Leucocyte Counts in Normal Young Rabbits

Br. vet. ]. (1973), [29, 47

LEUCOCYTE COUNTS IN NORMAL YOUNG RABBITS By G. M.

PURVIS

*

AND

M. M. H.

SEWELL

Department of Animal Health, Royal (Dick) School of Veterinary Studies, Easter Bush, Roslin, Midlothian

SUMMARY

The results are presented of a study of the leucocytes in the peripheral blood of 33 male and 23 female newly weaned rabbits. The results were similar to those described for older rabbits by previous authors except that the numbers of lymphocytes were relatively high, while the numbers of heterophils were relatively low. There were significantly fewer lymphocytes in the blood from the female rabbits. The packed cell volumes were also lower than those described for older rabbits. INTRODUCTION

The reported values of the leucocyte counts in apparently normal rabbits vary widely, even if differences in techniques are discounted (Schalm, 1965; Archer, 1965; Schermer, 1967). The following data were obtained from a relatively homogeneous group of young rabbits prior to their use in experimental studies (Purvis, 197 I). Colour plates showing the morphology and staining characteristics of the cells described in this paper can also be found in this reference. MATERIALS AND METHODS

Rabbits. A total of 56 New Zealand cross rabbits (33 males, 23 females), aged between 7 and 9 weeks old was used. These had been obtained from the University of Edinburgh Centre for Laboratory Animals. Some of the rabbits were infected with small numbers of Passalurus ambiguus (Oxyuroidea) but this had no apparent effect on the blood values studied. All the animals had received coccidiostats before weaning and oocysts were only occasionally seen in the faeces. Collection of blood. Th~ rabbit's ear was shaved and the veins dilated by applying a little xylol to the tip with cotton wool. The peripheral ear vein was punctured half-way along the ear with a sterile disposable lancet (No. A805 Hawksley Cristalet, Hawksley and Sons Ltd.). Pressure was applied below the wound and, after the blood had been allowed to flow freely for some seconds, 1 ml of blood was collected into a 2·s-ml vial containing sequestrene (Stayne Laboratories Ltd.). The blood was immediately mixed by slowly inverting the

* Present address: Ministry of Agriculture, Fisheries & Food, Veterinary Investigation Centre, Coley Park, Reading, RGI 6DT.

BRITISH VETERINARY JOURNAL, 129, I

container several times. The bleeding was controlled by pressure and prolonged haemorrhage did not occur in any of the rabbits. Haematology. All haematological examinations commenced within three hours of collection, on blood that had just been mixed on a Matburn Cell Suspension Mixer (Matburn Ltd., London) for at least one minute. Packed cell volumes were determined by a microhaematocrit technique, the tubes being centrifuged for eight minutes. Total white cell counts were made in an improved Neubauer chamber by a modification of the method of Dacie & Lewis (I 963). The cells counted includ ed normoblasts, th e counts being later corrected from the results of the differential cell counts. Differential cell counts were carried out by the four fields battlement techniqu e (Dacie & Lewis, I963) on blood films made on chemically cleaned slides. The film s were dried in air at room temperature for four hours and stained by the Undritz modification of the p eroxidase staining technique (Undritz, I952; Archer, I 965), to avoid confusion between the heterophil and eosinophil cells, both of which contain reddish cytoplasmic granules. For this technique the smears are fixed with May-Grunwald stain (G.T. Gurr Ltd.) and then treated with a Giemsa-peroxidase solution. The peroxidase solution is stable for only three days and must usually be freshly prepared. It also contains benzidine and precautions must be taken to avoid contact with this carcinogen (Chester Beatty Research Institute, London, I 966). Both white cells and normoblasts wer e counted. RESULTS

The mean packed cell volume for all the rabbits was 36.6 ± 2'4, the range being from 3I to 42 per cent. No rabbit was obviously anaemic. The data for the absolute leucocyte counts are shown in Table I and the percentage figures are shown in Table II. There were no significant differences between the male and female rabbits in terms of the percentages of the various cell types, but the blood of the females contained fewer of each cell type. This difference was only significant for the total leucocyte count (F 32,22 = I '46, t = 2'58, P < 0'05) and for the numbers TABLE I A BSOL U TE VAL U ES FOR LE UCO CYTES IN THE PERIPHERAL BLOOD OF RABBITS

Number of eel/simms of blood (Mean ± Standard Deviation. Range in brackets) Cells Male

T ota l leucocytes Lymphocytes H eterophils Monocytes Basophils Eosinophils Plasma-type

8,430 5,230 2,27 2 4 08 399 84 36

± ± ± ± ± ± ±

1,819 (4,800-12,700) 1,3 17 (2,860-8,001 ) 906 (804-4,23 2) 233 (34- 893) 205 (62-1 , 138) 86 (0-375) 30 (0-1 88)

Female

7,245 4,393 2,157 30 4 29 8 78 16

± ± ± ± ± ± ±

1,505 (3,762- 10,600) 985 (1,862-5,966 ) 1,094 (891-5,406) 20 9 (4g-864) 145 ( 12 4-74 2) 87 (0-212 ) 20 (0-53 )

LEUCOCYTE COUNTS IN NORMAL YOUNG RABBITS

49

TABLE II PERCENTAGE DISTRIBUTION OF LEUCOCYTES AND NORMOBLASTS IN THE BLOOD OF RABBITS

Cells Lymphocytes Heterophils Monocytes Basophils Eosinophils Plasma-type Normoblasts

(klean

±

PERIPHERAL

Percentage of cells Standard Deviation. Range in brackets)

61 '3 27.6 4'4 4'4 1'0

0'3

1·6

± ± ± ± ± ± ±

9'9 9.6 2'5 1'9 1'0

0'5 3'2

(38-78'5) (12-5 1) (0'5- 10'5) (0'5- 12'5) (0-3'5) (0-2) (0-8'5)

of lymphocytes (F 32• 22 = 1·89, t = 2·63, P < 0'02). Both these parameters were normally distributed. Lymphocytes were usually the most numerous leucocytes. Most were small, about 6- 7 {tm in diameter, while a few were much larger. These latter forms were often difficult to differentiate from monocytes. The appearance of the lymphocytes after staining did not differ from those in other animal species. The nucleus stained purple and the small area of the cytoplasm surrounding it stained pale blue. Small vacuoles were occasionally visible in the cytoplasm but no granules could be seen. The heterophils, which are the equivalent of the neutrophils in man and most other domestic animals, differed from these by having small red granules in their cytoplasm. They were of medium size, about 9 {tm in diameter, and the nucleus stained purple. It was usually segmented with three to five individual segments linked to one another by thin filaments. The red granules did not usually fill the cytoplasm but were scattered about with a clear space in between each granule. The heterophil count was usually lower than the lymphocyte count. The monocytes were about 12-15 {tm in diameter, with a lobulated nucleus which stained purple. There was a broad zone of greyish-blue cytoplasm which often contained vacuoles of varying size but no granules. The basophils were about the same size as the heterophils and possessed a large lobulated nucleus which stained dark blue. The cytoplasm was also blue in colour and contained a variable number of small blue-black granules. The eosinophils were larger than the heterophils, being about 12 {tm in diameter. They possessed a big well-segmented nucleus. The cytoplasm was usually closely packed with large granules, lying side by side and apparently flattened against one another so that no intervening areas of cytoplasm could be seen. If the blood film was stained by a conventional Romanovsky stain, such as Leishmann, the difference between the eosinophils and the heterophils was not always obvious, since the granules of both stained reddish. It was to prevent such possible confusion between the two cells that the Undritz stain was used as this stains the granules of the eosinophil yellow-brown in colour while those of the heterophil remain red. Eighteen of the rabbits had zero counts.

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Plasma-type cells were rare and frequently absent. Thirty-one of the rabbits had zero counts. These cells varied in size from 7-12 ,urn in diameter. Different forms were observed but a characteristic feature of all of them was a dark blue cytoplasm with vacuoles but no granules. The nucleus was sometimes large and irregular and sometimes small and discreet, usually being situated in one corner of the cell. Normoblasts were easily differentiated from lymphocytes, as the nucleus was much smaller and darker and, although the cytoplasm was grey, it was otherwise similar to that of mature erythrocytes. As with the plasma-type cells, normoblasts were frequently absent. Twenty-nine of the rabbits had zero counts. DISCUSSION

The white cells of rabbits are unusual in several respects. Firstly, the heterophil is morphologically similar to an eosinophil yet appears to be functionally equivalent to the neutrophil of other mammals. Secondly, both basophils and monocytes are relatively common in rabbits and, finally, the occurrence of normoblasts is rare in the peripheral blood in non-anaemic animals of other species, which have not been severely bled. These observations agree with those of all previous authors (Bushnell & Bangs, 1926; Pearce & Casey, 1930; Scarborough, 1931; Gardner, 1947; Archer, 1965; Schalm, 1965; Schermer, 1967), as does the high degree of variation in the levels between similar individual rabbits. The total white cell counts are also similar to those previously described, as are the numbers and proportions of monocytes, eosinophils and basophils-except for Schermer (1967), who recorded basophil levels as high as 30 per cent. Previous authors have considered packed cell volumes of 50 per cent to be within the normal range and the lower values recorded here may be partly accounted for by the use of the microhaematocrit technique. Most other authors have also found that heterophils are the most numerous white cells, whereas in this study lymphocytes were usually more numerous. Both these differences may be related to the age of the rabbits studied, as most other authors used rabbits over six months old and Schermer (1967) states that lymphocyte levels decline in rabbits over five months of age. However, later observations in these animals up to 12 weeks of age showed little change in the haematological values, except for transient increases in both lymphocytes and normoblasts following the removal of 10 ml or more of blood (Purvis, 197 I). The significant sex difference in the numbers of lymphocytes has not previously been reported in rabbits. ACKNOWLEDGEMENTS

We wish to thank Professor Sir Alexander Robertson for research facilities, Dr D. L. Doxey for advice and Mr R. Munro for photographic services. One of us (G. M. Purvis) is grateful to the Animal Health Trust (Research Training Scholarship) and the Royal College of Veterinary Surgeons (Sir Frederick Smith Fellowship) for their financial support during these studies.

LEUCOCYTE COUNTS IN NORMAL YOUNG RABBITS

REFERENCES ARCHER, R. K. (1965). Haematological Techniquesfor Use on Animals. Oxford: Blackwell. BUSHNELL, L. D. & BANGS, E. F. (1926).]. infect. Dis. 39. 291. DACIE, J. V. & LEWIS, S. M. (1963). Practical Haematology, 3rd edn. London: Churchill. GARDNER, M. V. (1947). ]. Franklin Inst. 243. 251. PEARCE, L. & CASEY, A. E. (1930).]. expo Med. 51, 83. PURVIS, G. M. (1971). Ph.D. thesis, University of Edinburgh. SCARBOROUGH, R . A. (1931). Yale]. bioi. Med. 3.64. SCHALM, O. W. (1965). Veterinary Haematology, 2nd edn. London: Bailliere, Tindall & Cassell. SCHERMER, S. (1967). The Blood Morphology of Laboratory Animals, 3rd edn. Philadelphia: F. A. Davis. UNDRITZ, E. (19Y2). Sando;;; Atlas of Haematology, Basle, Switzerland, p. 28. (Accepted for publication 24 August 1972) Nunu'irations leucocytaires chez de jeunes lapins nonnaux (Purvis et Sewell) ResuDle. Les resultats sont presentes d'apres une etude des leucoctytes au niveau du sang peripherique de 33 m.Ues et 23 femelles lapins recemment sevres. Les resultats furent identiques a ceux decrits pour des lapins plus ages par de precedents auteurs excepte Ie fait que Ie nombre de lymphocytes etait relativement eleve, alors que Ie nombre de polynucIeaires etait relativement bas. II y await de maniere significative moins de lymphocytes dans de sang des femelles. Les volumes cellulaires etaient egalement moins eleves que ceux decrits pour les lapins plus ages. Leukozytenzahlung bei nonnalen jungen Kaninchen (Purvis und Sewell) ZusaDlDlenfassung. Resultate einer Leukozytenuntersuchung des peripheren Blutes 33 mannlicher und 23 weiblicher Kaninchen, die vor kurzem abgestillt worden waren. Die Resultate ahnelten denen, die bereits von anderen Autoren flir altere Kaninchen angegeben sind, abgesehen davon, dass die Anzahl der Lymphozyten relativ gross war, wahrend die Zahl der Heterophilen relativ klein war. 1m Blue weiblicher Kaninchen waren signifikant weniger Lymphozyten. Die Erythrozytenvolumina waren ebenfalls geringer als die bei alteren Kaninchen beobachteten . Recuento de leucocitos en conejos jovenes nonnales (Purvis y Sewell) ResuDlen. Se presentan los resultados obtenidos del recuento de leucocitos de sangre periferica en 33 machos y 23 hem bras de conejos acabados de destetar. Los resultados son semejantes a los descritos en conejos de mayor edad por otros autores excepto que el numero de linfocitos se mostro relativamente alto y el de heter6filos relativamente bajo. Tambien se observo un numero significativo mas bajo de linfocitos en la sangre de las hembras. El hematocrito tambien se mostro mas bajo que el descrito en animales de mayo edad.