the form of crumbles did not improve weight gain significantly when compared to birds fed mash. It appears that a part of the growth depressing effect of EDTA in the diet of chicks is related to utilization of trace minerals.
The addition of EDTA to a practical broiler starting ration containing 0.8% calcium significantly increased blood oxytetracycline and chlortetracycline levels in 28 day old chickens following oral administration of these antibiotics. A dietary level of 0.5% EDTA resulted in approximately a two-fold increase in blood antibiotic levels. This level of EDTA depressed weight gain significantly when fed continously for either 7 or 21 days. The growth depression was partially alleviated by increasing manganese and zinc fortification of the diet. ACKNOWLEDGMENTS
The authors wish to express gratitude to the Charles Pfizer Company and American Cyanamid Company for antibiotic analyses on blood serum and whole blood samples, respectively. REFERENCES Duncan, D. B., 1955. Multiple range and multiple F test. Biometrics, 11: 1-42. Eisner, H. J., F. E. Stein, A. C. Dornbusch and J. J. Oleson, 1953. The enhancement of serum levels of chlortetracycline in experimental animals. J. Pharmacol. Expt. Therap. 108: 442-449. Harms, R. H., and P. W. Waldroup, 1961. Potentiation of terramycin. I. Evaluation of terephthalic acid and low dietary calcium in broiler diets. Avian Diseases, 5: 183-188. Peterson, E. H., 1958. Potentiating effect of terephthalic acid upon absorption of chlortetracycline from the avian alimentary tract. Arkansas Agric. Expt. Sta. Report Series, 74. Price, K. E., Z. ZoUi, Jr. and J. H. Hare, 1959. Effect of dietary calcium phosphorus adjustment and/or supplementation with terephthalic acid upon serum antibiotics levels of chickens. Poultry Sci. 39: 233-235. Snedecor, G. W., 1957. Statistical Methods, 5th Edition, Iowa State College Press, Ames, Iowa.
JANUARY 13-15. CORNELL AGRICULTURAL WASTE MANAGEMENT CONFERENCE, HOTEL SYRACUSE, SYRACUSE, N.Y.
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9. 10.
6-26 Day 6-26 Day Gain (gm.) F/G
SUMMARY
F. G. PEOUDFOOT
Becker, W. A., 1964. The storage of White Leghorn hatching eggs in plastic bags. Poultry Sci. 4 3 : 1109-1112. Bohren, B. B., L. B. Crittenden and R. T. King, 1961. Hatching time and hatchability in the fowl. Poultry Sci. 40: 620-633. Kosin, I. L., 1964. Recent research trends in hatchability—related problems of the domestic fowl. World's Poultry Sci. J. 20: 254-268. Merritt, E. S., 1964. Pre-incubation storage effects on subsequent performance of chickens. Brit. Poultry Sci. 5: 67-73.
Proudfoot, F. G., 1964. The effects of plastic packaging and other treatments on hatching eggs. Can. J. Animal Sci. 44: 87-95. Proudfoot, F. G., 1966a. The use of sealed Cryovac and polyethylene case liners with and without nitrogen gas for the preservation of hatching eggs. Poultry Sci. 4 5 : 105-108. Proudfoot, F. G., 1966b. Hatchability of stored chicken eggs as affected by daily turning during storage and pre-warming and vacuuming eggs enclosed in plastic with nitrogen. Can. J. Animal Sci. 46:47-50.
D. E. GREENE, 2 K. E. RINEHART 2 AND E. L. STEPHENSON Department of Animal Sciences, University of Arkansas, FayeUevitte, Arkansas 72701 (Received for publication February 15, 1968) TABLE 1.—Composition of Basal Ration
C
ONSIDERABLE effort has been devoted to increasing the concentration of tetracycline compounds in the blood when a given level of the antibiotic is administered orally. Techniques used to date have not proven entirely satisfactory and thus the search has continued for a practical means of potentiating broad spectrum antibiotics in poultry rations. Price et al. (1959) demonstrated that the calcium ion inhibited absorption of orally administered tetracycline compounds. Reduction of the dietary calcium level from 1.18 to 0.18% resulted in more than a two-fold increase in serum levels of oxytetracycline (OTC) or chlortetracychne (CTC). Harms and Waldroup (1961) reported maximum blood OTC levels when
Ingredient Fine Ground Corn Soybean Meal (50% protein) Alfalfa Meal (17% protein) Fish Meal (65% protein) Animal Fat Sodium Chloride Trace Mineral Mix1 Dicalcium Phosphate Calcium Carbonate Vitamin Mix2 DL-Methionine
Gm./Kg. 552.0 323.0 20.0 50.0 30.0 4.0 0.5 10.0 5.0 5.0 0.5 1000.0
1
Supplies (in ppm), Mn, 50; Fe, 50: Zn, 50: Cu, 5; I, 1.37: Co, 0.5. 2 Suppliers per Kg. of feed: 4400 I.U. vit. A; 1320 I.C.U. vit. D 3 ; 4.4 mg. riboflavin; 22 mg. niacin; 8.8 mg. d-calcium pantothenate; 440 mg. choline chloride; 11 meg. vit. Bi 2 ; and 100 mg. ethoxyquin. Calculated Analyses: Protein (%), 24.6; Calcium (%), 0.82; Phosphorus (%), 0.69; Metabolizable Energy (kcal./kg.), 3113.
1
A portion of the data used herein was taken from a thesis submitted by K. E. Rinehart to the Graduate School, University of Arkansas, in partial fulfillment of the requirements for the degree of Master of Science. 2 Present address, Ralston Purina Company, St. Louis, Missouri.
the diet contained a suboptimum level of calcium. Peterson (1958) reported that the addition of terephthalic acid to a poultry mash containing CTC resulted in a four-fold in-
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 9, 2015
The Influence of E D T A on Blood Antibiotic Levels Following Oral Administration of Tetracycline Compounds1
EDTA
AND BLOOD ANTIBIOTIC LEVELS
crease in serum antibiotic levels. A report by Eisner et al. (1953) indicated that oral administration of certain organic acids enhanced absorption of tetracyclines from the gastrointestinal tract of rats, guinea pigs, and dogs. This group included sequestrene or ethylenediamine tetraacetic acid (EDTA). The purpose of the investigations reported in this manuscript was to determine the efficacy of EDTA as a potentiator for tetracycline compounds in the chick.
Antibiotic levels in blood serum and whole blood were determined by the Charles Pfizer Company, Terre Haute, Indiana, and The American Cyanamid Company, Princeton, New Jersey. Birds used for growth studies were fed a common diet from 0-6 days of age and assigned to treatment groups according to body weight in such a manner that the average starting weight for all groups was essentially the same. When both sexes were used the males and females were assigned separately with an equal number of each sex in all pens. Statements of probability in this report are based on the analysis of variance as outlined by Snedecor (1957) with significant differences between treatment means determined by the multiple range test of Duncan (1955). RESULTS AND DISCUSSION
Preliminary studies indicated EDTA to be more effective than some of its related salts in supporting maximum antibiotic concentrations in either blood serum or whole blood. Relatively high levels of EDTA were used in these preliminary evaluations, however, and optimal concentrations were not determined. Experiment 1 employed a wide range of levels of EDTA. Serum and whole blood OTC concentrations were significantly increased (Table 2) when EDTA was added to the diet. The 1.25% level of EDTA appeared to give maximum response. The pattern of OTC levels in serum and whole blood was similar. A narrower range of EDTA levels was used in experiment 2 with diets containing either OTC or CTC. Results (Table 3) indicate no significant response in serum CTC levels beyond the 0.25% level of EDTA. The 0.50% level of EDTA supported maximum whole blood CTC levels. Both serum and whole blood OTC values
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 9, 2015
EXPERIMENTAL
Broiler type chicks of both sexes were employed in all experiments except 2 and 7 where all females were used. Birds were reared in electrically heated battery brooders within a temperature controlled environment. In the initial experiment, birds were offered a standard cornsoybean meal ration (Table 1) plus tap water ad libitum from hatch to 28 days of age at which time they were given experimental rations containing EDTA and tetraclines ad libitum for three days. Three birds were selected at random from each experimental group of ten at the end of this three day period and blood samples were taken by cardiac puncture. Three milliliter samples from each of the three birds were pooled for analysis. In subsequent experiments, in which blood samples were taken, birds were fasted for four hours at the end of the three day feeding period and then re-fed for three hours to enhance uniformity of antibiotic intake just prior to bleeding. In experiments with both sexes two males and one female were bled for each pooled sample. Whole blood samples were heparinized, frozen immediately and held for antibiotic analysis. Pooled blood samples for serum analysis were allowed to stand for 12 hours at 5°C. (40°F.), centrifuged, serum collected and frozen until analyzed.
1501
1502
D. E. GREENE, K. E. RINEHART AND E. L. STEPHENSON
TABLE 2.—Effect of EDTA on blood OTC levels1 Experiment 1 Ration
Serum
2
Basal As"l"+0.625%EDTA A s " l " + 1.2S0%EDTA As " 1 " + 1 . 8 7 5 % EDTA As'T*+2.500%EDTA As"l"+3.125%EDTA
1. 2. 3. 4. 5. 6.
0.69 1.35 1.97 1.75 1.91 1.91
b a, b a a a a
Whole Blood 0.93 1.67 1.89 1.74 1.84 1.95
b a, b a a, b a, b a
1 2
Average of 3 replicates reported as /ug./ml. Contained 440 mg. OTC/kg. Values followed by the same superscript are not significantly different (P<0.01).
TABLE 3.—Effect of EDTA and NaSOt on blood antibiotic levels1 Experiment 2
Basal (CTC) 2 As 1 + 0 . 2 5 % As 1 + 0 . 5 0 % As 1 + 1.00% As 1 + 1.40% Basal (OTC)2 As 6 + 0 . 2 5 % As 6 + 0 . 5 0 % As 6 + 1 . 0 0 % As 6 + 1 . 4 0 %
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1
EDTA EDTA EDTA NaS0 4 EDTA EDTA EDTA NaSCu
Serum
Whole Blood
.585 d .790 b .872 b .860 b .782 b .378 e .679 c .011 a .973 a .558 d
.710 d .963 b .033 a ,020 a .785 c .335 g .450 f .610 e .653 e .470 f
Average of 4 replicates reported as ^g./ml. Contained 440 mg. OTC or CTC/kg. Values followed by the same superscript are not significantly different (p<0.01). 2
% Calcium - u ^ x n y.0 2
0 0.25 0.50 1.00 1 2
1.2
0.9
0.6 .353 .532 .672 .932
.302 .453 .552 .717
.225 .390 .430 .503
Each ration contained 440 mg. OTC/kg. Average of 4 replicates reported as /jg./ml.
rangement of treatments with three levels of calcium and four levels of EDTA. The data (Table 4) show a suppression in serum OTC levels as the calcium level of the diet increased within each level of EDTA. Serum OTC levels increased with each increment of EDTA up to 1.0% of the diet. Statistical analyses indicated significant (P < 0.01) effects of EDTA level and of calcium level with the interaction being nonsignificant. These results are not surprising if it is postulated that the mechanism of action of EDTA is that of binding calcium to prevent its interference with absorption of the tetracycline compound. Experiment 4 was an attempt to more closely define the optimal level of EDTA needed for potentiation of oxytetracycline (Table 5). A level of 0.2% EDTA signifiTABLE 5.—Effect of EDTA and NaSOi on OTC levels1 Experiment 4 Ration 1. 2. 3. 4. 5. 6. 7. 8. 1
Serum OTC
2
Basal As " l " + 0 . 2 0 % As " l " + 0 . 3 0 % As " l " + 0 . 4 0 % As " l " + 0 . 5 0 % As " 1 " + 1 . 0 0 % As " 1 " + 1.40% As "5*'+"7"
EDTA EDTA EDTA EDTA EDTA NaS0 4
.353 .530 .591 .651 .672 .868 .470 .685
e c, d b, c, d b, c b a d, e b
Average of 4 replicates reported as jug./ml. Contained 440 mg. OTC/kg. Values followed by same superscript are not significantly different (P<0.01). 2
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 9, 2015
were maximum when the diet contained 0.50% EDTA. The addition of 1.4% NaS0 4 significantly increased both serum and whole blood antibiotic levels but the response was somewhat less than obtained with 0.5% EDTA. Although absolute values differed slightly between OTC and CTC, the pattern of response to EDTA was essentially the same. The magnitude of the potentiating effect was greatest in serum OTC values which indicated greater sensitivity in this measurement than in other values. Therefore, subsequent studies employed OTC and only serum antibiotic levels were determined. Experiment 3 involved a factorial ar-
Ration
TABLE A.—Effect of dietary EDTA and calcium on serum OTC levels1 Experiment 3
EDTA
AND BLOOD ANTIBIOTIC LEVELS
1503
different (P<0.01).
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TABLE 7.—Effect of EDTA, zinc, and manganese cantly increased serum OTC level. The reon chick performance1 sponse appeared to plateau between 0.4 Experiment 6 and 0.5% EDTA; however, the 1.00% 7-28 Day 7-28 Day level gave significantly higher serum OTC Ration Gain (gm.) F/G levels than the 0.5% level of EDTA. It 532 a 1.80 a cannot be determined whether maximum 1. Basal 2. A s " l " + 0 . 5 0 % E D T A 454 d 2.09 b 2 potentiation was reached in this experi- 3. As " l " + 0 . 5 0 % EDTA 498 b 1.88 a ment. Sodium sulfate did not significantly 4. As " l " + 0 . 5 0 % E D T A + 100 p.p.m. Zinc 456 c, d 2 . 1 2 b increase blood OTC levels. The combina- 5. A s " l " + 0 . 5 0 % E D T A - | 100 p.p.m. Manganese 464 c, d 2.07 b tion of EDTA and NaS0 4 did not indicate 6. A s " l " + 0 . 5 0 % E D T A + an additive effect. 100 p.p.m. Zinc+lOO 100 p.p.m. Manganese 488 b, c 1.95 a, b With indications that 0.5% EDTA 1 would be an effective level for OTC potenAverage of 4 replicates of 10 birds each. 2 Fed 7-14 days; unsupplemented basal 14-28 tiation, experiment 5 was designed to deterdays. mine the effect of this compound on growth Values followed by the same superscript are not and feed consumption. It is interesting to significantly different (P<0.01). note (Table 6) that weight gain from 6 to 26 days of age decreased with each increment of EDTA. This was true in the pres- statistically significant differences among ence or absence of OTC. When OTC levels birds fed the various levels of EDTA. were combined for analysis, the effect of Birds were removed from the experimental each increment of EDTA was statistically rations in this test at 26 days of age and significant. Feed intake was also reduced fed a practical broiler ration with no with each increment of EDTA although EDTA or OTC until 47 days of age. All this did not appear to be sufficient to ac- birds appeared to gain at a similar rate folcount for the total effect on growth. Oxytet- lowing withdrawal of EDTA. Those that racycline did not significantly influence encountered suppression in weight before weight gain or feed per unit of gain. Bone 26 days of age were unable to make comash analyses on tibias from ten birds per pensatory gains during the 26-47 day petreatment at 26 days of age did not show riod. It was postulated that the effect of EDTA on growth might be that of binding certain trace minerals rendering them unTABLE 6.—Effect of EDTA on chick performance1 Experiment 5 available to the bird. Experiment 6 was designed to determine the effect of adding «T26 Fd. Bone «T472 supplemental manganese and/or zinc to Ration g& Intake F/G Ash § £ (gm % (gm.) -> (gm.) diets containing EDTA. Growth rate (Table 994 443 a 799 a, b 1.81 4 0 . 6 8 1. Basal 7) was significantly (P < 0.01) reduced 2. A s " l " + 0 . 2 5 % EDTA 417 a, h 790 a, b 1.89 38.56 993 when birds were fed diets containing 3. A s " l " + 0 . 5 0 % EDTA 379 b, c. 785 a, b, c 2 . 0 8 39.80 952 0.50% EDTA for 21 days or when fed this 4. A s " l " + 1 . 0 0 % ration for a 7-day period followed by the EDTA 317 d 723 c, d 2.15 39.24 919 5. A s " l " + O T O 425 a 816 a 1.94 4 0 . 0 8 1049 953 control ration from 12-28 days of age. ft. A s " 2 " + 0 T C 3 407 a, b 786 a, b, c 1.94 39.12 748 b, c, d 2 . 0 3 38.64 941 7. A s " 3 " + 0 T C 3 368 c Supplementing the diet with 100 ppm zinc 703 d 2.32 39.76 835 8 . A s " 4 " + O T O > 303 d or manganese alone had no effect on weight 1 Average of 5 replicates of 5 males and 5 females per pen. 2 gain, however, gains were significantly (P All birds fed diet 1 from 26-47 days of age. 3 Contained 440 mg./kg. of diet. < 0.01) increased when the two were Values followed by same superscript are not significantly
1504
D. E. GREENE, K. E. RINEHART AND E. L. STEPHENSON
TABLE 8.—Effect of selected mineral elements on performance of birds fed EDTA1 Experiment 7 Ration 1. 2. 3. 4. 5. 6. 7. 8.
Basal As"l"+1.0%EDTA As "2" 2 As "2" crumbled As "2"+100 p.p.m. Zinc As "5"+100 p.p.m. Manganese As "6"+100 p.p.m. Magnesium As " 7 " + 5 0 p.p.m. Copper As " 8 " + 1 . 0 % CaCOs As " 8 " + 1.5% Dicalcium Phosphate
366 317 337 334 257
a b a, b a, b c
2.04 2.10 2.10 1.86 2.24
a, b a, b a, b b a
341a, b
1.99 a, b
348 a, b
2.03 a, b
336 a, b 340 a, b
1.93 b 1.89 b
272 c
2.07 a, b
1
Average of 5 replicates of 7 females each. As " 2 " for 6-13 days and as " 1 " for 14-26 days. Values followed by the same superscript are not significantly different (P<0.05). 2
added in combination. Following the observation that the addition of zinc and manganese improved performance of birds fed EDTA, an additional experiment was designed to test these in combination with copper, magnesium, calcium, and phosphorus. Results (Table 8) confirmed that a combination of zinc and manganese was effective in partially alleviating the adverse effect of EDTA on growth and feed per unit of gain. No additional improvement in gain was noted when magnesium, copper, calcium, and phosphorus were added. Feeding the ration containing EDTA in the form of crumbles did not improve weight gain significantly when compared to birds fed mash. It appears that a part of the growth depressing effect of EDTA in the diet of chicks is related to utilization of trace minerals.
The addition of EDTA to a practical broiler starting ration containing 0.8% calcium significantly increased blood oxytetracycline and chlortetracycline levels in 28 day old chickens following oral administration of these antibiotics. A dietary level of 0.5% EDTA resulted in approximately a two-fold increase in blood antibiotic levels. This level of EDTA depressed weight gain significantly when fed continously for either 7 or 21 days. The growth depression was partially alleviated by increasing manganese and zinc fortification of the diet. ACKNOWLEDGMENTS
The authors wish to express gratitude to the Charles Pfizer Company and American Cyanamid Company for antibiotic analyses on blood serum and whole blood samples, respectively. REFERENCES Duncan, D. B., 1955. Multiple range and multiple F test. Biometrics, 11: 1-42. Eisner, H. J., F. E. Stein, A. C. Dornbusch and J. J. Oleson, 1953. The enhancement of serum levels of chlortetracycline in experimental animals. J. Pharmacol. Expt. Therap. 108: 442-449. Harms, R. H., and P. W. Waldroup, 1961. Potentiation of terramycin. I. Evaluation of terephthalic acid and low dietary calcium in broiler diets. Avian Diseases, 5: 183-188. Peterson, E. H., 1958. Potentiating effect of terephthalic acid upon absorption of chlortetracycline from the avian alimentary tract. Arkansas Agric. Expt. Sta. Report Series, 74. Price, K. E., Z. ZoUi, Jr. and J. H. Hare, 1959. Effect of dietary calcium phosphorus adjustment and/or supplementation with terephthalic acid upon serum antibiotics levels of chickens. Poultry Sci. 39: 233-235. Snedecor, G. W., 1957. Statistical Methods, 5th Edition, Iowa State College Press, Ames, Iowa.
JANUARY 13-15. CORNELL AGRICULTURAL WASTE MANAGEMENT CONFERENCE, HOTEL SYRACUSE, SYRACUSE, N.Y.
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9. 10.
6-26 Day 6-26 Day Gain (gm.) F/G
SUMMARY