A Study of the Influence of Ambient Temperature on the Respiratory Metabolism and Certain Blood Constituents in Non-Hibernating Mammals, Hibernating Mammals, and the Bat

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P U R D U E U N IV E R S IT Y

THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION

by________________D

ENTITLED

o n a ld

E d w a rd S t u l l k e n

A. S lu dy^ o f "fclis I n f l u s n c s

of

ijn b ie n t

TsDijDSPat/Uros

on t h e R e s p i r a t o r y R e t a b o l i sir a n d C e r t a i n B l o o d C o n s t i t u ­ e n t s i n N o n - H i b e r n a t i n g IVammals , H i b e r n a t i n g H a m m a ls , a n d t he R a t . ________________________________________________________

COMPLIES WITH THE UNIVERSITY REGULATIONS ON GRADUATION THESES

AND IS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS

FOR THE DEGREE OF

D o c to r

o f P h ilo s o p h y

tOFESsoR i n C h a r g e o f T h e s i s

H

Ja n u a ry 20,

ead of

School

or

D epartment

1950

TO THE LIBRARIAN:----THIS THESIS IS NOT TO BE REGARDED AS CONFIDENTIAL,

f

.

C3- ce

HC1 to titrate Ba(0H)2 wash

no. 2

sf = sample

factor (20 for 200 cc wash,

5 for

50 cc wash) Tf- time factor (3 for 20 minutes, 4 for 15 minutes) 1.17:

1 cc ü/lO HC1 = 1.17 ccOOg

j wt. = wt of mouse In one experiment three adult male white mice and three adult

male black mice

the effect

of lowered

(natural color) were usedto determine ambient temperature on OO2production.

They were kept in individual cages until the time of the determination when they were moved into the animal chamber of the COg measuring device.

They were allowed enough

time to become quiet before the measurement was made.

After

several determinations had been made at room temperature the animals were moved to the cold room which varied be­ tween 6-10°C, and several additional determinations were made at varying intervals. The effect of slowly lowering the environmental tem­ perature on the COg production was determined on three bats freshly caught in August,

The animals were placed

in the animal chamber as soon as they arrived in the lab­ oratory and allowed about 12 hours to become quiet before

31 the first determination was made.

The chamber was then

cooled to the next desired temperature and 24 hours later another determination was made*

Blood Determinations As far as possible, all blood determinations to be made were taken from a single sample of blood collected by decapitation (mice and bats) or by cardiac puncture (ground squirrels).

Decapitation was performed with a sharp razor

blade in a holder and the blood allowed to flow onto a clean watch glass.

It was then drawn into calibrated

pipettes and measured into the various solutions desired. Because of the very small volume of blood in bats there was not always enough blood to make all the determinations desired.

The awake ground squirrels had a tendency to be­

come quite excited when restrained and it washecessary to /

anaesthetize them lightly with ether before making a cardiac puncture.

It was very easy to make a cardiac puncture on a

hibernating ground squirrel, and careful handling sometimes failed to rouse the animal from torpor.

The blood drawn in

cardiac punctures, usually 1/2 cc., was expelled gently onto a watch glass and then taken up in calibrated pipettes. Erythrocyte counts were made using standard techniques. It was necessary to recalibrate an ordinary red cell pipette so that the blood could be diluted 1:400 times instead of the usual 1:200 because of the very high counts obtained for bats under certain conditions.

The diluting fluid used

32 was 3*0^ KaF. Blood hemoglobin was determined by the Sheard-Sanford method in which 0*1 cc of blood was diluted with 20 cc of 0*1% ÎTa2 C03 and the light transmission of the resulting mixture determined by a photelometer*

This figure was com­

pared with a standard curve to give gms of Hb/100 ml blood* Blood sugars were determined by the Folin-Wu micro method (68), In some instances specific gravity of the blood was determined.

When this was done. Hammerschlag*s chloroform-

benzol method for specific gravity was used. Three groups of ten adult white mice were decapitated and blood samples collected for erythrocyte number, hemoglobin, and blood sugar determinations.

The first

group was a control and had not been subjected to any tem­ perature changes other than the natural fluctuation of the laboratory room temperature (230-26°C),

The second group

wa^placed individually in the refrigerator at 5°G for 15 minutes immediately prior to decapitation, and the third group was kept in the cold room at 5°C for 5 days before decapitati on. Erythrocyte counts, hemoglobin, blood sugar, and specific gravity determinations were made on thirteenlined ground squirrels during various phases of the hiber­ nating cycle (awake in summer, awake in winter, just after entering hibernation, and after long hibernation) and at different environmental temperatures.

In most cases at

33 least two weeks time was allowed to elapse between successive bleedings in order to allow the animal to recover from the loss of blood, however a few animals were bled after one seven day interval. The blood of summer-caught and winter-caught bats was analyzed for number of eyrthrocytes, hemoglobin content, and blood sugar content following subjection to normal and low temperatures.

Bats caught in the summer were either

kept in the laboratory over night or in the cold room at 5°C for five days before decapitation.

The bats captured

in the winter were in hibernation at the time of capture and were placed in the cold room at 5°C as soon as possible after capture, where they were kept for six to fourteen days before use.

Some were decapitated before they were

removed from the cold room while others were moved to a warm room (210-24°C) for three to twenty-four hours so that they would "warm up" and become active before decapita­ tion. One group of twelve bats (E. fuscus) were forceably given water by mouth with a pipette four and two days be­ fore decapitation to see if it would be possible to relieve the condition of hemoconeentrâtion.

One of these animals

died, apparently of drowning. Another group of three bats (E. fuscus) was caught while in hibernation and decapitated as soon as they were brought into the laboratory to find out if hemoconeentrâtion

34 was a result of keeping the hats confined in the cold room, or if it occurred in naturally hibernating hats also.

Insulin Injections in Bats Seven summer and six winter-caught hats (E. fuscus ) were given intraperitoneal injections of insulin (Lilly’s "Iletin” ) in doses ranging from 1/6 to 5 units. observed for signs of hypoglycemic shock.

They were

Three of the

summer, and one winter-caught bat which had received one, three, five, and five units of insulin respectively were decapitated and their blood sugar determined.

35 RESULTS AML DISCUSSION Mice The immediate and suhsequent effects of suddenly lowering the environmental temperature from near thermal neutrality* to 4 0-7°C on the Og consumption of normal white mice are given in Table 1.

Within the first ten minutes

at the lower temperature there is a 283% increase in Og consumption.

Because of the rapidity with which this re­

sponse take Enlace it is reasonable to assume that some short acting type of regulatory mechanism is responsible. The nervous system undoubtedly takes some part, because it has been noted that the mice invariably shiver upon first being placed in the cold, and increased muscle tonus would increase the Og consumption. Adrenalin release from the adrenal medulla probably aids this reaction.

This is indicated by an increase in

blood sugar which also occurs shortly after exposure to cold (see below),

Cannon et al (12) have shown that the

adrenal medulla of rats is activated during short exposure to cold* Following this great rise, the Og consumption falls for four or five hours to its lowest point during prolonged exposure to temperatures of 40-7°C (Figure 3.),

At this

point and for the next five or six hours the 0^ consumption is increased about 180% above that near thermal neutrality, *Thi environmental temperâtureiiaving the least effect on metabolic rate.

36

Table 1.

The Effect of Suddenly Decreased Environmental

Temperature from Near Thermal Neutrality to 40-7°C on the 0^ Consumption of Mice. Elapsed time

Male Mice

hours

Temperature

Og Consumption

No. wt-gms_______ °C__________ cc/gm/hr 1

22.5

28

1.85

2

22

30

1.81

At least

3

25

28

1.93

24 hours

4

21.5

30

1.80

within 3 degrees

5

22

31

1.81

of temperature.

6-15

________________ 16-20 0.0

247

26

1 2 1 _______ 30__________

2.02 1.91__

Placed in cold room.

0.10

1

22.5

5

7.42

0.10

2

22

6

7.63

0.10

3

25

5

7.45

0.10

4

21.5

5

7.58

0.10

5

22

6

7.66

0.15

6-15

248

7

7.56

0.15_________ 16-20

121__________ 7_____________ 7.52_____

1.0

1

22.5

5

6,89

1.0

2

22.0

5

6.38

1.0

3

25.0

6

6.76

1.0

4

21.5

6

6.7°

1.0

5

22

6

6.65

6-15

247

7

7.01

16-20

120

7

6.88

1.25 1.0

37 Table 1 (cont.), Elapsed time

Male Mice

Temperature

h o u r s ______ No» wt-gras_______ PC_______

Og Consumption cc/gm/hr

2.0

1

22.5

4

6.42

2.0

2

22.0

5

6.40

2.0

3

25.0

5

6.3°

2.0

4

21.5

5

6.40

2.0

5

22.0

5

6.37

2.0

6-15

246.0

7

6.50

2.0

16-20

120.0

6

6.46

3.0

6-15

245.0

7

6.11

3.0

16-20

119.0

6

6.06

4.0

1

22.5

5

5.51

4.0

2

22.0

5

5.13

4.0

3

25.0

6

5.58

4.0

4

21.5

5

5.31

4.0

5

22.0

5

5.27

4.0

6-15

244.0

7

5.61

4.0

16-20

118.0

5

5.50

5.0

6-15

243.0

7

5. 58

5.0

16-20

119.0

6

5.59

6.0

1

22.0

5

5.67

6.0

2

22.5

5

5.44

6.0

3

24.5

5

5.48

6.0

4

21.5

6

5.35

6.0

5

22.5

6

5.36

6.0

6-15

242.0

6

5.60

38

Table 1 (cont.). Elapsed time

Temperature

Mo. wt-gms

°C

Og Consumption cc/gni/hr

6.0

16-20

118.0

6

5.54

8.0

10

244.0

7

5.55

8.0

5

117.0

6

5.59

9.0

1

22.0

5

5.59

9.0

2

22.0

4

5.53

9.0

3

24.0

6

5.37

9.0

4

22.0

5

5.38

9.0

5

22.0

6

5.42

10.0

6-15

248.0

7

5.58

16-20

121.0

7

5.53

12.0

1

23.0

4

5.59

12.0

2

22.0

4

5.56

12.0

3

24.5

5

5.51

12.0

4

22.0

5

5. 46

12.0

5

21.5

4

5.38

12.0

6-15

244.5

7

5.59

12.0

16-20

120.0

6

5.59

15.0

6-15

247.0

6

5.56

H Oi o

hours

Male Mice

16-20

122.5

6

5.58

18.0

1

22.5

5

5.59

18.0

1

22.5

5

5.59

18.0

1

23.0

4

5.48

18.0

1

22.0

5

5.51

18.0

1

21.5

5

5.54

_ 1 0 . 0 ______

39 Table 1 (cont.). Elapsed time hours

Male Mice Fo. wt-gms

Temperature t

0g Consumption cc/gm/hr

6-15

250.0

7

5.60

18.0

16-20

121.0

6

5.61

24.0

1

23.0

5

5.61

24.0

2

22.5

6

5.62

24.0

3

24.0

5

5.60

24.0

4

22.5

6

5.62

24.0

5

22.0

5

5.60

24.0

6-15

246.5

7

5.68

24.0

16-20

119.0

6

5.67

30.0

1

22.5

5

5.67

30.0

2

22.5

5

5.65

30.0

3

24.0

5

5.66

30.0

4

22.0

5

5.70

o . to1

I

18.0

5

21.5

5

5.66

36.0

1

22.0

6

5.75

36.0

2

22.5

5

5.69

36.0

3

24.5

5

5.76

36.0

4

22.5

6

5.74

36.0

5

22.0

5

5.71

36.0

6-14

223.0

6

5.79

36.0

16-20

120.5

6

5.80

42.0

1

22.5

5

5.79

42.0

2

22.0

6

5.80

42.0

3

25.0

5

5.81

40

Table 1 (cont.) Elapsed time

Male Mice

Temperature

hours_________ No. wt-gms

Og Consumption

fç______ cc/gra/hr

42.0

4

22.5

5

5.77

.42.0

5

21.5

5

5.77

48

1

22.5

5

5.83

48

2

22.5

5

5.85

48

3

24.5

5

5.87

48

4

22.0

6

5.79

48

5

22.0

5

5.83

48

6-14

225.0

7

5.89

50

16-20

123.5

7

5.88

60

1

23.0

5

5.83

60

2

22.0

6

5.83

60

3

24.5

6

5.81

60

4

22.0

6

5.84

60

5

21.5

6

5.86

60

6-14

226.5

7

5.86

60

16-20

123.5

8

5.80

72

1

22.5

6

5.81

72

2

22.5

5

5.86

72

3

25.0

6

5.86

72

4

22.5

5

5.82

72

5

21.5

5

5. 85

72

6-14

224.5

7

5.90

72

16-20

122.0

6

5.89

41 Table 1 (c ont,)• Elapsed time hours

Male Mice No, wt-gms

Temperature °C

Og Consumption cc/gm/hr

96

1

22.0

5

5.85

96

2

22.5

5

5.83

96

3

24.5

5

5.86

96

4

22.0

5

5.80

96

5

22.0

5

5.82

96

6-14

225.0

7

5.88

96

16-20

123.0

5

5.90

120

1

22.0

5

5.81

120

2

22.0

5

5.84

120

4

22.0

5

5.82

120

5

22.5

5

5.88

120

16-20

122.5

6

5.87

42

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