Leptin levels in rat offspring are modified by the ratio of linoleic to α-linolenic acid in the maternal diet
2002; Elsevier BV; Volume: 43; Issue: 10 Linguagem: Inglês
10.1194/jlr.m200105-jlr200
ISSN1539-7262
AutoresMarina Korotkova, Britt G. Gabrielsson, Malin Lönn, Lars-Åke Hanson, Birgitta Strandvik,
Tópico(s)Fatty Acid Research and Health
ResumoThe supply of polyunsaturated fatty acids (PUFA) is important for optimal fetal and postnatal development. We have previously shown that leptin levels in suckling rats are reduced by maternal PUFA deficiency. In the present study, we evaluated the effect of maternal dietary intake of (n-3) and (n-6) PUFA on the leptin content in rat milk and serum leptin levels in suckling pups. For the last 10 days of gestation and throughout lactation, the rats were fed an isocaloric diet containing 7% linseed oil (n-3 diet), sunflower oil (n-6 diet), or soybean oil (n-6/n-3 diet). Body weight, body length, inguinal fat pad weight, and adipocyte size of the pups receiving the n-3 diet were significantly lower during the whole suckling period compared with n-6/n-3 fed pups. Body and fat pad weights of the n-6 fed pups were in between the other two groups at week one, but not different from the n-6/n-3 group at week 3. Feeding dams the n-3 diet resulted in decreased serum leptin levels in the suckling pups compared with pups in the n-6/n-3 group. The mean serum leptin levels of the n-6 pups were between the other two groups but not different from either group. There were no differences in the milk leptin content between the groups.These results show that the balance between the n-6 and n-3 PUFA in the maternal diet rather than amount of n-6 or n-3 PUFA per se could be important for adipose tissue growth and for maintaining adequate serum leptin levels in the offspring. The supply of polyunsaturated fatty acids (PUFA) is important for optimal fetal and postnatal development. We have previously shown that leptin levels in suckling rats are reduced by maternal PUFA deficiency. In the present study, we evaluated the effect of maternal dietary intake of (n-3) and (n-6) PUFA on the leptin content in rat milk and serum leptin levels in suckling pups. For the last 10 days of gestation and throughout lactation, the rats were fed an isocaloric diet containing 7% linseed oil (n-3 diet), sunflower oil (n-6 diet), or soybean oil (n-6/n-3 diet). Body weight, body length, inguinal fat pad weight, and adipocyte size of the pups receiving the n-3 diet were significantly lower during the whole suckling period compared with n-6/n-3 fed pups. Body and fat pad weights of the n-6 fed pups were in between the other two groups at week one, but not different from the n-6/n-3 group at week 3. Feeding dams the n-3 diet resulted in decreased serum leptin levels in the suckling pups compared with pups in the n-6/n-3 group. The mean serum leptin levels of the n-6 pups were between the other two groups but not different from either group. There were no differences in the milk leptin content between the groups. These results show that the balance between the n-6 and n-3 PUFA in the maternal diet rather than amount of n-6 or n-3 PUFA per se could be important for adipose tissue growth and for maintaining adequate serum leptin levels in the offspring. An adequate supply of polyunsaturated fatty acids (PUFA) during pregnancy and lactation is crucial for optimal fetal and postnatal development. However, neither the role of nor the requirements for individual PUFA are yet established (1Innis S.M. Essential fatty acids in infant nutrition: lessons and limitations from animal studies in relation to studies on infant fatty acid requirements.Am. J. Clin. Nutr. 2000; 71: 238S-244SGoogle Scholar). Over the last 20 years the consumption of saturated fat from animal food and n-6 PUFA from oil seeds has been substantially increased, while the dietary intake of n-3 PUFA from plants and marine products has declined in industrialized countries (2Uauy R. Mena P. Valenzuela A. Essential fatty acids as determinants of lipid requirements in infants, children and adults.Eur. J. Clin. Nutr. 1999; 53: S66-S77Google Scholar, 3Sanders T.A. Polyunsaturated fatty acids in the food chain in Europe.Am. J. Clin. Nutr. 2000; 71: 176S-178SGoogle Scholar). As a consequence, the ratio n-6/n-3 PUFA in the diet has raised to 10:1–15:1 (3Sanders T.A. Polyunsaturated fatty acids in the food chain in Europe.Am. J. Clin. Nutr. 2000; 71: 176S-178SGoogle Scholar, 4Spector A.A. Essentiality of fatty acids.Lipids. 1999; 34: S1-S3Google Scholar). Since the maternal diet is the most important variable determining milk fatty acid composition (5Fidler N. Koletzko B. The fatty acid composition of human colostrum.Eur. J. Nutr. 2000; 39: 31-37Google Scholar), this shift in dietary intake of PUFA results in rising concentrations of n-6 PUFA and reduction of n-3 PUFA levels in human milk (3Sanders T.A. Polyunsaturated fatty acids in the food chain in Europe.Am. J. Clin. Nutr. 2000; 71: 176S-178SGoogle Scholar). The ratio n-6/n-3 PUFA in the milk is of importance, as n-6 and n-3 PUFA compete for the synthesis and the incorporation of long-chain PUFA into the cell membranes and they also have different functional roles (4Spector A.A. Essentiality of fatty acids.Lipids. 1999; 34: S1-S3Google Scholar). In animals and man, variations of the ratio n-6/n-3 PUFA in the milk are followed by changes in growth (6Carlson S.E. Werkman S.H. Peeples J.M. Cooke R.J. Tolley E.A. Arachidonic acid status correlates with first year growth in preterm infants.Proc. Natl. Acad. Sci. USA. 1993; 90: 1073-1077Google Scholar, 7Amusquivar E. Ruperez F.J. Barbas C. Herrera E. Low arachidonic acid rather than alpha-tocopherol is responsible for the delayed postnatal development in offspring of rats fed fish oil instead of olive oil during pregnancy and lactation.J. Nutr. 2000; 130: 2855-2865Google Scholar), neural and retinal development (2Uauy R. Mena P. Valenzuela A. Essential fatty acids as determinants of lipid requirements in infants, children and adults.Eur. J. Clin. Nutr. 1999; 53: S66-S77Google Scholar, 8Saste M.D. Carver J.D. Stockard J.E. Benford V.J. Chen L.T. Phelps C.P. Maternal diet fatty acid composition affects neurodevelopment in rat pups.J. Nutr. 1998; 128: 740-743Google Scholar), and immune responsiveness (9Rayon J.I. Carver J.D. Wyble L.E. Wiener D. Dickey S.S. Benford V.J. Chen L.T. Lim D.V. The fatty acid composition of maternal diet affects lung prostaglandin E2 levels and survival from group B streptococcal sepsis in neonatal rat pups.J. Nutr. 1997; 127: 1989-1992Google Scholar) of the offspring and might also have additional effects.We have previously shown that the maternal deficiency of PUFA in rats affects the serum leptin levels in their offspring (10Korotkova M. Gabrielsson B. Hanson L. Strandvik B. Maternal essential fatty acid deficiency depresses serum leptin levels in suckling rat pups.J. Lipid Res. 2001; 42: 359-365Google Scholar) and alters milk leptin concentration (11Korotkova M. Gabrielsson B. Hanson L.A. Strandvik B. Maternal dietary intake of essential fatty acids affects adipose tissue growth and leptin mRNA expression in suckling rat pups.Pediatr. Res. 2002; 52: 78-84Google Scholar). Leptin is an adipose tissue-derived hormone that regulates food intake and energy expenditure, and is involved in several physiological and pathological processes (12Ahima R.S. Flier J.S. Leptin.Annu. Rev. Physiol. 2000; 62: 413-437Google Scholar). Moreover, possible developmental roles of leptin in the perinatal period have been suggested (13Ahima R.S. Prabakaran D. Flier J.S. Postnatal leptin surge and regulation of circadian rhythm of leptin by feeding. Implications for energy homeostasis and neuroendocrine function.J. Clin. Invest. 1998; 101: 1020-1027Google Scholar, 14Hassink S.G. de Lancey E. Sheslow D.V. Smith-Kirwin S.M. O'Connor D.M. Considine R.V. Opentanova I. Dostal K. Spear M.L. Leef K. Ash M. Spitzer A.R. Funanage V.L. Placental leptin: an important new growth factor in intrauterine and neonatal development?.Pediatrics. 1997; 100: E1-E6Google Scholar). During early development, leptin is produced by the placenta and by fetal and neonatal adipose tissues (14Hassink S.G. de Lancey E. Sheslow D.V. Smith-Kirwin S.M. O'Connor D.M. Considine R.V. Opentanova I. Dostal K. Spear M.L. Leef K. Ash M. Spitzer A.R. Funanage V.L. Placental leptin: an important new growth factor in intrauterine and neonatal development?.Pediatrics. 1997; 100: E1-E6Google Scholar, 15Matsuda J. Yokota I. Iida M. Murakami T. Yamada M. Saijo T. Naito E. Ito M. Shima K. Kuroda Y. Dynamic changes in serum leptin concentrations during the fetal and neonatal periods.Pediatr. Res. 1999; 45: 71-75Google Scholar), and is also provided via maternal milk (16Casabiell X. Pineiro V. Tome M.A. Peino R. Dieguez C. Casanueva F.F. Presence of leptin in colostrum and/or breast milk from lactating mothers: a potential role in the regulation of neonatal food intake.J. Clin. Endocrinol. Metab. 1997; 82: 4270-4273Google Scholar). In humans, leptin levels in early life predict weight gain later in infancy (17Ong K.K. Ahmed M.L. Dunger D.B. The role of leptin in human growth and puberty.Acta Paediatr. Suppl. 1999; 88: 95-98Google Scholar, 18Harigaya A. Onigata K. Nako Y. Nagashima K. Morikawa A. Role of serum leptin in the regulation of weight gain in early infancy.Biol. Neonate. 1999; 75: 234-238Google Scholar). These studies suggest that circulating leptin levels during the perinatal period could be important for normal development and health.The aim of the present study was to investigate the effects of maternal dietary intake of n-6 and n-3 PUFA on the serum leptin levels in rat pups during the suckling period and on the leptin content in rat milk.MATERIAL AND METHODSAnimalsPregnant Sprague-Dawley rats (BK Universal, Stockholm, Sweden) were received on day 7 of gestation and kept in our animal facility under constant conditions of humidity (70–80%), temperature (22–25°C), and light (12-h light and dark cycle). The rats were housed individually in plastic cages with food and water available ad libitum. Ten days before delivery, the rats were assigned to one of three groups (n = 9–10 in each group) receiving either a diet containing both n-6 and n-3 essential fatty acids (n-6/n-3), a n-3 essential fatty acids-enriched (n-3) diet, or a n-6 essential fatty acids-enriched (n-6) diet. Litter size was adjusted to 10 pups per litter. Pups (n = 10–16) randomized from each litter were used at each time point. Body weight and length of pups were recorded every week. The animals were killed by decapitation in the morning (09 AM–11 AM) at 1 or 3-weeks-of-age. Truncal blood was collected and sera were kept frozen (−20°C) until analyses of leptin, glucose, protein, cholesterol, and triglycerides. Pairs of subcutaneous (inguinal) fat pads were removed, weighed, and kept frozen (−20°C) until analyses of fatty acid (FA) composition of total lipids and phospholipids. Fat pads from parallel sets of animals were placed into the RNAlater™ (Ambion, Austin, TX) and stored at −20°C until analysis by RT-PCR.Milk samples were collected from dams at 3 weeks of lactation. After separation from the pups for 30 min, dams were anaesthetized i.p. with pentobarbital (35 mg/kg body weight) and injected ip with 4 IU of oxytocin (Sigma Chemical Co., St. Louis, MO) to stimulate milk flow. Milking was initiated 5 min after oxytocin injection and milk was collected by hand expression. The milk samples were stored at −20°C until analyses of leptin and of FA composition of total lipids.The study was approved by the Animal Ethics Committee of Göteborg University.DietsThe dams were fed one of three experimental pellet diets (Morinaga Milk Industry Co. LTD, Tokyo, Japan) for the last 10 days of gestation and throughout lactation. The diets differed only by lipid composition: 7% soybean oil (diet contains both n-6 and n-3 PUFA), sunflower oil (n-6 PUFA-enriched, n-6 diet), or linseed oil (n-3 PUFA-enriched, n-3 diet). The composition of the three diets is given in Table 1. The data on major components, salt, and vitamins have been obtained from the manufacturer. The FA composition was determined in our laboratory with the method described below. The ratio n-6/n-3 fatty acids in the n-6/n-3 diet was 9 and in the n-6 and n-3 diets, 216 and 0.4, respectively. The total metabolizable energy of the diets was 13.9 MJ/kg.TABLE 1Composition of the dietsDietn-3 Dietn-6/n-3 Dietn-6 Diet%Casein20.020.020.0Potato starch54.054.054.0Glucose10.010.010.0Cellulosal flour4.04.04.0Mineral mixaSalt mixture containing (wt%): KH2PO4 (34.1); CaCO3 (35.9); KCl (2.5); NaCl (18); MgSO4 × H2O (5.1); FeC6H5O7 × 5H2O (3.3); MnO (0.27); Cu2C6H4O7 × 2.5H2O (0.06); ZnCO3 (0.04); CoCl2 × 6H2O (0.002); KAl(SO4)2 × 2H2O (0.008); NaF (0.025); KIO3 (0.009); Na2B4O7 × 10H2O (0.002); Na2SeO3 × 5H2O (0.001); Na2MoO4 × 2H2O (0.001).4.04.04.0Vitamin mixbVitamin mixture containing: Vit A 11.9 IU/g; Vit D3 1.5 IU/g; Vit B1 4 μg/g; Vit B2 12 μg/g; Vit B6 5 μg/g; Ca-pantotenate 45% 11 μg/g; Niacin 40 μg/g; Vit B12 0.02 μg/g; Vit K3 7.75 μg/g; Biotin 2% 3 μg/g; Vit C 500 μg/g; Inositol 30 μg/g; Vit E 42 μg/g; Choline chloride 50% 1 mg/g; Folic acid 0.5 μg/g.1.01.01.0Linseed oil7.0Soybean oil7.0Sunflower oil7.0Fatty acidsmol%16:0 14 12816:10.30.10.118:06.13.94.218:1 32 21 2218:2 14 56 6520:00.30.40.318:3 336.20.3a Salt mixture containing (wt%): KH2PO4 (34.1); CaCO3 (35.9); KCl (2.5); NaCl (18); MgSO4 × H2O (5.1); FeC6H5O7 × 5H2O (3.3); MnO (0.27); Cu2C6H4O7 × 2.5H2O (0.06); ZnCO3 (0.04); CoCl2 × 6H2O (0.002); KAl(SO4)2 × 2H2O (0.008); NaF (0.025); KIO3 (0.009); Na2B4O7 × 10H2O (0.002); Na2SeO3 × 5H2O (0.001); Na2MoO4 × 2H2O (0.001).b Vitamin mixture containing: Vit A 11.9 IU/g; Vit D3 1.5 IU/g; Vit B1 4 μg/g; Vit B2 12 μg/g; Vit B6 5 μg/g; Ca-pantotenate 45% 11 μg/g; Niacin 40 μg/g; Vit B12 0.02 μg/g; Vit K3 7.75 μg/g; Biotin 2% 3 μg/g; Vit C 500 μg/g; Inositol 30 μg/g; Vit E 42 μg/g; Choline chloride 50% 1 mg/g; Folic acid 0.5 μg/g. Open table in a new tab Fatty acid analysisTotal lipids of white adipose tissue, representing mainly triglycerides, and milk were extracted according to Folch et al. (19Folch J.L.M. Sloane-Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J Biol Chem. 1957; 226: 497-509Google Scholar). The total lipids from adipose tissue were fractionated on a single SEP-PAK aminopropyl cartridge (Waters Corp., MA) and the fraction of phospholipids was analyzed. Milk total lipids were not fractionated. The FA methyl esters were separated by capillary gas-liquid chromatography in a Hewlett-Packard 6890 gas chromatograph according to the method described previously (10Korotkova M. Gabrielsson B. Hanson L. Strandvik B. Maternal essential fatty acid deficiency depresses serum leptin levels in suckling rat pups.J. Lipid Res. 2001; 42: 359-365Google Scholar). The separation was recorded with HP GC Chem Station software (HP GC, Wilmington, DE). The FA 21:1 was used as internal standard and the FA methyl esters identified by comparison with retention times of pure reference substances (Sigma Aldrich Sweden AB, Stockholm, Sweden). The unsaturation index (USI) was calculated as ratio Σ (mol% each unsaturated FA × number of double bonds of the same FA)/saturated fatty acids (SFA).Analysis of leptinLeptin concentrations in serum and milk were measured by a rat leptin radioimmunoassay (RIA; Linco Research Ltd., St. Charles, MO) and all samples from one experiment were analyzed in duplicates in the same assay. The intra-assay coefficient of variation (CV) at 0.25 ng/ml was 2.4%, and at 20 ng/ml 1.6%. Milk samples were thawed at 37°C and vortexed vigorously before pipetting. The samples were diluted in assay buffer (1:2 to 1:5) before sonication (five bursts, five s/burst with cooling on ice between each burst, 80% power) to ensure homogenous samples. To control for possible matrix effects in the individual milk samples, each sample was divided into three tubes and a standard addition procedure was employed by adding 1 ng and 2 ng leptin, respectively, to the second and third tube. The original leptin content was then calculated using linear regression. An effect of non-specific background in the milk on the leptin data was evaluated by comparing added leptin with measured leptin. There was a matrix effect in the milk in both the diet groups. Therefore, we used the leptin values calculated from the standard addition procedure.Analysis of glucose, protein, cholesterol, and triglyceride levels in serumSerum glucose was determined by a quantitative glucose oxidase/PAP assay (ABX Diagnostics, Parc Evromedicine, Montpellier, France). Serum protein was determined by a quantitative colorimetric assay (Biuret reaction) (ABX Diagnostics). Serum cholesterol was determined by a quantitative enzymatic colorimetric assay (InfinityTM cholesterol reagent, Sigma Diagnostics Inc., St. Louis, MO). Serum triglycerides were determined by a quantitative GPO (glycerol peroxidase)/PAP assay (ABX Diagnostics).RNA extraction and analysis by RT-PCRTotal RNA was isolated from the adipose tissue of each individual rat with the RNeasy Mini Kit (QIAGEN, Valencia, CA) and used for analysis of leptin mRNA (mRNA) by RT-PCR. The RNA samples were treated with DNase (DNA-freeTM, Ambion, Austin, TX) according to the manufacturer's instructions. The concentration of RNA was determined spectrophotometrically (OD260) and its integrity was verified by agarose gel electrophoresis, with visualization by ethidium bromide (EtBr) staining.Synthesis of cDNA was performed in a volume of 30 μl using 0.7 μg of total RNA and 3.3 μM random hexamers (Pharmacia Biotech, Uppsala, Sweden) in a solution containing 1× First strand buffer (Life Technologies, Gaitherburg, MD), dNTPmix (0.5mM each of dATP, dGTP, dCTP, and dTTP; Ultrapure dNTP Set, Pharmacia Biotech), RNase-inhibitor (1 U/μl, rRNasin, Promega, Madison, WI, USA) and Reverse Transcriptase (13.3 U/μl, SuperscriptTMII RT, Life Technologies). The mixture was incubated at room temperature for 10 min and at 42°C for 60 min followed by 10 min at 70°C. The cDNA was stored at −70°C.Multiplex relative RT-PCR was used for the analysis of differences in mRNA abundance. The leptin gene was co-amplified with invariant endogenous control. The cDNA was amplified by PCR using specific primers for the rat leptin cDNA (20Jin L. Zhang S. Burguera B.G. Couce M.E. Osamura R.Y. Kulig E. Lloyd R.V. Leptin and leptin receptor expression in rat and mouse pituitary cells.Endocrinology. 2000; 141: 333-339Google Scholar). The primer pairs: 5′ CCT GTG GCT TTG GTC CTA TCT G 3′ (nucleotides 87–108. GenBank accession number D4582) and 5′ AGG CAA GCT GGT GAG GAT CTG 3′ (nucleotides 310–330) generated a single 244 base pair (bp) product. QuantumRNA 18S internal standard (Ambion) was used as internal control and generated a single 489 bp product. Linear range and optimal ratio of 18S primers/competimers were determined. The PCR reaction was carried out in a final volume of 50 μl with 2 μl of cDNA product, 1× PCR buffer, 2.0 mM MgCl2, 0.4 μM of each primer, 0.2 mM of each dNTPs, and 1.25 U AmpliTaqGold (Applied Biosystems, Foster City, CA). PCR was performed using the GeneAmp PCR System 9600 (Applied Biosystems) and following conditions: 94°C (12 min) for 1 cycle, 94°C (30 s), 60°C (30 s), 72°C (30 s) for 30 cycles, 72°C (7 min). The negative control consisted of omission of the reverse transcriptase for each sample, which resulted in no bands after RT-PCR.The PCR products were separated on 2% EtBr agarose gel and were subsequently visualized and quantified using IPLab Gel Scientific Image processing (Signal Analitics, Vienna, VA). The intensity obtained for leptin amplicon was related to that of 18S in each individual sample.Adipocyte cell size and numberAdipocyte and stroma-vascular (S-V) fractions were prepared following the procedure outlined by Smith et al. (21Smith U. Sjostrom L. Bjornstorp P. Comparison of two methods for determining human adipose cell size.J. Lipid Res. 1972; 13: 822-824Google Scholar). Briefly, about 0.5 g adipose tissue (n = 4 in each group) was cut into smaller pieces and transferred to a plastic vial containing prewarmed 10 ml Parker medium 199 (SBL, Stockholm, Sweden) supplemented with 4% BSA and 0.8 mg/ml collagenase type A (Roche Diagnostics, Bromma, Sweden). The vials were incubated for 1 h at 37°C in a shaking water bath. The cells were filtered through a 250 μm nylon mesh and the adipocytes were allowed to float to the surface for 5 min before aspiration of the medium. The adipocytes were washed twice with 5 ml medium, allowing the cells to float to the surface each time. After the final wash, the adipocytes were resuspended in fresh medium (20% cells and 80% medium) to yield the final cell suspension. The suspension was gently mixed before placing 3–4 drops on a glass slide onto which two layers of adhesive tape had been attached to form a small chamber and a cover slip was placed on top. Cell diameter was measured with a Zeiss microscope at 10× magnification (Axioplan2 imaging, Carl Zeiss, Göttingen, Germany). Digital images were captured with a video camera mounted on the microscope and transferred to a computerized image analysis system, KS400 (Carl Zeiss). By introducing conditions on the roundness of the cell areas as well as smoothness of the contours, the program could identify healthy fat cells and automatically calculated the diameter.Statistical analysisValues are presented as mean ± SD. The data were analyzed by one-way ANOVA (Fisher's PLSD). Differences within individuals were determining using paired t-test. When the number of observations was limited, non-parametric statistical methods were used (Kruskal-Wallis test). A value of P < 0.05 was considered statistically significant.RESULTSFA composition of total milk lipidsThere was no difference in the total amount of PUFA or USI in milk from rats fed the different diets (Table 2). The levels of SFA were higher in the milk from dams fed the n-3 diet, while the levels of monounsaturated fatty acids (MUFA) were higher in the milk of the dams fed the n-6/n-3 diet. Feeding the n-6 diet to the dams generated decreased levels of α-linolenic 18:3(n-3) and docosahexoenoic 22:6(n-3) acids in the milk lipids, whereas the levels of linoleic 18:2(n-6) and γ-linolenic 18:3(n-6) acids were enhanced compared with those in the dams fed the n-6/n-3 diet. Feeding the n-3 diet to lactating animals induced marked changes in the total milk lipid FA composition. The contents of 18:2(n-6), eicosadienoic acid 20:2(n-6), and arachidonic 20:4(n-6) acids were significantly lower compared with the other diets, while the levels of 18:3(n-3) were markedly higher in the n-3 group compared with the n-6/n-3 group (P < 0.05). The content of 22:6(n-3) was reduced in the n-3 group compared with the n-6/n-3 group.TABLE 2The fatty acid composition of the milk total lipids from the rats fed different diets at 3 weeks of lactationFatty Acidsn-3n-6/n-3n-6mol%12:012.9 ± 1.1aValues with unlike superscript letters are significantly different (P < 0.05).10.3 ± 2.1bValues with unlike superscript letters are significantly different (P < 0.05).13.4 ± 1.9aValues with unlike superscript letters are significantly different (P < 0.05).14:012.6 ± 1.4aValues with unlike superscript letters are significantly different (P < 0.05).8.0 ± 2.5bValues with unlike superscript letters are significantly different (P < 0.05).10.8 ± 2.6aValues with unlike superscript letters are significantly different (P < 0.05).16:018.9 ± 1.2aValues with unlike superscript letters are significantly different (P < 0.05).19.1 ± 2.0aValues with unlike superscript letters are significantly different (P < 0.05).16.6 ± 1.9bValues with unlike superscript letters are significantly different (P < 0.05).16:1(n-7)0.68 ± 0.26aValues with unlike superscript letters are significantly different (P < 0.05).1.1 ± 0.4bValues with unlike superscript letters are significantly different (P < 0.05).0.63 ± 0.34aValues with unlike superscript letters are significantly different (P < 0.05).18:03.7 ± 0.3aValues with unlike superscript letters are significantly different (P < 0.05).4.7 ± 0.5bValues with unlike superscript letters are significantly different (P < 0.05).4.2 ± 0.4cValues with unlike superscript letters are significantly different (P < 0.05).18:1(n-9)14.9 ± 1.4aValues with unlike superscript letters are significantly different (P < 0.05).19.4 ± 2.0bValues with unlike superscript letters are significantly different (P < 0.05).15.8 ± 2.4aValues with unlike superscript letters are significantly different (P < 0.05).18:2(n-6)9.4 ± 0.8aValues with unlike superscript letters are significantly different (P < 0.05).31.1 ± 2.7bValues with unlike superscript letters are significantly different (P < 0.05).35.1 ± 2.3cValues with unlike superscript letters are significantly different (P < 0.05).18:3(n-6)0.04 ± 0.02aValues with unlike superscript letters are significantly different (P < 0.05).0.10 ± 0.02aValues with unlike superscript letters are significantly different (P < 0.05).0.23 ± 0.11bValues with unlike superscript letters are significantly different (P < 0.05).18:3(n-3)26.3 ± 1.6aValues with unlike superscript letters are significantly different (P < 0.05).3.1 ± 0.4bValues with unlike superscript letters are significantly different (P < 0.05).0.0 ± 0.0cValues with unlike superscript letters are significantly different (P < 0.05).20:2(n-6)0.14 ± 0.04aValues with unlike superscript letters are significantly different (P < 0.05).0.60 ± 0.12bValues with unlike superscript letters are significantly different (P < 0.05).0.57 ± 0.13bValues with unlike superscript letters are significantly different (P < 0.05).20:4(n-6)0.31 ± 0.06aValues with unlike superscript letters are significantly different (P < 0.05).1.6 ± 0.4bValues with unlike superscript letters are significantly different (P < 0.05).1.4 ± 0.3bValues with unlike superscript letters are significantly different (P < 0.05).24:00.05 ± 0.01aValues with unlike superscript letters are significantly different (P < 0.05).0.08 ± 0.03aValues with unlike superscript letters are significantly different (P < 0.05).0.13 ± 0.02bValues with unlike superscript letters are significantly different (P < 0.05).24:1(n-9)0.02 ± 0.02aValues with unlike superscript letters are significantly different (P < 0.05).0.53 ± 0.14bValues with unlike superscript letters are significantly different (P < 0.05).0.44 ± 0.22bValues with unlike superscript letters are significantly different (P < 0.05).22:6(n-3)0.45 ± 0.1aValues with unlike superscript letters are significantly different (P < 0.05).0.62 ± 0.1bValues with unlike superscript letters are significantly different (P < 0.05).0.24 ± 0.13cValues with unlike superscript letters are significantly different (P < 0.05).20:4(n-6)/22:6(n-3)0.7 ± 0.1aValues with unlike superscript letters are significantly different (P < 0.05).2.5 ± 0.6aValues with unlike superscript letters are significantly different (P < 0.05).8.1 ± 4.7bValues with unlike superscript letters are significantly different (P < 0.05).n-69.8 ± 0.9aValues with unlike superscript letters are significantly different (P < 0.05).33.4 ± 2.9bValues with unlike superscript letters are significantly different (P < 0.05).37.3 ± 2.3cValues with unlike superscript letters are significantly different (P < 0.05).n-326.7 ± 1.6aValues with unlike superscript letters are significantly different (P < 0.05).3.75 ± 0.3bValues with unlike superscript letters are significantly different (P < 0.05).0.2 ± 0.1cValues with unlike superscript letters are significantly different (P < 0.05).n-6/n-30.4 ± 0.0aValues with unlike superscript letters are significantly different (P < 0.05).8.9 ± 0.7aValues with unlike superscript letters are significantly different (P < 0.05).206.3 ± 101.5bValues with unlike superscript letters are significantly different (P < 0.05).SFA48.1 ± 2.3aValues with unlike superscript letters are significantly different (P < 0.05).42.3 ± 4.2bValues with unlike superscript letters are significantly different (P < 0.05).45.3 ± 4.8bValues with unlike superscript letters are significantly different (P < 0.05).MUFA15.6 ± 1.6aValues with unlike superscript letters are significantly different (P < 0.05).21.0 ± 2.4bValues with unlike superscript letters are significantly different (P < 0.05).16.8 ± 2.9aValues with unlike superscript letters are significantly different (P < 0.05).PUFA36.6 ± 2.0aValues with unlike superscript letters are significantly different (P < 0.05).37.1 ± 3.0aValues with unlike superscript letters are significantly different (P < 0.05).37.6 ± 2.5aValues with unlike superscript letters are significantly different (P < 0.05).USI2.5 ± 0.2aValues with unlike superscript letters are significantly different (P < 0.05).2.5 ± 0.4aValues with unlike superscript letters are significantly different (P < 0.05).2.4 ± 0.7aValues with unlike superscript letters are significantly different (P < 0.05).SFA, saturated fatty acids; MUFA monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; USI, unsaturation index. Values given as mean ± SD (n = 7–8).a Values with unlike superscript letters are significantly different (P < 0.05).b Values with unlike superscript letters are significantly different (P < 0.05).c Values with unlike superscript letters are significantly different (P < 0.05). Open table in a new tab FA composition of total lipids and phospholipids in white adipose tissue from the pupsTotal lipid FA composition of white adipose tissue in the suckling pups at 3-weeks-of-age generally reflected that of the total milk lipids (Table 2 and 3). White adipose tissue total lipid FA composition of pups suckling dams on the n-6/n-3 and on the n-6-diet was similar, except the significantly lower levels of (n-3) PUFA in the n-6-group (Table 3). The ratios of 20:4(n-6) to 22:6(n-3), and of n-6 to n-3 FA in the n-6-group were higher compared both to the n-3 and n-6/n-3 groups.TABLE 3The fatty acid composition of white adi
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