Reticulocyte Counts in the Chicken1

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R. L. KEMP AND W. M.

atitis). U. S. Dept. Agr., Bur. An. Ind. Bull. 8: 1-38. Smith, T., 1910. Amoeba meleagridis. Science, 32: 509-512. Smith, T., 1915. Further investigations into the etiology of the protozoan disease of turkeys known as blackhead, enterohepatitis, typhlitis, etc. J. Med. Res. 3 3 : 243-270. Tyzzer, E. E., 1920. The flagellate character and reclassification of the parasite producing "blackhead" in turkeys—Histomonas (gen. nov.) meleagridis (Smith). J. Parasitol. 6: 124-131. Tyzzer, E. E., 1934. Studies on histomoniasis or "blackhead" infection in the chicken and the turkey. Proc. Am. Acad. Arts Sci. 69: 189-264. Walker, R. V. L., 1948. Enterohepatitis (blackhead) in turkeys. I. Pentatrichonomas associated with enterohepatitis and its propagation in developing chick embryos. Canad. J. Comp. Med. Vet. Sci. 12 : 43-46. Wiley, J. V., 1961. Personal communication. Whitmoyer Laboratories, Myerstown, Penn. Young, G., 1958. The process of invasion and persistence of Candida albicans injected intraperitoneally into mice. J. Infect. Dis. 102: 114-120.

Reticulocyte Counts in the Chicken1 VlONA COATES AND B . E . MARCH Department of Poultry Science, The University of British Columbia, Vancouver 8, Canada (Received for publication April 4, 1966)

T

HERE is little information available regarding the numbers of reticulocytes present in the blood of chickens of different ages. Orten (1934) concluded from a survey of the literature to 1934 that nearly all of the nucleated erythrocytes of birds, fish and reptiles show the reticulum. However, this statement does not seem to be borne out in the case of birds by the data reported by some other investigators. Vaughan et al. (1930) found that pigeons normally have reticulocyte counts below 12 percent. Wills (1932) showed that successive reticulocyte counts in pigeons may fluctuate considerably over a range of from J

T h e research was supported by a grant from the Muscular Dystrophy Association of Canada.

less than 10 percent to as high as 60 percent in response to dietary modifications which appear to have involved either induction or relief of anaemia. Peabody and Neale (1933) used reticulocyte levels in the pigeon to measure the hematopoietic response to liver extracts and reported that the birds which had levels of about 20 percent when first caged showed a decrease to about 10 percent after 25 days of confinement, and thereafter came to a fairly constant level in the range of 11-13 percent. Magath and Higgins (1934) found the level of reticulocytes in adult mallard ducks to be in the range of 16.6 to 27.7 percent. Lucas and Jamroz (1961), on the basis of their studies of blood of normal

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entero-hepatitis. Medy. Weterynaryjna 12: 201204. Kuprowski, M., 1960. Zur Pathogenese und Morphologie der Moniliasis der Huhnervogel. Deutsche Tierarzt. Woch. 67 : 185-189. Lund, E. E., 1963. Histomonas wenrichi n. sp. (Mastigophora: Mastigamoebidae), a nonpathogenic parasite of gallinaceous birds. J. Protozool. 10: 401-404. Lund, E. E., and M. M. Farr, 1965. Enterohepatitis (blackhead) in H. E. Biester and L. H. Schwarte, Diseases of Poultry. 5th Ed. Iowa State Univ. Press, Ames. p. 1096-1110. Malewitz, T. D., 1958. The pathology of experimentally produced histomoniasis in turkeys. Amer. J. Vet. Res. 19: 181-185. Ohara, T., and W. M. Reid, 1961. Histomoniasis in chickens: Age of greatest susceptibility and pathogenicity studies. Avian Dis. 5: 355-361. Schiefer, B., and B. Mehnert, 1963. Untersuchungen zur Atiologie und Pathogenese der Typhlohepatitis der Huhnervogel. Zentralb. f. Veterinarmed. Reihe B, Band X heft 1, 28-48. Smith, T., 1895. An infectious disease among turkeys caused by protozoa (infectious entero-hep-

REID

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RETICULOCYTE COUNTS

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adult chickens, state that reticulocytes are "extremely rare." Robertson et cd. (1947) reported a level of about 10 percent of reticulocytes to be normal in 4-week-old chicks. Since there seemed to be so little data regarding the number of reticulocytes present in the blood of chickens, the following study was undertaken to establish the normal levels of reticulocytes in chickens of different ages, and determine if there are any variations in the reticulocyte numbers in mature birds of different breeds. METHODS Blood was drawn from a wing vein into tubes containing 0.1 ml. of heparin solution per ml. of blood taken. The heparin solution was 0.1 percent in 0.8S percent sodium chloride solution. One ml. of heparinized blood and 1 ml. of brilliant cresyl blue solution (1 percent in 0.85 percent sodium chloride solution) were mixed and incubated in a water bath at 25°C. for 30 minutes. The tubes were inverted to mix thoroughly. Smears were then made from the stained preparation and counterstained with Wright's stain. The slides were examined microscopically and the numbers of reticulocytes were

determined on the basis of a counted thousand red blood cells. Two sets of determinations of reticulocyte numbers were carried out. The first was made on blood samples taken from White Leghorn chicks of different ages from hatching to 15 weeks of age. The first determinations were made on blood drawn from chicks immediately after they were taken from the incubator. Chicks from the same hatch were reared in a battery brooder. At intervals, blood samples were taken from five birds. Birds which had been bled were not returned to the population. The second set of determinations was made on mature birds of different breeds.

TABLE 1.—Reticulocyte counts in the chick from hatching to 15 weeks of age. Five chicks were examined at each age Age of chicks

Av. reticulocyte count %

Range

newly hatched 1 day 3 days 1 week 2 weeks 3 weeks 6 weeks 8 weeks 10 weeks 15 weeks

34.8 36.7 30.8 28.4 27.1 28.8 12.5 11.5 10.8 10.1

31.0-36.6 35.3-38.7 28.9-33.0 21.5-36.1 24.5-32.2 20.5-36.7 10.3-17.0 8.1-14.2 8.2-14.2 8.2-12.2

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FIG. 1. Erythrocytes showing rlecreasing reticulation from cell 1 to cell 5.

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V. COATES AND B . E . MARCH TABLE 2.—Reticulocyte counts in mature chickens of different breeds No. of birds examined

Av. reticulocyte count %

Range

male female female female

10 5 5 5

10.1 11.9 8.5 9.1

7.8-12.3 10.1-13.3 7.4-13.3 7.0-11.5

New Hampshire New Hampshire

male female

IS 5

8.1 7.2

5.4-14.1 6.0-10.4

White Rock Cornish Minnesota heavy linef Black Australorp Fayoumi

male male male male male

10.S 20.5 17.8 12.6 12.3

7.1-14.4 18.3-22.0 14.2-20.4 9.1-14.9 9.4-15.2

White White White White

Leghorn Leghorn Leghorn Leghorn

strain strain strain strain

A A B C

8 6* 8 8 8

* Two birds examined from this flock showed counts of 42.9 and 44.1 percent respectively and were not included in the average. t This is a line developed by Dr. R. N. Shoffner at the University of Minnesota for broiler production.

RESULTS AND DISCUSSION

Figure 1 shows a series of reticulocytes with varying degrees of reticulation ranging from a heavily-staining network in cell 1 to a few isolated dots in cell 5. The counts given in Tables 1 and 2 represent the number of cells showing reticular material regardless of the amount present. This designation of reticulocytes reduces subjectivity of reticulocyte counting. It is felt that much of the variation in the reticulocyte counts reported in the literature for chicken blood is due either to differences in staining technique or to differences in the description of cells identified as reticulocytes. The reticulocyte count in the newly hatched chick (Table 1) was found to be approximately 3S percent. The count gradually decreased and reached the adult level at 8 to 10 weeks of age. The decline in reticulocyte number as the birds became older is similar to the effect of age reported by Orten and Smith (1934) in the albino rat. They found that the rat at birth had reticulocyte counts of 99 percent, at weaning 25 percent, and at about 10 weeks adult levels of 1 to 3 percent. The reticulocyte counts determined in

mature chickens of several breeds are given in Table 2. The range of values observed within each breed indicates considerable individual variation. Counts on birds of three strains of White Leghorns showed less variation among strains than was evident among the different breeds. Of the breeds tested, New Hampshires had the lowest and Cornish had the highest counts (7.6 and 20.5 percent respectively). REFERENCES Lucas, A. M., and C. Jamroz, 1961. Atlas of Avian Hematology, p. 28. Agriculture Monograph 25. U.S. Department of Agriculture. Magath, T. B., and G. M. Higgins, 1934. The blood of the normal duck. Fol. Haem. 51: 230241. Orten, J. M., 1934. The properties and significance of the reticulocyte. Yale J. Bio. Med. 6: 520539. Orten, J. M., and A. H. Smith, 1934. The proportion of reticulocytes in the blood of albino rats. Am. J. Physiol. 108: 66-73. Peabody, W. A., and R. C. Neale, 1933. The pigeon as a hematopoietic test animal. J. Am. Pharm. Assoc. 22: 1231-1237. Robertson, E. I., G. F. Fiala, M. L. Scott, L. C. Norris and G. F. Heuser, 1947. Response of anemic chicks to pteroylglutamic acid. Proc. Soc. Exp. Biol. Med. 64: 441-443. Vaughan, J. M., G. L. Muller and L. Zetrel, 1930.

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Sex

Breed

RETICULOCYTE COUNTS The response of grain-fed pigeons to substances effective in pernicious anaemia. Brit. J. Exp. Path. 1 1 : 456-468.

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Wills, L., 1932. Spontaneous fluctuations in the reticulocyte counts in pigeon's blood. Brit. J. Exp. Path. 13 : 172-175.

The Effects of Arsanilic Acid on Laying Hens At Three Dietary Protein Levels1 PART 2. FERTILITY, HATCHABILITY, CHICK GROWTH AND BLOOD SPOT INCIDENCE

(Received for publication April 4, 1966)

E

ARLIER investigation by Libby et al. (1953) determined the presence of residual arsenic in the egg to be of little consequence and well below the limits allowed by the Food and Drug Administration. Neither they nor Wharton and Fritz (1953) found any significant effects of the arsonic acids on hatchability or early progeny growth. Lillie et al. (1957) concluded after 3 years of experiments with dual purpose breeds that arsanilic acid alone or in combination with fat and antibiotics in 15% protein diets did not influence fertility or hatchability but some positive maternal diet influence was noted in the chicks. Carlson (1957) adding 90 gms. of arsanilic acid per ton to 12, 15 and 16% protein rations reported improved production and hatachability but found no consistent effects on progeny growth. Later, Carlson (1959) continued the life cycle experiments with small numbers of chicks with beneficial but still inconclusive results. Kurnick et al. (1960) feeding 17% protein diets and arsanilic acid recorded 1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. 2 Present address: Extension Poultry Specialist, Western Washington Experiment Station, Puyallup, Washington. This work was supported, in part, by a grant from Abbott Laboratories, North Chicago, Illinois.

improved growth at 48 days of age in progeny from dams fed arsanilic acid although fertility or hatchability were unaffected. EXPERIMENTAL PROCEDURE

General management and procedure were similar to those discussed by Andrews et al. (1966). Fertility experiments were primarily conducted with the cage birds using weekly inseminations of .05 cc. of pooled semen from caged New Hampshire males. As a further check, 2 experiments were conducted with artificial insemination of the floor hens by the same males. Fertility was measured by breaking out all undeveloped eggs at 7 days and examining the germinal disc to see if life had started. Additional breakouts were made at the 22nd day of incubation. Two groups of chick experiments were conducted. Hatch 1 and hatch 2 were from the first experiment while hatches 3, 4, and 5 were from the second one. All chicks were pedigree hatched, grouped in lots and reared in electric chick batteries. RESULTS AND DISCUSSION

Fertility. Eggs from the cage treatments were set during the winters of both experiments. Floor pen eggs were set from February to April of the second experiment. Arsanilic acid treated hens, at all dietary

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D. K. ANDREWS,2 H. R. BIRD AND M. L. SUNDE Department of Poultry Science, University of Wisconsin, Madison, Wisconsin