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Exercise during rat pregnancy and lactation: Maternal effects and offspring growth
Physiology& Behavior,Vol. 47, pp. 427--433. ©Pergamon Press plc, 1990. Prin~d in the U.S.A.
0031-9384/90 $3.00 + .DO
Exercise During Rat Pregnancy and Lactation: Maternal Effects and Offspring Growth G E N E V I E V E T. C O U R A N T 1 A N D S U S A N I. B A R R 2
School of Family and Nutritional Sciences, University of British Columbia 2205 East Mall, Vancouver, B.C., Canada, V6T 1 W5 R e c e i v e d 5 S e p t e m b e r 1989
COURANT, G. T. AND S. I. BARR. Exerciseduring rat pregnancy and lactation: Maternaleffects and offspring growth. PHYSIOL BEHAV 47(3) 427-433, 1990.--Eighty Spragne-Dawley rats were divided into exercised (E) and sedentary (S) groups. E rats were trained to run on a treadmill (30 m/min, 2 hr/day). Within each group, two subgroups were mated and three served as virgin time controls. Of the mated subgroups, one was terminated within 24 hours of delivery and the other on day 14 of lactation. Subgroups of virgin S and E controls were terminated at times corresponding to the mating, delivery and lactation day 14 of mated rats. MANOVA revealed that exercise significantly affected food intake, body weight and body composition in both virgin and mated animals: generally, E rats ate more, gained more weight, and had less carcass fat than S controls. E rats did not store fat during pregnancy. At parturition, they were 7.0% fat, similar to both E (6.6%) and S (7.6%) controls prior to mating, and less than S rats at parturition (11.9%). Despite diminished fat stores at parturition in E rats, litter size and pup birthweight were similar in E and S rats, as was offspring growth during lactation (mean weights on day 14 of 28.9 g and 29.3 g, respectively). Remaining body fat and increased food intake were adequate to support normal pup growth. Exercise
Food intake
Body composition
Pregnancy
RAT pregnancy is characterized by weight gain, an increase in food intake, and increased fat deposition (19, 20, 30, 31). During lactation the additional fat stores are largely catabolized while food intake increases further to support the high energy cost of milk production (7, 14, 20). Similar to pregnancy, exercise in nongravid female rats generally results in increased food intake, but in contrast to pregnancy, body fat is usually decreased and weight may remain the same or decrease (2, 15, 21, 23). The effects of exercise on these parameters in pregnant and especially in lactating rats have not been well documented. This subject is of interest since exercise and pregnancy exert opposing effects on fat deposition. If exercise training is superimposed on rat pregnancy, would fat stores be reduced? If so, would lactation performance and offspring growth be compromised, or would food intake during pregnancy and/or lactation increase sufficiently to compensate for the lowered fat stores? While the majority of rat studies have found that, even at strenuous levels, maternal aerobic exercise does not significantly affect gross fetal development or pregnancy outcome, it does appear to lower maternal weight gain in most (4, 17, 18, 34) but not all (28,33) studies. The lower maternal weight gain has been suggested, though not directly demonstrated, to reflect a reduction in maternal fat stores (18). The food intake of the animals in this
Lactation
study was not reported: thus, it is not known whether changes in their food intake could have contributed to or modulated the observed weight decrements. In this study, we sought to determine the effects of moderate aerobic exercise on food intake, body composition, body weight and offspring growth in female nonpregnant, pregnant and lactating rats. In order to separate the effects of exercise from those of pregnancy and lactation, our study design included groups of exercising and sedentary pregnant and lactating rats, as well as groups of age-matched nongravid exercising and sedentary controls. METHOD
Experimental Design and Protocol A modified 2 x 2 × 3 factoriai design was used for the experiment. Rats were equally divided into exercised and sedentary groups (factor = "activity"). The exercised rats ran on a treadmill while the sedentary rats followed a normal laboratory routine. Each of these two groups was divided into five subgroups (n = 8/subgroup), two of which were mated and three of which were not (factor = "gravidity"). Of the mated subgroups, one was
~Present address: 33 rue de Geneve, St. Genis-Pouilly, France F-01630. 2Re.quests for reprints should be addressed to Susan I. Bart.
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COURANT AND BARR
ACCLIMATIZATION TRAINING
WEEK
0
1
2
MATING (I- Bdays)
3
0
RANDOMIZE
TRAINON
TERMINATE
RATS INTO GROUPS
TREADMILL EVT EVP
EVT SVT
EVL EMP EML
MATE EMP SMP
PREGNANCY
1
2
LACTATION
3
( DELIVERY)
1
2
(LACTATION DAY 14)
TERMINATE
EML SML
EVP EMP SVP SMP
TERMINATE EVL EML SVL SML
FIG. 1. Experimental protocol. E= exercised, S = sedentary ("activity" factor); V = virgin, M = mated ("gravidity" factor); T = terminated at time corresponding to end of training, P = terminated at time corresponding to parturition, L=terminated at time corresponding to day 14 of lactation ("stage" factor).
terminated within 24 hours of delivering its pups and the other was terminated after 14 days of lactation. The three unmated or virgin subgroups were terminated at the times corresponding to the mating, delivery, or day 14 of lactation of the mated animals (factor= "stage"). Thus sedentary and exercised virgin rats were used as age controls for these three time periods in the experiment. The experimental protocol is summarized in Fig. 1.
Animals Eighty virgin female Sprague-Dawley rats, weighing between 170 and 190 grams, were obtained from Charles River Breeding Laboratories, Hull, Canada. On arrival they were housed communally and two days later were randomized into the 10 experimental subgroups and housed individually in stainless steel screen bottom cages. During the five-day adjustment period, the animals were acclimatized to an inverted 12-hour on, 12-hour off light cycle (with lights on at 1:00 p.m.), and to a room temperature of 21°C. Pregnant rats were placed in individual plastic cages with corn cob bedding (San-i-cel, Paxton Processing, Paxton, IL) on day 19 of pregnancy, where they remained until terminated. Animals were given ad lib access to a ground commercial nonpurified diet (5001, Ralston Purina Canada, Longueil, Quebec) and fresh water, except during the exercise period, as described below.
Exercise Following the adjustment period, the rats assigned to exercising groups were trained progressively over a period of three weeks, six days per week, to run on a motor-driven treadmill. Training and subsequent exercise took place in the morning near the end of the dark cycle. At the end of the training period, the rats were capable of running at 30 rn/min on a 0% grade for two consecutive hours. The treadmill was equipped with a shock grid that delivered a mild electric shock when the animals did not run at speed. No animals were injured due to the use of the grid. After the training period, the rats continued to run at 30 m/min for two hours per day, five consecutive days per week until they were terminated. Pregnant rats ran up to and including day 20 of pregnancy, and if allowed to lactate, resumed exercise on day two of lactation. In order to prevent the possible overheating of the animals, an electric fan was directed over the treadmill. On the basis of measurements of oxygen consumption of rats running at various speeds and on various grades (5, 6, 29), it was
estimated that the rats in this study were consuming oxygen at the rate of approximately 60 ml/kg/min, or 70% VO2 max, a moderately strenuous aerobic workload. During the exercise period, access to food and water was removed from all animals. Sedentary lactating dams were also removed from their pups during this period.
Mating Following the training period, rats to be mated were housed overnight on a one-to-one basis with male Sprague-Dawley rats. The finding of a vaginal plug and/or the presence of sperm in the vaginal washings the following morning was taken as day 0 of pregnancy. Following delivery, litters of lactating dams were culled to eight, including four males and four females when possible.
Measurements Food intake was measured daily. Spillage, which was minimal due to the use of covered food cups, was accounted for. All animals were weighed every third day. Pregnant rats were also weighed on day 0 of pregnancy and then every third day. A postpartum weight was taken within 24 hours of delivery, and lactating dams were weighed every third day and on day 14 of lactation. Rat pups were weighed within 24 hours of birth and then every third day and on day 14.
Termination Animals were weighed, anaesthetized with ether and decapitated. Carcasses, including the head, were reweighed and stored individually in polyethylene bags at - 2 0 ° C .
Carcass Preparation and Analysis Preparation for sampling and analysis of the rat carcasses was based on the method of Hartsook and Hershberger (13). Frozen carcasses were individually autoclaved (Model STM-E, Market Forge Co., Everett, MA) in approximately 150 ml of distilled water for three hours at 115°C and two atmospheres pressure, and then were homogenized in a Waring blender at high speed for 10 minutes with enough distilled water to achieve approximately a 1:1 ratio of carcass weight to water added. The blender contents were then poured through a household wiremesh sieve to crudely separate the hair from the remainder of the mixture. The hair and adhering material, or " h a i r , " as it is hereafter referred to, was placed on a petri dish, weighed and stored at -20°(]. The remaining mixture, hereafter referred to as the homogenate, was quantitatively transferred back to the Wafing blender and blended again at high speed for 30 seconds to assure homogenization. (This separation allowed accurate sampling of the two fractions of the rat carcass. The composition of the whole carcass was then calculated from results of analysis of the two fractions.) Five samples of the homogenate were immediately taken using a large bore bulb syringe and placed in each of five petri dishes. These were weighed and stored at - 20°C. The frozen homogenate and " h a i r " were then freeze-dried (75150 Labconco Tray Freeze Dryer, Labconco Corp., Kansas City, MO) and the freeze-dried homogehate (not the " h a i r " ) was ground to a fine powder using a household coffee grinder (Moulinex) and stored at - 2 0 ° C . A measure of the water lost during freeze-drying of the homogenate and " h a i r " of each carcass and the weights recorded during the preparation of the carcass for analysis allowed the calculation of the rat's total body water content. Triplicate samples of both powdered homogenate and the " h a i r " were ashed in a
EXERCISE DURING RAT PREGNANCY AND LACTATION
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TABLE 1 WEEKLY FOODINTAKESOF RATS DURINGTHE TRAINING,PREGNANCYAND LACTATIONPERIODS Food Intake (g/week) Time Period
VSed
VEx
MANOVA
MSed
MEx
Act
Grav
AxG
ns * :~
----
----
Training
week l 2 3
120.8 -+ 13.8 126.6 - 11.6 126.1 ~ 14.6
120.8 --- 12.2 133.3 -+ 11.5 143.3 --- 15.2
Pregnancy
week 1 2 3
130.7 ~ 1 4 . 5 130.5 - 1 1 . 9 126.7 -*- 12.0
150.8 -+ 12.7 147.8 - 15.7 155.1 --- 18.3
155.3 - 14.6 156.8 _ 1 4 . 7 175.3 --- 1 9 . 8
167.3 ~ 15.8 168.6 _ 19.7 177.4 _ 21.7
~: :~ :~
~t :~ :~
ns ns t
Lactation
week 1 2
129.2 -- 8.0 131.1 _ 8.1
142.3 -4- 20.1 155.6 -+ 17.8
214.8 ___ 20.3 366.3 _+ 33.9
237.9 -+ 11.4 380.0 _ 31.5
~" *
:~ :~
ns ns
Results are presented as mean +__ S.D. for 8 animals per group. VSed=virgin, sedentary; VEx=virgin, exercised; MSed= mated, sedentary; MEx = mated, exercised. Significance of effects of Act (activity), Gray (gravidity) and A x G (activity by gravidity interaction) was as follows: *p<0.05; tp<0.01; :~p<0.00l.
muffle furnace (Thermolyne Model 2000, Thermolyne Corp., Dubuque, IA) at 650°C for 18 hours. The percent fat content of duplicate analysis of the powdered homogenate and " h a i r " was determined by the method of Atklnson et al. (3). Triplicate determinations of the nitrogen content of duplicate samples were obtained for both the rat homogenate and " h a i r " using the method of Fukomoto and Chang (11) and an ammonium sulfate solution for standards. The mean percentage of total body weight accounted for by analysis of water, fat, ash and protein was 99.5-+1.1%. It was expected to be less than 100% since the carbohydrate component of the carcass was not analyzed and therefore not included in the total. A rough approximation of the carcass energy content (CEC) was calculated, as follows, for each rat: CEC = [carcass wt. (g) x % fat/100 x 9.4 kcal/g] + [carcass wt.(g) × % protein/100 × 5.65 kcal/g] (25).
Statistical Analysis Statistical analyses were performed using programs available in SPSSx (22). Multivariate analysis of variance (MANOVA) was used to test for the main effects of activity and gravidity on food intake and body weight during each of the training, pregnancy and lactation periods. M A N O V A was also used to test for main effects of activity, and gravidity and stage on body composition (fat, water, ash and protein) and for the estimated carcass energy content. Because the modified factorial design did not permit a complete 2 x 2 × 3 analysis, a 2 × 2 x 2 analysis was performed, including only two levels of the stage factor (pregnancy and lactation). MANOVA was also performed on a 2 × 3 design of the experiment (activity vs. stage for each of the virgin and mated subgroups). This allowed the inclusion of the training stage factor excluded in the analysis of the 2 × 2 × 2 design, t-Tests were used to determine the effect of maternal activity on litter size and offspring growth. All results are presented as mean---standard deviation. The level of statistical significance was set at p<0.05. RESULTS
Food Intake Mean weekly food intakes and results of MANOVA are presented in Table 1. Both activity and gravidity significantly
affected food intake throughout the experiment, beginning at week two of training for activity and week one of pregnancy for gravidity. Exercised animals ate more than their sedentary counterparts, and pregnant animals consumed more than their nonpregnant counterparts. The only significant interaction occurred between activity and gravidity at week three of pregnancy. While both exercised and sedentary pregnant groups consumed more than their respective nonpregnant controls, the effect of activity appeared to be confined to the virgin animals. There was a large difference in the food intakes of the virgin sedentary and exercised animals, but no difference in food intakes of pregnant sedentary and exercised animals.
Body Weight As with food intake, results of MANOVA presented in Table 2 show that activity had a significant effect on body weight from week two of the training period to the end of the lactation period. Exercised virgin or mated animals consistently weighed more than sedentary controls. The effect of gravidity was significant beginning at week one of the pregnancy period, and with the exception of the parturition time point, remained so throughout the experiment. At parturition, the weight of mated rats decreased to a level which no longer differed significantly from that of the unmated controls. As lactation progressed, however, the mated rats gained more weight that their virgin controls, such that they were once again heavier.
Body Composition A summary of the body composition analyses for all rat groups is presented in Table 3. Results of MANOVA performed on a 2 × 2 × 2 design, in which the training stage was deleted from the analysis, revealed that percentage body fat was significantly affected by activity, F(1,56)= 55.7, p<0.001, gravidity, F(1,5) = 21.9, p<0.001, and stage, F(1,56) = 14.0, p<0.001. The effect of activity can be observed in the consistently lower values for percentage fat in both virgin and mated exercised rats than in sedentary controls. The effects of gravidity and stage, and the significant interaction, F(1,56) = 38.9, p<0.001, between these
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COURANT AND BARR
TABLE 2 BODY WEIGHTS OF RATS DURING THE TRAINING, PREGNANCY AND LACTATION PERIODS Body Weight (g) Time Period
VSed
± ± -----
7.9 10.0 13.3 16.8
Act
Grav
A×G
ns ns * $
-----
-----
195.8 212.6 224.9 234.6
Pregnancy ( n = 16/group)
week 1 2 3
255.2 --- 14.8 260.8 ± 16.6 268.2 --- 16.9
280.6 --- 16.2 287.4 ± 20.6 299.2 --- 25.5
273.0 ___ 12.2 305.5 ± 16.0 373.4 --+ 26.0
296.1 --- 16.1 327.2 ± 20.7 391.4 ± 33.3
$ :~ :~
:~ :~ $
ns ns ns
268.2 ± 21.6
298.7 -+ 25.1
281.9 ± 14.2
298.5 -+ 16.9
:~
ns
ns
279.3 -+ 17.8 284.0 --- 16.7
306.3 ± 19.1 311.4 ± 18.9
296.6 ± 16.0 327.4 ± 21.6
329.1 ± 15.9 359.0 ± 26.2
~: :~
t ~:
ns ns
week 1 2
193.3 211.9 231.6 253.3
MEx
week 0 1 2 3
Lactation (n=8/group)
10.4 10.7 11.7 13.3
MSed
Training (n=40/group)
parturition
± ± -----
VEx
MANOVA
Results are presented as mean ± S.D. VSed=virgin, sedentary; VEx=virgin, exercised; MSed=mated, sedentary; MEx = mated, exercised. Significance of effects of Act (activity), Grav (gravidity) and A × G (activity by gravidity interaction) are as follows: *p<0.05; tp<0.01; ~:p<0.001.
m a i n effects, are revealed by the tendency for percent b o d y fat to be similar or h i g h e r in m a t e d than virgin a n i m a l s during pregn a n c y , but lower during lactation. Finally, a three-way interaction a m o n g activity, gravidity and stage, F ( 1 , 5 6 ) = 8 . 2 , p < 0 . 0 1 , is explained by the finding that during the p r e g n a n c y stage, m a t i n g appeared to be associated with h i g h e r values for percent fat in sedentary but not in exercised animals, w h e r e a s during the lactation stage, m a t i n g was associated with lower values for b o d y fat in both activity groups. T h e pattern o f findings f r o m 2 × 2 × 2 M A N O V A of percentage b o d y water w a s very similar to those for percentage fat, but c h a n g e s were generally in the opposite direction. T h e significant effect o f activity, F ( 1 , 5 6 ) = 5 0 . 8 , p < 0 . 0 0 1 , is revealed in the
consistently higher values for percentage water in exercised animals. Significant effects o f gravidity, F(1,56) = 58.3, p < 0 . 0 0 1 , and stage, F ( 1 , 5 6 ) = 2 4 . 1 , p < 0 . 0 0 1 , and the significant interaction b e t w e e n these two effects, F ( 1 , 5 6 ) = 5 1 . 2 , p < 0 . 0 0 1 , are indicated by the finding that values for percentage water were similar in virgin and m a t e d a n i m a l s during the p r e g n a n c y stage, w h e r e a s during the lactation stage, they increased in the m a t e d a n i m a l s and tended to decrease in the virgin animals. A three-way interaction a m o n g activity, gravidity a n d stage, F ( 1 , 5 6 ) = 5 . 1 , p < 0 . 0 5 , is illustrated by the finding that b e t w e e n p r e g n a n c y and lactation, percentage water appeared to decrease in sedentary but not exercised virgin animals, w h e r e a s it increased in both groups of m a t e d animals.
TABLE 3 BODY COMPOSITION ANALYSIS SUMMARY FOR ALL RAT GROUPS Group
Fat
Water
Ash
Protein
%
Training VSed VEx
7.57 --- 1.21 6.59 ± 1.49
68.1 ± 2.0 69.5 4- 1.0
4.02 --- 0.31 3.83 ± 0.20
19.86 ± 1.00 18.75 ___ 1.02
Pregnancy (at parturition) VSed VEx
9.78 ± 2.43 7.56 ± 1.62
66.0 ± 2.1 68.0 --- 1.2
4.07 --- 0.25 4.16 --- 0.16
19.44 ___ 0.94 19.64 ± 1.46
11.86 ± 2.57 6.97 --- 2.16
65.3 --- 1.9 69.1 --- 1.9
3.70 ± 0.17 4.05 ± 0.13
19.14 --- 1.60 19.77 --- 0.73
12.38 ± 1.88 7.34 ± 1.96
64.4 ± 1.5 67.9 ± 1.3
3.92 +-- 0.17 4.09 ± 0.16
19.24 ___ 1.45 20.43 --- 0.71
5.72 --- 1.39 3.53 --- 0.66
71.0 ± 1.3 72.8 ± 0.7
3.55 ± 0.25 3.82 ± 0.26
18.64 ± 0.71 18.72 ± 0.69
MSed MEx Lactation (day 14) VSed VEx MSed MEx
Results are presented as mean --- S.D. for eight animals in each group.
EXERCISE DURING RAT PREGNANCY AND LACTATION
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TABLE 4 ESTIMATED CARCASS ENERGY CONTENT OF RATS TERMINATED AT, OR CORRESPONDING TO THE TIME OF MATING, AT PARTURITION AND ON DAY 14 OF LACTATION
Estimated Carcass Energy Content* (kcal) Termination Time Mating Parturition Lactation
VSed
VEx
MSed
MEx
430.2 --- 4 2 . 4 541.1 --- 83.0 639.2 --- 81.1
425.2 ± 42.0 543.7 --- 93.9 574.3 ± 65.0
619.1 --- 66.2 520.8 ± 44.9
529.0 ___86.5 498.8 ± 28.0
*Values are means --- S.D. for eight animalsin each group. VSed= virgin, sedentary; VEx = virgin, exercised; MSed= mated, sedentary; MEx = mated, exercised. Carcass energy content was estimated as [carcass wt. (g) x % fat/100 × 9.4 kcal/g] + [carcass wt. (g) × % proteirdl00 x 5.65 kcal/g] (25).
Activity, F(1,56)=20.9, p<0.001, gravidity, F(1,56)=40.8, p<0.001, and stage, F(1,56)= 12.5, p<0.001, also had significant effects on percentage ash. In general, exercised animals had higher values for percent ash than sedentary animals; mated animals had lower values than nonmated animals; and lactating animals had lower values than nonlactating animals. No significant interactions were detected. The only significant finding from the 2 × 2 × 2 MANOVA of percent protein was an effect of gravidity, F(1,56) = 5.1, p<0.05, with mated animals tending to have lower values for percent protein than nonmated animals. MANOVA was also performed on 2 × 3 designs of the study (activity vs. stage, within each of the virgin and mated groups), allowing for the inclusion of the training stage. Significant activity by stage interactions on percentage fat, not detected by the 2 × 2 × 2 MANOVA, were revealed in both virgin, F(2,42) = 5.3, p<0.01, and mated rats, F(2,42) -- 5.54, p<0.01. In both groups, percent fat increased in sedentary rats and remained unchanged in exercised rats from the training stage to the end of the pregnancy stage. During the lactation stage, however, the percent carcass fat decreased in mated sedentary and exercised rats, increased in virgin sedentary rats, and remained unchanged in virgin exercised rats.
Estimated Carcass Energy Content Table 4 presents the results of estimated carcass energy content (CEC). In general, exercised rats had lower values for CEC than sedentary rats, and a 2 × 2 × 2 MANOVA revealed a significant main effect of activity, F(1,56) = 8.9, p<0.01. A significant effect of gravidity, F(1,56)= 5.9, p<0.05, and a significant stage by gravidity interaction effect, F(1,56)= 16.4, p<0.001 were also detected. Finally, a three-way interaction was detected among activity, gravidity and stage, F(1,56) = 4.7, p<0.05. These interaction effects reveal differences in the pattern of change in CEC from parturition to the end of lactation. Compared to values at parturition, mean CEC values at day 14 of lactation were higher in virgin sedentary rats, similar in virgin exercised rats, and lower in both mated sedentary and mated exercised rats.
Pregnancy Outcome and Offspring Growth Among the 32 animals delivering litters, three exercised and three sedentary rats each had one stillborn pup. Litter size averaged 12.3 ± 3.3 pups for the eight sedentary rats that continued to lactate, not significantly different from the mean litter size
of 13.2---4.7 for the eight exercised rats that continued through lactation. Mean pup birthweight was similar for pups born to sedentary and exercised rats, averaging 5.9 ± 0.5 g and 5.7 ___0.4 g, respectively. During the lactation period, when litters were culled to eight pups per dam, two pups nursed by sedentary dams died and one pup nursed by an exercising dam died. Growth of pups nursed by sedentary and exercised dams was virtually identical during the lactation period. Mean weights of pups nursed by sedentary and exercised dams, respectively, were 8.4--.0.9 g and 8.3 --- 1.2 g on day 3; 13.2__.2.3 g and 12.7-4-1.5 g on day 6; 18.4±2.1 g and 18.6±2.0 g on day 9; 24.6±2.4 g and 24.6±2.1 g on day 12; and 29.3---2.8 and 28.9±2.3 g on day 14. DISCUSSION
This study was conducted to assess the effects of moderately strenuous aerobic exercise, during rat pregnancy and lactation, on food intake, body weight and composition, pregnancy outcome and offspring growth. Data were obtained from exercised and sedentary mated rats, and from nonmated exercised and sedentary controls, permitting the effects of exercise during pregnancy to be compared to the effects of exercise alone. We found that exercise in both virgin and mated rats was associated with increased food intake, greater body weight, and lower percentage body fat, and that pregnancy outcome and offspring growth were similar in exercised and sedentary rats. In comparing our findings to those of other authors, several factors which may influence the results must be considered. For example, Pitts has suggested that discrepancies in the effect of exercise on body weight may be due to differences in the age and strain of the animals, and in the mode, intensity and duration of the exercise bout (26). Other factors may include the time of day at which the exercise was performed, as rats are normally more active during the dark cycle (9), and whether or not control animals were removed from access to food and drink during the time period when exercise was performed. Failure to do this would result in control animals having greater access to food, and could explain larger food intakes and greater body weight. This step, which was included in our protocol, was described by the author of only one previous study (18). Previous work has demonstrated that food intake increases with exercise in virgin female rats (15, 21, 23, 26, 27, 32), and also increases in nonexercised animals during pregnancy and lactation (7, 20, 30). Our findings of elevated food intakes in virgin exercised animals and in sedentary pregnant and lactating animals
432
are thus confu'matory. Two studies which reported food intake of pregnant rats subjected to swimming exercise reported opposite results: Savard et al. (28) did not detect a difference in food intakes of trained and sedentary rats, while Treadway et al. (33) found that the animals who swam consumed significantly more food during pregnancy. These authors also reported that the food intake of the swimmers remained elevated during lactation, although the difference between exercised and nonexercised animals was less marked than during pregnancy. We found that, except during the final week of gestation when a significant activity by gravidity interaction occurred, activity exerted an independent effect on food intake of both pregnant and lactating animals. The reason for the lack of effect of exercise during the final week of pregnancy is not obvious. However, because cellular proliferation of the gastrointestinal tract (10) and progressive increases in the size and weight of the alimentary canal (7) are reported to occur during lactation rather than pregnancy, it could be speculated that an anatomical limit to food intake may occur. Similar to their effects on food intake, both activity and gravidity were associated with increased body weight in the animals we studied. As alluded to previously, there are discrepancies in the literature regarding the effect of exercise on body weight of both virgin and pregnant rats. Many studies in virgin rats report that weight is maintained at control levels (2, 15, 21, 23), while others report that weights are decreased (18,32) or increased (27). None of these authors removed access to food from control animals during the exercise period. Our findings support those of Ring et al. (27); however, as discussed later, differences in body composition may mean that body weight is not a valid index of body energy content in exercised animals. Our finding of increased body weight in exercised pregnant animals was unexpected, as previous studies found exercised animals to weigh the same (28,33) or less than sedentary controls (4, 17, 18, 34). While Treadway's (33) and Savard's (28) studies, which found no difference in body weight, used a different strain of rat (Wistar or Zucker) and a different exercise mode (swimming), the strain and approximate age of the animals studied by Bagnall et al. (4) and Mottola et al. (18) were similar to those in the present study. Moreover, Mottola removed access to food from pregnant controls during the exercise period, although unfortunately food intake was not reported. The exercise differed only in the degree of incline of the treadmill (18% versus 0% in our study); accordingly, their animals exercised at a greater intensity (6). This could contribute to an explanation of the discrepancy: Mayer's early study of exercised female rats indicated that energy intake increases with activity only within a certain zone or level of activity (15). With extreme exercise, food intake no longer compensated, and lower body weights were observed (15). Alternately, Mottola's animals may have experienced a greater level of stress in association with the exercise such that food intake was impaired (12). To date, studies on the effects of exercise during rat pregnancy have not exposed control animals to the shock grid. It could be suggested that stress resulting from shock exposure may have affected food intake, body weight and composition (1,8). Pairing an exercised animal with a nonexercised animal and delivering an equal number of stimuli to the nonexercised pair member would have controlled for shock exposure. However, we felt that this could be an ineffective control, as the effects of unavoidable shock (as would be experienced by the nonexercised animal) may be more severe than those of an avoidable shock (as experienced by the exercised animal) (1, 8, 16). Moreover, it is often found that severe stress reduces food intake and body weight (8), whereas our exercised rats ate more and gained more weight. Thus, our data do not suggest that excessive levels of stress hormones in the
COURANT AND BARR
exercised animals were responsible for the results. Part of the difficulty in interpreting studies comparing body weights of exercised and sedentary animals arises from the consideration that body weight may not be a valid index of body energy stores. In the present study, MANOVA indicated that exercised animals generally weighed more than sedentary animals, yet their estimated CEC tended to be lower. For example, at parturition the average weight of mated exercised animals was 391.4 g, greater then the average weights of both sedentary mated (373.4 g) and virgin controls (268.2 g). Yet their estimated CEC averaged 529.0 kcal, less than the estimated CEC of sedentary mated rats (619.1 kcal) and similar to that of virgin controls which weighed over 120 g less (541.1 kcal). Exercise during pregnancy appeared to suppress the fat accumulation that normally occurs during gestation (14, 19, 20, 31). At parturition, exercised pregnant rats had a mean body fat percentage of 6.97%, similar to those of virgin animals terminated at the end of the training period (6.59% and 7.57% for exercised and sedentary rats, respectively). In contrast, sedentary pregnant animals averaged 11.86% fat. Comparable body composition data could not be located in the literature. However, Savard et al. found that lean Zucker rats exercised intensely (swimming) during pregnancy had parametrial, retroperitoneat and inguinal fat pads with lower weights and cell sizes than those of sedentary pregnant rats on day 21 of gestation (28). Mottola et al. found that the "skin" component, which included subcutaneous fat, weighed less in rats that ran during pregnancy than in sedentary controls (18). These data tend to support the findings of the present study. Despite lowered body fat stores, exercise did not appear to interfere with growth of lean body mass in the pregnant animals. Percent protein did not differ between sedentary and exercised groups, and because exercised animals tended to weigh more, their total body protein content would have been similar to or somewhat greater than that of the sedentary animals. During lactation, despite gains in body weight, both sedentary and exercised mated rats catabolized body fat, such that by day 14 of lactation, they averaged 5.72% and 3.53% fat, respectively. As a result, estimated CEC tended to be lower at the end of lactation than at parturition. While no comparable data could be located in the literature in regard to exercise during lactation, our findings in sedentary rats are comparable to previous reports in sedentary dams (14,20). Our results on pregnancy outcome and offspring growth indicate that moderately strenuous aerobic exercise continued during pregnancy and lactation had no detectable adverse effects. Previous reports of pregnancy outcome of rats trained prior to and during pregnancy at either mild (17,24) or strenuous levels (4, 18, 28, 33) have also shown no significant differences in litter size or mean birthweight. The only previous report of offspring growth of rats exercised during lactation is that of Treadway and Lederman (33). The offspring of rats who swam weighed 25.8 g on day 15, which was nonsignificantlylower than the mean weight of 28.9 g of offspring of sedentary rats. However, pups and sedentary dams were not separated during the time period when the trained animals were swimming, thus resulting in a shorter access time for feeding in the pups of trained dams. Mean weights of pups nursed by exercised and sedentary dams in the present study were virtually identical through day 14 of lactation. Thus, it appeared that the exercised animals, despite starting lactation without an increase in fat stores, compensated for this deficit through a small increase in food intake and through mobilizing available fat. It is also possible that they may have reduced their spontaneous activity during the remainder of the day, but we did not evaluate this. Pup survival and growth, however, were not compromised as a result of maternal exercise.
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ACKNOWLEDGEMENTS We wish to thank Diana J. Pollock for exceptional technical assistance during this study. This research was funded in part by a grant from the Natural Sciences and Engineering Research Council of Canada.
REFERENCES 1. Abott, B. B.; Schoen, L. S.; Badia, P. Predictable and unpredictable shock: behavioral measures of aversion and physiological measures of stress. Psychol. Bull. 96:45-71; 1984. 2. Applegate, E. A.; Upton, D. E.; Stern, J. S. Food intake, body composition and blood lipids following treadmill exercise in male and female rats. Physiol. Behav. 28:917-920; 1982. 3. Atkinson, T.; Fowler, V. R.; Garton, G. A.; Lough, A. K. A rapid method for determination of lipid in animal tissues. Analyst 97: 562-568; 1972. 4. Bagnall, K. M.; Mottola, M. F,; McFadden, K. D. The effects of strenuous exercise on maternal rats and their developing fetuses. Can. J. Appl. Sport Sci. 8:254-259; 1983. 5. Bedford, T. G.; Tipton, C. M.; Wilson, N. C.; Oppliger, R.; Gisolfi, C. V. Maximum oxygen consumption of rats and its changes with various experimental procedures. J. Appl. Physiol. 47:1278-1283; 1979. 6. Brooks, G. A.; White, T. P. Determination of metabolic and heart rate responses of rats to treadmill exercise. J. Appl. Physiol. 45:10091015; 1978. 7. Cripps, A. W.; Williams, V. J. The effect of pregnancy and lactation on food intake, gastrointestinal anatomy and the absorptive capacity of the small intestine in the albino rat. Br. J. Nutr. 33:17-32; 1975. 8. Dess, N. K.; Minor, T. R.; Brewer, J. Suppression of feeding and body weight by inescapable shock: Modulation by quinine adulteration, stress reinstatement, and controllability. Physiol. Behav. 45: 975-983; 1989. 9. Eikelboom, R.; Mills, R. A microanalysis of wheel running in male and female rats. Physiol. Behav. 43:625--630; 1988. 10. Fell, B. F.; Smith, K. A.; Campbell, R. M. Hypertrophic and hyperplastic changes in the alimentary canal of the lactating rat. J. Pathol. Bacteriol. 85:179-188; 1963. 11. Fukomoto, H.; Chang, G. W. Manual salicylate-hypochlorite procedure for determination of ammonia in Kjeldahl digests. J. Assoc. Off. Anal. Chem. 65:1076-1079; 1982. 12. Guilland, J. C.; Moreau, D.; Genet, J. M.; Klepping, J. Role of catecholamines in regulation by feeding of energy balance following chronic exercise in rats. Physiol. Behav. 42:365-370; 1988. 13. Hartsook, E. W.; Hershberger, T. V. A simplified method for sampling small animal carcasses for analyses. Soc. Exp. Biol. Med. 113:973-977; 1963. 14. Kanto, U.; Clawson, A. J. Effect of energy intake during pregnancy and lactation on body composition in rat. J. Nutr. 110:1829--1839; 1980. 15. Mayer, J.; Marshall, N. B.; Vitale, J. J.; Christensen, J. H.; Mashayekhi, M. B.; Stare, F. J. Exercise, food intake and body weight in normal rats and genetically obese adult mice. Am. J. Physiol. 177:544-548; 1954. 16. Momede, P.; Dantzer, R.; Michand, B.; Kelley, K. W.; Le Moal, M. Influence of stressor predictability and behavioral control on lymphocyte reactivity, antibody responses and neuroendocrine activation in
rats. Physiol. Behav. 43:577-583; 1988. 17. Mottola, M.; Bagnall, K. M.; McFadden, K. D. The effects of maternal exercise on developing rat fetuses, Br. J. Sports Med. 17:117-121; 1983. 18. Mottola, M. F.; Bagnall, K. M.; Belcastro, A. N.; Foster, J.; Secord, D. The effects of strenuous maternal exercise during gestation on maternal body components in rats. J. Anat. 148:65-75; 1986. 19. Naismith, D. J. The requirement for protein, and the utilization of protein and calcium during pregnancy. Metabolism 15:582-595; 1966. 20. Nalsmith, D. J.; Richardson, D. P.; Pritchard, A. E. The utilization of protein and energy during lactation in the rat, with particular regard to the use of fat accumulated in pregnancy. Br. J. Nutr. 48:433--441; 1982. 21. Nance, D. M.; Bromley, B,; Barnard, R. J.; Gorski, R. A. Sexually dimorphic effects of forced exercise on food intake and body weight in the rat. Physiol. Behav. 19:155-158; 1977. 22. Nie, N. H. SPSSx user's guide. New York: McGraw-Hill Book Company; 1983. 23. Oscai, L. B.; Mole, P. A.; Krusack, L. M.; Holloszy, J. O. Detailed body composition analysis on female rats subjected to a program of swimming. J. Nutr. 103:412--418; 1973. 24. Parizkova, J.; Petrasek, R. The impact of daily work load during pregnancy on lipid metabolism in the liver of the offspring. Eur. J. Appl. Physiol. 39:81-87; 1978. 25. Pike, R. L.; Brown, M. L. Nutrition. An integrated approach. 3rd edition. New York: John Wiley & Sons; 1984. 26. Pitts, G. C. Body composition in the rat: interactions of exercise, age, sex, and diet. Am. J. Physiol. 246:R495-R501; 1984. 27. Ring, G. C.; Bosch, M.; Lo, C. Effects of exercise on growth, resting metabolism, and body composition of Fischer rats. Soc. Exp. Biol. Med. 133:1162-1165; 1970. 28. Savard, R.; Palmer, J. E.; Greenwood, M. R. C. Effects of exercise training on regional adipose tissue metabolism in pregnant rats. Am. J. Physiol. 250:R837-R844; 1986. 29. Shepherd, R. E.; Gollnick, P. D. Oxygen uptake of rats at different work intensities. Pflugers Arch. 362:219-222; 1976. 30. Shirley, B. The food intake of rats during pregnancy and lactation. Lab. Anim. Sci. 34:169-172; 1984. 31. Spray, C. M. A study of some aspects of reproduction by means of chemical analysis. Br. J. Nutr. 4:354-360; 1950. 32. Tokuyama, K.; Saito, M.; Okuda, H. Effects of wheel running on food intake and weight gain of male and female rats. Physiol. Behav. 28:899--903; 1982. 33. Treadway, J. L.; Lederman, S. A. The effects of exercise on milk yield, milk composition, and offspring growth in rats. Am. J. Clin. Nutr. 44:481-488; 1986. 34. Wilson, N. C.; Gisolfi, C. V. Effects of exercising rats during pregnancy. J. Appl. Physiol. 48:34-40; 1980.
0031-9384/90 $3.00 + .DO
Exercise During Rat Pregnancy and Lactation: Maternal Effects and Offspring Growth G E N E V I E V E T. C O U R A N T 1 A N D S U S A N I. B A R R 2
School of Family and Nutritional Sciences, University of British Columbia 2205 East Mall, Vancouver, B.C., Canada, V6T 1 W5 R e c e i v e d 5 S e p t e m b e r 1989
COURANT, G. T. AND S. I. BARR. Exerciseduring rat pregnancy and lactation: Maternaleffects and offspring growth. PHYSIOL BEHAV 47(3) 427-433, 1990.--Eighty Spragne-Dawley rats were divided into exercised (E) and sedentary (S) groups. E rats were trained to run on a treadmill (30 m/min, 2 hr/day). Within each group, two subgroups were mated and three served as virgin time controls. Of the mated subgroups, one was terminated within 24 hours of delivery and the other on day 14 of lactation. Subgroups of virgin S and E controls were terminated at times corresponding to the mating, delivery and lactation day 14 of mated rats. MANOVA revealed that exercise significantly affected food intake, body weight and body composition in both virgin and mated animals: generally, E rats ate more, gained more weight, and had less carcass fat than S controls. E rats did not store fat during pregnancy. At parturition, they were 7.0% fat, similar to both E (6.6%) and S (7.6%) controls prior to mating, and less than S rats at parturition (11.9%). Despite diminished fat stores at parturition in E rats, litter size and pup birthweight were similar in E and S rats, as was offspring growth during lactation (mean weights on day 14 of 28.9 g and 29.3 g, respectively). Remaining body fat and increased food intake were adequate to support normal pup growth. Exercise
Food intake
Body composition
Pregnancy
RAT pregnancy is characterized by weight gain, an increase in food intake, and increased fat deposition (19, 20, 30, 31). During lactation the additional fat stores are largely catabolized while food intake increases further to support the high energy cost of milk production (7, 14, 20). Similar to pregnancy, exercise in nongravid female rats generally results in increased food intake, but in contrast to pregnancy, body fat is usually decreased and weight may remain the same or decrease (2, 15, 21, 23). The effects of exercise on these parameters in pregnant and especially in lactating rats have not been well documented. This subject is of interest since exercise and pregnancy exert opposing effects on fat deposition. If exercise training is superimposed on rat pregnancy, would fat stores be reduced? If so, would lactation performance and offspring growth be compromised, or would food intake during pregnancy and/or lactation increase sufficiently to compensate for the lowered fat stores? While the majority of rat studies have found that, even at strenuous levels, maternal aerobic exercise does not significantly affect gross fetal development or pregnancy outcome, it does appear to lower maternal weight gain in most (4, 17, 18, 34) but not all (28,33) studies. The lower maternal weight gain has been suggested, though not directly demonstrated, to reflect a reduction in maternal fat stores (18). The food intake of the animals in this
Lactation
study was not reported: thus, it is not known whether changes in their food intake could have contributed to or modulated the observed weight decrements. In this study, we sought to determine the effects of moderate aerobic exercise on food intake, body composition, body weight and offspring growth in female nonpregnant, pregnant and lactating rats. In order to separate the effects of exercise from those of pregnancy and lactation, our study design included groups of exercising and sedentary pregnant and lactating rats, as well as groups of age-matched nongravid exercising and sedentary controls. METHOD
Experimental Design and Protocol A modified 2 x 2 × 3 factoriai design was used for the experiment. Rats were equally divided into exercised and sedentary groups (factor = "activity"). The exercised rats ran on a treadmill while the sedentary rats followed a normal laboratory routine. Each of these two groups was divided into five subgroups (n = 8/subgroup), two of which were mated and three of which were not (factor = "gravidity"). Of the mated subgroups, one was
~Present address: 33 rue de Geneve, St. Genis-Pouilly, France F-01630. 2Re.quests for reprints should be addressed to Susan I. Bart.
427
428
COURANT AND BARR
ACCLIMATIZATION TRAINING
WEEK
0
1
2
MATING (I- Bdays)
3
0
RANDOMIZE
TRAINON
TERMINATE
RATS INTO GROUPS
TREADMILL EVT EVP
EVT SVT
EVL EMP EML
MATE EMP SMP
PREGNANCY
1
2
LACTATION
3
( DELIVERY)
1
2
(LACTATION DAY 14)
TERMINATE
EML SML
EVP EMP SVP SMP
TERMINATE EVL EML SVL SML
FIG. 1. Experimental protocol. E= exercised, S = sedentary ("activity" factor); V = virgin, M = mated ("gravidity" factor); T = terminated at time corresponding to end of training, P = terminated at time corresponding to parturition, L=terminated at time corresponding to day 14 of lactation ("stage" factor).
terminated within 24 hours of delivering its pups and the other was terminated after 14 days of lactation. The three unmated or virgin subgroups were terminated at the times corresponding to the mating, delivery, or day 14 of lactation of the mated animals (factor= "stage"). Thus sedentary and exercised virgin rats were used as age controls for these three time periods in the experiment. The experimental protocol is summarized in Fig. 1.
Animals Eighty virgin female Sprague-Dawley rats, weighing between 170 and 190 grams, were obtained from Charles River Breeding Laboratories, Hull, Canada. On arrival they were housed communally and two days later were randomized into the 10 experimental subgroups and housed individually in stainless steel screen bottom cages. During the five-day adjustment period, the animals were acclimatized to an inverted 12-hour on, 12-hour off light cycle (with lights on at 1:00 p.m.), and to a room temperature of 21°C. Pregnant rats were placed in individual plastic cages with corn cob bedding (San-i-cel, Paxton Processing, Paxton, IL) on day 19 of pregnancy, where they remained until terminated. Animals were given ad lib access to a ground commercial nonpurified diet (5001, Ralston Purina Canada, Longueil, Quebec) and fresh water, except during the exercise period, as described below.
Exercise Following the adjustment period, the rats assigned to exercising groups were trained progressively over a period of three weeks, six days per week, to run on a motor-driven treadmill. Training and subsequent exercise took place in the morning near the end of the dark cycle. At the end of the training period, the rats were capable of running at 30 rn/min on a 0% grade for two consecutive hours. The treadmill was equipped with a shock grid that delivered a mild electric shock when the animals did not run at speed. No animals were injured due to the use of the grid. After the training period, the rats continued to run at 30 m/min for two hours per day, five consecutive days per week until they were terminated. Pregnant rats ran up to and including day 20 of pregnancy, and if allowed to lactate, resumed exercise on day two of lactation. In order to prevent the possible overheating of the animals, an electric fan was directed over the treadmill. On the basis of measurements of oxygen consumption of rats running at various speeds and on various grades (5, 6, 29), it was
estimated that the rats in this study were consuming oxygen at the rate of approximately 60 ml/kg/min, or 70% VO2 max, a moderately strenuous aerobic workload. During the exercise period, access to food and water was removed from all animals. Sedentary lactating dams were also removed from their pups during this period.
Mating Following the training period, rats to be mated were housed overnight on a one-to-one basis with male Sprague-Dawley rats. The finding of a vaginal plug and/or the presence of sperm in the vaginal washings the following morning was taken as day 0 of pregnancy. Following delivery, litters of lactating dams were culled to eight, including four males and four females when possible.
Measurements Food intake was measured daily. Spillage, which was minimal due to the use of covered food cups, was accounted for. All animals were weighed every third day. Pregnant rats were also weighed on day 0 of pregnancy and then every third day. A postpartum weight was taken within 24 hours of delivery, and lactating dams were weighed every third day and on day 14 of lactation. Rat pups were weighed within 24 hours of birth and then every third day and on day 14.
Termination Animals were weighed, anaesthetized with ether and decapitated. Carcasses, including the head, were reweighed and stored individually in polyethylene bags at - 2 0 ° C .
Carcass Preparation and Analysis Preparation for sampling and analysis of the rat carcasses was based on the method of Hartsook and Hershberger (13). Frozen carcasses were individually autoclaved (Model STM-E, Market Forge Co., Everett, MA) in approximately 150 ml of distilled water for three hours at 115°C and two atmospheres pressure, and then were homogenized in a Waring blender at high speed for 10 minutes with enough distilled water to achieve approximately a 1:1 ratio of carcass weight to water added. The blender contents were then poured through a household wiremesh sieve to crudely separate the hair from the remainder of the mixture. The hair and adhering material, or " h a i r , " as it is hereafter referred to, was placed on a petri dish, weighed and stored at -20°(]. The remaining mixture, hereafter referred to as the homogenate, was quantitatively transferred back to the Wafing blender and blended again at high speed for 30 seconds to assure homogenization. (This separation allowed accurate sampling of the two fractions of the rat carcass. The composition of the whole carcass was then calculated from results of analysis of the two fractions.) Five samples of the homogenate were immediately taken using a large bore bulb syringe and placed in each of five petri dishes. These were weighed and stored at - 20°C. The frozen homogenate and " h a i r " were then freeze-dried (75150 Labconco Tray Freeze Dryer, Labconco Corp., Kansas City, MO) and the freeze-dried homogehate (not the " h a i r " ) was ground to a fine powder using a household coffee grinder (Moulinex) and stored at - 2 0 ° C . A measure of the water lost during freeze-drying of the homogenate and " h a i r " of each carcass and the weights recorded during the preparation of the carcass for analysis allowed the calculation of the rat's total body water content. Triplicate samples of both powdered homogenate and the " h a i r " were ashed in a
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TABLE 1 WEEKLY FOODINTAKESOF RATS DURINGTHE TRAINING,PREGNANCYAND LACTATIONPERIODS Food Intake (g/week) Time Period
VSed
VEx
MANOVA
MSed
MEx
Act
Grav
AxG
ns * :~
----
----
Training
week l 2 3
120.8 -+ 13.8 126.6 - 11.6 126.1 ~ 14.6
120.8 --- 12.2 133.3 -+ 11.5 143.3 --- 15.2
Pregnancy
week 1 2 3
130.7 ~ 1 4 . 5 130.5 - 1 1 . 9 126.7 -*- 12.0
150.8 -+ 12.7 147.8 - 15.7 155.1 --- 18.3
155.3 - 14.6 156.8 _ 1 4 . 7 175.3 --- 1 9 . 8
167.3 ~ 15.8 168.6 _ 19.7 177.4 _ 21.7
~: :~ :~
~t :~ :~
ns ns t
Lactation
week 1 2
129.2 -- 8.0 131.1 _ 8.1
142.3 -4- 20.1 155.6 -+ 17.8
214.8 ___ 20.3 366.3 _+ 33.9
237.9 -+ 11.4 380.0 _ 31.5
~" *
:~ :~
ns ns
Results are presented as mean +__ S.D. for 8 animals per group. VSed=virgin, sedentary; VEx=virgin, exercised; MSed= mated, sedentary; MEx = mated, exercised. Significance of effects of Act (activity), Gray (gravidity) and A x G (activity by gravidity interaction) was as follows: *p<0.05; tp<0.01; :~p<0.00l.
muffle furnace (Thermolyne Model 2000, Thermolyne Corp., Dubuque, IA) at 650°C for 18 hours. The percent fat content of duplicate analysis of the powdered homogenate and " h a i r " was determined by the method of Atklnson et al. (3). Triplicate determinations of the nitrogen content of duplicate samples were obtained for both the rat homogenate and " h a i r " using the method of Fukomoto and Chang (11) and an ammonium sulfate solution for standards. The mean percentage of total body weight accounted for by analysis of water, fat, ash and protein was 99.5-+1.1%. It was expected to be less than 100% since the carbohydrate component of the carcass was not analyzed and therefore not included in the total. A rough approximation of the carcass energy content (CEC) was calculated, as follows, for each rat: CEC = [carcass wt. (g) x % fat/100 x 9.4 kcal/g] + [carcass wt.(g) × % protein/100 × 5.65 kcal/g] (25).
Statistical Analysis Statistical analyses were performed using programs available in SPSSx (22). Multivariate analysis of variance (MANOVA) was used to test for the main effects of activity and gravidity on food intake and body weight during each of the training, pregnancy and lactation periods. M A N O V A was also used to test for main effects of activity, and gravidity and stage on body composition (fat, water, ash and protein) and for the estimated carcass energy content. Because the modified factorial design did not permit a complete 2 x 2 × 3 analysis, a 2 × 2 x 2 analysis was performed, including only two levels of the stage factor (pregnancy and lactation). MANOVA was also performed on a 2 × 3 design of the experiment (activity vs. stage for each of the virgin and mated subgroups). This allowed the inclusion of the training stage factor excluded in the analysis of the 2 × 2 × 2 design, t-Tests were used to determine the effect of maternal activity on litter size and offspring growth. All results are presented as mean---standard deviation. The level of statistical significance was set at p<0.05. RESULTS
Food Intake Mean weekly food intakes and results of MANOVA are presented in Table 1. Both activity and gravidity significantly
affected food intake throughout the experiment, beginning at week two of training for activity and week one of pregnancy for gravidity. Exercised animals ate more than their sedentary counterparts, and pregnant animals consumed more than their nonpregnant counterparts. The only significant interaction occurred between activity and gravidity at week three of pregnancy. While both exercised and sedentary pregnant groups consumed more than their respective nonpregnant controls, the effect of activity appeared to be confined to the virgin animals. There was a large difference in the food intakes of the virgin sedentary and exercised animals, but no difference in food intakes of pregnant sedentary and exercised animals.
Body Weight As with food intake, results of MANOVA presented in Table 2 show that activity had a significant effect on body weight from week two of the training period to the end of the lactation period. Exercised virgin or mated animals consistently weighed more than sedentary controls. The effect of gravidity was significant beginning at week one of the pregnancy period, and with the exception of the parturition time point, remained so throughout the experiment. At parturition, the weight of mated rats decreased to a level which no longer differed significantly from that of the unmated controls. As lactation progressed, however, the mated rats gained more weight that their virgin controls, such that they were once again heavier.
Body Composition A summary of the body composition analyses for all rat groups is presented in Table 3. Results of MANOVA performed on a 2 × 2 × 2 design, in which the training stage was deleted from the analysis, revealed that percentage body fat was significantly affected by activity, F(1,56)= 55.7, p<0.001, gravidity, F(1,5) = 21.9, p<0.001, and stage, F(1,56) = 14.0, p<0.001. The effect of activity can be observed in the consistently lower values for percentage fat in both virgin and mated exercised rats than in sedentary controls. The effects of gravidity and stage, and the significant interaction, F(1,56) = 38.9, p<0.001, between these
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TABLE 2 BODY WEIGHTS OF RATS DURING THE TRAINING, PREGNANCY AND LACTATION PERIODS Body Weight (g) Time Period
VSed
± ± -----
7.9 10.0 13.3 16.8
Act
Grav
A×G
ns ns * $
-----
-----
195.8 212.6 224.9 234.6
Pregnancy ( n = 16/group)
week 1 2 3
255.2 --- 14.8 260.8 ± 16.6 268.2 --- 16.9
280.6 --- 16.2 287.4 ± 20.6 299.2 --- 25.5
273.0 ___ 12.2 305.5 ± 16.0 373.4 --+ 26.0
296.1 --- 16.1 327.2 ± 20.7 391.4 ± 33.3
$ :~ :~
:~ :~ $
ns ns ns
268.2 ± 21.6
298.7 -+ 25.1
281.9 ± 14.2
298.5 -+ 16.9
:~
ns
ns
279.3 -+ 17.8 284.0 --- 16.7
306.3 ± 19.1 311.4 ± 18.9
296.6 ± 16.0 327.4 ± 21.6
329.1 ± 15.9 359.0 ± 26.2
~: :~
t ~:
ns ns
week 1 2
193.3 211.9 231.6 253.3
MEx
week 0 1 2 3
Lactation (n=8/group)
10.4 10.7 11.7 13.3
MSed
Training (n=40/group)
parturition
± ± -----
VEx
MANOVA
Results are presented as mean ± S.D. VSed=virgin, sedentary; VEx=virgin, exercised; MSed=mated, sedentary; MEx = mated, exercised. Significance of effects of Act (activity), Grav (gravidity) and A × G (activity by gravidity interaction) are as follows: *p<0.05; tp<0.01; ~:p<0.001.
m a i n effects, are revealed by the tendency for percent b o d y fat to be similar or h i g h e r in m a t e d than virgin a n i m a l s during pregn a n c y , but lower during lactation. Finally, a three-way interaction a m o n g activity, gravidity and stage, F ( 1 , 5 6 ) = 8 . 2 , p < 0 . 0 1 , is explained by the finding that during the p r e g n a n c y stage, m a t i n g appeared to be associated with h i g h e r values for percent fat in sedentary but not in exercised animals, w h e r e a s during the lactation stage, m a t i n g was associated with lower values for b o d y fat in both activity groups. T h e pattern o f findings f r o m 2 × 2 × 2 M A N O V A of percentage b o d y water w a s very similar to those for percentage fat, but c h a n g e s were generally in the opposite direction. T h e significant effect o f activity, F ( 1 , 5 6 ) = 5 0 . 8 , p < 0 . 0 0 1 , is revealed in the
consistently higher values for percentage water in exercised animals. Significant effects o f gravidity, F(1,56) = 58.3, p < 0 . 0 0 1 , and stage, F ( 1 , 5 6 ) = 2 4 . 1 , p < 0 . 0 0 1 , and the significant interaction b e t w e e n these two effects, F ( 1 , 5 6 ) = 5 1 . 2 , p < 0 . 0 0 1 , are indicated by the finding that values for percentage water were similar in virgin and m a t e d a n i m a l s during the p r e g n a n c y stage, w h e r e a s during the lactation stage, they increased in the m a t e d a n i m a l s and tended to decrease in the virgin animals. A three-way interaction a m o n g activity, gravidity a n d stage, F ( 1 , 5 6 ) = 5 . 1 , p < 0 . 0 5 , is illustrated by the finding that b e t w e e n p r e g n a n c y and lactation, percentage water appeared to decrease in sedentary but not exercised virgin animals, w h e r e a s it increased in both groups of m a t e d animals.
TABLE 3 BODY COMPOSITION ANALYSIS SUMMARY FOR ALL RAT GROUPS Group
Fat
Water
Ash
Protein
%
Training VSed VEx
7.57 --- 1.21 6.59 ± 1.49
68.1 ± 2.0 69.5 4- 1.0
4.02 --- 0.31 3.83 ± 0.20
19.86 ± 1.00 18.75 ___ 1.02
Pregnancy (at parturition) VSed VEx
9.78 ± 2.43 7.56 ± 1.62
66.0 ± 2.1 68.0 --- 1.2
4.07 --- 0.25 4.16 --- 0.16
19.44 ___ 0.94 19.64 ± 1.46
11.86 ± 2.57 6.97 --- 2.16
65.3 --- 1.9 69.1 --- 1.9
3.70 ± 0.17 4.05 ± 0.13
19.14 --- 1.60 19.77 --- 0.73
12.38 ± 1.88 7.34 ± 1.96
64.4 ± 1.5 67.9 ± 1.3
3.92 +-- 0.17 4.09 ± 0.16
19.24 ___ 1.45 20.43 --- 0.71
5.72 --- 1.39 3.53 --- 0.66
71.0 ± 1.3 72.8 ± 0.7
3.55 ± 0.25 3.82 ± 0.26
18.64 ± 0.71 18.72 ± 0.69
MSed MEx Lactation (day 14) VSed VEx MSed MEx
Results are presented as mean --- S.D. for eight animals in each group.
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TABLE 4 ESTIMATED CARCASS ENERGY CONTENT OF RATS TERMINATED AT, OR CORRESPONDING TO THE TIME OF MATING, AT PARTURITION AND ON DAY 14 OF LACTATION
Estimated Carcass Energy Content* (kcal) Termination Time Mating Parturition Lactation
VSed
VEx
MSed
MEx
430.2 --- 4 2 . 4 541.1 --- 83.0 639.2 --- 81.1
425.2 ± 42.0 543.7 --- 93.9 574.3 ± 65.0
619.1 --- 66.2 520.8 ± 44.9
529.0 ___86.5 498.8 ± 28.0
*Values are means --- S.D. for eight animalsin each group. VSed= virgin, sedentary; VEx = virgin, exercised; MSed= mated, sedentary; MEx = mated, exercised. Carcass energy content was estimated as [carcass wt. (g) x % fat/100 × 9.4 kcal/g] + [carcass wt. (g) × % proteirdl00 x 5.65 kcal/g] (25).
Activity, F(1,56)=20.9, p<0.001, gravidity, F(1,56)=40.8, p<0.001, and stage, F(1,56)= 12.5, p<0.001, also had significant effects on percentage ash. In general, exercised animals had higher values for percent ash than sedentary animals; mated animals had lower values than nonmated animals; and lactating animals had lower values than nonlactating animals. No significant interactions were detected. The only significant finding from the 2 × 2 × 2 MANOVA of percent protein was an effect of gravidity, F(1,56) = 5.1, p<0.05, with mated animals tending to have lower values for percent protein than nonmated animals. MANOVA was also performed on 2 × 3 designs of the study (activity vs. stage, within each of the virgin and mated groups), allowing for the inclusion of the training stage. Significant activity by stage interactions on percentage fat, not detected by the 2 × 2 × 2 MANOVA, were revealed in both virgin, F(2,42) = 5.3, p<0.01, and mated rats, F(2,42) -- 5.54, p<0.01. In both groups, percent fat increased in sedentary rats and remained unchanged in exercised rats from the training stage to the end of the pregnancy stage. During the lactation stage, however, the percent carcass fat decreased in mated sedentary and exercised rats, increased in virgin sedentary rats, and remained unchanged in virgin exercised rats.
Estimated Carcass Energy Content Table 4 presents the results of estimated carcass energy content (CEC). In general, exercised rats had lower values for CEC than sedentary rats, and a 2 × 2 × 2 MANOVA revealed a significant main effect of activity, F(1,56) = 8.9, p<0.01. A significant effect of gravidity, F(1,56)= 5.9, p<0.05, and a significant stage by gravidity interaction effect, F(1,56)= 16.4, p<0.001 were also detected. Finally, a three-way interaction was detected among activity, gravidity and stage, F(1,56) = 4.7, p<0.05. These interaction effects reveal differences in the pattern of change in CEC from parturition to the end of lactation. Compared to values at parturition, mean CEC values at day 14 of lactation were higher in virgin sedentary rats, similar in virgin exercised rats, and lower in both mated sedentary and mated exercised rats.
Pregnancy Outcome and Offspring Growth Among the 32 animals delivering litters, three exercised and three sedentary rats each had one stillborn pup. Litter size averaged 12.3 ± 3.3 pups for the eight sedentary rats that continued to lactate, not significantly different from the mean litter size
of 13.2---4.7 for the eight exercised rats that continued through lactation. Mean pup birthweight was similar for pups born to sedentary and exercised rats, averaging 5.9 ± 0.5 g and 5.7 ___0.4 g, respectively. During the lactation period, when litters were culled to eight pups per dam, two pups nursed by sedentary dams died and one pup nursed by an exercising dam died. Growth of pups nursed by sedentary and exercised dams was virtually identical during the lactation period. Mean weights of pups nursed by sedentary and exercised dams, respectively, were 8.4--.0.9 g and 8.3 --- 1.2 g on day 3; 13.2__.2.3 g and 12.7-4-1.5 g on day 6; 18.4±2.1 g and 18.6±2.0 g on day 9; 24.6±2.4 g and 24.6±2.1 g on day 12; and 29.3---2.8 and 28.9±2.3 g on day 14. DISCUSSION
This study was conducted to assess the effects of moderately strenuous aerobic exercise, during rat pregnancy and lactation, on food intake, body weight and composition, pregnancy outcome and offspring growth. Data were obtained from exercised and sedentary mated rats, and from nonmated exercised and sedentary controls, permitting the effects of exercise during pregnancy to be compared to the effects of exercise alone. We found that exercise in both virgin and mated rats was associated with increased food intake, greater body weight, and lower percentage body fat, and that pregnancy outcome and offspring growth were similar in exercised and sedentary rats. In comparing our findings to those of other authors, several factors which may influence the results must be considered. For example, Pitts has suggested that discrepancies in the effect of exercise on body weight may be due to differences in the age and strain of the animals, and in the mode, intensity and duration of the exercise bout (26). Other factors may include the time of day at which the exercise was performed, as rats are normally more active during the dark cycle (9), and whether or not control animals were removed from access to food and drink during the time period when exercise was performed. Failure to do this would result in control animals having greater access to food, and could explain larger food intakes and greater body weight. This step, which was included in our protocol, was described by the author of only one previous study (18). Previous work has demonstrated that food intake increases with exercise in virgin female rats (15, 21, 23, 26, 27, 32), and also increases in nonexercised animals during pregnancy and lactation (7, 20, 30). Our findings of elevated food intakes in virgin exercised animals and in sedentary pregnant and lactating animals
432
are thus confu'matory. Two studies which reported food intake of pregnant rats subjected to swimming exercise reported opposite results: Savard et al. (28) did not detect a difference in food intakes of trained and sedentary rats, while Treadway et al. (33) found that the animals who swam consumed significantly more food during pregnancy. These authors also reported that the food intake of the swimmers remained elevated during lactation, although the difference between exercised and nonexercised animals was less marked than during pregnancy. We found that, except during the final week of gestation when a significant activity by gravidity interaction occurred, activity exerted an independent effect on food intake of both pregnant and lactating animals. The reason for the lack of effect of exercise during the final week of pregnancy is not obvious. However, because cellular proliferation of the gastrointestinal tract (10) and progressive increases in the size and weight of the alimentary canal (7) are reported to occur during lactation rather than pregnancy, it could be speculated that an anatomical limit to food intake may occur. Similar to their effects on food intake, both activity and gravidity were associated with increased body weight in the animals we studied. As alluded to previously, there are discrepancies in the literature regarding the effect of exercise on body weight of both virgin and pregnant rats. Many studies in virgin rats report that weight is maintained at control levels (2, 15, 21, 23), while others report that weights are decreased (18,32) or increased (27). None of these authors removed access to food from control animals during the exercise period. Our findings support those of Ring et al. (27); however, as discussed later, differences in body composition may mean that body weight is not a valid index of body energy content in exercised animals. Our finding of increased body weight in exercised pregnant animals was unexpected, as previous studies found exercised animals to weigh the same (28,33) or less than sedentary controls (4, 17, 18, 34). While Treadway's (33) and Savard's (28) studies, which found no difference in body weight, used a different strain of rat (Wistar or Zucker) and a different exercise mode (swimming), the strain and approximate age of the animals studied by Bagnall et al. (4) and Mottola et al. (18) were similar to those in the present study. Moreover, Mottola removed access to food from pregnant controls during the exercise period, although unfortunately food intake was not reported. The exercise differed only in the degree of incline of the treadmill (18% versus 0% in our study); accordingly, their animals exercised at a greater intensity (6). This could contribute to an explanation of the discrepancy: Mayer's early study of exercised female rats indicated that energy intake increases with activity only within a certain zone or level of activity (15). With extreme exercise, food intake no longer compensated, and lower body weights were observed (15). Alternately, Mottola's animals may have experienced a greater level of stress in association with the exercise such that food intake was impaired (12). To date, studies on the effects of exercise during rat pregnancy have not exposed control animals to the shock grid. It could be suggested that stress resulting from shock exposure may have affected food intake, body weight and composition (1,8). Pairing an exercised animal with a nonexercised animal and delivering an equal number of stimuli to the nonexercised pair member would have controlled for shock exposure. However, we felt that this could be an ineffective control, as the effects of unavoidable shock (as would be experienced by the nonexercised animal) may be more severe than those of an avoidable shock (as experienced by the exercised animal) (1, 8, 16). Moreover, it is often found that severe stress reduces food intake and body weight (8), whereas our exercised rats ate more and gained more weight. Thus, our data do not suggest that excessive levels of stress hormones in the
COURANT AND BARR
exercised animals were responsible for the results. Part of the difficulty in interpreting studies comparing body weights of exercised and sedentary animals arises from the consideration that body weight may not be a valid index of body energy stores. In the present study, MANOVA indicated that exercised animals generally weighed more than sedentary animals, yet their estimated CEC tended to be lower. For example, at parturition the average weight of mated exercised animals was 391.4 g, greater then the average weights of both sedentary mated (373.4 g) and virgin controls (268.2 g). Yet their estimated CEC averaged 529.0 kcal, less than the estimated CEC of sedentary mated rats (619.1 kcal) and similar to that of virgin controls which weighed over 120 g less (541.1 kcal). Exercise during pregnancy appeared to suppress the fat accumulation that normally occurs during gestation (14, 19, 20, 31). At parturition, exercised pregnant rats had a mean body fat percentage of 6.97%, similar to those of virgin animals terminated at the end of the training period (6.59% and 7.57% for exercised and sedentary rats, respectively). In contrast, sedentary pregnant animals averaged 11.86% fat. Comparable body composition data could not be located in the literature. However, Savard et al. found that lean Zucker rats exercised intensely (swimming) during pregnancy had parametrial, retroperitoneat and inguinal fat pads with lower weights and cell sizes than those of sedentary pregnant rats on day 21 of gestation (28). Mottola et al. found that the "skin" component, which included subcutaneous fat, weighed less in rats that ran during pregnancy than in sedentary controls (18). These data tend to support the findings of the present study. Despite lowered body fat stores, exercise did not appear to interfere with growth of lean body mass in the pregnant animals. Percent protein did not differ between sedentary and exercised groups, and because exercised animals tended to weigh more, their total body protein content would have been similar to or somewhat greater than that of the sedentary animals. During lactation, despite gains in body weight, both sedentary and exercised mated rats catabolized body fat, such that by day 14 of lactation, they averaged 5.72% and 3.53% fat, respectively. As a result, estimated CEC tended to be lower at the end of lactation than at parturition. While no comparable data could be located in the literature in regard to exercise during lactation, our findings in sedentary rats are comparable to previous reports in sedentary dams (14,20). Our results on pregnancy outcome and offspring growth indicate that moderately strenuous aerobic exercise continued during pregnancy and lactation had no detectable adverse effects. Previous reports of pregnancy outcome of rats trained prior to and during pregnancy at either mild (17,24) or strenuous levels (4, 18, 28, 33) have also shown no significant differences in litter size or mean birthweight. The only previous report of offspring growth of rats exercised during lactation is that of Treadway and Lederman (33). The offspring of rats who swam weighed 25.8 g on day 15, which was nonsignificantlylower than the mean weight of 28.9 g of offspring of sedentary rats. However, pups and sedentary dams were not separated during the time period when the trained animals were swimming, thus resulting in a shorter access time for feeding in the pups of trained dams. Mean weights of pups nursed by exercised and sedentary dams in the present study were virtually identical through day 14 of lactation. Thus, it appeared that the exercised animals, despite starting lactation without an increase in fat stores, compensated for this deficit through a small increase in food intake and through mobilizing available fat. It is also possible that they may have reduced their spontaneous activity during the remainder of the day, but we did not evaluate this. Pup survival and growth, however, were not compromised as a result of maternal exercise.
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ACKNOWLEDGEMENTS We wish to thank Diana J. Pollock for exceptional technical assistance during this study. This research was funded in part by a grant from the Natural Sciences and Engineering Research Council of Canada.
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