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Blood Cells in the Bone Marrow of the Chick Before and After Hatching
Blood Cells in the Bone Marrow of the Chick Before and After Hatching B. R. BURMESTER, J. M. SEVERENS, AND E. ROBERTS Department of Animal Husbandry, University of Illinois, Urbana, Illinois (Received for publication January 10, 1941)
URING the course of certain studies on the resistance of chicks to the pullorum disease (Roberts, Severens, and Card, 1939), data were collected on the blood cell activity in the bone marrow of chicks during the period of six days before hatching and seven days after hatching. The data presented here show the quantative changes in the blood cells of the bone marrow and circulation during this critical period in the development of the chick. METHODS
Blood samples for cell counts of the circulating blood were drawn from the vitelline vein in the case of embryos and from the wing vein in case of hatched chicks. The chicks were killed with ether and both femurs and tibiae were dissected from the chicks and embryos. As soon as all adhering tissue had been removed from the bones, they were split in half along their longitudinal axis and immediately immersed in oxalated plasma which had previously been prepared from hen's blood. The marrow cells were suspended in the plasma by gently shaking the bones with the plasma for one minute in a small stoppered vial. The suspension was filtered through several layers of gauze into a graduated 15 c.c. centrifuge tube. When the bone marrow was hyperplastic very little residue remained on the gauze. The shaking and filtering was repeated with fresh plasma. A third shaking was preceded by scraping out the spongy red bone at the epiphysis.
The volume of the suspension was noted and total counts were made by the ordinary methods used for circulating blood. For the blood smear, Wright's staining method was used to make the differential counts. The vital-dye method could not be used because it was found that the marrow cells did not take up the vital dye after the above treatment.1 RESULTS
Data on the cells of the bone marrow were obtained from embryos and chicks of the White Leghorn and Rhode Island Red breeds, but inasmuch as no significant differences were found, only the data for the White Leghorn chicks will be given here. These data, together with cell counts of the circulating blood, are summarized in Table 1. It is recognized that a high proportion of immature cells of all types are to be found in the bone marrow and circulating blood of embryos; however, in order to simplify the counting procedure, we have used only the mature cell classification for making the white cell counts, and have included the young or immature cells in the class to which they appeared to belong on the basis of their morphological characteristics and staining reactions. The red cells of the bone marrow were classified into mature and immature cells. The latter include the basophil and polychromatophil ' I t should be understood that this is an arbitrary method and results are of value only in following a relative change or a growth.
[391]
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 30, 2015
D
White Neutro- Lymphophils cells cytes as% as % of as % of white White Neutro- Lympho- of all white phils cytes cells cells cells cells
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Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 30, 2015
Blood celis of the circulation
B. R . BURMESTER, J. M . S E V E R E N S , AND E . ROBERTS
Blood cells of the bone marrow
392
BLOOD CELLS I N BONE MARROW OF C H I C K BEFORE AND AFTER HATCHING
phils to total white cells decreases markedly throughout the whole period. On the other hand, the concentration of lmphocytes and the proportion of lymphocytes to total white cells in the circulating blood
The counts show that the number of blood cells in the four long bones used increase rapidly after hatching time. This is true of both the red cells and the white cells. The proportion of red cells to white cells does not shift materially during this period except that there appears to be a little increase in the proportion of white cells from the eighteenth clay of incubation to the first day after hatching. During the same period there appears to be a slight decrease in the percentage of immature red cells. There is also an increase in the proportion of neutrophils which reaches its maximum on the second day after hatching and deceases again. The percentage of lymphocytes in the bone marrow appears to remain fairly constant although there is some indication that a low point is reached just after hatching time.
FIG. 1. Changes in the hemopoeitic activity of the bone marrow as related to growth and development of the chick.
The red cells in the circulation reach a concentration of about iy> millions per cmm. during the twentieth day of incubation, which remains unchanged during the remaining period investigated. During this period the white cells increase more rapidly than the red cells, since the mean of the percent white cells during the seven days of incubation is 0.39, and that for the seven days after hatching is 0.72. The concentration of neutrophils in the circulation reaches a maximum level at about the same time as the increase of neutrophils of the bone marrow takes place; however, the proportion of neutro-
increases greatly during the period investigated. The embryonic weight as given by Byerly (1930) and the early chick weight without yolk sac as reported by Heywang (1940) were used with blood cell counts (three-day moving average) on the marrow of the four long bones in preparing the graph of Figure 1. This graph shows the proportionate increase in total body weight and the hemopoeitic activity of the bone marrow investigated. The curves indicate that the increase in the blood cells of the bone marrow follow rather closely the in-
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 30, 2015
erythroblast. The white cells designated here as neutrophils are designated by others as heterophils (Cook and Dearstyne, 1934), polymorphonuclear eosinophils (Forkner, 1929), and polymorphonuclear cells with eosinophilic rods (Blain, 1928). The polymorphonuclear eosinophils with granules, the basophils, the monocytes, and the unclassified individually make up a very small fraction of the total white cells, hence are not included in this table.
393
394
B. R. BURMESTER, J. M. SEVERENS, AND E. ROBERTS
SUMMARY The total number of red cells and the various types of white cells in the marrow of the four long bones of the chick were determined for the period from the fifteenth day of incubation to the seventh day after hatching. The most significant change found was a rapid rise in the number of red and white cells of the bone marrow from the time of hatching to the fifth day thereafter. This increase was greatly out of proportion to the increase in body weight (cited data)
during the same period. The concentration of neutrophils in the circulation and the total number in the marrow of the four long bones reach their maxima at about the second day after hatching; however, the proportion of neutrophils to total white cells decreases markedly throughout the whole period. The concentration of lymphocytes and the proportion of lymphocytes to total white cells in the circulation increases greatly during the period investigated. ACKNOWLEDGMENT The technical assistance of Doris Cook is gratefully acknowledged. REFERENCES
Blain, D., 1928. A study of the white blood cells of the normal fowl by supravital technic. Anat. Rec. 39(2):285-293. Byerly, T. C , 1930. Effect of breed on growth of chick embryo. Jour. Morph. 50:341. Cook, F. W., and R. S. Dearstyne, 1934. Hematology of the fowl. North Carolina Experiment Station Tech. Bull. 44. Forkner, C. E., 1929. Blood and bone marrow cells of the domestic fowl. Jour. Exp. Med. 50:121-141. Heywang, B. W., 1940. The effect of cold drinking water on chick growth and yolk absorption. Poult. Sci. 19:201-204. Roberts, E., J. M. Severens, and L. E. Card, 1939. Nature of the heredity factors for resistance and susceptibility to pullorum disease in the domestic fowl. Proc. 7th World's Poultry Congress 52-54.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 30, 2015
crease in body weight (data of Byerly) before hatching time. After hatching there is a rapid increase in the blood cells of the bone marrow which is greatly out of proportion to the increase in body weight (data of Heywang). From the day of hatching to the sixth day the number of red cells in the bone marrow increases 11 times, whereas the body weight increases only 0.5 times during the same period. This rapid rise apparently terminates during the fifth or sixth day after hatching. Although the concentration of red cells in the circulation has reached the posthatching level by the twentieth day of incubation and the white cells by the day after hatching, it is probable that the rapid increase in the blood cells of the bone marrow is related to the changes in the embryo while it emerges from the egg.
URING the course of certain studies on the resistance of chicks to the pullorum disease (Roberts, Severens, and Card, 1939), data were collected on the blood cell activity in the bone marrow of chicks during the period of six days before hatching and seven days after hatching. The data presented here show the quantative changes in the blood cells of the bone marrow and circulation during this critical period in the development of the chick. METHODS
Blood samples for cell counts of the circulating blood were drawn from the vitelline vein in the case of embryos and from the wing vein in case of hatched chicks. The chicks were killed with ether and both femurs and tibiae were dissected from the chicks and embryos. As soon as all adhering tissue had been removed from the bones, they were split in half along their longitudinal axis and immediately immersed in oxalated plasma which had previously been prepared from hen's blood. The marrow cells were suspended in the plasma by gently shaking the bones with the plasma for one minute in a small stoppered vial. The suspension was filtered through several layers of gauze into a graduated 15 c.c. centrifuge tube. When the bone marrow was hyperplastic very little residue remained on the gauze. The shaking and filtering was repeated with fresh plasma. A third shaking was preceded by scraping out the spongy red bone at the epiphysis.
The volume of the suspension was noted and total counts were made by the ordinary methods used for circulating blood. For the blood smear, Wright's staining method was used to make the differential counts. The vital-dye method could not be used because it was found that the marrow cells did not take up the vital dye after the above treatment.1 RESULTS
Data on the cells of the bone marrow were obtained from embryos and chicks of the White Leghorn and Rhode Island Red breeds, but inasmuch as no significant differences were found, only the data for the White Leghorn chicks will be given here. These data, together with cell counts of the circulating blood, are summarized in Table 1. It is recognized that a high proportion of immature cells of all types are to be found in the bone marrow and circulating blood of embryos; however, in order to simplify the counting procedure, we have used only the mature cell classification for making the white cell counts, and have included the young or immature cells in the class to which they appeared to belong on the basis of their morphological characteristics and staining reactions. The red cells of the bone marrow were classified into mature and immature cells. The latter include the basophil and polychromatophil ' I t should be understood that this is an arbitrary method and results are of value only in following a relative change or a growth.
[391]
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 30, 2015
D
White Neutro- Lymphophils cells cytes as% as % of as % of white White Neutro- Lympho- of all white phils cytes cells cells cells cells
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<
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Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 30, 2015
Blood celis of the circulation
B. R . BURMESTER, J. M . S E V E R E N S , AND E . ROBERTS
Blood cells of the bone marrow
392
BLOOD CELLS I N BONE MARROW OF C H I C K BEFORE AND AFTER HATCHING
phils to total white cells decreases markedly throughout the whole period. On the other hand, the concentration of lmphocytes and the proportion of lymphocytes to total white cells in the circulating blood
The counts show that the number of blood cells in the four long bones used increase rapidly after hatching time. This is true of both the red cells and the white cells. The proportion of red cells to white cells does not shift materially during this period except that there appears to be a little increase in the proportion of white cells from the eighteenth clay of incubation to the first day after hatching. During the same period there appears to be a slight decrease in the percentage of immature red cells. There is also an increase in the proportion of neutrophils which reaches its maximum on the second day after hatching and deceases again. The percentage of lymphocytes in the bone marrow appears to remain fairly constant although there is some indication that a low point is reached just after hatching time.
FIG. 1. Changes in the hemopoeitic activity of the bone marrow as related to growth and development of the chick.
The red cells in the circulation reach a concentration of about iy> millions per cmm. during the twentieth day of incubation, which remains unchanged during the remaining period investigated. During this period the white cells increase more rapidly than the red cells, since the mean of the percent white cells during the seven days of incubation is 0.39, and that for the seven days after hatching is 0.72. The concentration of neutrophils in the circulation reaches a maximum level at about the same time as the increase of neutrophils of the bone marrow takes place; however, the proportion of neutro-
increases greatly during the period investigated. The embryonic weight as given by Byerly (1930) and the early chick weight without yolk sac as reported by Heywang (1940) were used with blood cell counts (three-day moving average) on the marrow of the four long bones in preparing the graph of Figure 1. This graph shows the proportionate increase in total body weight and the hemopoeitic activity of the bone marrow investigated. The curves indicate that the increase in the blood cells of the bone marrow follow rather closely the in-
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 30, 2015
erythroblast. The white cells designated here as neutrophils are designated by others as heterophils (Cook and Dearstyne, 1934), polymorphonuclear eosinophils (Forkner, 1929), and polymorphonuclear cells with eosinophilic rods (Blain, 1928). The polymorphonuclear eosinophils with granules, the basophils, the monocytes, and the unclassified individually make up a very small fraction of the total white cells, hence are not included in this table.
393
394
B. R. BURMESTER, J. M. SEVERENS, AND E. ROBERTS
SUMMARY The total number of red cells and the various types of white cells in the marrow of the four long bones of the chick were determined for the period from the fifteenth day of incubation to the seventh day after hatching. The most significant change found was a rapid rise in the number of red and white cells of the bone marrow from the time of hatching to the fifth day thereafter. This increase was greatly out of proportion to the increase in body weight (cited data)
during the same period. The concentration of neutrophils in the circulation and the total number in the marrow of the four long bones reach their maxima at about the second day after hatching; however, the proportion of neutrophils to total white cells decreases markedly throughout the whole period. The concentration of lymphocytes and the proportion of lymphocytes to total white cells in the circulation increases greatly during the period investigated. ACKNOWLEDGMENT The technical assistance of Doris Cook is gratefully acknowledged. REFERENCES
Blain, D., 1928. A study of the white blood cells of the normal fowl by supravital technic. Anat. Rec. 39(2):285-293. Byerly, T. C , 1930. Effect of breed on growth of chick embryo. Jour. Morph. 50:341. Cook, F. W., and R. S. Dearstyne, 1934. Hematology of the fowl. North Carolina Experiment Station Tech. Bull. 44. Forkner, C. E., 1929. Blood and bone marrow cells of the domestic fowl. Jour. Exp. Med. 50:121-141. Heywang, B. W., 1940. The effect of cold drinking water on chick growth and yolk absorption. Poult. Sci. 19:201-204. Roberts, E., J. M. Severens, and L. E. Card, 1939. Nature of the heredity factors for resistance and susceptibility to pullorum disease in the domestic fowl. Proc. 7th World's Poultry Congress 52-54.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 30, 2015
crease in body weight (data of Byerly) before hatching time. After hatching there is a rapid increase in the blood cells of the bone marrow which is greatly out of proportion to the increase in body weight (data of Heywang). From the day of hatching to the sixth day the number of red cells in the bone marrow increases 11 times, whereas the body weight increases only 0.5 times during the same period. This rapid rise apparently terminates during the fifth or sixth day after hatching. Although the concentration of red cells in the circulation has reached the posthatching level by the twentieth day of incubation and the white cells by the day after hatching, it is probable that the rapid increase in the blood cells of the bone marrow is related to the changes in the embryo while it emerges from the egg.