Download INFLUENCE OF AMBIENT TEMPERATURE STRESS ON

I N F L U E N C E OF A M B I E N T T E M P E R A T U R E STRESS ON
RESPONSE OF P R E G N A N T RATS TO D I E T A R Y
P R O T E I N LEVELS
J. L. Cox, 1, 2 B. G. HARMON, D. E. BECKER, H. W. NORTON AND A. H. JENSEN
Illinois Agricultural Experiment Station, Urbana
set-point. Two constant-running 25 cm. fans
circulated the air within a chamber and an
metabolism. The endogenous urinary nitrogen exhaust fan provided ventilation. Within the
excretion of rats was increased after an ex- chambers the rats were housed in stainless
posure to a cold environment (Treichler and steel metabolism cages with wire mesh floors
Mitchell, 1941). Similarly, men consuming and backs and solid sides and fronts. Lights
low-nitrogen-low-energy diets were in nega- were automatically turned on at 6 a.m. and
tive nitrogen balance during and for several off at 6 p.m. daily in each chamber.
The glucose-casein diets used in these trials
days after exposure to 8 ~ C. (Issekutz et al.,
1962). Klain et al. (1962) using growing contained 5, 10, 20 and 30% protein. Table 1
rats showed that cold exposure overcame the shows the composition of the 5 % protein diet;
decreased food intake associated with amino higher protein diets were formed by subacid imbalanced diets. Subsequently, the ira- stituting casein and methionine for glucose.
balancing amino acids were found to be Methionine was maintained at 1.3% of the
metabolized more rapidly than other dietary dietary protein in all diets. Daily dietary intakes were equalized within temperatures and
amino acids (Klain and Winders, 1964).
However, rats at 7 or 23.5 ~ C. both rejected were 18, 12 and 9 gin. per rat at 7, 23 and
an amino acid imbalanced diet in favor of a 33 ~ C., respectively, for each trial. Results of
nitrogen-free diet (Harper and Rogers, 1966). a preliminary trial had indicated that most
The dearth of information regarding the rats would consistently consume these
nutrition of pregnant animals maintained amounts with little or no refusal. Any diet
under temperature stress led to the trials re- refused was collected and weighed when the
ported herein. The objective of these trials rat was weighed. Between feedings, the diets
was to assess the influence of dietary protein were stored in plastic bags at 3 ~ C.
Experiment 1. Seventy-two rats were fed
levels on the reproductive performance of
rats maintained at one of three different a stock diet ad libitum through the 4th day
post coitum. On the 5th day the rats were
ambient temperatures.
weighed and randomly allotted to temperature
and dietary treatments. Experimental diets
Experimental
were fed ad libitum during the first 4 days of
Mature female Sprague-Dawley rats were temperature exposure; thereafter, rats were
randomly assigned to cages at the time of ar- fed the amounts indicated above. On the 12th
rival. TO schedule mating, the estrous cycle day of gestation the rats were weighed and a
of each female was charted, using the
7-day collection of urine and feces begun.
vaginal smear technique. Mating was con- Finally, on the 19th day of gestation the
firmed by the presence of sperm in the vaginal animals were weighed and sacrificed for obsmear the morning after cohabitation with a servation of the fetuses and corpora lutea.
male.
Experiment 2. One wk. before mating was
At specified times after mating, females begun, 100 female rats were randomly diswere randomly assigned to environmental
tributed between the four diets. During this
chambers having either 7, 23 or 33 ~ C. air period the rats were ad libitum fed. Within a
temperature and 50% relative humidity.
dietary treatment, the first 18 rats to mate
Temperature control was within 1 ~ C. of the were randomly assigned to temperature treatment at the time of mating and exposed to
t Present address: Merck Institute for Therapeutic Research,
Rahway, New Jersey,
temperature on day 1 or 4 of gestation, at
~This paper represents part of a thesis submitted by the
which time equalized feeding began. The
senior author to tl~e Graduate College of the University of
Illinois as partial fulfillment of the requirements for the Ph.D.
experimental period was divided into three
degree.
650
P O S I N G the nonpregnant animal to cold
E Xstress
has been shown to alter nitrogen
P R O T E I N LEVELS FOR P R E G N A N T RATS
TABLE
1. C O M P O S I T I O N
PROTEIN DIET
Ingredient
Glucose
Casein a
DL-methionine
Vitamin mix b
Mineral mix ~
C o r n oil
Cellulose a
Vitamin A and D ~
OF 5%
%
76. 815
6. 020
0. 065
1. 0 0 0
4. 0 0 0
10.0(30
2. 000
0.100
a Vitamin extracted.
bSupplied in mg./lO0 gin. diet: thiamine HCt, 0.26; riboflavin, 0.66; niacin, 4.40; panthothenic acid, 2.64; pyrido~ine,
0.25; choline chloride, 220.0; vitamin K, 0.02; vitamin E,
12.0; folic acid, 0.20; biotin, 0.02; ascorbic acid, 30.0; vitamin
Blz, 4.40 mcg./100 gin. diet.
e Supplied in rag./100 gin. diet: NaC1, 432; KaCoH~O~*H20,
946; K2HPO~, 309; CaPHO4*2H20, 1422; CaCOa, 654;
MgCOa, 164; FeC~H.~OT*3H.20, 64; CuSO4oSH~O, 0.7;
MnSO,*HeO, 5.5; KI, 0.2; ZnCO8, 1.8.
a Solka floc BW-40: Brown and Company, Boston, Mass.
e Quadrex, 4000 I.U. vitamin A and 500 I.U. vitamin D
per gram: Nopco Chemical Co., Harrison, N. J.
consecutive collection periods of 5, 5 and 7
days. The animals were weighed at the beginning of each period and were weighed and
sacrified on the 17th day of exposure.
Experiment 3. Seventy-two female rats were
randomly assigned to the four diets as in
experiment 2. As the first 12 rats fed each
diet were mated they were randomly assigned
and immediately exposed to either 7 or 23 ~ C.
They continued on the previously assigned
diet and equalized feeding was begun. Urine
and feces were collected for the first 20 days
of gestation in five periods of 4 days each.
The animals were weighed at mating and at
the end of each collection period and sacrificed
on the 20th day of gestation.
Nitrogen retention was determined for each
female at specific intervals during her exposure to temperature treatment. Within a
collection period, daily urine collections for
each rat were pooled and stored at 3 ~ C. until
analyzed. Feces were collected at the end of a
collection period and dried in a forced-draft
oven at 45 ~ C. for at least 7 days. The
nitrogen content of urine, feces and diets was
determined by manual Kjeldahl in experiment
1, and by the method described by Cox and
Harmon (1966) in experiments 2 and 3.
Statistical analysis was by the method of
fitting constants. Only significant ( P < . 0 5 )
constants were retained in the model, and the
standard error of the estimate is reported to
indicate the random variation.
R e s u l t s and Discussion
The temperature and dietary treatments of
these experiments had little consistent effect
651
on litter size, fetal weight or fetal survival as
indicated by the difference between number
of corpora lutea and number of fetuses. This
suggests that the dam was not stressed by the
treatments sufficiently to interfere with fetal
development. Others have demonstrated that
temperature stress or low-protein diets are
capable of interrupting fetal development.
Pregnant rats exposed to 35 ~ C. throughout
gestation had 58% loss of implanted fetuses
compared to 7% for controls at 25 ~ C. (Macfarlane et al., 1957). Fernandez-Cano (1958)
exposed rats to eitller 40 or --3.3 ~ C. for 5
hours on two consecutive days during early
gestation. Embryonic mortality was greater
in the temperature stressed animals than in
controls, but greater loss occurred in rats subjected to 40 than --3.3 ~ C. exposure. Similarly, embryo survival was reduced in rats
fed ad libitum amounts of diets containing
less than 5~o protein (Nelson and Evans,
1953).
In experiment 2, all rats of the day-4 group
fed the 10% protein diet died on the 19th day
of gestation. Each of these rats was pregnant
with a large litter, and the fetuses appeared
normal for the stage of gestation. The exact
cause of death was not discernible, but it was
not considered a treatment effect.
Weight gain of the dam was significantly
influenced by both temperature and dietary
protein level in all experiments (table 2).
T h o s e animals at 23 ~ C. gained significantly
( P < . 0 5 ) more than animals at either 7 or
33 ~ C. There was a significant linear ( P < .05)
and curvilinear ( P < . 0 5 ) effect of protein
level on weight gain. Rats fed the higher protein diets at 7 and 23 ~ C. gained more than
those fed less dietary protein. However, rats
at 33 ~ C. did not show this response, and in
experiments 1 and 2 the interaction of temperature and protein level was significant
(P<.05).
The nitrogen retention of rats in experiment I is shown in table 3. Litter size was
significantly ( P < . 0 5 ) correlated with nitrogen retention and was included in the model
for statistical analysis. There were significant
linear ( P < . 0 5 ) and curvilinear ( P < . 0 5 )
effects of both temperature and protein level
on nitrogen retention. However, the interaction of protein and temperature was significant
( P < . 0 5 ) . At 7 ~ C., nitrogen retention increased directly with protein level; whereas, it
reached a maximum at 20% protein for rats
at 23 and 33 ~ C. Also, rats at 33 ~ C. retained
the least nitrogen at all protein levels. These
652
COX ET AL.
TABLE
2. A V E R A G E
WEIGHT
GAIN OF PREGNANT
RATS, GM. (EXPERIMENTS
1-3)
Protein level, %
E x p e r i m e n t "' ~'
5
T e m p e r a t u r e , ~ C.
10
20
30
1
7
23
33
6
20
22
(6) c
(6)
(6)
22
38
25
(6)
(5)
(6)
14
48
28
(6)
(6)
(5)
28
44
22
(6)
(6)
(6)
2 ( d a y 1) a
7
23
33
25
15
28
(2)
(2)
(3)
36
43
29
(1)
(3)
(3)
30
35
31
(2)
(3)
(2)
38
39
35
(3)
(3)
(2)
2 ( d a y 4) a
7
23
33
13
35
16
(2)
(2)
(2)
25
46
.,
(3)
(2)
(0)
46
64
54
(3)
(3)
(1)
57
66
52
(3)
(3)
(2)
3
7
23
3
28
(3)
(6)
27
44
(5)
(6)
25
56
(6)
(5)
20
57
(6)
(6)
aGain of rat from day 5 19 of gestation in Exp. 1, 1 17 or 4 20 in Exp. 2 and 1-20 in Exp. 3. Average initial weight:
Exp 1, 238 gm., Exp. 2, 238 gin. Exp. 3, 208 gin.
bStandard error of estimate: Exp. 1, 7.74; Exp. 2, 7.95; Exp. 3, 9.77.
e Number of rats per treatment.
a Day of gestation rats exposed to temperature treatments.
e All rats died on day 19 of gestation.
rats were consuming less food daily than those
subjected to 7 and 23 ~ C. Thus, they would
have obtained relatively less net energy from
non-protein dietary components necessitating
some utilization of protein to meet energy
needs and reducing nitrogen retention. In
experiment 2, nitrogen retention (table 4)
during the first collection period was correlated directly with protein level and inversely correlated to temperature. Thus,
nitrogen retention was greatest in those
animals consuming the largest quantities of
dietary protein. In the second collection period, the interaction of time, temperature and
protein was significant ( P < . 0 5 ) . I t appeared
that nitrogen retention was maximized to a
greater extent at lower protein levels in day-1
than day-4 rats at all temperatures. However, rats at 33 ~ C. retained as much nitrogen
as rats at 23 ~ C. when exposed to temperature on day 1, but lesser amounts when exposed on day 4. During collection period 3,
the interaction of time with protein level was
significant ( P < . 0 5 ) and indicates that the
amount of nitrogen retained from the high
T A B L E 3. A V E R A G E N I T R O G E N R E T E N T I O N
O F P R E G N A N T R A T S , ~' b M G . / D A Y
(EXPERIMENT
1)
P r o t e i n level, %
Temperature, o C.
5
10
20
30
7
23
33
26.1
19.7
10.1
80.9
78.4
28.6
125.7
125.1
69.3
150.3
119.9
49.0
a Day 13 to 19 gestation.
t, Standard error of estimate, 33.23.
protein diets was greater in the day-4 than
the day-1 rats. Both day-1 and day-4 rats retained similar amounts of nitrogen when fed
the 5 and 10% protein diets.
Figure 1 shows graphically the nitrogen
retention of rats in experiment 3. In the first
4-day collection period in both environments,
nitrogen retention was directly ( P ~ . 0 5 ) related to amount of protein consumed. In periods 2 and 3, nitrogen retention also reflected
the dietary protein level. However, the amount
of nitrogen retained by rats fed 10, 20 or
30% protein at 7 ~ C. decreased successively
in periods 2 and 3. This also occurred in rats
fed 20 and 30% protein at 23 ~ C. A similar
decrease in nitrogen retention continued into
collection period 4 for rats fed 20 and 30%
protein. During this time, nitrogen retention
was greatest in rats fed 10% protein at both
temperatures, however, the interaction of
protein and temperature was significant
( P < . 0 5 ) . This interaction was due to the
different response of rats to the 20 and 30%
protein diets at the two temperatures. Compared to rats fed 10% protein, rats at 23 ~ C.
had reduced nitrogen retention when fed the
two highest protein diets, whereas the rats at
7 ~ C. did not. Nitrogen retention increased
during period 5 compared to period 4, and was
greatest during this time when the 20% protein diet was fed although the increase over
that resulting from the 10% diet was small at
7~
It appears from these results that nitrogen
retention during the first few days of gestation
is related to protein intake regardless of
temperature. Since nitrogen retention in
653
P R O T E I N LEVELS FOR P R E G N A N T RATS
TABLE
4. A V E R A G E N I T R O G E N R E T E N T I O N OF R A T S D U R I N G D I F F E R E N T
P E R I O D S OF G E S T A T I O N , M G . / D A Y ( E X P E R I M E N T
2)
D a y of g e s t a t i o n ~ a n d t e m p e r a t u r e
1
Protein, %
7 ~ C.
4
23 o C.
33 ~ C.
7 o C.
23 o C.
33 ~ C.
36.6
49.0
117.1
123.9
24.1
55.0
72 .4
57.2
4.9
26.5
78.7
45.5
49.6
31.3'
76,2
101,0
31.2
42.9
66.3
78.7
3.7
a
....
54.6
45.9
50.1
76.3
21.3
23.4
27.9
60.0
107.7
57.9
24.8
47.1
111.3
62.2
37.3
33.4
95.2
37.3
37.1
58.2
54.8
90.7
11.3
.... a
42.5
69.1
26.7
41.2
92.5
--4.6
45.0
75.1
59.3
57.5
43.4
52.2
60.4
21.8
30.7
43.0
76.4
102.8
31.9
54.9
116.6
83.2
28.8
.... a
99.2
75.4
P e r i o d 1 b, c
5
10
20
30
Period 2
5
10
20
30
Period 3
5
10
20
30
a Day of gdstation rats exposed to temperature.
b Collection',periods 1, 2 and 3, were, respectively, 5, 5 and 7 days.
~'Standard error of estimate: period 1, 25.54; period 2, 34.32; period 3, 31.41
a All animals died.
experiments 2 and 3 was unrelated to litter
size at any stage of gestation, differences in
retention between treatments presumably reflect changes in maternal tissue nitrogen.
Thus these results are consistent with the results of Poo et al. (1940) who reported that
the total quantity of maternal-tissue protein
formed during pregnancy was dependent on
protein intake. Also, the nitrogen retention of
protein-depleted male rats was increased immediately when a 20% protein diet was
offered at 2 or 22 ~ C. (Beaton, 1963b). However, in nondepleted males, nitrogen retention was reduced from the 2nd to 6th day of
exposure to 2 ~ C. (Beaton, 1963a). The
animals in both of these studies were ad
libitum fed and this indicates that the effects
of cold on nitrogen retention are altered in
animals actively synthesizing tissue protein
in response to protein repletion. A similar
response by the pregnant rats in experiments
2 and 3 could have been the explanation for
those at 7 ~ C. retaining more nitrogen in
early gestation than rats consuming the same
diet at 23 ~ C.
There may have been an increase in the
energy requirement of the rats during the
time represented by the 2nd to 4th collection
periods of experiment 3. This could account
for the decreased nitrogen retention during
this time. However, more than this may have
been involved, since the animals fed the 5%
protein diet did not retain more nitrogen at 7
than at 23 ~ C. According to Payne and Jacob
(1965), tissue synthesis in the growing rat
requires similar amounts of energy regardless
of ambient temperature. Thus, unless dietary
energy was limiting, increased nitrogen consumption by the rats at 7 ~ C. should have
resulted in increased nitrogen retention. That
the 57o protein diet was not deficient in
energy is supported by the fact that feeding
the 10% protein diet increased nitrogen retention at both temperatures. Since the two
diets were isocaloric and fed in equal amounts
within a temperature, it seems plausible that
the increased nitrogen retention of rats fed
the 10% protein diet was due to the increased
protein consumed. Therefore, it is not clear
why the greater consumption of the 5% protein diet at 7 ~ C. did not result in increased
nitrogen retention.
In experiments 2 and 3 the nitrogen retention of rats increased greatly during the period that included the 20th day of gestation.
Beaton et al. (1954) has shown that nitrogen
retention of the pregnant rat increases
markedly after the 15th day of gestation. In
all experimental periods in which the rats
were past the 15th day of gestation for a
majority of the time, the rats fed the 20%
protein diet at 23 and 33 ~ C. retained the
most nitrogen. However, for rats at 7 ~ C.
results of the three experiments were not con-
654
COX E T AL.
180
. . . .
.....
...........
140
I00
60
5%
PROTEIN
I0% PROTEIN
20%
PROTEIN
30% PROTEIN
ROOM TEMP. 7~
%%""~176
%%%%,
%,%%,'
,%s%163
'"'.
%
-''', """..~t~
..~""
I=
I.o
z
I---
z
t.i,_l
(,..o
o
20
i
I
I
180
~'~1 ''~
. . . .
. . . .
..........
,
140
I
5%
I0%
20%
30%
ROOM TEMP 23~
I.-z
I00
.:
60
20 p
f
I
I
I
f
2
3
4
COLLECTION PERIOD
5
Figure 1. Average nitrogen retention of pregnant rats during each of
five consecutive 4-day periods. Standard error of the estimate for each
period was: period 1, 30.90; period 2, 34.60; period 3, 32.25; period 4, 22.22;
period 5, 24.55.
sistent. In experiment 1 and for the third
period of rats exposed to temperature on day
4 in experiment 2, nitrogen retention was
greatest when the 30eft, protein diet was fed.
In the fifth collection period of experiment 3,
there was little difference in nitrogen retention of rats fed 10, 20 or 30% protein diets
at 7 ~ C. 3"his discrepancy may be due to litter
size since the average litter size was 12.0,
l I . l and 9.0 for experiments 1, 2 and 3,
respectively.
Summary
Three experiments involving 192 pregnant
rats were conducted to determine the influence
of temperature on the response to protein
levels. Dietary protein levels were 5, 10, 20
and 3 0 ~ in all experiments. Temperature
treatments were 7, 23 or 33 ~ C. in experiments
1 and 2, and 7 and 23 ~ C. in experiment 3.
Temperature, protein level and time of initial
exposure were observed to have little in-
PROTEIN
LEVELS
FOR PREGNANT
fluence on e m b r y o survival. W e i g h t gain of
the d a m was less at 7 and 33 t h a n at 23 ~ C.
and increased w i t h increasing p r o t e i n levels
except at 33 ~ C.
N i t r o g e n r e t e n t i o n of the d a m was l i n e a r l y
correlated w i t h p r o t e i n i n t a k e during early
gestation. T h e a m o u n t of n i t r o g e n retained b y
the rats decreased as gestation progressed w i t h
rats c o n s u m i n g the high protein diets showing
the g r e a t e s t decline. T h i s decline in n i t r o g e n
retention m a y reflect an increased energy requirement during midgestation.
I n the l a t t e r stages of gestation, the a m o u n t
of n i t r o g e n r e t a i n e d increased over the preceding period. N i t r o g e n r e t e n t i o n was the
greatest in rats fed the 2 0 % protein diet a t
23 and 33 ~ C. H o w e v e r , at 7 ~ C. it a p p e a r e d
t h a t c o n s u m p t i o n of the 2 0 % p r o t e i n diet did
n o t always s u p p o r t the greatest n i t r o g e n retention. H o w e v e r , feeding the 10% p r o t e i n
diet generally resulted in greater n i t r o g e n retention t h a n feeding the 5 % protein diet a t
all stages of gestation at each t e m p e r a t u r e .
Literature
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McHenry. 1954. Protein metabohsm in the pregnant
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Beaton, J. R. 1963b. Protein repletion in rats during
RATS
655
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