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The Effect of the Protein Level of the Ration Upon Certain Blood Constituents of the Hen*
T h e Effect of the Protein Level of the Ration Upon Certain Blood Constituents of the Hen* WALTER C. RUSSELL AND ALBERT L. WEBER New Jersey Agricultural Experiment Station, New
Brunswick
(Received for Publication January 6, 1934)
HE extensive use of chemical methods of blood analysis in studies of human diseases has led to their application in the study of the blood chemistry of domestic and experimental species. Studies of the effect of dietary or seasonal changes upon certain constituents of the blood of the chicken have been made by Thompson and Carr (1923), Thompson and Powers (1925), Hogan, Shrewsbury and Kempster (1929),Horvath (1930),ElvehjemandNeu (1932) and others. It was the objective of this investigation to determine whether the amount of protein in the ration affected the level of non-protein nitrogen, uric acid, urea nitrogen, creatine, and blood sugar in the blood of hens. If an effect were demonstrated, the methods employed might serve as a rapid means of studying protein feeding. ! EXPERIMENTAL
Two groups of S. C. White Leghorn hens which had received rations differing in their protein content for more than six months were available for blood studies. The term "low protein" is used to designate a ration which contained 12.71 percent of protein by analysis and which is representative of a ration composed chiefly of cereals. The "high-protein" type of ration contained 19.14 percent of protein, meat scrap having •Journal Series paper of the New Jersey Experiment Station.
been incorporated to the extent of IS percent. All-mash feeding was used and the birds were kept in confinement throughout the experiment. The low-protein ration had the following composition: ground yellow corn 62 percent, wheat bran 10 percent, red dog flour 10 percent, pin head oats 10 percent, ground bone 5 percent, dried skimmed milk 2 percent, and salt 1 percent. The high-protein ration contained IS percent meat scrap (55-60 percent protein), introduced at the expense of the bone meal and part of the yellow corn. One pound of cod liver oil was added to each 100 pounds of the ration. Blood was obtained from the heart with an 18 gauge needle and a 10 cc. hypodermic syringe. The needle and syringe was moistened with a 20 percent solution of potassium oxalate and the test tube into which the syringe was emptied contained 20 mg. of the oxalate. The sample withdrawn was usually 7 to 8 cc. The hens were bled at intervals of not less than five days and usually of seven days. Each individual was bled ten time during the experiment. Judging from weight and general appearance the bleeding did not harm the birds. The blood samples were prepared for analysis and the determination of non-protein nitrogen, urea nitrogen and creatine made according to the system of Folin and Wu (1919). Uric acid was determined according to Benedict (1922) and blood sugar
[376]
Downloaded from http://ps.oxfordjournals.org/ by guest on April 30, 2015
T
NOVEMBER,
• 1934.
VOL.
377
X I I I , No. 6
by the Folin (1926) modification of the Folin-Wu (1920) method. On account of the low concentration in the blood, in the determination of urea nitrogen, 0.1 mg. of nitrogen in the form of standard ammonium sulfate solution was added to the unknown to increase the color depth and permit a more accurate comparison with the standard. For the same reason 0.03 mg. of creatinine was added to the creatine samples. RESULTS AND DISCUSSION The values in Table 1 were obtained at approximately weekly intervals during a
decrease as more eggs are produced. How far the increase in egg production is actually responsible for these trends would have to be determined by more frequent observations, timed to the production of individual eggs. From the statistical standpoint no significant differences prevail between the averages for the low and high protein groups. In fact, greater differences exist among the values in each group than are found between the averages of the groups. Furthermore, in no instance are the values for one group consistently higher or lower than those of the
Determination No.
1 2 3 4 5 6 7 8 9 10 Aver.
Non-protein nitrogen Low protein
High protein
mg. 49.3 42.8 45.2 38.6 45.2 37.3 34.0
mg. 35.1 43.4 42.3 41.5 28.7 32.3
35.2 31.5 39.9
30.8 31.9 43.1 36.5
Uric acid Low protein mg. 4.6 4.0 3.4 3.6 3.3 3.7 3.3 2.9 3.9 3.8 3.7
High protein mg. 2.9 3.8 4.2 4.8 3.6 3.0 4.3 4.8 4.3 5.7 4.1
Urea nitrogen
Creatine*
Blood sugar
Low protein
High protein
Low protein
High protein
Low protein
mg. 5.3 6.9 10.6 8.7 4.8 8.4 4.1 6.1 6.5 7.6 6.9
mg. 4.5 8.8 6.9 6.3 3.3 7.2 7.6 10.2 8.5 9.4 7.1
mg. 6.0 6.1 6.2 5.7 6.0 5.8 5.9 5.6 4.6 4.4 5.6
mg. 5.0 5.4 4.2 6.3 5.1 5.1 6.1 3.2 3.9 3.3 4.8
mg. 180 184 186 191 202 183 201 203 195 202 193
High protein mg. 179 194 190 188 189 180 202 182 183 189 187
* Includes trace of creatinine.
2 to 2.5 month period. Each value is anL average of S individual determinations andI the same five hens were used in each group» throughout the experiment. At the beginning! of the experimental period the hens were; passing from a molt into egg production. Although space does not permit the presentation of the egg production and the blood1 analysis record of each hen, an examinationl of the individual records did not reveal anyj consistent variation of the blood picture; with egg production. The non-protein nitrogen values (Table 1) show a tendency to3 become lower as egg production increasess and the creatine levels show quite a markedi
other. Hence, the determination of the blood contituents under consideration does not appear to afford a means of ascertaining the effect of the protein level of the ration upon the blood picture when the protein levels studied are those which might prevail in practical feeding. SUMMARY
The non-protein nitrogen, uric acid, urea nitrogen, creatine and blood sugar of the blood of hens fed at a low-protein level were essentially the same as found when a highprotein level was fed. The greatest percentage difference prevailed between the creatine
Downloaded from http://ps.oxfordjournals.org/ by guest on April 30, 2015
TABLE 1.—Summary of non-protein nitrogen, uric acid, urea nitrogen, creatine and blood sugar values m mg. per 100 cc. of blood
378
POULTRY
values, the higher value being observed for the low-protein ration. The determination of the above-named blood constituents does not appear to afford a method for the study of the effect of different protein levels on the hen. ACKNOWLEDGMENT. The authors are indebted to Prof. C. S. Piatt, of the Department of Poultry Husbandry, for placing the experimental groups at their disposal. REFERENCES
, 1920. A system of blood analysis. Supplement I. A simplified and improved method for determination of sugar. J. Biol. Chem. 41:367-374. Folin, O., 1926. The determination of sugar in blood and in normal urine. J. Biol. Chem. 67:357-370. Hogan, A. G., C. L. Shrewsbury, and H. L. Kempster, 1929. The effect of inadequate rations on the composition of the blood and of the bone of chicks. Mo. Agr. Exp. Sta. Res. Bui. 124:117. Horvath, A. A., 1930. Changes in hen's blood produced by a diet of sprouted soy beans. Am. Jour. Physiol. 94:65-68. Thompson, T. J. and I. L. Carr, 1923. The relation of certain blood constituents to a deficient diet. Biochem. J. 17:373-375. Thompson, T. J. and H. W. Powers, 1925. The variation of certain blood constituents of chickens during the molting season. Poul. Sci. 4:186188.
Heterotaxia in Chick Embryos
D
URING the second day of incubation a rotation of the embryo begins which normally results in the turning of the chick to lie on its left side. A reverse movement causing the chick to lie on its right side is known as heterotaxia and is often associated with inversion of the viscera (Lillie1). Fol and Warynski 2 experimentally produced reversal of the unequal development of the two sides of the body and an assumption of the heterotaxic position by application of heat from a cauter held above exposed 36-hour chick embryos. Dareste3 stated that heterotaxia normally was extremely rare but cites evidence for its production by unequal heating of parts of the embryo. Taylor, Gunns, and Moses4 experimenting on the effect of inter1 Lillie, F. R., 1919. The Development of the Chick. Holt & Co., New York. 2 Fol H., and S. Warynski, 1883. Sur la production de l'inversion viscerale ou heterotaxie chez des embryons de poulet. Compt. Rend. 96 :1674-1676. 3 Dareste, C , 1891. Production artificielle des monstruosites. Reinwald & Cie, Paris. 4 Taylor, L. W., C. A. Gunns and B. D. Moses, 1933. The effect of current interruption in electrical incubation. Calif. Agr. Exp. Sta. Bull. 550:3-19.
rupted incubation found heterotaxia in 0.20 and 0.08 percent of dead embryos from chilled and control lots respectively. In terms of fertile eggs, the incidence was 0.115 and 0.029 percent respectively. However, heterotaxic embryos which lived to late incubation stages or which hatched were not identified in the procedure of the experiment and the above percentages were doubtless too low. While determining the orientation of chick embryos by candling after five days of incubation, embryos in the heterotaxic position were noted in normally incubated eggs during three successive years. The incidence of heterotaxia in 14,073 fertile eggs was 23, or 0.163 percent, with yearly range of from 0.144 to 0.185 percent. One embryo was destroyed in a broken egg and 11, or 50 percent, of the remainder, hatched. No significant association between heterotaxia and breed, family, embryonic orientation, position of the egg during the first week of incubation, or other malpositions was found. Evidently, heterotaxia in chick embryos occurs spontaneously with great rarity in eggs incubated normally and causes no apparent reduction in hatchability. L. W. TAYLOR
University of California
Downloaded from http://ps.oxfordjournals.org/ by guest on April 30, 2015
Benedict, S. R., 1922. The determination of uric acid in blood. J. Biol. Chem. 51:187-207. Elvehjem, C. A. and V. F. Neu, 1932. Studies in vitamin A avitaminosis in the chick. J. Biol. Chem. 97:71-82. Folin, 0. and H. Wu, 1919. A system of blood analysis. J. Biol. Chem. 38:81-110.
SCIENCE
Brunswick
(Received for Publication January 6, 1934)
HE extensive use of chemical methods of blood analysis in studies of human diseases has led to their application in the study of the blood chemistry of domestic and experimental species. Studies of the effect of dietary or seasonal changes upon certain constituents of the blood of the chicken have been made by Thompson and Carr (1923), Thompson and Powers (1925), Hogan, Shrewsbury and Kempster (1929),Horvath (1930),ElvehjemandNeu (1932) and others. It was the objective of this investigation to determine whether the amount of protein in the ration affected the level of non-protein nitrogen, uric acid, urea nitrogen, creatine, and blood sugar in the blood of hens. If an effect were demonstrated, the methods employed might serve as a rapid means of studying protein feeding. ! EXPERIMENTAL
Two groups of S. C. White Leghorn hens which had received rations differing in their protein content for more than six months were available for blood studies. The term "low protein" is used to designate a ration which contained 12.71 percent of protein by analysis and which is representative of a ration composed chiefly of cereals. The "high-protein" type of ration contained 19.14 percent of protein, meat scrap having •Journal Series paper of the New Jersey Experiment Station.
been incorporated to the extent of IS percent. All-mash feeding was used and the birds were kept in confinement throughout the experiment. The low-protein ration had the following composition: ground yellow corn 62 percent, wheat bran 10 percent, red dog flour 10 percent, pin head oats 10 percent, ground bone 5 percent, dried skimmed milk 2 percent, and salt 1 percent. The high-protein ration contained IS percent meat scrap (55-60 percent protein), introduced at the expense of the bone meal and part of the yellow corn. One pound of cod liver oil was added to each 100 pounds of the ration. Blood was obtained from the heart with an 18 gauge needle and a 10 cc. hypodermic syringe. The needle and syringe was moistened with a 20 percent solution of potassium oxalate and the test tube into which the syringe was emptied contained 20 mg. of the oxalate. The sample withdrawn was usually 7 to 8 cc. The hens were bled at intervals of not less than five days and usually of seven days. Each individual was bled ten time during the experiment. Judging from weight and general appearance the bleeding did not harm the birds. The blood samples were prepared for analysis and the determination of non-protein nitrogen, urea nitrogen and creatine made according to the system of Folin and Wu (1919). Uric acid was determined according to Benedict (1922) and blood sugar
[376]
Downloaded from http://ps.oxfordjournals.org/ by guest on April 30, 2015
T
NOVEMBER,
• 1934.
VOL.
377
X I I I , No. 6
by the Folin (1926) modification of the Folin-Wu (1920) method. On account of the low concentration in the blood, in the determination of urea nitrogen, 0.1 mg. of nitrogen in the form of standard ammonium sulfate solution was added to the unknown to increase the color depth and permit a more accurate comparison with the standard. For the same reason 0.03 mg. of creatinine was added to the creatine samples. RESULTS AND DISCUSSION The values in Table 1 were obtained at approximately weekly intervals during a
decrease as more eggs are produced. How far the increase in egg production is actually responsible for these trends would have to be determined by more frequent observations, timed to the production of individual eggs. From the statistical standpoint no significant differences prevail between the averages for the low and high protein groups. In fact, greater differences exist among the values in each group than are found between the averages of the groups. Furthermore, in no instance are the values for one group consistently higher or lower than those of the
Determination No.
1 2 3 4 5 6 7 8 9 10 Aver.
Non-protein nitrogen Low protein
High protein
mg. 49.3 42.8 45.2 38.6 45.2 37.3 34.0
mg. 35.1 43.4 42.3 41.5 28.7 32.3
35.2 31.5 39.9
30.8 31.9 43.1 36.5
Uric acid Low protein mg. 4.6 4.0 3.4 3.6 3.3 3.7 3.3 2.9 3.9 3.8 3.7
High protein mg. 2.9 3.8 4.2 4.8 3.6 3.0 4.3 4.8 4.3 5.7 4.1
Urea nitrogen
Creatine*
Blood sugar
Low protein
High protein
Low protein
High protein
Low protein
mg. 5.3 6.9 10.6 8.7 4.8 8.4 4.1 6.1 6.5 7.6 6.9
mg. 4.5 8.8 6.9 6.3 3.3 7.2 7.6 10.2 8.5 9.4 7.1
mg. 6.0 6.1 6.2 5.7 6.0 5.8 5.9 5.6 4.6 4.4 5.6
mg. 5.0 5.4 4.2 6.3 5.1 5.1 6.1 3.2 3.9 3.3 4.8
mg. 180 184 186 191 202 183 201 203 195 202 193
High protein mg. 179 194 190 188 189 180 202 182 183 189 187
* Includes trace of creatinine.
2 to 2.5 month period. Each value is anL average of S individual determinations andI the same five hens were used in each group» throughout the experiment. At the beginning! of the experimental period the hens were; passing from a molt into egg production. Although space does not permit the presentation of the egg production and the blood1 analysis record of each hen, an examinationl of the individual records did not reveal anyj consistent variation of the blood picture; with egg production. The non-protein nitrogen values (Table 1) show a tendency to3 become lower as egg production increasess and the creatine levels show quite a markedi
other. Hence, the determination of the blood contituents under consideration does not appear to afford a means of ascertaining the effect of the protein level of the ration upon the blood picture when the protein levels studied are those which might prevail in practical feeding. SUMMARY
The non-protein nitrogen, uric acid, urea nitrogen, creatine and blood sugar of the blood of hens fed at a low-protein level were essentially the same as found when a highprotein level was fed. The greatest percentage difference prevailed between the creatine
Downloaded from http://ps.oxfordjournals.org/ by guest on April 30, 2015
TABLE 1.—Summary of non-protein nitrogen, uric acid, urea nitrogen, creatine and blood sugar values m mg. per 100 cc. of blood
378
POULTRY
values, the higher value being observed for the low-protein ration. The determination of the above-named blood constituents does not appear to afford a method for the study of the effect of different protein levels on the hen. ACKNOWLEDGMENT. The authors are indebted to Prof. C. S. Piatt, of the Department of Poultry Husbandry, for placing the experimental groups at their disposal. REFERENCES
, 1920. A system of blood analysis. Supplement I. A simplified and improved method for determination of sugar. J. Biol. Chem. 41:367-374. Folin, O., 1926. The determination of sugar in blood and in normal urine. J. Biol. Chem. 67:357-370. Hogan, A. G., C. L. Shrewsbury, and H. L. Kempster, 1929. The effect of inadequate rations on the composition of the blood and of the bone of chicks. Mo. Agr. Exp. Sta. Res. Bui. 124:117. Horvath, A. A., 1930. Changes in hen's blood produced by a diet of sprouted soy beans. Am. Jour. Physiol. 94:65-68. Thompson, T. J. and I. L. Carr, 1923. The relation of certain blood constituents to a deficient diet. Biochem. J. 17:373-375. Thompson, T. J. and H. W. Powers, 1925. The variation of certain blood constituents of chickens during the molting season. Poul. Sci. 4:186188.
Heterotaxia in Chick Embryos
D
URING the second day of incubation a rotation of the embryo begins which normally results in the turning of the chick to lie on its left side. A reverse movement causing the chick to lie on its right side is known as heterotaxia and is often associated with inversion of the viscera (Lillie1). Fol and Warynski 2 experimentally produced reversal of the unequal development of the two sides of the body and an assumption of the heterotaxic position by application of heat from a cauter held above exposed 36-hour chick embryos. Dareste3 stated that heterotaxia normally was extremely rare but cites evidence for its production by unequal heating of parts of the embryo. Taylor, Gunns, and Moses4 experimenting on the effect of inter1 Lillie, F. R., 1919. The Development of the Chick. Holt & Co., New York. 2 Fol H., and S. Warynski, 1883. Sur la production de l'inversion viscerale ou heterotaxie chez des embryons de poulet. Compt. Rend. 96 :1674-1676. 3 Dareste, C , 1891. Production artificielle des monstruosites. Reinwald & Cie, Paris. 4 Taylor, L. W., C. A. Gunns and B. D. Moses, 1933. The effect of current interruption in electrical incubation. Calif. Agr. Exp. Sta. Bull. 550:3-19.
rupted incubation found heterotaxia in 0.20 and 0.08 percent of dead embryos from chilled and control lots respectively. In terms of fertile eggs, the incidence was 0.115 and 0.029 percent respectively. However, heterotaxic embryos which lived to late incubation stages or which hatched were not identified in the procedure of the experiment and the above percentages were doubtless too low. While determining the orientation of chick embryos by candling after five days of incubation, embryos in the heterotaxic position were noted in normally incubated eggs during three successive years. The incidence of heterotaxia in 14,073 fertile eggs was 23, or 0.163 percent, with yearly range of from 0.144 to 0.185 percent. One embryo was destroyed in a broken egg and 11, or 50 percent, of the remainder, hatched. No significant association between heterotaxia and breed, family, embryonic orientation, position of the egg during the first week of incubation, or other malpositions was found. Evidently, heterotaxia in chick embryos occurs spontaneously with great rarity in eggs incubated normally and causes no apparent reduction in hatchability. L. W. TAYLOR
University of California
Downloaded from http://ps.oxfordjournals.org/ by guest on April 30, 2015
Benedict, S. R., 1922. The determination of uric acid in blood. J. Biol. Chem. 51:187-207. Elvehjem, C. A. and V. F. Neu, 1932. Studies in vitamin A avitaminosis in the chick. J. Biol. Chem. 97:71-82. Folin, 0. and H. Wu, 1919. A system of blood analysis. J. Biol. Chem. 38:81-110.
SCIENCE