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An Estimation of Percentage Trapped Plasma in Normal Chicken Microhematocrit, Using Cr51
An Estimation of Percentage Trapped Plasma in Normal Chicken Microhematocrit, Using Cr51 ROBERT R.
COHEN*
Department of Biology, University of Colorado, Bottlder, Colorado 80302 (Received for publication June 27, 1966)
T
* Present address, Department of Biology, University of Saskatchewan, Saskatoon, Canada.
A dye or a radioisotope has commonly been used in both the direct and indirect methods for trapped plasma. Gregersen and Shiro (1938) used T-1824 dye to estimate that plasma constituted 4%, by volume, of the packed erythrocyte mass of human blood in the Wintrobe (Wintrobe, 1933a) hematocrit method. This procedure uses a centrifugal force of approximately 1.5 X 103 g. Similar values were found with this force of centrifugation by Gregersen et al. (1950), using T-1824, and by Vazquez et al. (1952) using the radioisotope I 131 . In comparison to this value, much lower percentages trapped plasma were found using the centrifugal forces employed in micro-capillary hematocrit methods. Furth (1956), using Cr51 and 10 X 103 g, found 2% trapped plasma with human blood; O'Brien et al. (1957) found the same value for goat blood using 15 X 103 g, with I131tagged plasma and Cr51-tagged erythrocytes; Garby and Vuille (1961), using I 131 and approximately 10 X 103 g, found 1.31% trapped plasma with human blood. Very few determinations of trapped plasma have been made on avian blood. A number of investigators have used the correction factor for human blood at the same centrifugal force; however, the studies of Furth (1956), Chien et al. (1965), and others show that percentage trapped plasma can vary significantly with the size and shape of the erythrocytes. In studies on chicken blood, Gourley (1957) found values of 10.2% and 5.4% at 1.3 X 103 g, using P 32 and T-1824, respectively, and Bell (1957) estimated 6.1% trapped plas-
219
Downloaded from http://ps.oxfordjournals.org/ at Western Michigan University on May 5, 2015
HE determination of hematocrit has been used for several decades as a simple and accurate measure of blood erythrocyte levels. The rapid micro-capillary method for hematocrit is now widely employed in mammalian blood studies and is being used to an increasing extent in avian work (see Cohen, 1967). The present paper describes the determination of a correction factor for trapped plasma in microhematocrit determinations on chicken blood. Hedin (1895) was perhaps the first to postulate that the packed erythrocyte mass, after centrifugation, contained a certain amount of plasma trapped between the erythrocytes. The true erythrocyte volumepercent would therefore be derived by correcting for this trapped plasma. Small amounts of trapped plasma were detected in mammalian hematocrits by Keith et al. (1915), Ege (1920), Millar (1925), Kennedy and Millikan (1938), and others. These and other early studies have been reviewed by Owen and Power (1953). Hlad and Holmes (1953) have considered the theoretical aspects of plasma trapping. The procedures used to determine the amount of trapped plasma include "indirect" methods, where the total volume of plasma in the centrifuged blood sample is determined by the dye-dilution principle, and "direct" methods, which employ the measurement of the amount of plasma marker in the packed erythrocyte mass (Owen and Power, 1953).
220
R. R. COHEN
ma at 3.2 X 103 g with a protein-precitation method. Reports of avian trapped plasma at higher centrifugal forces appear to be lacking. In the work described below, percentage trapped plasma was determined in chicken blood centrifuged at 12.8 X 103 g. This approximates the force reached in the International Model MB Microhematocrit Centrifuge.
The radioisotope Cr51 in the Cr3+ form was used as a plasma tag. In contrast to Cr042", Cr34 apparently can not enter the cells of the blood but will bind to the plasma proteins (Gray and Sterling, 1950). Accordingly, Furth (19S6) added Cr51 as CrCl3 to the plasma of human blood in a determination of percent trapped plasma. In the present study, Cr51 was obtained in aqueous solution (Abbott Laboratories, Oak Ridge, Tennessee) as Na 2 Cr0 4 and was reduced to Cr3+ by the addition of ascorbic acid in stoichiometric excess. According to Read and Gilbertsen (1957), part of the Cr3+ produced in this reaction remains complexed with ascorbate. Blood samples were taken from the brachial vein of each of six 10-week old male White Leghorn chickens averaging 1.0 kg. in body weight. The sampling procedure was modified from that of Fredrickson et al. (1958). After binding the legs together, the bird was placed on its side and the wing extended to expose its ventral side. Any contour feathers in the brachial vein area were cut off near their base and any down feathers were moistened and spread away from the vein (see Cohen, 1967). The area was swabbed with 95% ethanol. A 5-ml. Yale B-D Luer-Lok syringe, fitted with a 21-gauge needle and the dead-space filled with 1 X 103 units/ml. aqueous heparin solution, was used to remove 3.0 ml. blood from the vein. Before
Downloaded from http://ps.oxfordjournals.org/ at Western Michigan University on May 5, 2015
MATERIALS AND METHODS
retracting the needle, a cotton wad was pressed on the vein at the point of needle entry. The wad was held in place for 1 to 2 min. to prevent subcutaneous hemorrhage. The heparinized blood sample was ejected into a glass vial and approximately 100 [iA. of Cr3+ solution, containing 5 |ic. Cr51, was added with mixing. The sample was divided into halves and each was placed in a polyethylene centrifuge tube 8 cm. in length, constructed from polyethylene tubing (7 mm. O.D., 3 mm. I.D.). After the tubes were stoppered they were placed in a Servall Enclosed Superspeed Centrifuge, into rubber adaptors designed to hold 12ml. glass centrifuge tubes. The tubes were centrifuged for 10 min. at 12.8 X 103 g, the force reached in the International Model MB Microhematocrit Centrifuge. In the calculations involving RPM's and centrifugal force, the effective radii were taken as the radii to the midpoints of the packed erythrocyte columns. Following centrifugation, a 170 pd. sample of plasma was removed from the tube with a micropipette and added with rinsing to 2.0 ml. isotonic NaCl in a 1-dram glass shell vial. The tube was then sectioned several millimeters below the buffy coat. A 170 \iA. sample of the packed erythrocyte mass was obtained from a point several millimeters below the fluid surface and added with rinsing to a vial of saline solution as described above. A Baird-Atomic well-type scintillation counter was used to measure the relative Cr51 activities of the plasma and packed-erythrocyte samples. All counts were corrected for background .activity. To eliminate errors due to variation in counting geometry, all samples were mixed well at the time of counting. At least 7.0 X 103 counts were recorded in each case, giving a total counting standard deviation of 1.2%. Preliminary studies, involving the addi-
221
MlCEOHEMATOCRIT TRAPPED PLASMA
CPM packed erythrocyte mass/100 fil. CPM plasma/100 jul. RESULTS The calculated percentages trapped plasma are listed in Table 1, along with the mean value for the sample from each bird. The grand mean, with the 95% confidence interval (5 degrees of freedom) is 2.12 ± 0.09%. According to this value, the true hematocrit (Hct) of chicken blood, in microcapillary determinations at 12.8 X 103 g, may be calculated by the equation Hct =
L c (0.979)
,
Lx where Lc = length of packed erythrocyte column in capillary tube and LT = total length of blood sample in capillary tube. DISCUSSION The percentage trapped plasma found here (2.1%) is similar to those found in mammalian blood at high centrifugal force by Furth (1956), O'Brien et al. (1957), and Garby and Vuille (1961). As expected, it is distinctly lower than the values of 10.2% and 5.4% (Gourley, 1957) and 6.1% (Bell, 1957) found in chicken blood at 1.3 X 108 g and 3.2 X 103 g, respectively. A definite source of error in determining percentage trapped plasma was described by Chaplin and Mollison (1952) and by Owen and Power (1953). These investiga-
TABLE 1.—Amount of plasma in packed erythrocyte column of chicken blood centrifuged at 12.SX103 g* Sample**
Percentage tr plasma
1A IB
2.17 2.25
2A 2B
2.11 2.26
3A 3B
2.26 1.99
4A 4B
2.26 2.04
5A 5B
2.01 2.03
6A 6B
2.00 2.00
* Expressed as percentage of packed erythrocyte volume. ** Duplicate blood samples from each of six birds.
tors found that at low forces of centrifugation (1.6 X 103 g and 2.2 X 103 g, respectively) the percentage trapped plasma varied significantly along the packed erythrocyte column; the percentage was greatest just below the buffy coat and least at the bottom of the tube. However, according to Garby and Vuille (1961), no such differential erythrocyte packing is found at the higher force of approximately 10 X 103 g. The applicability of the correction factor found here to microhematocrit determinations on other avian species will depend upon the relative average erythrocyte size of those species. According to the erythrocyte-size data of Wintrobe (1933b), Barsch et al. (1937), Bennett and Chisholm (1964) and others, a correction factor of 2.1% would probably be applicable to the blood of most non-passerine species and of the larger passerines. The more gross approximation of 2% trapped plasma would likely be valid for most of the smaller passerines, which have relatively small erythrocytes.
Downloaded from http://ps.oxfordjournals.org/ at Western Michigan University on May 5, 2015
tion of Cr51, centrifuging, and washing the erythrocytes once in isotonic NaCl solution, had shown that an insignificant amount of Cr51 was bound to the erythrocytes in this procedure. Calculation of the percent trapped plasma therefore involved a direct ratio-comparison of the plasma and packed-erythrocyte Cr51 activities in counts per min. (CPM) per unit volume of sample: % trapped plasma
222
R. R. COHEN SUMMARY 51
ACKNOWLED GMENTS
This work was partially supported by grants from the Department of Biology, University of Colorado, and funds from an NDEA Fellowship Program. I wish to thank Drs. Charles H. Norris and Paul W. Winston for counsel during the work, and Drs. B. M. Rever, P. R. Sweeny, and M. W. Zink, of the Department of Biology, University of Saskatchewan, for reviewing the manuscript. REFERENCES Bartsch, P. W., W. H. Ball, W. Rosenzweig and L. Salman, 1937. Size of red blood corpuscles and their nuclei in fifty North American birds. Auk, 54: 516-519. Bell, D. J., 1957. The distribution of glucose between the plasma water and the erythrocyte water in hens' blood. Quart J. Exptl. Physiol. 42: 410-416. Bennett, G. F., and A. E. Chisholm, 1964. Measurements on the blood cells of some wild birds of North America. Wildl. Dis. 38: 1-22. Chaplin, H., Jr., and P. L. Mollison, 19S2. Correction for plasma trapped in the red cell col-
Downloaded from http://ps.oxfordjournals.org/ at Western Michigan University on May 5, 2015
The radioisotope Cr was used as a plasma tag to estimate the amount of plasma trapped in the packed erythrocyte mass in microhematocrit determinations on chicken blood. Cr51 was added to the plasma of whole heparinized blood in the form of Cr3+. The blood was centrifuged at the force used in the microhematocrit centrifuge, and samples of plasma and of packed erythrocyte mass were assayed for Cr51 activity. Determinations on six birds gave an average of 2.12% trapped plasma by volume, with a 95% confidence interval of ± 0.09%. This value compares favorably with previous avian determinations at lower centrifugal force and with mammalian determinations at a similar force. The applicability of this correction factor to the blood of other avian species is discussed.
umn of the hematocrit. Blood, 7: 1127-1238. Chien, S., R. J. Dellenback, S. Usami and M. I. Gregersen, 1965. Plasma trapping in hematocrit determination. Proc. Soc. Exptl. Biol. Med. 119: 1155-1158. Cohen, R. R., 1967. Anticoagulation, centrifugation time, and sample replicate number in the microhematocrit method for avian blood. Poultry Sci. 46: 214-218. Ege, R., 1920. Uber die Bestimmungen des Blutkoperchenvolums. Biochem. Ztschr. 109: 241248. Frederickson, T. N., H. L. Chute and D. C. O'Meara, 1958. A simple improved method for drawing blood from chickens. J. Am. Vet. Med. Assoc. 132 : 390-391. Furth, F. W., 1956. Effect of spherocytosis on volume of trapped plasma in red cell column of capillary and Wintrobe hematocrits. J. Lab. Clin. Med. 48: 421-430. Garby, L., and J. C. Vuille, 1961. The amount of trapped plasma in high-speed micro-capillary hematocrit centrifuge. Scand. J. Clin. Lab. Invest. 13: 642-645. Gourley, D. R. H., 1957. Phosphate transfer in chicken erythrocytes. Am. J. Physiol. 190: 536-542. Gray, S. J. and K. Sterling, 1950. The tagging of red cells and plasma proteins with radioactive chromium. J. Clin. Invest. 29: 1604-1613. Gergersen, M. I., A. A. Boyden and J. B. Allison, 1950. Direct comparison in dogs of plasma volume measured with T-1824 and antigens. Am. J. Physiol. 163 : 517-528. Gergersen, M. I., and H. Schiro, 1938. The behavior of the dye T-1824 with respect to its absorption by red blood cells and its fate in blood undergoing coagulation. Am. J. Physiol. 121: 284-292. Hedin, S. G., 1895. Ueber die Einwirkung einiger Wasserlosungen auf das Volumen der rothen Blutkorperchen. Skand. Arch. Physiol. 5: 207231. Hlad, C. J., Jr., and J. H. Holmes, 1953. Factors affecting hematocrit determinations: Trapped plasma, its amount and distribution. J. Appl. Physiol. S: 457-470. Keith, N. M., L. G. Rountree and J. T. Geraghty, 1915. A method for the determination of plasma and blood volume. Arch. Int. Med. 16: 547-576. Kennedy, J. A., and G. A. Millikan, 1938. A micro blood volume method using a blue dye and photocell. J. Physiol. 9 3 : 276-284.
MlCEOHEMATOCEIT TRAPPED PLASMA
Med. 100: 259-262. Vazquez, O. N., K. Newerly, R. S. Yalow and S. A. Berson, 1952. Determination of trapped plasma in the centrifuged erythrocyte volume of normal human blood with radioiodinated (I131) human serum albumin and radiosodium (Na 24 ). J. Lab. Clin. Med. 39: 595-604. Wintrobe, M. M., 1933a. Macroscopic examination of the blood. Am. J. Med. Sci. 185: 5871. Wintrobe, M. M., 1933b. Variations in the size and hemoglobin content of erythrocytes in the blood of various vertebrates. Fol. Haem. 51: 32-49.
Calcium and Vitamin D in Broiler Rations* JACOB BIELY AND B. E. MARCH Department of Poultry Science, The University of British Columbia, Vancouver 8, Canada (Received for publication Tune 28, 1966)
W
ITH the modifications that have been made in the energy content of chick starter and broiler rations the requirements for some of the nutrients may be different than those determined on the basis of diets commonly formulated some years ago. For example, the inclusion of fat in poultry rations has been shown by Biely and March (1954, 1957), Sunde (1956), and Davidson (1956) to be practicable only when the level of protein is increased to maintain a proportion between the energy level and the protein level in the diet. The relation of dietary fat and energy level to requirements of other nutrients is not so clear. Although from our present knowledge of calcium, phosphorus, and vitamin D interrelationships, it should be possible to define calcium requirements within fairly close limits, the calcium requirement has been * Research conducted under a grant from The National Research Council of Canada.
reported within recent years to be as low as 0.5 percent (Simco and Stephenson, 1961) and as high as 1.07 percent (Edwards et al., 1963) of the diet. A number of factors such as genetic differences in calcium utilization, environmental differences, and differences in the composition of the basal diets used may be responsible for the variations reported by different laboratories. McDougall (1938) and Knudson and Floody (1940) showed that the vitamin D requirement of rats was reduced when fat or oil was added to the diet. There do not seem to have been comparable experiments with chicks, however, pertaining to the fats commonly added to broiler diets. Depending upon the nature of the fat, high levels of dietary fat may result in poor utilization of calcium. Many years ago, Givens (1917) reported that fecal calcium excretion in dogs was proportional to the utilization of the fat which was fed. Further evidence of interacting effects between dietary
Downloaded from http://ps.oxfordjournals.org/ at Western Michigan University on May 5, 2015
Millar, W. G., 1925. Observations on the haematocrite method of measuring the volume of erythrocytes. Quart. J. Exptl. Physiol. 15: 187-190. O'Brien, W. A., D. L. Howie and W. H. Crosby, 1957. Blood volume studies in wounded animals. J. Appl. Physiol. 11: 110-114. Owen, C. A., Jr., and M. H. Power, 1953. Intercellular plasma of centrifuged human erythrocytes as measured by means of iodo131-albumin. J. Appl. Physiol. 5: 322-329. Read, R. C , and S. Gilbertsen, 1957. Radioactive sodium chromate and the measurement of red cell and plasma volume in man. Arch. Int.
223
COHEN*
Department of Biology, University of Colorado, Bottlder, Colorado 80302 (Received for publication June 27, 1966)
T
* Present address, Department of Biology, University of Saskatchewan, Saskatoon, Canada.
A dye or a radioisotope has commonly been used in both the direct and indirect methods for trapped plasma. Gregersen and Shiro (1938) used T-1824 dye to estimate that plasma constituted 4%, by volume, of the packed erythrocyte mass of human blood in the Wintrobe (Wintrobe, 1933a) hematocrit method. This procedure uses a centrifugal force of approximately 1.5 X 103 g. Similar values were found with this force of centrifugation by Gregersen et al. (1950), using T-1824, and by Vazquez et al. (1952) using the radioisotope I 131 . In comparison to this value, much lower percentages trapped plasma were found using the centrifugal forces employed in micro-capillary hematocrit methods. Furth (1956), using Cr51 and 10 X 103 g, found 2% trapped plasma with human blood; O'Brien et al. (1957) found the same value for goat blood using 15 X 103 g, with I131tagged plasma and Cr51-tagged erythrocytes; Garby and Vuille (1961), using I 131 and approximately 10 X 103 g, found 1.31% trapped plasma with human blood. Very few determinations of trapped plasma have been made on avian blood. A number of investigators have used the correction factor for human blood at the same centrifugal force; however, the studies of Furth (1956), Chien et al. (1965), and others show that percentage trapped plasma can vary significantly with the size and shape of the erythrocytes. In studies on chicken blood, Gourley (1957) found values of 10.2% and 5.4% at 1.3 X 103 g, using P 32 and T-1824, respectively, and Bell (1957) estimated 6.1% trapped plas-
219
Downloaded from http://ps.oxfordjournals.org/ at Western Michigan University on May 5, 2015
HE determination of hematocrit has been used for several decades as a simple and accurate measure of blood erythrocyte levels. The rapid micro-capillary method for hematocrit is now widely employed in mammalian blood studies and is being used to an increasing extent in avian work (see Cohen, 1967). The present paper describes the determination of a correction factor for trapped plasma in microhematocrit determinations on chicken blood. Hedin (1895) was perhaps the first to postulate that the packed erythrocyte mass, after centrifugation, contained a certain amount of plasma trapped between the erythrocytes. The true erythrocyte volumepercent would therefore be derived by correcting for this trapped plasma. Small amounts of trapped plasma were detected in mammalian hematocrits by Keith et al. (1915), Ege (1920), Millar (1925), Kennedy and Millikan (1938), and others. These and other early studies have been reviewed by Owen and Power (1953). Hlad and Holmes (1953) have considered the theoretical aspects of plasma trapping. The procedures used to determine the amount of trapped plasma include "indirect" methods, where the total volume of plasma in the centrifuged blood sample is determined by the dye-dilution principle, and "direct" methods, which employ the measurement of the amount of plasma marker in the packed erythrocyte mass (Owen and Power, 1953).
220
R. R. COHEN
ma at 3.2 X 103 g with a protein-precitation method. Reports of avian trapped plasma at higher centrifugal forces appear to be lacking. In the work described below, percentage trapped plasma was determined in chicken blood centrifuged at 12.8 X 103 g. This approximates the force reached in the International Model MB Microhematocrit Centrifuge.
The radioisotope Cr51 in the Cr3+ form was used as a plasma tag. In contrast to Cr042", Cr34 apparently can not enter the cells of the blood but will bind to the plasma proteins (Gray and Sterling, 1950). Accordingly, Furth (19S6) added Cr51 as CrCl3 to the plasma of human blood in a determination of percent trapped plasma. In the present study, Cr51 was obtained in aqueous solution (Abbott Laboratories, Oak Ridge, Tennessee) as Na 2 Cr0 4 and was reduced to Cr3+ by the addition of ascorbic acid in stoichiometric excess. According to Read and Gilbertsen (1957), part of the Cr3+ produced in this reaction remains complexed with ascorbate. Blood samples were taken from the brachial vein of each of six 10-week old male White Leghorn chickens averaging 1.0 kg. in body weight. The sampling procedure was modified from that of Fredrickson et al. (1958). After binding the legs together, the bird was placed on its side and the wing extended to expose its ventral side. Any contour feathers in the brachial vein area were cut off near their base and any down feathers were moistened and spread away from the vein (see Cohen, 1967). The area was swabbed with 95% ethanol. A 5-ml. Yale B-D Luer-Lok syringe, fitted with a 21-gauge needle and the dead-space filled with 1 X 103 units/ml. aqueous heparin solution, was used to remove 3.0 ml. blood from the vein. Before
Downloaded from http://ps.oxfordjournals.org/ at Western Michigan University on May 5, 2015
MATERIALS AND METHODS
retracting the needle, a cotton wad was pressed on the vein at the point of needle entry. The wad was held in place for 1 to 2 min. to prevent subcutaneous hemorrhage. The heparinized blood sample was ejected into a glass vial and approximately 100 [iA. of Cr3+ solution, containing 5 |ic. Cr51, was added with mixing. The sample was divided into halves and each was placed in a polyethylene centrifuge tube 8 cm. in length, constructed from polyethylene tubing (7 mm. O.D., 3 mm. I.D.). After the tubes were stoppered they were placed in a Servall Enclosed Superspeed Centrifuge, into rubber adaptors designed to hold 12ml. glass centrifuge tubes. The tubes were centrifuged for 10 min. at 12.8 X 103 g, the force reached in the International Model MB Microhematocrit Centrifuge. In the calculations involving RPM's and centrifugal force, the effective radii were taken as the radii to the midpoints of the packed erythrocyte columns. Following centrifugation, a 170 pd. sample of plasma was removed from the tube with a micropipette and added with rinsing to 2.0 ml. isotonic NaCl in a 1-dram glass shell vial. The tube was then sectioned several millimeters below the buffy coat. A 170 \iA. sample of the packed erythrocyte mass was obtained from a point several millimeters below the fluid surface and added with rinsing to a vial of saline solution as described above. A Baird-Atomic well-type scintillation counter was used to measure the relative Cr51 activities of the plasma and packed-erythrocyte samples. All counts were corrected for background .activity. To eliminate errors due to variation in counting geometry, all samples were mixed well at the time of counting. At least 7.0 X 103 counts were recorded in each case, giving a total counting standard deviation of 1.2%. Preliminary studies, involving the addi-
221
MlCEOHEMATOCRIT TRAPPED PLASMA
CPM packed erythrocyte mass/100 fil. CPM plasma/100 jul. RESULTS The calculated percentages trapped plasma are listed in Table 1, along with the mean value for the sample from each bird. The grand mean, with the 95% confidence interval (5 degrees of freedom) is 2.12 ± 0.09%. According to this value, the true hematocrit (Hct) of chicken blood, in microcapillary determinations at 12.8 X 103 g, may be calculated by the equation Hct =
L c (0.979)
,
Lx where Lc = length of packed erythrocyte column in capillary tube and LT = total length of blood sample in capillary tube. DISCUSSION The percentage trapped plasma found here (2.1%) is similar to those found in mammalian blood at high centrifugal force by Furth (1956), O'Brien et al. (1957), and Garby and Vuille (1961). As expected, it is distinctly lower than the values of 10.2% and 5.4% (Gourley, 1957) and 6.1% (Bell, 1957) found in chicken blood at 1.3 X 108 g and 3.2 X 103 g, respectively. A definite source of error in determining percentage trapped plasma was described by Chaplin and Mollison (1952) and by Owen and Power (1953). These investiga-
TABLE 1.—Amount of plasma in packed erythrocyte column of chicken blood centrifuged at 12.SX103 g* Sample**
Percentage tr plasma
1A IB
2.17 2.25
2A 2B
2.11 2.26
3A 3B
2.26 1.99
4A 4B
2.26 2.04
5A 5B
2.01 2.03
6A 6B
2.00 2.00
* Expressed as percentage of packed erythrocyte volume. ** Duplicate blood samples from each of six birds.
tors found that at low forces of centrifugation (1.6 X 103 g and 2.2 X 103 g, respectively) the percentage trapped plasma varied significantly along the packed erythrocyte column; the percentage was greatest just below the buffy coat and least at the bottom of the tube. However, according to Garby and Vuille (1961), no such differential erythrocyte packing is found at the higher force of approximately 10 X 103 g. The applicability of the correction factor found here to microhematocrit determinations on other avian species will depend upon the relative average erythrocyte size of those species. According to the erythrocyte-size data of Wintrobe (1933b), Barsch et al. (1937), Bennett and Chisholm (1964) and others, a correction factor of 2.1% would probably be applicable to the blood of most non-passerine species and of the larger passerines. The more gross approximation of 2% trapped plasma would likely be valid for most of the smaller passerines, which have relatively small erythrocytes.
Downloaded from http://ps.oxfordjournals.org/ at Western Michigan University on May 5, 2015
tion of Cr51, centrifuging, and washing the erythrocytes once in isotonic NaCl solution, had shown that an insignificant amount of Cr51 was bound to the erythrocytes in this procedure. Calculation of the percent trapped plasma therefore involved a direct ratio-comparison of the plasma and packed-erythrocyte Cr51 activities in counts per min. (CPM) per unit volume of sample: % trapped plasma
222
R. R. COHEN SUMMARY 51
ACKNOWLED GMENTS
This work was partially supported by grants from the Department of Biology, University of Colorado, and funds from an NDEA Fellowship Program. I wish to thank Drs. Charles H. Norris and Paul W. Winston for counsel during the work, and Drs. B. M. Rever, P. R. Sweeny, and M. W. Zink, of the Department of Biology, University of Saskatchewan, for reviewing the manuscript. REFERENCES Bartsch, P. W., W. H. Ball, W. Rosenzweig and L. Salman, 1937. Size of red blood corpuscles and their nuclei in fifty North American birds. Auk, 54: 516-519. Bell, D. J., 1957. The distribution of glucose between the plasma water and the erythrocyte water in hens' blood. Quart J. Exptl. Physiol. 42: 410-416. Bennett, G. F., and A. E. Chisholm, 1964. Measurements on the blood cells of some wild birds of North America. Wildl. Dis. 38: 1-22. Chaplin, H., Jr., and P. L. Mollison, 19S2. Correction for plasma trapped in the red cell col-
Downloaded from http://ps.oxfordjournals.org/ at Western Michigan University on May 5, 2015
The radioisotope Cr was used as a plasma tag to estimate the amount of plasma trapped in the packed erythrocyte mass in microhematocrit determinations on chicken blood. Cr51 was added to the plasma of whole heparinized blood in the form of Cr3+. The blood was centrifuged at the force used in the microhematocrit centrifuge, and samples of plasma and of packed erythrocyte mass were assayed for Cr51 activity. Determinations on six birds gave an average of 2.12% trapped plasma by volume, with a 95% confidence interval of ± 0.09%. This value compares favorably with previous avian determinations at lower centrifugal force and with mammalian determinations at a similar force. The applicability of this correction factor to the blood of other avian species is discussed.
umn of the hematocrit. Blood, 7: 1127-1238. Chien, S., R. J. Dellenback, S. Usami and M. I. Gregersen, 1965. Plasma trapping in hematocrit determination. Proc. Soc. Exptl. Biol. Med. 119: 1155-1158. Cohen, R. R., 1967. Anticoagulation, centrifugation time, and sample replicate number in the microhematocrit method for avian blood. Poultry Sci. 46: 214-218. Ege, R., 1920. Uber die Bestimmungen des Blutkoperchenvolums. Biochem. Ztschr. 109: 241248. Frederickson, T. N., H. L. Chute and D. C. O'Meara, 1958. A simple improved method for drawing blood from chickens. J. Am. Vet. Med. Assoc. 132 : 390-391. Furth, F. W., 1956. Effect of spherocytosis on volume of trapped plasma in red cell column of capillary and Wintrobe hematocrits. J. Lab. Clin. Med. 48: 421-430. Garby, L., and J. C. Vuille, 1961. The amount of trapped plasma in high-speed micro-capillary hematocrit centrifuge. Scand. J. Clin. Lab. Invest. 13: 642-645. Gourley, D. R. H., 1957. Phosphate transfer in chicken erythrocytes. Am. J. Physiol. 190: 536-542. Gray, S. J. and K. Sterling, 1950. The tagging of red cells and plasma proteins with radioactive chromium. J. Clin. Invest. 29: 1604-1613. Gergersen, M. I., A. A. Boyden and J. B. Allison, 1950. Direct comparison in dogs of plasma volume measured with T-1824 and antigens. Am. J. Physiol. 163 : 517-528. Gergersen, M. I., and H. Schiro, 1938. The behavior of the dye T-1824 with respect to its absorption by red blood cells and its fate in blood undergoing coagulation. Am. J. Physiol. 121: 284-292. Hedin, S. G., 1895. Ueber die Einwirkung einiger Wasserlosungen auf das Volumen der rothen Blutkorperchen. Skand. Arch. Physiol. 5: 207231. Hlad, C. J., Jr., and J. H. Holmes, 1953. Factors affecting hematocrit determinations: Trapped plasma, its amount and distribution. J. Appl. Physiol. S: 457-470. Keith, N. M., L. G. Rountree and J. T. Geraghty, 1915. A method for the determination of plasma and blood volume. Arch. Int. Med. 16: 547-576. Kennedy, J. A., and G. A. Millikan, 1938. A micro blood volume method using a blue dye and photocell. J. Physiol. 9 3 : 276-284.
MlCEOHEMATOCEIT TRAPPED PLASMA
Med. 100: 259-262. Vazquez, O. N., K. Newerly, R. S. Yalow and S. A. Berson, 1952. Determination of trapped plasma in the centrifuged erythrocyte volume of normal human blood with radioiodinated (I131) human serum albumin and radiosodium (Na 24 ). J. Lab. Clin. Med. 39: 595-604. Wintrobe, M. M., 1933a. Macroscopic examination of the blood. Am. J. Med. Sci. 185: 5871. Wintrobe, M. M., 1933b. Variations in the size and hemoglobin content of erythrocytes in the blood of various vertebrates. Fol. Haem. 51: 32-49.
Calcium and Vitamin D in Broiler Rations* JACOB BIELY AND B. E. MARCH Department of Poultry Science, The University of British Columbia, Vancouver 8, Canada (Received for publication Tune 28, 1966)
W
ITH the modifications that have been made in the energy content of chick starter and broiler rations the requirements for some of the nutrients may be different than those determined on the basis of diets commonly formulated some years ago. For example, the inclusion of fat in poultry rations has been shown by Biely and March (1954, 1957), Sunde (1956), and Davidson (1956) to be practicable only when the level of protein is increased to maintain a proportion between the energy level and the protein level in the diet. The relation of dietary fat and energy level to requirements of other nutrients is not so clear. Although from our present knowledge of calcium, phosphorus, and vitamin D interrelationships, it should be possible to define calcium requirements within fairly close limits, the calcium requirement has been * Research conducted under a grant from The National Research Council of Canada.
reported within recent years to be as low as 0.5 percent (Simco and Stephenson, 1961) and as high as 1.07 percent (Edwards et al., 1963) of the diet. A number of factors such as genetic differences in calcium utilization, environmental differences, and differences in the composition of the basal diets used may be responsible for the variations reported by different laboratories. McDougall (1938) and Knudson and Floody (1940) showed that the vitamin D requirement of rats was reduced when fat or oil was added to the diet. There do not seem to have been comparable experiments with chicks, however, pertaining to the fats commonly added to broiler diets. Depending upon the nature of the fat, high levels of dietary fat may result in poor utilization of calcium. Many years ago, Givens (1917) reported that fecal calcium excretion in dogs was proportional to the utilization of the fat which was fed. Further evidence of interacting effects between dietary
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