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The Effect of Dicumarol,1 Diphacinone2 and Pivalyl3 Upon Blood Prothrombin Time of Chicks4
387
METHIONINE SOURCES on growth of chicks fed amino acid diets. Poultry Sci. 43: 1113-1118. Marrett, L. E., and M. L. Sunde, 1965. The effect of other D-amino acids on the utilization of the isomers of methionine and its hydroxy analogue. Poultry Sci. 44: 957-964. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill
Book Company, Inc., New York, N.Y. Tipton, H. C , B. C. Dilworth, and E. J. Day, 1965. The relative biological value of DL-methionine hydroxy analogue in chick diets. Poultry Sci. 44: 987-992. Yankelowitz, S., 1960. Factors affecting the intestinal absorption of methionine. Thesis, Rutgers, The State University. New Brunswick, N J .
O. W. CHARLES, BEN C. DILWORTH, REX D. BUSHONG, JR. AND ELBERT J. DAY Department of Poultry Science, Mississippi State University, State College, Mississippi (Received for publication September 24, 1965)
NCREASED prothrombin time due to feeding dicumarol has been observed with the chick (Quick and Stefanini, 1948; Harms and Tarver, 1957) and laying hens (Waldroup and Harms, 1962; Day et al., 1964). The action of dicumarol in the inhibition of the blood clotting process is lessened by the presence of vitamin K in the diet according to Quick and Stefanini (1948). Although sulfaquinoxaline is used to induce hypoprothrombinemia in chick assays of vitamin K compounds (Perdue and Frost, 1961; Griminger and Daves, 1959), the use of other anticoagulants may prove of value in studies on vitamin K activity or the mechanism of blood clotting as related to the occurrence of blood spots in eggs. The experiments reported herein were designed to evaluate the effects of three anticoagulants on the prothrombin time of chicks.
I
1
3-3'-methylenebis-4 hydroxy coumarin. 2 diphenylacetyl-1, 3-indandione 1-hydrazone; Nease Chemical Co., Inc., State College, Pa. 3 2 pivalyl-1, 3-indandione; Nease Chemical Co., Inc., State College, Pa. 4 Mississippi Agricultural Experiment Station Journal Article No. 1318. 2
EXPERIMENTAL
Four experiments were conducted using broiler strain chicks obtained from a commercial hatchery. All chicks were wing banded and maintained in wire cages. A practical type broiler diet and tap water were provided ad libitum throughout each experiment. The chicks were six week old cockerels in Experiments 1 and 2, eleven week old cockerels in Experiment 3 and eleven week old chicks of both sexes in Experiment 4. The anticoagulants ere administered orally in no. 1 gelatin capsules. Whole blood prothrombin determinations were made according to the method of Woody et al. (1963). Treatment prothrombin time averages were obtained using 4, 4, 8 and 10 birds in Experiments 1, 2, 3, and 4, respectively. Control non-treated chicks were maintained in each experiment. Each of the three drugs was given orally at levels of 1, 2, 4, 8, 16 and 32 mg./chick in Experiment 1 and at 50, 100 and 150 mg./chick in Experiment 2. In Experiment 3, each drug was tested at the 50, 100, 200 and 500 mg./chick level. Sex, drug and post-dosage
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 22, 2015
The Effect of Dicumarol, 1 Diphacinone 2 and PivalyP Upon Blood Prothrombin Time of Chicks4
388
O. W. CHARLES, B. C. DILWORTH, R. D. BUSHONG, JR. AND E. J. DAY TABLE 1.—The effect of dosage level and three anticoagulants on the prothrombin time of broiler cockerels
Drug level, mg./bird
Prothrombin time, seconds1 Dicumarol
Diphac- Pivalyl inone
Average2 0 50 100 150 Average2
18 24 26 38 44 58 53
18 21 24 29 32 40 47
37"
—
Experiment 2 20 20 40 50 44 48 47 51
20 44 41 38
20 45 44 45
44»
41"
—
21"
33 b
50"
1
Prothrombin determinations were made 20 hours post-dosage. 2 Averages within each experiment without a common superscript are significantly different at the 5% level of probability.
time were the criteria studied in Experiment 4. In this latter trial, the dosage level of each drug was 100 mg./chick. The prothrombin determinations were made 20 hours post-dosage in Experiment 1 and 2. The rate at which each drug exerted its effect was examined in the last two experiments. Blood samples were collected 20, 24, 28 and 32 hours (Exp. 3) and 4, 8, 12, 16, 20, 24 and 28 hours (Exp. 4) following drug administration. Statistical examinations of the prothrombin time data were made according to Steel and Torrie (1960) and Duncan (1955). RESULTS AND DISCUSSION In Experiment 1 significant differences in prothrombin time were obtained twenty hours after administering oral doses of three anticoagulants. Diphacinone and pivalyl caused a significant increase in the prothrombin time as compared to the dicu-
In a third experiment, the prothrombin time was measured for all treatments at 20, 24, 28 and 32 hours post-dosage in order to compare the pharmacodynamics of these anticoagulants at different time intervals. Four dosage levels (50, 100, 200 and 500 mg./bird) of dicumarol, diphacinone and pivalyl were administered to cockerels and prothrombin times were determined at four hour intervals (Table 2). The effect of dicumarol tended to decline steadily after the twentieth hour for the three lower levels tested, while the highest level tended to maintain a constant prothrombin time through the 32nd hour (Fig. 1). In contrast, the prothrombin time of birds treated with diphacinone and pivalyl continued to rise from the 20th through the
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 22, 2015
Experiment 1 18 18 18 21 20 27 17 32 16 37 23 41 33 56
0 1 2 4 8 16 32
Average
marol treated birds. Only the highest level of dicumarol (32 mg./bird) elicited a significant increase in the prothrombin time; while diphacinone and pivalyl caused significant changes at a much lower level (4 mg./bird). Small increases in the prothrombin time were obtained with each increase in dosage level with all three drugs except for the highest level of pivalyl (Table 1). Much higher levels of the same three drugs were used in a second experiment in an attempt to determine the drug level that would elicit a maximum increase in prothrombin time (Table 1). It is evident from these data that the lowest dosage level (50 mg.) produced essentially the same effect on prothrombin time as the highest level (150 mg.) at 20 hours postdosage. Since the lowest drug level caused a maximum increase in the prothrombin time for all drugs, significant differences attributable to drugs were not found. It is evident from the data in Experiment 2 that the effect of anticoagulants on prothrombin time cannot be measured using high drug levels and a constant time post-dosage.
389
BLOOD PROTHROMBIN TIME TABLE 2.—The effect of drug level and lime on prothrombin time of chicks treated with three anticoagulants, Experiment 3 Hours post-dosage Drug Dicumarol
Pivalyl
Drug X'level
—
20
24
28
32
0 50 100 200 500
23 41 51 50 62
23 41 44 34 58
23 31 37 43 45 •
23 25 31 27 65
23 34 41 39 58
DrugXtime av.
45
40
36
34
39*
0 50 100 200 500
24 41 55 53 50
25 65 55 63 71
26 58 59 56 85
27 72 64 81 85
25 59 58 63 73
DrugXtime av.
45
56
56
66
56b
0 50 100 200 500
24 55 53 53 53
26 59 69 69 75
25 110 70 75 65
26 99 85 101 134
25 81 69 75 82
DrugXtime av.
48
59
69
89
66°
• Drug means without a common superscript are significantly different at the 5% level of probability.
32 nd hour. The increase in prothrombin time caused by pivalyl was much greater than that produced by diphacinone, (Fig. 2 and Table 2). Each increase in the level of dicumarol and diphacinone caused a corresponding increase in prothrombin time. In-
creasing the level of pivalyl did not always cause a prothrombin response since the lowest level used (50 mg./bird) approached the maximum response level (Fig. 1 and Table 2). PIVALyL
DIPHACINONE
28 32 HOURS POST-DOSAGE
FIG. 1. Drug X level interaction, Experiment 3.
FIG. 2. Drug X time interaction, Experiment 3.
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 22, 2015
Diphacinone
mg./bird
390
O. W. CHARLES, B. C. DILWORTH, R. D. BUSHONG, JR. AND E. J. DAY TABLE 3.—The effect of sex and time on prothrombin time of chicks treated with three anticoagulants, Experiment 4 Prothrombin time, seconds1
Hours post-dosage
Dicumarol2
Diphacinone2
PivalyP
Female
Male
Female
Male
Female
24 24 34 35 41 45 41 25
25 29 32 37 44 49 41 29
24 26 32 34 49 67 61 55
25 23 34 40 54 66 74 58
24 24 30 34 47 63 80 76
25 26 30 36 50 66 79
34
36
44
47
47
50
0 4 8 12 16 20 24 28 Drug X sex av.
35"
Drug av.
46
b
48°
Composite data SexesXtime
Male Female 1 2
0
4
8
12
16
20
24
28
Av.
72 75
74 78
96 96
103 113
137 148
175 181
182 194
156 175
41" 44 b
Drug averages without a common superscript are significantly different (P<.05). Each drug administered at the 100 mg. per bird level.
A statistical examination of these data revealed that all first order interactions, drug X level, drug X time and level X time were significant. The drug X level interaction was due primarily to the maximum prothrombin time obtained for pivalyl at the lowest treatment level (SO mg./bird) in contrast to the increasing prothrombin time of chicks given increasing doses of dicumarol (Fig. 1). The pharmacodynamics of anticoagulants in man have been studied by Weiner et al. (1950), Weiner (1962), Swintosky (1956), O'Reilly et al. (1961, 1962, 1963), Nelson (1964) and others. They found that variations in the prothrombin time response to different anticoagulants may be conditioned by a number of factors. Weiner et al. (1950) showed that the rate of elimination of dicumarol from the plasma is dependent on the size of the dose, while O'Reilly et al. (1963) found that the converse was true for warfarin. O'Reilly et al.
(1962, 1963) using human subjects treated with warfarin, found practically none of this drug excreted in the urine or feces. Nelson (1964) concluded that the elimination of warfarin from the plasma must occur almost exclusively by chemical transformation. The above observations along with the data in this experiment suggest that these drugs differ in either their rate of absorption or chemical transformation in the chick. Weiner (1962) has suggested that the prothrombinopenic response of a particular drug is related to the duration of maintenance of its plasma concentration. In Experiment 4 a single dosage level of 100 mg./bird was used for all drugs. Female birds exhibited highly significant increases in prothrombin time over male birds for all drugs and all times studied (Table 3). These findings are somewhat different from those of Metta et al. (1959), and Malhotra et al. (1963), who reported
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 22, 2015
Male
391
BLOOD PROTHROMBIN TIME PIVALYL
FEMALE 84 80
PIVALYL FEMALE"" MALE *
'», MALE " "
64 60 \
DIPHICANONE
56 52 48 §44 a. X O 40 36 32 28 24 20 12 16 HOURS POST-DOSAGE
20
24
28
FIG. 3. The effect of drug, sex and time on blood prothrombin time, Experiment 4.
male rats to be more susceptible to hemorrhagic diathesis than female rats. Since the average weight of the female and male chicks was 920 and 1083 grams, respectively, it is possible that the drug dose—body weight relationship was responsible for the significantly higher prothrombin time of the females. However, since the data from Experiment 3 indicate a maximum dosage response was attained at the 100 mg./bird
level as compared to the 200 mg./bird level, the differences in dosage response appear to be best explained as sex differences. When a constant level of each drug was used (100 mg./bird), dicumarol produced a maximum effect on prothrombin time in 20 hours for both sexes, while diphacinone caused a maximum effect in 20 hours for male and 24 hours for female birds. Male birds treated with pivalyl reached a peak
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 22, 2015
76 DIPHICANONE FEMALE MALE 72 DICUMAROL FEMALE " MALE *• 68
"* »
392
O. W. CHARLES, B. C. DILWORTH, R. D. BUSHONG, JR. AND E. J. DAY
SUMMARY Three anticoagulants were administered orally in gelatin capsules to broiler chickens. Graded levels of each anticoagulant (0-32mg./bird) produced a significant difference in the prothrombin time of broiler cockerels, 20 hours post-dosage. However, drug differences could not be detected at 20 hours post-dosage when the drug level approached maximum response level (50mg./bird). When prothrombin determinations were made at four hour intervals, 20, 24, 28, and
32 hours post-dosage, each drug elicited significant differences in chick prothrombin times. Significant interactions were found for drug X level, drug X time and level X time. The effect of dicumarol on prothrombin time is more rapid but less severe than diphacinone. Pivalyl requires a longer time and causes a more severe hyperprothrombinemia than either dicumarol or diphacinone. Dicumarol caused a higher prothrombin time in female than in male birds, although both sexes experienced peak hyperprothrombinemia simultaneously (100 mg./bird, 20 hours post-dosage). Diphacinone (100 mg./ bird) exerted a maximum effect on the prothrombin time of female birds 24 hours postdosage while male birds experienced a peak effect 20 hours following drug administration. REFERENCES Day, E. J., B. C. Dilworth and P. N. Dua, 1964. Reduced incidence of blood spots in eggs with dicumarol supplementation. Poultry Sci. 4 3 : 796-798. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 1 1 : 1-42. Griminger, P., and O. Daves, 1959. Potency of vitamin K and two analogues in counteracting the effects of dicumarol and sulfaquinoxaline in the chick. J. Nutrition, 60: 361-368. Harms, R. H., and F. R. Tarver, Jr., 1957. The influence of dicumarol upon blood clotting time and blood loss of young chickens. Poultry Sci. 36: 76-79. Metta, V. C , M. S. Mameesh and B. C. Johnson, 1959. Vitamin K deficiency in rats induced by the feeding of irradiated beef. J. Nutrition, 69: 18-22. Malhotra, O. P., and E. F. Reber, 1963. Methionine and testosterone effect on occurrence of hemorrhagic diathesis in rats. American J. Phys. 205: 1089-1092. Nelson, E., 1964. Kinetics of drug absorption, distribution, metabolism and excretion. J. Pharm. Sci. 50: 181. O'Reilly, R. A., P. M. Aggeler, M. S. Hoag and L. S. Leong, 1961. The assay of warfarin and its biological application. Blood: 18: 787-788.
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 22, 2015
prothrombin time in 24 hours while female birds did not reach a peak prothrombin time within the limits of this experiment (28 hours). The rate of response was essentially the same for all drugs through the 12 th hour, when the response to diphacinone and pivalyl increased sharply and continued at the accelerated rate through their peak effects. In contrast, dicumarol maintained a constant rate of increase through the peak response time (Fig. 3). It is not known whether the pharmacodynamic differences found for the three drugs studied are a function of absorption, elimination rates or some other effect. The prothrombin time is a function of prothrombin, factor V, factor VII and factor X and a deficiency of one or more of these factors will prolong the prothrombin time or decrease the rate (Malhotra and Reber, 1963). Depression of prothrombin can be explained by vitamin K deficiency but depression of factor V is not vitamin K dependent (Quick, 1957). Malhotra and Reber (1963) have said that factor V is decreased in liver damage. Should additional investigation prove that certain drugs produce liver injury they would be unsuitable for such use as sensitizing agents in vitamin K studies.
BLOOD PROTHROMBIN TIME
Febiger, Philadelphia, Pa. p. 379. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc. New York, N.Y. Swintosky, J. V., 1956. Illustrations and pharmaceutical interpretations of first-order drug elimination rate from the bloodstream. J. Amer. Pharm. Ass. Ed. 45: 395. Waldroup, P. W., and R. H. Harms, 1962. The influence of dicumarol on the incidence of blood spots in eggs. Poultry Sci. 4 1 : 510-512. Weiner, M., S. Shapiro, J. Axelrod, J. R. Cooper and B. B. Brodie, 1950. The physiological disposition of dicumarol in man. J. Pharmacal Exp Ther. 69 : 409. Weiner, M., 1962. Pharmacologic considerations of antithrombin therapy. Advanc. Pharmacal. 1: 277. Woody, R. C , E. J. Day and B. Glick, 1963. The effect of time and environment upon blood prothrombin time. Poultry Sci. 42: 1062-1063.
The Effect of Low Protein and High Fiber Grower Diets on the Performance of Broiler Pullets1 P. W. WALDROUP, B. L. DAMRON AND R. H. HARMS Florida Agricultural Experiment Stations, Gainesville, Florida (Received for publication September 27, 1965)
OULTRYMEN have long been seeking a method to alter the growth pattern of both egg-type and meat-type pullets in an attempt to improve performance during the laying period. Vondell (1943) reported that allowing feeders to become empty each day and withholding feed one day a week resulted in satisfactory pullets. Novikoff and Biely (1945) reported that restricting feed intake from 1 day to 12 months of age resulted in slower growth and lowered egg production.
P
1
Florida Agr. Exp. Sta. Journal Series No. 2293.
Many workers have found that restricting feed intake during the growing period results in delaying sexual maturity of pullets (Milby and Sherwood, 1953, 1956; Davis and Watts, 1955; Quisenberry, 1959; Fuller, 1960). Davis and Watts (1955) reported that restriction of feed intake of New Hampshire pullets during the growing period resulted in slightly larger initial eggs, while Milby and Sherwood (1956) and Quisenberry (1959) reported that egg size at any given age was not affected by the feeding program. Restriction of feed intake of grower pullets has been reported to decrease egg pro-
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 22, 2015
O'Reilly, R. A., P. M. Aggeler, M. S. Hoag, and L. S. Leong, 1962. Studies on the coumarin anticoagulant drugs: the assay of warfarin and its biological application. Thrombos. Diathes. Laemmon. (Stuttg) 8: 82-95. O'Reilly, R. A., P. M. Aggeler and L. S. Leong, 1963. Studies on the coumarin anticoagulant drugs: The pharmacodynamics of warfarin in man. J. Clinical Inv. 42: 1542-1561. Perdue, H. S., and D. V. Frost, 1960. Comparison of vitamin K and menadione sodium bisulfite complex as standards in chick diets suggested for vitamin K assay. Feedstuffs, 1: 23-60. Quick, A. J., and M. Stefanini, 1948. Experimentally induced changes in the prothrombin level of the blood. IV. The relation of vitamin K deficiency to the intensity of dicumarol action and to the effect of excess vitamin A intake; with a simplified method for vitamin K assay. J. Biol. Chem. 175:945-952. Quick, A. J., 1957. Hemorrhagic Disease. Lea and
393
METHIONINE SOURCES on growth of chicks fed amino acid diets. Poultry Sci. 43: 1113-1118. Marrett, L. E., and M. L. Sunde, 1965. The effect of other D-amino acids on the utilization of the isomers of methionine and its hydroxy analogue. Poultry Sci. 44: 957-964. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill
Book Company, Inc., New York, N.Y. Tipton, H. C , B. C. Dilworth, and E. J. Day, 1965. The relative biological value of DL-methionine hydroxy analogue in chick diets. Poultry Sci. 44: 987-992. Yankelowitz, S., 1960. Factors affecting the intestinal absorption of methionine. Thesis, Rutgers, The State University. New Brunswick, N J .
O. W. CHARLES, BEN C. DILWORTH, REX D. BUSHONG, JR. AND ELBERT J. DAY Department of Poultry Science, Mississippi State University, State College, Mississippi (Received for publication September 24, 1965)
NCREASED prothrombin time due to feeding dicumarol has been observed with the chick (Quick and Stefanini, 1948; Harms and Tarver, 1957) and laying hens (Waldroup and Harms, 1962; Day et al., 1964). The action of dicumarol in the inhibition of the blood clotting process is lessened by the presence of vitamin K in the diet according to Quick and Stefanini (1948). Although sulfaquinoxaline is used to induce hypoprothrombinemia in chick assays of vitamin K compounds (Perdue and Frost, 1961; Griminger and Daves, 1959), the use of other anticoagulants may prove of value in studies on vitamin K activity or the mechanism of blood clotting as related to the occurrence of blood spots in eggs. The experiments reported herein were designed to evaluate the effects of three anticoagulants on the prothrombin time of chicks.
I
1
3-3'-methylenebis-4 hydroxy coumarin. 2 diphenylacetyl-1, 3-indandione 1-hydrazone; Nease Chemical Co., Inc., State College, Pa. 3 2 pivalyl-1, 3-indandione; Nease Chemical Co., Inc., State College, Pa. 4 Mississippi Agricultural Experiment Station Journal Article No. 1318. 2
EXPERIMENTAL
Four experiments were conducted using broiler strain chicks obtained from a commercial hatchery. All chicks were wing banded and maintained in wire cages. A practical type broiler diet and tap water were provided ad libitum throughout each experiment. The chicks were six week old cockerels in Experiments 1 and 2, eleven week old cockerels in Experiment 3 and eleven week old chicks of both sexes in Experiment 4. The anticoagulants ere administered orally in no. 1 gelatin capsules. Whole blood prothrombin determinations were made according to the method of Woody et al. (1963). Treatment prothrombin time averages were obtained using 4, 4, 8 and 10 birds in Experiments 1, 2, 3, and 4, respectively. Control non-treated chicks were maintained in each experiment. Each of the three drugs was given orally at levels of 1, 2, 4, 8, 16 and 32 mg./chick in Experiment 1 and at 50, 100 and 150 mg./chick in Experiment 2. In Experiment 3, each drug was tested at the 50, 100, 200 and 500 mg./chick level. Sex, drug and post-dosage
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 22, 2015
The Effect of Dicumarol, 1 Diphacinone 2 and PivalyP Upon Blood Prothrombin Time of Chicks4
388
O. W. CHARLES, B. C. DILWORTH, R. D. BUSHONG, JR. AND E. J. DAY TABLE 1.—The effect of dosage level and three anticoagulants on the prothrombin time of broiler cockerels
Drug level, mg./bird
Prothrombin time, seconds1 Dicumarol
Diphac- Pivalyl inone
Average2 0 50 100 150 Average2
18 24 26 38 44 58 53
18 21 24 29 32 40 47
37"
—
Experiment 2 20 20 40 50 44 48 47 51
20 44 41 38
20 45 44 45
44»
41"
—
21"
33 b
50"
1
Prothrombin determinations were made 20 hours post-dosage. 2 Averages within each experiment without a common superscript are significantly different at the 5% level of probability.
time were the criteria studied in Experiment 4. In this latter trial, the dosage level of each drug was 100 mg./chick. The prothrombin determinations were made 20 hours post-dosage in Experiment 1 and 2. The rate at which each drug exerted its effect was examined in the last two experiments. Blood samples were collected 20, 24, 28 and 32 hours (Exp. 3) and 4, 8, 12, 16, 20, 24 and 28 hours (Exp. 4) following drug administration. Statistical examinations of the prothrombin time data were made according to Steel and Torrie (1960) and Duncan (1955). RESULTS AND DISCUSSION In Experiment 1 significant differences in prothrombin time were obtained twenty hours after administering oral doses of three anticoagulants. Diphacinone and pivalyl caused a significant increase in the prothrombin time as compared to the dicu-
In a third experiment, the prothrombin time was measured for all treatments at 20, 24, 28 and 32 hours post-dosage in order to compare the pharmacodynamics of these anticoagulants at different time intervals. Four dosage levels (50, 100, 200 and 500 mg./bird) of dicumarol, diphacinone and pivalyl were administered to cockerels and prothrombin times were determined at four hour intervals (Table 2). The effect of dicumarol tended to decline steadily after the twentieth hour for the three lower levels tested, while the highest level tended to maintain a constant prothrombin time through the 32nd hour (Fig. 1). In contrast, the prothrombin time of birds treated with diphacinone and pivalyl continued to rise from the 20th through the
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 22, 2015
Experiment 1 18 18 18 21 20 27 17 32 16 37 23 41 33 56
0 1 2 4 8 16 32
Average
marol treated birds. Only the highest level of dicumarol (32 mg./bird) elicited a significant increase in the prothrombin time; while diphacinone and pivalyl caused significant changes at a much lower level (4 mg./bird). Small increases in the prothrombin time were obtained with each increase in dosage level with all three drugs except for the highest level of pivalyl (Table 1). Much higher levels of the same three drugs were used in a second experiment in an attempt to determine the drug level that would elicit a maximum increase in prothrombin time (Table 1). It is evident from these data that the lowest dosage level (50 mg.) produced essentially the same effect on prothrombin time as the highest level (150 mg.) at 20 hours postdosage. Since the lowest drug level caused a maximum increase in the prothrombin time for all drugs, significant differences attributable to drugs were not found. It is evident from the data in Experiment 2 that the effect of anticoagulants on prothrombin time cannot be measured using high drug levels and a constant time post-dosage.
389
BLOOD PROTHROMBIN TIME TABLE 2.—The effect of drug level and lime on prothrombin time of chicks treated with three anticoagulants, Experiment 3 Hours post-dosage Drug Dicumarol
Pivalyl
Drug X'level
—
20
24
28
32
0 50 100 200 500
23 41 51 50 62
23 41 44 34 58
23 31 37 43 45 •
23 25 31 27 65
23 34 41 39 58
DrugXtime av.
45
40
36
34
39*
0 50 100 200 500
24 41 55 53 50
25 65 55 63 71
26 58 59 56 85
27 72 64 81 85
25 59 58 63 73
DrugXtime av.
45
56
56
66
56b
0 50 100 200 500
24 55 53 53 53
26 59 69 69 75
25 110 70 75 65
26 99 85 101 134
25 81 69 75 82
DrugXtime av.
48
59
69
89
66°
• Drug means without a common superscript are significantly different at the 5% level of probability.
32 nd hour. The increase in prothrombin time caused by pivalyl was much greater than that produced by diphacinone, (Fig. 2 and Table 2). Each increase in the level of dicumarol and diphacinone caused a corresponding increase in prothrombin time. In-
creasing the level of pivalyl did not always cause a prothrombin response since the lowest level used (50 mg./bird) approached the maximum response level (Fig. 1 and Table 2). PIVALyL
DIPHACINONE
28 32 HOURS POST-DOSAGE
FIG. 1. Drug X level interaction, Experiment 3.
FIG. 2. Drug X time interaction, Experiment 3.
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 22, 2015
Diphacinone
mg./bird
390
O. W. CHARLES, B. C. DILWORTH, R. D. BUSHONG, JR. AND E. J. DAY TABLE 3.—The effect of sex and time on prothrombin time of chicks treated with three anticoagulants, Experiment 4 Prothrombin time, seconds1
Hours post-dosage
Dicumarol2
Diphacinone2
PivalyP
Female
Male
Female
Male
Female
24 24 34 35 41 45 41 25
25 29 32 37 44 49 41 29
24 26 32 34 49 67 61 55
25 23 34 40 54 66 74 58
24 24 30 34 47 63 80 76
25 26 30 36 50 66 79
34
36
44
47
47
50
0 4 8 12 16 20 24 28 Drug X sex av.
35"
Drug av.
46
b
48°
Composite data SexesXtime
Male Female 1 2
0
4
8
12
16
20
24
28
Av.
72 75
74 78
96 96
103 113
137 148
175 181
182 194
156 175
41" 44 b
Drug averages without a common superscript are significantly different (P<.05). Each drug administered at the 100 mg. per bird level.
A statistical examination of these data revealed that all first order interactions, drug X level, drug X time and level X time were significant. The drug X level interaction was due primarily to the maximum prothrombin time obtained for pivalyl at the lowest treatment level (SO mg./bird) in contrast to the increasing prothrombin time of chicks given increasing doses of dicumarol (Fig. 1). The pharmacodynamics of anticoagulants in man have been studied by Weiner et al. (1950), Weiner (1962), Swintosky (1956), O'Reilly et al. (1961, 1962, 1963), Nelson (1964) and others. They found that variations in the prothrombin time response to different anticoagulants may be conditioned by a number of factors. Weiner et al. (1950) showed that the rate of elimination of dicumarol from the plasma is dependent on the size of the dose, while O'Reilly et al. (1963) found that the converse was true for warfarin. O'Reilly et al.
(1962, 1963) using human subjects treated with warfarin, found practically none of this drug excreted in the urine or feces. Nelson (1964) concluded that the elimination of warfarin from the plasma must occur almost exclusively by chemical transformation. The above observations along with the data in this experiment suggest that these drugs differ in either their rate of absorption or chemical transformation in the chick. Weiner (1962) has suggested that the prothrombinopenic response of a particular drug is related to the duration of maintenance of its plasma concentration. In Experiment 4 a single dosage level of 100 mg./bird was used for all drugs. Female birds exhibited highly significant increases in prothrombin time over male birds for all drugs and all times studied (Table 3). These findings are somewhat different from those of Metta et al. (1959), and Malhotra et al. (1963), who reported
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Male
391
BLOOD PROTHROMBIN TIME PIVALYL
FEMALE 84 80
PIVALYL FEMALE"" MALE *
'», MALE " "
64 60 \
DIPHICANONE
56 52 48 §44 a. X O 40 36 32 28 24 20 12 16 HOURS POST-DOSAGE
20
24
28
FIG. 3. The effect of drug, sex and time on blood prothrombin time, Experiment 4.
male rats to be more susceptible to hemorrhagic diathesis than female rats. Since the average weight of the female and male chicks was 920 and 1083 grams, respectively, it is possible that the drug dose—body weight relationship was responsible for the significantly higher prothrombin time of the females. However, since the data from Experiment 3 indicate a maximum dosage response was attained at the 100 mg./bird
level as compared to the 200 mg./bird level, the differences in dosage response appear to be best explained as sex differences. When a constant level of each drug was used (100 mg./bird), dicumarol produced a maximum effect on prothrombin time in 20 hours for both sexes, while diphacinone caused a maximum effect in 20 hours for male and 24 hours for female birds. Male birds treated with pivalyl reached a peak
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76 DIPHICANONE FEMALE MALE 72 DICUMAROL FEMALE " MALE *• 68
"* »
392
O. W. CHARLES, B. C. DILWORTH, R. D. BUSHONG, JR. AND E. J. DAY
SUMMARY Three anticoagulants were administered orally in gelatin capsules to broiler chickens. Graded levels of each anticoagulant (0-32mg./bird) produced a significant difference in the prothrombin time of broiler cockerels, 20 hours post-dosage. However, drug differences could not be detected at 20 hours post-dosage when the drug level approached maximum response level (50mg./bird). When prothrombin determinations were made at four hour intervals, 20, 24, 28, and
32 hours post-dosage, each drug elicited significant differences in chick prothrombin times. Significant interactions were found for drug X level, drug X time and level X time. The effect of dicumarol on prothrombin time is more rapid but less severe than diphacinone. Pivalyl requires a longer time and causes a more severe hyperprothrombinemia than either dicumarol or diphacinone. Dicumarol caused a higher prothrombin time in female than in male birds, although both sexes experienced peak hyperprothrombinemia simultaneously (100 mg./bird, 20 hours post-dosage). Diphacinone (100 mg./ bird) exerted a maximum effect on the prothrombin time of female birds 24 hours postdosage while male birds experienced a peak effect 20 hours following drug administration. REFERENCES Day, E. J., B. C. Dilworth and P. N. Dua, 1964. Reduced incidence of blood spots in eggs with dicumarol supplementation. Poultry Sci. 4 3 : 796-798. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 1 1 : 1-42. Griminger, P., and O. Daves, 1959. Potency of vitamin K and two analogues in counteracting the effects of dicumarol and sulfaquinoxaline in the chick. J. Nutrition, 60: 361-368. Harms, R. H., and F. R. Tarver, Jr., 1957. The influence of dicumarol upon blood clotting time and blood loss of young chickens. Poultry Sci. 36: 76-79. Metta, V. C , M. S. Mameesh and B. C. Johnson, 1959. Vitamin K deficiency in rats induced by the feeding of irradiated beef. J. Nutrition, 69: 18-22. Malhotra, O. P., and E. F. Reber, 1963. Methionine and testosterone effect on occurrence of hemorrhagic diathesis in rats. American J. Phys. 205: 1089-1092. Nelson, E., 1964. Kinetics of drug absorption, distribution, metabolism and excretion. J. Pharm. Sci. 50: 181. O'Reilly, R. A., P. M. Aggeler, M. S. Hoag and L. S. Leong, 1961. The assay of warfarin and its biological application. Blood: 18: 787-788.
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prothrombin time in 24 hours while female birds did not reach a peak prothrombin time within the limits of this experiment (28 hours). The rate of response was essentially the same for all drugs through the 12 th hour, when the response to diphacinone and pivalyl increased sharply and continued at the accelerated rate through their peak effects. In contrast, dicumarol maintained a constant rate of increase through the peak response time (Fig. 3). It is not known whether the pharmacodynamic differences found for the three drugs studied are a function of absorption, elimination rates or some other effect. The prothrombin time is a function of prothrombin, factor V, factor VII and factor X and a deficiency of one or more of these factors will prolong the prothrombin time or decrease the rate (Malhotra and Reber, 1963). Depression of prothrombin can be explained by vitamin K deficiency but depression of factor V is not vitamin K dependent (Quick, 1957). Malhotra and Reber (1963) have said that factor V is decreased in liver damage. Should additional investigation prove that certain drugs produce liver injury they would be unsuitable for such use as sensitizing agents in vitamin K studies.
BLOOD PROTHROMBIN TIME
Febiger, Philadelphia, Pa. p. 379. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc. New York, N.Y. Swintosky, J. V., 1956. Illustrations and pharmaceutical interpretations of first-order drug elimination rate from the bloodstream. J. Amer. Pharm. Ass. Ed. 45: 395. Waldroup, P. W., and R. H. Harms, 1962. The influence of dicumarol on the incidence of blood spots in eggs. Poultry Sci. 4 1 : 510-512. Weiner, M., S. Shapiro, J. Axelrod, J. R. Cooper and B. B. Brodie, 1950. The physiological disposition of dicumarol in man. J. Pharmacal Exp Ther. 69 : 409. Weiner, M., 1962. Pharmacologic considerations of antithrombin therapy. Advanc. Pharmacal. 1: 277. Woody, R. C , E. J. Day and B. Glick, 1963. The effect of time and environment upon blood prothrombin time. Poultry Sci. 42: 1062-1063.
The Effect of Low Protein and High Fiber Grower Diets on the Performance of Broiler Pullets1 P. W. WALDROUP, B. L. DAMRON AND R. H. HARMS Florida Agricultural Experiment Stations, Gainesville, Florida (Received for publication September 27, 1965)
OULTRYMEN have long been seeking a method to alter the growth pattern of both egg-type and meat-type pullets in an attempt to improve performance during the laying period. Vondell (1943) reported that allowing feeders to become empty each day and withholding feed one day a week resulted in satisfactory pullets. Novikoff and Biely (1945) reported that restricting feed intake from 1 day to 12 months of age resulted in slower growth and lowered egg production.
P
1
Florida Agr. Exp. Sta. Journal Series No. 2293.
Many workers have found that restricting feed intake during the growing period results in delaying sexual maturity of pullets (Milby and Sherwood, 1953, 1956; Davis and Watts, 1955; Quisenberry, 1959; Fuller, 1960). Davis and Watts (1955) reported that restriction of feed intake of New Hampshire pullets during the growing period resulted in slightly larger initial eggs, while Milby and Sherwood (1956) and Quisenberry (1959) reported that egg size at any given age was not affected by the feeding program. Restriction of feed intake of grower pullets has been reported to decrease egg pro-
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O'Reilly, R. A., P. M. Aggeler, M. S. Hoag, and L. S. Leong, 1962. Studies on the coumarin anticoagulant drugs: the assay of warfarin and its biological application. Thrombos. Diathes. Laemmon. (Stuttg) 8: 82-95. O'Reilly, R. A., P. M. Aggeler and L. S. Leong, 1963. Studies on the coumarin anticoagulant drugs: The pharmacodynamics of warfarin in man. J. Clinical Inv. 42: 1542-1561. Perdue, H. S., and D. V. Frost, 1960. Comparison of vitamin K and menadione sodium bisulfite complex as standards in chick diets suggested for vitamin K assay. Feedstuffs, 1: 23-60. Quick, A. J., and M. Stefanini, 1948. Experimentally induced changes in the prothrombin level of the blood. IV. The relation of vitamin K deficiency to the intensity of dicumarol action and to the effect of excess vitamin A intake; with a simplified method for vitamin K assay. J. Biol. Chem. 175:945-952. Quick, A. J., 1957. Hemorrhagic Disease. Lea and
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