• ISSN 1000-0615
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Volume 46 Issue 2
Feb.  2022
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Optimum dietary leucine requirement of juvenile sea cucumber Apostichopus japonicus

  • Corresponding author: WANG Jiying, ytwjy@126.com
  • Received Date: 2021-01-04
    Accepted Date: 2021-02-19
    Available Online: 2021-06-16
  • A 60-d feeding trial was conducted to determine the dietary leucine requirement of juvenile sea cucumber Apostichopus japonicus with initial body weight (16.40±0.14) g. Six experimental diets were formulated with the graded leucine levels 1.29% (D1, control group), 1.63% (D2), 1.98% (D3), 2.22% (D4), 2.58% (D5) and 2.97% (D6) dry diets. The results showed that: There were no differences in survival rate among all groups (96.00%-98.67%). Both weight growth rate (WGR) and the specific growth rate (SGR) increased with increasing leucine content until reaching peak levels at 1.98% dietary leucine, but decreased thereafter. The WGR and SGR of D3, D4 and D5 groups were significantly higher than D1 group. The WGR reached maximum value of 100.84% in the D3 group. There were no significant effects on ratio of intestine weight to body wall weight (IBR) and ratio of intestine length to body length (IBL) of sea cucumber. The crude lipid contents of body wall was increased when dietary leucine content increased from 1.29% to 1.98% but decreased when the dietary leucine content exceeded 1.98%, and D3 group was significantly higher than other groups, but there were no significant effects on moisture, crude protein and crude ash contents. Both methionine and leucine content of body wall were significantly increased by dietary leucine, but there were no significant effects on total amino acids (TAA). Both lipase and protease activities of intestinal were increased with increasing levels of leucine up to 1.98% diet and then decreased. The lipase of D3 group was significantly higher than other groups, meanwhile, the protease of D2, D3 and D4 groups were significantly higher than other groups. All of alanine aminotransferase (ALT), asparate aminotransferase (AST) and total antioxidant capacity (T-AOC) activities of intestinal were increased when dietary leucine content increased from 1.29% to 1.98%. There were no significant effects on AST activity when the dietary leucine content exceeded 1.98%, but the ALT and T-AOC activities were decreased. The T-AOC activity of D3 group was significantly higher than D1, D2, D5 and D6 groups. The catalase (CAT) and superoxide dismutase (SOD) activities reached maximum value when the dietary leucine content was 2.22%. The CAT activity of D4 was significantly higher than D3, D5 and D6 groups, The SOD activity of D4 was significantly higher than D1, D2 and D6 groups. The malondialdehyde (MDA) content was decreased when dietary leucine content increased from 1.29% to 1.98% but increased when the dietary leucine content exceeded 2.22%, the MDA contents of D3 and D4 groups were significantly lower than other groups. With WGR as evaluation indicator, quadratic regression analysis showed that the optimum dietary leucine requirement of sea cucumber with body weight 16.40 g was 2.11% diet (10.37% dietary protein).
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Optimum dietary leucine requirement of juvenile sea cucumber Apostichopus japonicus

    Corresponding author: WANG Jiying, ytwjy@126.com
  • 1. Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Centre for Research on Environmental Ecology and Fish Nutrion of the Ministry of Agriculture and Rural Affairs, National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
  • 2. Key Laboratory of Marine Ecological Restoration, Shandong Marine Resource and Environment Research Institute, Yantai 264006, China

Abstract: A 60-d feeding trial was conducted to determine the dietary leucine requirement of juvenile sea cucumber Apostichopus japonicus with initial body weight (16.40±0.14) g. Six experimental diets were formulated with the graded leucine levels 1.29% (D1, control group), 1.63% (D2), 1.98% (D3), 2.22% (D4), 2.58% (D5) and 2.97% (D6) dry diets. The results showed that: There were no differences in survival rate among all groups (96.00%-98.67%). Both weight growth rate (WGR) and the specific growth rate (SGR) increased with increasing leucine content until reaching peak levels at 1.98% dietary leucine, but decreased thereafter. The WGR and SGR of D3, D4 and D5 groups were significantly higher than D1 group. The WGR reached maximum value of 100.84% in the D3 group. There were no significant effects on ratio of intestine weight to body wall weight (IBR) and ratio of intestine length to body length (IBL) of sea cucumber. The crude lipid contents of body wall was increased when dietary leucine content increased from 1.29% to 1.98% but decreased when the dietary leucine content exceeded 1.98%, and D3 group was significantly higher than other groups, but there were no significant effects on moisture, crude protein and crude ash contents. Both methionine and leucine content of body wall were significantly increased by dietary leucine, but there were no significant effects on total amino acids (TAA). Both lipase and protease activities of intestinal were increased with increasing levels of leucine up to 1.98% diet and then decreased. The lipase of D3 group was significantly higher than other groups, meanwhile, the protease of D2, D3 and D4 groups were significantly higher than other groups. All of alanine aminotransferase (ALT), asparate aminotransferase (AST) and total antioxidant capacity (T-AOC) activities of intestinal were increased when dietary leucine content increased from 1.29% to 1.98%. There were no significant effects on AST activity when the dietary leucine content exceeded 1.98%, but the ALT and T-AOC activities were decreased. The T-AOC activity of D3 group was significantly higher than D1, D2, D5 and D6 groups. The catalase (CAT) and superoxide dismutase (SOD) activities reached maximum value when the dietary leucine content was 2.22%. The CAT activity of D4 was significantly higher than D3, D5 and D6 groups, The SOD activity of D4 was significantly higher than D1, D2 and D6 groups. The malondialdehyde (MDA) content was decreased when dietary leucine content increased from 1.29% to 1.98% but increased when the dietary leucine content exceeded 2.22%, the MDA contents of D3 and D4 groups were significantly lower than other groups. With WGR as evaluation indicator, quadratic regression analysis showed that the optimum dietary leucine requirement of sea cucumber with body weight 16.40 g was 2.11% diet (10.37% dietary protein).

  • 亮氨酸(Leu)是水生动物的必需氨基酸之一,分子式为C6H13NO2,与异亮氨酸(Ile)和缬氨酸(Val)被统称为支链氨基酸。亮氨酸是支链氨基酸中唯一的生酮氨基酸,在机体合成蛋白质、能量代谢、葡萄糖平衡等方面具有重要作用,是一种功能性氨基酸[1]。亮氨酸主要在肌肉部位氧化,是合成机体蛋白质的原料,可以抑制蛋白质降解和促进肌肉蛋白合成[2]。饲料中添加的游离氨基酸与蛋白态氨基酸吸收不同步是导致鱼虾类对游离氨基酸利用效果不佳的主要原因,对晶体氨基酸进行包膜缓释处理可改善晶体氨基酸的作用效果[3]。研究表明,亮氨酸能够增强团头鲂(Megalobrama amblycephala)[4]、尼罗罗非鱼(Oreochromis niloticus)[5]、大黄鱼(Larimichthys crocea)[6]、三疣梭子蟹(Portunus trituberculatus)[7]、斑节对虾(Penaeus monodon)[8]等水产动物的生长性能、抗氧化及免疫能力,并确定了其对亮氨酸的需求量。

    仿刺参(Apostichopus japonicus)属于棘皮动物门(Echinodermata)海参纲(Holothuroidea),被列为“海八珍”之一,具有很好的食用及药用价值,是我国渤海、黄海海域重要的海水养殖品种[9]。对仿刺参支链氨基酸的营养需求研究中,韩秀杰等[3]报道了其对异亮氨酸和缬氨酸的最适需求量,而亮氨酸作为支链氨基酸中唯一的生酮氨基酸,其对仿刺参生长、消化及免疫能力影响的研究尚未见报道。研究仿刺参对亮氨酸的最适需求,对配制营养均衡、环境友好的配合饲料有重要的指导意义。因此,本实验通过在饲料中添加不同含量的亮氨酸,研究其对仿刺参幼参的生长、消化酶活性及抗氧化能力的影响,从而为仿刺参配合饲料的合理配制提供依据。

1.   材料与方法
  • 本实验以鱼粉、藻粉和谷朊粉为主要蛋白源,以鱼油和大豆卵磷脂为主要脂肪源设计粗蛋白质为20.00%、粗脂肪为2.70%的基础饲料配方,在基础饲料中分别添加0.00%、0.80%、1.60%、2.40%、3.20%和4.00%的包膜亮氨酸,配成饲料亮氨酸含量分别为1.29%、1.63%、1.98%、2.22%、2.58%和2.97%的6组等氮等脂的实验饲料,命名为D1(对照组)、D2、D3、D4、D5和D6组(表1表2)。

    项目 items组别 groups
    D1D2D3D4D5D6
    原料/% ingredient
    鱼粉 fish meal 7.00 7.00 7.00 7.00 7.00 7.00
    藻粉 algae powder 30.00 30.00 30.00 30.00 30.00 30.00
    花生粕 peanut meal 5.00 5.00 5.00 5.00 5.00 5.00
    谷朊粉 wheat gluten 7.00 7.00 7.00 7.00 7.00 7.00
    包膜甘氨酸 coated glycine 4.80 4.00 3.20 2.40 1.60 0.80
    包膜亮氨酸 coated leucine 0.00 0.80 1.60 2.40 3.20 4.00
    维生素预混料 vitamins premix1) 1.00 1.00 1.00 1.00 1.00 1.00
    矿物质预混料 minerals premix2) 1.00 1.00 1.00 1.00 1.00 1.00
    抗氧化剂 antioxidant 0.10 0.10 0.10 0.10 0.10 0.10
    鱼油 fish oil 1.00 1.00 1.00 1.00 1.00 1.00
    大豆卵磷脂 soybean lecithin 1.00 1.00 1.00 1.00 1.00 1.00
    海泥 sea mud 42.10 42.10 42.10 42.10 42.10 42.10
    合计 total 100.00 100.00 100.00 100.00 100.00 100.00
    营养组成 (干物质) nutrient composition (dry matter basis)
    粗蛋白质/% crude protein 20.46 20.19 20.43 20.42 20.32 20.17
    粗脂肪/%  crude lipid 2.75 2.73 2.63 2.72 2.68 2.66
    粗灰分/% crude ash 53.87 53.81 55.11 54.48 55.41 54.52
    能量/(KJ/g) gross energy 7.90 7.95 7.96 8.02 8.13 8.21
    注:1)维生素预混料(mg/kg 或 IU/kg 饲料)为维生素A 7 500.00 IU,维生素D 1 500.00 IU,维生素E 60.00 mg,维生素K3 18.00 mg,维生素B1 12.00 mg,维生素B2 12.00 mg,维生素B12 0.10 mg,泛酸 48.00 mg,烟酸 90.00 mg,叶酸 3.70 mg,D-生物素 0.20 mg,吡哆醇 60.00 mg,维生素C 310.00 mg;2)矿物质预混料(mg/kg 饲料)为 锌 35.00 mg,锰 21.00 mg,铜 8.30 mg,铁 23.00 mg,钴 1.20 mg,碘 1.00 mg,硒 0.30 mg
    Notes: 1) Vitamin premix(mg/kg or IU/kg diet) are vitamin A 7 500.00 IU, vitamin D 1 500.00 IU, vitamin E 60.00 mg, vitamin K3 18.00 mg, vitamin B1 12.00 mg, vitamin B2 12.00 mg, vitamin B12 0.10 mg, pantothenate acid 48.00 mg, nicotinic acid 90.00 mg, folic acid 3.70 mg, D-biotin 0.20 mg, pyridoxine 60.00 mg, vitamin C 310.00 mg; 2) mineral premix (mg/kg diet) are Zn 35.00 mg, Mn 21.00 mg, Cu 8.30 mg, Fe 23.00 mg, Co 1.20 mg, I2 1.00 mg, Se 0.30 mg

    Table 1.  Composition and nutrient level of the experimental diets (air-dry basis)

    氨基酸 amino acids
    组别 groups
    D1D2D3D4D5D6
    亮氨酸 Leu 1.29 1.63 1.98 2.22 2.58 2.97
    甘氨酸 Gly 3.13 2.97 2.52 2.18 1.73 1.33
    天冬氨酸 Asp 1.33 1.29 1.37 1.28 1.35 1.36
    苏氨酸 Thr 0.66 0.67 0.69 0.64 0.66 0.67
    丝氨酸 Ser 0.82 0.85 0.86 0.84 0.84 0.86
    谷氨酸 Glu 3.86 4.01 4.17 4.00 4.08 4.09
    丙氨酸 Ala 1.08 0.85 1.02 0.84 1.09 0.86
    半胱氨酸 Cys 0.64 0.66 0.62 0.70 0.64 0.63
    缬氨酸 Val 0.69 0.74 0.75 0.73 0.71 0.75
    蛋氨酸 Met 0.27 0.22 0.14 0.32 0.20 0.09
    异亮氨酸 Ile 0.52 0.58 0.59 0.58 0.56 0.58
    酪氨酸 Tyr 0.89 0.93 0.87 0.90 0.87 0.88
    苯丙氨酸 Phe 1.19 1.20 1.21 1.20 1.21 1.22
    赖氨酸 Lys 0.76 0.76 0.79 0.76 0.76 0.77
    脯氨酸 Pro 1.05 1.14 1.06 1.11 1.10 1.10
    组氨酸 His 0.29 0.31 0.31 0.30 0.31 0.32
    精氨酸 Arg 0.88 0.92 0.93 0.91 0.91 0.90
    总氨基酸 TAA 19.34 19.69 19.87 19.50 19.59 19.39

    Table 2.  Amino acids composition of experimental diets (dry matter basis) %

    实验所用亮氨酸购自上海麦克林生化科技有限公司(纯度≥99%),β-环状糊精购自孟州市华兴生物化工有限责任公司,参考胡友军等[10]的方法对亮氨酸进行包被,包被后的有效亮氨酸含量约为48%。实验所用固体原料经超微粉碎后过200目标准筛,按配方配比称重,加入鱼油及适量蒸馏水充分混匀,用小型颗粒饲料挤压机制成直径为0.20 cm、长度为0.30 cm的颗粒,60 °C烘干,用小型粉碎机破碎,筛选粒度20~100目的颗粒备用。

  • 养殖实验在山东省海洋资源与环境研究院东营实验基地循环水养殖系统中进行,实验时间为2019年11月6日至2020年1月4日,共计60 d。实验用仿刺参幼参购自山东安源种业科技有限公司。正式实验前,将仿刺参幼参置于养殖系统中用对照组饲料驯养2周,停食24 h后,挑选参刺坚挺粗壮、体质量为[(16.40 ± 0.14) g]的仿刺参幼参450头随机分配到18个圆柱形循环水桶(h80 cm,h65 cm,d60 cm)中,每桶放置1个海参养殖筐。每组饲料随机投喂3个桶,每天定时(16:00)饱食投喂1次,初始投喂量为仿刺参幼参体质量的3%,每天观察仿刺参的摄食情况,根据摄食情况调整次日投喂量。每3天换水1次,换水量为养殖桶内水位的1/2,换水时用虹吸软管将桶底残饵及粪便吸出,养殖实验进行1个月时更换海参养殖筐1次并彻底清洗循环水桶。养殖期间,控制水流流速约2 L/min,水温13~17 °C,溶解氧含量>6 mg/L,pH 7.5~8.2,盐度 28,氨氮、亚硝酸盐含量≤0.05 mg/L,室内保持弱光环境。

    养殖实验结束后,停食24 h,将每桶的仿刺参全部捞出称总重并记录仿刺参数量,计算增重率、特定生长率、成活率等。每桶随机选取10头仿刺参,放置于白色托盘中,待其身体自然舒展后,测量其体长及体质量,然后解剖,收集体壁及肠道,测量体壁重、肠道重及肠道长,用于计算肠壁比及肠长比。体壁保存于−20 °C,肠道保存于−80 °C待测。

  • 成活率(survival rate, SR, %) = Nt/N0 × 100%;

    增重率(weight gain rate, WGR, %) = (WtW0)/W0 × 100%;

    特定生长率(specific growth rate, SGR, %/d) = (lnWt−lnW0)/t × 100%;

    肠壁比(ratio of intestine weight to body wall weight, IBR, %) = Wi/Ww × 100%;

    肠长比(ratio of intestine length to body length, IBL) = Lg/Lb

    式中,Nt为仿刺参终末头数,N0为仿刺参初始头数,Wt为仿刺参终末体质量(final body weight, FBW)(g),W0为仿刺参初始体质量(initial body weight,IBW)(g),t为养殖实验天数(d),Wi为取样仿刺参肠道重 (g),Ww为取样仿刺参体壁重 (g),Lg为取样仿刺参肠道长度(cm),Lb为取样仿刺参体长度(cm)。

    实验饲料及仿刺参体壁水分含量采用105 °C恒重法测定(GB/T 6435—2014),粗蛋白质含量采用凯氏定氮法测定(GB/T 6432—2018),粗脂肪含量采用索氏抽提法测定(GB/T 6433—2006),粗灰分含量采用550 °C马弗炉灼烧法测定(GB/T 6438—2007),氨基酸含量采用酸水解法(GB/T 18246—2019)使用全自动氨基酸测定仪(Hitachi L-8900,Japan)测定,饲料能量采用燃烧法使用量热仪(IKA,C6000,Germany)测定。

    肠道中淀粉酶(amylase)、脂肪酶(lipase)、蛋白酶(protease)、酸性磷酸酶(acid phosphatase,ACP)、碱性磷酸酶(alkaline phosphatase,AKP)、谷草转氨酶(asparate aminotransferase,AST)、谷丙转氨酶(alanine aminotransferase,ALT)、总抗氧化能力(total antioxidant capacity,T-AOC)、过氧化氢酶(catalase,CAT)、丙二醛(malondialdehyde,MDA)和超氧化物歧化酶(superoxide dismutase,SOD)活性均使用南京建成生物工程研究所生产的试剂盒进行测定,各种酶活性单位参照试剂盒说明书表示。

  • 实验数据用SPSS Statistics 17.0软件进行单因素方差分析(One-Way ANOVA),并用Duncan氏检验进行多重比较分析,当P<0.05时表示具有显著性差异,统计结果用平均值±标准差(mean ± SD)表示。采用一元二次回归分析,确定仿刺参幼参对亮氨酸的最适需求量。

2.   结果
  • 各组仿刺参之间的成活率(SR)无显著性差异(P>0.05),成活率为96.00%~98.67%;随饲料亮氨酸含量从1.29%提高到1.98%,仿刺参幼参的增重率(WGR)和特定生长率(SGR)显著升高(P<0.05),而随着亮氨酸含量进一步提高,WGR和SGR呈现下降的趋势,WGR在D3组达到最大值100.84%,D3、D4和D5组WGR显著高于対照组(P<0.05),且3组之间无显著性差异(P>0.05)。各组间肠壁比(IBR)和肠长比(IBL)均无显著性差异(P>0.05)(表3)。

    项目 items组别 groups
    D1D2D3D4D5D6
    初始体质量/g IBW 16.40±0.019 16.40±0.015 16.41±0.009 16.41±0.011 16.40±0.006 16.39±0.012
    终末体质量/g FBW 31.05±0.43a 31.68±0.47ab 32.95±0.66c 32.48±0.41bc 32.15±0.67bc 30.65±0.43ab
    增重率/% WGR 89.32±2.77a 93.18±2.90ab 100.84±4.01c 97.91±2.39bc 96.08±3.97bc 86.99±2.97ab
    特定生长率/(%/d) SGR 1.06±0.03a 1.10±0.02ab 1.16±0.04c 1.14±0.02bc 1.12±0.04bc 1.04±0.02ab
    肠壁比/% IBR 5.73±0.39 5.65±0.50 5.83±0.35 5.77±0.42 5.81±0.42 5.78±0.54
    肠长比 IBL 3.49±0.15 3.47±0.15 3.53±0.18 3.50±0.20 3.53±0.18 3.53±0.21
    成活率/% SR 96.00±4.00 97.33±4.62 97.33±4.62 96.00±6.93 98.67±2.31 96.00±4.00
    注:同行数据不同上标字母表示差异显著(P<0.05),下同
    Notes: In the same row, different superscript letters represent significant differences (P<0.05), the same below

    Table 3.  Effects of dietary leucine on growth performance and body index of A. japonicus

    以增重率为评价指标,经一元二次回归分析得出,体质量为(16.40 ± 0.14) g的仿刺参幼参对饲料中亮氨酸的最适需求量为2.11%(10.37%饲料粗蛋白质)(图1)。

    Figure 1.  Regression analysis between dietary leucine level and weight gain rate of A. japonicus

  • 随饲料亮氨酸含量从1.29%提高到1.98%,仿刺参体壁的粗脂肪含量显著升高(P<0.05),在D3组达到最大值 (5.50 ± 0.15)%,且显著高于其他组(P<0.05),随亮氨酸含量进一步提高,仿刺参体壁粗脂肪含量显著降低(P<0.05);各组间水分、粗蛋白质和粗灰分含量均无显著性差异(P>0.05)(表4)。

    项目 items
    组别 groups
    D1D2D3D4D5D6
    水分 moisture 91.88±0.39 92.16±0.28 91.54±0.36 92.25±0.14 92.13±0.48 91.86±0.44
    粗蛋白质 crude protein 44.61±1.22 44.89±0.82 45.10±0.95 44.61±0.25 44.22±1.35 44.23±0.78
    粗脂肪 crude lipid 4.49±0.28a 4.89±0.37b 5.50±0.15c 4.18±0.03a 4.35±0.12a 4.21±0.13a
    粗灰分 crude ash 32.36±0.52 32.39±0.89 30.49±1.58 32.20±0.36 32.72±1.77 32.67±1.32
    注:粗灰分、粗蛋白质和粗脂肪含量为干基含量
    Notes: The crude ash, crude protein and crude lipid contents of body wall are based on dry basis

    Table 4.  Effects of dietary leucine on approximate composition of body wall of A. japonicus %

    随饲料亮氨酸含量的升高,仿刺参体壁蛋氨酸 (Met)含量显著提高(P<0.05),体壁酪氨酸(Tyr)、丙氨酸(Ala)和脯氨酸(Pro)含量呈先升高后降低趋势(P<0.05),而苯丙氨酸和甘氨酸呈先降低后升高趋势(P<0.05);各组间体壁总氨基酸含量无显著性差异(P>0.05)(表5)。

    项目 items
    组别 groups
    D1D2D3D4D5D6
    精氨酸 Arg 2.91±0.05ab 2.99±0.07b 2.92±0.04b 2.95±0.06b 3.00±0.02b 2.82±0.05a
    组氨酸 His 0.62±0.02ab 0.62±0.04ab 0.60±0.06a 0.65±0.01b 0.62±0.01ab 0.60±0.02a
    苏氨酸 Thr 2.37±0.04 2.39±0.06 2.45±0.09 2.38±0.05 2.42±0.03 2.38±0.07
    异亮氨酸 Ile 1.28±0.05ab 1.31±0.01ab 1.30±0.02ab 1.31±0.04ab 1.33±0.02b 1.26±0.06a
    亮氨酸 Leu 2.27±0.02a 2.30±0.03ab 2.31±0.02ab 2.32±0.01b 2.31±0.04ab 2.32±0.02b
    赖氨酸 Lys 1.92±0.06 1.96±0.07 1.91±0.07 1.99±0.01 1.93±0.05 1.91±0.05
    缬氨酸 Val 1.89±0.07 1.96±0.02 1.93±0.06 1.96±0.02 1.97±0.05 1.88±0.06
    蛋氨酸 Met 0.76±0.01a 0.85±0.06b 1.08±0.04cd 1.02±0.03c 1.02±0.05c 1.11±0.01d
    半胱氨酸 Cys 1.55±0.01 1.61±0.05 1.58±0.09 1.63±0.03 1.65±0.09 1.58±0.04
    苯丙氨酸 Phe 2.52±0.07d 1.61±0.03a 2.04±0.03b 2.03±0.03b 1.61±0.06a 2.28±0.09c
    谷氨酸 Glu 6.25±0.05bc 6.32±0.14c 6.09±0.10a 6.23±0.02b 6.26±0.02bc 6.24±0.03b
    甘氨酸 Gly 5.33±0.04bc 5.46±0.14c 5.11±0.12a 5.26±0.05ab 5.33±0.06bc 5.34±0.10bc
    酪氨酸 Tyr 2.14±0.08ab 2.12±0.04ab 2.23±0.08b 2.26±0.05b 2.15±0.09ab 2.06±0.08a
    天冬氨酸 Asp 4.36±0.08a 4.46±0.11ab 4.55±0.08b 4.46±0.08ab 4.47±0.05ab 4.40±0.10ab
    丙氨酸 Ala 2.53±0.06a 2.63±0.06bc 2.68±0.07c 2.59±0.05abc 2.56±0.02ab 2.51±0.04a
    脯氨酸 Pro 2.89±0.08c 3.01±0.06d 2.76±0.07ab 2.83±0.01bc 2.84±0.03bc 2.70±0.06a
    丝氨酸 Ser 2.28±0.04a 2.32±0.02a 2.52±0.07b 2.26±0.05a 2.25±0.03a 2.33±0.09a
    总氨基酸 TAA 43.87±0.78 43.95±0.57 44.06±0.84 44.14±0.37 43.70±0.48 43.72±0.33

    Table 5.  Effects of dietary leucine on amino acid composition of body wall of A. japonicus %

  • 饲料亮氨酸含量在2.22%及以下时,仿刺参肠道淀粉酶活性呈现平稳的趋势,当亮氨酸含量达到2.97%时,淀粉酶活性显著降低(P<0.05),D2、D3和D4组淀粉酶活性显著高于D6组(P<0.05);随饲料亮氨酸含量从1.29%提高到1.98%,仿刺参肠道脂肪酶和蛋白酶活性显著提高(P<0.05),随饲料亮氨酸含量进一步提高,脂肪酶和蛋白酶活性均显著降低(P<0.05),D3组脂肪酶活性显著高于其他组(P<0.05),D2、D3和D4组蛋白酶活性显著高于其他组(P<0.05)(表6)。

    项目 items组别 groups
    D1D2D3D4D5D6
    淀粉酶/(U/mg prot) amylase 1.04±0.05ab 1.14±0.07b 1.14±0.11b 1.14±0.10b 1.06±0.11ab 0.96±0.07a
    脂肪酶/(U/g prot) lipase 1.05±0.05a 1.20±0.10b 1.95±0.06e 1.35±0.04d 1.27±0.07bc 1.29±0.03cd
    蛋白酶/(U/mg prot) protease 187.97±17.77a 210.01±18.27b 223.89±15.97b 216.06±17.36b 187.16±19.25a 168.61±8.67a
    酸性磷酸酶/(U/g prot) ACP 93.81±1.22abc 95.09±8.51bc 100.93±10.35c 99.22±8.03bc 91.24±5.45ab 85.62±5.06a
    碱性磷酸酶/(U/g prot) AKP 349.87±15.00b 359.82±42.99b 379.53±23.44b 362.98±27.28b 308.62±26.92a 302.77±16.05a
    谷草转氨酶/(U/g prot) AST 0.80±0.06a 0.92±0.07b 1.11±0.08c 1.16±0.15c 1.12±0.13c 1.06±0.11c
    谷丙转氨酶/(U/g prot) ALT 2.55±0.15a 3.19±0.16c 3.80±0.32d 2.97±0.18bc 3.08±0.18c 2.80±0.18b
    总抗氧化能力/(nmol/mg prot) T-AOC 75.99±1.47a 81.83±1.53ab 101.19±4.23d 95.30±7.98cd 89.47±3.72bc 88.01±5.37bc
    过氧化氢酶/(U/g prot) CAT 500.05±22.80bc 497.71±26.85bc 492.34±22.80b 524.61±24.16c 447.00±22.87a 434.02±21.95a
    丙二醛/(nmol/mg prot) MDA 3.38±0.14d 2.40±0.17b 1.82±0.18a 1.98±0.18a 2.36±0.10b 3.09±0.14c
    超氧化物歧化酶/(U/mg prot) SOD 15.43±0.71a 16.02±1.27a 16.24±0.71ab 17.54±1.41b 16.22±1.22ab 16.05±1.14a

    Table 6.  Effects of dietary leucine on intestinal antioxidant and digestive enzymes of A. japonicus

    饲料亮氨酸含量不超过2.22%时,仿刺参肠道ACP和AKP活性各组间无显著性差异(P>0.05),当超过2.22%时,ACP和AKP活性显著降低(P<0.05),ACP和AKP活性均在D3组有最大值,D3组ACP活性显著高于D5、D6组(P<0.05),D5、D6组AKP活性显著低于其他组(P<0.05);随饲料亮氨酸含量从1.29%提高到1.98%,AST、ALT和T-AOC活性显著升高(P<0.05),超过1.98%后,AST活性趋于平稳,而ALT和T-AOC活性显著降低(P<0.05),各实验组AST活性显著高于对照组(P<0.05),D3组ALT活性显著高于其他组(P<0.05),D3组T-AOC活性显著高于D1、D2、D5和D6组(P<0.05);当饲料亮氨酸含量为2.22%时,CAT和SOD活性有最大值,D4组CAT活性显著高于D3、D5和D6组(P<0.05),SOD活性显著高于D1、D2和D6组(P<0.05);随饲料亮氨酸含量从1.29%提高到1.98%,MDA含量显著降低(P<0.05),当饲料亮氨酸含量超过2.22%后,MDA含量显著升高(P<0.05),D3和D4组MDA含量显著低于其他组(P<0.05)(表6)。

3.   讨论
  • 亮氨酸是水产动物的必需氨基酸,许多研究表明,饲料中添加适量亮氨酸可提高水产动物的生长性能[11]。本实验中,随饲料亮氨酸含量从1.29%提高到1.98%,仿刺参幼参的WGR和SGR均显著升高,当饲料中亮氨酸含量达到1.98%时,WGR达到最大值100.84%。这是因为饲料中缺乏亮氨酸,影响氨基酸平衡,体内过多的其他氨基酸无法被有效利用而被机体代谢,抑制了体蛋白的合成,不利于水产动物的生长,饲料中添加适量的亮氨酸能改善氨基酸平衡,促进其生长。而随着亮氨酸含量进一步提高,仿刺参幼参的WGR和SGR呈现下降的趋势,仿刺参幼参生长受到抑制,在吉富罗非鱼 (GIFT O. niloticus)[12]、卵形鲳鲹(Trachinotus ovatus)[13]、中国花鲈(Lateolabrax maculatus)[14]、三疣梭子蟹[7]和斑节对虾[8]等水产动物的研究中也得到类似的结果,饲料中添加适量的亮氨酸有利于其生长,而过量的亮氨酸会抑制其生长性能。其原因可能:①亮氨酸与异亮氨酸、缬氨酸统称为支链氨基酸,支链氨基酸之间存在拮抗作用,过量的亮氨酸在小肠壁处与异亮氨酸和缬氨酸竞争载体,影响了异亮氨酸和缬氨酸的吸收和转运,降低了氨基酸的利用率;②亮氨酸过量再次影响了氨基酸平衡,过量亮氨酸提高了支链氨基酸转氨酶和支链氨基酸脱氢酶活性,使机体消耗能量将多余的亮氨酸代谢及排出,同时这2种酶也增强了异亮氨酸和缬氨酸的代谢,引起异亮氨酸和缬氨酸缺乏,影响机体生长[15];③亮氨酸是生酮氨基酸,过量的亮氨酸在代谢过程中产生了较多的酮类和其他有害代谢物,从而抑制了水产动物的生长[11]。但是虹鳟(Oncorhynchus mykiss)[16]、草鱼(Ctenopharyngodon idella)[15]和异育银鲫(Carassius auratus gibelio)[17]的研究显示,过量的亮氨酸并没有对其生长性能产生显著的抑制作用,这可能是不同物种对亮氨酸的耐受力不同导致的。因此,饲料中亮氨酸含量对不同水产动物生长性能影响的作用机理有待进一步研究。

    以WGR为评价指标,经一元二次回归分析得出,(16.40 ± 0.14) g的仿刺参幼参对饲料中亮氨酸的最适需求量为2.11%,占饲料粗蛋白质的10.37%。该数值(占饲料粗蛋白质)远高于一般鱼、虾、蟹类水产动物对亮氨酸的需求[18],如大黄鱼(6.79%)[6]、斑节对虾(4.3%)[8]、凡纳滨对虾(Litopenaeus vannamei)(5.71%)[19]、三疣梭子蟹(5.14%)[7]、中华绒螯蟹(Eriocheir sinensis)(5.88%)[2]等,该结果与仿刺参对精氨酸[20]、赖氨酸[21]和缬氨酸[3]等氨基酸需求量的研究结果相似。其原因可能:①仿刺参对饲料粗蛋白质的需求低于一般鱼、虾、蟹类水产动物[22-23],而在仿刺参体壁的10种必需氨基酸中,亮氨酸是含量最高的3种氨基酸之一,仅次于精氨酸,与苏氨酸含量相近,补充大量的亮氨酸才能满足仿刺参的快速增长[20];②不同物种对亮氨酸需求量的差异,可能与实验对象、生长阶段、评价指标、蛋白质来源和饲养管理等因素有关;③仿刺参与一般鱼、虾、蟹类相比,具有独特的摄食习性—舔食性,摄食活动持续时间长,饲料与水体接触面积大,且在水中停留时间远远长于鱼、虾、蟹类饲料,实验所用晶体亮氨酸,存在一定的溶失现象。

  • 已有研究证明,亮氨酸可以激活mTOR通路,促进氨基酸合成蛋白质,并降低机体蛋白质的分解代谢,从而起到蛋白质沉积的作用,提高水产动物机体蛋白质的含量[24-25]。在中华绒螯蟹[2]、吉富罗非鱼[12]和卵形鲳鲹[13]的研究中,饲料中添加适量亮氨酸,都显著提高了全鱼和肌肉的粗蛋白质含量。但也有研究得出不同的结论,如在凡纳滨对虾[19]、日本花鲈 (L. japonicus)[26]和大黄鱼[26]上的研究表明,饲料亮氨酸水平对其机体粗蛋白质含量无显著影响,这与本实验结果一致,表明仿刺参蛋白质的合成不仅受亮氨酸水平的影响,仿刺参机体还具有维持体壁蛋白质恒定的能力。在不同水产动物的亮氨酸需求的研究中,出现以上不同实验结果的原因可能是实验对象、生长阶段、生长速率等因素的差异。支链氨基酸对于机体的脂肪代谢具有重要的调控作用,能够显著影响机体的脂肪代谢和沉积。机体缺乏亮氨酸会激活脂肪组织的解偶联蛋白1(UCP-1)和激素敏感性脂肪酶(HSL),促进脂肪脂解产热,导致机体脂肪含量减少[27],而过量亮氨酸会影响氨基酸平衡,导致机体过度消耗能量从而减少机体脂肪的沉积[3]。本研究也证明了这一点,亮氨酸缺乏或过量,均显著降低仿刺参体壁粗脂肪含量,适量的亮氨酸具有提高仿刺参体壁粗脂肪含量的作用。

    随饲料亮氨酸含量的增加,仿刺参体壁必需氨基酸中蛋氨酸含量的增长最为显著;另外,饲料中添加适量的亮氨酸,显著提高了仿刺参体壁酪氨酸、丙氨酸和脯氨酸含量,降低了苯丙氨酸和甘氨酸含量。饲料亮氨酸含量不超过2.58%时,仿刺参体壁精氨酸含量各组间无显著性差异,当亮氨酸含量超过2.58%后精氨酸含量才显著降低,说明只有饲料亮氨酸含量变化比较明显时,部分氨基酸之间才会表现出明显的协同或拮抗作用。各组仿刺参体壁的总氨基酸含量无显著性差异,说明仿刺参机体对自身的氨基酸模式具有一定的调节能力,有维持机体蛋白质恒定的能力。

  • 有研究表明亮氨酸在代谢过程中能够给谷氨酸和谷氨酰胺提供碳源和氮源[28],而谷氨酰胺能够促进蛋白酶、脂肪酶的分泌[29],且亮氨酸代谢产物β-羟基-β-甲基戊二烯二酰CoA是胆固醇的前体物质,而胆固醇可保持细胞膜的流动性,有利于维持肠上皮细胞的完整性,从而保证消化酶的分泌[30]。亮氨酸缺乏显著降低了青鱼(Mylopharyngodon piceus)肠道α-淀粉酶、胰蛋白酶、糜蛋白酶和弹性蛋白酶的活性[31];饲料中添加适量亮氨酸,吉富罗非鱼胃蛋白酶、肠蛋白酶、肠脂肪酶和肠Na+-K+-ATP等酶的活性均得到提高[12],这些研究结果也证明了饲料亮氨酸可以影响水产动物消化酶的活性。本实验中,随饲料亮氨酸含量从1.29%提高到1.98%,仿刺参肠道脂肪酶和蛋白酶活性显著提高,饲料亮氨酸含量进一步提高时,脂肪酶和蛋白酶活性均显著降低,脂肪酶和蛋白酶活性均在D3组达到最大值,表明饲料中添加适量亮氨酸能显著改善仿刺参肠道的消化能力,有利于蛋白质和脂肪的消化吸收利用。同时,因亮氨酸经过包膜处理,延缓了其在水中的溶失及在消化道中的吸收速度,有利于外源添加晶体氨基酸与蛋白态氨基酸的同步吸收,改善其吸收利用能力,从而促进了仿刺参的生长[3]。饲料亮氨酸含量在2.22%及以下时,仿刺参肠道淀粉酶活性呈现平稳的趋势,当亮氨酸含量达到2.97%时,淀粉酶活性显著降低,表明亮氨酸对仿刺参淀粉代谢影响较小,只有亮氨酸含量过高时,才会抑制仿刺参的淀粉代谢,这可能是由于饲料中糖类物质含量较少及仿刺参对糖类物质利用较弱造成的。

    ACP和AKP是重要的水解酶,能催化磷酸单脂的水解及磷酸基团的转移反应,在免疫反应中发挥作用,对动物的生存具有重要的意义,一直以来都是动物免疫学中的一个重要指标[32]。本实验中,饲料亮氨酸含量不超过2.22%时,仿刺参肠道ACP和AKP活性各组间无显著性差异,当亮氨酸含量超过2.22%时,ACP和AKP活性显著降低,ACP和AKP活性均在D3组有最大值,说明只有亮氨酸含量变化明显时,才会显著影响仿刺参肠道ACP和AKP活性,过量亮氨酸能抑制仿刺参的非特异性免疫能力。AST和ALT是氨基酸代谢中起重要作用的2种酶,其活性与氨基酸代谢强弱有关[17]。肝脏是一般鱼类重要营养物质的合成代谢器官,所以在一般鱼类机体中,肝脏中的AST和ALT活性较高。仿刺参作为一种低营养等级的舔食性底栖生物,不具备肝脏等功能性器官[33],因此,其营养物质的合成代谢与鱼类存在较大差异。本实验中,随饲料亮氨酸含量从1.29%提高到1.98%,仿刺参肠道AST和ALT活性显著升高,亮氨酸含量超过1.98%后,AST活性趋于平稳,而ALT活性显著降低,且各实验组AST和ALT活性均显著高于对照组,这表明仿刺参肠道参与了仿刺参营养物质的合成代谢,同时表明了饲料中添加适量亮氨酸能显著增强仿刺参的营养物质代谢能力。

    生物机体正常代谢会产生大量的活性氧,活性氧族如超氧阴离子自由基(${\rm{O}}_2^ - \cdot$)、过氧化氢(H2O2)、羟基自由基(${\rm{HO}}^{-}\cdot$)和单线态氧(1O2)都是有氧代谢的副产物,线粒体是活性氧的一个重要来源,线粒体消耗的氧中有2%被转化成氧自由基[34]。活性氧会对动物机体造成氧化损伤,如对核酸和蛋白质造成氧化损伤,甚至会损伤细胞膜上的多不饱和脂肪酸,使其发生脂质过氧化反应,破坏细胞膜完整性。水生动物的抗氧化能力主要通过机体的自身免疫反应实现,T-AOC、SOD和CAT是生物机体抗氧化酶系统中3个重要的酶,能清除机体内部的活性氧,减轻活性氧毒害,在机体抗氧化中起关键作用[35]。本实验中,当饲料亮氨酸含量为2.22%时,CAT和SOD活性有最大值,D4组CAT活性显著高于D3、D5和D6组,SOD活性显著高于D1、D2和D6组;T-AOC活性随饲料亮氨酸含量的升高呈先升高后降低趋势,D3组T-AOC活性显著高于D1、D2、D5和D6组。这表明在饲料中添加适量的亮氨酸能提高仿刺参抗氧化酶的活性,从而提高仿刺参清除氧自由基能力和抗氧化能力。生物膜中含有大量的多不饱和脂肪酸,极易受氧自由基攻击而形成脂质过氧化产物MDA,所以水产动物体内的MDA含量能间接反映机体的受损伤程度和抗氧化能力[36]。本实验中,随饲料亮氨酸含量从1.29%提高到1.98%,MDA含量显著降低,当饲料亮氨酸含量超过2.22%后,MDA含量显著升高,D3和D4组MDA含量显著低于其他组,这表明饲料中添加适量亮氨酸能减轻活性氧对仿刺参机体的损伤,在团头鲂[4]、卵形鲳鲹[13]和三疣梭子蟹[7]上的研究也得到类似的结果。

4.   结论
  • 综上所述,本实验条件下,以增重率为评价指标,体质量为(16.40±0.14) g的仿刺参幼参对饲料中亮氨酸的最适需求量为2.11%(10.37%饲料粗蛋白质)。饲料中添加适量亮氨酸能有效增强仿刺参幼参对营养物质的消化能力,提高抗氧化及免疫能力,进而促进仿刺参的生长。

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