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The effect of vitamin B12 deficiency on erythrocyte counts and hemoglobin levels of chick embryo blood
The Effect of Vitamin B12 Deficiency on Erythrocyte Counts and Hemoglobin Levels of Chick Embryo Blood1v2 Jeng Mein Hsu, Joel R. Stern and James McGinnis From the Department
of Poultry
Science, The State College of Washington, Washington
Received
November
Pullman,
13, 1951
INTRODUCTION
In a previous publication (I), it was reported that hemoglobin and hematocrit and erythrocyte counts of vitamin Blz-deficient chicks were not affected by an uncomplicated deficiency of this vitamin. However, when the Blz deficiency was aggravated by injecting the chicks with phenylhydrazine hydrochloride, the hemoglobin levels and hematocrit and erythrocyte counts in the deficient birds were significantly lower than those of chicks receiving supplementary Blz. It was demonstrated that vitamin Blz does function to some extent in maintaining hemoglobin levels and the volume of red blood cells. Carver and McGinnis (2) reported that hatchability was low in the case of eggs laid by hens fed a vitamin Blz-deficient diet. A recent observation concerning vitamin Blz-deficient eggs has been the high mortality that occurs mainly during the latter part of incubation (3). The present study was conducted to observe hemoglobin levels and erythrocyte counts of normal and of vitamin Blz-deficient chicken embryos during the period between the 18th and 21st days of incubation when most of the embryo mortality of deficient eggs occurs. PROCEDURE In the first trial, 350 White Leghorn eggs were used; half were from hens maintained on a vitamin BIP-deficient diet and half from hens fed a diet containing supplemental B12. These diets are shown in Table I. Erythrocyte counts, using the i Scientific Paper No. 1065, Institute of Agricultural Sciences, The State College of Washington, Pullman, Project 1033. 2 This investigation was supported in part by funds provided for biological and medical research by the State College of ‘Washington, Initiative 171. 261
262
JENG
MEIN
HSU,
JOEL
R.
STERN
TABLE Composition Ingredients
AND
JAMES
MCGINNIS
I
of Basal Diet
Diet 1
Diet 2
Diet 3 -
Whole wheat Ground yellow corn Ground wheat Ground oats Ground barley Millrun Soybean oil meal (solvent) Dehydrated alfalfa Ground limestone Dicalcium phosphate Iodized salt Vitamin A and D supplement (4000 I.U. A; 1000 U. D/g.) Riboflavin concentrate (500 rg./g.) MnSO( Vitamin Bit premix (1 Kdg.)
%
%
%
28.5 10.0 10.0 5.0 10.00 10.00 14.5 5.0 3.25 3.25 0.50
40.0 17.35 2.08 2.08 13.19 15.90 3.00 2.60 1.90 0.60
40.0 16.50 2.10 2.10 13.40 15.90 3.00 2.60 2.90 0.60
0.10
0.20
0.20
0.5 5.65 g/100 lb. -
0.6 11.40 g./lOO lb. 0.50
0.60 11.60 g./lOO lb. -
method of Olson (4), were begun after the eggs had been incubated for 18 days and were continued daily until the chicks hatched. Shell and membranes were cleared from the embryo which was sacrificed in the process, Blood was drawn directly from the heart with a syringe and placed on a clean, dry slide from which the sample was drawn into a diluting pipet. No anticoagulant was used. The diluted samples were kept under refrigeration for 24 hr., then were placed in the chambers on both sides of a hemocytometer and the average of the two counts was used in computing the results. In the second trial, 200 eggs from White Leghorn hens fed a vitamin Bls-deficient diet (diet 1, Table I) were used. A small hole was drilled into the large end of half these eggs after washing the spot with alcohol, and 0.1 ml. of a sterilized solution containing 20 pg./ml. of vitamin Blz was injected into the albumin through a 22-gauge needle. The holes were then sealed with paraffin and the eggs placed in a conventional electric incubator. Blood samples were obtained from embryos at the 18th day of incubation and from newly hatched chicks (21 days after the start of incubation). Erythrocyte counts were made as described above, and hemoglobin determinations were made by the method of Wu (5) using a hemin reference standard prepared according to the directions of EIvehj em (6). Four different groups of hens laid the eggs which were used in the third trial.
B12
AND
EMBRYO
263
HEMATOLOGY
Eggs from the first group were vitamin Brz-deficient and those from the second group were from hens fed diet 3, Table I, supplemented with 0.1 y0 of a vitamin Bit concentrate containing 12.5 mg. of B&b. Hens fed terramycin at a level of 15 mg./kg. of feed produced the eggs for group 3, and hens fed the same level of terramycin, plus the above level of Br,, produced eggs for group 4. Hemoglobin determinations and erythrocyte counts were made, as previously described, only on the day that the chicks hatched. RESULTS
The average erythrocyte counts of the chick embryos studied in trial 1 are shown in Table II. Each figure is an average of two counts on blood from six embryos; the values for the 21st day were obtained TABLE Trial
1: E$ect of Vitamin
II
BIZ on Erythrocyte Count Hatchability
of Chick Embryo Blood and on Erythrocyte Diet 1
Day of incubation
x l@/cu.
18 19 20 21 (blood drawn from hatched chicks) Av. Hatchability: per cent of fertile eggs Vitamin Blz supplement
mm.
1.58 1.61 1.59 1.83 1.65 45 (157)” None
counts Diet 2
x ios/cu. mm.
2.26 2.15 2.12 2.38 2.230 75 (157)b 5 rg.k.
6P < 0.01. b Number of eggs.
on blood from newly hatched chicks. An analysis of variance of the data disclosed that a highly significant increase in erythrocyte numbers resulted from supplementation with vitamin B12. The hatchability of eggs from hens fed the Blz-supplemented diet was higher than that of eggs from vitamin B,,-deficient hens. The results of erythrocyte counts and hemoglobin determinations on blood of chick embryos injected with vitamin Blz are indicated in Table III. (Again, values for the 21st day were obtained from the blood of newly hatched chicks.) Both on the 18th and 21st days, embryos and chicks from eggs injected with vitamin Blz had higher hemoglobin levels and erythrocyte counts than those which did not receive vitamin Blz.
264
JENG
MEIN
HSU,
JOEL
R.
STERN
TABLE Trial
2: Effect
of Vitamin
AND
JAMES
MCGINNIS
III
B,, on Hemoglobin Levels and Erythrocvte I ” Embryo and Chick Blood Hemoglobin
Erythrocyte
Counts of
count
Treatment Embryo5
Chicka
Embryo”
HfS%Lof eggs
Chicka
g/100
None
Av.
Vitamin Blz injection, 2 pg./egg
g./lOO ml.
x lO~/CU. mm.
x 1o=/cu. mm.
11.0 9.5 9.3 9.5 8.2 8.8
ml.
14.0 13.9 12.0 14.9 12.4 14.0
1.89 1.64 1.40 1.60 1.49 1.15
1.81 1.80 1.75 2.15 2.22 1.97
9.4
13.6
1.53
1.95
15.1 13.6 13.4 11.0 9.3 10.5
15.6 16.9 17.5 16.0 15.6 16.4
2.52 2.36 2.34 2.28 1.61 1.70
2.46 2.83 2.85 2.40 2.49 2.21
16.4”
2.130
2.540
Av. 12.2~
%
45 (157)”
_. 82 (82)C
(1P < 0.01. b Age of embryos: 18 days; age of chicks: c Number of fertile eggs set.
day of hatching.
Hemoglobin levels and erythrocyte counts of day-old chicks hatched from hens fed diets varying in vitamin B,, and terramycin are shown in Table IV. It was found that hemoglobin levels and erythrocyte counts were significantly higher in chicks from hens receiving vitamin Blz than in chicks from hens which did not receive Blz. The terramycin added to the hen diet was without effect on erythrocyte or hemoglobin level, but did increase hatchability. DISCUSSION
The importance of vitamin Blz in hatchability was shown by Carver and McGinnis (7) and by Petersen et al. (8). Data presented in this paper show that vitamin Blz deficiency decreased the number of red blood cells and level of hemoglobin in chick embryos in addition to
B12
AND
EMBRYO
TABLE
265
HEMATOLOGY
IV
Trial 3: The Effect of Hen Diet on Hemoglobin Newly
Levels and Erythrocyte Hatched Chicks
Hemoglobin
Treatment
count
Hatch of fertile eggs
x lO~/cu.mm. 1.67 1.84 2.42 2.83
31728)b 79 (24) 82 (30) 100 (30)
Erythrocyte
Q./l00 ml. 10.2 10.8 13.5 13.9
None Terramycin Vitamin Btz Terramycin + B,,
Counts in
Significance of mean differences Effect or interaction
Vitamin B12 Terramycin b B1* X terramycin
Hemoglobin
3.2” 0.5 -0.1
Erythrocyte
counts
0.87” 0.28 0.12
a P < 0.01. b Number of fertile eggs.
decreasing hatchability. Unpublished data obtained in this laboratory showed that B12-deficient embryos. liberated less CO2 than B12-supplemented embryos in the late periods of incubation. This difference in CO2 liberation was possibly caused by a difference in number of red cells with a corresponding difference in hemoglobin levels, perhaps leading, therefore, to a difference in rate of respiration. It was surprising that terramycin increased hatchability without affecting red blood cell numbers or hemoglobin level. Other workers found (9), that penicillin increased hatchability, and suggested that this effect was due to increased vitamin Bi2 synthesis since the B12 content of egg yolks increased following feeding of penicillin to the hens. It should be recalled, however, that blood determinations made on the 21st day of incubation were on blood from newly hatched chicks. It is possible that red cell counts and hemoglobin levels of blood from embryos which did not hatch might have been lower than for the chicks which did hatch. If this were the case, it might be postulated that the effect of terramycin was through increased production or availability of vitamin B12. It should be noted (Table III) that the hemoglobin level from chicks
266
JENG MEIN HSU, JOEL R. STERN AND JAMES MCGINNIS
which did not receive an injection of vitamin Bla was 13.6 g./lOO ml., whereas, the hemoglobin level of chicks hatched from eggs laid by hens receiving vitamin Blz in the diet was 13.5 g./lOO ml. (Table IV). Although the hens which supplied the eggs used in the study shown in Table III were fed a vitamin Blz-deficient diet, it is possible that some of these hens had a sufficient Blz storage to transmit the vitamin to the embryo in the amount sufficient for normal hemopoiesis. The chicks which hatched from these eggs would then have normal hemoglobin levels. Chicks which failed to hatch would probably have had lower hemoglobin levels. Since blood was obtained only from hatched chicks, only those blood samples which might be expected to contain normal hemogIobin concentrations were obtained. SUMMARY
Experiments were conducted to investigate the effect of vitamin Blz deficiency upon hemoglobin levels and erythrocyte counts in White Leghorn chicks and embryos. Hemoglobin and red cell counts on vitamin Blz-supplemented embryo blood were significantly higher than on blood of BIz-deficient embryos from the 18th to 21st day of incubation. Vitamin Blz injected into the egg was as effective in raising embryo hemoglobin levels and red cell counts as BIS added to the diet of the laying hens. Terramycin added to a B1,-deficient diet for hens did not improve hemoglobin levels or erythrocyte counts in hatched chicks in spite of the fact that hatchability was markedly improved. The relation between B12, terramycin, and hatchability is discussed. REFERENCES 1. STERN, J. R., Hsu, J. M., AND MCGINNIS, J., J. Biol. Chem. 194, 191 (1952). 2. CARVER, J. S., AND MCGINNIS, J., Poultry Sci. 29, 307 (1950). 3. PEELER, H. T., MILLER, R. F., CARLSON, C. W., NORRIS, L. C., AND HEUSER, G. F., Poultry Sci. 30, 11 (1951). 4. OLSON, C., JR., in BIESTER, H. E., AND SCHWARTE, L. H., Diseases of Poultry. Iowa State College Press, Ames, Iowa, 1948. 5. WV, H., J. Biochem. (Japan) 2, 173 (1922). 6. ELVEHJEX, C. A., J. Biol. Chem. QQ, 203 (1931). 7. CARVER, J. S., and MCGINNIS, J., Poultry Sci. 29, 752 (1950). 8. PETERSEN, C. F., WIESE, A. C., LAMPMAN, C. E., AND DAHLSTBOM, R. V., Poultry Sci. 29, 618 (1950). 9. ELM, J. E., GEE, L. L., AND COUCH, J. R., Proc. Sot. Ezptl. Biol. Med. ‘77, 209
(1951).
of Poultry
Science, The State College of Washington, Washington
Received
November
Pullman,
13, 1951
INTRODUCTION
In a previous publication (I), it was reported that hemoglobin and hematocrit and erythrocyte counts of vitamin Blz-deficient chicks were not affected by an uncomplicated deficiency of this vitamin. However, when the Blz deficiency was aggravated by injecting the chicks with phenylhydrazine hydrochloride, the hemoglobin levels and hematocrit and erythrocyte counts in the deficient birds were significantly lower than those of chicks receiving supplementary Blz. It was demonstrated that vitamin Blz does function to some extent in maintaining hemoglobin levels and the volume of red blood cells. Carver and McGinnis (2) reported that hatchability was low in the case of eggs laid by hens fed a vitamin Blz-deficient diet. A recent observation concerning vitamin Blz-deficient eggs has been the high mortality that occurs mainly during the latter part of incubation (3). The present study was conducted to observe hemoglobin levels and erythrocyte counts of normal and of vitamin Blz-deficient chicken embryos during the period between the 18th and 21st days of incubation when most of the embryo mortality of deficient eggs occurs. PROCEDURE In the first trial, 350 White Leghorn eggs were used; half were from hens maintained on a vitamin BIP-deficient diet and half from hens fed a diet containing supplemental B12. These diets are shown in Table I. Erythrocyte counts, using the i Scientific Paper No. 1065, Institute of Agricultural Sciences, The State College of Washington, Pullman, Project 1033. 2 This investigation was supported in part by funds provided for biological and medical research by the State College of ‘Washington, Initiative 171. 261
262
JENG
MEIN
HSU,
JOEL
R.
STERN
TABLE Composition Ingredients
AND
JAMES
MCGINNIS
I
of Basal Diet
Diet 1
Diet 2
Diet 3 -
Whole wheat Ground yellow corn Ground wheat Ground oats Ground barley Millrun Soybean oil meal (solvent) Dehydrated alfalfa Ground limestone Dicalcium phosphate Iodized salt Vitamin A and D supplement (4000 I.U. A; 1000 U. D/g.) Riboflavin concentrate (500 rg./g.) MnSO( Vitamin Bit premix (1 Kdg.)
%
%
%
28.5 10.0 10.0 5.0 10.00 10.00 14.5 5.0 3.25 3.25 0.50
40.0 17.35 2.08 2.08 13.19 15.90 3.00 2.60 1.90 0.60
40.0 16.50 2.10 2.10 13.40 15.90 3.00 2.60 2.90 0.60
0.10
0.20
0.20
0.5 5.65 g/100 lb. -
0.6 11.40 g./lOO lb. 0.50
0.60 11.60 g./lOO lb. -
method of Olson (4), were begun after the eggs had been incubated for 18 days and were continued daily until the chicks hatched. Shell and membranes were cleared from the embryo which was sacrificed in the process, Blood was drawn directly from the heart with a syringe and placed on a clean, dry slide from which the sample was drawn into a diluting pipet. No anticoagulant was used. The diluted samples were kept under refrigeration for 24 hr., then were placed in the chambers on both sides of a hemocytometer and the average of the two counts was used in computing the results. In the second trial, 200 eggs from White Leghorn hens fed a vitamin Bls-deficient diet (diet 1, Table I) were used. A small hole was drilled into the large end of half these eggs after washing the spot with alcohol, and 0.1 ml. of a sterilized solution containing 20 pg./ml. of vitamin Blz was injected into the albumin through a 22-gauge needle. The holes were then sealed with paraffin and the eggs placed in a conventional electric incubator. Blood samples were obtained from embryos at the 18th day of incubation and from newly hatched chicks (21 days after the start of incubation). Erythrocyte counts were made as described above, and hemoglobin determinations were made by the method of Wu (5) using a hemin reference standard prepared according to the directions of EIvehj em (6). Four different groups of hens laid the eggs which were used in the third trial.
B12
AND
EMBRYO
263
HEMATOLOGY
Eggs from the first group were vitamin Brz-deficient and those from the second group were from hens fed diet 3, Table I, supplemented with 0.1 y0 of a vitamin Bit concentrate containing 12.5 mg. of B&b. Hens fed terramycin at a level of 15 mg./kg. of feed produced the eggs for group 3, and hens fed the same level of terramycin, plus the above level of Br,, produced eggs for group 4. Hemoglobin determinations and erythrocyte counts were made, as previously described, only on the day that the chicks hatched. RESULTS
The average erythrocyte counts of the chick embryos studied in trial 1 are shown in Table II. Each figure is an average of two counts on blood from six embryos; the values for the 21st day were obtained TABLE Trial
1: E$ect of Vitamin
II
BIZ on Erythrocyte Count Hatchability
of Chick Embryo Blood and on Erythrocyte Diet 1
Day of incubation
x l@/cu.
18 19 20 21 (blood drawn from hatched chicks) Av. Hatchability: per cent of fertile eggs Vitamin Blz supplement
mm.
1.58 1.61 1.59 1.83 1.65 45 (157)” None
counts Diet 2
x ios/cu. mm.
2.26 2.15 2.12 2.38 2.230 75 (157)b 5 rg.k.
6P < 0.01. b Number of eggs.
on blood from newly hatched chicks. An analysis of variance of the data disclosed that a highly significant increase in erythrocyte numbers resulted from supplementation with vitamin B12. The hatchability of eggs from hens fed the Blz-supplemented diet was higher than that of eggs from vitamin B,,-deficient hens. The results of erythrocyte counts and hemoglobin determinations on blood of chick embryos injected with vitamin Blz are indicated in Table III. (Again, values for the 21st day were obtained from the blood of newly hatched chicks.) Both on the 18th and 21st days, embryos and chicks from eggs injected with vitamin Blz had higher hemoglobin levels and erythrocyte counts than those which did not receive vitamin Blz.
264
JENG
MEIN
HSU,
JOEL
R.
STERN
TABLE Trial
2: Effect
of Vitamin
AND
JAMES
MCGINNIS
III
B,, on Hemoglobin Levels and Erythrocvte I ” Embryo and Chick Blood Hemoglobin
Erythrocyte
Counts of
count
Treatment Embryo5
Chicka
Embryo”
HfS%Lof eggs
Chicka
g/100
None
Av.
Vitamin Blz injection, 2 pg./egg
g./lOO ml.
x lO~/CU. mm.
x 1o=/cu. mm.
11.0 9.5 9.3 9.5 8.2 8.8
ml.
14.0 13.9 12.0 14.9 12.4 14.0
1.89 1.64 1.40 1.60 1.49 1.15
1.81 1.80 1.75 2.15 2.22 1.97
9.4
13.6
1.53
1.95
15.1 13.6 13.4 11.0 9.3 10.5
15.6 16.9 17.5 16.0 15.6 16.4
2.52 2.36 2.34 2.28 1.61 1.70
2.46 2.83 2.85 2.40 2.49 2.21
16.4”
2.130
2.540
Av. 12.2~
%
45 (157)”
_. 82 (82)C
(1P < 0.01. b Age of embryos: 18 days; age of chicks: c Number of fertile eggs set.
day of hatching.
Hemoglobin levels and erythrocyte counts of day-old chicks hatched from hens fed diets varying in vitamin B,, and terramycin are shown in Table IV. It was found that hemoglobin levels and erythrocyte counts were significantly higher in chicks from hens receiving vitamin Blz than in chicks from hens which did not receive Blz. The terramycin added to the hen diet was without effect on erythrocyte or hemoglobin level, but did increase hatchability. DISCUSSION
The importance of vitamin Blz in hatchability was shown by Carver and McGinnis (7) and by Petersen et al. (8). Data presented in this paper show that vitamin Blz deficiency decreased the number of red blood cells and level of hemoglobin in chick embryos in addition to
B12
AND
EMBRYO
TABLE
265
HEMATOLOGY
IV
Trial 3: The Effect of Hen Diet on Hemoglobin Newly
Levels and Erythrocyte Hatched Chicks
Hemoglobin
Treatment
count
Hatch of fertile eggs
x lO~/cu.mm. 1.67 1.84 2.42 2.83
31728)b 79 (24) 82 (30) 100 (30)
Erythrocyte
Q./l00 ml. 10.2 10.8 13.5 13.9
None Terramycin Vitamin Btz Terramycin + B,,
Counts in
Significance of mean differences Effect or interaction
Vitamin B12 Terramycin b B1* X terramycin
Hemoglobin
3.2” 0.5 -0.1
Erythrocyte
counts
0.87” 0.28 0.12
a P < 0.01. b Number of fertile eggs.
decreasing hatchability. Unpublished data obtained in this laboratory showed that B12-deficient embryos. liberated less CO2 than B12-supplemented embryos in the late periods of incubation. This difference in CO2 liberation was possibly caused by a difference in number of red cells with a corresponding difference in hemoglobin levels, perhaps leading, therefore, to a difference in rate of respiration. It was surprising that terramycin increased hatchability without affecting red blood cell numbers or hemoglobin level. Other workers found (9), that penicillin increased hatchability, and suggested that this effect was due to increased vitamin Bi2 synthesis since the B12 content of egg yolks increased following feeding of penicillin to the hens. It should be recalled, however, that blood determinations made on the 21st day of incubation were on blood from newly hatched chicks. It is possible that red cell counts and hemoglobin levels of blood from embryos which did not hatch might have been lower than for the chicks which did hatch. If this were the case, it might be postulated that the effect of terramycin was through increased production or availability of vitamin B12. It should be noted (Table III) that the hemoglobin level from chicks
266
JENG MEIN HSU, JOEL R. STERN AND JAMES MCGINNIS
which did not receive an injection of vitamin Bla was 13.6 g./lOO ml., whereas, the hemoglobin level of chicks hatched from eggs laid by hens receiving vitamin Blz in the diet was 13.5 g./lOO ml. (Table IV). Although the hens which supplied the eggs used in the study shown in Table III were fed a vitamin Blz-deficient diet, it is possible that some of these hens had a sufficient Blz storage to transmit the vitamin to the embryo in the amount sufficient for normal hemopoiesis. The chicks which hatched from these eggs would then have normal hemoglobin levels. Chicks which failed to hatch would probably have had lower hemoglobin levels. Since blood was obtained only from hatched chicks, only those blood samples which might be expected to contain normal hemogIobin concentrations were obtained. SUMMARY
Experiments were conducted to investigate the effect of vitamin Blz deficiency upon hemoglobin levels and erythrocyte counts in White Leghorn chicks and embryos. Hemoglobin and red cell counts on vitamin Blz-supplemented embryo blood were significantly higher than on blood of BIz-deficient embryos from the 18th to 21st day of incubation. Vitamin Blz injected into the egg was as effective in raising embryo hemoglobin levels and red cell counts as BIS added to the diet of the laying hens. Terramycin added to a B1,-deficient diet for hens did not improve hemoglobin levels or erythrocyte counts in hatched chicks in spite of the fact that hatchability was markedly improved. The relation between B12, terramycin, and hatchability is discussed. REFERENCES 1. STERN, J. R., Hsu, J. M., AND MCGINNIS, J., J. Biol. Chem. 194, 191 (1952). 2. CARVER, J. S., AND MCGINNIS, J., Poultry Sci. 29, 307 (1950). 3. PEELER, H. T., MILLER, R. F., CARLSON, C. W., NORRIS, L. C., AND HEUSER, G. F., Poultry Sci. 30, 11 (1951). 4. OLSON, C., JR., in BIESTER, H. E., AND SCHWARTE, L. H., Diseases of Poultry. Iowa State College Press, Ames, Iowa, 1948. 5. WV, H., J. Biochem. (Japan) 2, 173 (1922). 6. ELVEHJEX, C. A., J. Biol. Chem. QQ, 203 (1931). 7. CARVER, J. S., and MCGINNIS, J., Poultry Sci. 29, 752 (1950). 8. PETERSEN, C. F., WIESE, A. C., LAMPMAN, C. E., AND DAHLSTBOM, R. V., Poultry Sci. 29, 618 (1950). 9. ELM, J. E., GEE, L. L., AND COUCH, J. R., Proc. Sot. Ezptl. Biol. Med. ‘77, 209
(1951).