ORIGINAL / Investigación animal

 

Assessments of body composition and bone parameters of lactating rats treated with diet containing flaxseed meal (Linum usitatissinum) during post-weaning period

Evaluaciones de composición corporal y parámetros óseos en ratas lactantes tratadas con dietas a base de linaza (Linum usitatissinum) durante el periodo de destete

 

 

Danielle Cavalcante Ribeiro, Paula Cristina Alves da Silva, Aline D'Avila Pereira, Bianca Ferolla da Camara Boueri, Carolina Ribeiro Pessanha, Maíra Duque Coutinho de Abreu, Henrique Saldanha Melo, Letícia Rozeno Pessoa, Carlos Alberto Soares da Costa and Gilson Teles Boaventura

Laboratory of Experimental Nutrition. Departament of Nutrition and Dietetics. Fluminense Federal University. Rio de Janeiro. Brazil.

Correspondence

 

 

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ABSTRACT

Introduction: There are few studies on body composition and the effects of diet on weight postpartum women. The aim was to evaluate the body composition and bone parameters in lactating rats treated with diet containing flaxseed flour during postweaning period.
Methods: After weaning, the lactating rat were divided in control (n = 6) and experimental (F, n = 6) group, treated with 25% flaxseed flour diet. After 30 days, body composition by dual-energy X-ray absorptiometry, serum analysis, organs and intra-abdominal fat mass, femur and lumbar vertebra parameters were determined.
Results: The groups showed similar food intake, body mass and bone parameters. While F group showed the following: lower body (-5%), gonadal (-17%), mesenteric (-23%) and intra-abdominal (-6%) fat mass. Increase of HDL-cholesterol (+10%) and lower glucose (-15%), triglycerides (P < 0.05, -37%) and cholesterol (P < 0.05, -21%).
Conclusions: The findings highlight the effects of flaxseed for control of adiposity and to maintain a healthy biochemical profile during the postnatal period.

Key words: Flaxseed flour. Rats. Postweaning period. Adiposity. Bone.

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RESUMEN

Introducción: Hay pocos estudios sobre la composición corporal y los efectos de la dieta en mujeres en el periodo postparto. El objetivo consistió en evaluar la composición corporal y los parámetros óseos en ratas lactantes tratadas con dietas a base de linaza durante el periodo de destete.
Métodos: Después del destete, las ratas lactantes fueron divididas en un grupo de control (n = 6) y un grupo experimental (F, n = 6), tratadas con una dieta a base de harina de lino al 25%. Al cabo de 30 días, se midieron los parámetros corporales mediante absorciometría de rayos X de doble energía, se realizó un análisis sérico, y se evaluó órganos y masa grasa intra-abdominal así como los parámetros en fémur y vértebras lumbares.
Resultados: El grupo mostró una ingesta alimenticia similar, así como parámetros óseos y de masa corporal. Mientras que el grupo F mostró los porcentajes siguientes en masa grasa: parte inferior del cuerpo (-5%), gonadal (-17%), mesentérica (-23%) e intra-abdominal (-6%). Aumento de HDL-colesterol (+10%) y disminución de glucosa (-15%), triglicéridos (P < 0,05, -37%) y colesterol (P < 0,05, -21%).
Conclusiones: Los resultados destacan los efectos del lino para el control de la adiposidad y para mantener un perfil bioquímico sano durante el periodo postnatal.

Palabras clave: Harina de lino. Ratas. Periodo pos-destete. Adiposidad. Huesos.

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Introduction

According to the World Health Organization (WHO), there are periods of life considered vulnerable to the development of future obesity, as women in the reproductive period, with successive pregnancies.¹ The period of pregnancy and lactation are phases that produce a redirection of nutrients to the maternal tissues. The relationship between pregnancy and changes in adipose tissue was analyzed in a longitudinal study in which the results indicated that there is an association between pregnancy and increased adiposity in women.^2,3 Reports and studies of women with severe obesity have pointed to pregnancy as a cause of their excess weight.⁴

It is too often mothers face challenges related to changes common to postpartum period, such as weight gain, decreasing levels of physical activity and increased food consumption.^5-7 Furthermore, during pregnancy occur changes in bone physiology to compensate for the increasing needs of minerals and developing fetuses. During lactation, there is an increase in the rate of bone resorption, and loss of calcium from the skeleton feeding. Although the rate of bone formation is increased during this time, is exceeded by the rate of bone resorption, resulting in a rapid decrease in bone mass.⁸ However, there are few studies on body composition and the effects of diet on weight postpartum women.

Preventive and therapeutic strategies, including nutritional interventions are possible ways to avoid the appearance of changes in metabolism.^2,9 In this context, flaxseed (Linum usitatissimum) has distinguished between the effects foods with disease prevention, in addition to being an adjunct in combating obesity and overweight.^10,11

Flaxseed (Linum usitatissimum) is made up of 41% lipids (50-55% as α-linolenic acid, and 15-18% as linoleic acid), 28% fibers, 21% protein, 4% minerals and 6% carbohydrates distributed among phenolic acids, sugars, lignan and hemicelluloses.¹² Previous studies showed protective effect of flaxseed intake in experimental models.^13-16 Nevertheless, there is a lack of data on the lactating rats. Thus, the aim of this study was to evaluate the body composition and bone parameters in lactating rats treated with diet containing flaxseed flour during postweaning period.

 

Materials and methods

The protocol used to deal with experimental animals was approved by Ethics Committee on Animal Research of Fluminense Federal University, Niteroi-RJ, Brazil. All procedures are in accordance with the provisions of Brazilian Society of Science and Laboratory's Animals.

Wistar rats were kept in a room with controlled temperature (23 ± 1^o C) and with an artificial dark-light cycle (lights on from 07:00 to 19:00 hours). Virgin female rats (3 months old) were caged with male rats and after mating each female was placed in an individual cage with free access to water and food. Within 24h of birth excess pups were removed, so that only six pups were kept per dam. This procedure maximizes lactation performance.¹⁷ During 21 days of lactation, rat dams were continued on an ad libitum diet of standard laboratory food (Nuvilab^®, Paraná, Brazil).

After the 21 days of lactation, at weaning, the lactating rat were randomized divided in control (C, n = 6) and experimental (F, n = 6) group. Both groups were treated with semi-purified diet based on American Institute of Nutrition (AIN-93M) recommendations.¹⁸ The control diet containing 14 g of casein, 4 ml of soybean oil and 5 g of fiber/100 g. While the experimental diets containing 8 g of casein and 25 g of flaxseed flour. The groups received the same amounts of vitamins and minerals per gram of diet (table I). The flaxseeds were crushed to obtain the flour that was weighed and used immediately for the diet preparation. The experimental diet had a concentration of 25% of flaxseed that aimed to meet the entire recommended fiber intake and it was not necessary to add oil because this seed is source of this component. Food intake (g) and body mass (g) were evaluated every 3 days. The female rats had free access to diet and water.

 

 

At the end of the nutritional period, 30 days post-weaning, after 8 h of fasting, rats were anesthetized with Thiopentax^® (Tiopental, 0.1 mg/100 g) and subjected to dual-energy X-ray absorptiometry (DXA)^19,20 using a Lunar IDXA 200368 GE instrument (Lunar, Wisconsin, USA) with specific software (encore 2008. Version 12.20 GE Healthcare). Total lean (g), fat mass (g), and bone analysis (bone mineral density- BMD (g/cm²); bone mineral content- BMC (g); and bone area (cm²)) were measured for each rat. The DXA technician did not know about the experimental protocol.

After DXA, the length (cm, measured as the distance from tip of the nose to the tip of the tail) was evaluated. And blood was collected by cardiac puncture. Blood samples were centrifuged and serum was stored at -80^o C for posterior analyze of glucose, triglycerides, cholesterol, HDL-cholesterol, calcium, phosphorus, magnesium (mg/dL, respectively) and albumin (g/dL) by colorimetric method (Bioclin BS-120, Belo Horizonte, MG, Brazil). Liver, heart, kidney, pancreas and intra-abdominal fat mass (retroperitoneal, mesenteric and gonadal) were excised and weighted (g).

Right femur and lumbar vertebra (LV4) were collected and cleaned of soft tissue and preserved at -80^o C for posterior analyze. Bone dimension: the distance between epiphysis and the medial point width of the diaphysis were measured using calipers with a readability of 001mm. After drying overnight, femur and LV4 were weighed. Before, bone mineral density (BMD), in each femur and LV4, was determined by DXA.²¹ After DXA analyses the bones were carried out in order to make the mineral composition where the bone samples were dried at 105^o until reaching stable weight, then it were submitted to higher temperatures to 550^o in Muffle Quimis Microprocessor - Q318M until they become ashes and liquefied them in thermostat at 80^o with nitric acid to 70% in order to analysis minerals of calcium, phosphorus and magnesium by colorimetric method (Bioclin, Belo Horizonte, MG, Brazil).

Statistical analyses were carried out using the Graph Pad Prism statistical package version 5.00, 2007 (San Diego, CA, USA). Food intake and body mass were analyzed by two-way ANOVA, followed by Bonferroni post-test. The other data were analyzed by Student's t test. All results are expressed as means ± SEM with significance level of 0.05.

 

Results

During the nutritional period, food intake and body mass no differs between groups. In regard to body length, control (41.05 ± 0.63 cm) and experimental (42.42 ± 1.37 cm) groups showed no significantly differences in the end of experimental period (fig. 1).

 

 

In regard to body composition, total lean, body BMD, BMC and bone area the groups showed similar results. Meantime, the experimental group showed lower fat mass (-5%) to control (table II).

 

 

When evaluated the organs mass at 30 days post-weaning, liver, heart, kidney and pancreas mass were similar between control and experimental groups. The adiposity showed lower gonadal (-17%) and mesenteric (-23%) and intra-abdominal (-6%) fat mass in the experimental group (table III).

 

 

Serum analyzes showed no differences to calcium, phosphorus, magnesium and albumin. However, the experimental rats showed increase of HDL-cholesterol (+10%) and smaller glucose (-15%), triglycerides (P < 0.05, -37%) and cholesterol (P < 0.05, -21%) compared to control group (table IV).

 

 

Femur and lumbar vertebra (LV4) analyzes showed no differences to mass, bone dimensions and bone mineral density. When evaluated the bone mineral composition in femur and lumbar vertebra, the groups showed no differences to calcium, phosphorus and magnesium concentrations (table V).

 

 

Discussion

To our knowledge, this study to evaluate the effects of diet containing flaxseed flour on the maternal physiology after lactation in rats. Our results showed that, associated with a significant reduction in the serum cholesterol and triglycerides, the flaxseed flour intake contributes to control of body and intra-abdominal adiposity.

The nutritive demands of lactation are considerably greater than those of pregnancy. The nutrient intake of lactating women affects the nutrient content of breast-milk and maternal health. Thus, nutritional requirements for lactating women are higher compared to women who do not breastfeed.^2,22 However, there is little information that evaluates the nutrient intake and body composition of women after lactation period. In the present study, both groups received diet based on AIN-93M, to be used during adult maintenance.¹⁸ And to our surprise, regardless of flaxseed meal in the experimental diet, the groups showed similar food intake and body mass development. Previous studies related that the presences of fibers from flaxseed, as their bioactive compounds, are able to promote the satiety sensation, helping to reduce body mass.^14,23 Nevertheless, seems that the diet containing flaxseed did not affect satiety and the balance of the body mass, in the post-lactation period.

Excessive weight gain during pregnancy and retention of weight in the post-partum period are risk factors for obesity in later life.^24,25 Strategies to prevent post-partum obesity include behaviors associated with improved diet and weight control.²⁶ In present study, the fat tissue analyzes, highlighted a lower percentage of body, gonadal, mesenteric and intra-abdominal fat mass in the female rats treated with experimental diet. Flaxseed contain relevant concentration of lipids, and previous studies support the concept that fats do not affects body fat compartments equally.^27-29 Thereby, the flaxseed flour intake may be associated with a decrease of adipose mass in body and intra-abdominal compartments.

In regard to bone structure, during lactation there is an increase in the rate of maternal bone resorption and in calcium losses from the maternal skeleton. However, when offspring are weaned and milk production is halted, the bone mineral density (BMC) recovers by ~6 months after lactation. In the experimental models, BMC returns to the prepregnancy baseline value after 2-3 weeks in mice,³⁰ 2-6 weeks in rats.^31,32 Nevertheless, the multi-parity and lactation were risk factors for osteoporosis.^8,32,33 Several studies have documented the link between the intake of dairy foods and osteoporosis and indicate that polyunsaturated fatty acids may influence bone health.^21,34-36 The flaxseed contains high concentration of α-linolenic acid and some studies have reported a bone protective effect of this fatty acid; whereas, others have reported no effect.^37-40 The body composition, femur and lumbar vertebra (LV4) analysis demonstrated that female rats of the experimental group followed during ~4 weeks (30 days) after lactation, probably showed the bone parameters recovery regardless of the flaxseed flour.

Among the women, the cardiovascular diseases are responsible for almost half of deaths. Pregnancy brings a physiological stress that can uncover an underlying propensity for chronic diseases, addition to osteoporosis. Thus, the access to postnatal care constitutes a good opportunity for disease prevention.^41,42 In this context, the diet containing flaxseed flour contributed to the reduction of cholesterol and triglycerides. Although no significant differences have been observed, the experimental group showed higher HDL-cholesterol and lower glucose serum concentration. The findings corroborate previous reports^12,23 that whole flaxseed contributes to the health lipid profile in hyper-lipidemic subjects and in rats with normal biochemical parameters, reducing risks for chronic diseases.

In summary, despite of preliminary analysis, this study described the changes in body composition, bone structure and growth of maternal rats after weaning. Furthermore the findings highlight the contribution of flaxseed flour intake for control of adiposity and to maintain a healthy biochemical profile during the postnatal period.

 

References

1. World Health Organization. Obesity: preventing and managing the global epidemic. Report of a WHO Consultation on Obesity. WHO Technical Report Series no. 894. Geneva: WHO, 1998.         [ Links ]

2. Picciano MF. Pregnancy and lactation: physiological adjustments, nutritional requirements and the role of dietary supplements. Am S Nutr Sc 2003; 133: 1997-2002.         [ Links ]

3. Boardley DJ, Sargent RG, Coker AL, et al. The relationship between diet, activity, and other factors, and postpartum weight change by race. Obstetrics and Gynecology 1995; 86: 834-8.         [ Links ]

4. Gunderson EP, Abrams B. Epidemiology of gestational weight gain and body weight changes after pregnancy. Epidemiol Revi 1999; 21: 261-75.         [ Links ]

5. Hirani SAA, Karmaliani R. Evidence based workplace interventions to promote breastfeeding practices among pakistani working mothers. Women and Birth 2013; 26: 10-6.         [ Links ]

6. Leslie WS, Gibson A, Hankey CR. Prevention and management of excessive gestational weight gain: a survey of overweight and obese pregnant women. BMC Pregnancy and Childbirth 2013; 13: 1-7.         [ Links ]

7. Gindri TB, Duarte PF, Costa NCG. The working mother as a factor influence on the practice of breastfeeding: a literature review. REMENFE 2010; 1: 78-85.         [ Links ]

8. Liu XS, Ardeshirpour L, Vanhouten JN, et al. Site-specific changes in bone microarchitecture, mineralization, and stiffness during lactation and after weaning in mice. J Bone Miner Res 2012; 27: 865-75.         [ Links ]

9. Brant LHC, Cardozo LFMF, Velarde LGC, et al. Impact of flaxseed intake upon metabolic syndrome indicators in female wistar rats. Acta Cir Bras 2012; 27: 537-43.         [ Links ]

10. Cardozo LFMF, Boaventura GT, Brant LHC, et al. Prolonged consumption of flaxseed flour increases the 17b-estradiol hormone without causing adverse effects on the histomorphology of Wistar rats penis. Food Chem Toxicol 2012; 50: 4092-6.         [ Links ]

11. Troina AA, Figueiredo MS, Moura EG, et al. Maternal flaxseed diet during lactation alters milk composition and programs the offspring body composition, lipid profile and sexual function. Food Chem Toxicol 2010; 48: 697-703.         [ Links ]

12. Cardozo LFMF, Soares LL, Chagas MA, et al. Maternal consumption of flaxseed during lactation affects weight and hemoglobin level of offspring in rats. J Pediatr 2010; 86: 126-30.         [ Links ]

13. Almeida KCL, Boaventura GT, Guzman-Silva MA. Flaxseed (Linum usitatissimum) as a source of α;-linolenic acid in the development of the myelin sheath. Rev Nutr 2009; 22: 747-54.         [ Links ]

14. Daleprane JB, Batista A, Pacheco JT, et al. Dietary flaxseed supplementation improves endothelial function in the mesenteric arterial bed. Food Res Int 2010; 43: 2052-6.         [ Links ]

15. Cardozo LFMF, Soares LL, Brant LHC, et al. Hematologic and immunological indicators are altered by chronic intake of flaxseed in wistar rats. Nutr Hosp 2011; 26: 1091-6.         [ Links ]

16. Leite CDFC, Almeida KCL, Guzmân-Silva MA, et al. Flaxseed and its contribution to body growth and brain of wistar rats during chidhood and adolescence. Nutr Hosp 2011; 26: 415-20.         [ Links ]

17. Fishbeck KL, Rasmussen KM. Effect of repeated cycles on maternal nutritional status, lactational performance and litter growth in ad libitum-fed and chronically food-restricted rat. J Nutr 1987; 117: 1967-75.         [ Links ]

18. Reeves PG. Components of the AIN-93 diets as improvements in the AIN-76A diet. J Nutr 1997; 127: 838-41.         [ Links ]

19. Lukaski HC, Hall CB, Marchello MJ, et al. Validation of dual x-ray absorptiometry for body-composition assessment of rats exposed to dietary stressors. Nutrition 2001; 17: 607-13.         [ Links ]

20. Tsujio M, Mizorogi T, Kitamura I, et al. Bone mineral analysis through dual x-ray absorptiometry in laboratory animals. J Vet Med Sci 2009; 71: 1493-7.         [ Links ]

21. Costa CAS, Carlos AS, Gonzalez GD, et al. Diet containing low n-6/n-3 polyunsaturated fatty acids ratio, provided by canola oil, alters body composition and bone quality in young rats. Eur J Nutr 2012; 51: 191-8.         [ Links ]

22. Chen H, Wang P, Han Y, et al. Evaluation of dietary intake of lactating women in china and its potential impact on the health of mothers and infants. BMC Women's Health 2012; 12: 18.         [ Links ]

23. Pacheco JT, Daleprame JB, Boaventura GT. Impact of dietary flaxseed (linum usitatissimum) supplementation on biochemical profile in healthy rats. Nutr Hosp 2011; 26: 798-802.         [ Links ]

24. Lovelady C. Balancing exercise and food intake with lactation to promote post-partum weight loss. Proc Nutr Soc 2011; 70: 181-4.         [ Links ]

25. Maturi MS, Afshary P, Abedi P. Effect of physical activity intervention based on a pedometer on physical activity level and anthropometric measures after childbirth: a randomized controlled trial. BMC Pregnancy Childbirth 2011; 11: 103.         [ Links ]

26. Ostbye T, Peterson B, Krause KM, et al. Predictors of post-partum weight change among overweight and obese women: results from the active mothers postpartum study. J Womens Health 2012; 21: 215-22.         [ Links ]

27. Costa CAS, Alves EG, Gonzalez GP, et al. Computed tomography in the evaluation of abdominal fat distribution associated with a hyperlipidic diet in previously undernourished rats. Radiol Bras 2007; 40: 337-40.         [ Links ]

28. Costa CAS, Alves EG, Gonzalez GP, et al. Evaluation of body development, fat mass and lipid profile in rats fed with high-pufa and -mufa diets, after neonatal malnutrition. Brit J Nutr 2009; 101: 1639-44.         [ Links ]

29. Heredia FP, Larque E, Portillo MPP, et al. Age-related changes in fatty acids from different adipose depots in rat and their association with adiposity and insulin. Nutrition 2008; 24: 1013-22.         [ Links ]

30. Kirby BJ, Ardeshirpour L, Woodrow JP, et al. Skeletal recovery after weaning does not require PTHrP. J Bone Miner Res 2011; 26: 1242-51.         [ Links ]

31. Bowman BM, Miller SC. Skeletal mass, chemistry, and growth during and after multiple reproductive cycles in the rat. Bone 1999; 25: 553-9.         [ Links ]

32. Bowman BM, Miller SC. Greatly increased cancellous bone formation with rapid improvements in bone structure in the rat maternal skeleton after lactation. J Bone Miner Res 2002; 17: 1954-60.         [ Links ]

33. Keramat A, Larigani B, Adibi H. Risk factors for spinal osteoporosis as compared with femoral osteoporosis in urban Iranian women. Iranian J Publ Health 2012; 41: 52-9.         [ Links ]

34. Griel AE, Kris-Etherton PM, Hilpert KF, et al. An increase in dietary n-3 fatty acids decreases a marker of bone resorption in humans. Nutr J 2007; 6: 2.         [ Links ]

35. Hsu Y, Venners SA, Terwedow HA, et al. Relation of body composition, fat mass, and serum lipids to osteoporotic fractures and bone mineral density in Chinese men and women. Am J Clin Nutr 2006; 83: 146-54.         [ Links ]

36. Watkins B, Yong L, Allen K, et al. Dietary ratio of (n-6)/(n-3) polyunsaturated fatty acids alters the fatty acid composition of bone compartments and biomarkers of bone formation in rats. J Nutr 2000; 130: 2274-84.         [ Links ]

37. Lukas R, Gigliotti JC, Smith BJ, et al. Consumption of different sources of omega-3 polyunsaturated fatty acids by growing female rats affects long bone mass and microarchitecture. Bone 2011; 49: 455-62.         [ Links ]

38. Sakaguchi K, Morita I, Murota S. Eicosapentaenoic acid inhibits bone loss due to ovariectomy in rats. Prostaglandins Leukot Essent Fatty Acids 1994; 50: 81-4.         [ Links ]

39. Claassen N, Coetzer H, Steinmann CML, Kruger MC. The effect of different n-6/n-3 essential fatty acids ratios on calcium balance in rats. Prostaglandins Leukot Essent Fatty acids 1995; 53: 13-9.         [ Links ]

40. Poulsen RC, Moughan PJ, Kruger MC. Long-chain polyunsaturated fatty acids and the regulation of bone metabolism. Exp Biol Med (Maywood) 2007; 232: 1275-88.         [ Links ]

41. Alves E, Henriques A, Correia S, et al. Cardiovascular risk profile of mothers of a Portuguese birth cohort: a survey 4 years after delivery. J Prev Med 2013; 57: 494-9.         [ Links ]

42. McBride CM, Emmons KM, Lipkus IM. Understanding the potential of teachable moments: the case of smoking cessation. Health Educ Res 2003; 18: 156-70.         [ Links ]

 

 

Correspondence:
Gilson Teles Boaventura.
Laboratory of Experimental Nutrition.
Departament of Nutrition and Dietetics.
Fluminense Federal University.
Rio de Janeiro. Brazil.
E-mail: gilsontb@gmail.com

Recibido: 14-V-2014.
Aceptado: 14-VI-2014.