Journal of Exercise Physiologyonline

October 2019Volume 22 Number 5

Editor-in-ChiefTommy Boone, PhD, MBAReview BoardTodd Astorino, PhDJulien Baker, PhDSteve Brock, PhDLance Dalleck, PhDEric Goulet, PhDRobert Gotshall, PhDAlexander Hutchison, PhDM. Knight-Maloney, PhDLen Kravitz, PhDJames Laskin, PhDYit Aun Lim, PhDLonnie Lowery, PhDDerek Marks, PhDCristine Mermier, PhDRobert Robergs, PhDChantal Vella, PhDDale Wagner, PhDFrank Wyatt, PhDBen Zhou, PhD

Official Research Journal of the American Society of

Exercise Physiologists

ISSN 1097-9751

Official Research Journal of the American Society of Exercise Physiologists

ISSN 1097-9751

JEPonline

The Effects of Omega-3 and Melatonin Supplementation Associated with Moderate Physical Exercise on Biochemical Parameters, Hippocampus Morphometry, and Memory in Wistar Rats

Danielle Dutra Pereira1,5, Wanessa Noadya Ketruy de Oliveira1, Gilberto Vieira Fialho2, Wedja Stephany de Assis Lima2, Jeine Emanuele Santos da Silva1, Laíse de Souza Elias1, Leandro Álvaro Aguiar1, Thaís Heloise da Silva Almeida1, Raphael Fabricio de Souza4,5, Joaquim Evêncio-Neto1

1Department of Animal Morphology and Physiology, Federal Rural University of Pernambuco,Recife, Brasil. 2University Center Maurício de Nassau, Recife, Brasil. 4Department of Physical Education, Federal University of Sergipe - UFS, São Cristovão, Brazil. 5Group of Studies and Research of Performance, Sport, Health and Paralympic Sports - GEPEPS, the Federal University of Sergipe - UFS, São Cristovão, Sergipe, Brazil

ABSTRACT

Pereira DD, Oliveira WNK, Fialho GV, Lima WSA, Siva JESS, Elias LS, Aguiar LA, Almeida THS, de Souza RF, Evêncio-Neto J. The Effects of Omega-3 and Melatonin Supplementation Associated with Moderate Physical Exercise on Biochemical Parameters, Hippocampus Morphometry, and Memory in Wistar Rats. JEPonline 2019;22(5):200-219. The purpose of this study was to evaluate the effects of omega-3 and melatonin supplementation associated with moderate exercise. Sixty-day-old rats were divided into 8 Experimental Groups (n = 5 animals per group) according to the treatment: (1) A, omega-3; (2) B, melatonin; (3) C, physical exercise; (4) D, omega-3 and melatonin; (5) E, omega-3 and exercise; (6) F, melatonin and exercise; (7) G, omega-3, melatonin, and exercise; and (8) H, Control. The body mass at 30 days postnatal (M30) of the animals in B Group (42.47 ± 1.23) was lower compared to all

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Groups. At 60 days postnatal (M60), A (198.4 ± 4.15), B (213.3 ± 3.63), and C Groups (208.8 ± 6.11) presented higher body weight than the F Group (161.5 ± 6.37) and the H group (151.8 ± 4.80). The G group (172.4 ± 6.42) had a lower mean body weight than the B Group. At 30 days of age, VLDL (79.08 ± 12.89) and triglyceride (395.4 ± 64.43) in the F Group were higher than the B Group (30.44 ± 3.34 and 152.2 ± 16.9 respectively). After 60 days, the HDL levels of the F Group (77.13 ± 13.32) had increased higher than the D Group (25.98 ± 2.51). Regarding the glycemic response, the D Group (248.2 ± 24.48) was higher than all the Groups, while the H Group (155.6 ± 16.80) differed from the A, C, and F Groups. Regarding hippocampal morphometry at M30, only the D Group differed from the A and G Groups, presenting a higher neuronal density. At M60, the B Group presented a higher number of neurons than the A, C, E, F, and H Groups. The C group demonstrated neuronal reduction in relation to the A, D, E, F, and G Groups. When D was observed, it demonstrated a greater number of neurons than the A, E, F, and H Groups. Finally, the H Group had lower neuronal density when compared to the A, E, F, and G Groups. For the recognition test, there were no differences between the evaluated treatments at 30 and 60 days of age. Physical exercise related to omega-3 and melatonin supplementation after 30 and 60 days resulted in changes in body weight, biochemical parameters, and hippocampus morphometry, but did not interfere with the memory of Wistar Rats.

Key Words: Exercise, Hippocampus, Melatonin, Memory, Omega-3

INTRODUCTION

In the perinatal period, physiological processes such as hyperplasia, hypertrophy, myelination, and neuronal migration occur at maximum speed (7). Rapid development is observed and the brain becomes more vulnerable to environmental demands (58). The level of neurogenesis in adult individuals can also be positively or negatively modulated by environmental conditions (41). The combination of various stimuli can increase neurogenesis and neuron survival (62).

Neurogenesis is important for maintaining cognitive health (3). Hippocampus is a much studied structure of the central nervous system (CNS), which plays an important role in learning and memory (17). Neurogenesis in this brain region is well established and more intense during embryonic development (52) and persists throughout adulthood (21).

Hippocampus is formed by two main regions, the dentate gyrus (DG) and the Ammon’s horn (cornu ammonis, CA), the latter being anatomically and functionally differentiated into distinct subfields named CA1, CA2, CA3, and CA4 (13). The CA1 pyramidal cells originate from the primary efferences of the hippocampal trisynaptic circuit. The cells are fundamental to the formation of declarative memories as part of a reverberant and functionally active circuit that involves the entorhinal cortex, GD and CA3 (2).

Proper nutrition during early life is an essential factor in helping to ensure proper brain development (58). Likewise, a rational diet that meets the needs of the body and the preventive properties of some nutrients are determinant of a healthy lifestyle for people of different age groups (75). Considerable emphasis has been placed on omega-3 (ω-3), eicosapentaenoic (EPA, 20:5n-3), and docosahexaenoic (DHA, 22:6n-3), and polyunsaturated fatty acids (PUFA) act on the physicochemical properties of the cell

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membrane, such as fluidity, permeability, and viscosity (69). Each acid plays an important role in axonal and dendritic growth (4). EPA and DHA are important components involved in neurogenesis (8) to help ensure proper brain development. The contribution of each is critical to cell structure and function in the nervous system (29). Omega-3 fatty acids have been shown to be effective in enhancing learning and memory, and preventing memory deficit under various experimental conditions (36).

In addition to a balanced diet, regular physical activity has been shown to be important in health promotion (19), and its benefits extend to neurocognitive aspects (12). Physical exercise promotes vascularization and energy metabolism, increases neurogenesis and synaptogenesis (14), and improves memory, learning (18), brain activity, and cognitive function (6). However, there is evidence that exercise intensity may determine memory responsiveness (9). For example, moderate exercise intensity protects hippocampal cells from ischemic damage (64) and, therefore, is closely linked to cognitive improvements (38). Paradoxically, high intensity physical exercise is characterized by systemic fatigue, which may result in a decline in cognitive performance, tasks that require attention, memory, and learning (72).

In neurogenesis, melatonin also plays a crucial role (15) by acting on neural stem cells, increasing proliferation (67), viability, survival, and differentiation of these cells (54). Thus, melatonin lays the foundation to form larger neurons and, to lesser extent the astrocytes (33). Melatonin contributes to the maturation and complexity of new neuron dendrites (78) that facilitates their integration into pre-existing circuits (8).

The purpose of the present study was determine the effects of omega-3 and melatonin supplementation in association with moderate intensity exercise on body weight, biochemical parameters, hippocampal morphometry, and memory in the Wistar rats. METHODS

A total of 120 Rattus norvegicus, Albinus variety, Wistar strain from the Department of Animal Morphology and Physiology of the Federal Rural University of Pernambuco were kept in a temperature controlled environment (22 ± 2ºC) in 12-hr light-dark cycle with water and food (Presence® commercial ration) ad libitum. Of the total animals, 40 were females (F0) and 80 were males (F1).

The animals (F0) were divided into 8 experimental groups (n = 5 animals per group) according to treatment kind: (1) A, omega-3; (2) B, melatonin; (3) C, physical exercise; (4) D, omega-3 and melatonin; (5) E, omega-3 and exercise; (6) F, melatonin and exercise; (7) G, omega-3, melatonin, and exercise; and (8) H, control. Protocols were started from 60 days of age and continued until weaning offspring (165 days of age). The offspring (n = 10 males per group) were subjected to the same protocol as their mothers from weaning to 60 days of age.

ProceduresSupplementation with omega-3 was performed using Naturalis® fish oil capsules (1000 mg) containing 180 mg EPA and 120 mg DHA) administered by gavage (10). Melatonin was

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administered at a dose of 0.5 mg∙kg-1 body weight between 18:00 and 19:00 hrs (70) subcutaneously. Crystalline melatonin (Sigma Chemical Co., Et. Louis, Mo., USA) was dissolved in 0.1 mL 0.9% NaCl containing 5% ethanol. The Control Group received daily injections of 0.1 mL subcutaneous vehicle (56) and 0.9% NaCl saline solution by gavage.

Moderate swimming exercise was performed in a tank with water maintained at a controlled temperature of 31 ± 1°C. The training lasted 60 min, performed at frequency of 5 d∙wk -1 (59). Initially, the animals were submitted to adaptation to the aqueous environment for 5 consecutive days, increasing the time of the sessions gradually until reaching the stipulated in the protocol. From the 1st to the 4th day of adaptation, the animals remained in the tank for 15, 30, 45, and 60 min, respectively. On the 5th day, the same time as the previous day (60 min) was repeated and used until the end of the experiment.

At 30 (M30) and 60 (M60) days old, F1 generation animals were weighed on an analytical balance to assess body weight. Five animals from each Group were randomly selected and anesthetized with the combination of ketamine (50 mg) and xylazine (20 mg) at a dose of 0.1 mL for each 100 g of live weight administered intramuscularly (37). Then, the animals were euthanized by the cardiac puncture exsanguination method. The data for lipid profile (triglycerides, total cholesterol, HDL, and LDL) and serum glycemic were obtained from blood samples (0.5 mL) collected in tubes without anticoagulant with the aid of Bioplus 200f®.

Subsequently, the animals were perfused with physiological solution (0.9% NaCl) with 10% formaldehyde in phosphate buffer (pH 7.4). The brains were collected and fixed in a 10% formaldehyde buffered solution, dehydrated in increasing concentrations of ethyl alcohol (70% to P.A.), diaphanized in xylol, impregnated and embedded in paraffin. Coronal 4-mm sections of the hippocampus were obtained using a Leica® rotary microtome. The sections were adhered to histological slides, stained with Cresil violet and covered with glass coverslips. Photomicrographs were obtained under a Leica® light microscope in a 40X objective, using ImageJ® software for morphometric evaluation of the hippocampal CA1 region.

Fifteen areas were randomly delimited in each histological section in a 40x magnification and, with the aid of a grid (21 x 21), the intersection points of the straight lines that affected the hippocampal CA1 pyramidal neurons were counted bilaterally, obtaining the mean for each histological section (Figure 1). These data allowed for determining the relative density of the area (%) of neurons in this region in rats aged 30 and 60 days. Area density in the hippocampal region CA1 was calculated (28)

For the Spontaneous Object Recognition Test (SORT), a neutral and opaque color test arena (dimensions 65x45x45 cm) was used, and triplicate objects, approximately 200 mL in volume and heavy enough not to be moved. In the first trial, 2 identical objects (A1 and A2) were placed in opposite corners of the arena, separated by sufficient space for exploration of all their faces. The animal was positioned in the middle region of the arena with its back to the objects and was able to explore them freely for 3 min. In the second rehearsal, the arena objects were exchanged for an identical one to the previous ones (A3) and for a new object called B. After a predetermined time interval of 24 hrs, the rat was again placed in the testing arena for 3 min to explore the new pair of objects (A3 and B) in the so-called retest session. The simple measure of recognition or discrimination was the difference between time spent

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exploring the new stimulus (B) and time spent exploring the family stimulus (A3), i.e., B-A3 (23).

Figure 1. Photomicrographs of Rat Hippocampus. A = Coronal section of hippocampus with indications of CA1, CA2, CA3, CA4, and GD regions. B = Region CA1. The yellow boxes represent the areas where cell density counts were made. C = Grid (21 x 21) used to count the intersection points of lines that focused on the pyramidal neurons. Cresil violet: 400X.

Statistical Analyses

The Kolmogorov-Smirnov test was applied to verify the normal distribution of the data. For the data that followed a Gaussian distribution, an analysis of variance (ANOVA) and a post-hoc Tukey test were performed. The data with non-Gaussian distribution were analyzed by Kruskal-Wallis test and Dunn post-hoc test. All tests were performed using GraphPad Prism 5.1 software. The statistical significance was set at an alpha level of P<0.05%.

RESULTS

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It was observed that at moments M30 and M60, the different treatments interfered with metabolism, resulting in a change in weight gain (Figure 2). The mean value for body mass in M30 of the Group B animals (42.47 ± 1.23) was lower than the body mass of Groups A (72.71 ± 3.06), C (71.14 ± 2.52), D (66.69 ± 1.88), E (53.91 ± 0.87), F (67.18 ± 3.02), G (62.00 ± 5.02), and H. When compared to Group E, Groups A, C, D, and F had a higher mean body weight value. Group H had lower average weight than Groups A and C.

At M60, Groups A (198.4 ± 4.15), B (213.3 ± 3.63), and C (208.8 ± 6.11) had higher body weight than Groups F (161.5 ± 6.37) and H (151.8 ± 4.80); also reducing the weight of Group G (172.4 ± 6.42) in relation to Group B.

Total cholesterol, HDL, LDL, VLDL, triglycerides, and glucose at M30 are shown in Figure 3. Matrix and offspring treatment modified only the VLDL and triglyceride parameters at M30 of the data analysis (P<0.05). VLDL (79.08 ± 12.89) and triglyceride (395.4 ± 64.43) from Group F showed a significant difference when compared to VLDL (30.44 ± 3.34) and triglyceride (152.2 ± 16.9) from Group B.

Figure 2. Body Weight (mean ± standard deviation) of the Animals Evaluated at Thirty (M30) and Sixty (M60) Days of Age. A = Omega-3; B = Melatonin; C = Physical Exercise; D = Omega-3 and Melatonin; E = Omega-3 and Physical Exercise; F = Melatonin and Physical Exercise; G = Omega-3, Melatonin and Exercise; H = Control. Different letters at the top of the column indicate significant difference between groups (P<0.05).

At time M60, the experimental protocols resulted in modifications of the serum lipid profile between treatments (Figure 4). The HDL data indicated that Group F (77.13 ± 13.32) showed a significant difference when compared to Group D (25.98 ± 2.51).

When the VLDL and triglyceride tests were performed, it was observed that Group F differed from Groups E and D, presenting an average value of VLDL of 56.00 ± 4.40 and triglyceride of 280.00 ± 22.00, while It presented an average value of 31.49 ± 4.74 for VLDL and 157.4 ± 23.68 for triglyceride, and D presented VLDL of 27.12 ± 1.78 and triglyceride of 135.6 ± 8.90.

In the glycemic test, Group D (248.2 ± 24.48) was different from Groups A (39.20 ± 14.39), B (95.60 ± 14.39), C (39.40 ± 9.14), E (131.4 ± 28.95), F (20.60 ± 4.57), and G (40.75 ± 9.30), while Group H (155.6 ± 16.80) differed from Groups A, C, and F. There was a still significant difference between Group E and F.

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Figure 3. Serum Biochemical Profile of Animals Evaluated at 30 Days of Age (M30). A = Omega-3; B = Melatonin; C = Physical Exercise; D = Omega-3 and Melatonin; E = Omega-3 and Physical Exercise; F = Melatonin and Physical Exercise; G = Omega-3, Melatonin and Exercise; H = Control. Median ± interquartile range. Different letters at the top of the column indicate significant difference between groups (P<0.05).

Figure 4. Serum Biochemical Profile of Animals Evaluated at 60 Days of Age (M60). A = Omega-3; B = Melatonin; C = Physical Exercise; D = Omega-3 and Melatonin; E = Omega-3 and Physical Exercise; F = Melatonin and Physical Exercise; G = Omega-3, Melatonin and Exercise; H = Control. Median ± interquartile range. Different letters at the top of the column indicate significant difference between groups (P<0.05).

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Data regarding hippocampal morphometry for the different treatments can be seen in Table 1. The analysis showed that supplementation with omega-3 and melatonin and submission to physical exercise interfered with the number of hippocampal CA1 neurons evaluated by area density at different ages (M30 and M60) for the treatments performed. At M30, only Group D differed significantly from Groups A and G, presenting a higher neuronal density.

Table 1. Area Density (%) of Pyramidal Neurons of the Hippocampal CA1 Region of Rats Evaluated at 30 and 60 Days of Age

Groups M30 M60

A 19.68 ± 1.03 a 20.38 ± 0.35 a

B 20.10 ± 0.69 abc 22.63 ± 0.34 b

C 20.72 ± 0.48 abc 15.61 ± 0.58 c

D 24.24 ± 1.3 bc 23.22 ± 0.46 bd

E 20.36 ± 0.61 abc 20.50 ± 0.43 ae

F 20.23 ± 0.59 abc 19.91 ± 0.42 aef

G 19.00 ± 0.45 ac 21.14 ± 0.35 abdef

H 21.17 ± 0.55 abc 16.97 ± 0.40 c

A = Mothers and offspring supplemented with omega-3; B = Melatonin-supplemented mothers and offspring; C = Mothers and offspring submitted to physical exercise; D = Mothers and offspring supplemented with omega-3 and melatonin; E = Mothers and offspring supplemented with omega-3 and submitted to swimming; F = Mothers and offspring supplemented with melatonin and submitted to physical exercise; G = Mothers and offspring supplemented with omega-3 and melatonin and submitted to physical exercise; H = Control group. Different letters in the same column indicate statistically significant difference between groups (P<0.05).

The treatments performed altered the behavior of the pyramidal neuron population in the CA1 region of offspring hippocampus at 60 days of age. Group B had a higher number of neurons than Groups A, C, E, F and H. In Group C, a smaller neuronal population was observed compared to Groups A, D, E, F and G. When Group D was observed, this one presented higher number of neurons than Groups A, E, F and H. Finally, Group H presented lower neuronal density when compared to Groups A, E, F and G (Figure 5).

The SORT data were compared. The analysis showed that supplementation with omega-3 and melatonin and submission to physical exercise did not interfere with this type of test (Figure 6).

M30 M60

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Figure 5. Photomicrographs of Rat Hippocampus at M30 and M60 Days of Age. A = Omega-3; B = Melatonin; C = Physical Exercise; D = Omega-3 and Melatonin; E = Omega-3 and Physical Exercise; F = Melatonin and Physical Exercise; G = Omega-3, Melatonin and Exercise; H = Control. Cresil violet: 400X.midal neuron population in the CA1 region.

Figure 6. Spontaneous Object Recognition Test at M30 and M60. A = Omega-3; B = Melatonin; C = Physical Exercise; D = Omega-3 and Melatonin; E = Omega-3 and Physical Exercise; F = Melatonin and Physical Exercise; G = Omega-3, Melatonin and Exercise; H = Control. (P>0.05).DISCUSSION

The animals presented weight variation due to the kind of treatment they were submitted. At M30, the animals that received exogenous melatonin (Group B) had lower body mass when compared to the Control group and all other kinds of treatment used. Similarly, animals treated with omega-3 and submitted to swimming (Group E) presented a reduction to those

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supplemented with omega-3 (Group A), submitted to swimming (Group C), and treated with melatonin and submitted to swimming (Group D). At M60, the animals in the omega-3 supplemented groups (Group A), treated with melatonin (Group B) and submitted to physical exercise (Group C) presented higher body weight than the group treated with melatonin and submitted to physical exercise (Group F) and the Control group (Group H).

At M30, the average body weight of Group B was lower when compared to the other Groups. This finding may be associated with the use of melatonin, as pointed out in research by Rasmussen et al. (55), who demonstrated that daily administration of melatonin in middle-aged male rats fed a high-calorie diet decreased body weight, suppressed visceral fat deposition, and restored leptin and insulin levels. According to Cipolla-Neto (16), melatonin regulates biological aspects of adipocytes that influence energy metabolism, lipidemia and body weight, such as lipolysis, adipocyte differentiation and fatty acid uptake that presents an antiobesogenic effect.

The increase in body weight of animals supplemented with omega-3 in both M30 and M60 corroborate with the fatty acid ratio were involved in adipogenesis. A study by Águila et al. (1) showed that the average body weight of animals in the omega-3 canola oil-treated group was higher (11.8%) than in the Control group. Also, it is understood that PUFA-rich diets can modulate protein expression in adipose tissue and may act as transcriptional regulators of some genes related to lipid metabolism (50).

In our findings, however, Group E in M30 supplemented with fish oil associated with physical training had the opposite effect to Group A. Here, it is reasonable to infer that the reduction in body weight of animals in Group E may be associated with physical exercise. This fact also occurred in group F in M60. Improved aerobic conditioning triggers a series of physiological stimuli that enhance oxygen uptake and the use of fatty acids as an energy source, which reduces body fat deposits and decreases obesity rates (60). In addition, regular exercise can lead to changes in body composition, increase muscle mass and reduce body fat that may have happened to Group C at time M60. Data from studies by Pellizzon et al. (48) showed that rats fed normolipid diet and trained with swimming for 6 wks were heavier than rats with hyperlipid diet that were also trained, assuming that frequent swimming exercise was able to increase muscle mass of normolipid diet animals.

In the analysis of the biochemical profile, a significant difference was found only in the VLDL and triglyceride parameters of the animals treated with exogenous melatonin and submitted to swimming (Group F) compared to the animals treated with melatonin only (Group B), at time M30. At time M60, different treatment types resulted in a significant difference in serum HDL value of Group F animals when compared to omega-3 supplemented and melatonin-treated animals (Group D). The values of VLDL and triglyceride also showed significant difference between Group F when compared to animals supplemented with omega-3 and treated with melatonin (Group D), and animals supplemented with omega-3 and submitted to swimming (Group E). For glucose values, the animals from Group D showed higher values than the animals from all other experimental groups, except for the Control group, which also presented high values compared to the animals supplemented with omega-3 (A), who were submitted to exercise (Group C), and who were treated with melatonin and swimming (Group F). The animals from Group E also showed differences in glycemic levels when compared to Group F.

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The triglyceride results can be explained by the reduction of VLDL fractions, thus minimizing the hepatic triglyceride production, by the possible decrease in free fatty acids. The high HDL value of group F in M60 may be due to exercise. Studies have shown that exercise is effective in raising HDL by up to 25% (25), which is observed as a function of the frequency and intensity of aerobic exercise (34). This outcome is a function of the stimulation of lipoprotein lipase, considering that the generation of HDL particles is a process inherent in the metabolism of triglyceride-rich lipoproteins (21) as well as a reduction in the catabolism of apolipoprotein A-I and the decrease in esterified cholesterol transfer protein activity (27).

However, the role of exercise in elevating plasma HDL concentration appears to be conditioned by several factors, such as an improvement in insulin resistance, reduced body weight, as well as triglyceridemia, gender, age, previous lipid profile, enzyme genetic polymorphisms, and proteins involved in HDL metabolism (5). Such factors are responsible for the high variability of the HDL cholesterol response to physical exercise. As observed in our study among the Experimental Groups, several studies indicated that supplementation with ω-3 PUFA is not able to modify HDL levels (42). The study by Pedersen et al. (46), however, points to an increase in these levels. When analyzing total cholesterol and LDL, most studies do not observe significant differences between groups supplemented with ω-3 and groups submitted to normal diet (73). However, the effects of omega-3 fatty acids on total cholesterol, HDL and LDL are still controversial. Also, the existing literature on the impact of omega-3 supplementation on LDL subfractions is inconsistent (45).

Our results are consistent with previous studies showing that supplementation with ω-3 fatty acids causes favorable lipid changes in serum and tissues. The most consistent finding is the reduction in serum concentration of triglycerides and free fatty acids in the postprandial state (40). The results observed in this study are similar to the recent findings by Skulas-Ray et al. (66) who observed a 27% reduction in triglyceride concentrations after 60-day ingestion of 3.4 g of omega-3 fatty acids by healthy subjects with moderate hypertriglyceridemia. Oelrich et al. (45) also demonstrated a decrease of 26% in triglyceride concentrations after ingestion of 4 g of EPA and DHA for 3 months in hypertriglyceridaemic subjects.

The decrease in triacylglycerol concentration is related to the reduction of hepatic lipogenesis caused by the direct inhibition of the activity of liver enzymes related to the synthesis of fatty acids, such as fatty acid synthase, glucose-6-phosphate dehydrogenase and triacylglycerol lipase (32), and an increased activity of enzymes related to fatty acid oxidation, such as the liver carnitine palmitoyl transferase enzyme (74). There is a dose-response relationship between triglyceride levels and ω-3 fatty acid intake (26).

Our findings indicate a tendency to increase glucose values in rats submitted to fish oil supplementation. In fact, the results are similar to those described by Mori et al. (43) who observed a significant increase in fasting insulin and a tendency to increase fasting glucose values in hyperlipemic men after the ingestion of fish oil. The same researchers also reported that patients with type 2 diabetes who had consumed these fatty acids decreased blood pressure and triacylglycerol concentration, although there was an increase in fasting glucose concentration (76).

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Glycemic metabolism is directly proportional to ω-3 fatty acid dosages. The most likely mechanism to explain the adverse effects of these PUFAs on glycemic control would be an increase in liver glucose production, which may be related to increased flow of hepatic precursors of gluconeogenesis (76). Serum glucose elevation observed in groups submitted to omega-3 diets, such as Groups D and E, may also be related to fatty acid overload that results in the adipose tissue hypertrophy. This fact is associated with the production of inflammatory cytokines that can trigger hyperglycemia and insulin resistance (35).

Another fact is that the unsaturation of PUFA favors the action of free radicals, consequently increasing lipoperoxidation. The study by Pedersen et al. (46), reports that ω-3 PUFA supplementation is associated with increased formation of reactive oxygen species - (ROS). A correlation between hyperglycemia and increased lipid peroxidation has been reported (47).

The reason why group E had a high glycemic level compared to group F may be linked to the absence of exogenous administration of melatonin. As noted by Picinato et al. (49), melatonin plays a role in controlling circadian rhythms involved in the regulation of glucose homeostasis and in insulin secretion. Several studies indicate that the action of melatonin on the glycemic profile may be associated with its effect on improving glucose uptake by increasing insulin sensitivity that promotes changes in gene expression of glucose protein transporter-4 (GLUT-4) (77). Increased energy expenditure by skeletal muscles as a result of physical exercise promotes an increase in intracellular AMP: ATP ratio, which in turn stimulates the adenosine monophosphate kinase (AMPK) pathway that promotes the translocation of GLUT-4 containing vesicles, which results in the facilitation of glucose transport to the muscle that is similar to insulin (44).

Physical exercise related to omega-3 and melatonin supplementation interfered with the number of hippocampal CA1 neurons. At time M30, only the animals supplemented with omega-3 and treated with melatonin (Group D) differed significantly from the animals in the group supplemented with omega-3 (group A) and the group supplemented with exercise-associated omega-3 and melatonin (Group G), thus presenting a higher neuronal density. At 60 days of age (M60), the melatonin-treated group (Group B) had higher neuronal density than Groups A, C, E, F, and H while the group undergoing physical exercise (Group C) had a lower neuronal population in relation to Groups A, D, E, F, and G.

The fact that group A had a low density of neurons in the CA1 region at M30 may be linked to the relationship between ω-3 PUFA and oxidative stress, which has been addressed in the literature (65). The unsaturation of PUFA favors the action of free radicals, consequently, an increase in lipoperoxidation. Erdogan et al. (24) reported that supplementation with ω-3 PUFA is related to the increase in the formation of ROS, which are extremely reactive molecules and are able to combine nonspecifically with several molecules of the cell structure resulting in tissue damage (61).

When fish oil supplementation is combined with melatonin administration (Group D), the result is a higher neuronal density compared to fish oil supplementation alone (Group A) in M60 and M30. These data are in agreement with the findings from Chu et al. (15) who reported that melatonin, through its antioxidant action, has a neuroprotective effect. The same was not seen in Group G, which in addition to the supplementation with fish oil and

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melatonin underwent physical exercise that together with omega-3 may contributed to the production of ROS due to accelerated energy metabolism. In fact, these oxidative damage-inducing reactive species may contribute to neurodegeneration (53).

The action of melatonin reinforces the claim that the neuronal membrane becomes more sensitive to oxidative damage if not adequately counterbalanced by antioxidant defense (73). According to Crupi et al. (20), melatonin also exerts action on neural stem cells increasing their proliferation, viability, survival, and differentiation. Studies by Rennie et al. (58) found that pinealectomy causes a decrease in the number of pyramidal neurons in the hippocampus of adult rats, which can be reversed after the replacement of melatonin in the drinking water of animals.

The smaller population of neurons observed in Group C (Figure 5-2C) at M60 may be associated with the increase in ROS production that occurs during physical exercise due to the increased cellular metabolism and the uptake and utilization of oxygen by the peripheral tissues (66). Sports that obtain energy through aerobic metabolism are more likely to promote the release of ROS compared to those that obtain energy through anaerobic metabolism (30).

A recent study by Soya et al. (68) reported that while moderate-intensity exercise increases the activation of hippocampal neurons, there is a significant increase in blood lactate levels and other markers of biological stress response. In fact, they observed that hippocampal neurogenesis is more significant in low intensity exercise with no lactate production or biological stress. In particular, spatial memory is known to be dependent on hippocampal activity in rats (31). Although morphological changes in the hippocampus were observed between the different Experimental Groups, these did not result in significant differences in SORT.

Similar to our results, a recent study by Mello et al. (39) shows that there was no difference in learning, emotionality, and exploratory activity with the use of PUFA. When comparing the intragroup relationship through the discrimination index, it was observed that memory was preserved in all Groups, regardless of the protocol used, achieving recognition of the new object as to its form and location (51). Interestingly, the memory test data disagree with the studies conducted with young rats fed a diet rich in omega-3 PUFA that had positive effects on learning ability (11).

Corroborating our results regarding physical exercise and its effect on SORT, Mello et al. (39) rats underwent forced exercise for 8 wks on a treadmill. The results indicate no significant difference when compared to the Control Group, which shows no effect of physical activity on the spatial memory test. Interestingly, the same type of exercise applied for 2 wks resulted in impaired rat performance in the same test.

CONCLUSIONS

Supplementation with omega-3, melatonin, and the submission to exercise interfered with the metabolism of the animals at 30 and 60 days, which resulted in weight gain alteration. The EPA, DHA, PUFA, melatonin, and physical exercise had an effect on the animals' glucose, HDL, VLDL, and triglycerides levels.

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The duration of omega-3 and melatonin supplementation in the rats and physical training significantly interfered with the density of pyramidal neurons of the offspring hippocampus CA1 evaluated at 30 and 60 days of age, which indicated that the different protocols used have an influence on brain tissue development.

Physical exercise and fish oil and melatonin supplementation during the experimental period did not influence the learning processes. The present research provides evidence that the effects induced by the association between omega-3, melatonin, and exercise on learning and memory are not always beneficial and depends on the intensity, the developmental period, and the duration of the interventions.

ACKNOWLEDGMENTSThe Graduate Program in Animal Bioscience of Federal Rural University of Pernambuco and Coordination of Improvement of Higher Level Personnel (CAPES) Brazil, for the financial aid and the Graduate Scholarship.

Address for correspondence: Danielle Dutra Pereira – Avenida Jerônimo Gueiros, nº 328, Bairro Centro – CEP 53510-335 – Abreu e Lima/ PE – (81) 996643402 Email: dani.dutra15@ hotmail.com

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