TWO DIETARY BETAINE SUPPLEMENTATIONS ON GROWTH, FEED UTILIZATION, LIPID METABOLISM, AND IMMUNE RESPONSE OF WHITE SHRIMP (LITOPENAEUS VANNAMEI) FED TWO LEVELS OF COMPOUND ATTRACTANTS
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摘要:
实验旨在研究两种含不同水平复合诱食剂(8%, 0)的基础饲料中添加甜菜碱对凡纳滨对虾(Litopenaeus vannamei)生长性能、饲料利用率、脂质代谢和免疫应答的影响。共制备原料型复合诱食剂对照组(P)、豆粕替代原料型诱食剂组(N)及在二者基础上分别添加0.6%甜菜碱的PB组和NB组等四组饲料, 喂养幼虾[初始体重(0.71±0.00) g] 7周。结果显示, 饲料中添加原料型复合诱食剂可提高对虾肝胰腺中fas、acc、sod、dorsal和relish及肠道toll和sod的表达量, 而肠道中relish表达量则相反。饲料中添加甜菜碱可提高对虾肌肉灰分含量, 上调对虾肝胰腺中fas、acc、sod和dorsal及肠道中sod的表达量, 而降低摄食量、肌肉粗脂肪和血淋巴中低密度脂蛋白胆固醇和丙二醛的含量及肝胰腺总一氧化氮合酶活性, 下调肝胰腺中acox、cpt-1、myd88和relish的表达量。原料型复合诱食剂与甜菜碱在对虾的饲料系数、肝胰腺总一氧化氮合酶活力、ampk、acc、cpt-1、sod、myd88和relish表达量等指标上呈现显著的交互作用。由此可见, 饲料中添加1.30%酵母提取物+ 4.80%鱿鱼内脏粉+ 1.90%鱼溶浆提高了对虾的免疫力和抗氧化能力。饲料中添加0.6%甜菜碱降低了对虾摄食量, 促进了脂质利用, 并上调了免疫相关基因的表达。
Abstract:Feed attractants have been widely used to improve the palatability of aquatic animal feeds, but there are few studies on the effects of feed attractants on growth and health of Litopenaeus vannamei. This study aims to investigate the effects of betaine supplementations on growth performance, feed utilization, lipid metabolism, and immune response of Litopenaeus vannamei fed two levels of raw feed attractants (8%, 0). Juvenile shrimp [initial body weight (0.71±0.00) g] were randomly distributed into four groups, each group included four fiberglass tanks [30 shrimp in each tank (0.3 m³)]. The shrimp were fed with raw feed attractants group (P), soybean meal replacing feed attractants in P (N), raw feed attractants + betaine group (PB) and betaine supplementation group (NB) for 7 weeks, respectively. Results showed that the raw feed attractants enhanced the expression levels offas, acc, sod, dorsal, relish in hepatopancreas, as well as toll and sod in the intestine of shrimp, while the expression levels of relish in the intestine were reversed. Betaine increased ash content in muscle, upregulated fas,acc, sod, and dorsal in hepatopancreas, as well as sod expression in the intestine, while it decreased feed intake, muscle crude lipid, low density lipoprotein cholesterol, malondialdehyde contents and total nitric oxide synthase activity in hepatopancreas, as well as the expression levels of acox, cpt-1,myd88 and relish in hepatopancreas. The interaction of raw feed attractants and betaine had significant effects on feed conversion ratio, hepatopancreas total nitric oxide synthase activity, ampk, acc, cpt-1, sod, myd88 and relish. In conclusion, a diet with 1.30% yeast extract +4.80% squid viscera powder+1.90% fish soluble improves the immunity and antioxidant capacity of shrimp. Dietary 0.6% betaine reduces feed intake and promotes lipid utilization, and up-regulates the expression of immune-related genes of shrimp. This study showed that two dietary betaine supplementations on growth, feed utilization, lipid metabolism, and immune response of white shrimp (Litopenaeus vannamei) fed two levels of compound attractants, which provides reference value for white shrimp culture.
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Keywords:
- Feed attractants /
- Betaine /
- Growth performance /
- Lipid metabolism /
- Immune response /
- Litopenaeus vannamei
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水产动物饲料不仅要具有促进生长和发育的丰富的营养物质, 还应具有足够吸引水产动物摄食的外观和气味。因而, 诱食剂被广泛应用于水产动物饲料中, 以更好地促进水生动物的摄食和生长。诱食剂分为原料型诱食剂和化合物型诱食剂两种。原料型诱食剂从自然界中获得, 通常包括中草药、大蒜和动物原料(如墨鱼膏、酵母提取物、鱿鱼膏、干贝粉提取物、鱿鱼副产品及鱼溶浆)等, 含多种化合物, 多种组分共同起作用; 化合物型诱食剂包括甜菜碱、氨基酸、DMPT和硫代甜菜碱等, 经过人工加工或者合成获得, 为单一组分[1]。酵母提取物是从发酵的酵母中提取的, 含有丰富的寡糖、小肽和氨基酸, 对水生动物的生长和发育有益[2, 3]。先前的研究表明, 饲料中添加2%酵母提取物可有效提高凡纳滨对虾(Litopenaeus vannamei)的采食量和饲料利用率[2]。此外, 酵母提取物还能增加凡纳滨对虾肠道菌群的多样性[4], 增强其免疫力[5]。鱿鱼内脏粉和鱼溶浆具有浓郁的腥味, 引起水生动物的味觉刺激从而促进摄食。鱿鱼内脏粉从鱿鱼中提取, 富含氨基酸[6], 添加到饲料中可以提高云斑尖塘鳢(Oxyeleotris marmorata)[7]和凡纳滨对虾[8]的摄食量。鱼溶浆的总固体含量为40%—50%, 通常粗蛋白质含量约为30%[9], 可以有效地促进黑鲷(Acanthopagrus schlegelii)[10]、草鱼(Ctenopharyngodon idella)[11]、黄颡鱼(Pelteobagrus fulvidraco)[12]和凡纳滨对虾[13]的生长。然而, 单一的诱食剂长时间使用可能造成水产动物厌食和健康等问题[1], 同时也会受到自身可用性、成本和成分波动等的因素的限制[6]。饲料中添加复合诱食剂可在一定程度上解决单一诱食剂存在的问题。研究表明, 饲料中添加复合诱食剂可促进大菱鲆幼鱼(Scophthalmus maximus L.)[14]、鳜(Siniperca chuatsi)[15]和凡纳滨对虾[16]的生长和摄食。
甜菜碱是一种广泛存在于植物、动物和微生物中的氨基酸, 可以有效地改善水生动物饲料的适口性[17－19]。已有研究表明, 饲料中添加甜菜碱可使凡纳滨对虾(0.5%)[20]、罗氏沼虾(Macrobrachium rosenbergii; 0.5%)[21]和印度鲤幼鱼(Labeo rohita; 0.25%)[22]的生长和摄食效果最佳, 使凡纳滨对虾(1.0%)获得最佳的饲料利用率[20]。其两性离子特性能增加水产饲料的水稳性[19, 23－25]。此外, 已有研究表明, 饲料中添加甜菜碱可以改善肠屏障功能[26], 促进水产动物脂肪代谢、减少脂肪沉积[27, 28], 并能够调节nf-κb炎症通路[29], 从而改善水产动物的健康。
凡纳滨对虾是世界养殖量最高的对虾品种, 具有生长速度快、环境适应性强和经济价值高等特点[30－32]。而饲料中原料型诱食剂与甜菜碱的效果差异及其组合利用效果对凡纳滨对虾的生长和健康的影响尚未见报道。本实验旨在研究原料型组合诱食剂与甜菜碱对凡纳滨对虾生长性能、饲料利用、脂质代谢及免疫应答的影响。
1. 材料与方法
1.1 实验饲料配方
设计4种实验饲料: 以商品对虾饲料(P)为阳性对照, 利用豆粕替代P中的诱食剂作为阴性对照(N), 分别在阳性对照和阴性对照的基础上分别添加甜菜碱(PB和NB)。按如下步骤配制饲料。首先, 原料用锤式粉碎机粉碎(SF-320, 苏中制药机械有限公司, 中国江苏)后通过80目筛网。其次, 称取原料并混合均匀, 然后加入油和水并在霍巴特式搅拌机(M-256, 华南理工大学, 中国广州)中充分混合。最后, 使用双螺杆挤出机(F-26, 华南理工大学, 中国广州)将混合物压制成直径为1.0和1.5 mm的饲料, 并在电热鼓风干燥箱中65℃干燥30min并进而风干, 使用前置于−20℃保存。饲料配方及营养成分分析见表 1。
表 1 实验饲料配方(湿物质基础)Table 1. Formulation of supplement experimental diets (%Wet basis)配方Formula 组别Group P N PB NB 鱼粉 Fish meal 21.53 21.53 21.53 21.53 鸡肉粉 Chicken by-product meal 4.00 4.00 4.00 4.00 花生粕 Peanut meal 6.00 6.00 6.00 6.00 鱼排粉 Fish by-product meal 7.00 7.00 7.00 7.00 虾粉 Shrimp meal 6.00 6.00 6.00 6.00 玉米蛋白粉 Corn protein powder 5.00 5.00 5.00 5.00 棉籽蛋白 Cottonseed protein 4.00 4.00 4.00 4.00 豆粕 Soybean meal 7.00 15.00 7.00 15.00 小麦面粉 Wheat flour 17.20 17.20 16.60 16.60 氯化胆碱 Choline chloride 0.31 0.31 0.31 0.31 磷酸二氢钙 Monocalcium phosphate 1.23 1.23 1.23 1.23 氯化钙 Calcium chloride 0.98 0.98 0.98 0.98 贝壳粉 Shell powder 0.62 0.62 0.62 0.62 酵母提取物 Yeast extract 1.30 0 1.30 0 鱿鱼内脏粉 Squid visceral powder 4.80 0 4.80 0 鱼溶浆 Fish soluble 1.90 0 1.90 0 甜菜碱Betaine 0 0 0.60 0.60 大豆磷脂 Soybean phospholipid 1.23 1.23 1.23 1.23 赖氨酸 Lysine 0.37 0.37 0.37 0.37 矿物质预混料 Mineral premix 2.46 2.46 2.46 2.46 维生素预混料 Vitamin premix 0.92 0.92 0.92 0.92 发酵豆粕 Fermented soybean meal 6.15 6.15 6.15 6.15 营养成分 Proximate composition (% wet weight) 粗蛋白 Crude protein 43.00 42.07 43.53 43.54 粗脂肪 Crude lipid 6.60 5.84 6.91 4.72 水分 Moisture 6.78 6.44 8.51 6.86 灰分 Ash 16.13 15.97 15.83 16.10 注: 所有原料均由福州海马饲料有限公司提供 Note: All raw materials are provided by Grobest Group Holdings Limited (CN), Fuzhou, China 1.2 实验虾和饲养
凡纳滨对虾幼虾从广东恒兴饲料实业有限公司(中国湛江)种苗基地获得, 用商业饲料饲养1周, 以适应实验条件。试验选用480尾健康、均匀的初始体重(0.70±0.00) g对虾, 随机分为4组, 每组4个重复(300 L玻璃纤维桶), 每个重复30尾虾。每天7:00、12:00、17:00和21:00投喂4次, 饵料重量为对虾体重的5%—10%, 共喂养7周。在前4周, 投喂直径为1.0 mm的饲料, 在最后3周, 投喂直径为1.5 mm的饲料。在实验过程中, 每3—4天用二氧化氯消毒过的海水换掉桶内60%的水, 以维持水质。每天测量水温和盐度, 养殖期间分别为24—28℃和26‰—29‰。
1.3 取样及化学分析
在饲养试验结束后, 禁食24h, 随后称重计数。从每个重复中随机选取5尾虾, 剥去外壳和头尾, 置于−20℃的冰箱中保存, 用于分析肌肉成分。使用1 mL注射器从围心腔中抽取5尾虾的血淋巴, 然后在4℃条件下离心(4000 r/min)10min。收集上清液并置于−80℃下保存。每个重复取2尾虾的肝胰腺和肠, 置于RNAlater中, −80℃放置保存, 用于实时荧光定量。每个重复取2尾虾的肝胰腺, 置于−80℃保存, 用于酶活分析。
按标准方法[33]测定饲料和肌肉样品的水分、粗蛋白质、粗脂肪和灰分含量。水分通过105℃烘干法测定, 粗蛋白质含量用杜马斯定氮仪(KjeltecTM 8400, 瑞典)测定; 粗脂肪含量采用XT15脂肪仪(Ankom, USA)测定, 粗灰分含量利用马弗炉于550℃下灰化6h测定。
血淋巴中总蛋白(TP)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)甘油三酯(TG)、总胆固醇(T-CHO)和丙二醛(MDA)的含量, 以及肝胰腺中的丙二醛含量、总一氧化氮合酶(T-NOS)、总抗氧化能力(T-AOC)和总超氧化物歧化酶(T-SOD)酶活均采用南京建成生物工程研究所生产的试剂盒进行测定, 按照试剂盒的说明书方法进行操作。
使用TransZol Up Plus RNA试剂盒(TransGen, 中国)提取肝胰腺和肠道中的RNA。琼脂糖凝胶电泳和分光光度分析(Nanodrop 2000)用于评估RNA质量和浓度。根据制造商(TaKaRa, 日本)的说明, 使用PrimeScript™RT-PCR试剂盒进行cDNA合成。实时荧光PCR用LightCycler 480(Roche Diagnostics, Switzerland)进行测定, 反应体积10 μL, 包含5 μL的SYBR®Green Pro Taq HS、1 μL的cDNA、0.4 μL的正向和反向引物, 以及3.2 μL灭菌双蒸水。反应中每个样品3个重复。PCR 的反应条件为: 95℃ 2min, 40个反应循环(95℃ 15s, 60℃ 15s, 72℃ 20s), 随后进行融解曲线分析并冷却到4℃。根据我们对内参基因评价的初步实验结果, 以基因延伸因子1α(ef-1α)为内参基因, 相对基因表达用2–ΔΔCT法[34]进行分析。所有引物均使用PrimerQuest工具(National Center for Biotechnology Information, USA)设计, 引物序列如表 2所示。
表 2 实时荧光定量PCR引物序列Table 2. Primer sequence used for quantitative Real-time PCR引物
Primer序列Sequence (5′—3′) 登录号
GenBank No.ef-1a-R GTATTGGAACAGTGCCCGTG JF288785.1 ef-1a-F ACCAGGGACAGCCTCAGTAAG ampk-R TCAGAGGAGGAGCAGGAAC KP272117.1 ampk-F CCCGAGGTCTAATAGGCAC toll-R CCTCGCACATCCAGGACTTTTA / toll-F GACCATCCCTTTTACACCAGACT sod-R CAGAGCCTTTCACTCCAACG / sod-F GCAATGAATGCCCTTCTACC myd88-R GGGAGTGGCAGAAACTTATC / myd88-F GTGCACCAGAGTCATTGTAG dorsal-R CGTAACTTGAGGGCATCTTC FJ998202.1 dorsal-F TGGGGAAGGAAGGATGG relish-R GGCTGGCAAGTCGTTCTCG EF432734 relish-F CTACATTCTGCCCTTGACTCTGG fas-R GGTGACTAGCTCGGCTACATGGTT XM_027367955.1 fas-F CAGGTGGAGATGCTCCTCGTGTT acox-R GTAGCGTCCGAGGCACTGA XM_027370700.1 acox-F CCCGGGTGTCCACTGACA acc-R TTTGACACCTGAGCCAGACC XM_027360190.1 acc-F TGCATAGAAACGGCATTGCG cpt-1-R GTCGGTCCACCAATCTTC XM_027361888.1 cpt-1-F CAACTTCTACGGCACTGAT 注: F表示正向引物, R表示反向引物; ef-1α. 延伸因子1α; ampk. AMP-激活蛋白激酶; toll. toll蛋白; sod. 超氧化物歧化酶; myd88. 髓系分化因子88; dorsal. 转录因子p65样; relish. 核因子NF-kappa-B p105亚基样; fas. 脂肪酸合成酶; acox. 酰基辅酶A氧化酶; acc. 乙酰辅酶A羧化酶; cpt-1. 肉碱棕榈酰转移酶 Note: F presents forward primer, R presents reverse primer. ef-1α. elongation factor 1α; ampk. AMP-activated protein kinase; toll. protein toll; sod. superoxide dismutase; myd88. myeloid differentiation primary response gene 88; dorsal. transcription factor p65-like; relish. nuclear factor NF-kappa-B p105subunit-like; fas. fatty acid synthase; acox. acyl-coenzyme A oxidase; acc. acetyl CoA carboxylase;cpt-1. carnitine palmitoyl transferase 1.4 计算公式及统计分析
本研究结果以均值±标准误(SE)表示。采用双因素方差分析(Two-way Anova), P<0.05代表有显著性差异。终末体质量(Final body weight, FBW)、成活率(Survival rate, SR)、饲料系数(Feed conversion ratio, FCR)、增重率(Weight gain, WG)、特定生长率(Specific growth rate, SGR)和摄食量(Feed intake, FI)等指标的计算公式如下:
成活率(SR, %)=100×试验结束时虾数量/试验开始时虾数量;
增重率(WGR, %)=100×[终末体质量(g)−初始体质量(g)]/初始体质量(g);
饲料系数(FCR)=摄食饲料干物质质量×100/(终末体质量−初始体质量);
特定生长率(SGR, %/d)=100×[ln平均终末体质量(g)−ln平均初始体质量(g)/实验天数(D)];
摄食量(FI, g)=饲料摄食总量(g)/虾数量×100。
2. 结果
2.1 生长性能和饲料利用
生长性能和饲料利用见表 3。原料型复合诱食剂对对虾的SR、FBW、FI、FCR、WGR和SGR无显著影响(P>0.05)。甜菜碱显著降低了对虾的FI(P<0.05); 对SR、FBW、FCR、WGR和SGR无显著影响(P>0.05)。原料型复合诱食剂与甜菜碱的互作作用对对虾的FCR有显著影响(P<0.05), 对对虾的SR、FBW、FCR、WGR和SGR无显著影响(P>0.05)。
表 3 两种复合诱食剂水平的基础饲料中添加甜菜碱对凡纳滨对虾生长性能和饲料利用率的影响Table 3. Effects of dietary betaine supplementations on growth performance and feed utilization of the Litopenaeus vannamei fed two levels of compound attractants组别Group 成活率SR (%) 终末体质量FBW (g) 摄食量FI (g) 饲料系数FCR 增重率WGR (%) 特定生长率SGR (%/d) P 97.49±0.83 8.23±0.14 10.05±0.11b 1.33±0.03 1066.04±20.56 4.90±0.03 N 93.33±1.35 8.44±0.34 10.52±0.16b 1.36±0.03 1094.04±47.85 4.95±0.08 PB 92.22±4.84 8.03±0.25 9.05±0.44a 1.37±0.06 1038.81±35.42 4.73±0.13 NB 93.33±1.35 8.27±0.12 9.17±0.16a 1.21±0.01 1070.81±19.03 4.91±0.03 双因素方差分析P值 P-value of two-factor ANOVA 组合 Combination 0.49 0.35 0.43 0.22 0.38 0.19 甜菜碱 Betaine 0.25 0.44 0.00 0.08 0.46 0.23 交互作用 Interaction 0.25 0.96 0.35 0.04 0.95 0.41 注: 平均值±标准误(n=4); 相同字母上标或同一行无字母上标表示无显著差异(P>0.05), 不同字母表示存在显著差异(P<0.05)。A和B为原料型复合诱食剂对对虾的显著差异, a和b为甜菜碱对对虾的显著差异; “组合 Combination”表示“双因素方差分析原料型复合诱食剂P值”, “甜菜碱 Betaine”表示“双因素方差分析甜菜碱P值”, “交互作用 Interaction”表示“原料型复合诱食剂与甜菜碱的交互作用P值”, 下同 Note: Mean±SE (n=4); The same letter superscript or no letter superscript in the same line indicates no significant difference (P>0.05), while different letters indicate a significant difference (P<0.05). A and B are the significant differences of raw feed attractants toLitopenaeus vannamei; a and b are the significant differences of betaine to Litopenaeus vannamei, the same applies below 2.2 肌肉成分
肌肉成分见表 4。原料型复合诱食剂对肌肉水分、灰分、粗脂肪和粗蛋白质均无显著影响(P>0.05)。甜菜碱显著提高了对虾肌肉灰分含量(P<0.05), 显著降低了肌肉粗脂肪含量(P<0.05), 对水分和粗蛋白质含量无显著影响(P>0.05)。原料型复合诱食剂与甜菜碱的交互作用对肌肉的水分、灰分、粗脂肪和粗蛋白无显著影响(P>0.05)。
表 4 两种原料型复合诱食剂水平的基础饲料中添加甜菜碱对凡纳滨对虾肌肉组成的影响(湿重)Table 4. Effects of dietary betaine supplementations on muscle composition of the Litopenaeus vannamei fed two levels of compound attractants (wet basis) (%)组别
Group水分
Moisture灰分
Ash粗脂肪
Crude lipid粗蛋白
Crude proteinP 73.86±0.81 0.57±0.08a 1.08±0.15b 22.02±0.41 N 74.51±0.82 0.55±0.04a 0.93±0.06b 21.83±0.33 PB 74.76±0.48 1.48±0.02b 0.65±0.04a 22.27±0.21 NB 74.84±0.33 1.48±0.02b 0.64±0.04a 22.55±0.11 双因素方差分析P值 P-value of two-factor ANOVA 组合 Combination 0.58 0.84 0.08 0.99 甜菜碱 Betaine 0.36 0.00 0.00 0.12 交互作用 Interaction 0.67 0.84 0.15 0.55 2.3 血淋巴生化指标
血淋巴生化指标见表 5。原料型复合诱食剂对血淋巴生化指标无显著影响(P>0.05)。甜菜碱显著降低了血淋巴中LDL-C和MDA含量(P<0.05); 对HDL-C、T-CHO、TP和TG含量无显著影响(P>0.05)。原料型复合诱食剂与甜菜碱的交互作用对血淋巴生化指标无显著影响(P>0.05)。
表 5 两种原料型复合诱食剂水平的基础饲料中添加甜菜碱对凡纳滨对虾血淋巴生化指标的影响Table 5. Effects of dietary betaine supplementations on hemolymph biochemical indexes of Litopenaeus vannamei fed two levels of compound attractants组别
Group低密度脂蛋白胆固醇
LDL-C (mmol/L)高密度脂蛋白胆固醇
HDL-C (mmol/L)总胆固醇T-CHO
(mmol/L)总蛋白TP
(g/L)甘油三酯TG
(mmol/L)丙二醛MDA
(nmol/L)P 11.55±0.88b 3.38±0.59 0.46±0.07 71.38±8.63 0.32±0.03 5.33±0.70b N 14.56±3.95b 2.32±0.63 0.47±0.10 67.66±3.54 0.32±0.10 5.99±0.52b PB 8.53±1.69a 5.07±0.59 0.26±0.01 69.59±2.55 0.34±0.04 3.74±0.54a NB 6.91±0.21a 3.59±1.21 0.41±0.08 71.45±5.50 0.35±0.02 3.77±0.39a 双因素方差分析P值 P-value of two-factor ANOVA 组合 Combination 0.77 0.14 0.31 0.87 0.88 0.56 甜菜碱 Betaine 0.03 0.09 0.13 0.86 0.70 0.00 交互作用 Interaction 0.30 0.79 0.41 0.62 0.95 0.59 2.4 肝胰腺酶活
肝胰腺酶活见表 6。原料型复合诱食剂对肝胰腺酶活无显著影响(P>0.05)。甜菜碱显著降低了对虾肝胰腺一氧化氮合酶活性(P<0.05)。甜菜碱对对虾肝胰腺MDA含量、T-SOD和T-AOC活性无显著影响(P>0.05)。原料型复合诱食剂与甜菜碱的交互作用对T-NOS、T-SOD、MDA和T-AOC活性无显著影响(P>0.05)
表 6 两种原料型复合诱食剂水平的基础饲料中添加甜菜碱对凡纳滨对虾肝胰腺抗氧化相关参数的影响Table 6. Effects of dietary betaine supplementations on hepatopancreas anti-oxidative related parameters of Litopenaeus vannamei fed two levels of compound attractants组别
Group总一氧化氮合酶
T-NOS
(U/mg prot)总超氧化物歧化酶
T-SOD
(U/mg prot)丙二醛
MDA
(nmol/mg prot)总抗氧化能力T-AOC
(mmol/g)P 2.54±0.19b 21.65±3.16 4.04±0.36 0.28±0.04 N 3.48±0.19b 21.19±1.48 5.55±0.48 0.27±0.10 PB 2.86±0.22a 24.49±1.41 4.92±1.09 0.36±0.05 NB 1.71±0.14a 23.75±4.40 4.81±0.68 0.23±0.05 双因素方差分析P值 P-value of Two-factor ANOVA 组合 Combination 0.60 0.84 0.34 0.37 甜菜碱 Betaine 0.00 0.37 0.92 0.77 交互作用 Interaction 0.00 0.96 0.27 0.38 2.5 肝胰腺基因表达
肝胰腺脂质代谢及免疫相关基因表达如图 1所示, 原料型复合诱食剂显著上调了对虾肝胰腺fas和acc的表达量(P<0.05); 对ampk、acox和cpt-1的表达量无显著影响(P>0.05)。甜菜碱显著上调了肝胰腺fas和acc的表达量(P<0.05), 并且显著下调了ampk的表达量(P<0.05)。原料型复合诱食剂与甜菜碱的交互作用对ampk、acc和cpt-1的表达量有显著影响(P<0.05)。原料型复合诱食剂显著上调了对虾肝胰腺sod、dorsal和relish的表达量(P<0.05); 对toll和myd88的表达量无显著影响(P>0.05)。甜菜碱显著上调了肝胰腺sod和dorsal的表达量(P<0.05), 并且显著下调了myd88和relish的表达量(P<0.05); 对toll的表达量无显著影响(P>0.05)。原料型复合诱食剂与甜菜碱的交互作用对sod、myd88和dorsal的表达量有显著影响(P<0.05), 对toll和relish的表达量无显著影响(P>0.05)。
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图 1 肝胰腺脂质代谢及免疫相关基因表达平均值±标准误(n=4); 柱状图中相同字母上标或同一行无字母上标表示无显著差异(P>0.05), 不同字母表示存在显著差异(P<0.05)。图中“组合 Combination (P)”表示“双因素方差分析原料型复合诱食剂P值”, “甜菜碱 Betaine (P)”表示“双因素方差分析甜菜碱P值”, “交互作用 Interaction (P)”表示“原料型复合诱食剂与甜菜碱的交互作用P值”, 下同Figure 1. Hepatopancreas lipid metabolism and immune-related gene expressionMean±SE (n=4) are presented as bar charts, the same letter superscript or the same row without letter superscript indicates no significant difference (P>0.05), while different letters indicate a significant difference (P<0.05). “Combination (P)”, “Betaine (P)”, and “Interaction (P)” means “P value of raw feed attractants in two-way ANOVA”, “P value of betaine in two-way ANOVA”, and “P value of interaction between feedstock raw feed attractants and betaine”, respectively, the same applies below2.6 肠道免疫相关基因表达
肠道免疫相关基因表达如图 2所示。原料型复合诱食剂添加组的toll和sod表达量显著高于未添加组(P<0.05),relish表达量显著低于未添加组(P<0.05); 对dorsal表达量无显著影响(P>0.05)。甜菜碱添加组的sod表达量显著高于未添加组(P<0.05),toll显著低于未添加组(P<0.05); 对relish和dorsal表达量无显著影响(P>0.05)。原料型复合诱食剂与甜菜碱的交互作用对toll、relish和sod表达量有显著影响(P<0.05), 对dorsal的表达量无显著影响(P>0.05)。
3. 讨论
营养的摄入及水生动物健康状况是影响其正常生长发育的重要因素。在本实验研究中, 原料型诱食剂添加组饲料(P, PB)与未添加组饲料(N, NB)的不同在于8%的原料型复合诱食剂被8%的豆粕替代, 而原料型复合诱食剂对对虾的SR、FBW、FI、FCR、WGR和SGR无显著影响。而饲料中添加0.6%甜菜碱显著降低对虾的FI, 对SR、FBW、FCR、WGR和SGR无显著影响。已有研究表明, 适宜的植物蛋白量替代鱼粉后对凡纳滨对虾幼虾的生长发育和采食量无不良影响[35, 36]。此外, 当饲料配方中的动物蛋白含量并不低时, 诱食剂的添加对水产动物的摄食和生长也无显著影响[37]。本实验配方中的豆粕替代原料型复合诱食剂对于凡纳滨对虾的生长和饲料利用并没有负面影响, 可能是饲料配方中动物蛋白含量较高, 已满足了对虾生长和摄食的需求。之前的研究表明, 饲料中甜菜碱添加量为0.01%—0.075%时, 凡纳滨对虾的FCR、SR、SGR和WGR与未添加组无显著差异[20], 饲料中添加量为0.2%时, 凡纳滨对虾获得很好的生长性能和饲料转化率; 另外一个研究结果与本实验结果相近, 甜菜碱添加量达到0.5%时, 对虾生长性能并没有明显改善, 反而有所下降[38]; 也有研究表明甜菜碱添加量为4%时, 凡纳滨对虾的FI、SR、SGR和FCR无显著变化[24]。然而, 也有研究报道饲料中甜菜碱添加量(0.5%)和(0.8%)可显著提高罗氏沼虾[28]和印度白对虾(Fenneropenaeus indicus)[39]的存活率, 饲料中5、10和15 g/kg 的甜菜碱添加量均可提高罗氏沼虾的增重、采食量和饲料利用率[21]。导致这些不同结果的原因可能是不同浓度的甜菜碱对不同养殖时期的不同物种刺激是不同的, 从而使得水产动物对饲料的敏感度不同[40]。本研究中, NB组对虾的FCR比其余3组低, 可能饲料中添加0.6%的甜菜碱(P=0.08)有提高无原料型复合诱食剂添加组对虾的饲料利用率的趋势。
肌肉粗脂肪含量是虾类品质的重要指标。在本研究中, 甜菜碱降低了对虾肌肉的脂质含量, 提高了对虾肌肉的灰分。与我们的研究结果相似, 饲料中添加0.5%的甜菜碱可显著降低罗氏沼虾肌肉的粗脂肪含量[28]。脂质代谢是生命活动的重要代谢途径, 肝脏中脂质的合成和分解的动态平衡是其健康的重要保证。脂肪酸合成的关键在于脂肪酸合成酶(fas)催化乙酰辅酶A转化为丙二酰辅酶A, 乙酰辅酶A羧化酶(acc)进一步催化长链脂肪酸的合成[41], 酰基辅酶A氧化酶(acox)将长支链脂肪酸氧化成短链脂肪酸, 且通过氧化胆汁酸的辅酶A酯中间体, 催化胆固醇转化为胆汁酸。当机体的能量较低时, ampk可以调控促进分解脂质的过程, 增加ATP合成, 从而维持机体能量平衡[42]。肉碱棕榈酰转移酶(cpt-1)则是脂肪酸分解的重要酶, 催化脂酰辅酶A进入线粒体基质彻底分解产生能量[43]。在本研究中, 饲料中添加原料型复合诱食剂或甜菜碱均显著上调了fas和acc的表达量, 饲料中添加甜菜碱下调ampk、acox和cpt-1的表达量。添加原料型诱食剂可能会倾向于增加对虾脂肪合成, 肌肉粗脂肪也呈现出类似的结果, 添加原料型诱食剂后对虾肌肉粗脂肪有增加的趋势(P=0.08)。结合肌肉粗脂肪结果, 饲料中添加甜菜碱可能增强了脂肪分解供能。而生物体的脂质代谢平衡是正常生命活动的保证, 当脂质分解活动到达某一程度时, 机体会负反馈抑制脂质分解, 促进脂质合成, 以保持脂质代谢平衡[41]。因而饲料中添加甜菜碱使脂肪酸合成相关基因上调可能是生物体内的脂肪分解已经到达正常范围的最大限度, 从而使得脂肪酸合成反弹性增加。尽管甜菜碱在对虾的脂质代谢机制方面的研究较少, 但在陆生动物上有大量的相关报道, 如甜菜碱对大鼠[44]和小型猪[45]的降脂作用通过调节其脂质代谢机制达到, 本研究也证实了甜菜碱对降低对虾肌肉脂肪含量, 调节脂质代谢的功能, 但具体的作用机制有待进一步研究。血淋巴生化指标可以在一定程度上反映水生动物代谢和健康状况, 主要体现在水生动物的生理、营养和病理等方面[46, 47]。LDL是血液循环中主要的胆固醇载体, 它的生理功能是将胆固醇运送到细胞, HDL在胆固醇的逆向运输中起着关键作用, 它的功能与LDL相反[16]。在本研究中, 甜菜碱显著降低血淋巴中LDL-C含量, 而HDL-C、TP、T-CHO和TG含量无显著变化, 说明饲料中添加甜菜碱使对虾将肝胰腺中胆固醇运送到细胞的频率降低, 暗示虾体内脂质分解增强[48]。总之, 饲料中添加甜菜碱有利于凡纳滨对虾脂质分解代谢。
超氧化物歧化酶(SOD)是生物体内重要的抗氧化酶, 具有抗氧化和抗衰老的作用, 其作用机理主要是清除对机体有害的超氧阴离子自由基(
${\rm{O}}^{-}_2 $ )[49]。在本研究中, 原料型复合诱食剂和甜菜碱均能显著上调对虾肝胰腺和肠道中sod表达量。MDA是多不饱和脂肪酸(PUFAs)的脂质过氧化分解产物, 具有细胞毒性作用[50]。在本实验中, 饲料中添加甜菜碱可使虾的血淋巴中MDA含量显著降低。因此可以推断, 饲料中添加原料型复合诱食剂或甜菜碱均有利于虾肝胰腺抗氧化基因的表达, 从而提高虾的抗氧化能力[48]。此外, 甜菜碱有利于虾的血淋巴中脂质的氧化代谢或过氧化代谢废物的清除[28]。一氧化氮合酶(NOS)催化产生一氧化氮(NO)杀死细菌和病毒毒素, 从而提高虾的免疫力[51], 然而, 当细胞内的NOS的浓度过高时, 可以对细胞产生毒害作用[52, 55]。在本研究中, 饲料中添加甜菜碱显著降低肝胰腺中T-NOS活性, 对T-SOD和T-AOC活性以及MDA含量无显著影响。已有研究表明, NO也能够和O2–作用生成过氧亚硝基(ONOO–), 它的强氧化和硝基化作用能够氧化酶蛋白, 使之硝基化并灭活酶活性, 还可通过细胞膜扩散导致细胞的代谢功能紊乱, 此外, 病原体还能够产生一系列酶并利用抗氧化物和自由基清除剂等物质, 以抵抗宿主的抗病反应[52]。在本研究中, 尽管甜菜碱降低了T-NOS的活性, 但对T-SOD和T-AOC活性及MDA含量无显著影响, 因而我们推测可能这种变化对对虾的健康并无影响。无脊椎动物被认为完全依赖它们的先天免疫来抵御病原体, 因为它们缺乏适应性免疫[53]。toll是一种重要的先天免疫受体, 在机体对病原体感染的免疫应答中起着至关重要的作用, 它通过与受体复合物结合诱导nf-κb信号通路实现[54]。而myd88、relish和dorsal是nf-κb信号通路的主要组成部分, 参与调节许多炎症反应, 其中myd88又被认为是激活先天免疫toll通路下游信号的中心连接子[55]。在本研究中, 原料型复合诱食剂显著上调虾肝胰腺中dorsal和relish及肠道中的toll的表达量, 显著下调肠道relish的表达量。这意味着饲料中添加复合诱食有利于提高虾肝胰腺的免疫相关基因的表达, 从而增强对虾免疫应答。饲料中添加甜菜碱显著上调肝胰腺dorsal的表达量, 显著下调myd88和relish的表达量; 同时显著下调肠道toll的表达量。饲料中添加甜菜碱也可能提高对虾的免疫应答能力[29]。4. 结论
在本试验中, 饲料中添加8%原料型复合诱食剂不影响凡纳滨对虾的生长性能、肌肉成分和血淋巴生化指标, 但促进了凡纳滨对虾肝胰腺中免疫基因的表达; 饲料中添加0.6%甜菜碱不影响凡纳滨对虾的生长性能, 但显著降低了对虾肌肉粗脂肪的含量, 促进对虾的抗氧化能力和脂质代谢, 提高肠道免疫相关基因表达。原料型复合诱食剂与甜菜碱的交互作用对对虾的饲料系数、总一氧化氮合酶、脂质代谢相关基因和免疫相关基因有显著影响。
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图 1 肝胰腺脂质代谢及免疫相关基因表达
平均值±标准误(n=4); 柱状图中相同字母上标或同一行无字母上标表示无显著差异(P>0.05), 不同字母表示存在显著差异(P<0.05)。图中“组合 Combination (P)”表示“双因素方差分析原料型复合诱食剂P值”, “甜菜碱 Betaine (P)”表示“双因素方差分析甜菜碱P值”, “交互作用 Interaction (P)”表示“原料型复合诱食剂与甜菜碱的交互作用P值”, 下同
Figure 1. Hepatopancreas lipid metabolism and immune-related gene expression
Mean±SE (n=4) are presented as bar charts, the same letter superscript or the same row without letter superscript indicates no significant difference (P>0.05), while different letters indicate a significant difference (P<0.05). “Combination (P)”, “Betaine (P)”, and “Interaction (P)” means “P value of raw feed attractants in two-way ANOVA”, “P value of betaine in two-way ANOVA”, and “P value of interaction between feedstock raw feed attractants and betaine”, respectively, the same applies below
表 1 实验饲料配方(湿物质基础)
Table 1 Formulation of supplement experimental diets (%Wet basis)
配方Formula 组别Group P N PB NB 鱼粉 Fish meal 21.53 21.53 21.53 21.53 鸡肉粉 Chicken by-product meal 4.00 4.00 4.00 4.00 花生粕 Peanut meal 6.00 6.00 6.00 6.00 鱼排粉 Fish by-product meal 7.00 7.00 7.00 7.00 虾粉 Shrimp meal 6.00 6.00 6.00 6.00 玉米蛋白粉 Corn protein powder 5.00 5.00 5.00 5.00 棉籽蛋白 Cottonseed protein 4.00 4.00 4.00 4.00 豆粕 Soybean meal 7.00 15.00 7.00 15.00 小麦面粉 Wheat flour 17.20 17.20 16.60 16.60 氯化胆碱 Choline chloride 0.31 0.31 0.31 0.31 磷酸二氢钙 Monocalcium phosphate 1.23 1.23 1.23 1.23 氯化钙 Calcium chloride 0.98 0.98 0.98 0.98 贝壳粉 Shell powder 0.62 0.62 0.62 0.62 酵母提取物 Yeast extract 1.30 0 1.30 0 鱿鱼内脏粉 Squid visceral powder 4.80 0 4.80 0 鱼溶浆 Fish soluble 1.90 0 1.90 0 甜菜碱Betaine 0 0 0.60 0.60 大豆磷脂 Soybean phospholipid 1.23 1.23 1.23 1.23 赖氨酸 Lysine 0.37 0.37 0.37 0.37 矿物质预混料 Mineral premix 2.46 2.46 2.46 2.46 维生素预混料 Vitamin premix 0.92 0.92 0.92 0.92 发酵豆粕 Fermented soybean meal 6.15 6.15 6.15 6.15 营养成分 Proximate composition (% wet weight) 粗蛋白 Crude protein 43.00 42.07 43.53 43.54 粗脂肪 Crude lipid 6.60 5.84 6.91 4.72 水分 Moisture 6.78 6.44 8.51 6.86 灰分 Ash 16.13 15.97 15.83 16.10 注: 所有原料均由福州海马饲料有限公司提供 Note: All raw materials are provided by Grobest Group Holdings Limited (CN), Fuzhou, China 
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表 2 实时荧光定量PCR引物序列
Table 2 Primer sequence used for quantitative Real-time PCR
引物
Primer序列Sequence (5′—3′) 登录号
GenBank No.ef-1a-R GTATTGGAACAGTGCCCGTG JF288785.1 ef-1a-F ACCAGGGACAGCCTCAGTAAG ampk-R TCAGAGGAGGAGCAGGAAC KP272117.1 ampk-F CCCGAGGTCTAATAGGCAC toll-R CCTCGCACATCCAGGACTTTTA / toll-F GACCATCCCTTTTACACCAGACT sod-R CAGAGCCTTTCACTCCAACG / sod-F GCAATGAATGCCCTTCTACC myd88-R GGGAGTGGCAGAAACTTATC / myd88-F GTGCACCAGAGTCATTGTAG dorsal-R CGTAACTTGAGGGCATCTTC FJ998202.1 dorsal-F TGGGGAAGGAAGGATGG relish-R GGCTGGCAAGTCGTTCTCG EF432734 relish-F CTACATTCTGCCCTTGACTCTGG fas-R GGTGACTAGCTCGGCTACATGGTT XM_027367955.1 fas-F CAGGTGGAGATGCTCCTCGTGTT acox-R GTAGCGTCCGAGGCACTGA XM_027370700.1 acox-F CCCGGGTGTCCACTGACA acc-R TTTGACACCTGAGCCAGACC XM_027360190.1 acc-F TGCATAGAAACGGCATTGCG cpt-1-R GTCGGTCCACCAATCTTC XM_027361888.1 cpt-1-F CAACTTCTACGGCACTGAT 注: F表示正向引物, R表示反向引物; ef-1α. 延伸因子1α; ampk. AMP-激活蛋白激酶; toll. toll蛋白; sod. 超氧化物歧化酶; myd88. 髓系分化因子88; dorsal. 转录因子p65样; relish. 核因子NF-kappa-B p105亚基样; fas. 脂肪酸合成酶; acox. 酰基辅酶A氧化酶; acc. 乙酰辅酶A羧化酶; cpt-1. 肉碱棕榈酰转移酶 Note: F presents forward primer, R presents reverse primer. ef-1α. elongation factor 1α; ampk. AMP-activated protein kinase; toll. protein toll; sod. superoxide dismutase; myd88. myeloid differentiation primary response gene 88; dorsal. transcription factor p65-like; relish. nuclear factor NF-kappa-B p105subunit-like; fas. fatty acid synthase; acox. acyl-coenzyme A oxidase; acc. acetyl CoA carboxylase;cpt-1. carnitine palmitoyl transferase
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表 3 两种复合诱食剂水平的基础饲料中添加甜菜碱对凡纳滨对虾生长性能和饲料利用率的影响
Table 3 Effects of dietary betaine supplementations on growth performance and feed utilization of the Litopenaeus vannamei fed two levels of compound attractants
组别Group 成活率SR (%) 终末体质量FBW (g) 摄食量FI (g) 饲料系数FCR 增重率WGR (%) 特定生长率SGR (%/d) P 97.49±0.83 8.23±0.14 10.05±0.11b 1.33±0.03 1066.04±20.56 4.90±0.03 N 93.33±1.35 8.44±0.34 10.52±0.16b 1.36±0.03 1094.04±47.85 4.95±0.08 PB 92.22±4.84 8.03±0.25 9.05±0.44a 1.37±0.06 1038.81±35.42 4.73±0.13 NB 93.33±1.35 8.27±0.12 9.17±0.16a 1.21±0.01 1070.81±19.03 4.91±0.03 双因素方差分析P值 P-value of two-factor ANOVA 组合 Combination 0.49 0.35 0.43 0.22 0.38 0.19 甜菜碱 Betaine 0.25 0.44 0.00 0.08 0.46 0.23 交互作用 Interaction 0.25 0.96 0.35 0.04 0.95 0.41 注: 平均值±标准误(n=4); 相同字母上标或同一行无字母上标表示无显著差异(P>0.05), 不同字母表示存在显著差异(P<0.05)。A和B为原料型复合诱食剂对对虾的显著差异, a和b为甜菜碱对对虾的显著差异; “组合 Combination”表示“双因素方差分析原料型复合诱食剂P值”, “甜菜碱 Betaine”表示“双因素方差分析甜菜碱P值”, “交互作用 Interaction”表示“原料型复合诱食剂与甜菜碱的交互作用P值”, 下同 Note: Mean±SE (n=4); The same letter superscript or no letter superscript in the same line indicates no significant difference (P>0.05), while different letters indicate a significant difference (P<0.05). A and B are the significant differences of raw feed attractants toLitopenaeus vannamei; a and b are the significant differences of betaine to Litopenaeus vannamei, the same applies below
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表 4 两种原料型复合诱食剂水平的基础饲料中添加甜菜碱对凡纳滨对虾肌肉组成的影响(湿重)
Table 4 Effects of dietary betaine supplementations on muscle composition of the Litopenaeus vannamei fed two levels of compound attractants (wet basis) (%)
组别
Group水分
Moisture灰分
Ash粗脂肪
Crude lipid粗蛋白
Crude proteinP 73.86±0.81 0.57±0.08a 1.08±0.15b 22.02±0.41 N 74.51±0.82 0.55±0.04a 0.93±0.06b 21.83±0.33 PB 74.76±0.48 1.48±0.02b 0.65±0.04a 22.27±0.21 NB 74.84±0.33 1.48±0.02b 0.64±0.04a 22.55±0.11 双因素方差分析P值 P-value of two-factor ANOVA 组合 Combination 0.58 0.84 0.08 0.99 甜菜碱 Betaine 0.36 0.00 0.00 0.12 交互作用 Interaction 0.67 0.84 0.15 0.55
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表 5 两种原料型复合诱食剂水平的基础饲料中添加甜菜碱对凡纳滨对虾血淋巴生化指标的影响
Table 5 Effects of dietary betaine supplementations on hemolymph biochemical indexes of Litopenaeus vannamei fed two levels of compound attractants
组别
Group低密度脂蛋白胆固醇
LDL-C (mmol/L)高密度脂蛋白胆固醇
HDL-C (mmol/L)总胆固醇T-CHO
(mmol/L)总蛋白TP
(g/L)甘油三酯TG
(mmol/L)丙二醛MDA
(nmol/L)P 11.55±0.88b 3.38±0.59 0.46±0.07 71.38±8.63 0.32±0.03 5.33±0.70b N 14.56±3.95b 2.32±0.63 0.47±0.10 67.66±3.54 0.32±0.10 5.99±0.52b PB 8.53±1.69a 5.07±0.59 0.26±0.01 69.59±2.55 0.34±0.04 3.74±0.54a NB 6.91±0.21a 3.59±1.21 0.41±0.08 71.45±5.50 0.35±0.02 3.77±0.39a 双因素方差分析P值 P-value of two-factor ANOVA 组合 Combination 0.77 0.14 0.31 0.87 0.88 0.56 甜菜碱 Betaine 0.03 0.09 0.13 0.86 0.70 0.00 交互作用 Interaction 0.30 0.79 0.41 0.62 0.95 0.59
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表 6 两种原料型复合诱食剂水平的基础饲料中添加甜菜碱对凡纳滨对虾肝胰腺抗氧化相关参数的影响
Table 6 Effects of dietary betaine supplementations on hepatopancreas anti-oxidative related parameters of Litopenaeus vannamei fed two levels of compound attractants
组别
Group总一氧化氮合酶
T-NOS
(U/mg prot)总超氧化物歧化酶
T-SOD
(U/mg prot)丙二醛
MDA
(nmol/mg prot)总抗氧化能力T-AOC
(mmol/g)P 2.54±0.19b 21.65±3.16 4.04±0.36 0.28±0.04 N 3.48±0.19b 21.19±1.48 5.55±0.48 0.27±0.10 PB 2.86±0.22a 24.49±1.41 4.92±1.09 0.36±0.05 NB 1.71±0.14a 23.75±4.40 4.81±0.68 0.23±0.05 双因素方差分析P值 P-value of Two-factor ANOVA 组合 Combination 0.60 0.84 0.34 0.37 甜菜碱 Betaine 0.00 0.37 0.92 0.77 交互作用 Interaction 0.00 0.96 0.27 0.38
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