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Experimental Physiology ? Research Paper Can physical exercise during gestation attenuate the effects of a maternal perinatal low-protein diet on oxygen consumption in rats? Marco Fidalgo Amorim1, Jose´ Antonio dos Santos2, Sandro Massao Hirabara3, Elizabeth Nascimento1, Sandra Lopes de Souza4, Raul Manha?es de Castro1, Rui Curi5 and Carol Go´is Leandro6 1Department of Nutrition, 2Superior School of Physical Education and 4Department of Anatomy, Federal University of Pernambuco, Brazil 3Institute of Physical Activity Sciences and Sports, Cruzeiro do Sul University, Brazil 5Institute of Biomedical Science, University of Sao Paulo, Brazil 6Department of Nutrition, Centro Academico Vitoria, Federal University of Pernambuco, Brazil A protocol of physical exercise, based on maximal oxygen uptake (V?O2max), for female rats before and during pregnancy was developed to evaluate the impact of a low-protein diet on oxygen consumption during gestation and growth rate of the offspring. Virgin female Wistar rats were divided into four groups as follows: untrained (NT, n = 5); trained (T, n = 5); untrained with low-protein diet (NT+LP, n = 5); and trained with low-protein diet (T+LP, n = 5). Trained rats were submitted to aprotocol ofmoderatephysical trainingona treadmill over aperiodof 4 weeks (5 days week?1 and 60 min day?1, at 65% of V?O2max). At confirmation of pregnancy, the intensity and duration of the exercise was reduced. Low-protein groups received an 8% casein diet, and their peers received a 17%casein diet. The birthweight and growth rate of the pups up to the 90th day were recorded. Oxygen consumption (V?O2), CO2 production and respiratory exchange ratio (RER) were determined using an indirect open-circuit calorimeter. Exercise training increased V?O2max by about 20% when compared with the initial values (45.6 ± 1.0 ml kg ?1 min?1). During gestation, all groups showed a progressive reduction in the resting V?O2 values. Dams in the NT+LP group showed lower values of resting V?O2 than those in the NT group. The growth rate of pups from low-protein-fed mothers was around 50% lower than that of their respective controls. The T group showed an increase in body weight from the 60th day onwards, while the NT+LP group presented a reduced body weight from weaning onwards. In conclusion, physical training attenuated the impact of the low-protein diet on oxygen consumption during gestation and on the growth rate of the offspring. (Received 26 February 2009; accepted after revision 14 May 2009; first published online 29 May 2009) Correspondingauthor C. G. Leandro: Rua Prof. Moraes Rego 1235, CEP 50670-901, Departamento de Nutric¸a?o, Cidade Universita´ria, Recife, PE, Brasil. Email: carolleandro22@yahoo.com.br Fetal growth and development depend primarily on the embryonic genome, the maternal?placental?fetal unit, fetal hormones and maternal milieu, and adequate nutrient and oxygen supply to the developing fetus (Harding, 2001). Poor nutrition during early life leads to low birth weight and consequently changes in long-lasting phenotype. Perinatal undernutrition influences brain growth spurt, feeding behaviour, ontogeny of reflexes, skeletal muscle mechanical properties and locomotor activity in adult rats (Barros et al. 2006; Lopes de Souza et al.2008; Toscano et al.2008a,b; Orozco-Solis et al.2009). Undernutrition also reduces fetal O2 delivery, causing retardation in intrauterine growth and abnormalities of cardiovascular function in adult offspring (Barker, 1999a). ?Programming? is the term used to explain that during early ontogeny, the developing organism passes through a ?critical window? of sensitivity or plasticity, during which environmental factors generate long-lasting adjustments to the phenotype (Lucas, 1991). The environmental factors lead to transcriptional changes in proteins of metabolic and growth pathways (Burdge et al. 2007). Some of these changes are induced by epigenetic regulation that can also respond to several environmental stimuli, such as maternal physical activity. DOI: 10.1113/expphysiol.2009.047621 C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society ) at CAPES - Usage on July 19, 2010ep.physoc.orgDownloaded from Exp Physiol ( Exp Physiol 94.8 pp 906?913 Maternal physical exercise and undernutrition during gestation 907 Maternal lifestyle modulates several maternal physiological adaptations to pregnancy involved in feto-placental growth by enhancing nutrient and oxygen availability to the fetus (Clapp et al. 2004; Haakstad et al. 2007). However, the effects of maternal exercise on fetal oxygenation and feto-placental growth are controversial. There are different responses according to the type of exercise, frequency, physical fitness of the mother, the point of time in the pregnancy when the exercise is carried out, and the duration and intensity of the exercise (Clapp et al. 2002; Clapp, 2003). Physical exercise is considered intense when the percentage of effort is higher than 75% V?O2max (Leandro et al. 2007). Intense physical exercise and high daily physical activity workload during pregnancy have been associated with low birth weight (Clapp et al. 2002; Rao et al. 2003). Studies in India have demonstrated an inverse relationship between intense physical activity and birth weight in rural and urban women who had high levels of physical activity related to agricultural and domestic duties (Rao et al. 2003; Dwarkanath et al. 2007). In contrast, low to moderate physical exercise (40?70% of V?O2max) is associated with improved cardiorespiratory fitness, increased metabolic rate (reduction of body weight) and elevated lean body mass, all of which are recommend for aged, injured, obese and pregnant humans (Leandro et al. 2007). In women submitted to physical training, the rate of placental bed blood flow increases at rest, and more glucose and oxygen delivery to the placental site are observed (Clapp, 2003). An epidemiological study found that moderate physical exercise during pregnancy is associated with a 100?150 g increase in the birth weight (Hatch et al. 1993). Energy expenditure during pregnancy can therefore be an important factor affecting the relationship between maternal nutrition and the size of the fetus at birth (Clapp et al. 2000). In the present study, a protocol of moderate physical exercise was developed, based on maximal oxygen consumption (V?O2max) for non-pregnant rats. The intensity of the exercise was classified as moderate, and the trained females were given exercise routines according to their physical fitness before pregnancy. At the confirmation of pregnancy, oxygen consumption was monitored weekly during the programme of physical training in order to control the low intensity of physical exercise. Our hypothesis is that exercise-induced physiological changes during gestation could attenuate or modulate the impact of a low-protein diet on the oxygen consumption during gestation and the growth rate of the offspring. Methods The experimental protocol was approved by the Ethical Committee of the Biological Sciences Center, Federal University of Pernambuco, Brazil and followed the Guidelines for the Care and Use of Laboratory Animals (Bayne, 1996). Animals Virgin female albino Wistar rats (Rattus novergicus) aged 60 days and weighing 180 ± 11 g (mean ± S.E.M.) were obtained from the Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sa?o Paulo, Brazil. Female rats were maintained at a room temperature of 22 ± 1?C with a controlled light? dark cycle (dark 06.00?18.00 h). Standard laboratory chow (52% carbohydrate, 21% protein, 4% lipids; Nuvilab CR1-Nuvital R©, Curitiba, Parana, Brazil) and water were given ad libitum. Animals were randomly divided into two groups: untrained rats (NT, n = 10) and trained rats (T, n = 10). Trained rats were submitted to a training programme of moderate running over a period of 4 weeks (5 days week?1 and 60 min day?1) on a treadmill (Millennium Inbramed, Porto Alegre, Brazil) at a controlled intensity based on their V?O2max. Untrained rats were handled daily in order to reproduce the handling conditions to which trained rats were submitted. After the 4 week training period, the rats were mated (2 females for 1 male). The day on which spermatozoa were present in a vaginal smear was designated as the day of conception, day 0 of pregnancy. Pregnant rats were then transferred to individual cages. Half of the rats from each group received a 17% casein diet and the other half received an 8% casein isocaloric diet (low-protein group, LP) ad libitum (Table 1). Thus, two more groups were formed, as follows: untrained (NT, n = 5); trained (T, n = 5); untrained with low-protein diet (NT+LP, n = 5); and trained with low-protein diet (T+LP, n = 5). The exercise programme was maintained during gestation, with a progressive reduction of intensity until day 19 of gestation. The mother?s body weight was determined weekly throughout the experiment. At the time of delivery, the litter size and birth weight of the pups were recorded. During the suckling period, the offspring were kept in groups of six pups. Offspring of low-protein- fed mothers remained on an 8% low-protein diet and the control groups received a 17% casein diet (Table 1). After weaning (on the 22th day of age), male offspring only were divided into four groups according to their mother?s manipulations (NTp, n = 17, pups from NT mothers; Tp, n = 13, pups from T mothers; NT+LPp, n = 17, pups from NT-LP mothers; and T-LPp, n = 13, pups from T-LP mothers). The offspring were kept in a collective cage (one cage per litter) and received standard laboratory chow (52% carbohydrate, 21% protein and 4% lipids; Nuvilab CR1-Nuvital R©; Reeves et al. 1993) ad libitum. Body weight and growth rate were recorded up to the 90th day of life. Rats were killed by decaptation at 150th day of life. C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society ) at CAPES - Usage on July 19, 2010ep.physoc.orgDownloaded from Exp Physiol ( 908 M. F. Amorim and others Exp Physiol 94.8 pp 906?913 Table 1. Composition of the diets Amount per 1 kg of diet Ingredient Low-protein diet Control diet (8% protein) (17% protein) Casein 79.3 g 179.3 g Vitamin mix? 10 g 10 g Mineral mixture? 35 g 35 g Cellulose 50 g 50 g Choline bitartrate 2.5 g 2.5 g DL-Methionine 3.0 g 3.0 g Soya oil 70 ml 70 ml Corn starch 750.2 g 650.2 g ? Vitamin mixture contained the following (in mg kg?1 of diet): retinol, 12; cholecalciferol, 0.125; thiamine, 40; riboflavin, 30; pantothenic acid, 140; pyridoxine, 20; inositol, 300; cyanocobalamin, 0.1; menadione, 80; nicotinic acid, 200; choline, 2720; folic acid, 10; p-aminobenzoic acid, 100; and biotin, 0.6. ? Mineral mixture contained the following (in mg kg?1 of diet): CaHPO4, 17 200; KCl, 4000; NaCl, 4000; MgO, 420; MgSO4, 2000; Fe2O2, 120; FeSO4.7H2O, 200; and trace elements, 400 (MnSO4.H2O, 98; CuSO4.5H2O, 20; ZnSO4.7H2O, 80; CoSO4.7H2O, 0.16; and KI, 0.32; with sufficient starch to bring to 40 g [per kg of diet]). Protocol of the maximal effort test Both trained and untrained animals were submitted to a V?O2max test according to the protocol suggested by Leandro et al. (2007). Briefly, the initial treadmill speed was 0.3 km h?1. Every 3 min, the speed was increased by 0.3 km h?1. The tests were stopped when the animals were unable to keep running on the treadmill, and the V?O2max was then determined. At every stage of effort, blood was collected from the tail vein and blood lactate was measured using an Accutrend Lactate testing system (Accutrend, Indianapolis, IN, USA). At a speed of 1.8 km h?1, rats reached the maximal values of effort (V?O2 = 48.76 ± 3.3 ml kg ?1 min?1 and blood lactate = 5.1 ± 1.5 mg dl?1; Fig. 1A and B). Protocol of physical training The V?O2max values of the rats assigned to the T group were used to adjust the 4 week programme of moderate running training. The training was effective for all animals in the trained group because of their homogeneity in terms of motor behaviour (Leandro et al. 2007). The speed of the treadmill (for each session of exercise) was adjusted according to the percentage of V?O2max. This adjustment was possible because oxygen consumption was measured during the different stages of the training programme. During the weeks of training, animals of the trained group performed physical exercise in an open- circuit calorimeter (Oxymax Deluxe System, Columbus Instruments, Columbus, OH, USA). The speed of the treadmill was modified according to the performance of animals, and their oxygen consumption reached 65% V?O2max (Table 2). The first week was dedicated to the adaptation of the animals. During this week, the speed was very low and the duration of the exercise session was for 20 min per day (Table 2). In the second and subsequent weeks, the protocol was divided into four progressive stages: (1) warm-up (5 min); (2) intermediary (10 min); (3) training (30 min); and (4) cool-down period (5 min). The treadmill speed in the warm-up and cool-down periods was around 0.4 km h?1 (corresponding to around 40% of V?O2max). In the intermediary period, the animals ran two sets of 10 min, except in the second week when they ran one set only. Relative to the training period, in the second week the speed of the treadmill corresponded to 65% of V?O2max (Table 2). During pregnancy, rats ran at a progressively reducing intensity of effort. The maximal effort test was not performed in pregnant rats. The adjustment of the speed of the treadmill was based on the last values of the V?O2max test (in the fourth week). Pregnant rats ran at 40% V?O2max Rest 0.3 0.6 0.9 1.2 1.5 1.8 2.1 20 30 40 50 60 A Speed (Km.h-1) V O 2 (m l k g-1 m in - 1 ) Rest 0.3 0.6 0.9 1.2 1.5 1.8 0 1 2 3 4 5 6 7 B Speed (Km.h-1) Bl o o d La ct at e co n ce n tr at io n (m g dl - 1 ) Figure 1. Oxygen consumption and Blood Lactate concentration during maximal exercise test A, oxygen consumption (V?O2 ) during a maximal exercise test on a treadmill, based on the protocol for rats suggested by Leandro et al. (2007). B, blood lactate concentration during the test to evaluate V?O2max. All rats were submitted to the maximal exercise test (n = 20). The values are presented as means ± S.E.M. C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society ) at CAPES - Usage on July 19, 2010ep.physoc.orgDownloaded from Exp Physiol ( Exp Physiol 94.8 pp 906?913 Maternal physical exercise and undernutrition during gestation 909 Table 2. Treadmill training programme according to speed, inclination and duration of each session, for the 4 week period of training Time Speed Percentage of V?O2max Duration of each Total duration of (weeks) (km h?1) (means ± S.E.M.) stage (min) each session (min) Initial (adaptation) 0.3 36.1 ± 2.7 5 20 0.4 38.7 ± 2.9 5 ? 0.5 37.8± 1.9 5 ? 0.3 35.7 ± 3.1 5 ? 2nd week 0.4 42.5 ± 1.7 5 50 0.5 47.8 ± 3.1 10 ? 0.6 57.3± 5.0 30 ? 0.4 54.4 ± 2.4 5 ? 3rd week 0.4 32.4 ± 2.0 5 60 0.5 41.8 ± 3.0 10 ? 0.6 51.1 ± 2.8 10 ? 0.8 64.0 ± 3.3 30 ? 0.4 57.2 ± 3.4 5 ? 4th week 0.5 26.8 ± 1.7 5 60 0.6 43.4 ± 3.9 10 ? 0.8 49.1 ± 5.1 10 ? 0.9 65.3 ± 4.7 30 ? 0.5 57.8 ± 3.9 5 ? Table 3. Training programme of running according to speed, inclination and duration of each session for the 3 weeks of training during gestation Time Speed Percentage of V?O2max Duration of each Total duration of (weeks) (km h?1) (means ± S.E.M.) stage (min) each session (min) 1st week 0.4 52.9 ± 3.4 5 50 0.5 57.8 ± 4.4 10 ? 0.6 63.1 ± 1.4 10 ? 0.8 66.4± 4.9 20 ? 0.4 62.0 ± 2.6 5 ? 2nd week 0.4 42.0 ± 1.3 5 30 0.5 47.8 ± 5.1 10 ? 0.6 43.5 ± 5.7 10 ? 0.4 42.1 ± 3.2 5 ? 3rd week 0.3 36.7 ± 1.8 5 20 0.4 32.2 ± 2.9 5 ? 0.5 32.8 ± 1.7 5 ? 0.3 29.9 ± 2.6 5 ? until the 19th day of gestation (Table 3). There was no physical training during the lactation period. Resting V?O2 and respiratory exchange ratio (RER) measurements The V?O2 and RER were measured using an indirect, open-circuit calorimeter. This system monitors oxygen concentration by volume at the inlet and outlet ports of a chamber through which a known flow of air is being forcibly ventilated. Air was drawn from the front end at a constant rate (2.5 l min?1) and passed through a silica column to absorb water. The system was constructed with stable gas sensors optimized for sensing concentrations near ambient conditions. The measurement was accurately made from values that differed by as little as 0.1% from inlet to outlet. The flow rates were adjusted according to the weight of the animal to ensure that the changes in the composition of the expired gases were more than 0.05%. The flowmeter was controlled and calibrated with gases of known concentrations (CO2 offset = 0.001; CO2 gain = 0.65%; and O2 = 21.2%). Measurements were performed at approximately 10.00 h after 3 h of fasting. After a 10 min stabilization period of the gases in the interior of the chamber, O2 and CO2 consumptions and RER were determined every 1 min during a 15 min period. Controlling software (7400 Oxymax Single Chamber, Columbus Instruments, Columbus, OH, USA) provided specific calorimetric measurement and recording of V?O2 and RER values minute by minute. The V?O2 and RER of rats from all groups were recorded every week before mating. During pregnancy, V?O2 and RER were recorded on the 2nd, 10th and 19th days. C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society ) at CAPES - Usage on July 19, 2010ep.physoc.orgDownloaded from Exp Physiol ( 910 M. F. Amorim and others Exp Physiol 94.8 pp 906?913 Table 4. Resting oxygen consumption and respiratory exchange ratio (RER) of untrained (NT, n = 10) and trained rats (T, n = 10) Time (weeks) Group V?O2 (ml kg ?1 min?1) RER ( V?CO2 / V?O2 ) 1st week NT 23.3 ± 1.9 0.854 ± 0.09 T 22.9 ± 1.1 0.798 ± 0.1 2nd week NT 25.0 ± 2.0 0.881 ± 0.06 T 23.0 ± 1.7 0.825 ± 0.06 3rd week NT 22.8 ± 1.3 0.862 ± 0.09 T 23.9 ± 2.1 0.870 ± 0.1 4th week NT 22.1 ± 1.1 0.823 ± 0.07 T 25.4 ± 1.0? 0.779 ± 0.1? Values are presented as means ± S.E.M. ?P < 0.05 when compared with untrained as indicated by two-way ANOVA and Bonferroni?s post hoc test. Abbreviations: V?O2 , oxygen uptake; and V?CO2 , carbon dioxide output. Offspring body mass and growth rate Body weight of the pups was recorded daily throughout the experiment. Body weight gain was calculated as follows: Percentage weight gain = [body weight (g) × 100/birthweight (g)] ? 100. Growth rate was calculated by the gain of grams per day (g day?1; Bayol et al. 2004). Statistical analysis Results are presented as means ± S.E.M. Student?s unpaired t test was used to compare groups (trained and untrained). One-way ANOVA was used to examine intergroup differences. Post hoc differences among the means from experimental groups were determined by Tukey?s test. Figure 2. Body weight gain during gestation by untrained (NT, n = 5), trained (T, n = 5), untrained + low protein (NT+LP, n = 5) and trained + low protein rats (T+LP, n = 5) Analysis was performed in each third of gestation, relative to the body mass on the first day of pregnancy. The values are presented as means + S.E.M. ?P < 0.05 compared with NT group using two-way ANOVA and Bonferroni?s post hoc test. Pearson?s correlation coefficient was used to correlate body weight and V?O2max. For measurements of body weight and growth rate, two-way repeated measures ANOVA followed by Bonferroni?s post hoc test were used. Significance was set at P < 0.05. Results The protocol of the maximal effort test was repeated every week (once a week, on a non-training day) in order to adjust the exercise intensity in the zone of training at around 50?65% of the V?O2max (Table 2). From the third week on, trained animals consistently increased the rate of maximal oxygen consumption with respect to the speed for each week (first week, 45.6 ± 2.7 ml kg?1 min?1; second week, 47.9 ± 3.9; third week, 50.8 ± 3.0; and fourth week, 54.7 ± 3.9; P < 0.05). This observation was also compared with untrained rats, which progressively diminished their V?O2max level over the third week. Untrained rats decreased their maximal oxygen consumption levels over the weeks compared with their initial values (first week, 44.9 ± 2.7 ml kg?1 min?1; second week, 43.5 ± 2.6; third week, 41.6 ± 3.8; and fourth week, 41.1 ± 2.4; P < 0.05) . In the fourth week, trained rats presented a lower body mass gain within that time compared with untrained rats (T, 13 ± 3.9% and NT, 17% ± 3.8; P < 0.05). Both groups showed a significant negative correlation (r2 = ?0.65, P < 0.001) between gain of body mass and the V?O2max in each maximal test over 4 weeks. Resting V?O2 and RER differed only in the fourth week (Table 4). After confirmation of pregnancy, groups were subdivided according to their dietary intake. Trained rats (T) and trained + low-protein diet (T+LP) groups kept running at a lower intensity of effort (Table 3). Gain of body mass was lower in the last third of gestation in the groups submitted to a low-protein diet (NT, 36.9 ± 6.1; T, 38.2 ± 5.5; NT+LP, 30.1 ± 5.1; and T+LP, 31.5 ± 6.1; P < 0.05; Fig. 2. Data were adjusted for the number of pups born to each dam [NT, 10.5 (10?13); T, 9.5 (9?14); NT+LP, 10.0 (8?11); and T+LP, 10.0 (8?10); values expressed as median (minimum and maximum)], and the correlation coefficient between number of pups and body weight gain of the mother was not significant (r2 = 0.31). During gestation, rats were submitted to measurement of oxygen consumption at rest. All groups showed a progressive reduction in the values of resting V?O2 . However, in T dams, this reduction was less pronounced than in NT dams (P < 0.05; Fig. 3]. The NT+LP dams showed lower values of resting V?O2 than those of the NT group, while T+LP dams showed no difference from those of the NT group (Fig. 2). The values of RER did not change when groups were compared with the NTgroup (NT, 0.870 ± 0.03; T, 0.883 ± 0.08; NT+LP, 0.821 ± 0.06; and T+LP, 0.840 ± 0.09; P > 0.05). C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society ) at CAPES - Usage on July 19, 2010ep.physoc.orgDownloaded from Exp Physiol ( Exp Physiol 94.8 pp 906?913 Maternal physical exercise and undernutrition during gestation 911 Table 5. Growth rate of the offspring Growth rate (g day?1) NTp Tp NT+LPp T+LPp 1?21 days 1.3 ± 0.09 1.8 ± 0.1? 0.6 ± 0.07? 0.8 ± 0.07? 22?30 days 4.1 ± 0.1 3.83 ± 0.1 2.8 ± 0.1? 3.3 ± 0.1? 31?60 days 6.1 ± 0.2 6.6 ± 0.2 5.3 ± 0.1? 5.9 ± 0.1 61?90 days 2.6 ± 0.1 2.9 ± 0.1 2.6 ± 0.1 2.5 ± 0.1 During gestation, the dams were in untrained (n = 17), trained (n = 13), untrained+low protein (n = 17) and trained + low protein groups (n = 13). The values are presented as means ± S.E.M. ?P < 0.05 when compared with untrained as indicated by two-way ANOVA and Bonferroni?s post hoc test. 2nd 12th 19th 16.0 18.5 21.0 23.5 26.0 NT T NT+LP T + LP * * * * Days of gestation O xy ge n co ns um pt io n (m l K g-1 m in - 1 ) Figure 3. Resting oxygen consumption by untrained (n = 5), trained (n = 5), untrained + low protein (n = 5) and trained + low protein rats (n = 5) during gestation The values are presented as means ± S.E.M. ?P < 0.05 when compared with untrained as indicated by two-way ANOVA and Bonferroni?s post hoc test. At weaning, the growth rate of pups from the trained group was higher than that of pups in the untrained group (Table 5). The growth rate of LPp and T+LPp groups was around 50% lower than that of their respective control groups. At 22?30 days of age, the NTp and Tp groups increased their growth rate by 110% compared with the lactation period. The highest growth rate was observed between the 31st and 60th day of life (4.0?6.0 g day?1). In this period, T+LPp did not show a difference when compared with NTp. There was no difference in the growth rate among groups at the period between the 61st and Figure 4. Body weight of offspring during growth Rats are the offspring of untrained dams (NTp offspring, n = 17), offspring of trained dams (Tp offspring, n = 13), untrained + low protein dams (NT+LPp offspring, n = 17) and trained + low protein dams (T+LPp offspring, n = 13). The values are presented as means + S.E.M. ?P < 0.05 compared with NTp group using two-way ANOVA and Bonferroni?s post hoc test. 90th days. Birth weight and the body weight at weaning were not different among groups, except for NT+LPp, which showed a lower body weight when compared with control animals (Fig. 4). Body weight of NT+LPp was lower from the weaning on when compared with the NTp group, while T+LPp rats showed a difference from the 30th day onwards. From the 60th day onwards, the Tp group showed higher body weight when compared with the NTp group. Discussion The interactions amongst maternal exercise, fetal oxygenation and feto-placental growth are complex because the effects of exercise on the maternal physiological parameters vary with the type, frequency, duration and intensity of the exercise. In the present study, we used an animal model to investigate the values of V?O2max in response to a protocol of physical training during gestation in order to control some confounding variables. The resulting adaptations can therefore be more confidently attributed to the exercise programme itself, allowing us to extrapolate for humans as we consider the type, duration and intensity of the exercise. Our data showed that trained animals increased their rate of oxygen consumption after the fourth week of training, showing a positive effect on the cardiorespiratory fitness. In females, physical training for 4 weeks (2 h day?1, 5 days week?1, with exercise level adjustments according toV?O2max every week) resulted in an increase in V?O2max C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society ) at CAPES - Usage on July 19, 2010ep.physoc.orgDownloaded from Exp Physiol ( 912 M. F. Amorim and others Exp Physiol 94.8 pp 906?913 (Wisloff et al. 2001). In the present study, the intensity of the load required to induce improved performance was adjusted throughout the protocol of training, and it was also useful to reduce the intensity of training during pregnancy. Previous studies have shown that regular moderate-intensity exercise in early pregnancy enhances feto-placental growth rate and birth size of the offspring (Clapp et al. 2000). During gestation, half of the animals of each group were submitted to a low-protein diet. All groups showed a progressive reduction in the values of resting V?O2 during gestation. In fact, V?O2 has an inverse relationship with gain of body weight (Leandro et al.2007). However, in dams previously submitted to a protocol of physical training, this reduction was less pronounced when compared with untrained dams. Physical exercise during pregnancy induces an increase in placental volume by enhancing the terminal villi, the finger-like structures formed in the placenta whose function is to fill with maternal blood and pass nutrients and oxygen on to the fetus (Thomas et al. 2008). In addition, since physical training can induce an increase in maternal lean body mass, the dietary low-protein-induced catabolism can be attenuated by a mechanism that includes an increase in availability of protein (Clapp, 2003). Thus, physical exercise can modulate the effect of a low-protein diet on the resting consumption of oxygen. Beginning a moderate-intensity exercise routine early in the course of pregnancy, during the hyperplastic phase of placental growth, may be an important mechanism for improving placental functional capacity, which in turn increases nutrient and oxygen delivery and overall growth rate of the fetus later in gestation (Clapp et al. 2000). Our results showed that pups from trained mothers showed a higher growth at weaning than their peers and a high body weight as they grew. In a previous study, there were no differences found in body composition of pups from running trained dams (before mating, 5 days week?1, for 2.0 h day?1 at 31 m min?1 up an 8 deg incline for 8 weeks; and during gestation, 27 m min?1 up a 5 deg incline, 1 h day?1; Treadway et al. 1986). The differences in the exercise protocols could justify the results. Higher intensity exercise over a long duration during gestation can induce negative outcomes for pups. In the present study, we maintained the intensity of exercise around 50?70% of V?O2max of the mothers. The physiological mechanism can be associated with an increase of uterine blood flow that occurs during moderate-intensity exercise, as well as with the expanded blood volume found in regularly exercising pregnant women (Saintonge & Rosso, 1981; Clapp et al. 2000; Clapp, 2003). Nutrient deficiency and oxygen supply during fetal growth and development are associated with long-lasting repercussions in adult life, which are the most studied programming factors acting on the critical periods of development (Barker, 1999b). Various animal models of maternal protein restriction were developed to validate the nutritional programming concept (Ozanne et al. 2003; Lopes de Souza et al. 2008; Passos et al. 2008). Fetal growth in late gestation is normally limited by maternal size and the capacity to provide nutrients for the fetus, a phenomenon known as constraint (Harding, 2001). In the present study, the NT+LPp offspring showed a reduced body weight from weaning onwards. Previous studies have demonstrated that maternal protein restriction (8% protein-restricted diet) causes changes in milk composition and volume of the lactating rats and can be related to the future body weight of their offspring (Passos et al., 2008). Neonatal maternal protein restriction is associated with lower stores of maternal nutrients and, subsequently, less transfer of nutrients to the offspring, which is related to reduced postnatal growth (Passos et al. 2008). It has been recently suggested that malnutrition during lactation may program growth hormone mRNA expression patterns in adulthood and that these changes could be responsible for differences in growth patterns (de Moura et al. 2007). Moderate exercise during gestation may have beneficial effects on postnatal growth rate in the offspring from undernourished mothers. The underlying mechanisms of these effects are supposed to be related to metabolic changes, redistribution of blood flow and changes in the production of fetal and placental hormones which control growth (de Moura et al. 2007). In summary, a protocol of physical training was developed for rats during gestation, based on maximal oxygen consumption. During gestation, resting oxygen consumption was measured in order to evaluate the effects of physical training in rats submitted to a low-protein diet. Physical training attenuated the negative impact of the low-protein diet on the oxygen consumption during gestation and on the growth rate of offspring. References Barker DJ (1999a). Early growth and cardiovascular disease. Arch Dis Child 80, 305?307. Barker DJ (1999b). Fetal origins of cardiovascular disease. Ann Med 31(Suppl 1), 3?6. Barros KM, Manha?es-De-Castro R, Lopes-De-Souza S, Matos RJ, Deiro´ TC, Cabral-Filho JE & Canon F (2006). 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