S O M E B I O C H E M I C A L A N D E L E C T R O - C A R D I O G R A P H I C A L D A T A

O N B A D G E R S B Y

B E N G T J O H A N S S O N From the Cardiological Laboratory, Department of Medicine, University of Lund, Allmanna

Sjukhuset, Malmo, Sweden. (Head : Professor Jan Waldenstrom).

In the last few years there has been a steadily increasing interest in hypo- thermia because of its usefulness in certain kinds of surgery. A great number of investigations concerning the physiology of man and homoiothermic animals in the hypothermic state have recently appeared. Such investigations have also been extended to include the hibernators, i.e. animals which have the ability of decreasing their body temperature in the winter to only a few degrees above zero. Thus in our laboratory the hedgehog has been subject to study (1-5). Another animal of interest in this connection is the badger. The winter sleep (6) of this animal resembles that of the hibernators in immobility and loss of body weight, while the body temperature-on the other hand-is at a constant homoiothermic level throughout the winter (6). - I t seems, however, as if very few studies have been made on the physiology and biochemistry of badgers. We had the opportunity to get some captured animals on which we carried out some biochemical and electrocardiographical studies, which are presented below.

METHODS AND MATERIAL

At the beginning we had access to four badgers (Meles meles L) which were used for studies from June 1955 to May 1956.

Blood samples were taken in June 1955 (no. I) , in August 1955 (nos. 2, 3 and 4) in October 1955 (nos. 2, 3 and 4), in February-March 1956 (nos 2, 3 and 4) and in May 1956 (nos. 3 and 4) by venous puncture after the animals had been anesthesized with ether and 2 cc nembutal (Abbott) intramuscularly. One animal (no. I ) died in June 1955 because of an overdosage of ether in connection with the taking of a blood sample. Another (no. 2) was found dead one morning in March 1956. The cause of death is unknown. These deaths are the reason why only results from two animals are given in the tables from the latter part of the investigation.

Acta Zoologica I9.57. Bd. X X X V I I I

206 BENGT JOHANSSON

The methods used in determining hemoglobin, red and white blood corpuscles, differential count, thrombocytes, total eosinophils, reticulocytes, hematocrit, plasma proteins, protein-bound iodine, serum sodium, serum potassium and blood sugar have been reported earlier (3). Determination of serum calcium was made acc. to a method by NIELSEN (7) (somewhat modified) and of serum cholesterol acc. to a method by ABELL et a1 (8).

The body temperature was determined in centigrades by a thermometer introduced 7 to 8 cm into the rectum.

The animals received kitchen garbage and water all the year. One animal (no, 4) was kept in a room where the air temperature during winter was about 16O C. The others were kept outdoors (nos. I, 2 and 3).

The two animals which remained in June 1956 were cooled in a specially built refrigeration box (9) after anesthesia with ether and nembutal. During the whole refrigeration period the animals were hyperventilated with a respirator (Lundia) at a rate of 24 respirations/min.

Electrocardiograms were obtained with an electrocardiograph (Elema Triplex). During refrigeration tracings were recorded for every two degrees decrease of the body temperature.

RESULTS AND DISCUSSION

Data on body weight and temperature are given in table I. The animals showed an average increase in body weight of 3.1 kg from August to October but afterwards decreased again in weight. No. 2. showed only a slight decrease. This animal was found dead shortly afterwards. The other animal which was out of doors (no. 3) lost much more in weight (4 kg) than the animal (no. 4) which was kept in a warm room all the winter (1.4 kg) although both animals received as much food as they wanted. In May only the weight of one animal (no. 3) was determined. This value was a little lower than the one found in February.

Although some early investigators (10, 11, 12) considered the badger to be a hibernator, it is now generally accepted (13, 14, 15) that this is not the case. A hibernator is a mammal which during the winter can decrease its body temperature to that of the surroundings if this temperature is within the range of oo to 15O centigrades. In this state, which is interrupted with rather long intervals, the animals are almost motionless, the heart rate is very slow but fairly regular and the metabolic rate reflected in the oxygen consumption is very much decreased. - As the body temperature of the badgers in the winter never decreased to that of the surroundings (table I) and the animals were found walking about quite often, it is obvious that the captured badger is not hibernating.

2

207 SOME BIOCHEMICAL AND ELECTROCARDIOGRAPHICAL

HEMATOLOGICAL RESULTS

Animals nos. 3 and 4 showed a slight decrease of the hemoglobin value in October while the red corpuscle value decreased in no. 3 and was mainly unchanged in no. 4. When the winter was over the hemoglobin values increased ; in no. 4 this increase appeared already at the end of February. At his time there was also a great increase in the number of red corpuscles. Animal no. 2 differed from the others (table I). The low values in March can with great probability be looked upon as a symptom of the disease from which it died shortly afterwards.

The data on the number of white blood corpuscles (table I) did not show any consistent changes. Thus, unlike in the hedgehog, no decrease was seen in the winter. The differential count (table I) was mainly unchanged although there was a tendency of the neutrophils to be slightly increased in October and May at the expense of the lymphocytes.

The number of total eosinophils and thrombocytes (table I ) showed great variations but no consistent change could be found. The values were lower than those seen in dogs (16, 17) and non-hibernating hedgehogs (3). - No consistent changes were found in the reticulocytes either (table I). The high value seen in animal no. 2 was probably secondary to the anemia.

Hedgehogs in hibernation showed an increase of the hematocrit in January. In March this increase was followed by a decrease. In one badger (no. 4) there was an increase in February but in the other (no. 3) no change was found (table 11).

The serum sodium concentration remained mainly unchanged during the different seasons in accordance with the results found in hedgehogs (3). The serum calcium concentration remained unchanged in no. 4 and showed an increase in August in nos. z and 3. The serum potassium concentration (table II), in October, showed a decrease, which persisted in February-March but in May it was restored to the level found in August. This resembles Suoma- lainen’s findings in hedgehogs which we, however, have not been able to verify.

Typical of most hibernators is a decrease in blood sugar during hiber- nation (3). In the badgers the values were rather a little higher in February- March than in other seasons (table 11).

The protein-bound iodine values (table 11) were of the same order as those found in other animals. They did not show any consistent changes in different seasons. This is in contrast with hedgehogs which show a decrease during hibernation (3).

The electrophoreses (table 11) of the badgers’ plasma proteins in most cases differed from those of certain other animals like the hedgehog and the dog, having only five distinct fractions with the method used. Of course it is open for

3

T A B L E

I8/&55 25/8-55 29/8-55

24/1-55 26/1-55 20/1-55

5/3-56 1/3-56

23/2-56

22/6-55 28/5-56 3 1 /5-56

28/5-56 31/5-56

4 m .g g c

I8/GSS 25/8-55 29/8-55

24/1-55 26/1-55 20/1*55

5/3-56 1/3-56

23/2-56

22/6-55 28/5-56 31/5-56

28/5-56 31/5-56

2

3 4

2

3 4

2

3 4

I

4 3

4 3

2

3 4

2

3 4

2

3 4

I

4 3

4 3

12.0

13.5 13.5

14.5 16.5 17-4

14.0 12.5 16.0

12.0

38.0 37.4

35.5 36.5 36.3

37.0 37.0 36.0

36.0 37.0

18.0 14.0

3.2 4-2 4.0

2.5 4.0 3.2

3.7 3.5

3.8 4 4 4.3

5.4 4.3

93 98 96

93 84 86

50

104

71

97

104 94

84

I02

4.8 5.3 5.0

4.0

4.9

4.7 4.0 4.8

4.6

5.1

4.3

5.0 3.9

4 8

44.4

35.0

43.4 37.0

40.6

39.8

28.0 36.8 49.2

28.0 45.5 44.1

50.7 41.8

7.3 9.1 7.5

7.7 7.5 7.7

4.0 11.3 11.9

6.0 8.6 8.4

7.5 9.0

3.1

4 3

3.6

5.2 2.7 3.4

3.1 3.3 3.8

2.9 4.0

7300 4.000 4.900

3.900 5.900 9.500

5.700 6.600 4.500

7.400 4.500 4.000

1.300 700

T A B L E

0.106 0.125

0.135 0.072 0.262

0. I 84 0.152 0- 1 74

0. I00

0.0g0

0.080 0.110

I

counted cells) Dii

m 3

2 8 a a zg

18

- 54 59 53

70 62 60

57 60 56

67 76 60

57

42

44

30

39

40 37 43

31

39

38

36

21

38 30

3 3 2

- 2 I

3 2 I

2 2 I

5 2

I60 16

1 70

16 I02

I I 2

26 I9 12

16 200 I2

2

4

272.000 268.000 750.000

270.000

478.- 487.000

548.000 421.ooo 3 I 8.000

522.000 182.000 402.000

1.8oo.000 488.000

0 I

I5

6 5 44

246 16 7

53 2

0

0

2 68

I1

n Y

h u u n 0 0 n V V

E: .A v

E .d v

E .rl v

G .d v

6.5 6.0 7.1

6.7 6.7 6.4

7.7 6.5 7.6

7.6 6.7

7.6 6.3

3.67 3.70 4.27

3.96 3.63 3.78

4.63 4.30 4.5 1

4.30 3.80

4.34 3.58

0.84 0.42 0.50

0.65 0.69 0.50

0.56 0.27 0.5 I

0.55 0.32

0.53 0.58

0.33 0.38 0.35

0.32 0.45 0.40

0.46

0.15 0.20

0.28 0.60

0.37 0.23

0.85 0.78 1.11

0.84 0.92 0.95

I .oj 0.87 1.17

1.22 0.93

1 .og I .oo

0.813 0.72 0.87

0-93 1.01

0.77

I .oo 0.86 I .26

I .25 1.05

1.27 0.96

14 A . 2. I951

210 BENGT JOHANSSON

discussion what to call these fractions but a comparison indicates that it is an a-globulin fraction that is lacking. We have therefore decided to call the dif- ferent fractions as follows: the albumin-, a-, /I1-, pa- and y-fraction. - The total protein and the albumin values were slightly higher in February-March, thus in accordance with values found in hedgehogs (3). This increase persisted in May as regards the total protein values. The /?- and y-fractions did not show any consistent changes although the individual alterations were rather large. In this respect, then, the badger differs from true hibernators like the hedgehog which show a decrease in the /I- and y-fractions during hibernation.

The observation has been made that the concentration of plasma cholesterol is higher in man than in other mammalian species (18) ; this has been offered as an evidence for the etiologic role of the blood lipids in the pathogenesis of atherosclerosis. Recently, however, BRAGDON ( 19) has made serum cholesterol determinations in a hibernator, Citellus columbianus, and has found compara- tively high values. WILBER and MUSACCHIA who analyzed Alaskan ground squirrels (Citellus barrowensis) found a total cholesterol value of about the same order as that found in human beings (20). They were not able to find any seasonal variations, SUOMALAINEN (6) reported a high total cholesterol content (455 mg per 100 cc) in the serum of hibernating hedgehogs, while the value was much less in early summer (250 mg per 100 cc). - In table I1 it can be seen that at least in October the values found in badgers were high, averaging 385 mg per IOO cc. In May the average value found was 153 rng per 100 cc.

ELECTROCARDIOGRAPHIC RESULTS

The heart rate was rather varying in different animals in different seasons. These changes were probably caused by a different anesthetic depth, although we tried to keep it as constant as possible. No consistent differences between the seasons were observed and the mean of all the determinations was 179 beats/min. It is thus of about the same size as that found in hedgehogs-181 beats/min. (5)-with a skin temperature of 3 2 . ~ ~ C, and a little higher than the value found in dogs-146 beats/min.-with a body temperature of about 38O C and a body weight of 2-35 kg (I). The slight difference in heart rate between the hedgehogs and the badgers was astonishing as the body weights differ much (0.5-1.0 kg and 12-17 kg respectively). The slightly lower body temperature in the hedgehogs is probably not the only reason, but also a superficial anesthetic level in the badgers during the ECG recording may have played a rde. The P-R, QRS, and Q-T intervals averaged 0.084, 0.055, and 0,201 secs., respectively. Fig. I a shows a typical ECG.

EFFECTS OF INDUCED HYPOTHERMIA

Two animals (nos. 3 and 4) were cooled by surface cooling in May 1956 in order to find out the animals’ reaction to cold. A true hibernator is able to

6

PI1 SOME BIOCHEMICAL AND ELECTROCARDIOGRAPHICAL

Fig. I b Fig. I a

Fig. I c

7

212

BENGT JOHANSSON

Fig. I d

Fig. I e

8

213 SOME BIOCHEMICAL AND ELECTROCARDIOGKAPHICAL

Fig. ~f

Fig. I . ECGs from a badger at different temperatures. before refrigeration. Body temperature 37" C. 26.5" C. Note the Osborn wave (J). 19.5" C. Large diphasic Osborn wave. T wave positive in leads 2 and 3. 19.0' C. Large Osborn wave. No T wave is seen. 15.5' C. Ventricular fibrillation. 14.5' C. The ventricular fibrillation has disappeared spontaneously.

The figure shows the three standard leads. The distance between two heavy lines is 0.1 sec.

tolerate a decrease of the body temperature to just above zero with the heart still beating spontaneously, while the heart of a non-hibernating mammal stops beating at about 13O C (21). One badger (no. 4) was cooled to 19O C; at this temperature the heart rate was 43 beats/min. (at 22O C it was 21 bcats/min.). After this the animal was rewarmed but died soon after the beginning of this procedure. The other animal when being cooled to 19.5~ C showed a heart rate of 13 beats/min. and the rhythm was a nodal one. At 17O C the heart rate had increased to 32 beats/rnin., total block was persisting and auricular and ventri- cular premature beats appeared rather frequently. A few minutes later ventri- cular fibrillation was registered, which, however, had disappeared spontaneously at 14.5~ C when the heart rate was 12 beats/min. (fig. I). At 14O C no de- flections were registered electrocardiographically. When after opening of the thorax the immobile heart was touched with the finger or pinched with a forceps a slow contraction appeared. In Fig. 2 the heart rate, P-R, QRS, and Q-T durations have been compared with corresponding values in dogs and hedgehogs. - An interesting finding was the very large Osborn wave ( I ) ; at the lower temperatures this was dominating. The T wave persisted to 1 9 . 5 ~ C

9

BENGT JOHANSSON

A d o 9 --- hibernating hedgehog --- ----- b a d g e r

r e f r ; g e r a f e d hedge hog

1 I I I I

3 10 15 20 25 30 35 L)O temp. Fig. z i z

do9

badger

--- hibernating hedgehog - - - r e f r i g e r a t e d hedgehog -----

\ \ \ \

1 I

5 10 15 20 25 30 35 4 O t e m p . Fig. z B

and showed an increasing positivity. Below 19.5' C the T wave disappeared and only the large Osborn wave was seen (Fig. I ) .

In tables I and I1 the hematologic findings at the lowest body temperature are given. Some consistent changes were observed : the number of leucocytes decreased, a fact observed both in hibernating hedgehogs (3) and cooled dogs (16). Furthermore the eosinophils decreased and the thrombocytes increased; the same changes have also been observed in hedgehogs during hibernation (3).

215 SOME BIOCH EMICAL AND ELECTROCARDIOGRAPHICAL

Heart rate

C

- dog --- hi bernoting hedgehoq --- re f r i ge ra ted bed9e h i9 badger -----

5 10 15 20 25 30 35 W,+ernp. Fig. 2 C

COMPARISON WITH DATA FROM HEDGEHOGS A N D DOGS

In this connection it may be of interest to compare the hematologic and electrocardio- graphic changes found in a hibernator when cooled with those found in the badger. Five hedgehogs (Erinaceus europaeus) were given 15-25 mg chlorpromazine (hibernal, Leo) intraperitoneally and were put into a refrigemtion box (9) where cold air (0' C ) was circulating. Electrocardiograms were registered with needle electrodes (see (I) for details) at different temperatures and some hematologic determinations were made before and after the refrigeration. The cooling was stopped at a skin abdominal temperature of +7" C. - More pronounced and consistent changes were found in the hemoglobin and red corpuscle values in hedgehogs than in badgers. Thus in the former there was an average decrease from 97 per cent to 81 per cent hemoglbbin and from 10.1 millions/mm3

I1

216 BENGT JOHANSSON

D. do9 - --- hibernating hedgehog --- retr igeroted hedgehog ------ badger

,

\ \

5 10 15 20 25 30 35 40 temp. Fig. z D

Fig. 2. Hear? rate (fig. C), P-R (fig. B), QRS (fig. A), and Q-T interval (fig. D) from dog (- ), badger ( - - * * *), hedgehog arising from hibernation (----) and hedgehog rendered hypothermic (- . -. - . - -). Abscissa : temperature in centigrades. Ordinata: heart rate in beats/niin., P-R, QRS, and Q-T in secs.

to 8.4 millions/mm3, respectively. As in the badgers there was a great decrease in the number of white corpuscles and total eosinophils from 10360 to 4140/mm3 and from 192 to 51/mm3, respectively. - The heart rate, P-R, QRS, and Q-T durations are given in Fig. 2. It is seen that the values from the badger and the dog do not differ much. The values from the refrigerated hedgehogs and those from the hedgehogs arisen from hibernation are also similar, but different from the dog and badger values. This may at least in part be explained by the difference in body weight, the weight for the badgers (12-17 kg)

I2

217 SOME BIOCHEMICAL AND ELECTROCARDIOGRAPHICAL

and the dogs (20-35 kg) being rather similar and greater than that of hedgehogs (0.5-1 kg). - As for the hedgehogs differences are seen in the heart rates a t 27.5“ C (a higher value in the refrigerated animals) and in the Q-T intervals. Below 15O-20~ C the Q-T is shorter in the refrigerated animals than in the hibernating ones. - When comparing ECGs from arctic ground squirrels, NARDONE (22) found a “left bundle branch block” in the hibernating specimens and a “right bundle branch block” in hypothermic specimens. No such changes could be discovered in the hedgehogs.

CONCLUSIONS

In conclusion it can be stated that some of the recorded data indicate con- ditions in our badgers similar to those of a true hibernator. Thus during winter the animals are rather motionless, the body weight shows a fairly large decrease and the albumin fraction of the electrophoresis shows an increase. There are differences too, however, and these are more conspicuous. Firstly, the body temperature does not decrease and it is not probable that this is caused by the captured state since captured hedgehogs hibernate. Secondly, most of the changes in the hematologic values do not agree with those found in hedgehogs. The lack of decrease in white blood corpuscles, and blood sugar is particularly worth mentioning. Thirdly, when cooled, the badger’s heart stops beating at about 14O C which is typical of non-hibernating mammals.

SUMMARY

Biochemical and electrocardiographical data have been collected in four badgers (Meles meles) in different seasons and have been compared with values from other animals.

The body weight decreased during the winter. The hemoglobin and red corpuscle values increased in winter or spring. The serum potassium values were slightly lower in October and persisted so to February-March. The total protein and albumin fraction was higher in February-March as was the blood sugar in two of the three recorded animals. Very high cholesterol values were found in October. No consistent changes were found in the other hematologic data (tables I and 11).

Electrocardiograms were alike in different seasons ; the heart rate averaged 179 beats/min., P-R 0.084 secs., QRS 0.055 secs., and Q-T 0.201 secs.

Refrigeration revealed interesting electrocardiographic changes : a large Osborn wave; a T wave which increased in positivity to 19.5’ and after- wards became isoelectric.

It is concluded that in some aspects badgers are similar to hibernators but that most findings agree with those found in nonchibernators.

218 BENGT JOHANSSON

The author is greatly indebted to Drs G. Biorck and Ivar Borg for providing the animals. Thanks are due also to Mr. R. Prahl for technical assistance, to the head and personal of the Chemical and Medical Laboratory, to Dr. B. Skanse and Mr. I. Hedenskog for their help with the chemical analyses, and to Dr. W. von Studnitz for help with the electrophoreses.

REFERENCES

I. BIORCK, G. and JOHANSSON, B. Acta Physiolog. Scand. 34: 257, 1955. 2. BIORCK, G., JOHANSSON, B. and SCHMID, H. Acta Physiolog. Scand. 37: 71, 1956. 3. BIORCK, G., JOHANSSON, B. and VEIGE, S. Acta Physiolog. Scand. 37: 281, 1956. 4. BIORCK, G. and JOHANSSON, B. Cardiologia. 30: 344, 1957. 5. JOHANSSON, B. Cardiologia. 30 : 37, 1957. 6. SUOMALAINEN, P. Proc. Finn. Acad. Science and Letters 1953. Helsinki 1954. 7. NIELSEN, H. Nord. Med. 48: 1059, 1952. 8. ABELL, L. L., LEVY, B., BRODIE, B. and KENDELL, F. E. J. Biol. Chem. 195: 357, 1952. 9. BIORCK, G., DAMCAARD-NIELSEN, M., HBCER, K., RYD, H. and WULFF, H. R. Scand. J.

Clin. Lab. Invest. 4 : 6, 1954 10. BUDDENBROCK, V. Grundriss der vergleichenden Physiologie 11. Berlin 1928. I I. MERZBACHER, L. Ergebn. d. Physiol. 3 : 214, 1904. 12. BARKOW, H. G. L. Der Winterschlaf nach seinen Erscheinungen in1 Tierreich. Berlin

13. BECHMANN, L. Der Zoologische Garten. 7: 317, 1866. 14. ADOLPHI, P. R. Der Zoologische Garten. 3: 170, 1862. 15. EISENTRAUT, M. Z. Saugetierk. 6: 152, 1931. 16. HELMSWORTH, J. A,, STILES, W. J. and ELSTUN, W. Proc. SOC. Exp. Biol. Med. 90 : 474,

17. ALBRFTTON, E. L. Standard values in blood. W. B. Saunders Co. Philadelphia 1952. 18. GOULD, R. Am. J. Med. 11: zog, 1951. rg. BRAGDON, J. Circulation Research. z : 520, 1954. 20. WILBER, C. G, and MUSACCHIA, X. J. J. Mammalogy. 31: 304, 1950. 21. BROOKS, CH. McC., HOFFMAN, B. F., SUCKLING, E. and ORIAS, 0. Excitability of the

22. NARDONE, R. M. Am. J. Physiol. 182: 364, 1955.

1846.

1955.

heart. Grune and Stratton. New York 1955.