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Effect of Ascorbic Acid Injections on the Amount in the Blood Plasma of Laying Hens
Effect of Ascorbic Acid Injections on the Amount in the Blood Plasma of Laying Hens G. HOWARD SATTERFIELD, THOMAS A. BELL, 1 AND F. W. COOK 2 AND ARTHUR D. HOLMES
Massachusetts State College and Agricultural Experiment Station, Amherst (Received for publication, July 24, 1944)
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ARLIER papers of this series have been concerned with the ascorbic acid content of the blood plasma of chickens and laying hens fed typical poultry rations. Holmes, Tripp, and Satterfield (1938), reported that the ascorbic acid content of plasma of rapidly growing eight-week old Rhode Island Red chicks varied from 1.56 mg, to 2.23 mg., and averaged 2.04 mg. percent, whereas that of 12-week old chicks varied from 1.46 mg. to 2.43 mg., and averaged 2.05 mg. percent. They found no consistent relationship between the plasma ascorbic acid values of the different sexes. Later these authors (1939) studied the ascorbic acid content of the blood plasma of Rhode Island Red pullets which received a typical poultry ration during a 15-month laying period. The ascorbic acid content of the blood plasma of the pullets at the various stages of the experiment was 1.84 mg. percent at the beginning of the laying period when the birds were 140 days old, 1.13 mg. percent at 211 days of age, 1.63 mg. at 255 days, 1.06 mg. at 503 days,
1.29 mg. at 509 days, i.07 percent at 506 days and 1.91 mg. percent at 600 days of age. These results show very little correlation between the ascorbic acid content of the blood plasma and egg production, body weight, food intake, or age of the birds. In the present study data were . collected concerning the ascorbic acid content of the blood of hens which had received large injections of ascorbic acid. EXPERIMENTAL
For the purpose of this study, 12 normal, well-developed Barred Plymouth Rock hens weighing about 3,200 gms., were divided into two groups of six birds each. Bdth groups received the same mash and scratch ration. The mash consisted of No. 2 yellow corn meal 25.2 percent, standard wheat middlings 20 percent, v No. 2 heavy oats finely ground 20 percent, standard wheat bran 10 percent, meat scrap (55 percent protein) 6.2 percent, fish meal (55 percent protein) 6.2 percent, dried skimmilk 7 percent, steamed bone meal 1 percent and cod liver oil (400 vitamin D and 3,000 vitamin A units per gram) 0.33 percent. The 1 Nutrition Officer, U.. S.. A. scratch ration contained equal parts of 8 United States Army. Massachusetts Agricultural Experiment Station. Contribution No. 524. cracked corn and wheat. The birds in the 139
Downloaded from http://ps.oxfordjournals.org/ at University of Western Ontario on April 11, 2015
University of North Carolina, Raleigh
140
G. H. SATTERFIELD, T. A. BELL, F. W. COOK, AND A. D. HOLMES
were taken from the heart of each bird of the experimental and control groups with a syringe that contained heparin as an anticoagulant. The ascorbic acid in the blood was determined by a modification of the Satterfield, Perlzwieg, and Dann (1937) method. The blood was transferred to a 15 cc. centrifuge tube which had previously been coated with 5 mg. of sodium cyanide, that served as an anti-oxidant. The sample was centrifuged 20 minutes at
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Minu+ea a f t i r injection." FIG.
experiment, a total of 246 injections for the six birds. The ascorbic acid was injected subcutaneously under the thigh. During the first week the injections contained 100 mg. of ascorbic acid and thereafter the injections contained 50 mg. of ascorbic acid, in 2 cc. of sterile Ringer's solution as described by Guyer (1936). Thus the total amount of ascorbic acid injected was 2,150 mg. per bird. Every three weeks samples of blood
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2,000 r.p.m. Then 2 cc. of the strawcolored plasma was pipetted into a 25 cc. Erlenmeyer flask and 3 cc. of reboiled distilled water was added, followed by 5 cc. of 4 percent metaphosphoric acid solution, and the mixture was rotated. The milky precipitate was filtered off and 5 cc. of the clear filtrate was titrated against the standardized 2-6 dichlorobenzenoneindophenol solution until a faint pink color remained for 30 seconds.
Downloaded from http://ps.oxfordjournals.org/ at University of Western Ontario on April 11, 2015
experimental group consumed 68.0 gms. of mash and 54.8 gms. of scratch feed, whereas those in the control group ate 70.4 gms. of mash and 49.5 gms. of scratch feed per bird per day, during the four month experimental period (FebruaryJune). Each hen in the experimental group received two injections of ascorbic acid per week for the first 9 weeks, and four injections 'per week for the balance of the
ASCORBIC ACID INJECTIONS IN LAYING HENS
RESULTS
was no detectable difference in the physical appearance or the vitality of the birds of the experimental and control groups during the experiment. The experimental group laid 455 eggs and the control group 460 eggs but one-third of the experimental group did not produce any eggs. No toxic effect could be detected when as much as 200 mg. of ascorbic acid was injected into birds during a week. SUMMARY
The data obtained in this study show that the ascorbic acid content of the blood of laying hens can be greatly increased by subcutaneous injections of ascorbic acid. However, this effect is very transitory. In two hours the amount of ascorbic acid in the hens' blood was nearly the same as before the injection of 50 mg. of ascorbic acid. No toxic effect was noted when 200 mg. was injected within a week. The subcutaneous injection of ascorbic acid did not affect the physical appearance, vitality or egg production of the hens. REFERENCES
Guyer, M. F., 1936. Animal micrology. 4th ed., p. 264. University of Chicago Press. Holmes, Arthur P., Francis Tripp, and G. Howard Satterfield, 1938. The Ascorbic Acid Content of Chick Blood. J. Nutrition 16:407-416. —, 1939. The ascorbic acid content of blood plasma of laying hens. Poultry Sci. 18:192-200. Satterfield, G. Howard, W. A. Perlzwieg and W. J. Dann, 1937. Unpublished data. Duke Medical School.
Downloaded from http://ps.oxfordjournals.org/ at University of Western Ontario on April 11, 2015
At the beginning of the experimental period the average ascorbic.acid content of the blood plasma (Figure 1) of the experimental group of hens was slightly higher than that of the control group, i.e., 1.6 mg. and 1.3 mg. per 100 cci of plasma respectively. The ascorbic acid content of the blood plasma of the control group remained practically constant throughout the experiment. In contrast, the amount of ascorbic acid in the blood of the experimental hens increased very rapidly following the injection and reached a maximum of 5.7 mg. per 100 cc. of blood plasma in about 25 minutes, an amount nearly four times the initial value. Following this the ascorbic acid decreased but the rate of decrease was not as rapid as the increase. After the second 25minute interval the blood contained 4.1 mg. of ascorbic acid per 100 cc. of blood plasma; during the second hour the decrease in the ascorbic acid content was still slower. Two hours after injection the amount of ascorbic acid in the blood plasma was 1.8 mg. or only 0.2 mg. above the initial level. Twenty-four hours after the injection the ascorbic acid content had returned to the initial level. However the amount of ascorbic acid continued to decrease slowly for three additional days and four days after injection was 1.0 mg. per 100 cc. of blood plasma or 0.6 mg. lower than at the start of the experiment. There
141
Massachusetts State College and Agricultural Experiment Station, Amherst (Received for publication, July 24, 1944)
E
ARLIER papers of this series have been concerned with the ascorbic acid content of the blood plasma of chickens and laying hens fed typical poultry rations. Holmes, Tripp, and Satterfield (1938), reported that the ascorbic acid content of plasma of rapidly growing eight-week old Rhode Island Red chicks varied from 1.56 mg, to 2.23 mg., and averaged 2.04 mg. percent, whereas that of 12-week old chicks varied from 1.46 mg. to 2.43 mg., and averaged 2.05 mg. percent. They found no consistent relationship between the plasma ascorbic acid values of the different sexes. Later these authors (1939) studied the ascorbic acid content of the blood plasma of Rhode Island Red pullets which received a typical poultry ration during a 15-month laying period. The ascorbic acid content of the blood plasma of the pullets at the various stages of the experiment was 1.84 mg. percent at the beginning of the laying period when the birds were 140 days old, 1.13 mg. percent at 211 days of age, 1.63 mg. at 255 days, 1.06 mg. at 503 days,
1.29 mg. at 509 days, i.07 percent at 506 days and 1.91 mg. percent at 600 days of age. These results show very little correlation between the ascorbic acid content of the blood plasma and egg production, body weight, food intake, or age of the birds. In the present study data were . collected concerning the ascorbic acid content of the blood of hens which had received large injections of ascorbic acid. EXPERIMENTAL
For the purpose of this study, 12 normal, well-developed Barred Plymouth Rock hens weighing about 3,200 gms., were divided into two groups of six birds each. Bdth groups received the same mash and scratch ration. The mash consisted of No. 2 yellow corn meal 25.2 percent, standard wheat middlings 20 percent, v No. 2 heavy oats finely ground 20 percent, standard wheat bran 10 percent, meat scrap (55 percent protein) 6.2 percent, fish meal (55 percent protein) 6.2 percent, dried skimmilk 7 percent, steamed bone meal 1 percent and cod liver oil (400 vitamin D and 3,000 vitamin A units per gram) 0.33 percent. The 1 Nutrition Officer, U.. S.. A. scratch ration contained equal parts of 8 United States Army. Massachusetts Agricultural Experiment Station. Contribution No. 524. cracked corn and wheat. The birds in the 139
Downloaded from http://ps.oxfordjournals.org/ at University of Western Ontario on April 11, 2015
University of North Carolina, Raleigh
140
G. H. SATTERFIELD, T. A. BELL, F. W. COOK, AND A. D. HOLMES
were taken from the heart of each bird of the experimental and control groups with a syringe that contained heparin as an anticoagulant. The ascorbic acid in the blood was determined by a modification of the Satterfield, Perlzwieg, and Dann (1937) method. The blood was transferred to a 15 cc. centrifuge tube which had previously been coated with 5 mg. of sodium cyanide, that served as an anti-oxidant. The sample was centrifuged 20 minutes at
0
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£_.C jotxc I_G uoup JO zo 30 "to &o GO TO e>o so soo //o /$o ...
Minu+ea a f t i r injection." FIG.
experiment, a total of 246 injections for the six birds. The ascorbic acid was injected subcutaneously under the thigh. During the first week the injections contained 100 mg. of ascorbic acid and thereafter the injections contained 50 mg. of ascorbic acid, in 2 cc. of sterile Ringer's solution as described by Guyer (1936). Thus the total amount of ascorbic acid injected was 2,150 mg. per bird. Every three weeks samples of blood
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-.22:
KjJeca Qf_fl
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2,000 r.p.m. Then 2 cc. of the strawcolored plasma was pipetted into a 25 cc. Erlenmeyer flask and 3 cc. of reboiled distilled water was added, followed by 5 cc. of 4 percent metaphosphoric acid solution, and the mixture was rotated. The milky precipitate was filtered off and 5 cc. of the clear filtrate was titrated against the standardized 2-6 dichlorobenzenoneindophenol solution until a faint pink color remained for 30 seconds.
Downloaded from http://ps.oxfordjournals.org/ at University of Western Ontario on April 11, 2015
experimental group consumed 68.0 gms. of mash and 54.8 gms. of scratch feed, whereas those in the control group ate 70.4 gms. of mash and 49.5 gms. of scratch feed per bird per day, during the four month experimental period (FebruaryJune). Each hen in the experimental group received two injections of ascorbic acid per week for the first 9 weeks, and four injections 'per week for the balance of the
ASCORBIC ACID INJECTIONS IN LAYING HENS
RESULTS
was no detectable difference in the physical appearance or the vitality of the birds of the experimental and control groups during the experiment. The experimental group laid 455 eggs and the control group 460 eggs but one-third of the experimental group did not produce any eggs. No toxic effect could be detected when as much as 200 mg. of ascorbic acid was injected into birds during a week. SUMMARY
The data obtained in this study show that the ascorbic acid content of the blood of laying hens can be greatly increased by subcutaneous injections of ascorbic acid. However, this effect is very transitory. In two hours the amount of ascorbic acid in the hens' blood was nearly the same as before the injection of 50 mg. of ascorbic acid. No toxic effect was noted when 200 mg. was injected within a week. The subcutaneous injection of ascorbic acid did not affect the physical appearance, vitality or egg production of the hens. REFERENCES
Guyer, M. F., 1936. Animal micrology. 4th ed., p. 264. University of Chicago Press. Holmes, Arthur P., Francis Tripp, and G. Howard Satterfield, 1938. The Ascorbic Acid Content of Chick Blood. J. Nutrition 16:407-416. —, 1939. The ascorbic acid content of blood plasma of laying hens. Poultry Sci. 18:192-200. Satterfield, G. Howard, W. A. Perlzwieg and W. J. Dann, 1937. Unpublished data. Duke Medical School.
Downloaded from http://ps.oxfordjournals.org/ at University of Western Ontario on April 11, 2015
At the beginning of the experimental period the average ascorbic.acid content of the blood plasma (Figure 1) of the experimental group of hens was slightly higher than that of the control group, i.e., 1.6 mg. and 1.3 mg. per 100 cci of plasma respectively. The ascorbic acid content of the blood plasma of the control group remained practically constant throughout the experiment. In contrast, the amount of ascorbic acid in the blood of the experimental hens increased very rapidly following the injection and reached a maximum of 5.7 mg. per 100 cc. of blood plasma in about 25 minutes, an amount nearly four times the initial value. Following this the ascorbic acid decreased but the rate of decrease was not as rapid as the increase. After the second 25minute interval the blood contained 4.1 mg. of ascorbic acid per 100 cc. of blood plasma; during the second hour the decrease in the ascorbic acid content was still slower. Two hours after injection the amount of ascorbic acid in the blood plasma was 1.8 mg. or only 0.2 mg. above the initial level. Twenty-four hours after the injection the ascorbic acid content had returned to the initial level. However the amount of ascorbic acid continued to decrease slowly for three additional days and four days after injection was 1.0 mg. per 100 cc. of blood plasma or 0.6 mg. lower than at the start of the experiment. There
141