The animal protocol was approved by the Animal Ethics Committee of the Fluminense Federal University (Protocol Number CEA 882/2016), and the procedures were in accordance with the guidelines for experimentation with animals (NIH Publication Nº. 85-23, revised 1996). Female wistar rats (3 months old) from the Centre of Laboratory Animals of Fluminense Federal University were housed under a controlled temperature (21 ± 1ºC), humidity (60 ± 10%) and 12 h light / dark cycle, with free access to water and food.

Initially, 24 female rats were mated with 12 males (2:1 ratio) and fed a commercial rodent chow (Nuvilab®, Nuvital Ltda, Paraná, Brazil). During pregnancy, all rats were kept in individual cages. After the offspring was born, for the lactation period, the rat dams were divided into two groups (n=12 per group), following the order that the female rats gave birth: the first rat to give birth was allocated into one group, the second was allocated to the other, successively. A control group (CG) was fed a control diet and filtered water, and the flaxseed group (FG) received the diet added with 25% brown flaxseed flour, and filtered water. Diets were manufactured and stored as pellets at 4 °C in agreement with the American Institute of Nutrition (AIN-93G) recommendations for rodent diets ¹⁷ (Table1). A concentration of 25% of flaxseed flour in the diet (25g/ 100g diet) would meet the recommended fiber intake. The addition of oil was not necessary, as the seed itself is a source of oil.

Table 1

	Diets
Nutrients (g/kg)	Control	Flaxseed Flour
Casein (≥85% of protein)	190	141
Corn starch	539.5	458.5
Sucrose	100	100
Soybean oil	70	0
Flaxseed	0	250
Fiber (cellulose)	50	0
Vitamin mix (AIN-93G)	10	10
Mineral mix (AIN-93)	35	35
L-Cystine	3	3
Choline	2.5	2.5
Tert-butylhydroquinone	0.014	0.014
Total	1000	1000
Carbohydrate (% of total kcal)	64	59
Protein (% of total kcal)	19	19
Fat (% of total kcal)	17	22
Energy (Kcal/kg)	3950	4009

AIN: American Institute of Nutrition, Casein: mineral and vitamin mix, L-cystine and choline bitartrate: Pragsoluções®; cornstarch and fiber: FARMOS®; soybean: Liza®; commercial sucrose: União®; flaxseed flour: Mãe terra®. Formulated based on the AIN-93G recommendations for rodent diets

At weaning, two male offspring from each mother started to receive commercial rodent chow with a normal lipid and protein content (Nuvilab®, Nuvital Ltda, Paraná, Brazil), containing 22% protein (main protein sources are meat, fish, soy and amino acids), 66% carbohydrate and 11% lipid and allocated into one of the eight experimental groups with six animals each one (n=6) until they were 180 days old:

The number of six animals per group followed the 3 Rs principle, which aims to reduce the number of animals used in the experiment. According to Damy et al., ¹⁸ the reduced number of animals in biomedical research does not affect the detection of biological effects.

For analysis of body mass gain throughout the study, body mass of the groups was measured once a week on a digital scale (precision 0.01g) (Filizola®). Food intake of the groups was measured three times a week. For feed intake calculation, individual intake was obtained by subtracting the remaining feed from the amount offered. Water and 1% NaCl solution intake were measured three times a week with the aid of a graduated test tube with 0.5mL precision. The amount supplied and the amount remained were quantified individually to determine water and NaCl intake. Sodium intake calculation was made adding the sodium intake from the 1% NaCl solution with the sodium contained in the commercial rat chow given to the animals (Nuvilab®, Nuvital Ltda, Paraná, Brazil) (2,700 mg Na / kg of rat chow).

At 90 days of life, exercise training was performed on a motorized treadmill (AvsProjetos®, Brazil) for 12 weeks, 5 times a week for 60 min each session, in individual lanes. Exercise training was started at a speed of 0.3 km/h in the first session, increasing progressively to a final speed of 1.1 km / h, according to each animal’s performance. This exercise intensity corresponded to 70% to 80% of the maximum VO ₂ . ¹⁹

When the animals were three months of age, systolic blood pressure (SBP) was measured (in mmHg) once a month until six months of age, using the non-invasive method of tail-cuff plethysmography (tail plethysmograph V1.10-Insight, Brazil).

After preconditioning in a chamber, the animals were pre-heated in the chamber to 35±2°C for five minutes. The official SBP of each animal was calculated by averaging three consecutive stable measurements (with a difference of about one minute among them).

At the end of the experimental protocol (180 days), after a 6-hour fasting period, the animals were anesthetized with a solution containing 80mg of ketamine (10% ketamine hydrochloride, Syntec Tecnologia Farmacêutica Aplicada à Medicina Veterinária, Brazil) and 10mg of xylazine (2% xylazine hydrochloride, Syntec Tecnologia Farmacêutica Aplicada à Medicina Veterinária, Brazil); a thoracotomy was performed and the heart and the aorta were removed. The artery was dissected from the aortic arch, and the aorta was sectioned transversely with transverse cuts in the distal end of the sections. Later, the pieces of aorta were fixed in buffered formalin at 10% (Millonnig formalin) for 24 hours, and processed with a standard technique of paraffin inclusion, as described by Pereira et al. ²⁵ Subsequently, the paraffin blocks containing the pieces of aorta were cut using a CUT 4050 microtome (Microtec®), in sections of 5µm, stained with hematoxylin and eosin, and Weigert’s Resorcin-Fuchsin for later evaluation.

The measurements were made using the ImageJ® software, with digital images obtained by the cellSens program with an optronics digital video camera and BX51 Olympus microscope. The images were captured at 4x magnification for measurement of the area, at 40x for analysis of thickness, and at 20x for quantification (%) of elastic lamellae and elastic fiber. All images were digitalized in .tiff format. The aortic wall area was measured by the difference between the external and internal areas of the arterial wall. For intima-media thickness, four different regions of the same diametrically opposite cut were analyzed for better precision. The number of lamellae was quantified in four different regions of the same cut, and the aortic elastin content (%) was determined by analysis of Weigert's Resorcin-Fuchsin slides using the ImageJ® software with a 20x magnification using the plugin Color Segmentation.

Data were presented as mean ± standard deviation. Data were tested for normality and homogeneity of variances (Kolmogorov-Smirnov test) and the differences among groups were tested, when appropriated, with one-way analysis of variance (ANOVA), followed by a Holm-Sidak post hoc test or non-parametric Kruskal-Wallis test, followed by Dunn’s post hoc test. Variables that did not present normal distribution were presented as median and interquartile range (box plot graphs). Regarding body mass and blood pressure, the two-way ANOVA test was performed, followed by the Bonferroni post hoc test. P value < 0.05 was considered statistically significant (GraphPad Prism v. 6.01 for Windows, GraphPad Software, San Diego, CA, USA).

At weaning (21 days of age), no difference was found in body mass between the groups, however, there was a tendency of the offspring of the dams of the flaxseed group to be smaller than control offspring (-16.7%, p=0.0686). Regarding body mass gain, it was noted that the ENaCLFG showed smaller body mass than the CG (-14%, p<0.05) and the ECG (-14.9%, p<0.05) from the 12 ^th week and the 16 ^th week, respectively, to the end of the study period. The ENaClFG also showed lower body mass than the NaClCG between weeks 18 and 21 (-10.3%, p<0.05) ( Figure 1 ). Daily feed intake was not different between the groups (p=0.1330) ( Table 2 ). During the study period, the animals receiving 1% NaCl solution had a higher fluid intake than animals receiving water (p<0.0001) ( Table 2 ). Regarding daily sodium intake, the animals of the groups NaClCG, ENaClCG, NaClFG and ENaClFG consumed a higher amount of sodium than their respective groups that consumed water (+935.5%, +845.3%, +833.4, +795.2%, respectively, p<0.0001) ( Table 2 ).

Figure 1

Table 2

Parameters / Groups	CG	NaClCG	ECG	ENaClCG	FG	NaClFG	EFG	ENaClFG	p-value
Diet (g/day/animal)	21.5±3.4	22.6±1.4	23.7±0.9	23.2±1.6	22.7±2.0	22.3±1.6	22.5±1.5	20.5±1.5	0.1330
Water or 1% NaCl solution (mL/day/animal)	31.7±5.8	54.0±2.6*	35.3±2.0 †	54.3±6.4* ,‡	33.5±4.2 †,§	51.2±6.9* ,‡,//	33.2±2.9 †,§,¶	48.9±3.9* ,‡,//,¶,#	<0.0001
Sodium (Na)(mg/day/animal)	58.0±9.3	601.3±30.0*	64.1±2.5 †	606.3±69.0* ,‡	61.4±5.4 †,§	573.1±72.7* ,‡,//	60.8±4.3 †,§,¶	545.1±40.7* ,‡,//,#	<0.0001

Data were presented as mean ± SD, with significance level of p<0.05 (one-way ANOVA, Holm-Sidak post-hoc test). * ≠ CG; † ≠ NaClCG; ‡ ≠ ECG; § ≠ ENaClCG; // ≠ FG; ¶ ≠ NaClFG; # ≠ EFG; CG: control group; NaClCG: sodium chloride control group; ECG: exercise training control group; ENaClCG: sodium chloride + exercise training control group; FG: flaxseed group; NaClFG: sodium chloride flaxseed group; EFG: exercise training + flaxseed group; ENaClFG: sodium chloride + exercise training + flaxseed group

Chronic consumption of 1% NaCl solution did not increase blood pressure in the study groups, and neither exercise training nor the flaxseed diet affected blood pressure during the lactation period (p=0.5016) ( Figure 2 ).

Figure 2

At the end of the study, no difference was found in aortic wall area (p=0.9364) or the aortic lumen area between the groups (p=0.8817) (Figures 3 and 4). However, the chronic use of 1% NaCl solution and exercise training increased the aortic intima-media thickness in the CG (10.4% and 13.3%, respectively, p<0,0001) either alone or combined (+ 17.7%, p <0.0001). In male offspring of female rats who consumed flaxseed during lactation, the chronic use of 1% NaCl alone did not increase the aortic intima-media thickness (which was not different from the CG and the FG). Nevertheless, exercise training increased the aortic intima-media thickness in the EFG compared to the CG and FG (12.8% and 9.7%, respectively, p <0.0001), and the use of the 1% NaCl solution combined with exercise training also increased aortic intima-media thickness when compared to CG and FG (+ 17.8% and 14.5%, respectively, p <0.0001) (Fig.3 and 4). Regarding the elastic component of the arterial wall, the chronic use of 1% NaCl solution by the CG caused a reduction in the number of elastic lamellae (-8.1%, p <0.0001), the FG showed a lower number of elastic lamellae than all CGs (p<0,0001), despite that, no difference amongst the groups were observed regarding the amount of elastin in the aortic intima-media (p=0.1629, Figures 3 and 4).

Figure 3

Figure 4