DIETARY SUPPLEMENTATION OF DIFFERENT FATTY ACIDS ON GROWTH, LIPID METABOLISM AND NON-SPECIFIC IMMUNITY OF TURBOT (SCOPHTHALMUS MAXIMUS L.)
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摘要:
为揭示不同链长和不饱和度的脂肪酸对水产动物生长、脂代谢和非特异性免疫的影响, 研究挑选初始体重为(8.00±0.20) g的大菱鲆(Scophthalmus maximus L.)幼鱼作为实验对象, 设置了添加不同链长和不饱和度脂肪酸的8种等氮等脂配合饲料: 对照组(CON)、棕榈酸组(PA)、硬脂酸组(SA)、油酸组(OA)、亚油酸组(LA)、亚麻酸组(ALA)、花生四烯酸组(ARA)和二十二碳六烯酸/二十碳五烯酸组(DHA/EPA), 在18℃的循环水养殖系统中进行为期8周的养殖实验。结果表明: 随着饲料中添加脂肪酸链长和不饱和度的增加, 大菱鲆幼鱼增重率(WGR)、特定生长率(SGR)和蛋白质效率(PER)均有增高的趋势, ARA组WGR和SGR最高, DHA/EPA组PER最高。饲料中添加不同脂肪酸对大菱鲆幼鱼鱼体的灰分和水分没有显著性影响(P>0.05), 但鱼体的粗蛋白含量随着脂肪酸链长和不饱和度的增加而增加, 粗脂肪含量随着脂肪酸链长和不饱和度的增加而降低。随着脂肪酸链长和不饱和度的增加, 大菱鲆幼鱼血浆胆固醇(T-CHO)含量逐渐降低, 而血浆甘油三酯(TG)含量在SA组最高, 在DHA/EPA组最低; 血浆低密度脂蛋白(LDL-C)含量随着添加脂肪酸链长和不饱和度的增加而降低, 高密度脂蛋白(HDL-C)呈相反的趋势。进一步对大菱鲆肝脏脂代谢相关基因检测得出, 饲料中添加不同脂肪酸可以通过调控脂代谢相关基因表达(FAS、PPARγ、SREBP1、PPARα和ACOX1)进而控制大菱鲆幼鱼鱼体的脂肪合成和分解。PA组的肝脏总抗氧化能力(T-AOC)最低, 且显著低于其他各处理组(P<0.05)。DHA/EPA组的肝脏超氧化物歧化酶(SOD)和过氧化氢酶(CAT)含量最高, 肝脏丙二醛(MDA)含量最低。进一步对肝脏免疫基因定量得出, PA、SA组显著提高了促炎因子(IL-1β、IL-8、MyD88、NF-κB p65、TLR2、TLR8、TLR9和TNF-α)的基因表达量, 且显著降低了抗炎因子的表达量(TGF-β), 而LA、ALA、ARA和DHA/EPA组则呈现出相反的趋势。研究结果表明, 随着饲料中添加脂肪酸链长和不饱和度的增加, 可以显著影响大菱鲆幼鱼的生长、脂代谢和非特异性免疫。
Abstract:Due to the continued strain on global fish oil resources, the utilization of alternative fats such as vegetable oil in aquaculture feed has gradually increased. However, there are differences in the types and concentrations of fatty acids between vegetable oil and fish oil. In order to reveal the effects of fatty acids with different chain lengths and degrees of unsaturation on the growth, lipid metabolism, and non-specific immunity of aquatic animals, juvenile turbot (Scophthalmus maximus L.) with an initial body weight of (8.00±0.20) g were selected as the experimental subjects in this study. Eight isonitrogenous and isolipidic compound diets with different chain lengths and unsaturated levels of fatty acids were set up: control group (CON), palmitic acid group (PA), stearic acid group (SA), oleic acid group (OA), linoleic acid group (LA), linolenic acid group (ALA), arachidonic acid group (ARA), and docosahexaenoic acid/eicosapentaenoic acid group (DHA/EPA). The experiment was conducted in a recirculating aquaculture system at 18℃ for 8 weeks. The results showed that with an increase in dietary fatty acid chain length and unsaturated degree, the weight gain rate (WGR), specific growth rate (SGR), and protein efficiency ratio (PER) of juvenile turbot exhibited an increasing trend. Notably, the ARA group displayed the highest WGR and SGR, while the DHA/EPA group exhibited the highest PER. Dietary variations in fatty acids had no significant effects on the ash and water content of juvenile turbot (P>0.05). However, crude protein content increased with rising fatty acid chain length and unsaturation degree, whereas crude fat content decreased. Moreover, as fatty acid chain length and unsaturated degree increased, the plasma cholesterol (T-CHO) content of juvenile turbot gradually decreased, while the plasma triglyceride (TG) content was the highest in SA group and the lowest in DHA/EPA group. Plasma low density lipoprotein (LDL-C) content decreased with the increasing fatty acid chain length and unsaturation degree, whereas the high density lipoprotein (HDL-C) showed an opposite trend. Further analysis of lipid metabolism-related genes in turbot liver showed that dietary fatty acids could control lipid synthesis and decomposition by regulating the expression of lipid metabolism-related genes (FAS, PPARγ, SREBP1, PPARα, and ACOX1). The total antioxidant capacity (T-AOC) of liver in PA group was the lowest, and was significantly lower than that in other treatment groups (P<0.05). The liver superoxide dismutase (SOD) and catalase (CAT) contents were the highest in DHA/EPA group, while the liver malondialdehyde (MDA) content was the lowest. Furthermore, gene expressions of pro-inflammatory factors (IL-1β, IL-8, MyD88, NF-κB p65, TLR2, TLR8, TLR9, and TNF-α) in PA and SA groups were significantly increased, whereas the expression of anti-inflammatory factor (TGF-β) was significantly decreased. Conversely, in LA, ALA, ARA, and DHA/EPA groups, the trend was reversed. These results suggest that dietary supplementation of fatty acids with longer chain lengths and higher unsaturated degree can significantly affect the growth, lipid metabolism and non-specific immunity of juvenile turbot.
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Keywords:
- Different fatty acid /
- Growth /
- Lipid metabolism /
- Antioxidant /
- Nonspecific immunity /
- Juvenile turbot
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脂质在生物体的生长和发育过程中扮演着极其重要的角色, 不仅能够为机体提供能量, 同时也能够提供一些机体自身所不能合成必需脂肪酸, 除此之外, 脂质还是生物膜的重要组成成分[1]。而饲料中的脂质是鱼类所必需的重要营养成分, 不仅构成了鱼体的重要组成部分, 还能够促进吸收各种脂溶性的维生素, 同时维持多种生物学过程的正常运行[2, 3]。脂肪主要是由脂肪酸组成的, 只有当鱼类摄入适宜脂肪酸组成的饲料时, 才能保证其正常进行生长、发育和繁殖等各项生命活动, 同时还能起到节约蛋白的作用。也有相关研究表明饲料中的脂肪酸组成不适宜会导致鱼体内过多的脂肪在肝脏和腹腔积累, 从而减少可食用部分并降低肉质品质等一系列问题产生[4－6]。
随着水产养殖业的快速发展, 鱼油成本高、产量低, 作为优质的脂肪源已经变得越来越稀缺[7]。这对整个水产养殖行业的发展造成了严重影响, 而植物油(如: 大豆油、棕榈油、菜籽油等)具有产量高, 成本低的特点。因此, 使用植物油代替鱼油已被证明是解决这一问题的有效方法[8－10]。由于植物油与鱼油相比, 其所含脂肪酸的成分不同, 特别是在高比例的不饱和脂肪酸及EPA、DHA等长链多不饱和脂肪酸的含量方面存在差异[11]。饲料中的脂肪酸含量和脂肪酸比例对鱼体生长、脂代谢、免疫和抗氧化能力起着至关重要的作用, 不同的脂肪酸对机体也起着不同的生理作用。DHA和EPA可以通过调节机体的能量代谢, 维持细胞通透性等作用来维持鱼体的代谢, 促进鱼体的生长等[12, 13]。花生四烯酸对水产动物、哺乳动物和人的生长、免疫和繁殖发育都发挥着不可或缺的作用[14－16]。一些海水鱼类和广盐性鱼类不能将亚麻酸转换成长链多不饱和脂肪酸(Long-chain polyunsaturated fatty acid, LC-PUFA ), 或者转换的能力不足[3, 17]。
大菱鲆(Scophthalmus maximus L.), 又名欧洲比目鱼, 俗称多宝鱼, 具有生长快、肉质细腻可口、营养价值高等特点。目前, 它已成为中国北方沿海最重要的海水养殖产业之一[18]。已有研究表明, DHA、EPA是大菱鲆的营养需求所必需的脂肪酸, 且大菱鲆DHA、EPA需求量为1%—3.5%[19]。饲料中的脂肪酸, 尤其是HUFAs会显著影响大菱鲆幼鱼的生长、存活率等[20]。因此, 通过探讨饲料中添加不同脂肪酸对大菱鲆幼鱼生长、脂代谢及免疫的影响, 旨在丰富大菱鲆的营养生理理论并提供基础数据, 深入研究脂肪酸在鱼体内反应的机制, 以科学指导方式推动水产养殖业可持续发展, 并为开发营养均衡的大菱鲆配合饲料提供支持。
1. 材料与方法
1.1 试验饲料
鱼粉、酪蛋白和谷朊粉为本实验饲料的主要的蛋白源, 主要的脂肪源为鱼油和大豆卵磷脂, 在基础饲料能够满足大菱鲆n-3 LC-PUFA需求的基础上, 分别添加0.5%的不同脂肪酸, 配置成蛋白含量为50%, 脂肪含量为12%的等氮等脂的配合饲料。根据添加的脂肪酸不同, 设置了8个不同的处理组, 分别为添加甘油三酯的对照组(CON)、棕榈酸组(PA, C16﹕0)、硬脂酸组(SA, C18﹕0)、油酸组(OA, C18﹕1)、亚油酸组(LA, C18﹕2n-6)、α-亚麻酸组(ALA, C18﹕3n-3)、花生四烯酸组(ARA, C20﹕4n-6)和二十二碳六烯酸(DHA, C22﹕6n-3)/二十碳五烯酸(EPA, C20﹕5n-3)的添加比例为2/1组(DHA/EPA)。实验具体的饲料组成和营养成分如表 1所示。通过气相色谱仪(GS, HP6890, 美国)分析饲料中脂肪酸组成, 具体脂肪酸组成如表 2所示。
表 1 饲料配方及近似组成(干物质)Table 1. Formulation and proximate composition of diets (dry matter)原料
Ingredient不同脂肪酸饲料Different fatty acid diets CON PA SA OA LA ALA ARA DHA/EPA 鱼粉Fish meal1 40.00 40.00 40.00 40.00 40.00 40.00 40.00 40.00 酪蛋白Casein2 11.00 11.00 11.00 11.00 11.00 11.00 11.00 11.00 谷朊粉Vital gluten3 14.65 14.65 14.65 14.65 14.65 14.65 14.65 14.65 木薯淀粉Cassava starch 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00 微晶纤维素Microcrystalline cellulose 6.17 6.17 6.17 6.17 6.17 6.17 6.17 6.17 鱼油Fish oil4 6.18 6.18 6.18 6.18 6.18 6.18 6.18 6.18 甘油三酯Triglyceride 0.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 棕榈酸Palmitic acid 0.00 0.50 0.00 0.00 0.00 0.00 0.00 0.00 硬脂酸Stearic acid 0.00 0.00 0.50 0.00 0.00 0.00 0.00 0.00 油酸Oleic acid 0.00 0.00 0.00 0.50 0.00 0.00 0.00 0.00 亚油酸Linoleic acid 0.00 0.00 0.00 0.00 0.50 0.00 0.00 0.00 α-亚麻酸Alpha linolenic acid 0.00 0.00 0.00 0.00 0.00 0.50 0.00 0.00 花生四烯酸Arachidonic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.50 0.00 DHA/EPA=2/1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.50 大豆卵磷脂Soybean lecithin 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 维生素预混料Vitamin premix5 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 矿物质预混料Mineral premix6 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 其他Others7 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 营养成分(干物质, %) Nutrient composition (dry matter, %) 水分Moisture 3.74 3.81 3.67 3.69 3.92 3.88 3.79 3.85 粗蛋白Crude protein 51.22 51.37 51.33 51.26 51.34 51.41 51.33 51.32 粗脂肪Crude fat 11.56 11.44 11.47 11.36 11.53 11.44 11.40 11.49 灰分Ash 10.71 10.63 10.74 10.77 10.57 10.66 10.72 10.69 注: 1鱼粉 Fish meal: 粗蛋白 Crude protein, 68.87%; 粗脂肪 Crude fat, 7.93%; 2酪蛋白 Casein: 粗蛋白 Crude protein, 92.13%; 粗脂肪 Crude fat, 0.38%; 3谷朊粉 Vital gluten: 粗蛋白 Crude protein, 84.05%; 粗脂肪 Crude fat, 0.72%; 4鱼油 Fish oil: 饱和脂肪酸 Saturated fatty acids (SFA), 25.39%; 单不饱和脂肪酸 Monounsaturated fatty acids (MUFA), 20.58%; n-6多不饱和脂肪酸 n-6 Polyunsaturated fatty acids (n-6PUFA), 6.82%; n-3多不饱和脂肪酸 n-3 Polyunsaturated fatty acids (n-3PUFA), 34.45%; 5维生素预混料 Vitamin premix (mg/kg): α-生育酚 α-Tocopherol (50%), 240; 核黄素 Vitamin B2 (80%), 45; 维生素B12 Vitamin B12 (1%), 10; 硫胺素 Vitamin B1(98%), 25; 泛酸 Vitamin B5 (98%), 60; 烟酸 Vitamin B3 (99%), 200; 叶酸 Folate (98%), 20; 维生素D3 Vitamin D3 (1.25%), 5; 维生素K3 Vitamin K3 (51%), 10; 生物素 Biotin (2%), 60; 盐酸吡哆醇 Pyridoxine hydrochloride (99%), 20; 醋酸维生素A Vitamin A acetate (15%), 32; 肌糖 Inosine (98%), 800; 维生素C Vitamin C (35%), 2000; 稻壳粉 Rice husk meal (100%), 6470; 抗氧化剂 Antioxidant (100%), 3; 6矿物质预混料 Mineral premix (mg/kg): 亚硒酸钠 Na2SeO3 (1%), 20; 硫酸锰 MnSO4·H2O (31.80%), 45; 硫酸铜 CuSO4·5H2O (25%), 10; 硫酸锌 ZnSO4·H2O (34.50%), 50; 氯化钴 CoCl2·6H2O (1%), 50; 硫酸镁 MgSO4·7H2O (15%), 1200; 碘化钙 Cal2 (1%), 60; 硫酸铁 FeSO4·H2O (30%), 80; 沸石粉 Zeolite, 3485; 7其他 Others (%): 氯化胆碱 Choline chloride (99%), 0.25; 磷酸二氢钙 Calcium dihydrogen phosphate, 0.5; 丙酸钙 Calcium propionate, 0.1; 乙氧基喹啉 Ethoxyquinoline, 0.05; 诱食剂 Food attractant (甜菜碱﹕丙酸噻亭﹕甘氨酸﹕丙氨酸﹕5-磷酸肌苷=4﹕2﹕2﹕1﹕1) (Betaine﹕Thietine propionate﹕Glycine﹕Alanine﹕Inosine 5-phosphate=4﹕2﹕2﹕1﹕1), 1; 黏合剂(海藻酸钠) Binding agent (sodium alginate), 0.5; 三氧化二钇 Y2O3, 0.1 表 2 试验饲料脂肪酸组成Table 2. Fatty acid composition of test diet脂肪酸
Fatty acid不同脂肪酸饲料Different fatty acid diets CON PA SA OA LA ALA ARA DHA/EPA C14﹕0 4.92 4.80 4.43 4.87 4.58 4.40 4.19 4.17 C16﹕0 22.43 24.49 21.12 21.40 21.48 20.88 21.07 21.25 C18﹕0 5.69 5.10 7.50 4.82 5.59 5.26 5.22 5.67 ∑SFA1 33.04 34.39 33.05 31.09 31.65 30.54 30.48 31.09 C16﹕1n-9 4.39 4.56 4.27 4.77 4.39 4.31 3.98 4.28 C18﹕1n-9 15.27 15.12 15.13 18.68 15.09 15.67 14.56 15.73 C20﹕1n-9 0.26 0.24 0.27 0.24 0.30 0.27 0.32 0.30 ∑MUFA2 19.92 19.92 19.67 23.69 19.78 20.25 18.86 20.31 C18﹕2n-6 9.57 9.88 9.48 10.25 13.31 10.83 9.96 10.57 C20﹕4n-6 0.53 0.55 0.58 0.41 0.72 0.60 2.51 0.60 ∑n-6PUFA3 10.10 10.43 10.06 10.66 14.03 11.43 12.47 11.17 C18﹕3n-3 3.29 3.48 3.77 3.60 4.03 5.55 3.35 3.85 C20﹕5n-3 4.17 3.86 3.91 3.59 4.16 4.00 3.93 5.11 C22﹕6n-3 6.36 5.98 6.09 5.71 5.52 6.26 6.03 8.32 ∑n-3PUFA4 13.82 13.32 13.77 12.9 13.71 15.81 13.31 17.28 注: 1SFA: 饱和脂肪酸Saturated fatty acids; 2MUFA: 单不饱和脂肪酸Monounsaturated fatty acids; 3n-6 PUFA: n-6系列多不饱和脂肪酸 n-6 Polyunsaturated fatty acids; 4n-3 PUFA: n-3系列多不饱和脂肪酸 n-3 Polyunsaturated fatty acids 在试验饲料制作时, 所有的原料均经过安装80目筛网的粉碎机粉碎后, 按照配方由少到多, 逐级混匀。而后加入大豆卵磷脂和鱼油充分混匀后过60目的筛网, 揉搓均匀后, 将氯化胆碱溶入水中, 添加到饲料里。用饲料机制作成直径为3 mm的颗粒饲料, 放入温度为55℃的烘箱中, 烘24h后放入–20℃的冰箱中保存使用。
1.2 养殖试验
大菱鲆幼鱼自山东省烟台市国信东方循环水养殖科技有限公司购买, 养殖试验在山东烟台国信东方海水鱼营养与饲料联合研究中心循环水养殖系统中进行。将所购置的所有大菱鲆放入循环水系统, 进行为期2周的暂养驯化, 暂养期间投喂8种饲料的混合料, 而后挑选出体型规格均一的健康大菱鲆幼鱼(8.00±0.20) g, 随机分配到各养殖桶(250 L)中, 每个处理组设置3个重复桶, 每桶放置40尾鱼。在试验期间, 循环系统水温为(18±0.5)℃, 水中溶氧为8 mg/L, 循环水pH为7.5—8, 盐度为盐度29‰—31‰, 氨氮<0.1 mg/L, 亚盐<0.01 mg/L。养殖试验为期8周, 每天固定投喂2次(7:00和19:00), 每次投喂时按照少量多次进行投喂, 至大菱鲆表观饱食, 投喂1h后换水5%。
1.3 样品采集
在养殖结束后, 将大菱鲆进行饥饿24h, 而后捞取每个桶的所有鱼进行称重并记录数量和总重, 用于生长和存活率的指标计算, 而后每个桶随机取4尾鱼保存于–20℃冰箱用于全鱼体组成测定分析。在饥饿48h后, 每个桶随机取2尾鱼用丁香酚(1﹕10000)麻醉后测量体重、体长, 并解剖称量内脏团和肝脏重量, 而后取血浆、肝脏、中后肠放入1.5 mL离心管中用液氮快速冷冻后并转移到–80℃的冰箱保存方便后续的分析(血浆为所取尾静脉血, 抽出的血置于肝素钠抗凝管中, 放于冰上暂时保存, 待取样结束后用离心机(转速5000×g)离心10min, 吸取上层血清后分装, 置–80℃冰箱中保存待用)。
1.4 饲料和鱼体组成分析
饲料常规和鱼体组成测定方法参照AOAC[21], 将待测样品放置烘箱105℃烘干至恒定重量, 测定水分含量。用杜马斯定氮仪(D200, 海能仪器)测定样品干物质粗蛋白含量, 用脂肪测定仪(SOX606, 海能仪器)测定样品干物质粗脂肪含量, 在马弗炉(SX470, 永光明医疗仪器)中550℃高温充分灼烧16h测定粗灰分含量。
1.5 肝脏和血清酶活力的测定
甘油三酯(TG, 货号A10-1-1)、总胆固醇(T-CHO, 货号A111-1-1)、低密度脂蛋白(LDL-C, 货号A1113-1-1)、高密度脂蛋白(HDL-C, 货号A112-1-1)、总抗氧化能力(T-AOC, 货号A015-2-1)、丙二醛(MDA, 货号A003-1-2)、过氧化氢酶(CAT, 货号A007-1-1)、超氧化物歧化酶(SOD, 货号A001-1-2)均采用南京建成生物公司试剂盒测定, 具体操作步骤严格按照试剂盒所提供的说明书上的要求操作。
1.6 RNA的提取和实时荧光定量PCR(qRT-PCR)
肝脏RNA的提取按照碧云天生物公司的RNAeasyTM动物RNA抽提试剂盒(离心柱式)里所提供的说明书操作步骤操作, 提取出肝脏中的RNA, 使用NanoDrop 2000分光光度计(Thermo Fisher Scientific, 美国)而后检测RNA纯度, 同时使用1.2%琼脂糖胶检测提取RNA的质量。合格的RNA用DEPC水稀释成同一浓度, 用PrimeScriptTM RT试剂盒(TaKaRa, 日本)将RNA反转录成cDNA。使用SYBR® Premix Ex TaqTM(TaKaRa, 日本)试剂和qRT-PCR定量仪(CFX96TM Real-Time System, BIO-RAD, 美国)进行定量逆转录聚合酶链式反应检测。内参基因为β-actin, 并采用2−∆∆Ct方法计算各目的基因的相对表达量。qRT-PCR的具体引物序列见表 3。
表 3 实时荧光定量 PCR 引物序列Table 3. Primer sequences used for qRT-PCR基因Gene 引物Primer (5′—3′) FAS GGCAACAACACGGATGGATAC
CTCGCTTTGATTGACAGAACACPPARγ AAGTGACGGAGTTCGCCAAGA
GTTCATCAGAGGTGCCATCASREBP1 GCCATTGACTACATCCGTTAC
CATCAGCCTGTCCATCTACTTCPPARα CGATCAGGTGACCCTGTTAA
TGGAACTTGGGCTCCATCACOX1 AGTCCTCGCCCAGCTTTACT
GGCTTCACATAGGTTCCGTCTTLR2 AGGAGCCAAGGGAGACCGAT
GGCGCTCATGATGTTGTCCTLR8 ACAGATCCTTGAACTCCCCG
TCCAATCCCTCTCCTCCAGATLR9 AAGGCTCTGAGGGGAAAGAC
TTCTTCACAGAGCTGAGGGGMyD88 CCCAATGGTAGCCCTGAGAT
CATCTCGGTCGAACACACACNF-κB p65 ATGCCTTTGAGGACCTTTT
GTGTTCTGGGATGCTGTGTIL-1β TACCTGTCGTGCCAACAGGAA
TGATGTACCAGTTGGGGAAIL-8 GACAGAGAGCAAACCCATC
CCAGTCAAGTACATTCAAGTNF-α CCCTTATCATTATGGCCCTT
TCCGAGTACCGCCATATCCTTGF-β CTGCAGGACTGGCTCAAAGG
CATGGTCAGGATGTATGGTGGTβ-actin GTAGGTGATGAAGCCCAGAGCA
CTGGGTCATCTTCTCCCTGT注: FAS. 脂肪酸合成酶, KC189927; PPARγ. 过氧化物酶体增殖物激活受体γ, KC189932; SREBP1. 固醇调节元件结合蛋白1, MH174964.1; PPARα. 过氧化物酶体增殖物激活受体α, XM035614759.2; ACOX1. 酰基辅酶A氧化酶1, KC189925; TLR2. toll样受体2, KU746963.1; TLR8. toll样受体8, KX708702.1; TLR9. toll样受体9, KU746969.1; MyD88: 髓样分化因子88, KP985236; NF-κB p65. 核因子-κB p65, MF370855; IL-1β. 白细胞介素1β, AJ295836.2; IL-8. 白细胞介素8, XM035638412.1; TNF-α. 肿瘤坏死因子α, FJ654645; TGF-β. 转化生长因子β, KU238187.1; β-actin. 内参基因, EU686692.1Note: FAS. fatty acid synthetase, KC189927; PPARγ. peroxisome proliferators activated receptor γ, KC189932; SREBP1. sterol-regulatory element-binding protein 1, MH174964.1; PPARα. peroxisome proliferators activated receptor α, XM035614759.2; ACOX1. acyl-CoA oxidase 1, KC189925; TLR2. toll-like receptor 2, KU746963.1; TLR8. toll-like receptor 8, KX708702.1; TLR9. toll-like receptor 9, KU746969.1; MyD88. myeloid differentiation factor 88, KP985236; NF-κB p65. nuclear factor-κB p65, MF370855; IL-1β: interleukin-1β, AJ295836.2; IL-8. interleukin-8, XM035638412.1; TNF-α. tumor necrosis factor-α, FJ654645; TGF-β. transforming growth factor-β, KU238187.1; β-actin. beta-actin, EU686692.1 1.7 生长指标公式
增重率(Weight gain rate, WGR, %)=[鱼末重(g)–鱼初重(g)]/鱼初重(g)×100;
饲料系数(Feed conversion ratio, FCR)=摄食量(g)/[鱼末重(g)–鱼初重(g)];
特定生长率(Specific growth rate, SGR, %/d)=[ln鱼末重(g)–ln鱼初重(g)]/养殖天数(d)×100;
摄食率(Feed intake, FI, %BW/d)=摄食量(g)/[(鱼末重(g)+鱼初重(g))/2]/养殖天数(d)×100;
蛋白质效率(Protein efficiency rate, PER)=[鱼末重(g)–鱼初重(g)]/蛋白质摄入量(g);
存活率(Survival rate, SR, %)=最终存活尾数(尾)/初始尾数(尾)×100;
1.8 数据分析
实验数据用平均值±标准误(mean±SE)来表示, 所得数据经过SPSS25.0软件分析进行单因素方差分析(ANOVA), 若各处理间存在显著差异, 则采用Tukey’s法进行多重比较, 以P<0.05表示差异显著。
2. 结果
2.1 饲料中添加不同脂肪酸对大菱鲆幼鱼生长性能的影响
如表 4所示, 在饲料中添加不同脂肪酸, 大菱鲆幼鱼的存活率(SR)和摄食率(FI)没有显著差异(P>0.05)。随着添加脂肪酸碳链长度和不饱和度的增加, 增重率(WGR)和特定生长率(SGR)呈现上升的趋势, ARA组和DHA/EPA组的WGR、SGR最高。DHA/EPA组的蛋白质效率(PER)显著高于PA组(P<0.05)。而DHA/EPA组的饲料系数(FCR)显著低于PA、OA和 LA组(P<0.05), 与其他组没有显著差异(P>0.05)。
表 4 饲料中添加不同脂肪酸对大菱鲆幼鱼生长指标的影响Table 4. Effects of different fatty acids on growth of juvenile turbot处理组
Treatment生长指标Growth index 增重率WGR (%) 饲料系数FCR 特定生长率SGR (%/d) 摄食率FI (%BW/d) 蛋白质效率PER 存活率SR (%) CON 569.27±9.50ab 0.72±0.00ab 3.40±0.03ab 1.89±0.01 2.80±0.01ab 100±0.00 PA 551.57±7.56ab 0.73±0.00b 3.35±0.02ab 1.92±0.01 2.73±0.02a 100±0.00 SA 554.80±5.49ab 0.72±0.00ab 3.35±0.01ab 1.90±0.01 2.77±0.01ab 100±0.00 OA 565.59±7.07ab 0.73±0.00b 3.39±0.02ab 1.91±0.01 2.76±0.02ab 100±0.00 LA 536.37±10.47a 0.73±0.00b 3.30±0.03a 1.89±0.01 2.75±0.02ab 100±0.00 ALA 562.16±9.37ab 0.71±0.00ab 3.38±0.03ab 1.87±0.01 2.82±0.01ab 100±0.00 ARA 585.88±7.13b 0.71±0.01ab 3.44±0.02b 1.89±0.01 2.82±0.02ab 100±0.00 DHA/EPA 580.59±8.57b 0.70±0.01a 3.42±0.02b 1.86±0.02 2.86±0.05b 100±0.00 注: 同一列内不同上标字母表示存在显著差异(P<0.05); 下同Note: Values in the same column labeled with different superscript letters are significantly different (P<0.05). The same applies below 2.2 饲料中添加不同脂肪酸对大菱鲆幼鱼体组成的影响
通过对大菱鲆幼鱼体组成成分分析(表 5): 在饲料中添加不同脂肪酸, 大菱鲆幼鱼的水分和灰分各组间均无显著差异(P>0.05)。随着脂肪酸碳链长度和不饱和度的增加, 鱼体的粗蛋白含量有所增加, 粗脂肪含量有所降低。在所有处理组中, DHA/EPA组粗蛋白含量最高, 且显著高于CON、PA、SA、OA、LA和ALA组(P<0.05), PA和SA组粗蛋白含量显著低于ALA、ARA和DHA/EPA组(P<0.05), ARA组粗蛋白含量显著高于CON、PA和SA组(P<0.05), 其他各组间差异不显著(P>0.05); 随着脂肪酸碳链长度和不饱和度的增加, 各处理组鱼体粗脂肪含量逐渐降低, DHA/EPA和ARA组粗脂肪含量显著低于CON、PA和SA组(P<0.05), 其他各组间粗脂肪含量差异不显著(P>0.05)。
表 5 饲料中添加不同脂肪酸对大菱鲆体组成的影响Table 5. Effects of different fatty acids on body composition of turbot处理组
Treatment体组成 Body composition (干物质) 水分
Moisture
(%)粗蛋白
Crude protein
(%)粗脂肪
Crude lipid
(%)灰分
Crude ash
(%)CON 76.24±0.57 64.75±0.47ab 22.89±0.17c 12.71±0.44 PA 76.32±0.38 63.96±0.38a 22.52±0.54bc 12.41±0.30 SA 76.46±0.47 64.11±0.41a 22.01±0.26bc 12.77±0.42 OA 76.52±0.20 65.55±0.55abc 21.07±1.15ab 12.87±0.52 LA 76.21±0.52 64.96±0.66abc 21.43±0.44abc 12.49±0.15 ALA 76.72±0.62 65.99±0.79bc 21.64±0.59abc 12.47±0.27 ARA 76.76±0.61 66.64±0.84cd 20.03±0.40a 12.64±0.39 DHA/EPA 76.94±0.55 68.02±0.83d 19.96±0.80a 12.57±0.25 2.3 饲料中添加不同脂肪酸对大菱鲆幼鱼血浆生化指标的影响
通过对大菱鲆幼鱼血浆生化指标分析(表 6): 在饲料中添加不同脂肪酸, 大菱鲆幼鱼的血浆总胆固醇(T-CHO)含量随着添加脂肪酸的链长和不饱和度的增加显著降低(P<0.05), ALA、ARA和DHA/EPA组T-CHO含量显著低于PA组和SA组(P<0.05), 其中DHA/EPA组的T-CHO含量最低, 显著低于CON、PA、SA和OA组(P<0.05), PA组T-CHO含量最高。DHA/EPA组甘油三酯(TG)含量显著低于LA、PA、SA和CON组(P<0.05), CON、PA和SA组TG含量显著高于ALA和DHA/EPA组(P<0.05), 其余各组间TG含量没有显著差异(P>0.05)。随着饲料脂肪酸链长和不饱和度的增加, 血浆中的低密度脂蛋白(LDL-C)显著降低(P<0.05), DHA/EPA组血浆低密度脂蛋白含量最低, 而PA组含量最高。血浆中的高密度脂蛋白(HDL-C)含量在DHA/EPA组最高, 而在PA组最低, 并且DHA/EPA、ALA和ARA组显著高于CON、PA、SA、OA和LA组(P<0.05), 但DHA/EPA与ALA和ARA组没有显著性差异(P>0.05)。OA和LA组显著高于PA组(P<0.05), 但与CON和SA组没有显著差异(P>0.05)。
表 6 饲料中添加不同脂肪酸对大菱鲆血浆生化指标的影响Table 6. Effects of different fatty acids on plasma biochemical indices of turbot处理组
Treatment血浆生化指标 Plasma biochemical index 甘油三酯
TG
(mmol/L)总胆固醇
T-CHO
(mmol/L)低密度脂
蛋白LDL-C
(mmol/L)高密度脂
蛋白HDL-C
(mmol/L)CON 3.02±0.25cd 1.95±0.22bcd 0.83±0.04d 2.02±0.10ab PA 3.58±0.08d 2.42±0.31d 0.96±0.04e 1.82±0.06a SA 3.66±0.21d 2.17±0.31cd 0.90±0.02e 1.88±0.05ab OA 2.63±0.25abc 1.95±0.40bcd 0.80±0.05d 2.05±0.09b LA 2.81±0.18bcd 1.60±0.40abc 0.71±0.03c 2.27±0.14b ALA 2.11±0.24ab 1.52±0.10ab 0.66±0.03c 2.44±0.06cd ARA 2.28±0.32abc 1.47±0.33ab 0.57±0.06b 2.43±0.04cd DHA/EPA 1.86±0.16a 1.19±0.31a 0.47±0.04a 2.54±0.03d 2.4 饲料中添加不同脂肪酸对大菱鲆幼鱼脂代谢相关基因表达量的影响
饲料中添加不同脂肪酸对大菱鲆幼鱼脂代谢相关基因表达量的结果如图 1所示: ALA和DHA/EPA组脂代谢相关基因FAS的表达量显著低于PA、SA和OA组(P<0.05; 图 1A), 但与ARA和CON组差异不显著(P>0.05); ARA和DHA/EPA组PPARγ的基因表达量显著低于PA、SA组(P<0.05), 但与LA、ALA和CON组差异不显著(P>0.05; 图 1B); ALA和DHA/EPA组SREBP1的表达量显著低于PA、SA、OA和LA组(P<0.05), 但与ARA和CON组差异不显著(P>0.05; 图 1C); DHA/EPA组PPARα基因表达量最高, 且显著高于其他各组(P<0.05; 图 1D); ALA和DHA/EPA组ACOX1基因表达量显著高于CON、PA、SA、OA和LA组(P<0.05; 图 1E)。
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图 1 饲料中添加不同脂肪酸对大菱鲆幼鱼脂代谢相关基因的影响CON. 对照组; PA. 棕榈酸组; SA. 硬脂酸组; OA. 油酸组; LA. 亚油酸组; ALA. 亚麻酸组; ARA. 花生四烯酸组; DHA/EPA.二十二碳六烯酸/二十碳五烯酸组。A—C. 饲料中添加不同脂肪酸对大菱鲆幼鱼肝脏脂肪合成相关基因表达影响; D—E. 饲料中添加不同脂肪酸对大菱鲆幼鱼肝脏脂肪分解相关基因表达影响; 不同字母代表组间差异显著(P<0.05)Figure 1. Effects of different fatty acids on fat metabolism-related genes of juvenile turbotCON. control group; PA. palmitic acid group; SA. stearic acid group; OA. oleic acid group; LA. linoleic acid group; ALA. linolenic acid group; ARA. arachidonic acid group; DHA/EPA. docosahexaenoic acid/eicosapentaenoic acid group. A—C. effects of dietary supplementation with different fatty acids on the expression of genes related to liver fat synthesis of juvenile turbot; D—E. effects of dietary supplementation with different fatty acids on the expression of genes related to liver fat decomposition of juvenile turbot; different letters represent significant differences between groups (P<0.05)2.5 饲料中添加不同脂肪酸对大菱鲆幼鱼肝脏抗氧化应激指标的影响
如表 7所示: 随着饲料中添加脂肪酸碳链长度和不饱和度的增加, 大菱鲆幼鱼的总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)酶活性均呈现增加的趋势, 而丙二醛(SOD)酶活性呈现下降的趋势。就总抗氧化能力而言, DHA/EPA组T-AOC最高, 且显著高于CON、PA、SA、OA和LA组(P<0.05), 而PA组T-AOC最低, 显著低于其他各处理组(P<0.05), ARA组T-AOC显著高于PA、SA和LA组, 其他处理组之间差异不显著(P>0.05); DHA/EPA组的SOD酶活性最高, 显著高于CON、PA、SA、OA、LA和ALA处理组(P<0.05), 但与ARA组之间差异并不显著(P>0.05); PA、SA、OA、LA和CON组之间的CAT酶活性差异并不显著(P>0.05), 但显著低于ARA和DHA/EPA组(P<0.05); PA组的MDA酶活性最高, 显著高于OA、LA、ALA、ARA和DHA/EPA组(P<0.05), DHA/EPA组与ARA组之间MDA酶活性差异不显著(P>0.05), 但显著低于其他各处理组(P<0.05)。
表 7 饲料中添加不同脂肪酸对大菱鲆幼鱼肝脏抗氧化酶活性的影响Table 7. Effects of different fatty acids on the activity of antioxidant enzymes in liver of juvenile turbot处理组
Treatment抗氧化指标Antioxidant index 总抗氧化
能力T-AOC
(mmol/
g prot)丙二醛
MDA
(nmol/
mg prot)超氧化物歧
化酶SOD
(U/mg prot)过氧化氢酶
CAT
(U/mg prot)CON 1.62±0.07bc 3.34±0.09cde 71.00±2.19bc 9.29±0.66ab PA 1.39±0.02a 3.61±0.12e 67.23±1.60ab 8.43±0.37a SA 1.58±0.03b 3.45±0.06de 65.87±2.50a 8.44±1.20a OA 1.64±0.05bc 3.19±0.18bcd 70.94±0.68bc 8.54±0.81a LA 1.59±0.04b 3.11±0.14bcd 69.72±0.64abc 9.80±0.51ab ALA 1.65±0.12bcd 3.08±0.13bc 71.48±0.90bc 10.85±0.99bc ARA 1.72±0.08cd 2.88±0.04ab 73.93±2.30cd 12.80±0.52cd DHA/EPA 1.76±0.03d 2.58±0.18a 76.99±1.81d 12.93±0.35d 2.6 饲料中添加不同脂肪酸对大菱鲆肝脏免疫相关基因表达量的影响
饲料中添加不同脂肪酸对大菱鲆幼鱼肝脏免疫相关基因表达量的结果如图 2所示: DHA/EPA组的肝脏免疫基因IL-1β表达量最低, PA组的IL-1β基因表达量最高, 并且随着添加脂肪酸链长和不饱和度的增加, IL-1β基因表达量呈现降低的趋势。饲料中添加不同脂肪酸, 各组间肝脏免疫基因IL-8表达量无显著差异(P>0.05); DHA/EPA组的MyD88基因表达量最低, 显著低于CON、PA、SA、OA和LA组(P<0.05), PA组MyD88基因表达量最高, 显著高于其他各处理组(P<0.05); 随着添加脂肪酸链长和不饱和度的增加, 大菱鲆幼鱼肝脏NF-κB p65基因表达量逐渐降低, DHA/EPA组NF-κB p65基因表达量显著低于PA、SA、OA和LA组(P<0.05), 但与其他组之间差异并不显著(P>0.05), PA组NF-κB p65基因表达量最高, PA和SA组NF-κB p65基因表达量显著高于其他各处理组(P<0.05); 随着添加脂肪酸链长和不饱和度的增加, 大菱鲆幼鱼肝脏TLR2和TLR9基因表达量逐渐降低, DHA/EPA组肝脏TLR2基因表达量最低, 而PA组TLR2基因表达量最高; ALA、ARA和DHA/EPA组TLR8基因表达量显著低于PA组(P<0.05); ALA、ARA、DHA/EPA组TLR9基因表达量显著低于CON、PA、SA和OA组(P<0.05); PA组TNF-α基因表达量最高, DHA/EPA组最低, 且显著低于其余各处理组(P<0.05); 随着脂肪酸链长和不饱和度的增加, 大菱鲆幼鱼肝脏TGF-β基因表达量呈现上升的趋势, 在PA组最低, 在DHA/EPA组最高。
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图 2 饲料中添加不同脂肪酸对大菱鲆幼鱼免疫相关基因的影响CON. 对照组; PA. 棕榈酸组; SA. 硬脂酸组; OA. 油酸组; LA. 亚油酸组; ALA. 亚麻酸组; ARA. 花生四烯酸组; DHA/EPA.二十二碳六烯酸/二十碳五烯酸组Figure 2. Effects of different fatty acids on immune-related genes of juvenile turbotCON. control group; PA. palmitic acid group; SA. stearic acid group; OA. oleic acid group; LA. linoleic acid group; ALA. linolenic acid group; ARA. arachidonic acid group; DHA/EPA. docosahexaenoic acid/eicosapentaenoic acid group3. 讨论
3.1 饲料中添加不同脂肪酸对大菱鲆幼鱼生长及体组成的影响
脂质主要是由脂肪酸组成的, 不同脂肪酸对机体有着不同的生理作用, 尤其是一些长链多不饱和脂肪酸, 例如DHA、EPA等, 作为一些海水鱼的必需脂肪酸, 对海水鱼的生长有着促进作用[22]。本研究通过在饲料中添加不同脂肪酸发现, 随着添加脂肪酸的链长和不饱和度的增加, 大菱鲆幼鱼的存活率和摄食率没有显著影响, 但大菱鲆幼鱼的增重率和特定生长率总体上呈现上升的趋势, 这说明长链多不饱和脂肪酸作为海水鱼的必需脂肪酸, 自身合成能力不足, 需要从饮食中摄取, 并且对鱼类有着促进生长的作用[23]。从生长结果来看, DHA/EPA组和ARA组高于其他处理组, 这说明DHA、EPA和ARA作为海水鱼的必需脂肪酸, 对鱼体的生长有着促进作用[24, 25], 当摄入一定量的必需脂肪酸时, 可以提高大菱鲆幼鱼的增重率和特定生长率。这与花鲈(Lateolabrax maculatus)、欧洲鲈(Dicentrarchus labrax)和鳕(Macullochella peelii)结果相一致[26—28]。DHA/EPA组的饲料系数最低, 而蛋白质效率最高, 这可能说明在饲料中添加必需脂肪酸, 大菱鲆幼鱼通过促进饲料中蛋白消化吸收, 进而被自身生长所利用来提高蛋白质效率、降低饲料系数, 从而促进鱼体生长。
在本研究中, 不同脂肪酸的处理组并没有对大菱鲆幼鱼鱼体的水分和灰分造成显著差异, 但随着脂肪酸链长和不饱和度的增加, 鱼体的粗蛋白含量在显著增加, 而鱼体的粗脂肪含量却显著降低, DHA/EPA组和ARA组显著增加了鱼体的粗蛋白含量, 并且显著降低了鱼体的粗脂肪含量, 这可能是由于脂肪酸链长和不饱和度决定了脂肪酸的利用和分解速率, 随着脂肪酸链长和不饱和度的增加, 通过激活脂肪合成和分解基因来控制脂肪的合成和分解, 进而控制鱼体的脂肪含量[29]。而鱼体的粗蛋白含量也与脂肪酸的链长和不饱和度有关, 可能是不同脂肪酸从某种程度上可以激活鱼体蛋白合成, 尤其是一些n-3长链多不饱和脂肪酸可以通过调节蛋白合成相关信号通路和基因表达, 促进蛋白质的合成和转运[30, 31]。
3.2 饲料中添加不同脂肪酸对大菱鲆幼鱼血浆生化指标的影响
血浆脂蛋白可以分为乳糜微粒(CM)、极低密度脂蛋白(VLDL)、低密度脂蛋白(LDL)和高密度脂蛋白(HDL), HDL的主要作用是将体内其他地方多余的胆固醇转运到肝脏中, 进而在肝脏中进一步代谢分解, 因此HDL具有胆固醇清除剂的作用, 也常称之为“优质蛋白”。而LDL的主要作用是将肝脏中的胆固醇转运到脂肪、肌肉及肾上腺等肝外组织中, 且LDL常常与动脉粥样硬化有着很强的关联性, 尤其是当血管壁受到损伤时, LDL会促进胆固醇在损伤的血管壁沉积。在本研究中, 血浆中的甘油三酯(TG)和总胆固醇(T-CHO)含量随着添加脂肪酸链长和不饱和度的增加而显著降低, 血浆HDL含量显著升高, 血浆LDL含量显著下降, 尤其是ALA、ARA和DHA/EPA组的HDL含量显著高于其他各组, 这与一些哺乳动物上的研究结果相一致, n-3长链多不饱和脂肪酸能够显著降低血浆TG和T-CHO含量而显著提高血浆HDL含量[32]。血浆T-CHO含量也与血浆LDL含量相关, 有研究表明, PUFA可以通过降低血浆LDL含量来降低T-CHO含量[33], 这与本实验的研究结果相一致。并且在大西洋鲑(Salmo salar L.)的研究中有着相同的结果, 用植物油替代鱼油, 植物油组中的大西洋鲑血浆TG含量和HDL含量均显著低于鱼油组[34]。
3.3 饲料中添加不同脂肪酸对肝脏脂代谢相关基因的影响
作为脂质代谢主要核受体的过氧化物酶体增殖物激活受体(PPARs), 在集体的脂质转运、分解和合成中起着至关重要的作用, 能够精准地调控脂质代谢[35, 36]。PPARα作为PPARs家庭成员中的一员, 调控脂质的分解代谢主要通过脂肪的β氧化和调节编码过氧化物酶体酶的靶基因来促进脂肪的分解[37, 38]。ACOX1作为PPARα的下游的脂肪酸氧化基因, 也受其调控[39, 40]。FAS是脂肪合成的相关基因, PPARγ通过降低脂肪分解的速度, 从而促进脂肪合成, 同时也能够促进FAS基因的表达, 而SREBP1与PPARγ共同促进脂肪合成[41－43]。本研究通过在饲料中添加不同脂肪酸发现, DHA/EPA和ALA作为n-3长链多不饱和脂肪酸, 能够显著降低脂肪的合成基因, 而显著提高脂肪的分解基因, 这与一些其他报道的结果一致[44, 45]。
3.4 饲料中添加不同脂肪酸对肝脏抗氧化指标的影响
当活性氧自由基(ROS)过多生成时, 会损伤机体的蛋白质分子、细胞膜和DNA分子等, 从而使生物体的生长发育受到一定影响。丙二醛是活性氧自由基与脂质发生过氧化反应而生成的产物, 具有强烈的细胞毒性。因此, MDA含量可以作为一项用于评估机体遭受氧化损伤的程度的重要指标[46－48]。而在本实验中, 饲料中添加0.5%的ARA和DHA/EPA为2/1的脂肪酸显著提高了肝脏过氧化氢酶含量, 显著降低了丙二醛含量, 这与一些其他研究结果相一致[49, 50]。并且DHA/EPA组的总抗氧化能力和超氧化物歧化酶活性均最高, 这与我们先前得出的结论相一致, 一定量的必需脂肪酸可以提高鱼体的抗氧化能力。饲料中添加0.5%的PA可以降低鱼体的抗氧化能力, 而在肉鸡饲粮中添加棕榈油可以显著降低丙二醛含量, 但对总抗氧化能力无显著差异, 这与我们的研究结果相反[51],可能是不同物种之间存在差异所导致的, 棕榈酸作为一种饱和脂肪酸, 对机体肝脏抗氧化能力仍存在一些争议。
3.5 饲料中添加不同脂肪酸对肝脏免疫相关基因的影响
肿瘤坏死因子α (TNF-α)是一种具有调节炎症反应的重要细胞因子, 对于维持机体免疫稳态起到有效的作用[52]。而白细胞介素-1β (IL-1β)通过增强吞噬细胞的抗菌特性, 启动辅助性T淋巴细胞1 (Th1)和辅助性T淋巴细胞17 (Th17)适应性免疫反应, 有助于宿主防御感染, IL-1β和IL-8是免疫调节中两种重要的促炎因子[53]。有研究表明, 摄入富含n-3 LC-PUFA的鱼油可以显著降低白细胞介素-1 (IL-1β)和肿瘤坏死因子α (TNF-α)的表达, 从而增强机体的免疫功能[54]。同样, 本实验的研究结果也显示, ALA、ARA和DHA/EPA组显著降低了TNF-α和IL-1β的基因表达, 这表明LC-PUFA能够有效提高大菱鲆幼鱼的非特异性免疫。
Toll样受体(TLRs)家族在微生物的先天免疫识别中起着重要作用, 可识别微生物成分的特定模式, 称为病原体相关分子模式(PAMP)。TLR 依赖性识别PAMP导致先天免疫系统的激活, 进而导致抗原特异性适应性免疫的激活。TLR介导的信号通路包括MyD88依赖性通路和TRIF依赖性通路, 两者都诱导基因表达[55]。MyD88通过诱导核因子-κB (NF-κB)的表达, 并最终激活机体的免疫应答从而抵御病原体的入侵[56]。在本研究中, TLR2、TLR8、TLR9、MyD88和NF-κB p65基因表达量随着脂肪酸的链长和不饱和度的增加呈现降低的趋势, PA、SA组的基因表达量最高, 而有研究表明, 饱和脂肪酸(SFA)能够激活TLR介导的促炎信号通路, 且PUFA能够抑制促炎信号通路的表达, 这和我们的研究结果一致[57]。转化生长因子(TGF-β)是一种具有免疫调控的重要细胞因子, 能够对炎症的发生产生抑制作用。在本研究中LA、ALA、ARA和DHA/EPA组均显著提高了TGF-β基因的表达量, 这与先前的研究, PUFA能够促进抗炎因子TGF-β表达结果一致[58]。有研究证明, NF-κB的激活可以抑制抗炎细胞因子TGF-β的表达, 而脂肪酸诱导的炎症反应可能是通过激活TLRs/NF-κB信号通路来实现的[59]。
4. 结论
通过在饲料中添加不同脂肪酸, 探究大菱鲆在生长性能、肝脏脂代谢和非特异性免疫指标, 从而得出在饲料中添加0.5%的不同脂肪酸, 能够从不同方面影响大菱鲆幼鱼的生长和代谢。随着添加脂肪酸链长和不饱和度的增加, 大菱鲆的生长呈现出上升的趋势。肝脏脂代谢也受添加脂肪酸的影响, 长链多不饱和脂肪酸显著降低脂肪合成基因的表达, 而升高了脂肪分解基因的表达, 饱和脂肪酸则相反。随着脂肪酸的链长和不饱和度的增加显著提高了大菱鲆幼鱼的非特异性免疫能力, 本研究结果为饲料中添加不同脂肪酸提供了一定的数据依据, 为大菱鲆配合饲料的开发提供了新的方向。
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图 1 饲料中添加不同脂肪酸对大菱鲆幼鱼脂代谢相关基因的影响
CON. 对照组; PA. 棕榈酸组; SA. 硬脂酸组; OA. 油酸组; LA. 亚油酸组; ALA. 亚麻酸组; ARA. 花生四烯酸组; DHA/EPA.二十二碳六烯酸/二十碳五烯酸组。A—C. 饲料中添加不同脂肪酸对大菱鲆幼鱼肝脏脂肪合成相关基因表达影响; D—E. 饲料中添加不同脂肪酸对大菱鲆幼鱼肝脏脂肪分解相关基因表达影响; 不同字母代表组间差异显著(P<0.05)
Figure 1. Effects of different fatty acids on fat metabolism-related genes of juvenile turbot
CON. control group; PA. palmitic acid group; SA. stearic acid group; OA. oleic acid group; LA. linoleic acid group; ALA. linolenic acid group; ARA. arachidonic acid group; DHA/EPA. docosahexaenoic acid/eicosapentaenoic acid group. A—C. effects of dietary supplementation with different fatty acids on the expression of genes related to liver fat synthesis of juvenile turbot; D—E. effects of dietary supplementation with different fatty acids on the expression of genes related to liver fat decomposition of juvenile turbot; different letters represent significant differences between groups (P<0.05)
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图 2 饲料中添加不同脂肪酸对大菱鲆幼鱼免疫相关基因的影响
CON. 对照组; PA. 棕榈酸组; SA. 硬脂酸组; OA. 油酸组; LA. 亚油酸组; ALA. 亚麻酸组; ARA. 花生四烯酸组; DHA/EPA.二十二碳六烯酸/二十碳五烯酸组
Figure 2. Effects of different fatty acids on immune-related genes of juvenile turbot
CON. control group; PA. palmitic acid group; SA. stearic acid group; OA. oleic acid group; LA. linoleic acid group; ALA. linolenic acid group; ARA. arachidonic acid group; DHA/EPA. docosahexaenoic acid/eicosapentaenoic acid group
表 1 饲料配方及近似组成(干物质)
Table 1 Formulation and proximate composition of diets (dry matter)
原料
Ingredient不同脂肪酸饲料Different fatty acid diets CON PA SA OA LA ALA ARA DHA/EPA 鱼粉Fish meal1 40.00 40.00 40.00 40.00 40.00 40.00 40.00 40.00 酪蛋白Casein2 11.00 11.00 11.00 11.00 11.00 11.00 11.00 11.00 谷朊粉Vital gluten3 14.65 14.65 14.65 14.65 14.65 14.65 14.65 14.65 木薯淀粉Cassava starch 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00 微晶纤维素Microcrystalline cellulose 6.17 6.17 6.17 6.17 6.17 6.17 6.17 6.17 鱼油Fish oil4 6.18 6.18 6.18 6.18 6.18 6.18 6.18 6.18 甘油三酯Triglyceride 0.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 棕榈酸Palmitic acid 0.00 0.50 0.00 0.00 0.00 0.00 0.00 0.00 硬脂酸Stearic acid 0.00 0.00 0.50 0.00 0.00 0.00 0.00 0.00 油酸Oleic acid 0.00 0.00 0.00 0.50 0.00 0.00 0.00 0.00 亚油酸Linoleic acid 0.00 0.00 0.00 0.00 0.50 0.00 0.00 0.00 α-亚麻酸Alpha linolenic acid 0.00 0.00 0.00 0.00 0.00 0.50 0.00 0.00 花生四烯酸Arachidonic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.50 0.00 DHA/EPA=2/1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.50 大豆卵磷脂Soybean lecithin 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 维生素预混料Vitamin premix5 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 矿物质预混料Mineral premix6 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 其他Others7 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 营养成分(干物质, %) Nutrient composition (dry matter, %) 水分Moisture 3.74 3.81 3.67 3.69 3.92 3.88 3.79 3.85 粗蛋白Crude protein 51.22 51.37 51.33 51.26 51.34 51.41 51.33 51.32 粗脂肪Crude fat 11.56 11.44 11.47 11.36 11.53 11.44 11.40 11.49 灰分Ash 10.71 10.63 10.74 10.77 10.57 10.66 10.72 10.69 注: 1鱼粉 Fish meal: 粗蛋白 Crude protein, 68.87%; 粗脂肪 Crude fat, 7.93%; 2酪蛋白 Casein: 粗蛋白 Crude protein, 92.13%; 粗脂肪 Crude fat, 0.38%; 3谷朊粉 Vital gluten: 粗蛋白 Crude protein, 84.05%; 粗脂肪 Crude fat, 0.72%; 4鱼油 Fish oil: 饱和脂肪酸 Saturated fatty acids (SFA), 25.39%; 单不饱和脂肪酸 Monounsaturated fatty acids (MUFA), 20.58%; n-6多不饱和脂肪酸 n-6 Polyunsaturated fatty acids (n-6PUFA), 6.82%; n-3多不饱和脂肪酸 n-3 Polyunsaturated fatty acids (n-3PUFA), 34.45%; 5维生素预混料 Vitamin premix (mg/kg): α-生育酚 α-Tocopherol (50%), 240; 核黄素 Vitamin B2 (80%), 45; 维生素B12 Vitamin B12 (1%), 10; 硫胺素 Vitamin B1(98%), 25; 泛酸 Vitamin B5 (98%), 60; 烟酸 Vitamin B3 (99%), 200; 叶酸 Folate (98%), 20; 维生素D3 Vitamin D3 (1.25%), 5; 维生素K3 Vitamin K3 (51%), 10; 生物素 Biotin (2%), 60; 盐酸吡哆醇 Pyridoxine hydrochloride (99%), 20; 醋酸维生素A Vitamin A acetate (15%), 32; 肌糖 Inosine (98%), 800; 维生素C Vitamin C (35%), 2000; 稻壳粉 Rice husk meal (100%), 6470; 抗氧化剂 Antioxidant (100%), 3; 6矿物质预混料 Mineral premix (mg/kg): 亚硒酸钠 Na2SeO3 (1%), 20; 硫酸锰 MnSO4·H2O (31.80%), 45; 硫酸铜 CuSO4·5H2O (25%), 10; 硫酸锌 ZnSO4·H2O (34.50%), 50; 氯化钴 CoCl2·6H2O (1%), 50; 硫酸镁 MgSO4·7H2O (15%), 1200; 碘化钙 Cal2 (1%), 60; 硫酸铁 FeSO4·H2O (30%), 80; 沸石粉 Zeolite, 3485; 7其他 Others (%): 氯化胆碱 Choline chloride (99%), 0.25; 磷酸二氢钙 Calcium dihydrogen phosphate, 0.5; 丙酸钙 Calcium propionate, 0.1; 乙氧基喹啉 Ethoxyquinoline, 0.05; 诱食剂 Food attractant (甜菜碱﹕丙酸噻亭﹕甘氨酸﹕丙氨酸﹕5-磷酸肌苷=4﹕2﹕2﹕1﹕1) (Betaine﹕Thietine propionate﹕Glycine﹕Alanine﹕Inosine 5-phosphate=4﹕2﹕2﹕1﹕1), 1; 黏合剂(海藻酸钠) Binding agent (sodium alginate), 0.5; 三氧化二钇 Y2O3, 0.1 
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表 2 试验饲料脂肪酸组成
Table 2 Fatty acid composition of test diet
脂肪酸
Fatty acid不同脂肪酸饲料Different fatty acid diets CON PA SA OA LA ALA ARA DHA/EPA C14﹕0 4.92 4.80 4.43 4.87 4.58 4.40 4.19 4.17 C16﹕0 22.43 24.49 21.12 21.40 21.48 20.88 21.07 21.25 C18﹕0 5.69 5.10 7.50 4.82 5.59 5.26 5.22 5.67 ∑SFA1 33.04 34.39 33.05 31.09 31.65 30.54 30.48 31.09 C16﹕1n-9 4.39 4.56 4.27 4.77 4.39 4.31 3.98 4.28 C18﹕1n-9 15.27 15.12 15.13 18.68 15.09 15.67 14.56 15.73 C20﹕1n-9 0.26 0.24 0.27 0.24 0.30 0.27 0.32 0.30 ∑MUFA2 19.92 19.92 19.67 23.69 19.78 20.25 18.86 20.31 C18﹕2n-6 9.57 9.88 9.48 10.25 13.31 10.83 9.96 10.57 C20﹕4n-6 0.53 0.55 0.58 0.41 0.72 0.60 2.51 0.60 ∑n-6PUFA3 10.10 10.43 10.06 10.66 14.03 11.43 12.47 11.17 C18﹕3n-3 3.29 3.48 3.77 3.60 4.03 5.55 3.35 3.85 C20﹕5n-3 4.17 3.86 3.91 3.59 4.16 4.00 3.93 5.11 C22﹕6n-3 6.36 5.98 6.09 5.71 5.52 6.26 6.03 8.32 ∑n-3PUFA4 13.82 13.32 13.77 12.9 13.71 15.81 13.31 17.28 注: 1SFA: 饱和脂肪酸Saturated fatty acids; 2MUFA: 单不饱和脂肪酸Monounsaturated fatty acids; 3n-6 PUFA: n-6系列多不饱和脂肪酸 n-6 Polyunsaturated fatty acids; 4n-3 PUFA: n-3系列多不饱和脂肪酸 n-3 Polyunsaturated fatty acids
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表 3 实时荧光定量 PCR 引物序列
Table 3 Primer sequences used for qRT-PCR
基因Gene 引物Primer (5′—3′) FAS GGCAACAACACGGATGGATAC
CTCGCTTTGATTGACAGAACACPPARγ AAGTGACGGAGTTCGCCAAGA
GTTCATCAGAGGTGCCATCASREBP1 GCCATTGACTACATCCGTTAC
CATCAGCCTGTCCATCTACTTCPPARα CGATCAGGTGACCCTGTTAA
TGGAACTTGGGCTCCATCACOX1 AGTCCTCGCCCAGCTTTACT
GGCTTCACATAGGTTCCGTCTTLR2 AGGAGCCAAGGGAGACCGAT
GGCGCTCATGATGTTGTCCTLR8 ACAGATCCTTGAACTCCCCG
TCCAATCCCTCTCCTCCAGATLR9 AAGGCTCTGAGGGGAAAGAC
TTCTTCACAGAGCTGAGGGGMyD88 CCCAATGGTAGCCCTGAGAT
CATCTCGGTCGAACACACACNF-κB p65 ATGCCTTTGAGGACCTTTT
GTGTTCTGGGATGCTGTGTIL-1β TACCTGTCGTGCCAACAGGAA
TGATGTACCAGTTGGGGAAIL-8 GACAGAGAGCAAACCCATC
CCAGTCAAGTACATTCAAGTNF-α CCCTTATCATTATGGCCCTT
TCCGAGTACCGCCATATCCTTGF-β CTGCAGGACTGGCTCAAAGG
CATGGTCAGGATGTATGGTGGTβ-actin GTAGGTGATGAAGCCCAGAGCA
CTGGGTCATCTTCTCCCTGT注: FAS. 脂肪酸合成酶, KC189927; PPARγ. 过氧化物酶体增殖物激活受体γ, KC189932; SREBP1. 固醇调节元件结合蛋白1, MH174964.1; PPARα. 过氧化物酶体增殖物激活受体α, XM035614759.2; ACOX1. 酰基辅酶A氧化酶1, KC189925; TLR2. toll样受体2, KU746963.1; TLR8. toll样受体8, KX708702.1; TLR9. toll样受体9, KU746969.1; MyD88: 髓样分化因子88, KP985236; NF-κB p65. 核因子-κB p65, MF370855; IL-1β. 白细胞介素1β, AJ295836.2; IL-8. 白细胞介素8, XM035638412.1; TNF-α. 肿瘤坏死因子α, FJ654645; TGF-β. 转化生长因子β, KU238187.1; β-actin. 内参基因, EU686692.1Note: FAS. fatty acid synthetase, KC189927; PPARγ. peroxisome proliferators activated receptor γ, KC189932; SREBP1. sterol-regulatory element-binding protein 1, MH174964.1; PPARα. peroxisome proliferators activated receptor α, XM035614759.2; ACOX1. acyl-CoA oxidase 1, KC189925; TLR2. toll-like receptor 2, KU746963.1; TLR8. toll-like receptor 8, KX708702.1; TLR9. toll-like receptor 9, KU746969.1; MyD88. myeloid differentiation factor 88, KP985236; NF-κB p65. nuclear factor-κB p65, MF370855; IL-1β: interleukin-1β, AJ295836.2; IL-8. interleukin-8, XM035638412.1; TNF-α. tumor necrosis factor-α, FJ654645; TGF-β. transforming growth factor-β, KU238187.1; β-actin. beta-actin, EU686692.1
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表 4 饲料中添加不同脂肪酸对大菱鲆幼鱼生长指标的影响
Table 4 Effects of different fatty acids on growth of juvenile turbot
处理组
Treatment生长指标Growth index 增重率WGR (%) 饲料系数FCR 特定生长率SGR (%/d) 摄食率FI (%BW/d) 蛋白质效率PER 存活率SR (%) CON 569.27±9.50ab 0.72±0.00ab 3.40±0.03ab 1.89±0.01 2.80±0.01ab 100±0.00 PA 551.57±7.56ab 0.73±0.00b 3.35±0.02ab 1.92±0.01 2.73±0.02a 100±0.00 SA 554.80±5.49ab 0.72±0.00ab 3.35±0.01ab 1.90±0.01 2.77±0.01ab 100±0.00 OA 565.59±7.07ab 0.73±0.00b 3.39±0.02ab 1.91±0.01 2.76±0.02ab 100±0.00 LA 536.37±10.47a 0.73±0.00b 3.30±0.03a 1.89±0.01 2.75±0.02ab 100±0.00 ALA 562.16±9.37ab 0.71±0.00ab 3.38±0.03ab 1.87±0.01 2.82±0.01ab 100±0.00 ARA 585.88±7.13b 0.71±0.01ab 3.44±0.02b 1.89±0.01 2.82±0.02ab 100±0.00 DHA/EPA 580.59±8.57b 0.70±0.01a 3.42±0.02b 1.86±0.02 2.86±0.05b 100±0.00 注: 同一列内不同上标字母表示存在显著差异(P<0.05); 下同Note: Values in the same column labeled with different superscript letters are significantly different (P<0.05). The same applies below
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表 5 饲料中添加不同脂肪酸对大菱鲆体组成的影响
Table 5 Effects of different fatty acids on body composition of turbot
处理组
Treatment体组成 Body composition (干物质) 水分
Moisture
(%)粗蛋白
Crude protein
(%)粗脂肪
Crude lipid
(%)灰分
Crude ash
(%)CON 76.24±0.57 64.75±0.47ab 22.89±0.17c 12.71±0.44 PA 76.32±0.38 63.96±0.38a 22.52±0.54bc 12.41±0.30 SA 76.46±0.47 64.11±0.41a 22.01±0.26bc 12.77±0.42 OA 76.52±0.20 65.55±0.55abc 21.07±1.15ab 12.87±0.52 LA 76.21±0.52 64.96±0.66abc 21.43±0.44abc 12.49±0.15 ALA 76.72±0.62 65.99±0.79bc 21.64±0.59abc 12.47±0.27 ARA 76.76±0.61 66.64±0.84cd 20.03±0.40a 12.64±0.39 DHA/EPA 76.94±0.55 68.02±0.83d 19.96±0.80a 12.57±0.25
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表 6 饲料中添加不同脂肪酸对大菱鲆血浆生化指标的影响
Table 6 Effects of different fatty acids on plasma biochemical indices of turbot
处理组
Treatment血浆生化指标 Plasma biochemical index 甘油三酯
TG
(mmol/L)总胆固醇
T-CHO
(mmol/L)低密度脂
蛋白LDL-C
(mmol/L)高密度脂
蛋白HDL-C
(mmol/L)CON 3.02±0.25cd 1.95±0.22bcd 0.83±0.04d 2.02±0.10ab PA 3.58±0.08d 2.42±0.31d 0.96±0.04e 1.82±0.06a SA 3.66±0.21d 2.17±0.31cd 0.90±0.02e 1.88±0.05ab OA 2.63±0.25abc 1.95±0.40bcd 0.80±0.05d 2.05±0.09b LA 2.81±0.18bcd 1.60±0.40abc 0.71±0.03c 2.27±0.14b ALA 2.11±0.24ab 1.52±0.10ab 0.66±0.03c 2.44±0.06cd ARA 2.28±0.32abc 1.47±0.33ab 0.57±0.06b 2.43±0.04cd DHA/EPA 1.86±0.16a 1.19±0.31a 0.47±0.04a 2.54±0.03d
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表 7 饲料中添加不同脂肪酸对大菱鲆幼鱼肝脏抗氧化酶活性的影响
Table 7 Effects of different fatty acids on the activity of antioxidant enzymes in liver of juvenile turbot
处理组
Treatment抗氧化指标Antioxidant index 总抗氧化
能力T-AOC
(mmol/
g prot)丙二醛
MDA
(nmol/
mg prot)超氧化物歧
化酶SOD
(U/mg prot)过氧化氢酶
CAT
(U/mg prot)CON 1.62±0.07bc 3.34±0.09cde 71.00±2.19bc 9.29±0.66ab PA 1.39±0.02a 3.61±0.12e 67.23±1.60ab 8.43±0.37a SA 1.58±0.03b 3.45±0.06de 65.87±2.50a 8.44±1.20a OA 1.64±0.05bc 3.19±0.18bcd 70.94±0.68bc 8.54±0.81a LA 1.59±0.04b 3.11±0.14bcd 69.72±0.64abc 9.80±0.51ab ALA 1.65±0.12bcd 3.08±0.13bc 71.48±0.90bc 10.85±0.99bc ARA 1.72±0.08cd 2.88±0.04ab 73.93±2.30cd 12.80±0.52cd DHA/EPA 1.76±0.03d 2.58±0.18a 76.99±1.81d 12.93±0.35d
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