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Volume 45 Issue 12
Dec.  2021
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Effects of threonine supplementation in poultry by-product meal (petfood grade) replacement diet on growth performance, intermediary metabolism and immunity response in largemouth bass (Micropterus salmoides)

  • Corresponding author: SONG Fei, songfei@m.scnu.edu.cn
  • Received Date: 2020-11-21
    Accepted Date: 2020-12-29
    Available Online: 2021-08-12
  • In order to investigate the effects of different dietary threonine levels on growth performance, glucose and lipid metabolism, immunity and utilization rate of poultry by-product meal (petfood grade) in largemouth bass(Micropterus salmoides), three isonitrogenous and isolipid diets were designed using M. salmoides with an initial body weight of (4.16±0.06) g as the research subjects (TC diet: threonine control group, LT diet: low threonine group and TS diet: threonine supplementation group) were cultured for for 8 weeks. Present results showed that compared with the TC diet, the LT diet significantly decreased the growth performance, feed efficiency and body nutrient composition, threonine addition could promote those differential effects caused by fishmeal totally substituted by poultry by-product meal. Moreover, the LT diet also suppressed the concentration of plasma nutrients and free amino acids compared with the control diet. TS diet had significant effects on elevating the plasma free amino acids concentration and made no notable difference compared to the TC diet. The mRNA expression level demonstrated that threonine supplementation markedly ameliorated the inhibition of anabolism, aggravation of catabolism and cellular inflammation caused by complete replacement of fishmeal with poultry by-product meal. Studies have shown that appropriate threonine supplementation could promote the growth performance by improving nutrient anabolism and immune response and inhibiting catabolism in M. salmoides.
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Effects of threonine supplementation in poultry by-product meal (petfood grade) replacement diet on growth performance, intermediary metabolism and immunity response in largemouth bass (Micropterus salmoides)

    Corresponding author: SONG Fei, songfei@m.scnu.edu.cn
  • 1. College of Life Science, South China Normal University, Institute of Modern Aquaculture Science and Engineering (IMASE), Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, Guangzhou    510631, China
  • 2. Southern Marine Science and Engineering Guangdong Laboratory, Zhuhai    519000, China

Abstract: In order to investigate the effects of different dietary threonine levels on growth performance, glucose and lipid metabolism, immunity and utilization rate of poultry by-product meal (petfood grade) in largemouth bass(Micropterus salmoides), three isonitrogenous and isolipid diets were designed using M. salmoides with an initial body weight of (4.16±0.06) g as the research subjects (TC diet: threonine control group, LT diet: low threonine group and TS diet: threonine supplementation group) were cultured for for 8 weeks. Present results showed that compared with the TC diet, the LT diet significantly decreased the growth performance, feed efficiency and body nutrient composition, threonine addition could promote those differential effects caused by fishmeal totally substituted by poultry by-product meal. Moreover, the LT diet also suppressed the concentration of plasma nutrients and free amino acids compared with the control diet. TS diet had significant effects on elevating the plasma free amino acids concentration and made no notable difference compared to the TC diet. The mRNA expression level demonstrated that threonine supplementation markedly ameliorated the inhibition of anabolism, aggravation of catabolism and cellular inflammation caused by complete replacement of fishmeal with poultry by-product meal. Studies have shown that appropriate threonine supplementation could promote the growth performance by improving nutrient anabolism and immune response and inhibiting catabolism in M. salmoides.

  • 寻求适宜的非鱼粉蛋白源替代鱼粉仍然是水产养殖业至今面临的重要营养学课题。过去的几十年,水产动物研究学者就这一问题开展了大量的探究工作,研究发现宠物级鸡肉粉由于其具有蛋白质含量高、消化吸收率高及适口性好等特点,成为替代鱼粉的优质蛋白源[1-3]。然而,在对肉食性鱼类的研究中发现,高比例宠物级鸡肉粉替代鱼粉后显著降低了水产动物的生长性能和饲料利用效率,究其原因,发现与鱼粉相比,宠物级鸡肉粉缺乏必需氨基酸是造成这一现象的重要原因之一[4]。因此,在饲料中合理补充必需氨基酸,提高非鱼粉蛋白源的利用效率成为解决鱼粉替代问题的重要途径之一。

    苏氨酸 (threonine,Thr) 是哺乳动物以及鱼体自身不能合成的必需氨基酸,也是许多饲料原料的第二、第三限制性氨基酸[5]。苏氨酸不仅可以作为底物直接参与蛋白质的合成,还可以作为营养分子参与机体生长发育、免疫机能和代谢等过程的调控。Zhao等[6]的研究发现,饲料中苏氨酸水平可显著提高杂交鲇[Pelteobagrus vachelli (♀)×Leiocassis longirostris (♂)]垂体生长激素 (GH) 和肝脏胰岛素样生长因子-1 (IGF-1) 的表达,从而促进生长性能。Dong等[7-8]研究表明,苏氨酸缺乏会降低草鱼(Ctenopharyngodon idella)幼鱼的抗病能力,并损伤鱼鳃和肠道的免疫和生理屏障。在对哺乳动物的研究中发现,日粮中苏氨酸缺乏或过量都会降低机体组织中蛋白质的合成,进而影响机体的生长发育[9],鱼类中的研究也得到了同样的结果[10]。因此,适量苏氨酸的摄入是鱼类正常生长发育的重要保障。

    大口黑鲈 (Micropterus salmoides) 俗称“加州鲈”,1983年引入广东省,因其具有生长速率快、抗病性强、肉质鲜美、耐运输等优势成为我国重要的经济鱼类,也成为了近年来产量增速较快的物种之一[11]。然而,前期大口黑鲈的池塘养殖生产主要以投喂冰鲜鱼为主,饵料利用率极低,且水体污染严重[12]。因此,高效配合饲料的开发与优化势在必行。利用氨基酸微平衡技术提高宠物级鸡肉粉在大口黑鲈饲料中的利用率成为解决大口黑鲈营养学产业问题的重要途径之一。本实验旨在研究饲料中宠物级鸡肉粉完全替代鱼粉后苏氨酸添加与否对大口黑鲈饲料利用效率及机体生长、代谢和免疫的影响,以期阐明苏氨酸调控大口黑鲈机体生长代谢的机制,为宠物级鸡肉粉在大口黑鲈饲料产业中的高效利用提供新思路,也为替代蛋白源在水产饲料中的开发与利用提供理论基础。

1.   材料与方法
  • 本实验饲料以鱼粉和宠物级鸡肉粉为主要蛋白源 (其必需氨基酸组成见表1),鱼油和大豆卵磷脂为主要脂肪源,配制3种含不同水平苏氨酸的等氮 (粗蛋白约54.7%) 等脂 (粗脂肪约12.6%) 饲料 (表2)。其中,苏氨酸对照组 (TC)包含65%鱼粉;低水平苏氨酸组 (LT)使用宠物级鸡肉粉完全替代鱼粉,补充除苏氨酸以外的所有必需氨基酸 (EAA) 至鱼粉组水平;苏氨酸添加组 (TS)为LT组饲料补充苏氨酸至TC组水平。饲料原料经320 μm筛网筛选后逐一加入搅拌机中混合,并加入油脂和适量的水后混合均匀。使用制粒机 (华南理工大学F-26,中国广州) 将混合物制成颗粒。饲料颗粒在45 °C下干燥至水分含量为10%左右,储存在−20 °C冰箱中待用。检测3种饲料氨基酸组成 (表3)。

    必需氨基酸
    essential amino acids
    原料 ingredient
    鱼粉
    fish meal
    宠物级鸡肉粉
    poultry by-product meal
    赖氨酸 Lys 5.38 3.88
    蛋氨酸 Met 1.91 1.26
    亮氨酸 Leu 4.88 4.33
    苏氨酸 Thr 2.88 2.45
    异亮氨酸 Ile 2.89 2.46
    缬氨酸 Val 3.30 2.93
    苯丙氨酸 Phe 2.78 2.55

    Table 1.  Essential amino acid composition of the fishmeal and poultry by-product meal (dry weight) %

    项目
    items
    饲料 diets
    TCTSLT
    原料 ingredients
    鱼粉 fish meal 65.00 0.00 0.00
    宠物级鸡肉粉 poultry by-product meal 0.00 65.00 65.00
    低筋麦粉 wheat meal 5.00 5.00 5.00
    谷朊粉 wheat gluten meal 12.00 12.00 12.00
    鱼油 fish oil 4.50 1.50 1.50
    大豆卵磷脂 soybean lecithin 1.50 1.50 1.50
    氨基酸混合物 AA mixture1 0.00 2.79 2.79
    L-苏氨酸 L-threonine 0.00 0.22 0.00
    丙氨酸 alanine 3.01 0.00 0.22
    磷酸一钙 monocalcium phosphate 1.50 1.50 1.50
    VC+VE (1∶1) 0.10 0.10 0.10
    氯化胆碱 choline chloride 0.60 0.60 0.60
    多维 vitamin premix2 0.50 0.50 0.50
    多矿 mineral premix3 1.50 1.50 1.50
    抗氧化剂 antioxidant 0.05 0.05 0.05
    防霉剂 mould inhibitor 0.10 0.10 0.10
    纤维素 cellulose 4.64 7.64 7.64
    营养成分 nutritional ingredient
    干物质 dry matter 89.54 89.91 89.41
    粗蛋白质 crude protein 54.49 54.82 54.49
    粗脂肪 crude lipid 12.13 12.74 12.88
    灰分 ash 15.81 15.83 15.39
    注:鱼粉为蛋白质67.09%,粗脂肪9.47%;宠物级鸡肉粉为粗蛋白质66.10%,粗脂肪12.06%。1. 氨基酸混合物(%)为L-组氨酸0.55,L-异亮氨酸0.22,L-亮氨酸0.26,L-赖氨酸0.84,L-蛋氨酸0.37,L-苯丙氨酸0.10,L-色氨酸0.02,L-缬氨酸0.17,L-酪氨酸0.14,L-牛磺酸0.13。2. 维生素混合物 (mg/kg)为维生素A 16 000 IU,维生素B1 17.80,维生素B2 48.00,维生素B6 29.52,维生素B12 0.24,维生素C 800,维生素D3 8 000 IU,维生素E 160,维生素K3 14.72,烟酰胺79.20,叶酸6.40,泛酸钙73.60,生物素0.64,肌醇320,左旋肉碱100。3. 矿物质混合物 (mg/kg): Co (COCl2) 0.24,Cu (CuSO4) 2.00,Se (Na2SeO3) 0.18,Mn (MnSO4) 6.20,Fe (FeSO4) 21.10,Zn (ZnSO4) 34.40,I (Ca (IO3)2) 1.63,Mg (MgSO4·H2O) 52.70
    Notes: fishmeal is protein 67.09%, crude lipid 9.47%; poultry by-product meal (pet foodgrade) is crude protein 66.10%, crude lipid 12.06%. 1. AA mixture (%) are L-histidine 0.55, L-isoleucine 0.22, L-leucine 0.26, L-lysine 0.84, L-methionine 0.37, L-phenylalanine 0.10, L-tryptophan 0.02, L-valine 0.17, L-tyrosine 0.14, L-taurine 0.13. 2. vitamin Premix (mg/kg diet) are vitamin A 16 000 IU, vitamin B117.80, vitamin B2 48.00, vitamin B6 29.52, vitamin B12 0.24, vitamin C 800, vitamin D3 8 000 IU, vitamin E 160, vitamin K3 14.72, niacinamide 79.20, folic acid 6.40, calcium-pantothenate 73.60, biotin 0.64, inositol 320, L-carnitine 100. 3. mineral premix (mg/kg diet) are Co (COCl2) 0.24, Cu (CuSO4) 2.00, Se(Na2SeO3) 0.18, Mn (MnSO4) 6.20, Fe(FeSO4) 21.10, Zn (ZnSO4) 34.40, I (Ca (IO3)2) 1.63, Mg (MgSO4·H2O) 52.70

    Table 2.  Composition of experimental diets (dry weight) %

    氨基酸
    amino acids
    饲料 diets
    TCTSLT
    必需氨基酸 essential amino acids
    苏氨酸 Thr 2.03 2.00 1.78
    缬氨酸 Val 2.32 2.32 2.34
    蛋氨酸 Met 1.34 1.29 1.31
    异亮氨酸 Ile 1.93 1.90 1.94
    亮氨酸 Leu 3.54 3.54 3.53
    赖氨酸 Lys 3.46 3.30 3.31
    精氨酸 Arg 2.70 3.10 3.12
    苯丙氨酸 Phe 2.02 2.00 1.99
    总必需氨基酸 total EAA1 19.36 19.46 19.33
    非必需氨基酸 non-essential amino acids
    天冬氨酸 Asp 1.51 1.46 1.46
    组氨酸 His 4.1 3.56 3.56
    丝氨酸 Ser 1.83 1.86 1.83
    谷氨酸 Glu 7.37 7.51 7.53
    甘氨酸 Gly 2.86 4.44 4.49
    丙氨酸 Ala 5.69 3.07 3.06
    酪氨酸 Tyr 1.58 1.72 1.70
    脯氨酸 Pro 2.28 3.26 3.24
    总非必需氨基酸 total NEAA2 27.21 26.87 26.87
    总氨基酸 total AA3 46.57 46.32 46.20
    注:1. 游离必需氨基酸总和;2. 游离非必需氨基酸总和;3. 游离氨基酸总和,下同
    Notes: 1. EAA (sum of free essential amino acids); 2. NEAA (sum of free non-essential amino acids); 3. TAA (sum of free amino acids), the same below

    Table 3.  Amino acids compositions of experimental diets (dry weight) %

  • 实验所用的大口黑鲈购自广州市增城渔场,在华南师范大学水产养殖中心进行养殖实验。实验开始前,使用TC饲料每天投喂2次(8:00和17:00) 至表观饱腹,持续2周,使鱼逐渐适应实验环境。实验开始前,将所有鱼禁食24 h后进行称重,获得初始体质量。将初始体质量为(4.16±0.02) g的大口黑鲈随机放入300 L水族箱中,每缸喂养30尾,每个处理设置4个重复。每天2次(7:00和17:00) 投喂大口黑鲈直至明显饱腹,记录每天投喂量及水温,持续8周。养殖实验使用室内循环水养殖系统,实验期间水温维持在24 °C,溶解氧含量为6.0~7.0 mg/L,NH4+-N为68~100 μg/L。

  • 实验开始时,随机挑选20尾全鱼,用100 mg/L丁香酚 (上海医药有限公司,中国上海) 进行麻醉后取样,获得初始鱼的体成分组成。饲养8周后,所有大口黑鲈禁食24 h,首先对所有鱼称重计数,然后每缸随机抽取4条鱼用麻醉剂进行麻醉,测量其体质量、体长及内脏团质量、肝脏质量,并迅速对血液和组织 (肝脏和肾脏) 进行采样。血液采集采用尾静脉穿刺法,血液样本在3 000 ×g,4 °C下离心5 min,取上清液至新的离心管中,放入液氮暂时保存。解剖肝脏和肾脏组织,装入离心管中置于液氮保存。取样完毕后,所有样品放入−80 °C冰箱中储存待用。

  • 使用自动生化分析仪 (Sysmex公司,CHEMIX-800,日本神户) 对血浆中总蛋白质浓度 (TP)、甘油三酯 (TG)、胆固醇 (TCH) 的含量以及谷丙转氨酶 (ALT)、谷草转氨酶 (AST)、乳酸脱氢酶 (LDH) 的活性进行测定分析。

  • 测定游离氨基酸(FAA)所用的血浆样品制备方法参照许丹丹[13]的方法。将血浆放置在4 °C解冻后,取0.4 mL于2 mL离心管中,加入1.2 mL 10%磺基水杨酸,充分混匀后静置5 min,13 000 r/min,4 °C离心15 min,取上清液过0.22 µm滤膜,滤过后的液体转移至上样管中,使用全自动氨基酸检测仪 (L-8900,HITACHI,Japan) 检测血浆中氨基酸含量,色谱柱为锂离子交换柱。

  • 肝脏和肾脏的总RNA使用Trizol法 (中国Vazyme生物技术有限公司) 提取,分别使用NanoDrop 2000分光光度计 (Thermo,NanoDrop Technologies,USA) 和1.2%琼脂糖凝胶电泳法测定RNA的含量和质量。使用DEPC水将获得的cDNA模板稀释至80 ng/μL用于实时定量PCR分析。使用Hiff® qPCR SYBR Green MasterMix (上海翊圣生物科技有限公司,中国)进行qRT-PCR反应。qRT-PCR的热循环程序:95 °C 3 min,95 °C 15 s,60 °C 15 s,72 °C 20 s,40次循环。在每次PCR结束后进行熔解曲线分析,以确认这些反应中仅存在1种PCR产物。目的基因的相对表达水平使用比较Ct方法 (2−ΔΔCt法)计算[14],以TC组的表达量作为1。不同处理之间的β肌动蛋白 (β-actin) 表达量差异不显著,作为内参基因。

    利用Primer 5.0软件设计引物 (表4),qRT-PCR分析了包括糖代谢中的丙酮酸激酶 (PK) 和磷酸烯醇式丙酮酸激酶 (PEPCK),脂肪酸合成代谢中的过氧化物酶体增殖物激活受体α (PPAR-α)、过氧化物酶体增殖物激活受体β (PPAR-β) 和乙酰辅酶A羧化酶1 (ACC1),甘油三酯合成代谢中的二酰甘油酰基转移酶1 (DGAT1),以及脂肪合成与分解代谢中的磷脂磷酸水解酶1 (LPIN1)、脂蛋白脂肪酶 (LPL)、单酰甘油脂肪酶 (MGL) 和激素敏感脂肪酶 (HSL)。qRT-PCR还分析了肿瘤坏死因子α (TNF-α)、白细胞介素1β (IL-1β) 和白细胞介素8 (IL-8) 等促炎性细胞因子;白细胞介素10 (IL-10)、转化生长因子β1 (TGF-β1) 等抗炎细胞因子;用于细胞解毒的谷胱甘肽转移酶 (GST) 和超氧化物歧化酶1 (SOD1)。

    基因
    genes
    正向引物(5′~3′)
    forward primer (5′-3′)
    反向引物(5′~3′)
    reverse primer (5′-3′)
    目的
    purpose
    糖酵解的关键调节因子 key regulators in glycolysis
    PK CTCTTTCATCCGCAAAGC AATTCCCAGGTCACCACG RT-qPCR
    PEPCK GGAAACGGCCAACATTCT GCCAACCAGCAGTTCTCAT RT-qPCR
    脂质代谢的关键调节因子 key regulators in lipid metabolism
    PPAR-α CCACCGCAATGGTCGATATG TGCTGTTGATGGACTGGGAAA RT-qPCR
    PPAR-β AGCACCTCGCCATTTGTAATCT GGACCCCAATCTCCTTCGTC RT-qPCR
    AAC1 ATCCCTCTTTGCCACTGTTG GAGGTGATGTTGCTCGCATA RT-qPCR
    DGAT1 CACGCCTCTTCTTGGAGAAC AATGGTACCCACAGCCAGAC RT-qPCR
    LPIN1 TCCTACGTTCCCGAGAGAAA TACGAGGGAACCACTTCCTG RT-qPCR
    LPL TTCCTCGACCCTCTGAAAGA GGAGTCAAGTTTGCCAGGAA RT-qPCR
    MGL AAGGTTTTTCTGGCGAAGGT CGTGGAAGTTCAGCTCATCA RT-qPCR
    HSL ATCAGAGCTGGAGCACCCTA GCAGAGGAGAGCAGAAAGGA RT-qPCR
    免疫系统的关键调节因子 key regulators in immune system
    TNF-α CTTCGTCTACAGCCAGGCATCG TTTGGCACACCGACCTCACC RT-qPCR
    IL-1β CGTGACTGACAGCAAAAAGAGG GATGCCCAGAGCCACAGTTC RT-qPCR
    IL-8 CGTTGAACAGACTGGGAGAGATG AGTGGGATGGCTTCATTATCTTGT RT-qPCR
    IL-10 CGGCACAGAAATCCCAGAGC CAGCAGGCTCACAAAATAAACATCT RT-qPCR
    TGF-β1 GCTCAAAGAGAGCGAGGATG TCCTCTACCATTCGCAATCC RT-qPCR
    GST AACTTTTCGCTGGCTGATGT TCTTGTCCCTGTGGGTTCTC RT-qPCR
    SOD1 TGGCAAGAACAAGAACCACA CCTCTGATTTCTCCTGTCACC RT-qPCR

    Table 4.  Primer sequences used for real-time quantitative PCR

  • 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%

    饲料效率(feed efficiency ratio, FER, %)=(WtW0)/Wf×100%

    蛋白质效率(protein efficiency ratio, PER, %)=(WtW0)/(Wf×Wp)×100%

    蛋白质沉积率(protein deposition rate, PDR, %)=(Wt×CPtW0×CP0)/(Wf×Wp)×100%

    脂肪沉积率(fat deposition rate, FDR, %)=(Wt×CLtW0×CL0)/(Wf×Wl)×100%

    肥满度(condition factor, CF, g/cm3)=W/L3×100%

    脏体比(viscerosomatic index, VSI, %)=Wv/W×100%

    肝体比(hepatosomatic index, HSI, %)=Wh/W×100%

    式中,Nt为终末尾数,N0为初始尾数,Wt为终末体质量(g),W0为初始体质量(g),t为实验天数(d),CPt为终末粗蛋白含量(%),CP0为初始粗蛋白含量(%),CLt为终末粗脂肪含量(%),CL0为初始粗脂肪含量(%),Wl为饲料中的粗脂肪含量(%),Wf为摄入饲料量(g),Wh为鱼肝脏质量(g),W为鱼体质量(g),L为鱼体长(cm),Wp为饲料中的粗蛋白质含量(%)。

  • 所有统计分析使用SPSS 22软件进行,数据使用“平均值±标准误”(mean±SE) 表示,实验结果采用单因素方差分析 (One-Way ANOVA) 和Tukey氏检验进行组间差异显著性比较,P<0.05表示差异显著。

2.   结果
  • 饲料苏氨酸水平对大口黑鲈鱼体增重率、特定生长率、饲料效率、脂肪和蛋白质沉积率等均有显著影响 (表5)。与对照组相比,LT饲料组显著降低了大口黑鲈的终末体质量、增重率和特定生长率 (P<0.05),补充苏氨酸后大口黑鲈终末体质量显著升高 (P<0.05),但与对照组相比仍显著降低 (P>0.05)。与TC组相比,大口黑鲈摄食LT组饲料后,其饲料效率、蛋白质效率、脂肪和蛋白质沉积率也显著下降 (P<0.05),然而在TS处理组中,饲料效率、蛋白质效率、脂肪和蛋白质沉积率相比于LT处理组均显著上升,但与对照组相比显著降低 (P<0.05)。苏氨酸缺乏还显著提升鱼体的脏体比 (P<0.05),但对肥满度和肝体比无显著影响 (P>0.05)。就鱼体成分而言,与TC组相比,LT处理组显著降低鱼体蛋白质含量 (P<0.05),饲料中补充苏氨酸对鱼体的蛋白含量没有显著的提高作用 (P>0.05)。不同处理组间鱼体的灰分、脂肪和水分含量均无显著影响 (P>0.05)。

    项目
    items
    饲料 dietsP
    TCTSLT
    初始体质量/g initial body weight 4.18±0.04 4.10±0.07 4.19±0.06 0.50
    终末体质量/g final body weight 28.80±0.26a 26.93±0.14b 25.49±0.25c 0.00
    存活率/% SR 100.0±0.00 100.0±0.00 100.0±0.00
    增重率/% WGR 589.90±13.50a 557.34±7.41ab 508.22±13.43b 0.01
    特定生长率/(%/d) SGR 3.45±0.04a 3.36±0.02ab 3.22±0.04b 0.01
    饲料效率/% FER 1.19±0.01a 1.12±0.00b 1.04±0.02c 0.00
    蛋白质效率/% PER 2.41±0.02a 2.28±0.01b 2.14±0.03c 0.00
    脂肪沉积率/% FDR 88.87±2.14a 79.04±1.15b 70.49±1.63c 0.00
    蛋白质沉积率/% PDR 47.53±1.38a 43.33±0.95b 40.7±0.30c 0.00
    肥满度/(g/cm3) CF 2.09±0.04 2.10±0.03 2.12±0.07 0.90
    脏体比/% VSI 7.39±0.28a 8.20±0.25ab 8.33±0.14b 0.02
    肝体比/% HSI 4.60±0.33 4.63±0.15 4.00±0.11 0.12
    鱼体成分(湿重) proximate composition of whole body (wet weight)
    灰分/% ash 3.48±0.13 3.34±0.13 3.57±0.05 0.33
    粗脂肪/% crude lipid 6.84±0.08 6.77±0.07 6.70±0.13 0.60
    粗蛋白质/% crude protein 17.19±0.07a 16.79±0.13b 16.51±0.08b 0.00
    水分/% moisture 72.98±0.18 72.54±0.20 72.95±0.18 0.22
    注:同行数据上标字母不同表示差异显著(P<0.05),下同
    Notes: different superscripted letters in the same row indicate significant differences (P<0.05), the same below

    Table 5.  Effect of dietary threonine level on growth performanceandfeed utilization of M. salmoides

  • 饲料中苏氨酸水平对大口黑鲈血浆甘油三酯、胆固醇、谷丙转氨酶均有显著影响 (表6)。与对照组相比,LT处理组降低血浆中甘油三酯和胆固醇的含量 (P<0.05),苏氨酸添加对于提高血浆中甘油三酯和胆固醇水平无显著性作用 (P>0.05)。另一方面,与摄食TC组饲料的大口黑鲈相比,摄食LT组饲料的大口黑鲈其血浆中谷丙转氨酶的含量显著升高 (P<0.05),LT组饲料中添加晶体苏氨酸 (TS组) 对降低血浆中谷丙转氨酶的含量无显著作用 (P>0.05);不同处理组对血浆中总蛋白、谷草转氨酶和乳酸脱氢酶均无显著性影响 (P>0.05)。

    项目
    items
    饲料 dietsP
    TCTSLT
    总蛋白/(µg/µL) TP 48.77±0.59 47.90±0.12 47.80±0.61 0.38
    甘油三酯/(mmol/L) TG 15.49±0.21a 14.40±0.43ab 12.51±0.66b 0.01
    胆固醇/(mmol/L) TCH 13.10±0.21a 11.68±0.12b 11.61±0.06b 0.00
    谷丙转氨酶/(U/L) ALT 18.00±0.58b 23.33±0.33a 23.00±0.58a 0.00
    谷草转氨酶/(U/L) AST 138.67±1.33 136.67±2.19 124.00±8.33 0.16
    乳酸脱氢酶/(U/L) LDH 1248.33±11.39 1272.00±47.61 1263.67±102.55 0.97

    Table 6.  Effect of dietary threonine levels on plasma physiological and biochemical indexes of M. salmoides

  • 饲料中苏氨酸水平对大口黑鲈血浆游离氨基酸水平有显著影响 (表7)。根据检测结果发现,与TC处理组相比,LT处理组显著降低了血浆中EAA、NEAA和TAA的含量 (P<0.05),补充苏氨酸后显著提高血浆中NEAA和TAA的含量 (P<0.05),使之与TC处理组间无显著差异 (P>0.05)。除此之外,与摄食TC组饲料的大口黑鲈相比,摄食LT组饲料的大口黑鲈血浆中Thr、Met、Lys、His、Gly、Ala和Cys含量均显著下降 (P<0.05)。饲料中补充苏氨酸能够显著提高血浆中Thr、Met、Lys、Gly和Cys的游离氨基酸水平 (P<0.05),使之与TC处理组间无显著性差异 (P>0.05)。

    氨基酸
    amino acids
    饲料 dietsP
    TCTSLT
    必需氨基酸 essential amino acids
    苏氨酸 Thr 25.35±0.34a 19.15±0.30b 13.65±1.18c 0.00
    缬氨酸 Val 26.44±0.31 27.26±0.18 27.58±0.88 0.38
    蛋氨酸 Met 16.23±0.07a 14.88±0.13b 13.51±0.45c 0.00
    异亮氨酸 Ile 16.10±0.21 16.33±0.16 15.99±0.53 0.78
    亮氨酸 Leu 29.19±0.46 28.97±0.24 29.18±1.07 0.97
    苯丙氨酸 Phe 16.37±0.06 15.36±0.13 15.85±0.42 0.08
    赖氨酸 Lys 29.79±0.20a 29.29±0.12a 22.53±0.75b 0.00
    精氨酸 Arg 2.46±0.15b 2.72±0.07b 3.39±0.16a 0.01
    组氨酸 His 16.40±0.25a 12.20±0.22b 13.63±0.77b 0.00
    必需氨基酸 EAA 178.33±1.49a 166.16±0.87ab 155.31±5.71b 0.01
    非必需氨基酸 non-essential amino acids
    丝氨酸 Ser 6.80±0.09 7.16±0.02 6.64±0.19 0.06
    谷氨酸 Glu 12.66±0.29 12.52±0.09 12.48±1.12 0.98
    甘氨酸 Gly 83.22±0.67a 76.52±1.24b 58.15±1.80c 0.00
    丙氨酸 Ala 60.69±0.71a 53.30±1.23b 50.90±2.12b 0.01
    半胱氨酸 Cys 5.46±0.16a 5.21±0.06a 4.32±0.21b 0.01
    酪氨酸 Tyr 19.48±0.09 19.22±0.51 17.94±0.82 0.20
    非必需氨基酸 NEAA 188.30±1.80a 173.93±2.58a 150.42±5.96b 0.00
    总氨基酸 TAA 366.63±3.14a 340.09±3.02a 305.73±11.66b 0.00

    Table 7.  Effect of dietary threonine levels on plasma free amino acids level of M. salmoides μg/mL

  • 饲料中苏氨酸水平对大口黑鲈糖酵解、脂肪酸与甘油三酯代谢、脂肪合成与分解代谢中关键酶的表达均有显著影响(图1)。糖酵解关键酶中,与TC和LT组相比,TS处理组显著升高鱼体肝脏中PK mRNA水平的表达量 (P<0.05),然而3个处理组对PEPCK mRNA水平的表达无显著影响 (P>0.05)(图1-a)。饲料苏氨酸水平显著影响大口黑鲈脂肪酸、甘油三酯代谢和脂肪合成中关键酶基因表达 (图1-b),与TC组相比,LT处理组显著降低了肝脏中PPAR-αPPAR-βACC1和LPIN1的表达量 (P<0.05)。饲料中补充苏氨酸显著提高肝脏中PPAR-βACC1和LPIN1的表达量 (P<0.05),使其与LC组无显著差异 (P>0.05)。苏氨酸水平显著影响脂肪分解代谢中关键酶基因表达 (图1-c),与TC组相比,LT处理组显著提高肝脏中LPLMGL mRNA的表达水平,同时降低了HSL mRNA的表达水平 (P<0.05)。饲料中补充苏氨酸显著降低了肝脏中LPLMGL的基因表达水平,也提高了HSL的基因表达水平 (P<0.05),使之与LC组无显著差异 (P>0.05)。

    Figure 1.  Effect of threonine levels on gene expressions of the key enzymes in liver involved in glucose and lipid metabolism in M. salmoides

  • 与对照组相比,低苏氨酸处理组显著降低了肾脏中TNF-α的表达量,同时升高了肾脏中IL-1βIL-8的mRNA水平表达量 (P<0.05)。饲料中补充苏氨酸对于提高TNF-α的mRNA水平和降低肾脏中IL-1βIL-8的mRNA水平均有显著作用 (P<0.05)。3个处理组对肾脏中IL-10、TGF-β1、GSTSOD1的表达量均无显著调控作用 (P>0.05)(图2)。

    Figure 2.  Effect of dietary threonine level on gene expression related to renal immune response in M. salmoides

3.   讨论
  • 非鱼粉蛋白源中,氨基酸组成不平衡是导致其替代效率低下的重要原因之一。苏氨酸作为鱼类的必需氨基酸之一,是非鱼粉蛋白源替代鱼粉后最常见的限制性氨基酸[15]。饲料中苏氨酸水平对鱼类生长性能和饲料利用具有明显的调控作用。在评估了鸡肉粉替代鱼粉后,不同的氨基酸添加水平对大口黑鲈生长和代谢的影响不尽相同。本研究发现,宠物级鸡肉粉完全替代鱼粉后,苏氨酸缺乏可导致大口黑鲈的增重率、特定生长率均显著下降,这一结果可能与苏氨酸缺乏导致饲料效率、蛋白质和脂质沉积率的下降有关。同样,此研究结果在草鱼[16]、花鲈(Lateolabrax japonicus)[17]、杂交鲇[6]、银黑鲷(Sparidentex hasta)[18]、团头鲂(Megalobrama amblycephala)[10]、斑点叉尾鮰(Ictalurus punctatus)[19]的研究中也有发现。在鱼类的生长中,机体蛋白质沉积做出了很大的贡献[6],而苏氨酸缺乏的饲料会显著降低鱼体蛋白质含量,补充苏氨酸后,大口黑鲈的增重率、特定生长率和饲料效率等均得到了显著提高。本研究结果表明,宠物级鸡肉粉完全替代鱼粉后,苏氨酸的添加能够显著促进大口黑鲈的生长性能,并提高鸡肉粉的利用效率。

    血浆生理生化指标不仅能反映物种的特性,而且还能作为动物生理、病理研究的重要参考依据[20]。本研究结果表明,饲料中苏氨酸不足可显著提高血浆中ALT含量,但对血浆中AST的含量没有显著性影响。AST和ALT是肝脏中两种重要的转氨酶,与AST相比,ALT对鱼的营养状况变化更为敏感[18]。血浆中AST和ALT活性升高表明肝功能受到损伤[21],因此这一实验结果表明,饲料中苏氨酸缺乏会在一定程度上影响肝脏的健康状态。乳酸脱氢酶 (LDH) 是一种存在于很多组织中的细胞质酶,血清LDH水平的升高表明细胞溶解[22]。饲料中苏氨酸水平对血浆中LDH含量无显著影响,说明苏氨酸缺乏未必会造成细胞损伤。

    另外,检测血浆中的营养成分时发现,与对照组相比,LT饲料组导致鱼体血浆中甘油三酯和胆固醇含量均显著下降,但血浆中总蛋白水平无显著差异。血浆胆固醇水平可指示肝脏中脂质和脂蛋白代谢水平,甘油三酯也主要在肝脏中合成,它可以作为肝脏健康状态的标志物[23],这一研究结果表明,鸡肉粉完全替代鱼粉显著降低了血浆中脂类成分的含量,从而对肝脏的代谢水平有显著的影响。在鸡肉粉完全替代鱼粉的饲料中,补充晶体苏氨酸可以在一定程度上提高血浆中甘油三脂和胆固醇的含量,但是作用效果不显著。综合各项生理生化指标,发现饲料中鸡肉粉完全替代会对鱼体肝脏造成一定的损伤,补充苏氨酸后,各项生理生化指标也无显著性变化,说明了苏氨酸在改善鱼体肝脏损伤方面的作用不显著。

    本研究还发现,饲料中苏氨酸水平对血浆中游离氨基酸含量有显著的调控作用。饲喂未添加苏氨酸的饲料时,血浆中的苏氨酸含量与对照组相比呈现极显著下降趋势 (P<0.001)。除此之外,其他多数EAA和NEAA的含量也显著降低,游离氨基酸总量显著下降 (P<0.05)。这些结果与鱼类饲喂氨基酸受限日粮的结果一致[24],苏氨酸缺乏造成饲料氨基酸组成的不平衡,进而降低了其他多种氨基酸的转运和吸收。饲料中补充晶体苏氨酸后,显著升高了血浆中多数EAA和NEAA以及TAA的含量 (P<0.05)。已有研究证明,血浆中的游离氨基酸水平与饲料氨基酸含量具有高度相关性[25-26]。Yun等[27]通过测量血浆游离氨基酸和氨浓度,来确定虹鳟(Oncorhynchus mykiss)的饲料最佳苏氨酸需要量。此外,也有研究通过测定血浆游离氨基酸,以推断不同蛋白质来源饲料中的限制性氨基酸[28]。本研究的结果与上述研究一致,说明苏氨酸是宠物级鸡肉粉完全替代鱼粉饲料的限制性氨基酸。补充了苏氨酸后,大口黑鲈血浆中游离氨基酸水平呈现出显著的上升趋势,且多数氨基酸含量与对照组间无显著差异 (P>0.05)。

    在鱼类糖代谢过程中,涉及的主要途径是糖酵解和糖异生的过程[29]。目前关于氨基酸调控鱼类糖代谢的研究较少,仅见于亮氨酸[30]、蛋氨酸和赖氨酸[31]。本研究测定了饲喂大口黑鲈不同苏氨酸水平的饲料后,糖酵解途径关键酶mRNA的表达水平,以阐明苏氨酸调节鱼体糖代谢的潜在机制。结果表明,鸡肉粉完全替代鱼粉后对这两种酶的mRNA水平均无显著影响。然而,添加了苏氨酸后,肝脏中PK基因的表达水平显著上升。以上结果表明,宠物级鸡肉粉完全替代鱼粉对大口黑鲈糖酵解代谢过程无显著影响。

    与糖代谢不同,苏氨酸对大口黑鲈脂代谢的影响非常显著。在Song等[32]的研究中,氨基酸受限的鱼粉替代饲料能显著降低肝脏的脂肪合成代谢,增加脂肪的分解代谢,而Dai等[33]在对虹鳟的研究中也发现,高水平氨基酸能显著上调脂肪合成相关基因的表达。Liang等[30]的研究发现,亮氨酸不足会显著降低脂肪合成相关基因FASACC1的表达,而补充亮氨酸后可明显改善这一反应,说明单一氨基酸受限也能够影响鱼类的脂质代谢。与上述研究结果一致,本研究发现,苏氨酸不足能显著降低脂肪从头合成关键酶(PPAR-αPPAR-βACC1)的表达,脂肪沉积减少(LPIN1表达水平下降),而脂肪分解代谢关键酶(LPLMGL)表达显著上升。添加苏氨酸可明显促进脂肪的合成并抑制脂肪分解,使之与对照组无差异。这些结果与先前探究不同赖氨酸水平饲料对大口黑鲈脂质代谢影响的结果相似[31]。本研究证明,与亮氨酸、赖氨酸等相同,苏氨酸也是调控鱼类脂质代谢的必需营养素,宠物级鸡肉粉替代鱼粉时,苏氨酸的添加与否对脂质代谢具有重要的调节作用。

    除了对生长代谢的调控以外,饲料中适当添加氨基酸还能够增强机体的免疫机能,提高动物体的健康水平[34-35]。SOD是机体内重要的抗氧化酶之一[36],能够催化超氧化物自由基歧化为过氧化氢[37],在自卫和免疫系统中起着重要作用[38],被认为是生物体抗氧化状态的指标和氧化应激的生物标志物[39]。而GST具有将谷胱甘肽(GSH)与含有亲电中心的化合物结合的能力,是一类主要的解毒酶[40],SOD与GST共同作用,在细胞解毒和保护方面发挥着重要作用。在Zhou等[41]、Habte-Tsion等[39]和Dong[7]等的研究中,喂食低水平苏氨酸饲料时鱼类的SOD和GST活性下降,而适宜的苏氨酸水平会提升SOD和GST的活性。在本研究中,无论鱼粉替代饲料是否添加了苏氨酸,肾脏中SOD1和GST的mRNA水平均与对照组无显著差异,说明苏氨酸缺乏对大口黑鲈肾脏的氧化应激的影响不显著,在团头鲂幼鱼的研究中也有相似的结果[10]。炎症细胞因子在免疫反应中也起着重要的作用,包括促炎细胞因子(如TNF、IL-1β、IL-8)和抗炎细胞因子(如TGF-β1、IL-10)[42-44]。在鱼类中,促炎细胞因子的上调和抗炎细胞因子的下调均会导致炎症反应[45]。已有研究发现,苏氨酸缺乏时能上调促炎细胞因子TNF-αIL-1βIL-8 mRNA水平,下调抗炎细胞因子TGFIL-10 mRNA水平,从而减弱机体的免疫屏障功能[8],在此过程中可能有NF-κB和TOR信号的介导[7]。本研究结果显示,苏氨酸缺乏时,促炎细胞因子IL-8、IL-1β的mRNA水平均显著上升,这与以往已有的研究结果一致,添加了苏氨酸后,促炎细胞因子的mRNA水平明显下调,与对照组无显著差异。以上所有结果进一步论证了饲料中苏氨酸水平对大口黑鲈免疫的调节作用。

    本研究表明,使用宠物级鸡肉粉完全替代鱼粉后,苏氨酸的添加与否对大口黑鲈生长、肝脏糖脂代谢和免疫具有重要的调控作用。苏氨酸作为鱼类的必需营养素,适量补充能够提高大口黑鲈生长性能、促进脂肪合成和抑制脂肪分解,并增强免疫机能。本研究为宠物级鸡肉粉在大口黑鲈饲料中的高效利用提供了方法途径,并为使用苏氨酸提高非鱼粉蛋白质的利用效率提供了理论依据。

    综上所述,鸡肉粉替代鱼粉是一个综合性的问题,其替代后对机体生长和代谢的影响涉及多方面,如氨基酸不平衡、缺乏小分子活性物质等,本研究探讨了鸡肉粉替代鱼粉重要的限制因素之一,即氨基酸不平衡问题,评估了鸡肉粉替代鱼粉后单一添加苏氨酸对大口黑鲈生长性能和代谢的影响,发现鸡肉粉替代鱼粉后,单一添加苏氨酸对大口黑鲈生长性能、糖代谢和免疫有一定的促进作用,但添加后的效果还是显著低于鱼粉组的效果,将来可能需要从除氨基酸以外的其他方面,如添加小分子活性物质等,全面优化提高鸡肉粉替代鱼粉的策略及方案,为实际生产提供全面有力的理论依据。

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