Abstract

This study aimed at evaluating the levels of different maternal exercise intensities on maternal and fetal outcomes. Wistar rats were mated and the pregnant rats were distributed into four experimental groups (n = 13 animals/group): Control (Not exercise group - 0% of the anaerobic threshold- AT), mild (20%), moderate (80%), and heavy-exercise intensity (140% of AT). These AT were matched to the load of 0, 1, 4 and 7% of the body weight of the animal related to swimming-induced physical intensity. In pregnancy, biomarkers related to maternal blood gases, oxidative stress, metabolism, and reproductive performance, and outcomes of their offspring were analyzed. The mild and moderate-swimming caused no change on implantation, live fetus numbers and oxidative stress status. However, the rats submitted to mild-exercise presented respiratory alkalosis and the heavy-exercise group showed respiratory acidosis. In addition, fetuses of the heavy-exercise dams were smaller for gestational age and lower serum adiponectin levels compared to those of other groups. In conclusion, the moderate-exercise intensity caused beneficial effects for maternal environment and the mild and moderate-exercise presented similar fetal repercussions. Nevertheless, the heavy-exercise intensity caused maternal metabolic alterations that damaged the fetal growth. Therefore, these findings confirm that physical intensity should be carefully conducted to avoid maternal complications and, consequently, compromised fetal repercussions.

Key words
exercise intensities; swimming; pregnancy; fetuses

INTRODUCTION

Water immersion is one of the best resources to increase the aerobic capacity of pregnant women (Juhl et al. 2010JUHL M, KOGEVINAS M, ANDERSEN PK, ANDERSEN NB OLSEN J. 2010. Is swimming during pregnancy a safe exercise? Epidemiology 21(2): 253-258.). In the maternal organism, the application of physical exercise have showed an improvement of cardiovascular function, prevention of excessive body weight gain, reduction of fat retention and improvement of physical fitness (Clapp 2000CLAPP JF. 2000. Exercise during pregnancy. A clinical update. Clin Sports Med 19(2): 273-286.). Considering the most recent ACOG guidelines (2015)ACOG COMMITTEE OPINION. 2015. Physical activity and exercise during pregnancy and the postpartum period. Obstet Gynecol 126(6): e135-e142., all pregnant women, without obstetric and medical contraindications, should be encouraged to exercise in a manner similar to the guidelines that are given to non-pregnant women. They can practice aerobic and strength exercises with moderate intensity for at least 20 to 30 minutes a day on most days of the week (Perales et al. 2017PERALES M, ARTAL R LUCIA A. 2017. Exercise during pregnancy. JAMA 317(11): 1113-1114.). Furthermore, clarity regarding the definition of exercise intensity is still lacking.

Several biochemical markers of physical training provoked great interest and represent a significant advance in recent decades for a better understanding of the importance of physical exercise practice for the health of the population. Among these markers, enzymatic activity of citrate synthase is studied, as it is relevant for the evaluation of oxidative metabolism (Basset & Howley 2000BASSET DRJ HOWLEY ET. 2000. Limiting factors for maximum oxygen uptake and determinants of endurance performance. Med Sci Sports Exerc 32(1): 70-84.). Citrate synthase catalyzes the first reaction of the Krebs cycle, where condensation of acetyl coenzyme A (Acetyl-CoA) with oxaloacetate occurs to form citrate and coenzyme A (CoA) (Halliwell & Gutteridge 1998HALLIWELL B GUTTERIDGE JMC. 1998. Oxidative Stress: Adaptation, damage, repair and death. Oxford: Oxford University Press.). Several studies have used mitochondrial enzyme activity to confirm or not the influence of physical exercise on the oxidative adaptation of rat skeletal muscle (Goodyear et al. 1992GOODYEAR LJ, HIRSHMAN MF, VALYOU PM HORTON ES. 1992. Glucose transporter number, function, and subcellular distribution in rat skeletal muscle after exercise training. Diabetes 41(9): 1091-1099., Powers et al. 1994POWERS SK, CRISWELL D LAWLER J. 1994. Influence of exercise and fiber type on antioxidant enzyme activity in rat skeletal muscle. Am J Physiol 226: R375-R380., Silva et al. 1997SILVA GJJ, BRUM PC, NEGRÃO CE KRIEGER EM. 1997. Acute and chronic effects of exercise on baroreflexes in spontaneously hypertensive rats. Hypertension 2(30): 714-719., Bexfield et al. 2009BEXFIELD NA, PARCELL AC, NELSON WB, FOOTE KM MACK GW. 2009. Adaptations to high-intensity intermittent exercise in rodents. J Appl Physiol 107: 749-754., Moreira et al. 2013MOREIRA JB, BECHARA LR, BOZI LH, JANNIG PR, MONTEIRO AW, DOURADO PM, WISLØFF U BRUM PC. 2013. High- versus moderate-intensity aerobic exercise training effects on skeletal muscle of infarcted rats. J Appl Physiol 114: 1029-1041.). The increased activity of oxidative enzymes present in mitochondria is indicative of the optimization of aerobic metabolism (Halliwell & Gutteridge 1998HALLIWELL B GUTTERIDGE JMC. 1998. Oxidative Stress: Adaptation, damage, repair and death. Oxford: Oxford University Press.). Another marker used is creatine kinase (CK), an enzyme that plays a key role in energy formation in muscle cells, as it is responsible for maintaining adequate levels of adenosine triphosphate (ATP) during muscle contraction (Katirji & Al-Jaberi 2001KATIRJI B AL-JABERI MM. 2001. Creatine kinase revisited. J Clin Neuromusc Dis 2: 158-163.). CK is as a marker of muscle damage since, is cytoplasmic; and has no ability to cross the sarcoplasmic membrane barrier (Melin et al. 1997MELIN B, BOURDON L, JIMENEZ C, CHARPENET A BERNARD O. 1997. Plasma myosin and creatine kinase time-course after a concentric-eccentric field exercise. Arch Physiol Biochem 105: 27-31., Nosaka & Newton 2002NOSAKA K NEWTON M. 2002. Repeated eccentric bouts do not exacerbate muscle damage and repair. J Strength Cond Res 16(1): 117-122., Poprzęcki et al. 2004POPRZĘCKI S, STASZKIEWICZ A HÜBNER-WOŹNIAK E. 2004. Effect of eccentric and concentric exercise on plasma creatine kinase (ck) and lactate dehydrogenase (LDH) activity in healthy adults. Biol Sport 21(2): 193-203.). As a result, increased serum CK concentrations are used to indicate muscle membrane damage (Smith et al. 1994SMITH LL, FULMER MG, HOLBERT D, MCCAMMON MR, HOUMARD JA, FRAZER DD, NSIEN E ISRAEL RG. 1994. The impact of a repeated bout of eccentric exercise on muscular strength, muscle soreness and creatine kinase. Br J Sports Med 28(4): 267-271., Brown et al. 1997BROWN SJ, CHILD RB, DAY SH DONNELLY AE. 1997. Exercise-induced skeletal muscle damage and adaptation following repeated bouts of eccentric muscle contractions. J Sports Sci 15: 215-222., Close et al. 2005CLOSE GL, KAYANI A, VASILAKI A MCARDLE A. 2005. Skeletal muscle damage with exercise and aging. Sports Med 35: 413-427., Nosaka et al. 2005NOSAKA K, NEWTON M, SACCO P, CHAPMAN D LAVENDER A. 2005. Partial protection against muscle damage by eccentric actions at short muscle lengths. Med Sci Sports Exerc 37(5): 746-753.).

The effects of maternal physical activity on fetal four cannot be adequately investigated in human pregnancies for self-evident ethical reasons. Therefore, animal models are essential to fill in the gaps in our knowledge. Rat models, besides being time- and cost-efficient, are very well suited for mechanistic studies. In male rats, exercise intensity has been defined in terms of lactate levels, pH and anaerobic threshold. However, studies assessing the effect of physical activity on female rats do not provide clear definitions of exercise intensity levels (Uriu-Hare et al. 1989URIU-HARE JY, KEEN CL, APPLEGATE EA STERN JS. 1989. The influence of moderate exercise in diabetic and normal pregnancy on maternal and fetal outcome in the rat. Life Sci 45: 647-654., Simsek et al. 2005SIMSEK M, NAZIROGLU M ERDINÇ A. 2005. Moderate exercise with a dietary vitamin C and E combination protects against streptozotocin-induced oxidative damage to the kidney and lens in pregnant rats. Exp Clin Endocrinol Diabetes 113: 53-59., Volpato et al. 2009VOLPATO GT, DAMASCENO DC, KEMPINAS WG, RUDGE MVC CALDERON IMP. 2009. Effect of exercise on the reproductive outcome and fetal development of diabetic rats. Reprod Biomed Online 19: 852-858., Damasceno et al. 2011DAMASCENO DC, SINZATO YK, LIMA PH, DE SOUZA MS, CAMPOS KE, DALLAQUA B, CALDERON IM, RUDGE MV VOLPATO GT. 2011. Effects of exposure to cigarette smoke prior to pregnancy in diabetic rats. Diabetol Metab Syndr 3: 20.). In our research laboratory, rats submitted to swimming during pregnancy had conflicting maternal and fetal results (Volpato et al. 2009VOLPATO GT, DAMASCENO DC, KEMPINAS WG, RUDGE MVC CALDERON IMP. 2009. Effect of exercise on the reproductive outcome and fetal development of diabetic rats. Reprod Biomed Online 19: 852-858., Corvino et al. 2015aCORVINO SB, NETTO AO, SINZATO YK, CAMPOS KE, CALDERON IM, RUDGE MV, VOLPATO GT, ZAMBRANO E DAMASCENO DC. 2015a. Intrauterine growth restricted rats exercised at pregnancy: maternal-fetal repercussions. Reprod Sci 22(8): 991-999., bCORVINO SB, VOLPATO GT, RUDGE MV DAMASCENO DC. 2015b. Intrauterine growth restricted rats exercised before and during pregnancy: maternal and perinatal repercussions. Evid Based Complement Alternat Med 2015: 294850.), as there are benefits after swimming only to rats-mothers or only to offspring. Controversial results regarding the effects on offspring can be explained due to the intensity of physical exercise. In strenuous exercise, a state of fetal hypoxia may occur, leading to risks for its development (Bennell 2011BENNELL K. 2011. The female athlete. In: Brukner P and Khan K (Eds), Clinical sports medicine, Australian, McGraw-Hill, p. 674-699.). According to Bell (2002)BELL R. 2002. The effects of vigorous exercise during pregnancy on birth weight. J Sci Med Sport 5: 32-36., intense physical exercises with high frequency and maintained for long periods of gestation can result in low birth weight newborns. There are several explanations to justify the small fetuses for gestational age, such as altered secretion of adipocytokines, as adiponectin, which has been implicated in intrauterine growth (Gluckman et al. 1996GLUCKMAN PD, CUTFIELD W, HARDING JE, MILNER D, JENSEN E, WOODHALL S, GALLAHER B, BAUER M BREIER BH. 1996. Metabolic consequences of intrauterine growth retardation. Acta Paediatr 417(Suppl): 3-6.). Netto et al. (2017)NETTO AO, MACEDO NCD, GALLEGO FQ, SINZATO YK, VOLPATO GT DAMASCENO DC. 2017. Evaluation of anaerobic threshold in non-pregnant and pregnant rats. An Acad Bras Cienc 89: 2749-2756. used the lactate minimum test to determine the anaerobic threshold (the point during exercise when aerobic metabolism is switched to anaerobic metabolism) in non-pregnant and pregnant Wistar rats during swimming program. In pregnant rats, the exercise with mild, moderate and heavy-intensity levels correspond to 20%, 80% and 140% of the anaerobic threshold, respectively. Herein, we hypothesized that among the three intensities tested the most adequate is moderate because it should represent a load that it is possible to practice physical exercise with stabilization of blood lactate for better maternal and perinatal outcomes. Based on these data, this study aimed at assessing the effects of different maternal exercise intensity levels on maternal outcomes and their offspring.

MATERIALS AND METHODS

Animals

Male and female adult Wistar rats weighing approximately 200-240 g were used. The animals were purchased from Biological Research Centre (CEMIB), State University of Campinas (UNICAMP), adapted and maintained at the Laboratory of Experimental Research of Obstetrics and Gynecology, Unesp, under controlled conditions of temperature (22 ± 2°C), humidity (50 ± 10%), 12 hours light/dark cycle, and water and feed were provided ad libitum. The experimental design is showed in figure 1.

Figure 1
Experimental design.

Procedures and animal handling were performed in accordance with the guidelines provided by the Brazilian College of Animal Experimentation in agreement with the International Guiding Principles for Biomedical Research Involving Animals promulgated by the Society for the Study of Reproduction. The local Ethics Committee for animal experiments approved all protocols described in this study (Protocol Number 1050/2013).

Mating procedure and experimental groups

All female rats were mated overnight with male rats. The day when sperms were found in the vaginal smear was designated as gestational day 0 (GD0). The mating period consisted of 15 consecutive days until a replicate number of groups was obtained. However, during this period, nonmated female rats were considered infertile and were discarded from the study (Santos et al. 2015SANTOS TM, SINZATO YK, GALLEGO FQ, IESSI IL, VOLPATO GT, DALLAQUA B DAMASCENO DC. 2015. Extracellular HSP70 levels in diabetic environment in rats. Cell Stress Chaperones 20: 595-603.). The pregnant rats were submitted to 0%, 20%, 80% and 140% of the anaerobic threshold (AT) for the swimming practice. In previous study (Netto et al. 2017NETTO AO, MACEDO NCD, GALLEGO FQ, SINZATO YK, VOLPATO GT DAMASCENO DC. 2017. Evaluation of anaerobic threshold in non-pregnant and pregnant rats. An Acad Bras Cienc 89: 2749-2756.), the AT of the pregnant rats was identified in minimum load of 5% of the body weight. Then, for this investigation the pregnant rats were distributed into four experimental groups (n = 13 animals/group) - 1) Control: Not exercise group - no load (0% of AT); 2) Mild-exercise intensity: with 20% of AT, which corresponded to 1% of body weight of each rat; 3) Moderate-exercise intensity: with 80% of AT, which corresponded to 4% of body weight; 4) Heavy-exercise intensity: with 140% of AT, equivalent to 7% of body weight.

Physical exercise – swimming program

To familiarize the rats to the swimming system (water), the rats were daily exposed to water for 10 minutes in cylindrical tanks (100 x 30 cm) containing water at a depth of 10 cm at 31±1°C. The adaptation period corresponded to 80 days of life (10 days before the mating period) and after pregnancy positive diagnosis (the first week of pregnancy - from day 0 to day 6). The first day of exercise was carried out on the day 7 of pregnancy, corresponding to post-embryonic implantation period. The swimming was performed according to the exercise intensity and, therefore, each rat carried its respective load. Fishing pellets were used as loads and inserted into jackets, tied to the rat´s chest. The exercise was held in the morning for 30 minutes daily. The rats were subjected to swimming from day 7 to day 20 of pregnancy (Netto et al. 2017NETTO AO, MACEDO NCD, GALLEGO FQ, SINZATO YK, VOLPATO GT DAMASCENO DC. 2017. Evaluation of anaerobic threshold in non-pregnant and pregnant rats. An Acad Bras Cienc 89: 2749-2756.). An appropriate control with sedentary rats was made with the same time of exposure to water. However, the water was shallow enough for the rats to stand/walk to reduce the confusing variable of stress between treatment groups.

At term pregnancy – maternal analysis

On day 21 of pregnancy, the rats were anesthetized using isoflurane inhalation (1 mL/min dose) for collection of arterial blood through puncture of the aorta. When the animals were in anesthesia and with stable breathing, the laparotomy and the arterial blood collection were performed. A portion of the samples was collected in tubes containing anticoagulant (heparin) for measurement of analysis creatine kinase (CK) and oxidative stress markers [malondialdehyde (MDA) and reduced thiol groups (S-H)]. For oxidative stress analysis, the blood samples were processed according to a modified method of de Souza et al. (2010)DE SOUZA MS, SINZATO YK, LIMA PH, CALDERON IM, RUDGE MV, DAMASCENO DC. 2010. Oxidative stress status and lipid profiles of diabetic pregnant rats exposed to cigarette smoke. Reprod Biomed Online 20: 547-552. and the samples were stored at -80°C and analyzed by spectrophotometry. CK measurements were performed using a commercial kit (EnzyChromTM® Creatine kinase Assay Kit, USA). Another part of the arterial blood samples was used for measurement of temperature, pH (normality limit: 7.35 -7.45 – compensated status), pCO₂ (CO₂ partial pressure), HCO₃, lactate and SO₂ (oxygen saturation) in the i-Stat System® apparatus (Abbott®, USA). SO₂ was used as a parameter to confirm arterial blood samples and as the inclusion criterion. Thus, rats with values above 95% were included in gasometry analyses.

The gravid uterus was dissected to count dead and alive fetuses, resorption (embryonic death), and implantation site numbers. The percentage of post-implantation loss was calculated (Volpato et al. 2015VOLPATO GT, DAMASCENO DC, SINZATO YK, RIBEIRO VM, RUDGE MV CALDERON IM. 2015. Oxidative stress status and placental implications in diabetic rats undergoing swimming exercise after embryonic implantation. Reprod Sci 22: 991-998.). The gastrocnemius muscle samples were withdrawn, stored at -80°C, and used for analysis of citrate synthase activity according to protocol modified by Alp et al. (1976)ALP PR, NEWSHOLME EA ZAMMIT VA. 1976. Activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates. Biochem J 154: 689-700.

At term pregnancy - fetal and placental analyses

Following the collection of fetuses from the uterine horns, each fetus was weighed for classification of fetal body weight in comparison to control group at same pregnancy time. The fetuses were classified was small (SGA), adequate (AGA) or large (LGA) for gestational (pregnancy) age to the mean ±1.7 x standard deviation (SD) of body weights obtained in the control group according to Afiune et al. (2017)AFIUNE LAF, LEAL-SILVA T, SINZATO YK, MORAES-SOUZA RQ, SOARES TS, CAMPOS KE, FUJIWARA RT, HERRERA E, DAMASCENO DC VOLPATO GT. 2017. Beneficial effects of Hibiscus rosa-sinensis L. flower aqueous extract in pregnant rats with diabetes. PLoS ONE 12(6): e0179785.. The fetal blood pool samples were collected from trunk region for serum adiponectin levels according to manufacturer (Millipore® EZRADP-62K). The placentas were weighed to calculate the placental efficiency (fetal weight/placental weight) (Moraes-Souza et al. 2017MORAES-SOUZA RQ, SOARES TS, CARMO NOL, DAMASCENO DC, CAMPOS KE VOLPATO GT. 2017. Adverse effects of Croton urucurana B. exposure during rat pregnancy. J Ethnopharmacol 199: 328-333.).

Statistical analysis

The results of the maternal metabolic biomarkers and reproductive outcomes and fetal serum adiponectin levels were analyzed by comparison two-by-two using Student t test. The classification of fetal body weight was analyzed by affixing the categories (SGA, AGA, and LGA) for the comparison between the groups. Then, the analysis of the proportionate measures were performed by Fisher’s exact test. For all statistical comparisons, p<0.05 was considered statistically significant.

RESULTS

The Table I shows the number of rats that began the experiment, completed full pregnancy, submitted or not to swimming during total period and number of dead rats. All rats of control, mild and moderate-exercise groups had at term pregnancy, were able to swim the complete program, and presented no death in these groups, except one rat submitted to moderate swimming program that bled before swimming and was removed from the group. The heavy-exercise group began the experiment with 13 animals, and three of them died during the experiment, totalizing 10 rats at term. Of these 10 rats, two rats completed the swimming program but eight did not.

The rats submitted to mild-exercise intensity presented pH 7.5, characterizing alkalosis status, and heavy-exercise showed pH 7.5, which confirmed acidosis. The pCO₂ and HCO₃ levels were greater in heavy-exercise compared to those of other groups. The activity of plasma creatine kinase was increased in the heavy-exercise group in comparison to control group. All of animals presented SO₂ greater than 95%. The muscular citrate synthase activity, blood lactate, blood MDA and reduced thiol (S-H) group concentrations showed no significant difference among the groups at term pregnancy (p0.05) (Table II).

The data of maternal reproductive outcomes were shown in Table III. The rats submitted to heavy-exercise intensity during pregnancy gained less weight than control rats, and the litter weight was lower than the moderate-exercise group. The placental efficiency was reduced in all exercise groups in relation to control rats, but it was the lowest in heavily exercised rats (p0.05).

The mild-exercise group presented greater percentage of fetuses classified as LGA in relation to control group, and heavy-exercise dams had SGA fetuses compared with those of control, mild and moderate-exercise groups (Figure 2a). The serum adiponectin levels of fetuses of heavy-exercise mothers were lower in relation to those of fetuses of other dams (Figure 2b).

Figure 2
a - Classification of fetal body weight, b - Serum adiponectin levels of fetuses from rats submitted to mild, moderate and heavy-exercise intensity during pregnancy. Data are presented as percentage. SGA - Small for gestational age; AGA - Adequate for gestational age; LGA – Large for gestational age. *p0.05 - compared to Control group; #p0.05 -compared to Mild-exercise group; $p0.05 - compared to Moderate-exercise group (a- Fisher’s Exact Test; b- Student t test)

DISCUSSION

The mild and moderate- intensity swimming applied to rats did not affect the number of rats that reached full term pregnancy, but the heavy-exercise led to the death of two rats before the end of pregnancy. Only intense-swimming caused changes in muscle damage, as confirmed by greater levels of creatine kinase. However, regardless of the exercise intensities applied to the rats during pregnancy, there is no compromise in the number of implantations or live fetuses. In addition, there were no changes in embryo loss rates. Regard to fetal growth, intense-swimming caused an increased number of small fetuses, which was confirmed by the increase in the percentages of SGA and levels of serum adiponectin of these offspring. The other swimming intensities did not damage the growth of the fetuses. The moderate-exercise protocol used in this study was the most adequately applied to maternal metabolic parameters and fetal growth. The heavy program of swimming caused impairment for both mothers and their offspring.

The rats exposed to mild-exercise showed higher pH values (pH 7.45) and a lower level of pCO₂, indicating a respiratory alkalosis. Then, these pregnant rats performed a hyperventilation to compensate that alkalosis. In other studies, hyperventilation and respiratory alkalosis also occurred in different species submitted to in mild-exercise (Hastings et al. 1982HASTINGS AB, WHITE FC, SANDERS TM BLOOR CM. 1982. Comparative physiological responses to exercise stress. J Appl Physiol Respir Environ Exerc Physiol 52: 1077-1083., Smith et al. 1983SMITH CA, MITCHELL GS, JAMESON LC, MUSCH TI DEMPSEY JA. 1983. Ventilatory response of goats to treadmill exercise: grade effects. Respir Physiol 54: 331-341., Chin et al. 2007CHIN LM, LEIGH RJ, HEIGENHAUSER GJ, ROSSITER HB, PATERSON DH KOWALCHUK JM. 2007. Hyperventilation-induced hypocapnic alkalosis slows the adaptation of pulmonary O2 uptake during the transition to moderate-intensity exercise. J Physiol 583: 351-364.). Our findings showed rats submitted to heavy-exercise had lower pH values (pH 7.35) and higher pCO₂, characterizing a respiratory acidosis. In an attempt to compensate for that acidosis, an increase in HCO₃ arisen. Nevertheless, it was not enough to normalize this acid level. According to Kato et al. (2005)KATO T, TSUKANAKA A, HARADA T, KOSAKA M MATSUI N. 2005. Effect of hypercapnia on changes in blood pH, plasma lactate and ammonia due to exercise. Eur J Appl Physiol 95: 400-408., the hypercapnia-induced respiratory acidosis directed to a lower blood pH due to lower blood lactate concentration at maximal exercise.

The enzymatic activity of creatine kinase (CK) in serum or plasma is useful for the evaluation of muscle damage from physical exercise (Foschini and Prestes 2007FOSCHINI D PRESTES J. 2007. Acute hormonal and immune responses after a bi-set strength trainning. Fitness Perform J 6: 38-44.). In this study, there were no changes in the CK activities in mild and moderate-exercise intensity, suggesting these exercise intensities were appropriate. However, the rats submitted to heavy-exercise intensity presented increased CK activity, indicating muscle damage.

Several studies have used the activity of mitochondrial enzymes to analyze the influence of physical exercise on the oxidative adaptation of skeletal muscle in rats (Bexfield et al. 2009BEXFIELD NA, PARCELL AC, NELSON WB, FOOTE KM MACK GW. 2009. Adaptations to high-intensity intermittent exercise in rodents. J Appl Physiol 107: 749-754.). An increase in the citrate synthase activity has been reported in swimming exercise (Medeiros et al. 2004MEDEIROS A, OLIVEIRA EM, GIANOLLA R, CASARINI DE, NEGRÃO CE BRUM PC. 2004. Swimming training increases cardiac vagal activity and induces cardiac hypertrophyin rats. Braz J Med Biol Res 37: 1909-1917.). However, our findings showed the citrate synthase activities were not modified after swimming during pregnancy regardless of intensity. Previous studies have indicated that the increase in the enzyme citrate synthase activity correlates with the intensity (Bexfield et al. 2009BEXFIELD NA, PARCELL AC, NELSON WB, FOOTE KM MACK GW. 2009. Adaptations to high-intensity intermittent exercise in rodents. J Appl Physiol 107: 749-754.) or duration of applied exercise (Powers et al. 1994POWERS SK, CRISWELL D LAWLER J. 1994. Influence of exercise and fiber type on antioxidant enzyme activity in rat skeletal muscle. Am J Physiol 226: R375-R380.). Then, it was expected that the group of rats submitted to heavy-exercise showed an increased citrate synthase activity, but no difference in this marker has been found. This can be explained because 10 of the 13 rats in this group that achieved at term pregnancy performing the exercise program were unable to swim the time established in the protocol (30 min), making an average of 10 minutes from 7 to 14 days of pregnancy and approximately 3 minutes from 15 to 20 days de pregnancy. Therefore, the citrate synthase activity does not reflect actual impairment with the practice of heavy-exercise.

The oxidative stress status biomarkers presented no changes in the blood of exercised rats. Evidence shows there is an increase in reactive oxygen species during physical exercise. To protect organs from tissue damage, antioxidant enzymes respond adaptively, thereby increasing their activities (Reddy et al. 1999REDDY AVULA CP FERNANDES G. 1999. Modulation of antioxidant enzymes and lipid peroxidation in salivary gland and other tissues in mice by moderate treadmill exercise. Aging (Milano) 11: 246-252.). However, this finding was not observed in this study.

The rats submitted to swimming at a heavy intensity gained less weight at term pregnancy. Our findings showed that the mild and moderate-exercise intensities had no measurable adverse effects on the number of implantation, embryo death and live fetuses and no appearance of external fetal malformations, indicating that our swimming protocol has been safe in pregnancy. It was verified mild to moderate-exercise during pregnancy did not harm the mother-fetus relationship. Physical exercise is a safe activity with beneficial effects for the mother and fetus during pregnancy (Evenson et al. 2014EVENSON KR, BARAKAT R, BROWN WJ, DARGENT-MOLINA P, HARUNA M, MIKKELSEN EM, MOTTOLA MF, OWE KM, ROUSHAM EK YEO S. 2014. Guidelines for physical activity during pregnancy: Comparisons from around the world. Am J Lifestyle Med 8: 102-121.). Although the rats submitted to heavy-exercise presented no changes in maternal reproductive outcomes, three rats of this group died before at term pregnancy due to exercise overload, and the rats that reached full pregnancy presented an early exhaustion signs during training and were removed from the water to prevent their deaths. Then, these findings did not reflect the impaired performance of a complete exercise protocol. Thus, it is important that the exercise protocols are performed with professional guidance and moderation, i.e., the type of exercise, weekly frequency, duration of each session, and intensity must be well controlled (Department of Health and Human Services 2018DEPARTMENT OF HEALTH AND HUMAN SERVICES. 2018. Physical activity guidelines advisory committee report. Washington: U.S. Department of Health and Human Services, 779 p.). Regarding studies with experimental animals, the results remain inconsistent because of the variability in the species and experimental protocols used. It was verified that heavy-exercise was not adequate, but we suggested that might be due to no previous training to the mother. Further studies might be conducted with rats submitted to previous exercise to pregnancy to avoid damaging for mother and fetuses.

In our study, the classification of the fetal weight was predominantly considered as adequate for gestational age (AGA) in all groups, regardless of swimming intensity applied in their dams. However, the heavy-exercise group presented greater percentage of fetuses classified as small for gestational age (SGA). In addition, these fetuses presented lower levels of serum adiponectin. Adiponectin is a protein highly expressed in human adipose cells (Yannakoulia et al. 2003YANNAKOULIA M, YIANNAKOURIS N, BLUHER S, MATALAS AL, KLIMIS-ZACAS D MANTZOROS CS. 2003. Body fat mass and macronutrient intake in relation to circulating soluble leptin receptor, free leptin index, adiponectin, and resistin concentrations in healthy humans. J Clin Endocrinol Metab 88: 1730-1736., Chan et al. 2004CHAN TF, YUAN SS, CHEN HS, GUU CF, WU LC, YEH YT, CHUNG YF, JONG SB SU JH. 2004. Correlations between umbilical and maternal serum adiponectin levels and neonatal birthweights. Acta Obstet Gynecol Scand 83: 165-169.). This protein modulates insulin action and promotes anti-atherogenic and anti-inflammatory effects (Nishizawa et al. 2002NISHIZAWA H ET AL. 2002. Androgens decrease plasma adiponectin, an insulin-sensitizing adipocyte-derived protein. Diabetes 51: 2734-2741., Combs et al. 2003COMBS TP, BERG AH, RAJALA MW, KLEBANOV S, IYENGAR P, JIMENEZ-CHILLARON JC, PATTI ME, KLEIN SL, WEINSTEIN RS SCHERER PE. 2003. Sexual differentiation, pregnancy, calorie restriction, and aging affect the adipocyte-specific secretory protein adiponectin. Diabetes 52: 268-276.). The adiponectin levels are inversely proportional to body fat content and presents negative correlation with the insulin resistance (Yang et al. 2001YANG WS, LEE WJ, FUNAHASHI T, TANAKA S, MATSUZAWA Y, CHAO CL, CHEN CL, TAI TY CHUANG LM. 2001. Weight reduction increases plasma levels of an adipose-derived anti-inflammatory protein, adiponectin. J Clin Endocrinol Metab 86: 3815-3819., Nemet et al. 2003NEMET D, WANG P, FUNAHASHI T, MATSUZAWA Y, TANAKA S, ENGELMAN L COOPER DM. 2003. Adipocytokines, body composition, and fitness in children. Pediatr Res 53: 148-152.). The adipocytokines have been associated with intrauterine growth (Lapillonne et al. 1997LAPILLONNE A, BRAILLON P, CLARIS O, CHATELAIN PG, DELMAS PD SALLE BL. 1997. Body composition in appropriate and in small for gestational age infants. Acta Paediatr 86: 196-200.). Kamoda et al. (2004)KAMODA T, SAITOH H, SAITO M, SUGIURA M MATSUI A. Serum adiponectin concentrations in newborn infants in early postnatal life. 2004. Pediatr Res 56: 690-693. compared adiponectin levels between female and male neonates but found no differences in adiponectin levels between the genders. These authors verified lower adiponectin levels in SGA than in AGA infants.

However, there are further studies in the literature showing that exercise during pregnancy cause no change (Gilbert et al. 2012GILBERT JS, BANEK CT, BAUER AJ, GINGERY A DREYER HC. 2012. Placental and vascular adaptations to exercise training before and during pregnancy in the rat. Am J Physiol Regul Integr Comp Physiol 303: R520-R526.), decrease (Damasceno et al. 2013DAMASCENO DC, SILVA HP, VAZ GF, VASQUES-SILVA FA, CALDERON IM, RUDGE MV, CAMPOS KE VOLPATO GT. 2013. Diabetic rats exercised prior to and during pregnancy: Maternal reproductive outcome, biochemical profile, and frequency of fetal anomalies. Reprod Sci 20: 730-738.) or increase (Uriu-Hare et al. 1989URIU-HARE JY, KEEN CL, APPLEGATE EA STERN JS. 1989. The influence of moderate exercise in diabetic and normal pregnancy on maternal and fetal outcome in the rat. Life Sci 45: 647-654.) on weights in birth of rats, showing that this result is contradictory. The exercised dams, regardless of intensity, exhibited increased placental weights as a compensatory mechanism to increase maternal-fetal exchanges and the nutritional supply to the fetus during development. However, the placental efficiency was decreased in exercised groups, but was more reduced in heavy-exercise group, causing lower blood flux to fetuses and, consequently leading to intrauterine growth restriction, confirmed by higher percentage of fetuses classified as SGA.

In conclusion, the moderate-swimming intensity caused beneficial effects for the maternal environment and the mild and moderate-exercise presented similar fetal repercussions. Nevertheless, the heavy-exercise intensity caused maternal metabolic alterations impairing fetal growth. Therefore, these findings confirm that exercise should be recommended for specialized professionals to avoid maternal complications and repercussions on their offspring.

ACKNOWLEGMENTS

The authors are grateful to Talísia Moreto, technician of the Laboratory of Experimental Research on Gynecology and Obstetrics, to laboratory team for support and Prof Dr. José Eduardo Corrente, statistician of the Escritório de Apoio à Pesquisa da Faculdade de Medicina de Botucatu, Universidade Estadual Paulista (UNESP), for their valuable contributions in study design and statistical analysis. This study was supported by FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant number 2012/25168-9) and CNPq – Conselho Nacional de Desenvolvimento Científico e Tecnológico for financial support (Process Number 475073/2013-4) and for researcher fellowship to DC Damasceno. The CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) financed the AO Netto´s fellowship (Finance Code 001).

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