Determination of the Absolute Numbers of Leukocyte Cell Types in Chickens1

Determination of the Absolute Numbers of Leukocyte Cell Types in Chickens1

Determination of the Absolute Numbers of Leukocyte Cell Types in Chickens' T . K . S. M U K K U R 2 A N D R . E . BRADLEY Department of Veterinary Sc...

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Determination of the Absolute Numbers of Leukocyte Cell Types in Chickens' T . K . S. M U K K U R 2 A N D R . E . BRADLEY

Department of Veterinary Science, University of Florida, Gainsville, Florida 32601 (Received for publication May 1, 1973)

POULTRY SCIENCE 53: 221-223, 1974



ANY studies have been reported previously on the quantitation of various leukocytic cell types in chickens of different ages (Cook, 1937; Olson, 1937; Goff et al., 1953; Lucas and Jamroz, 1961). A large number of investigations were concerned with the effects of various parameters such as sex, diet, environment, disease and the different methods of staining, etc., on leukocytic counts, (Lucas and Jamroz, 1961; Sturkie, 1965). In the past, it had been extremely difficult to distinguish between thrombocytes and large lymphocytes in stained films of avian blood (Lucas and Jamroz, 1961) until the time a procedure was developed (Mukkur and Bradley, 1967) which permitted one to distinguish between these cells with great ease and accuracy. In addition it had also been previously recognized (Lundquist and Hedlung, 1925; Olson, 1937) that it was difficult to distinguish between various types of granulocytes such as polymorphonuclear neutrophilic leukocytes and

eosinophils with the possible exception of basophils and was attributed to artifacts developing as a result of the staining procedure used. For instance, the use of two different methods of staining on the same blood sample yielded different percentage of leukocytic cell types (Twisselmann, 1939) thus emphasizing the need for an accurate and reproducible staining procedure. Due to the availability of a procedure that permitted one to distinguish the thrombocytes from large lymphocytes, we undertook an investigation to determine the absolute values for various types of leukocytes in conventionally raised chickens. Since great difficulty was encountered in distinguishing the polymorphonuclear neutrophilic leukocytes from eosinophils and the fact that basophils form only a small percentage of the total leukocyte count (Lucas and Jamroz, 1961; Sturkie, 1965), it was decided to count all the neutrophilic leukocytes, eosinophils and basophils in one category, classed as granulocytes. PROCEDURE

1. Florida Agricultural Experiment Station Journal Series No. 4903. 2. Present address: Department of Biology, University of Windsor, Windsor, Ontario, Canada.

A total of 78 apparently healthy male chickens (White Leghorn) were used for this study. Six different chickens were bled at different intervals (indicated in Table 1) from


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ABSTRACT The absolute numbers of various leukocytic cell types in chicken blood were determined by the use of a modified Giemsa method which permitted one to distinguish between thrombocytes and large lymphocytes. It was not possible to distinguish between the polymorphonuclear neutrophilic leukocytes and eosinophils with any accuracy as observed previously with the use of conventional procedures. The absolute distribution of granulocytes, lymphocytes and monocytes in conventionally raised White Leghorn chickens, 28-73 days of age, was determined. A large variation in the absolute numbers of leukocyte cell types was observed within each group, thus making the differences between each group insignificant. The accumulated absolute mean (28-73 days) of various cell types revealed the following percent distribution: Granulocytes, 24%; Monocytes, 11%; Lymphocytes, 65%.



TABLE 1.—Statistical analysis of leukocyte cell types related to total leukocyte counts in chickens from 28-73 days of age White blood cell count x 378* ± standard deviation Total

Day of bleeding (age) 28 31 34 37 41 45 49 56 63 65 67 70 73

15.16 14.33 16.67 25.67 19.83 24.33 32.67 44.83 36.50 34.00 33.17 27.00 27.16

± ± ± ± ± ± ± ± ± ± ± ± ±

9.45 7.94 7.26 12.37 6.52 9.75 20.57 29.58 24.48 20.33 23.41 14.14 14.47




3.7 3.07 3.30 5.36 6.35 6.95 9.1 10.68 4.56 6.52 10.64 8.57 6.69

0.99 1.39 1.93 3.78 1.90 2.61 4.81 3.47 4.31 4.86 4.41 2.29 3.07

10.49 9.87 11.54 16.54 11.59 14.76 18.75 30.69 27.63 22.47 18.12 16.15 17.41

± 3.2 ± 1.78 ± 1.04 ± 3.84 ± 2.21 ±4.15 ± 7.73 ± 8.18 ± 5.82 ± 5.93 ± 12.24 ± 6.49 ± 5.10

± ± ± ± ± ± ± ± ± ± ± ± ±

0.68 0.70 1.13 1.88 1.10 0.86 4.48 2.86 7.29 5.99 6.00 1.45 2.91

± ± ± ± ± ± ± ± ± ± ± ± ±

5.93 5.94 6.93 11.29 6.52 6.78 10.21 19.44 17.20 12.81 8.55 9.42 9.61

*Dilution factor

RESULTS AND DISCUSSION The differences in the absolute numbers

of total leukocytes, granulocytes, monocytes and lymphocytes between different groups at various intervals were found to be statistically insignificant. However, a large standard deviation was observed within each group at each interval for various cell types including the total number of leukocytes indicating a great variation in the absolute numbers from one bird to the other (Table 1). Similar variation was also observed by other investigators (Cook, 1937; Olson, 1937; Twisselmann, 1939; Goff et al., 1953). Since the differences in absolute numbers for all these types at various intervals were statistically insignificant, the mean values for various cell types from 28-73 days inclusive were used to calculate the average relative percentage distribution of various types of cells during this entire period (Table 2). This data leads the authors to suggest that great care should be exercised before attributing any granulocytic cell changes in the peripheral blood of chick-

TABLE 2.—Mean percentage distribution of leukocyte types in chickens from 28-73 days of age. Cell type Granulocytes Monocytes Lymphocytes

Percentage 24 11 65

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28 to 73 days of age, by the intracardiac route. Blood smears were made immediately and stained with Giemsa's stain according to the method described previously (Mukkur and Bradley, 1967). The total leukocyte counts were made by a modification of Rees-Ecker method (Mukkur and Bradley, 1969), by the use of brilliant cresyl blue. A stock solution (sodium citrate, 3.8 gm.; brilliant cresyl blue, 0.5 gm. Ringer's solution, 100.0 ml.) was prepared and stored at 4° C. The stock solution was filtered and diluted 1:10 immediately before use with Ringer's solution which served as the diluent for leukocyte counts. Blood (0.02 ml.) was mixed well with diluted stain solution (3.0 ml.) in a Sahli pipette, allowed to stand for one hour at room temperature and the cells counted using the four corner squares (4/10 mm. 3 ) of a hemocytometer. Granulocytes, monocytes and lymphocytes were distinguished from each other according to the criteria set by Lucas and Jamroz (1961). Statistical analysis was performed by the use of Dunnett's multiple comparison test (1964) with an IBM 360-50 computer to obtain " t " values at a significance level of 1%.


ens to pathological conditions until the time that procedures for the definite distinction of various granulocytic cell types become available in literature. REFERENCES

sinophilen Leukozyten in Hunnerblut. Fol. Haem. 31: 253-264. Mukkur, T. K. S., and R. E. Bradley, 1967. Differentiation of avian thrombocytes from leukocytes by the use of Giesa's stain. Poultry Sci. 46: 1595-1596. Mukkur, T. K. S., and R. E. Bradley, 1969. Eimeria tenella: Packed blood cell volume, hemoglobin and serum proteins of chickens correlated with the immune state. Exptl. Parasitol. 26: 1-16. Olson, C , 1937. Variation in the cells and hemoglobin content in the blood of normal domestic chicken. Cornell Vet. 27: 235-263. Sturkie, P. D., 1965. Avian Physiology, Second Ed., Comstock Publishing Associates. Cornell University Press, Ithaca, New York. Twisselmann, N. M., 1939. A study of the cell content of blood of normal chickens with special reference to comparative differential leukocyte counts made with supravital and Wright's staining technics. Poultry Sci. 18: 151-159.

Influence of a Xanthophyll-Free Pullet Grower Diet on Subsequent Egg Yolk Pigmentationl C . F . HINTON, JACK L . F R Y AND R. H . HARMS

Florida Agricultural Experiment Station, Gainesville, Florida 32611 (Received for publication May 1, 1973)

ABSTRACT Two experiments were conducted to determine the importance of xanthophyll in the diet of growing egg-type pullets. The experiments were designed so that both the rate of xanthophyll depletion from the body and the amount of xanthophyll deposited in the yolk from feed sources could be determined. Pullet chicks were fed diets varying in xanthophyll content during the growing period. Birds fed a diet containing yellow corn and alfalfa were placed on a xanthophyll-free diet at sexual maturity. Birds grown on a xanthophyll-free diet were placed on a layer diet containing yellow corn and alfalfa either at 20 weeks of age or at the time of sexual maturity of the individual bird. Rate of depletion of yolk xanthophyll of birds placed on a xanthophyll-free layer diet at 24 weeks of age was most rapid during the first nine days of the experiment. Birds placed on a diet containing yellow corn and alfalfa at 20 weeks of age, after being grown on a xanthophyll-free diet, produced commercially acceptable eggs at sexual maturity. Those placed on the yellow corn-alfalfa diet at sexual maturity produced yolks essentially equivalent to those of the controls by the time they reached 30 weeks of age. POULTRY SCIENCE 53.: 223-226, 1974



EPLACEMENT pullet growers have been concerned that if their pullets were not deeply pigmented, egg producers would be reluctant to purchase them because they 1. Florida Agr. Exp. Sta. Journal Series No. 4904.

would appear to lack vigor or might produce eggs with pale yolks. It has been previously established that pigment does not contribute nutritionally to the bird's diet since xanthophylls are metabolically inactive (Palmer and Kemster, 1919). It is also generally accepted that an increase in xanthophyll level in the feed will be reflected in yolk color to its

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Cook, S. F., 1937. A study of blood picture of poultry and its diagnostic significance. Poultry Sci. 16: 291-296. Dunnett, C. W., 1964. New tables for multiple comparisons with a control. Biometrics, 20: 482-491. Goff, S., W. C. Russell and M. W. Taylor, 1953. Hematology of the chick in vitamin deficiencies. I. Riboflavin. Poultry Sci. 32: 54-60. Lucas, A. M., and C. Jamroz, 1961. Atlas of Avian Hematology, Agriculture Monograph 25. U.S.D.A., Washington, D.C. Lundquist, C. W., and B. Hedlung, 1925. Uber die verschiedenen Granulaformen der sog. Pseudoeo-