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The Influence of Dicumarol upon Blood Clotting Time and Blood Loss of Young Chickens*
The Influence of Dicumaro] upon Blood Clotting Time and Blood Loss of Young Chickens* R. H. HARMS AND F. R. TARVER, J R . University of Tennessee, Agricultural Experiment Station, Knoxville, Tennessee (Received for publication July 10, 1956)
T
or the relationship of this material to blood obtained in the killing operation. The composition of the three diets used is presented in Table 1. Data presented in Table 2 were secured from chicks fed diet 2 while data in Tables 3 and 4 were secured from chicks fed basal diet 3. Basal diet 1 was fed to 9 week old chicks and the data secured from these birds are presented in Table 5. All chicks received feed and water ad libitum. Thirty-six hours prior to the determination of blood clotting time and/or total blood yield, dicumarol in the amounts indicated in the various tables was added to the basal diet. The 36 hour feeding period was arbitrarily selected in an effort to afford the anticoagulant adequate time to express itself. Of the 256 chicks used in these studies, 178 Single Comb White TABLE 1.—Composition of basal diets
Lbs./Cwt. Ingredients Yellow Corn Soybean Oil Meal Alfalfa Leaf Meal Fish Meal Dried Whey De-Fluorinated Phosphate Ground Limestone Salt Vitamin Mix1
EXPERIMENTAL
A total of 256 chicks were used in this study to determine the effect that 350 milligrams, 750 milligrams, and 1,500 milligrams of dicumarol per pound of feed might have upon blood clotting time and/
Diet #1
Diet #2
Diet #3
59.7 28.0 3.0 3.0 3.0
61.2 32.5 3.0
64.2 32.5
— —
— —. —
1.0 1.2 0.5 0.6
1.0 1.2 0.5 0.6
1.0 1.2 0.5 0.6
1 Supplied per lb. of feed: 0.9 mg. riboflavin, 3 mg. calcium pantothenate, 5 mg. niacin, 3 meg. vitamin B12, 250 mg. choline chloride, 1,737 I.U. vitamin A, 1,050 I.C.U. vitamin D 3 , 0.08 gms. MnS04, 2 mg. procaine penicillin, and 45 mg. arsanilic acid.
* Published with the approval of the Director of Tennessee Agricultural Experiment Station.
76
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 21, 2015
HE blood loss of slaughtered poultry is of great concern to both the poultry processor and the consumer. The blood remaining in the vascular system of dressed and cut up poultry is unattractive to the consumer. Blood retained in the carcass also lowers the quality and grade of the product. Newell and Shaffner (1950) observed that the total loss of blood during the bleeding operation was 35 percent to 50 percent of the total blood volume. Davis and Cole (1954) found that when birds were beheaded or a single artery cut, the flow of blood was slower at the end of 20 seconds than from those birds which were debrained and both carotid arteries cut or from birds that were stunned and a single artery cut or from chickens that were stunned and both arteries cut. The purpose of this study was to determine the influence of an anticoagulant upon blood clotting time and total blood loss due to such treatment. Dicumarol was selected as the anticoagulant to be used since it was shown by Quick and Stefanini (1948) to increase prothrombin time in chicks fed a vitamin K deficient diet.
DlCUMAROL AND BLOOD CLOTTING
77
TABLE 2.—Blood clotting time of jive week old Single TABLE 3.—Blood clotting time of six week old Single Comb White Leghorn chicks as effected by feeding Comb White Leghorn cockerels fed various levels of dicumarol in a diet which did not contain alfalfa dicumarol in a diet containing three percent alfalfa leaf meal leaf meal
Test No.
Sex
No. birds/ treatment
1 2 3 4
Mixed 9 9 9
5 6 61 5
Treatment clotting time (minutes & seconds) (mg. dicumarol/lb. of feed) 0
— 2'12" 2'34' —
350
750
No. birds
Treatment
Average clotting time1 (minutes and seconds)
10 10 10 10
Control 375 mg. 7S0 mg. 1,500 mg.
3'40" B'lO"" 22'34" 28'19"
1500
5'26" 11'19' 16'56" — 7'20" 1 2 W " 8'12" 16'40"» 19'55"* 3'56" 4'23" 4'21"
Leghorn chicks were grown to determine the effective level of dicumarol to be used to prolong blood clotting time and the effect upon blood losses during slaughter. These chicks were grown on wire in an electrically heated battery brooder. Dicumarol was first fed when these chicks were either 5 or 6 weeks of age. Data secured from the these chicks are presented in Tables 2, 3, and 4. Other chicks used in this study consisted of 78 cross bred chicks which had been grown on the floor until 9 weeks of age at which time they were placed at random in laying cages. These chicks were fed basal diet 1. Two groups of 8 males and 8 females were maintained in a temperature controlled room at 52°F. + 4°. Two other groups consisting of 9 males
1
Approximate L.S.D. .05 = 10'05" .01 = 13'05"
and 8 females were placed in a temperature controlled room maintained with a temperature of 85°F.±4°. After these birds had been in the temperature controlled room for 36 hours one group of birds in each room received 1,500 milligrams of dicumarol per pound of feed. At the end of a three day period all birds were sacrificed and blood losses were determined. Blood clotting times were determined by the capillary tube method using blood obtained by puncturing the brachial vein of each chick. Blood losses during killing operations were made by severing the jugular vein and carotid arteries on the outside of the neck in such a way that the blood could be collected in a waxed paper sack. Blood was collected over a 2 minute period and
TABLE 4.-—Effect of feeding dicumarol upon the blood losses of six week old Single Comb White Leghorn chicks
Test No.
1 2 3 4
Sex
ci" d"
& 9
Average blood losses (grams)
Blood loss (percent of live weight)
No. birds/ treatment
Control
1,500 mg. dicumarol/lb. feed
Control
1,500 mg. dicumarol/lb. feed
10 10 8 10
27.3 31.5 31.9 32.6
30.4 31.5 30.3 30.6
3.44 3.80 3.84 3.75
3.83* 4.03 3.88 3.81
* Significant at 0.05 level of probability.
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 21, 2015
1 These birds are the same as used in Test No. 3 which were fed the basal diet for 48 hours and clotting time again determined. * Significant at 0.05 level of probability when compared to control group. ** Significant at 0.01 level of probability when compared to control group.
78
R. H. HARMS AND F. R. TARVER, J R . TABLE 5.—Effect
Treatment
of feeding dicumarol upon the blood losses of nine week old crossbred chicks when held at 52° or 85° Fahrenheit
No. of birds/treatment Males
Females
Average blood loss (grams)
Blood loss (percent of live weight) Av.
Males Females
Males
Females
Av.
-
9 9
9 9
Warm Room (85°F. ) 42.0 38.1 40.0 49.4 37.6 43.5
3.40 4.18
3.54 4.06
3.46 4.13**
Control Dicumarol (1,500 mgs.)
8 8
8 8
Cool Room (52°F.) 43.6 50.4 36.9 41.8 48.9 34.8
3.89 4.10
3.45 3.49
3.69 3.82**
:
Significant at 0.01 level of probability.
then weighed to the nearest 1/10 gram. All data were analyzed using analysis of variance according to Snedecor (1946). RESULTS AND DISCUSSION
The Effect of Feeding Dicumarol Upon Blood Clotting Time. A significant increase in blood clotting time was obtained (Table 2) with chicks fed 750 milligrams of dicumarol per pound of basal diet. The inclusion of 1,500 milligrams of dicumarol per pound of feed resulted in a highly significant increase in blood clotting time. It should be pointed out that these chicks were fed a diet containing 3 percent dehydrated alfalfa leaf meal, thus an adequate level of vitamin K was considered to be present. The feeding of dicumarol at a level of 750 milligrams per pound in a basal diet containing no alfalfa leaf meal resulted in a highly significant increase in blood clotting time when compared to the control groups (Table 3). The difference in amounts of dicumarol required to produce a prolonged blood clotting time in these tests may be attributed to the difference of the vitamin K content of the two basal diets. Data presented in Tables 2 and 3 show that when chicks received a diet contain-
ing alfalfa leaf meal and supplemented with 1,500 milligrams of dicumarol per pound of feed, for a period of 36 hours, a prolonged blood clotting time was obtained. Chicks receiving a diet containing no alfalfa leaf meal but containing 750 milligrams of dicumarol per pound of feed for a period of 36 hours prior to examination gave a significantly prolonged blood clotting time. Immediately after securing data from the 5 week old Single Comb White Leghorn chicks, as represented in Table 2, they were given, ad libitum, the basal diet. At the end of 48 hours blood clotting time had returned to approximately normal. The Effect of Feeding Dicumarol Upon Blood Loss. The birds in this portion of the study (Table 4) were fed a diet containing 1,500 milligrams of dicumarol per pound of feed 36 hours prior to slaughtering. In test 1 the blood losses from the birds receiving dicumarol were significantly greater than the blood losses from the birds receiving the control diet when blood losses were measured as a percent of live weight. Blood losses, as percent of live weight, were greater in test 2, 3, and 4 from the birds receiving dicumarol than were the losses of the respective control
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 21, 2015
Control Dicumarol (1,500 mgs.)
DlCTJMAROL AND BLOOD CLOTTING
SUMMARY
The feeding of dicumarol to growing chickens resulted in prolonging blood clotting time. A level of 750 milligrams per pound of feed was required to induce prolonged blood clotting time when the basal diet did not contain alfalfa leaf meal. The feeding of dicumarol for a period of 36 hours was found adequate to prolong blood clotting time. Blood losses of chicks were significantly
increased when blood clotting time was prolonged by the feeding of dicumarol. ACKNOWLEDGMENTS The dicumarol and arsanilic acid were supplied through the courtesy of Abbott Laboratories, North Chicago, 111. Calcium pantothenate, niacin, vitamin A, vitamin B12, choline chloride and procaine penicillin were supplied through the courtesy of Chas. F. Pfizer, & Co., Inc., Terre Haute, Indiana. REFERENCES Davis, L. L., and M. E. Cole, 1954. Bleeding of chickens during killings operations. Poultry Sci. 33:616-^19. Newell, G. W., and C. S. Shaffner, 1950. Blood lost by chickens during killing. Poultry Sci. 29: 271-275. 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. Snedecor, G. W., 1946. Statistical Methods. Iowa State College Press, Ames, Iowa.
The Immunological Response of Chickens After Treatment with Several Vaccines of Visceral Lymphomatosis B . R . BuRMESTER, W . G . WALTER AND A . K . FONTES U. S. Department of Agriculture, Agricultural Research Service, Regional Poultry Research Laboratory, East Lansing, Michigan (Received for publication July 13,1956)
INTRODUCTION
T
HE transfer of immune substances from dam to offspring plays a very important part in the protection of the new born from many infectious diseases. This is true not only for progeny that develop in the uterus, but also for those which hatch from eggs. This parental immunity is an important factor in the con-
trol of several viral diseases of poultry such as infectious bronchitis, laryngotracheitis, and Newcastle disease. Burmester (1955a) demonstrated this type of immunity in the progeny of chickens that had received repeated injections of the virus of visceral lymphomatosis (V.L.). The progeny were much more resistant to challenge inoculation than progeny of the
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 21, 2015
groups, however, these differences did not reach a level of statistical significance. A highly significant increase in the amount of blood loss was obtained from 9 week old birds fed 1,500 milligrams of dicumarol per pound of feed (Table 5). This increase in blood loss was evident whether the birds were kept at either 52°F. or 85°F. No significant difference could be detected between blood losses at these temperatures. This study indicated that males had higher blood losses than females, however, these differences were not found to be statistically significant.
79
T
or the relationship of this material to blood obtained in the killing operation. The composition of the three diets used is presented in Table 1. Data presented in Table 2 were secured from chicks fed diet 2 while data in Tables 3 and 4 were secured from chicks fed basal diet 3. Basal diet 1 was fed to 9 week old chicks and the data secured from these birds are presented in Table 5. All chicks received feed and water ad libitum. Thirty-six hours prior to the determination of blood clotting time and/or total blood yield, dicumarol in the amounts indicated in the various tables was added to the basal diet. The 36 hour feeding period was arbitrarily selected in an effort to afford the anticoagulant adequate time to express itself. Of the 256 chicks used in these studies, 178 Single Comb White TABLE 1.—Composition of basal diets
Lbs./Cwt. Ingredients Yellow Corn Soybean Oil Meal Alfalfa Leaf Meal Fish Meal Dried Whey De-Fluorinated Phosphate Ground Limestone Salt Vitamin Mix1
EXPERIMENTAL
A total of 256 chicks were used in this study to determine the effect that 350 milligrams, 750 milligrams, and 1,500 milligrams of dicumarol per pound of feed might have upon blood clotting time and/
Diet #1
Diet #2
Diet #3
59.7 28.0 3.0 3.0 3.0
61.2 32.5 3.0
64.2 32.5
— —
— —. —
1.0 1.2 0.5 0.6
1.0 1.2 0.5 0.6
1.0 1.2 0.5 0.6
1 Supplied per lb. of feed: 0.9 mg. riboflavin, 3 mg. calcium pantothenate, 5 mg. niacin, 3 meg. vitamin B12, 250 mg. choline chloride, 1,737 I.U. vitamin A, 1,050 I.C.U. vitamin D 3 , 0.08 gms. MnS04, 2 mg. procaine penicillin, and 45 mg. arsanilic acid.
* Published with the approval of the Director of Tennessee Agricultural Experiment Station.
76
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 21, 2015
HE blood loss of slaughtered poultry is of great concern to both the poultry processor and the consumer. The blood remaining in the vascular system of dressed and cut up poultry is unattractive to the consumer. Blood retained in the carcass also lowers the quality and grade of the product. Newell and Shaffner (1950) observed that the total loss of blood during the bleeding operation was 35 percent to 50 percent of the total blood volume. Davis and Cole (1954) found that when birds were beheaded or a single artery cut, the flow of blood was slower at the end of 20 seconds than from those birds which were debrained and both carotid arteries cut or from birds that were stunned and a single artery cut or from chickens that were stunned and both arteries cut. The purpose of this study was to determine the influence of an anticoagulant upon blood clotting time and total blood loss due to such treatment. Dicumarol was selected as the anticoagulant to be used since it was shown by Quick and Stefanini (1948) to increase prothrombin time in chicks fed a vitamin K deficient diet.
DlCUMAROL AND BLOOD CLOTTING
77
TABLE 2.—Blood clotting time of jive week old Single TABLE 3.—Blood clotting time of six week old Single Comb White Leghorn chicks as effected by feeding Comb White Leghorn cockerels fed various levels of dicumarol in a diet which did not contain alfalfa dicumarol in a diet containing three percent alfalfa leaf meal leaf meal
Test No.
Sex
No. birds/ treatment
1 2 3 4
Mixed 9 9 9
5 6 61 5
Treatment clotting time (minutes & seconds) (mg. dicumarol/lb. of feed) 0
— 2'12" 2'34' —
350
750
No. birds
Treatment
Average clotting time1 (minutes and seconds)
10 10 10 10
Control 375 mg. 7S0 mg. 1,500 mg.
3'40" B'lO"" 22'34" 28'19"
1500
5'26" 11'19' 16'56" — 7'20" 1 2 W " 8'12" 16'40"» 19'55"* 3'56" 4'23" 4'21"
Leghorn chicks were grown to determine the effective level of dicumarol to be used to prolong blood clotting time and the effect upon blood losses during slaughter. These chicks were grown on wire in an electrically heated battery brooder. Dicumarol was first fed when these chicks were either 5 or 6 weeks of age. Data secured from the these chicks are presented in Tables 2, 3, and 4. Other chicks used in this study consisted of 78 cross bred chicks which had been grown on the floor until 9 weeks of age at which time they were placed at random in laying cages. These chicks were fed basal diet 1. Two groups of 8 males and 8 females were maintained in a temperature controlled room at 52°F. + 4°. Two other groups consisting of 9 males
1
Approximate L.S.D. .05 = 10'05" .01 = 13'05"
and 8 females were placed in a temperature controlled room maintained with a temperature of 85°F.±4°. After these birds had been in the temperature controlled room for 36 hours one group of birds in each room received 1,500 milligrams of dicumarol per pound of feed. At the end of a three day period all birds were sacrificed and blood losses were determined. Blood clotting times were determined by the capillary tube method using blood obtained by puncturing the brachial vein of each chick. Blood losses during killing operations were made by severing the jugular vein and carotid arteries on the outside of the neck in such a way that the blood could be collected in a waxed paper sack. Blood was collected over a 2 minute period and
TABLE 4.-—Effect of feeding dicumarol upon the blood losses of six week old Single Comb White Leghorn chicks
Test No.
1 2 3 4
Sex
ci" d"
& 9
Average blood losses (grams)
Blood loss (percent of live weight)
No. birds/ treatment
Control
1,500 mg. dicumarol/lb. feed
Control
1,500 mg. dicumarol/lb. feed
10 10 8 10
27.3 31.5 31.9 32.6
30.4 31.5 30.3 30.6
3.44 3.80 3.84 3.75
3.83* 4.03 3.88 3.81
* Significant at 0.05 level of probability.
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 21, 2015
1 These birds are the same as used in Test No. 3 which were fed the basal diet for 48 hours and clotting time again determined. * Significant at 0.05 level of probability when compared to control group. ** Significant at 0.01 level of probability when compared to control group.
78
R. H. HARMS AND F. R. TARVER, J R . TABLE 5.—Effect
Treatment
of feeding dicumarol upon the blood losses of nine week old crossbred chicks when held at 52° or 85° Fahrenheit
No. of birds/treatment Males
Females
Average blood loss (grams)
Blood loss (percent of live weight) Av.
Males Females
Males
Females
Av.
-
9 9
9 9
Warm Room (85°F. ) 42.0 38.1 40.0 49.4 37.6 43.5
3.40 4.18
3.54 4.06
3.46 4.13**
Control Dicumarol (1,500 mgs.)
8 8
8 8
Cool Room (52°F.) 43.6 50.4 36.9 41.8 48.9 34.8
3.89 4.10
3.45 3.49
3.69 3.82**
:
Significant at 0.01 level of probability.
then weighed to the nearest 1/10 gram. All data were analyzed using analysis of variance according to Snedecor (1946). RESULTS AND DISCUSSION
The Effect of Feeding Dicumarol Upon Blood Clotting Time. A significant increase in blood clotting time was obtained (Table 2) with chicks fed 750 milligrams of dicumarol per pound of basal diet. The inclusion of 1,500 milligrams of dicumarol per pound of feed resulted in a highly significant increase in blood clotting time. It should be pointed out that these chicks were fed a diet containing 3 percent dehydrated alfalfa leaf meal, thus an adequate level of vitamin K was considered to be present. The feeding of dicumarol at a level of 750 milligrams per pound in a basal diet containing no alfalfa leaf meal resulted in a highly significant increase in blood clotting time when compared to the control groups (Table 3). The difference in amounts of dicumarol required to produce a prolonged blood clotting time in these tests may be attributed to the difference of the vitamin K content of the two basal diets. Data presented in Tables 2 and 3 show that when chicks received a diet contain-
ing alfalfa leaf meal and supplemented with 1,500 milligrams of dicumarol per pound of feed, for a period of 36 hours, a prolonged blood clotting time was obtained. Chicks receiving a diet containing no alfalfa leaf meal but containing 750 milligrams of dicumarol per pound of feed for a period of 36 hours prior to examination gave a significantly prolonged blood clotting time. Immediately after securing data from the 5 week old Single Comb White Leghorn chicks, as represented in Table 2, they were given, ad libitum, the basal diet. At the end of 48 hours blood clotting time had returned to approximately normal. The Effect of Feeding Dicumarol Upon Blood Loss. The birds in this portion of the study (Table 4) were fed a diet containing 1,500 milligrams of dicumarol per pound of feed 36 hours prior to slaughtering. In test 1 the blood losses from the birds receiving dicumarol were significantly greater than the blood losses from the birds receiving the control diet when blood losses were measured as a percent of live weight. Blood losses, as percent of live weight, were greater in test 2, 3, and 4 from the birds receiving dicumarol than were the losses of the respective control
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 21, 2015
Control Dicumarol (1,500 mgs.)
DlCTJMAROL AND BLOOD CLOTTING
SUMMARY
The feeding of dicumarol to growing chickens resulted in prolonging blood clotting time. A level of 750 milligrams per pound of feed was required to induce prolonged blood clotting time when the basal diet did not contain alfalfa leaf meal. The feeding of dicumarol for a period of 36 hours was found adequate to prolong blood clotting time. Blood losses of chicks were significantly
increased when blood clotting time was prolonged by the feeding of dicumarol. ACKNOWLEDGMENTS The dicumarol and arsanilic acid were supplied through the courtesy of Abbott Laboratories, North Chicago, 111. Calcium pantothenate, niacin, vitamin A, vitamin B12, choline chloride and procaine penicillin were supplied through the courtesy of Chas. F. Pfizer, & Co., Inc., Terre Haute, Indiana. REFERENCES Davis, L. L., and M. E. Cole, 1954. Bleeding of chickens during killings operations. Poultry Sci. 33:616-^19. Newell, G. W., and C. S. Shaffner, 1950. Blood lost by chickens during killing. Poultry Sci. 29: 271-275. 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. Snedecor, G. W., 1946. Statistical Methods. Iowa State College Press, Ames, Iowa.
The Immunological Response of Chickens After Treatment with Several Vaccines of Visceral Lymphomatosis B . R . BuRMESTER, W . G . WALTER AND A . K . FONTES U. S. Department of Agriculture, Agricultural Research Service, Regional Poultry Research Laboratory, East Lansing, Michigan (Received for publication July 13,1956)
INTRODUCTION
T
HE transfer of immune substances from dam to offspring plays a very important part in the protection of the new born from many infectious diseases. This is true not only for progeny that develop in the uterus, but also for those which hatch from eggs. This parental immunity is an important factor in the con-
trol of several viral diseases of poultry such as infectious bronchitis, laryngotracheitis, and Newcastle disease. Burmester (1955a) demonstrated this type of immunity in the progeny of chickens that had received repeated injections of the virus of visceral lymphomatosis (V.L.). The progeny were much more resistant to challenge inoculation than progeny of the
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 21, 2015
groups, however, these differences did not reach a level of statistical significance. A highly significant increase in the amount of blood loss was obtained from 9 week old birds fed 1,500 milligrams of dicumarol per pound of feed (Table 5). This increase in blood loss was evident whether the birds were kept at either 52°F. or 85°F. No significant difference could be detected between blood losses at these temperatures. This study indicated that males had higher blood losses than females, however, these differences were not found to be statistically significant.
79