#11_XUE_bozza ! Ital. J. Food Sci., vol 28, 2016 - 683 PAPER COMPOSITION OF INTRAMUSCULAR PHOSPHOLIPID FATTY ACIDS OF INRA RABBIT AT DIFFERENT AGES SHAN XUE College of Biological Science and Technology, Minnan Normal University, Zhangzhou 363000, PR China *Corresponding author. Tel./Fax +86 18850285808 E-mail address: yixuanchenglion@sina.com; 281667860@qq.com ABSTRACT The composition of intramuscular phospholipids fatty acids in Longissimus dorsimuscle (LD), left-hind leg muscle (LL) and abdominal muscle (AM) of Inra rabbit slaughtered between 35 to 90 days old were investigated. Significant decreasing of intramuscular phospholipids (% total intramuscular lipids) was observed in three muscles as age increased (p < 0.05). The highest phospholipids content was found in LL in both male and female rabbits during the growth period, and the phospholipids content in three muscles of the males were higher than that of the females. Abundant amount of unsaturated fatty acids (UFA), especially polyunsaturated fatty acids (PUFA) characterised the fatty acid composition of the intramuscular phospholipids (32.94-55.79%), and the percentage of PUFA in the muscles were all significantly decreased during the growth of male and female Inra rabbit (p < 0.05). In addition, a significant reduction of PUFA/SFA ratio and a significant increase of SFA + MUFA were observed (p < 0.05). Major fatty acids, such as Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1n-9), linoleic acid (C18:2n-6) and arachidonic acid (C20:4) changed more obviously than other fatty acids. The analysis of partial least square regression (PLSR) showed that the composition of phospholipid fatty acids varied in age, muscle and gender, and the nutritional value of the phospholipid fatty acids decreased with age distinctly, the AM had better nutritional value of phospholipids. Keywords: rabbit, fatty acids, intramuscular phospholipids, composition ! Ital. J. Food Sci., vol 28, 2016 - 684 1. INTRODUCTION Functional foods are a tool that can be easily used in reducing public health costs. Compared to meats of other animal species, rabbit meat is characterized by high levels of polyunsaturated fatty acids (PUFA) and n-3 fatty acids, high levels of protein with essential amino acids, high digestibility value, lower cholesterol contents and significant source of vitamin B family (vitamins B2, B5, B6, B3, B12) etc. (DALLE ZOTTE and SZENDRÖ, 2011). Moreover, rabbit meat consumption could become a good way of providing bioactive compounds to human consumers, since the rabbit meat fatty acids profile may be favorably modified by the inclusion of raw materials rich in unsaturated fatty acids (UFA) in the diet (DAL BOSCO et al., 2004; HERNÁNDEZ, 2008; KOUBA et al., 2008). Rabbit meat is considered as dietetically healthy, relatively rich in n-3 PUFAs and with a lower n-6 to n-3 ratio (7�12) than pork, veal or chicken meats (DALLE ZOTTE, 2002; HERNÁNDEZ and GONDRET, 2006). Unlike pork or beef meat, it contains two important metabolites from α-linolenic acid (ALA), docosahexaenoic acid (DHA, C22:6n-3) and eicosapentaenoic acid (EPA, C20:5n-3) in detectable levels (COMBES and DALLE ZOTTE, 2005; EIBEN et al., 2010). Inra rabbit is imported from France and have high breeding efficiency. In recent years, interests have focused not only on the amount of intramuscular phospholipids but also on the composition of fatty acids. The intramuscular fat (IMF), which characterises the amount of fat, is one of the major factors affecting the palatability of meat (HOCQUETTE et al., 2010). Muscle lipids are composed of polar lipids, mainly phospholipids (rich in PUFA) located in the cell membranes, and triacylglycerols (high levels of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA)) along the muscle fibres (DE SMET et al., 2004). In the IMF, PUFA are restricted almost exclusively to the phospholipids fraction (WOOD et al., 2003). Thus, the amount of intramuscular phospholipids in the meat is an important factor (GRAY et al., 1996). Phospholipids consist of long-chain fatty acids attached to a phosphoryl group. Since the fatty acids chains can vary in length and degree of saturation, each phospholipid class possesses numerous molecular species with different chemical and biological properties (MARCO et al., 2004; WANG et al., 2009). CAMBERO et al. (1991) first analysed the phospholipid content and classes in rabbits and they provide important information on phospholipid prevalence according to breed and feeding, specifying that phospholipid differs also according to age and gender. Generally, the analysis of phospholipids is based the determination of the phospholipid classes (phosphatidyleth-anolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, sphingomyelin, lysophosphatidylcholine etc.) with high performance liquid chromatography (ALASNIER and GANDEMER, 1998; PETERSON and CUMMINGS, 2005; BOSELLI et al., 2008). However, practically limited literature data are available on the determination of the fatty acids in intramuscular phospholipids by gas chromatography after purification of the polar lipid fraction, especially the intramuscular phospholipids from Inra rabbit. Hence, in order to provide a database for the characterization of nutritional quality of Inra rabbit meat, the composition of intramuscular phospholipids fatty acids and the effect of ages, genders and muscles on the composition and nutritional value of fatty acids were investigated by gas chromatography. 2. MATERIALS AND METHODS 2.1. Sampling of Inra rabbit meat ! Ital. J. Food Sci., vol 28, 2016 - 685 A total of 200 35 days old weaned Inra rabbits (20 males+20 females per age) were provided by College of Animal Science and Technology, Southwest University. The ingredients and proximate chemical composition of the diet were shown in the Table 1. Table 1: The ingredients and proximate chemical composition of the diet. Item Diets Ingredients Proportion(%) Corn 24.2 Wheat bran 19 Soybean meal 10.82 Alfalfa meal 36 Corn germ cake 4 Rapeseed 3 Powder 0.5 Dicalcium 0.8 Lysine 0.07 Methionine 0.11 Salt 0.5 Premixa 1 Nutrition Proportion (%) Dry matter 89.8% Crude protein 16.0% Fat 3.3% Lysine 0.7 Methionine 0.6 Calcium 0.95 Phosphorus 0.59 Acid detergent fiber 33.2% Neutral detergent fiber 21.4% Digestible energyb 10.5 MJ/kg a The premix contains (per kg of diet): Vitamin A, 10000 IU; Vitamin D3, 1000 IU; Vitamin E, 30 mg; Vitamin K, 1 mg; Vitamin B1, 1 mg; Vitamin B2, 3.5 mg; Vitamin B6, 2 mg; Vitamin B12, 0.01 mg; niacin, 50 mg; folic acid, 0.3 mg; choline, 1000 mg; Zn, 30 mg; Cu, 5 mg; Mn, 15 mg; Fe, 30 mg; I, 1 mg. b Digestible energy (kcal/kg DM) = TDN×4400 (NRC, 1985). They were maintained in a closed building under natural environmental conditions in individual wire mesh cages, equipped with metal troughs and automatic nipple drinkers. The rabbits had free access to feed and water. The rabbits were bred under similar production system and slaughtered at the age of 35, 45, 60, 75, and 90 d in a local commercial slaughterhouse. The facilities of the slaughterhouse met the requirements of the Institute of Animal Care and Use Committee (IACUC), which is funded by the United States National Institutes of Health. After 24 h post-mortem, the longissimus dorsi muscle (LD), left-hind leg muscle (LL), and abdominal muscle (AM) (ventral musculus) of the carcass were removed and immediately vacuum- packed and frozen at -20°C until analyzed. ! Ital. J. Food Sci., vol 28, 2016 - 686 2.2. Intramuscular lipid content and fatty acid composition analysis Intramuscular lipids were extracted according to FOLCH et al. (1957). Total lipid content was measured by weighing after solvent evaporation. The content of IMF was expressed as percent of the muscle weight. Fractions of intramuscular phospholipids were prepared with silica cartridges (Sep-Pack, Waters, Milford, MA, USA) by the method of JUANEDA and ROCQUELIN (1985). Phospholipids were quantified by phosphorous determination (BARTLETT, 1959). The relative content of phospholpids was expressed as percent of the IMF weight, while the absolute content was expressed as percent of the muscle weight. The phospholipids were methylated with boron fluoride-methanol (Sigma Aldrich) according to MORRISON and SMITH (1964). The fatty acids methyl esters were analyzed by a QP-2010 gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and a split injector. One microliter of FA methyl esters was injected in split mode (5:1) onto a Rtx-Wax capillary column (Restek, Bellefonte, PA, USA; 30 m × 0.25 mm id × 0.25 µm film thickness). The temperature of the column was programmed as follows: 1 min at 140°C, increments of 8°C/min to 180°C and held at 180°C for 2 min, increments of 3°C/min to 210°C then increments of 5°C /min to 230°C and held at 230°C for 10 min. The temperature of the injector and the detector both were 250°C. The flow rate of the carrier gas (N2) was 1.5 mL/min. Identification of fatty acids was performed by comparison of the retention times with those of standards (Sigma). The results were expressed as percent of the total fatty acids methyl esters present. 2.3. Statistical analysis The Statistical Analysis System (1996) was used to determine means, standard errors and analysis of variance. Duncan’s multiple range test was used to compare differences among means. An alpha level of p < 0.05 was considered significant. The effect of ages, muscles and genders on the composition of intramuscular phospholipid fatty acids were performed by ANOVA-partial least squares regression (A-PLSR). Ten 0/1 indicators variables (35, 45, 60, 75, 90 d, female, male, LD, LL, AM), SFA+MUFA, and PUFA/SFA in the X-matrix and 21 kinds of fatty acids (C12:0 - C22: 6n-3 were represented by the number 1-21) in the Y-matrix. Ellipses represent R2=0.5 (50%) and 1.0 (100%). A PLSR was performed using the Unscrambler Software, version 9.7 (CAMO ASA, Trondheim, Norway). All data was centered and standardized before analysis. 3. RESULTS AND DISCUSSIONS 3.1. Variation of content of total intramuscular lipids and phospholipids of Inra rabbits 3.1.1. Variation of intramuscular lipid content The intramuscular lipid content (% muscle weight) of LD, LL and AM from male and female Inra rabbits were significantly increased (p <0.05) with age (Table 2). The intramuscular lipid content of the three muscles of male and female rabbits was all increased. During the growth of Inra rabbits, the AM showed the highest content of intramuscular lipid, followed by LL and LD. HERNÀNDEZ and DALLE ZOTTE (2010) reported that the leanest cut of meat in the rabbit carcass was the loin and the hindleg was the quantitatively important cut because of its low lipid content compared to the other meats, which was consistent with our investigation (male: 0.77-1.21%, female: 0.79-1.33%). ! Ital. J. Food Sci., vol 28, 2016 - 687 Hence, the lipid content depended greatly on the age, gender and muscle. During the growth period from 35 d to 90 d, the deposition degree of total intramuscular lipid of the females in AM (2.88% to 5.42%) was significantly higher than that in the LL (1.19% to 1.78%) and LD (0.79% to 1.33%). Table 2: Comparison of intramuscular lipid content of Inra rabbit at different agesa. LD a LL AM Male Female Male Female Male Female 35 dbc 0.77±0.02d 0.79±0.08C 1.16±0.12d 1.19±0.15C 2.41±0.16d 2.88±0.12E 45 d 0.80±0.08cd 0.82±0.06C 1.20±0.06d 1.24±0.12C 2.67±0.09c 3.18±0.08D 60 d 0.96±0.11bc 1.01±0.10BC 1.30±0.03c 1.45±0.13B 2.74±0.01c 3.58±0.20C 75 d 1.15±0.13ab 1.25±0.24AB 1.49±0.12b 1.64±0.07AB 4.60±0.03b 4.83±0.05B 90 d 1.21±0.13a 1.33±0.21A 1.66±0.02a 1.78±0.06A 5.16±0.20a 5.42±0.17A a LD, Longissimus dorsimuscle; LL, left-hind leg muscle; AM, abdominal muscle. b Results were expressed as means ± SE, data were means of three replicates. c Values in the same column with different letters were significantly different (p<0.05), male: a-d, female: A- E. 3.1.2. Variation of intramuscular phospholipids content The percentage of phospholipids in the total intramuscular lipids of both male and female Inra rabbits was significantly decreased at the three muscles with age (p <0.05) (Table 3). A higher percentage of phospholipids characterized the total lipids (22.35-53.81%), however, the phospholipid contents in the meat of both New Zealand white and the commercial hybrid ranged from 9% to 19% total lipid (CAMBERO et al., 1991). Table 3: Comparison of intramuscular phospholipids content (intramuscular lipid weight %) of Inra rabbit at different agesa. LDa LL AM Male Female Male Female Male Female 35 dbc 43.47±1.33a 42.11±1.96A 53.81±2.82a 51.03±2.22A 42.21±2.36a 37.01±1.98A 45 d 35.02±1.88b 33.64±1.53B 38.98±1.48b 36.78±1.65B 34.94±2.88b 31.99±1.68B 60 d 32.28±1.02b 31.14±1.92B 36.35±1.44b 33.87±1.60B 28.93±1.22c 25.31±1.16C 75 d 27.93±1.58c 24.13±0.17C 30.21±2.14c 27.84±2.39C 27.87±1.91c 23.36±2.96C 90 d 26.84±1.72c 23.52±0.86C 28.89±2.18c 26.78±1.16C 25.40±1.32c 22.35±1.29C a LD, Longissimus dorsimuscle; LL, left-hind leg muscle; AM, abdominal muscle. b Results were expressed as means ± SE, data were means of three replicates. c Values in the same column with different letters were significantly different (p<0.05), male: a-c, female: A- C. The highest relative phospholipids content was found in the LL in both male and female Inra rabbits at different ages. In addition, the relative content of phospholipids in Inra rabbit abdomen showed significant gender differences, whereas that in legs and back phospholipids content appeared more pronounced gender differences after the age of 60 d. However, the absolute percentage of intramuscular phospholipids in the three muscles ! Ital. J. Food Sci., vol 28, 2016 - 688 did not vary obviously (p > 0.05) (Fig. 1). Among the three muscles, the AM showed the maximum absolute percentage of intramuscular phospholipids, followed by LL and LD, and the absolute percentage of intramuscular phospholipids in males were higher than that in females. According to wood et al (2008), phospholipids remained constant or increase little, as muscle fatness increases, whilst triacylglycerols increased to a higher extent, which may explain this phenomenon. Figure 1: Comparison of intramuscular phospholipids content (muscle weight %) of Inra rabbit at different ages (A: male rabbits, B: female rabbits). 3.2. Effect of age, muscle and gender on composition of intramuscular phospholipids The comparative intramuscular phospholipids fatty acids in LD, LL and AM of both male and female Inra rabbits at 35, 45, 60, 75, 90 d were shown in Table 4 (LD), Table 5 (LL), and Table 6 (AM), respectively. High levels of UFA (the sum of PUFA and MUFA), especially the abundance of PUFA, including the long chain (C20-22) PUFA in muscle, were observed in all samples. In muscle, significant percentage is phospholipids, which has a much higher PUFA content in order to perform its function as a constituent of cellular membranes (Wood et al., 2008). The ratio of SFA and MUFA in LD, LL and AM increased significantly with age in both gender rabbits (p < 0.05), whereas the ratio of PUFA among the muscles were all significantly decreased (p < 0.05). In addition, a significant reduction of PUFA/SFA ratio and a significant increase of SFA + MUFA were observed (p < 0.05). During the growth, the phospholipids PUFA percentage (% intramuscular fatty acids) was significantly higher in the LL than that in the LD and AM of male rabbits, corresponding to the lowest MUFA percentage in male LL. The phospholipids PUFA percentage in female LD was the lowest, while the MUFA in female legs was the minimum. Compared to the LD and LL, the AM existed more obvious gender differences in SFA and UFA percentage of phospholipids. ! Ital. J. Food Sci., vol 28, 2016 - 689 Table 4: Composition of the fatty acids of intramuscular phospholipids (%) in Longissimus dorsi of Inra rabbit at different agesa. 35d 45d 60d 75d 90d male female male female male female male female male female C12:0b 0.10±0.02a 0.03±0.01C 0.05±0.01b 0.08±0.02B 0.12±0.03a 0.17±0.02A -d - - - C14:0 0.26±0.05e 0.28±0.05B 0.47±0.01d 0.52±0.15A 0.60±0.17c 0.57±0.07A 0.79±0.05b 0.58±0.05A 0.83±0.05a 0.53±0.04A C14:1 0.36±0.02b 0.41±0.02A 0.26±0.02c 0.46±0.04A 0.11±0.02d 0.41±0.06A 0.34±0.03b 0.46±0.11A 0.50±0.02a 0.46±0.02A C15:0 1.19±0.11d 1.46±0.22E 1.65±0.16c 1.57±0.10D 1.81±0.22b 2.47±0.31C 2.02±0.18a 3.20±0.32A 1.10±0.03d 2.79±0.13B C16:0 20.03±0.03e 17.16±0.19E 21.64±0.38d 20.30±0.05D 22.37±0.11c 21.25±0.09C 23.71±0.14b 23.34±0.12B 25.11±0.30a 28.22±0.02A C16:1n-7 0.24±0.03c 0.29±0.03D 0.31±0.02c 0.48±0.06A 0.34±0.03c 0.39±0.02C 0.52±0.02b 0.44±0.04B 1.75±0.24a 0.44±0.01B C17:0 0.50±0.02a 0.48±0.02A 0.42±0.03b 0.47±0.04A 0.38±0.02b 0.39±0.02B 0.48±0.02a 0.40±0.01B 0.41±0.01b 0.50±0.02A C17:1 0.56±0.04d 0.72±0.09E 0.82±0.05a 1.34±0.07A 0.92±0.07b 1.15±0.07B 0.66±0.05c 0.93±0.05D 0.41±0.01e 1.01±0.03C C18:0 11.04±0.03e 12.58±0.40D 11.72±0.07d 13.75±0.10C 12.56±0.13c 13.82±0.03C 13.11±0.02b 14.31±0.24B 13.50±0.03a 14.45±0.04A C18:1n-9 13.34±0.02e 13.54±0.15E 13.99±0.53d 13.69±0.15D 15.01±0.03c 14.43±0.03C 15.80±0.04b 14.61±0.06B 17.89±0.02a 17.75±0.07A C18:1n-7 1.63±0.03a 1.64±0.21A 1.52±0.31a 0.88±0.12D 1.00±0.03b 1.06±0.01C 0.86±0.02b 1.15±0.02B 0.65±0.02c 0.65±0.02E C18:2n-6 25.91±0.07a 25.00±0.23A 24.29±0.28b 20.24±0.13B 22.67±0.02c 19.14±0.10C 20.77±0.02d 17.65±0.11D 20.21±0.07e 15.09±0.08E C18:3n-3 0.27±0.03d 0.35±0.03B 0.41±0.02b 0.58±0.02A 0.48±0.02c 0.58±0.03A 0.78±0.06a 0.56±0.02A 0.37±0.02b 0.38±0.12B C20:0 0.05±0.01a 0.06±0.01B 0.08±0.03a 0.17±0.01A 0.11±0.03a 0.17±0.02A 0.09±0.02a 0.17±0.01A 0.13±0.02a 0.16±0.06A C20:1n-9 0.18±0.01ab 0.17±0.02B 0.11±0.03b 0.11±0.03C 0.18±0.06ab 0.22±0.02A 0.37±0.03a 0.14±0.01BC 0.16±0.02ab 0.10±0.02C C20:2n-6 0.68±0.03d 0.73±0.03E 0.71±0.03d 0.83±0.06D 1.19±0.02c 0.90±0.03C 1.65±0.03a 1.15±0.02B 1.74±0.03b 1.23±0.01A C20:3n-6 0.63±0.02e 0.68±0.04C 0.91±0.02d 1.21±0.07A 1.26±0.03c 1.20±0.06A 1.54±0.02b 1.21±0.04A 1.88±0.03a 0.82±0.05B C20:4n-6 13.80±0.08a 14.58±0.05A 11.78±0.08b 14.01±0.13B 9.72±0.14c 13.64±0.15C 9.47±0.04d 12.99±0.11D 8.81±0.05e 10.36±0.10E C20:5n-3 5.01±0.04a 5.53±0.03A 4.84±0.02b 5.03±0.12B 4.60±0.04c 4.60±0.03C 3.91±0.03d 4.04±0.06D 2.77±0.10e 3.40±0.02E C22:5n-3 2.58±0.02a 2.49±0.03A 2.47±0.01b 2.33±0.08B 2.34±0.02c 2.21±0.04C 1.84±0.01d 1.62±0.04D 1.12±0.03e 1.11±0.03E C22:6n-3 1.65±0.02a 1.83±0.02A 1.56±0.02b 1.41±0.04B 1.40±0.03c 1.23±0.01C 1.29±0.02d 1.03±0.01D 0.69±0.02e 0.56±0.03E SFAc 33.16±0.12e 32.06±0.06E 36.03±0.69d 36.86±0.78D 37.96±0.11c 38.83±0.34C 40.19±0.13b 41.99±0.26B 41.07±0.31a 46.65±0.07A PUFA 50.54±0.17a 51.18±0.24A 46.96±0.34b 45.64±0.07B 43.68±0.17c 43.51±0.35C 41.26±0.07d 40.27±0.30D 37.56±0.09e 32.94±0.09E MUFA 16.30±0.07e 16.76±0.28D 17.02±0.77d 17.50±0.85C 18.36±0.18c 17.66±0.06BC 18.55±0.19b 17.74±0.04B 21.37±0.22a 20.41±0.02A a Results were expressed as means ± SE, data were means of three replicates. b Values in the same column with different letters were significantly different (p<0.05), male: a-e, female: A-E. c SFA, total saturated fatty acids; MUFA, total monounsaturated fatty acids; PUFA, total polyunsaturated fatty acids. d ”-” : undetected. ! Ital. J. Food Sci., vol 28, 2016 - 690 Table 5: Composition of the fatty acids of intramuscular phospholipids (%) in left-hind leg muscle of Inra rabbit at different agesa. 35d 45d 60d 75d 90d male female male female male female male female male female C12:0b 0.05±0.01b 0.04±0.01B 0.04±0.01b 0.09±0.01A 0.27±0.02a 0.05±0.02B -d - - - C14:0 0.16±0.02b 0.23±0.06D 0.22±0.07b 0.41±0.11C 0.34±0.09b 0.54±0.13BC 0.77±0.16a 0.95±0.11A 0.26±0.01b 0.68±0.02B C14:1 0.35±0.03b 0.37±0.02B 0.22±0.02c 0.14±0.02C 0.14±0.01d 0.51±0.03A 0.23±0.04c 0.32±0.01B 0.52±0.01a 0.52±0.03A C15:0 1.12±0.25d 1.55±0.09B 1.46±0.35c 1.60±0.09B 1.51±0.36bc 2.39±0.47A 1.53±0.03b 1.77±0.13B 1.66±0.01a 1.84±0.07B C16:0 19.57±0.05e 16.98±0.13D 20.65±0.08d 20.48±0.07C 21.55±0.11c 21.95±0.06B 22.63±0.13b 22.01±0.22B 24.08±0.05a 24.66±0.20A C16:1n-7 0.34±0.02c 0.37±0.02D 0.32±0.02c 0.39±0.02CD 0.36±0.02c 0.41±0.02C 0.51±0.18b 0.75±0.02A 1.94±0.03a 0.48±0.01B C17:0 0.40±0.03c 0.43±0.01B 0.44±0.01b 0.36±0.01C 0.38±0.02c 0.50±0.02A 0.55±0.03a 0.40±0.03B 0.40±0.02c 0.49±0.01A C17:1 0.51±0.09bc 0.67±0.02B 0.94±0.12a 2.08±0.29A 0.86±0.13a 0.89±0.11B 0.57±0.01b 0.54±0.05B 0.35±0.02c 0.78±0.02B C18:0 11.79±0.06e 13.12±0.06E 13.15±0.04d 14.78±0.10D 14.61±0.03c 15.29±0.10C 15.97±0.04b 15.91±0.03B 16.12±0.02a 16.32±0.03A C18:1n-9 11.78±0.05e 12.09±0.12E 12.48±0.02d 12.34±0.06D 13.24±0.16c 13.60±0.12C 14.83±0.02b 13.94±0.15B 15.10±0.02a 15.75±0.07A C18:1n-7 1.48±0.06ab 1.39±0.03A 1.48±0.02ab 0.85±0.01C 1.41±0.10a 0.99±0.05B 1.53±0.02b 1.00±0.08B 0.61±0.01c 0.70±0.03D C18:2n-6 29.86±0.11a 26.34±0.02A 26.58±0.07b 20.62±0.25B 24.59±0.01c 18.77±0.72C 21.37±0.05d 18.21±0.02D 20.84±0.02e 15.45±0.05E C18:3n-3 0.37±0.02c 0.37±0.02CD 0.37±0.02c 0.72±0.06A 0.54±0.01ab 0.40±0.02C 0.83±0.37a 0.56±0.01B 0.50±0.03c 0.35±0.04D C20:0 0.05±0.01b 0.07±0.01A 0.06±0.01b 0.17±0.03A 0.13±0.03a 0.17±0.18A 0.09±0.02b 0.12±0.03A 0.08±0.01b 0.13±0.01A C20:1n-9 0.18±0.02b 0.18±0.02BC 0.17±0.02b 0.11±0.02D 0.27±0.02a 0.20±0.02B 0.28±0.03a 0.28±0.01A 0.27±0.01a 0.15±0.02C C20:2n-6 0.83±0.02d 0.83±0.02E 0.87±0.02c 0.93±0.01D 1.11±0.02b 1.03±0.02C 1.15±0.01b 1.41±0.02B 1.29±0.02a 1.53±0.02A C20:3n-6 0.83±0.02d 0.78±0.02E 0.93±0.02c 1.08±0.02D 1.08±0.04b 1.04±0.01C 1.18±0.02a 1.10±0.02B 0.95±0.02c 1.16±0.01A C20:4n-6 12.33±0.10a 15.29±0.07A 11.95±0.18b 14.42±0.07B 10.29±0.02c 13.94±0.05C 9.95±0.05d 13.56±0.09C 9.84±0.02d 12.59±0.07D C20:5n-3 3.77±0.06a 4.93±0.02A 3.58±0.03b 4.73±0.05B 3.34±0.03c 4.45±0.11C 3.12±0.01d 4.21±0.03D 3.09±0.02e 4.17±0.05D C22:5n-3 2.40±0.04a 2.33±0.02A 2.34±0.02b 2.31±0.03B 2.33±0.02b 2.06±0.04C 1.87±0.02c 1.79±0.03D 1.34±0.04d 1.59±0.08E C22:6n-3 1.82±0.15a 1.63±0.02A 1.75±0.03a 1.37±0.02B 1.66±0.03b 1.27±0.28C 1.05±0.01c 1.17±0.02D 0.83±0.02d 0.67±0.04E SFAc 33.14±0.29e 32.44±0.06D 36.02±0.56d 37.90±0.01C 38.77±0.11c 40.89±0.33B 41.53±0.23b 41.16±0.04B 42.60±0.07a 44.13±0.11A PUFA 52.22±0.45a 52.50±0.10A 48.37±0.26b 46.19±0.22B 41.95±0.07c 42.51±0.39C 40.51±0.35d 42.01±0.19D 38.67±0.03e 37.50±0.12E MUFA 14.64±0.16e 15.07±0.14D 15.61±0.09d 15.91±0.21C 16.28±0.14c 16.61±0.07B 17.96±0.13b 16.83±0.19B 18.72±0.08a 18.37±0.08A a Results were expressed as means ± SE, data were means of three replicates. b Values in the same column with different letters were significantly different (p<0.05), male: a-e, female: A-E. c SFA, total saturated fatty acids; MUFA, total monounsaturated fatty acids; PUFA, total polyunsaturated fatty acids. d ”-” : undetected. ! Ital. J. Food Sci., vol 28, 2016 - 691 Table 6: Composition of the fatty acids of intramuscular phospholipids (%) in abdominal muscle of Inra rabbit at different agesa. 35d 45d 60d 75d 90d male female male female male female male female male female C12:0b 0.17±0.01a 0.07±0.02A 0.07±0.02b 0.04±0.01B 0.07±0.02b 0.06±0.01AB -d - - - C14:0 0.53±0.16a 0.39±0.05D 0.38±0.07ab 0.65±0.19A 0.30±0.02b 0.41±0.06C 0.44±0.14ab 0.59±0.04B 0.27±0.02b 0.69±0.03A C14:1 0.25±0.02b 0.15±0.02D 0.21±0.02b 0.18±0.01C 0.60±0.35a 0.27±0.01B 0.24±0.02b 0.28±0.02B 0.47±0.05ab 0.56±0.19A C15:0 1.82±0.56a 1.36±0.02D 2.53±0.76a 1.68±0.39C 2.34±0.79a 1.80±0.57B 2.88±0.52a 1.83±0.51B 1.98±0.13a 1.94±0.04A C16:0 21.22±0.07c 12.65±0.01D 22.17±0.08bc 12.90±0.26D 22.67±0.31b 14.06±0.13C 22.97±0.06b 17.23±0.06B 23.87±1.09a 21.37±0.11A C16:1n-7 0.34±0.03d 0.34±0.02E 0.30±0.02d 0.48±0.02D 1.02±0.32b 0.65±0.02C 2.02±0.24a 1.05±0.02B 2.04±0.06a 1.56±0.12A C17:0 0.43±0.02bc 0.44±0.02A 0.44±0.01b 0.39±0.02B 0.41±0.03c 0.33±0.01C 0.52±0.01a 0.38±0.03B 0.37±0.02d 0.47±0.01A C17:1 0.82±0.23ab 0.61±0.11BC 0.93±0.28ab 1.27±0.14A 1.16±0.25a 0.74±0.19B 0.92±0.18ab 0.46±0.11C 0.65±0.03b 0.47±0.01C C18:0 10.00±0.08e 13.24±0.03E 10.58±0.02d 15.50±0.06D 10.95±0.28c 17.04±0.11C 11.42±0.34b 17.39±0.05B 13.48±1.35a 17.67±0.40A C18:1n-9 13.93±0.03b 13.22±0.02D 14.21±0.78b 13.65±0.50CD 14.66±0.24b 14.10±0.07C 16.42±0.28a 14.65±0.06B 16.89±0.85a 15.40±0.38A C18:1n-7 1.20±0.04a 1.42±0.03A 1.12±0.27a 0.90±0.19C 0.65±0.03b 0.71±0.07D 0.50±0.04b 1.13±0.04B 0.99±0.38ab 1.34±0.07A C18:2n-6 24.23±0.07a 25.26±0.10A 23.91±0.03b 22.09±0.09B 23.48±0.58b 20.80±0.19C 22.78±0.29bc 19.73±0.13D 22.56±0.99c 17.21±0.21E C18:3n-3 0.35±0.01c 0.35±0.02E 0.31±0.02d 0.60±0.02B 0.56±0.03b 0.51±0.02C 0.85±0.02a 0.67±0.02A 0.54±0.01b 0.45±0.02D C20:0 0.13±0.03a 0.10±0.02AB 0.07±0.01b 0.08±0.01B 0.11±0.01ab 0.11±0.02A 0.09±0.01b 0.12±0.01A 0.07±0.01b 0.13±0.01A C20:1n-9 0.26±0.01b 0.22±0.02B 0.17±0.02b 0.23±0.02B 0.30±0.02ab 0.27±0.02A 0.22±0.02b 0.27±0.02A 0.37±0.12a 0.20±0.03C C20:2n-6 0.96±0.02c 0.76±0.04E 1.11±0.03d 0.98±0.02D 1.14±0.03b 1.14±0.02B 1.23±0.07a 1.07±0.02C 1.08±0.05b 1.24±0.02A C20:3n-6 0.95±0.01d 0.83±0.02E 1.00±0.03c 1.13±0.02D 1.28±0.03b 1.43±0.01C 1.38±0.07a 1.78±0.05B 0.99±0.05cb 1.96±0.02A C20:4n-6 12.93±0.14a 17.88±0.08A 11.71±0.03b 17.40±0.15B 10.72±0.41c 16.08±0.12C 8.92±0.40d 13.64±0.10D 8.67±0.41d 11.22±0.17E C20:5n-3 5.65±0.06a 5.84±0.07A 5.23±0.02b 5.48±0.07B 4.44±0.15c 5.16±0.02B 3.48±0.10d 4.59±0.04C 2.88±0.14e 3.59±0.09D C22:5n-3 2.24±0.03a 2.96±0.03A 2.12±0.02b 2.73±0.02B 1.76±0.07d 2.72±0.02B 1.87±0.08c 1.91±0.07C 1.08±0.01e 1.56±0.03D C22:6n-3 1.60±0.14a 1.92±0.03A 1.45±0.01b 1.61±0.06B 1.37±0.08c 1.59±0.03B 1.08±0.04d 1.24±0.01C 0.76±0.01e 1.08±0.02D SFAc 34.30±0.30d 28.25±0.27E 36.22±0.98c 31.25±0.95D 36.84±0.73c 33.82±0.28C 38.31±0.35b 37.54±0.43B 40.03±0.50a 42.26±0.36A PUFA 48.91±0.45a 55.79±0.37A 46.84±0.14b 52.03±0.22B 44.75±0.21c 49.43±0.34C 41.59±0.82d 44.63±0.12D 38.56±1.65e 38.31±0.53E MUFA 16.79±0.16a 15.96±0.09B 16.94±0.77a 16.72±0.56C 18.41±0.48b 16.75±0.07D 20.10±0.49c 17.83±0.06B 21.41±1.28d 19.43±0.21A a Results were expressed as means ± SE, data were means of three replicates. b Values in the same column with different letters were significantly different (p<0.05), male: a-e, female: A-E. c SFA, total saturated fatty acids; MUFA, total monounsaturated fatty acids; PUFA, total polyunsaturated fatty acids. d ”-” : undetected. ! Ital. J. Food Sci., vol 28, 2016 - 692 In terms of fatty acids composition of intramuscular phospholipids of Inra rabbit, SFA among the muscles were mainly composed of palmitic (C16:0) and stearic (C18:0), MUFA were mainly represented by oleic (C18:1), whereas PUFA consisted of linoleic (C18:2) and arachidonic acid (C20:4). According to CAMBERO et al. (1991), the C16:0, C18:0, C18:1 and C18:2 were together representing more than 70% of the total fatty acids. A higher percentage of PUFA characterized the fatty acids composition of phospholipids (KANATT et al., 2006). In our study, the percentage of PUFA in males and females were accounted for 37.56-52.22% and 32.94-55.79% respectively in the intramuscular phospholipids during growth period from 35 d to 90 d. Long chain n-3 and n-6 PUFA were mainly found in phospholipids (ENSER et al., 2000; COOPER et al., 2004), which was also in good agreement with our investigation. However, the fatty acids composition was rarely detected in different rabbit muscles during different feeding days, especially on a particular rabbit species. Comparing the variety of intramuscular phospholipids from LD, LL and AM during the growth stages from 35 d to 90 d, C16:0, C18:0, palmitoleic acid methyl ester (C16:1n-7), C18:1n-9, cis-11,14-eicosadienoic acid methyl ester (C20:2n-6) and cis-8,11,14-eicosatrienoic acid methyl ester (C20:3n-6) increased significantly (p < 0.05) in both genders, whereas C18:2n-6, C20:4n-6, C20:5n-3, C22:5n-3 and C22:6n-3 decreased significantly (p < 0.05). The percentage of C16:0 and C18:0 in female-LD significantly increased (p < 0.05), and both C18:2n-6 and C20:4n-6 in female-LL significantly decreased (p < 0.05). Moreover, the percentage of C20:4n-6 in female-AM decreased faster than other samples during the test days. However, other fatty acids did not showed apparent changes. According to ALASNIER and GANDEMER (1998), the fatty acid composition of individual phospholipid classes was related to metabolic type of fibre in the rabbit, and the differences in fatty acid composition of phosphatidyl ethanolamine, phosphatidyl choline and cardiolipin explained a large part of the differences in fatty acid compositions of the total phospholipids of glycolytic and oxidative muscles. As the major ingredient of feeds for all species, the incorporation of C18:2n-6 into the muscles, in relation to the amount in the diet, was greatest among other fatty acids. C18:2n-6 was deposited in muscle phospholipids at a high level where it and its long chain products C20:4n-6 competed well for insertion into phospholipids molecules (WOOD et al., 2008). Comparing the changes of fatty acids in the LD, LL and AM, the deposition rate of C16:0 was faster in the LD of Inra rabbits (both males and females) than that in the LL and AM. However, the C16:0 had lowest percentage and slowest deposition rate in AM. In terms of C18:0, LL was sequentially higher than LD and AM in male rabbits, while AM was higher than LL and LD in female rabbits. Meanwhile, the percentage and deposition rate of C18:0 in AM were also higher in females than that in males. For the C18:1n-9 percentage, LD showed the highest and fastest deposition rate in both genders. The percentage of C18:2n-6 in LL was the highest, and decreased in the maximum levels with age. According to WOOD et al (2008), the higher percentage of C18:2n-6 in phospholipids compared with neutral lipids in all species mean that muscle from lean animals has relatively higher percentages of this major PUFA. In addition, during the growth period from 35 d to 90 d, the initial content of C20:4n-6 in LD was higher compared with other muscles, and showed the fastest reducing rate. No significantly variation was found among other fatty acids components. During the growth period of Inra rabbit, the n-6/n-3 values for LD, LL, and AM ranged from 4.02 to 6.61, 4.71 to 5.72 and 3.97 to 6.33 in males, and 3.88 to 5.05, 4.05 to 4.67 and 3.95 to 4.73 in females, respectively. There is an increasing recognition of the health benefits of PUFA in general, and of n-3 PUFAs in particular, because these fatty acids are essential for humans (ALESSANDRI et al., 1998; CONQUER et al., 2011). Nutritional value is determined primarily by the ratio between SFA and PUFA in meat and the balance between fatty acids of the n-6 and n-3 series. Unfortunately, Western diet is very high in n- ! Ital. J. Food Sci., vol 28, 2016 - 693 6 fatty acids relative to n-3 fatty acids (ENSER et al., 2000; HARGIS and VAN ELSWYK, 1993). Nutritionist recommendations are for a ratio of n-6/n-3 PUFA of less than 5 (WOOD et al., 2003; KOUBA et al., 2003), and a ratio of n-6/n-3 below about 4.0 is required in the diet to combat various ‘‘lifestyle diseases’’ such as coronary heart disease and cancers (SIMOPOULOS, 2004; WILLIAMS, 2000). According to FAO/WHO, the recommended dose of essential PUFA in a healthy daily diet is 5/1 to 10/1 (n-6/n-3) (DALLE ZOTTE and SZENDRÖ, 2011), and a lower ratio is more desirable in reducing the risk of many of chronic diseases, even if the optimal ratio may vary depending on the disease under consideration (SIMOPOULOS, 2002). Therefore, it can be suggested that the intramuscular phospholipids of Inra rabbits is recommended. ALASNIER and GANDEMER (1998) reported that the phosphatidyl ethanolamine of oxidative muscles contains less 18:2n-6 and more 18:0 and long chain PUFA of the n-6 and n-3 series than that of glycolytic ones; phosphatidyl choline of oxidative muscles contains more 18:0 and less 16:0 and 18:2n-6 than that of glycolytic ones; cardiolipin of the oxidative muscles contains less 18:2 n-6 than those of the glycolytic ones. In addition, they suggested that a part of the composition difference could be related to high mitochondria content of the oxidative muscles compared to the glycolytic ones. To check this hypothesis, further investigations are required to determinate the fatty acid composition of individual phospholipid classes of both mitochondria and microsomes in rabbit muscles. 3.3. Analysis of PLSR on composition of intramuscular phospholipids The analysis of PLSR showed that the first and second main ingredients explained 43% and 33% Y variables, respectively. From the nutritional point of view, the PUFA/SFA value is often used to evaluate the nutritional value of the meat, and higher value represents better nutritional value. However, the higher SFA + MUFA value of the meat are, the tenderness juiciness and the better flavor are (CAMERON and ENSER, 1991). On the contrary, if the content of PUFA is too high, the tenderness, flavor and juiciness of meat are poor. Hence, the SFA + MUFA value can be used to measure the quality indicators of samples after processing. The SFA + MUFA value of intramuscular phospholipid fatty acids of Inra rabbit was located in the bottom right renderings, indicating the higher the nutritional value of the sample in the bottom right, and the PUFA/SFA values were located in the top left of the renderings (Fig. 2). Thus, the better flavor of the sample after processing is closer to the top left. On the first principal component, the composition of phospholipid fatty acids showed obviously different in ages, genders and muscles. The LD was closer to the SFA + MUFA, indicating the better phospholipid processed-flavor after processing of LD. However, the AM was closer to PUFA/SFA, indicating the better phospholipid nutritional value of AM. On the second principal component, the composition of phospholipid fatty acid of the raw material showed obviously different in ages and genders. The nutritional value of the total lipid decreased with age. In addition, the 35 d-feedstock located in the top left oval, closely to 12 (C18: 2n-6), 18 (C20: 4n-6), 19 (C20: 5n-3), 20 (C22: 5n-3), 21 (C22: 6n-3) and other PUFAs, while the 90 d- feedstock located in the bottom right of the ellipse, closely to 6 (C16:1n-7,), 2 (C14:0), 3(C14:1n-6), 5 (C16:0), 10 (C18:1n-9) and some other saturated and monounsaturated fatty acids. Moreover, the intramuscular phospholipids of male rabbits was closely related to the 5 (C16:0) and 12 (C18:2n-6), that is the C16:0 and C18:2n-6 percentage in muscle was higher in male rabbits than that in the females. The intramuscular phospholipid of female rabbits was closely related to the percentage of 9 (C18:0)�14 (C20:0) and 18 (C20:4n-6), that is the C18:0, C20:0 and C20:4n-6 levels in muscle were higher in female rabbits than that in the males. Among the three sections, the AM had better phospholipid nutritional ! Ital. J. Food Sci., vol 28, 2016 - 694 value, which may be due to the higher percentage of PUFA, and the LD and LL showed better phospholipid flavor after processing, which may be due to the higher percentage of 13 (C18:3n-3) and 7 (C17:0), 15 (C20:1n-9), respectively. Figure 2: A PLSR correlation loadings plot for first 2 principal components (PCs). Ten 0/1 indicators variables (35 d, 45 d, 60 d, 75 d, 90 d, female, male, LD, LL, AM), SFA+MUFA, and PUFA/SFA in the X-matrix and 21 kinds of fatty acids (C12:0 - C22:6n-3 were represented by the number 1-21) in the Y-matrix. Ellipses represent R2=0.5 (50%) and 1.0 (100%). Overall, the composition of phospholipid fatty acids at different ages, genders and muscles showed significant difference. The effects of age, gender and muscle on the composition of phospholipid fatty acids mainly reflected on the first principal component. However, on the second principal component, only ages and genders showed the obvious difference of phospholipid fatty acids composition. 4. CONCLUSIONS Inra rabbits are a meat source of nutritious quality, containing low content of intramuscular lipids, low ratio of n-6/n-3, whilst high content intramuscular phospholipids (% lipid). A higher content of PUFA characterised the fatty acids composition of the phospholipids, and the significantly decrease of PUFA in intramuscular phospholipids during growth were observed. Among the fatty acids from intramuscular phospholipids, SFA mainly consists of C16:0 and C18:0, MUFA consist of C18:1, and PUFA consist of C18:2 and C20:4. There is a wide variation of total lipids content and fatty acid composition at different ages, genders and muscles in Inra rabbit. By the analysis of PLSR, the nutritional value of the phospholipid fatty acids decreased with age, and the AM showed better nutritional value than LD and LL. The absolute data analysis is important for recommendations and suggestion of the consumption of dietary ! Ital. J. Food Sci., vol 28, 2016 - 695 phospholipids from animal sources. Further investigation is necessary to explore the properties of processing and nutritional characteristics of different sections from Inra rabbit meat. 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