EFFECTS OF DIETARY β-1,3-GLUCAN ON GROWTH PERFORMANCE, SERUM METABOLITE, IMMUNE-RELATED GENE EXPRESSION AND RESISTANCE TO NITRITE STRESS IN LITOPENAEUS VANNAMEI
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摘要: 实验旨在研究β-1,3-葡聚糖的不同投喂方式对凡纳滨对虾(Litopenaeus vannamei)生长、血清代谢和抗亚硝酸氮应激能力的影响。选用480尾初体重(0.43±0.01) g的凡纳滨对虾, 随机分为4组, 即G0(全程投喂基础饲料)、G1组(全程投喂0.1%β-1,3-葡聚糖饲料)、G2组(0.1% β-1,3-葡聚糖饲料7d+基础饲料7d循环)和G3组(0.1% β-1,3-葡聚糖饲料14d+基础饲料14d循环)。在养殖84d后, 应用亚硝酸钠进行120h亚硝酸氮应激实验。结果显示, 各实验组凡纳滨对虾生长性能和全虾营养成分没有显著性差异。在养殖84d后, G2和G3组凡纳滨对虾肝胰腺脂肪酶活性显著高于G0和G1组(P<0.05), G1、G2和G3组凡纳滨对虾血清胆固醇和甘油三酯含量显著高于G0组(P<0.05), G3组凡纳滨对虾肌肉脂多糖/ß-1,3-葡聚糖结合蛋白(LGBP)、酚氧化物酶原(proPO)和超氧化物歧化酶(SOD)mRNA表达显著高于G0和G1组(P<0.05)。亚硝酸氮应激120h, G1、G2和G3组凡纳滨对虾累计死亡率显著低于G0组(P<0.05), G3组凡纳滨对虾累计死亡率显著低于G0、G1和G2组(P<0.05)。在亚硝酸氮应激120h后, 与G0组相比, G1、G2和G3组凡纳滨对虾血清总蛋白含量显著升高(P<0.05), 葡萄糖含量、谷丙转氨酶和谷草转氨酶活性显著降低(P<0.05); G3组凡纳滨对虾肌肉LGBP、proPO和SOD mRNA表达显著高于G0 (P<0.05), G1组凡纳滨对虾肌肉丝氨酸蛋白酶(SP)mRNA表达显著高于G0、G2和G3组(P<0.05)。结果表明, 14d间隔投喂0.1% β-1,3-葡聚糖可能通过促进能量代谢和LGBP、proPO和SOD mRNA表达提高凡纳滨对虾抗亚硝酸氮应激能力。Abstract: To investigate the effects of dietary β-1,3-glucan on growth performance, serum metabolite, immune-related gene and resistance to nitrite stress in Litopenaeus vannamei, 0.1% β-1,3-Glucan was supplemented to a basal diet (control, G0) with different feeding schedules: permanently β-1,3-glucan diet (G1), 7 days β-1,3 glucan diet-7 days of control diet (G2) and 14 days β-1,3 glucan diet-14 days of control diet (G3). After the feeding trial, these shrimps were exposed to nitrite-N (20 mg/L) for 120h. The results showed that dietary β-1,3-glucan had no significant effect on growth performance and whole-body composition after 84d feeding trial (P>0.05). The activity of lipase in hepatopancreas in G2 and G3 groups was significantly higher than that in G0 and G1 groups (P<0.05). The shrimp in G1, G2 and G3 groups had significantly higher hemolymph triacylglycerols and cholesterol levels than G0 group (P<0.05). Higher expression levels of LPS/b-glucan binding protein (LGBP), Prophenoloxidase (proPO) and superoxide dismutase (SOD) were observed in G3 group compared to G0 and G1 groups (P<0.05). After 120h nitrite-N stress, the mortality in G3 group was significantly lower than that of G0, G1 and G2 groups (P<0.05). Nitrite stress significantly reduced the levels of GLU, GOT and GPT and significantly enhanced hemolymph level. G3 group had significantly higher expression levels of LGBP, proPO and SOD compared to G0 group (P<0.05). The expression level of serine proteinase in G1 group was significantly higher than that in G0, G2 and G3 groups (P<0.05). L. vannamei fed diet G3 can improve nitrite resistance probably by accelerating energy metabolism and increasing expression levels of LGBP, proPO and SOD.
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Keywords:
- β-1,3-glucan /
- Litopenaeus vannamei /
- Growth /
- Serum metabolite /
- Immune-related gene /
- Nitrite stress
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凡纳滨对虾(Litopenaeus vannamei), 俗称南美白对虾, 具有环境适应性强、生长速度快、养殖周期短和市场需求量大等优点, 是我国养殖范围最广和产量最高的对虾种类[1]。然而, 随着对虾集约化养殖的迅速发展, 养殖水环境恶化和病害频发等诸多问题日益严峻, 严重影响了对虾养殖业的可持续发展。通过在饲料中添加功能性添加剂促进凡纳滨对虾营养物质代谢和免疫抗氧化功能是提高生长和抗病抗应激能力的重要途径[2, 3]。β-1,3-葡聚糖作为一种免疫增强剂已广泛应用在水产养殖业中, 并取得了显著成效。在饲料中添加β-1,3-葡聚糖能够促进凡纳滨对虾生长性能、提高免疫功能和抗环境应激能力[4]。
免疫刺激剂在生长和免疫反应中发挥重要作用, 但其作用效果与其添加剂量和时间相关。β-葡聚糖的添加剂量以0.1%—1%效果最佳, 高剂量可能降低鱼虾免疫力, 并产生免疫疲劳[5－8]。免疫刺激剂具有较短时间持续作用, 长期添加β-葡聚糖可能导致对虾产生免疫疲劳[9－12]。斑节对虾(Penaeus monodon)摄食含葡聚糖饵料3d即可显著提高酚氧化酶活性、血细胞总数和血细胞对哈维弧菌(Vibrio harveyi)的吞噬活力[10]。β-葡聚糖投喂斑节对虾亲虾24h后其白细胞吞噬活性大大增强, 超氧阴离子产量增加, 这种免疫作用可持续40d[11]。采用0.2%β-葡聚糖投喂凡纳滨对虾40d, 盐度应激24h后对虾酚氧化物酶水平下降, 可能与免疫疲劳有关[12]。长期连续摄食免疫刺激剂可能造成动物生理的应激, 影响生长、营养物质代谢或抗病抗胁迫能力。因此, 本实验观察了持续或间隔投喂β-1,3-葡聚糖84d对长期养殖凡纳滨对虾生长、血清代谢、免疫相关基因表达和抗亚硝酸氮应激能力的影响, 以期为β-1,3-葡聚糖在对虾饲料中的精准使用提供理论依据。
1. 材料与方法
1.1 实验饲料
以鱼粉和大豆浓缩蛋白等为主要蛋白源, 鱼油为主要脂肪源, 玉米淀粉为主要糖源配制等氮等能的基础饲料, 饲料配方及化学组成如表 1所示。在基础饲料中添加0.1% β-1,3-葡聚糖(Sigma公司, 纯度>99.0%), 配制1种实验饲料。实验按照不同投喂方式分为4个饲料组, 分别为: (1)对照组(G0): 基础饲料, 持续投喂84d; (2)处理组1(G1): 0.1%β-1,3-葡聚糖饲料, 持续投喂84d; (3)处理组2(G2): 0.1%β-1,3-葡聚糖饲料7d +基础饲料7d, 循环投喂84d。(4)处理组3(G3): 0.1%β-1,3-葡聚糖饲料14d+基础饲料14d, 循环投喂84d。饲料原料过60目筛粉碎, 混合均匀后用SLX-80型挤压机制成直径为1.0 mm的颗粒饲料, 在45℃下烘干。自然冷却后放入密封袋中于–20℃冰箱中保存待用。
表 1 饲料配方及化学成分(风干重)Table 1. Formulation and chemical composition (dry matter, %)原料Ingredient 含量Content 原料Ingredient 含量Content 秘鲁鳀鱼粉Peru anchovy fish meal 30.0 维生素预混料Vitamin premix1) 2.0 大豆浓缩蛋白Soy protein concentrate 30.0 矿物质预混料Mineral premix2) 0.5 玉米淀粉Corn starch 28.0 磷酸二氢钙Ca(H2PO4)2 1.5 鱼油Fish oil 5.0 α-纤维素α-cellulose 0.5 氯化胆碱Choline chloride (50%) 1.0 大豆卵磷脂Soybean lecithin (50%) 1.5 营养水平Nutrient level 营养水平Nutrient level 粗蛋白质Crude protein 39.1 灰分Ash 13.8 粗脂肪Crude lipid 11.2 水分Moisture 8.7 注: 1)维生素预混料为每千克饲料提供: 维生素A 60.0 mg; 维生素D 5000 IU; 维生素E 99.0 mg; 维生素K 5.0 mg; 维生素B1 50.0 mg; 维生素B2 40.0 mg; 维生素B6 100.0 mg; 维生素B12 0.1 mg; 泛酸钙 calcium pantothenate 120.0 mg; 烟酸 niacin 200.0 mg; 生物素 biotin 1.0 mg; 肌醇 inositol 300. 0 mg; 叶酸 folic acid 10.0 mg; 2)矿物质预混料为每千克饲料提供: 铁 12 mg; 铜 25 mg; 锌 32 mg; 锰 20 mg; 硒 0.05 mg; 碘 0.1 mg; 钴5 mg; 镁 0.06 mg; 钾 40 mgNote: 1)Vitamin premix composition (mg/kg feed): VA 60.0 mg; VD 5000 IU; VE 99.0 mg; VK 5.0 mg; VB1 50.0 mg; VB2 40.0 mg; VB6 100.0 mg; VB12 0.1 mg; calcium pantothenate 120.0 mg; niacin 200.0 mg; biotin 1.0 mg; inositol 300. 0 mg; folic acid 10.0 mg; wheat flour was used as the carrier. 2) Mineral premix composition (mg/kg feed): Fe 12 mg; Cu 25 mg; Zn 32 mg; Mn 20 mg; Se 0. 05 mg; I 0.1 mg; Co 5 mg; Mg 0.06 mg; K 40 mg 1.2 实验虾与饲养管理
养殖实验在广东省农业科学院动物科学研究所水产研究室室内循环水养殖系统中进行。养殖系统由圆柱形玻璃纤维桶(直径80 cm, 高70 cm)组成, 容水量300 L。实验用凡纳滨对虾先在室外循环水泥池中驯养2周, 期间投喂基础饲料。实验开始前禁食24h, 选取初始体均重为(0.43±0.01) g的凡纳滨对虾幼虾480尾, 随机分为4组, 每组设4个重复, 每个重复40尾虾, 分别以不同周期投喂2种饲料, 实验期为12周。实验期间定量投喂, 每日分别在09:00、14:30和20:30分3次投喂, 日投喂量为体重的4%—6%, 根据摄食情况调整投喂量。每天观察虾体健康状况, 记录死亡情况。养殖用水为进水速率为1.5 L/min的曝气自来水, 每天对养殖水体进行3次监测, 实验期间盐度5‰—6‰, 水温27—31℃, 氨氮<0.20 mg/L, 亚硝酸盐<0.01 mg/L, 溶氧>6.0 mg/L, pH 7.5—7.9。实验期间采取自然光源, 时间为2019年7月14日至2019年9月7日, 地点为广州市天河区五山。
1.3 样品采集
在饲养实验结束前, 禁食24h, 统计每桶对虾的存活数, 称量每桶虾体重, 计算末体重、特定生长率和存活率。每桶随机选取3尾置于–20℃保存, 用于检测全虾体组成。以无菌的1 mL注射器及针头自围心腔取血, 置于Eppenddorf离心管中, 静置2h, 3000 r/min离心10min, 取上清液作为待测血清, –78℃冷冻保存备用。采血后的对虾在冰盘上采集肌肉, 迅速投入液氮中速冻, 保存于–78℃冰箱用于肌肉血蓝蛋白(Haemocyanin)、脂多糖/ß-1,3-葡聚糖结合蛋白(LPS/b-glucan binding protein, LGBP)、抗菌肽(Penaedin-3)、酚氧化物酶原(Prophenoloxidase, proPO)、丝氨酸蛋白酶(Serine proteinase, SP)和超氧化物歧化酶(Superoxide dismutase, SOD)mRNA 表达的分析。
1.4 亚硝酸氮应激实验
在12周的饲养实验结束后, 每组保留60尾对虾进行应激实验。应激实验停止循环水, 每个实验组设4个应激实验缸, 应激持续120h, 应激源为亚硝酸钠(NaNO2)。150 L水体加入NaNO2, 使氮浓度达到20 mg/L。实验期间继续投喂实验饲料, 观察并记录各组对虾0、24h、48h、72h、96h和120h内死亡情况, 计算120h内的累计死亡率。和应激实验结束后, 采集对虾血清和肌肉样品, 置于–78℃冰箱冷冻保存备用。
累计计死亡率(Cumulative mortality rate, CMR, %)=应激后实验鱼死亡尾数/应激前实验鱼尾数×100
1.5 指标测定
生长性能计算 增重率(Weight gain rate, WGR, %)=100×(终末体重－初始体重)/初始体重; 特定生长率(Specific growth rate, SGR, %/d)=100×(ln终末体重－ln初始体重)/饲养天数; 存活率(Survival rate, SR, %)=100×实验结束时虾尾数/实验开始时放虾尾数。
实验饲料和实验虾营养成分测定 实验饲料和实验虾全虾营养成分测定参照AOAC(1984)[13]方法。水分含量采用105℃烘箱(HDG-9203A, 上海一恒科学仪器有限公司)烘干至恒重测定, 粗蛋白质含量采用凯氏自动定氮仪(KDN-103F, 上海纤检仪器有限公司)测定, 粗脂肪含量采用索氏抽提仪(SXT-06, 上海洪纪仪器设备有限公司)测定, 粗灰分含量采用550℃灼烧法(SX410, 天津泰斯特仪器有限公司)测定。
血清代谢指标分析 血清总蛋白(Total protein, TP)、葡萄糖(Glucose, GLU)、胆固醇(Cholesterol, CHO)、甘油三酯(Triglyceride, TG)、乳酸盐(lactate)含量及谷草转氨酶(Glutamic oxaloacetic transaminase, GOT)和谷丙转氨酶(Glutamic-pyruvic transaminase, GPT)活性采用Beckman Synchron CX5全自动生化分析仪测定。
基因mRNA表达量分析 取对虾肌肉组织在液氮中充分研磨成粉状, 加入裂解液按TaKaRa RNA提取试剂盒操作步骤提取总RNA。采用实时荧光定量PCR测定对虾肌肉LGBP、Haemocyanin、Penaedin-3a、proPO、SP和SOD mRNA相对表达量, 以β-肌动蛋白(β-actin)为内参基因。根据对虾上述6种基因的cDNA序列利用Primer5.0软件设计实时荧光定量PCR特异引物(表 2)。实时荧光定量PCR(SYBR Premix Ex Taq II, TaKaRa, 大连)扩增程序为93℃预变性3 min, 40个循环包括93℃ 30s, 55℃ 45s, 72℃ 45s。采用2−ΔΔCt法分析基因相对表达量[14]。
表 2 本实验的引物序列Table 2. Primers used in this study目的基因
Target gene引物Primer (5′—3′) 大小Size (bp) LPS/b-glucan binding protein ACCGCAGCATCAGTTATACCGTCATCGCCCTTCCAGTTG 77 Haemocyanin GTCTTAGTGGTTCTTGGGCTTGTCGGTCTCCGTCCTGAATGTCTCC 124 Penaedin-3a CACCCTTCGTGAGACCTTTGAATATCCCTTTCCCACGTGAC 141 Prophenoloxidase CGGTGACAAAGTTCCTCTTCGCAGGTCGCCGTAGTAAG 122 Serine proteinase ACGTTCTCACGACTGGTCACACTATGTAAGGCGCGTCGTTCTC 107 Superoxide dismutase TCATGCTTTGCCACCTCTCCCGCTTCAACCAACTTCTTC 143 β-actin CCACGAGACCACCTACAACAGCGAGGGCAGTGATTTC 142 1.6 数据分析
实验结果用平均值±标准误表示, 采用SPSS11.5软件进行数据分析和统计, 先对数据作单因素方差分析(One-way ANOVA), 若组间差异显著, 再用Duncan氏多重比较。显著性水平为P<0.05。
2. 结果
2.1 凡纳滨对虾生长性能
如表 3所示, G0、G1、G2和G3组凡纳滨对虾的终末均重、特定生长率和存活率没有显著性差异(P>0.05)。
表 3 β-1,3-葡聚糖对凡纳滨对虾生长性能的影响Table 3. Effects of dietary β-1,3-glucan on growth performance of litopenaeus vannamei项目Item G0 G1 G2 G3 初始均重Initial body weight (g) 0.43±
0.0010.42±
0.0020.43±
0.0020.43±
0.002终末均重Final body weight (g) 5.31±
0.195.62±
0.035.71±
0.215.13±
0.29增重率Weight gain rate (%) 1141±
471218±
181239±
471108±
63特定生长率Specific growth rate (%/d) 2.99±
0.0463.07±
0.0173.09±
0.412.96±
0.62存活率Survival rate (%) 64.82±
6.2379.98±
8.1280.12±
5.7576.56±
6.38注: 上标不同的同行数据间差异显著(P<0.05); 下同Note: Values in each row with different letters are significantly different (P<0.05). The same applies below 2.2 凡纳滨对虾肝胰腺消化酶活性
由表 4可知, G3组凡纳滨对虾肝胰腺的蛋白酶活性显著高于G1组(P<0.05)。G2和G3组凡纳滨对虾肝胰腺的脂肪酶活性显著高于G0和G1组(P<0.05)。各实验组凡纳滨对虾肝胰腺的淀粉酶活性没有显著性差异(P>0.05)。
表 4 β-1,3-葡聚糖对凡纳滨对虾肝胰腺消化酶活性的影响Table 4. Effects of dietary β-1,3-glucan on activities of digestive enzymes in hepatopancreas of Litopenaeus vannamei (U/mg prot)项目Item G0 G1 G2 G3 蛋白酶Protease 3553.77±
467.73ab2356.34±
616.84a3275.07±
359.06ab4250.97±
757.18b脂肪酶Lipase 6.76±1.57a 6.53±2.84a 9.69±1.01b 11.96±1.05c 淀粉酶Amylase 46.86±2.97 48.97±8.22 49.04±2.07 49.54±2.32 2.3 凡纳滨对虾体成分
如表 5所示, G0、G1、G2和G3组凡纳滨对虾的全虾水分、粗蛋白质、粗脂肪和灰分含量没有显著性差异(P>0.05)。
表 5 β-1,3-葡聚糖对凡纳滨对虾体成分的影响(%DM)Table 5. Effects of dietary β-1,3-glucan on body composition of Litopenaeus vannamei项目Item G0 G1 G2 G3 水分Moisture 76.68±
0.2476.98±
0.0976.12±
0.0876.74±
0.13粗蛋白Crude protein 70.85±
0.4471.00±
0.3470.26±
0.6169.73±
0.25粗脂肪Crude lipid 5.39±
0.426.00±
0.155.80±
0.235.87±
0.02灰分Ash 13.89±
0.3213.57±
0.2014.06±
0.4714.17±
0.192.4 亚硝酸氮应激120h累计死亡率
如图 1所示, 亚硝酸氮应激120h, G1、G2和G3组凡纳滨对虾累计死亡率显著低于G0组(P＜0.05), G3组凡纳滨对虾累计死亡率显著低于G0、G1和G2组(P < 0.05)。
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图 1 β-1,3-葡聚糖对凡纳滨对虾抗亚硝酸氮应激能力的影响Figure 1. Effects of dietary β-1,3-glucan on anti- nitrite-nitrogen stress ability of Litopenaeus vannamei2.5 凡纳滨对虾应激前后血清代谢指标
如图 2所示, 在养殖84d后, G1、G2和G3组凡纳滨对虾血清CHO和TG含量显著高于G0组(P<0.05)。各实验组凡纳滨对虾血清TP、GLU、LD、GOT和GPT活性无显著差异(P>0.05)。亚硝酸氮应激120h后, G1、G2和G3组凡纳滨对虾血清TP含量显著高于G0组(P<0.05)。G1和G3组凡纳滨对虾血清乳酸盐含量显著高于G0组(P<0.05)。凡纳滨对虾血清葡萄糖含量、谷丙转氨酶和谷草转氨酶活性在G0组达到最高, 显著高于G1、G2和G3组(P<0.05)。各实验组凡纳滨对虾血清CHO和TG含量无显著差异(P>0.05)。
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图 2 β-1,3-葡聚糖对凡纳滨对虾应激前后血清代谢指标的影响Figure 2. Effects of dietary β-1,3-glucan on hemolymph variables before (time 0) and after (120h) nitrite stress in Litopenaeus vannamei2.6 凡纳滨对虾应激前后免疫相关基因表达
如图 3所示, 在养殖84d后, G3组凡纳滨对虾肌肉LGBP和proPO mRNA表达显著高于G0、G1和G2组(P<0.05)。G3组凡纳滨对虾肌肉SOD mRNA表达显著高于G0和G1组(P<0.05)。在各实验组中凡纳滨对虾肌肉Haemocyanin、Penaedin-3a和SP mRNA表达量无显著差异(P>0.05)。在亚硝酸氮应激120h后, G3组凡纳滨对虾肌肉LGBP和proPO mRNA表达显著高于G0和G1组(P<0.05)。G1组凡纳滨对虾肌肉SP mRNA表达显著高于G0、G2和G3组(P<0.05)。G1、G2和G3组凡纳滨对虾SOD mRNA表达显著高于G0组(P<0.05)。各实验组凡纳滨对虾肌肉Haemocyanin和Penaedin-3a mRNA表达量无显著差异(P>0.05)。
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图 3 β-1,3-葡聚糖对凡纳滨对虾应激前后肌肉免疫相关基因表达的影响Figure 3. Effects of dietary β-1,3-glucan on expression of immune-related genes in muscle before (time 0) and after (120h) nitrite stress in L. vannamei3. 讨论
研究表明, 在饲料中短期添加β-葡聚糖可以提高凡纳滨对虾生长性能[3, 15]。摄食β-葡聚糖饲料56d显著提高低盐度养殖凡纳滨对虾生长性能、消化酶活性和存活率[3]。然而, 凡纳滨对虾连续摄食β-葡聚糖饲料36d造成免疫疲劳, 但间隔投喂β-葡聚糖饲料可以提高对虾免疫力[15]。在本实验中, 连续或间隔投喂β-1,3-葡聚糖84d对凡纳滨对虾生长性能和体成分没有显著影响, 但间隔投喂显著提高了凡纳滨对虾的脂肪酶活性及血清胆固醇和甘油三酯水平, 表明间隔投喂β-1,3-葡聚糖促进了对虾机体内的蛋白质和能量代谢。尼罗罗非鱼(Oreochromis niloticus)摄食实验饲料(2周、4周投喂或间隔7d投喂β-葡聚糖)56d, 对生长性能没有显著影响, 但间隔投喂显著提高罗非鱼免疫功能和抗Aeromonas hydrophila 和Flavobacterium columnare感染能力。这与本实验结果相似, 与未添加组相比, 投喂0.1% β-1,3-葡聚糖84d显著提高了对虾抗亚硝酸氮应激能力, 但14d间隔投喂β-1,3-葡聚糖组凡纳滨对虾的累计死亡率达到最低。
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β-1,3-葡聚糖通过结合甲壳动物的模式识别受体β-1,3-葡聚糖结合蛋白(BGBP)而连接到血淋巴颗粒细胞膜表面发生反应, 使颗粒细胞释放出丝氨酸, 丝氨酸继续激活酚氧化酶原发生级联放大反应, 使酶原变为具有活性的酚氧化酶, 反应产生大量黑色素和醌过氧化物等活性物质, 杀灭病原菌[27]。脂多糖-β-1,3-葡聚糖结合蛋白是一种多功能的模式识别受体, 能够识别革兰氏阴性菌的细胞壁成分脂多糖和真菌的细胞壁成分β-1,3-葡聚糖[28]。丝氨酸蛋白酶在级联放大反应中作为重要调控因子参与免疫反应。超氧化物歧化酶是生物体抗氧化防御系统重要的酶类, 清除体内活性氧自由基, 保护细胞膜及细胞内的核酸免受氧化应激的损害。饲料中加益生菌显著提高凡纳滨对虾抗副溶血弧菌感染的能力和上调对虾感染副溶血弧菌后先天免疫缺陷基因、抗菌肽、酚氧化酶原和溶菌酶mRNA表达[29]。凡纳滨对虾摄食蜈蚣藻多糖饲料显著提高肝胰腺血蓝蛋白、酚氧化酶原和热休克蛋白70基因mRNA表达量[30]。在本实验中, 14d间隔投喂0.1% β-1,3-葡聚糖显著上调凡纳滨对虾在亚硝酸氮应激前后的肌肉脂多糖/β-1,3-葡聚糖结合蛋白、酚氧化化酶原和超氧化物歧化酶表达, 表明β-1,3-葡聚糖通过刺激凡纳滨对虾的免疫相关基因的表达, 提高其抗亚硝酸氮应激能力。与本实验结果相似, Soraat等研究不同投喂方式对罗非鱼免疫功能和抗Aeromonas hydrophila 和Flavobacterium columnare感染能力的影响, 认为β-葡聚糖间隔投喂方式是最佳施加方法。斑节对虾摄食含褐藻酸钠饲料能够显著上调LPS/β-1,3-葡聚糖结合蛋白、超氧化物歧化酶和抗菌肽 mRNA表达, 但对酚氧化酶原没有影响[31]。在本实验中, 在饲料中添加0.1% β-1,3-葡聚糖对凡纳滨对虾肌肉血蓝蛋白和抗菌肽基因表达没有显著影响, 可能β-1,3-葡聚糖主要是通过作用于模式识别受体和抗氧化物酶基因发挥作用。
4. 结论
在84d养殖实验后, β-1,3-葡聚糖持续或间隔投喂对凡纳滨对虾生长性能和体成分没有显著影响, 但显著提高了凡纳滨对虾血清胆固醇、甘油三酯水平和降低凡纳滨对虾亚硝酸氮120h应激累计死亡率。7d或14d间隔投喂β-1,3-葡聚糖显著提高了凡纳滨对虾脂肪酶活性, 14d间隔投喂β-1,3-葡聚糖显著上调了凡纳滨对虾亚硝酸氮应激前后LGBP、proPO和SOD mRNA表达。饲料添加0.1% β-1,3-葡聚糖显著提高了凡纳滨对虾亚硝酸氮应激后血清TP含量, 并显著降低血清GLU含量、GOT和GPT活性。因此, 14d间隔投喂0.1% β-1,3-葡聚糖可能通过促进能量代谢和上调LGBP、proPO和SOD基因表达提高凡纳滨对虾抗亚硝酸氮应激能力。
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图 1 β-1,3-葡聚糖对凡纳滨对虾抗亚硝酸氮应激能力的影响
Figure 1. Effects of dietary β-1,3-glucan on anti- nitrite-nitrogen stress ability of Litopenaeus vannamei
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图 2 β-1,3-葡聚糖对凡纳滨对虾应激前后血清代谢指标的影响
Figure 2. Effects of dietary β-1,3-glucan on hemolymph variables before (time 0) and after (120h) nitrite stress in Litopenaeus vannamei
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图 3 β-1,3-葡聚糖对凡纳滨对虾应激前后肌肉免疫相关基因表达的影响
Figure 3. Effects of dietary β-1,3-glucan on expression of immune-related genes in muscle before (time 0) and after (120h) nitrite stress in L. vannamei
表 1 饲料配方及化学成分(风干重)
Table 1 Formulation and chemical composition (dry matter, %)
原料Ingredient 含量Content 原料Ingredient 含量Content 秘鲁鳀鱼粉Peru anchovy fish meal 30.0 维生素预混料Vitamin premix1) 2.0 大豆浓缩蛋白Soy protein concentrate 30.0 矿物质预混料Mineral premix2) 0.5 玉米淀粉Corn starch 28.0 磷酸二氢钙Ca(H2PO4)2 1.5 鱼油Fish oil 5.0 α-纤维素α-cellulose 0.5 氯化胆碱Choline chloride (50%) 1.0 大豆卵磷脂Soybean lecithin (50%) 1.5 营养水平Nutrient level 营养水平Nutrient level 粗蛋白质Crude protein 39.1 灰分Ash 13.8 粗脂肪Crude lipid 11.2 水分Moisture 8.7 注: 1)维生素预混料为每千克饲料提供: 维生素A 60.0 mg; 维生素D 5000 IU; 维生素E 99.0 mg; 维生素K 5.0 mg; 维生素B1 50.0 mg; 维生素B2 40.0 mg; 维生素B6 100.0 mg; 维生素B12 0.1 mg; 泛酸钙 calcium pantothenate 120.0 mg; 烟酸 niacin 200.0 mg; 生物素 biotin 1.0 mg; 肌醇 inositol 300. 0 mg; 叶酸 folic acid 10.0 mg; 2)矿物质预混料为每千克饲料提供: 铁 12 mg; 铜 25 mg; 锌 32 mg; 锰 20 mg; 硒 0.05 mg; 碘 0.1 mg; 钴5 mg; 镁 0.06 mg; 钾 40 mgNote: 1)Vitamin premix composition (mg/kg feed): VA 60.0 mg; VD 5000 IU; VE 99.0 mg; VK 5.0 mg; VB1 50.0 mg; VB2 40.0 mg; VB6 100.0 mg; VB12 0.1 mg; calcium pantothenate 120.0 mg; niacin 200.0 mg; biotin 1.0 mg; inositol 300. 0 mg; folic acid 10.0 mg; wheat flour was used as the carrier. 2) Mineral premix composition (mg/kg feed): Fe 12 mg; Cu 25 mg; Zn 32 mg; Mn 20 mg; Se 0. 05 mg; I 0.1 mg; Co 5 mg; Mg 0.06 mg; K 40 mg 
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表 2 本实验的引物序列
Table 2 Primers used in this study
目的基因
Target gene引物Primer (5′—3′) 大小Size (bp) LPS/b-glucan binding protein ACCGCAGCATCAGTTATACCGTCATCGCCCTTCCAGTTG 77 Haemocyanin GTCTTAGTGGTTCTTGGGCTTGTCGGTCTCCGTCCTGAATGTCTCC 124 Penaedin-3a CACCCTTCGTGAGACCTTTGAATATCCCTTTCCCACGTGAC 141 Prophenoloxidase CGGTGACAAAGTTCCTCTTCGCAGGTCGCCGTAGTAAG 122 Serine proteinase ACGTTCTCACGACTGGTCACACTATGTAAGGCGCGTCGTTCTC 107 Superoxide dismutase TCATGCTTTGCCACCTCTCCCGCTTCAACCAACTTCTTC 143 β-actin CCACGAGACCACCTACAACAGCGAGGGCAGTGATTTC 142
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表 3 β-1,3-葡聚糖对凡纳滨对虾生长性能的影响
Table 3 Effects of dietary β-1,3-glucan on growth performance of litopenaeus vannamei
项目Item G0 G1 G2 G3 初始均重Initial body weight (g) 0.43±
0.0010.42±
0.0020.43±
0.0020.43±
0.002终末均重Final body weight (g) 5.31±
0.195.62±
0.035.71±
0.215.13±
0.29增重率Weight gain rate (%) 1141±
471218±
181239±
471108±
63特定生长率Specific growth rate (%/d) 2.99±
0.0463.07±
0.0173.09±
0.412.96±
0.62存活率Survival rate (%) 64.82±
6.2379.98±
8.1280.12±
5.7576.56±
6.38注: 上标不同的同行数据间差异显著(P<0.05); 下同Note: Values in each row with different letters are significantly different (P<0.05). The same applies below
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表 4 β-1,3-葡聚糖对凡纳滨对虾肝胰腺消化酶活性的影响
Table 4 Effects of dietary β-1,3-glucan on activities of digestive enzymes in hepatopancreas of Litopenaeus vannamei (U/mg prot)
项目Item G0 G1 G2 G3 蛋白酶Protease 3553.77±
467.73ab2356.34±
616.84a3275.07±
359.06ab4250.97±
757.18b脂肪酶Lipase 6.76±1.57a 6.53±2.84a 9.69±1.01b 11.96±1.05c 淀粉酶Amylase 46.86±2.97 48.97±8.22 49.04±2.07 49.54±2.32
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表 5 β-1,3-葡聚糖对凡纳滨对虾体成分的影响(%DM)
Table 5 Effects of dietary β-1,3-glucan on body composition of Litopenaeus vannamei
项目Item G0 G1 G2 G3 水分Moisture 76.68±
0.2476.98±
0.0976.12±
0.0876.74±
0.13粗蛋白Crude protein 70.85±
0.4471.00±
0.3470.26±
0.6169.73±
0.25粗脂肪Crude lipid 5.39±
0.426.00±
0.155.80±
0.235.87±
0.02灰分Ash 13.89±
0.3213.57±
0.2014.06±
0.4714.17±
0.19
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