Johannesburg.) Johannesburg (6000 feet) it is customary to regard the normal red cell

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1 THE BLOOD COUNT AND BODY TEMPERATURE IN NORMAL RATS. By ARTHUR DIGHTON STAMMERS. (From the Department of Physiology, University of the Witwatersrand, Johannesburg.) IN connection with the maintenance of a colony of rats for experimental purposes, I have made observations to determine how far the height at which they live above sea-level, viz feet, influences the physiological processes. I give here observations on blood count and on body temperature. I. THE BLOOD COUNT. Red corpuscles. The figures which are available for comparison are few in number and, as far as I have been able to ascertain, relate to counts made at approximately sea-level. It is, of course, well known that, in man, the number of red cells normally present in the blood bears a definite relation to the altitude at which the individual is living. In Johannesburg (6000 feet) it is customary to regard the normal red cell count in man as 6 millions as opposed to 5 millions at sea-level, i.e. with a diminished barometric pressure of 140 mm. Hg. there is an increase of 20 p.c. in the red corpuscles. The references may be briefly cited as follows: Donaldson(1) quotes work by Rivas, in which the average red cell count in ten rats was 8-2 millions per c.mm. and by Margot, who found that adults aged 148 days had a count of 9.4 millions. Scott(2) states that, in his colony, the normal is 8-8 millions, while Bedson and Zilva(3) found 7-8, and Cramer, Drew and Mottram(4) between 9 and 10 millions. The average of all these works out at about 8*7 millions for adult animals. In these latter reports the number of cases examined is not stated. I have made counts in 60 rats over varying periods from ; the results are as follows: Cases observed Average red cell count 60 9x2 millions The lowest count observed was 7.4 and the highest 10x6 millions; the distribution is shown in the subjoined table.

2 330 A. D. STAMMERS. Red cells No. of Red cells No. of (millions) cases (millions) cases 7* * *7 4 9* o * * If it be assumed that an increase similar to that observed in man results from life at an altitude of 6000 feet, the average which might be expected would be in the region of 10-4 millions. There is, however, no a priori reason for this, since, as will be seen later, considerable differences exist between human and rat blood and normal conditions in the latter appear to permit far wider fluctuations in the cell content than in the case of human blood. Leucocytes. As regards the leucocyte count, the average numbers observed by different authors are summarised in the following table. These all refer to rats in the Wistar Institute colony, Philadelphia, during the period , in which Donaldson reports that the nutritional state was good. No. of cases Av. no. of Poly- Small Authors and sex leucocytes morphs lymphocytes Others Rivas p.c p.c p.c. Newrey ,, 12? Margot 3 & In observations on the 60 cases mentioned above, I obtained the following figures. The animals were all young healthy adults, weighing between 160 and 250 grm. and aged between 70 and 200 days. No. of cases Total Poly- Small and sex leucocytes morphs lymphocytes Others 30& p.c p.c. 6-5 p.c. 30? The differences between these and the figures obtained by the authors mentioned above are somewhat striking, particularly as regards the proportion of polymorphs to small lymphocytes. The explanation for this is somewhat obscure, since the nutritional state of the animals was good and they were apparently in perfect health. It has, however, been pointed out by Donaldson(s) that, in the experience of other observers (Kleineberger and Karl, Eyre, Cramer, Drew and

3 BLOOD COUNT AND TEMPERATURE IN RATS. 331 Mottram), the percentage values for small lymphocytes may be about twice those for the polymorphonuclear cells and it is concluded that the blood picture may vary within wide limits and yet the animals remain in good health. Blood platelets. The only comparative figures which are available on the blood platelets are those compiled by Cramer, Drew and Mottram(4), who state that the normal rats in their colony have an average count of about 0O8 million, and by Bedson and Zilva(3), whose normal rats gave a figure of about 1 million. Since the platelets disintegrate very readily in shed blood, the number found in a blood count depends upon the completeness of preservation and, in order to guard as far as possible against incomplete preservation, I have compared the results obtained with several preservative fluids. I have found no difficulty in distinguishing the platelets from bacteria, lipoid granules and other small particles and, since the platelets are about 3 /u in diameter, neither great magnification nor high resolving power (as has been pointed out by Bedson and Zilva) is required to see them. Cattoretti(6) states that digestion causes a decrease in the number of platelets in circulating blood lasting 1-2 hours; in order to avoid this possible source of variation, my counts in rats were invariably made about 3 hours after a meal. The technique adopted was a slight modification of that of Cramer, Drew and Mo t tram and was as follows: the rat was anaesthetised with ether and the tail immersed in warm water for a few moments and dried. It was then cut about 2 cm. from the end, whilst immersed in Toison's fluid. When the blood was flowing freely, the tail was transferred to another dish of Toison's fluid and the blood allowed to flow until a dilution suitable for counting was obtained. The tail was then removed, dried and blood taken in the ordinary way for a red cell count and for a differential film. The rat was then bled into 2 p.c. sodium citrate in normal saline and into 002 p.c. methyl violet saline in the same way as described for Toison's fluid. The blood was mixed in each dish with a platinum loop, a drop placed on a slide and a cover slip applied which was ringed with paraffin and set aside while the red cell count was made. The differential film was stained with Leishman's fluid. The proportion of red cells to platelets was estimated by means of an ocular micrometer ruled in squares and the red cell count was carried

4 .332 A. D. STAMMERS. out with the Standard American Haemocytometer with the Levy counting chamber. This was tested and found to give results concordant with those from the Thoma-Zeiss instrument used by Cramer, Drew and Mottram. Twenty-seven rats in all were examined in the way described and the results are summarised in the subjoined table. Not less than 50 platelets were counted in each case. Red cell Diff. Toison's Citrate Meth. violet count film fluid saline saline Maximum variation Minimum variation Average of total Red cell-platelet ratio The average variations are insignificant and well within the limits of experimental error. The differential films show a definitely lower count and this is, in my opinion, explained by the greater difficulty in counting the platelets by this method and to the fact that more uncertainty exists as to the identity of the true platelets. As a control, the number of platelets in human blood was also investigated. The finger was pricked and a platinum loopful of the blood was placed in each of the fluids already mentioned. The first six estimations gave the results tabulated below: Red cell Toison's Citrate Meth. violet count fluid saline saline Average of The differences observed suggested that delay was a factor in the production of the relatively high count in the case of the citrate and methyl violet saline fixatives. It was accordingly decided to make further estimations with each diluting solution and for this purpose to take blood from different fingers, thus ensuring that approximately the same time should elapse between pricking the finger and immersing the loopful of blood in the diluent. Ten further estimations gave the following figures: Red cell Toison's Citrate Meth. violet count fluid saline saline Average of It will be seen that a considerably closer approximation was obtained when the estimations were made in this manner. Since, in human blood, delay was found to increase, instead of, as might be expected, diminishing the number of platelets, the effect of

5 BLOOD COUNT AND TEMPERATURE IN RATS. 333 delay in fixing the blood was tried on the blood of rats. A loopful of blood was obtained from the tail of the rat at given intervals and placed ih 1 c.c. of citrate saline, the subsequent technique being as previously described. Twelve different rats were investigated in this way with the following results: At once After 30 secs. After 60 secs. Average of The figures obtained in this way seem to indicate definitely that a pronounced increase takes place in the platelet count as a result of delay in fixing the blood. Particles similar in appearance to platelets have been described as being produced in great numbers in plasma on cooling it and some observers indeed have considered that, in mammals, platelets are not normally present in the circulation. Whilst the results I have just given do not show that platelets are absent normally, they indicate that particles indistinguishable from platelets are readily produced in blood. II. BODY TEMPERATURE. In a recent communication, Price-Jones(7) states that an investigation into the body temperature of 200 normal rats showed a mean of F., the limits being 96-6 to A table is given in this author's paper, which shows the distribution of temperatures in intervals of 0.20 and a graph is also inserted, in which the numbers of animals are plotted against the temperature in 10 intervals. From these data, the standard deviation and the coefficient of variation are calculated, the figures given being 1 13 = standard deviation, and 1 10 = coefficient of variation. In connection with other investigations, it became necessary to determine the body temperature of my rats and the figures obtained are now presented. The average of 83 cases was , the range being from 96*8 to The distribution was as follows: Degrees F. No. of animals Degrees F. No. of animals * The standard deviation and the coefficient of variation, which were kindly worked out for me by one of my colleagues, Dr I. Liknaitzk,

6 334 A. D. STAMMERS. both give the figureo88. Forpurposesof comparisonwith Price-Jones results, a graph is appended, expressing the same relative information. Fig. 1. ok The ordinates indicate the number of animals, the abscisse the temperature groups (97 =96I-97j,etc.). The dotted line refers to Price-Jones'animals and the continuous line to the writer's. Both have been reduced to percentages of the totals. It may be mentioned that, as far as can be ascertained, the points in his graph do not agree with the figures in his table. These points occur as follows: Degrees F No. of animals

7 BLOOD COUNT AND TEMPERATURE IN RATS. 335 It is presumably intended that each full degree on the abscissa of the graph shall include temperatures half a degree below and above it, and in this case the figures would appear to be as follows: Degrees F. No. of animals The liberty has been taken of re-drawing Price-Jones' curve and including it in the graph illustrating the writer's results and, in order to make comparison possible, in view of the difference in numbers, both have been reduced to percentages of the totals. According to Pembrey(8), the average temperature of the adult albino rat is 99.50, while Macleod(s) finds a mean of It will thus be seen that the figures obtained by the writer as well as by Price- Jones exceed those previously reported. This increase, however, is not regarded as of any significance, since, as the latter author points out, the temperature of a rat may fluctuate within 2 or 30 F. under apparently the same conditions. The following figures bear out this statement. They were obtained by taking the temperatures on three successive days at as nearly as possible the same time. lst day 2nd day 3rd day Max. temperature Max. variation Max. variation ( ) (100*4-97.4) Min. temperature Min. variation Min. variation No variation. Two cases No variation. Four cases One animal showed a variation of only 0.20 throughout the three days. In each case the average for the day works out at The maximum range for any one animal in 48 hours is 40 and the minimum 020, the average being 1-1. The room temperature was on the first day and on the others. This investigation was made during June, which is usually the coldest month of the year in Johannesburg, the mean minimum temperature being recorded as 410 F. Other observers, according to Donaldson, have found that an increase in body temperature occurs with an increase in room temperature up to 860, the rate being for the body with 90 increase in room

8 336 A. D. STAMMERS. temperature. The temperature regulating mechanism in the rat will thus be seen to lack efficiency. SUMMARY. The normal red cell count in rats appears, from what few investigations have been made, to be in the region of about 8-7 millions per c.mm. at sea-level. The author's observations at an altitude of 6000 feet give an average count of 9-2 millions, which represents an increase of under 6 p.c. as compared with the 20 p.c. increase seen in normal human blood at this altitude. In the same animals the average leucocyte count was 9800, while the average reported by other workers is The proportion of polymorphonuclear leucocytes to small lymphocytes averaged The blood platelet count gave an average of just under 900,000. Comparisons were made with different diluents, since there appear to be complicating factors which influence the correct estimation of the platelets. No significant differences were observed with the three diluting solutions employed and a lower count obtained with a differential film is attributed to a greater difficulty in counting by this method. Delay in fixing the blood appears to be an important factor in determining the number of platelets. The range of body temperature of the rats in the writer's colony was '. The standard deviation and the coefficient of variation were both A graph illustrating the distribution of temperature as compared with that found by Price-Jones is appended. It seems that, under normal circumstances and apparently the same conditions, the temperature of the rat may vary within 2 or 30 F., and this animal may be regarded as being somewhat poikilothermous. The author desires to acknowledge assistance from the Research Grant Board of the Union of South Africa towards the expenses incurred in carrying out this work. REFERENCES. 1. Donaldson. The Rat, p. 72. Wistar Institute, Philadelphia Scott. Biochem. Jou.ii. 17. p Bedson and Zilva. Brit. Journ. Exp. Path. 4. p Cramer, Drew and Mottram. Ibid. 4. p Donaldson. The Rat, p Cattoretti. Atti. Soc. lomb. sc. med. e biol. 12. p Price-Jones. Journ. Path. Bact. 28. p Pembrey. This Journ. 18. p Macleod. Amer. Journ. Physiol. 18. p Donaldson. The Rat, p. 152.