DIETARY LYSINE LEVELS ON GROWTH PERFORMANCE, BIOCHEMICAL INDEXES, AND PROTEIN METABOLISM RELATED GENES OF JUVENILE RICE FIELD EEL (MONOPTERUS ALBUS)
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摘要:
为探究黄鳝(Monopterus albus)幼鳝对赖氨酸的需求量, 配制了6组赖氨酸实测值为2.31%、2.61%、2.91%、3.51%、4.71%和7.11%的等氮等能饲料, 饲喂初始体重为(7.64±0.04) g幼鳝90d。结果表明: (1)随饲料赖氨酸水平的升高, 幼鳝增重率(WGR)、特定生长率(SGR)、蛋白质沉积率(PRR)、蛋白质效率(PER)呈先升高后下降趋势, 饲料系数(FCR)呈先下降后上升趋势; 3.51%水平显著提高了幼鳝WGR、SGR、PRR、PER, 降低了FCR (P<0.05)。3.51%水平显著提高幼鳝肝脏mTOR、S6K1及IGF-1的相对表达量, 降低了eIF4E-BP2的相对表达量(P<0.05)。(2)随饲料赖氨酸水平的升高, 幼鳝肌肉粗蛋白质呈先升高后不变趋势, 粗脂肪呈先上升后下降趋势, 7.11%水平显著提高了幼鳝肌肉粗蛋白质、总氨基酸(TAA)、必需氨基酸(EAA)、非必需氨基酸(NEAA)含量(P<0.05), 3.51%水平显著提高了幼鳝肌肉粗脂肪含量、EAA/TAA和EAA/NEAA (P<0.05)。(3)随饲料赖氨酸水平的升高, 幼鳝血清总蛋白(TP)、白蛋白(ALB)呈升高趋势, 7.11%水平显著高于2.31%水平(P<0.05); 谷草转氨酶活性(GOT)及血氨(Sa)呈先下降后上升趋势, 溶菌酶(LZM)活性呈先上升后下降趋势; 3.51%水平Sa显著低于其余水平(P<0.05), 2.91%水平GOT显著低于2.31%水平(P<0.05), 4.71%水平LZM显著高于2.31%—2.91%水平(P<0.05)。(4)随饲料赖氨酸水平的升高, 幼鳝肝胰脏、前肠胰蛋白酶活性及前肠脂肪酶活性呈先升高后降低趋势, 2.91%—3.51%水平显著提高了幼鳝蛋白酶和脂肪酶活性(P<0.05)。综上, 饲料适宜赖氨酸水平能够提高幼鳝消化能力以及非特异性免疫能力, 通过促进GH/IGF及mTOR通路相关基因表达增强机体蛋白质沉积, 从而提高幼鳝生长性能。以WGR、SGR、FCR、PRR、PER为评价指标, 黄鳝幼鳝的适宜赖氨酸需求量为3.38%—3.55% (占饲料蛋白质6.57%—6.90%)。
Abstract:A 90d feeding trial was conducted to determine the dietary lysine requirement of juvenile rice field eel (Monopterus albus), with an initial weight of (7.64±0.04) g reared in indoor aerated tanks. Six isonitrogenous and isoenergetic practical diets were formulated with graded levels of lysine (2.31%, 2.61%, 2.91%, 3.51%, 4.71%, and 7.11% dry matter). The result showed that: (1) The weight gain rate (WGR), specific growth rate (SGR), protein retention rate (PRR), and protein efficiency ratio (PER) of M. albus increased firstly and then decreased with the increase of dietary lysine level, and the feed conversion ratio (FCR) decreased first and then increased. Conversely, the FCR decreased initially and then increased. A dietary lysine level of 3.59% significantly improved the WGR, SGR, PRR, and PER and reduced FCR (P<0.05). A level of 3.51% significantly increased the relative expression of mTOR, S6K1, and IGF-1 in the liver, while decreasing the relative expression of eIF4E-BP2 (P<0.05). (2) With the increase of dietary lysine level, the crude protein in the muscle of juvenile M. albus increased firstly and then plateaued, while crude fat increased firstly and then decreased. The 7.11% lysine level significantly increased the contents of crude protein, total amino acid (TAA), essential amino acid (EAA), and non-essential amino acid (NEAA) in muscle (P<0.05). The 3.51% level significantly increased the crude fat content, EAA/TAA, and EAA/NEAA (P<0.05). (3) With the increase of dietary lysine level, the serum total protein (TP) and albumin (ALB) showed an increasing trend, and the 7.11% level was significantly higher than the 2.31% level (P<0.05). Serum glutamic oxalacetic transaminase activity (GOT) and serum ammonia (Sa) decreased firstly and then increased, while lysozyme (LZM) activity increased firstly and then decreased. Sa at 3.51% was significantly lower than that at other levels (P<0.05). GOT at 2.91% was significantly lower than that at 2.31% (P<0.05), and LZM at 4.71% was significantly higher than the level between 2.31% and 2.91% (P<0.05). (4) The activity of trypsin in hepatopancreas and foregut, as well as lipase activity in foregut, increased firstly and then decreased with increasing dietary lysine levels. Levels between 2.91% and 3.51% significantly increased trypsin and lipase activity in juvenile M. albus (P<0.05). In summary, the appropriate dietary lysine level can improve the digestive ability and non-specific immunity, promote the expression of GH/IGF and mTOR pathway related genes, and enhance the protein deposition, thereby improving the growth performance of juvenile M. albus. The optimal lysine requirement of M. albus (7.64±0.04) g is estimated to be between 3.38% and 3.55% (6.57% to 6.90% of feed protein) based on a broken-line analysis of the relationships with WGR, SGR, FCR, PRR, and PER.
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赖氨酸, 是鱼类必需氨基酸之一, 在谷物食品中含量低, 且在加工过程中易被破坏, 常被认为是水产动物第一限制性氨基酸[1, 2]。在鱼类的生长[3]、营养摄入[4]、氮排泄[5]等方面发挥重要作用。已有不少研究表明, 饲料赖氨酸缺乏时会造成鱼类食欲下降、生长缓慢[6]、蛋白质沉积减少[7], 部分鱼类出现病理现象如体色异常、尾鳍糜烂, 甚至死亡率升高[8]。饲料赖氨酸过量也会破坏氨基酸的平衡[9]或与其他氨基酸拮抗[10]而造成鱼类的生长性能、饲料利用率和蛋白质沉积下降[11]。研究表明, 赖氨酸可激活mTOR通路从而上调S6K1, 抑制eIF4E-BP2及eIF4E表达, 从而促进生长和蛋白质沉积[9, 12]。关于水产动物对赖氨酸需求量的研究已经有很多, 如乌苏里拟鲿(Pseudobagrus ussuriensis)幼鱼的适宜赖氨酸需求量为3.64% (占饲料蛋白质8.95%)[13], 大黄鱼(Pseudosciaena crocea)仔鱼为3.34%—3.37% (6.49%—6.55%)[14], 卵形鲳鲹(Trachinotus ovatus)为2.94% (6.70%)[15], 草鱼(Ctenopharyngodon idella)幼鱼为2.07% (5.44%)[16], 军曹鱼(Rachycentron canadum)幼鱼为2.33% (5.30%)[17], 拟穴青蟹(Scylla paramamosain)仔蟹为2.03% (4.51%)[18], 黄颡鱼(Nibea albiflora)为2.49% (4.97%)[11]。
黄鳝(Monopterus albus), 因其营养丰富、肉质细腻而深受消费者喜爱, 2022年全国养殖量已达33万吨[19]。目前有关黄鳝的营养学需求研究大多集中在蛋白质[20, 21]、脂肪[22]、矿物质[23, 24]等方面, 关于氨基酸需求量的研究较少, 对赖氨酸需求量的研究还未见报道。本试验以人工繁养殖幼鳝为研究对象, 探究饲料中赖氨酸水平对幼鳝生长性能、生化指标、消化酶活性及蛋白质代谢相关基因的影响, 得出幼鳝对赖氨酸的适宜需求量, 以期为黄鳝人工配合饲料的精准配置提供理论依据。
1. 材料与方法
1.1 试验设计与饲料制作
以鱼粉、豆粕为主要蛋白源, 小麦粉为主要糖源, 豆油为脂肪源配制基础组饲料, 以黄鳝全鱼氨基酸模式为参考添加混合氨基酸, 向基础组中添加0.3%、0.6%、1.2%、2.4%和4.8%的DL-赖氨酸(DL-Lysine), 以L-丙氨酸(L-Alanine)调平, 配置6组等氮等能的饲料(Lys实测值为2.31%、2.61%、2.91%、3.51%、4.71%和7.11%)。饲料所用原料购于江西大佑农生物科技有限公司, 所有原料均粉碎过80目筛, 按配比(表 1)逐级混匀, 加入豆油, 20%左右水分, 制成1.5—2.0 mm膨化颗粒饲料, 避光风干, 置于阴凉避光环境备用。各组饲料氨基酸组成见表 2。
表 1 试验饲料组成及营养水平(干物质基础)Table 1. Composition and nutritional level of the experimental diets (DM basis)原料Ingredient (%) 饲料赖氨酸水平
Dietary lysine level (%)2.31 2.61 2.91 3.51 4.71 7.11 鱼粉Fish meal 30 30 30 30 30 30 复合蛋白粉Compound protein1 6 6 6 6 6 6 豆粕Soybean meal 16 16 16 16 16 16 玉米蛋白粉Corn protein powder 6 6 6 6 6 6 棉籽蛋白Cottonseed protein 6 6 6 6 6 6 小麦粉Wheat meal 20 20 20 20 20 20 碎米粉Crushed rice flour 2 2 2 2 2 2 豆油Soybean oil 4.35 4.35 4.35 4.35 4.35 4.35 DL-赖氨酸Lysine 0 0.3 0.6 1.2 2.4 4.8 L-丙氨酸Alanine 4.8 4.5 4.2 3.6 2.4 0 磷酸二氢钙Ca(H2PO4) 2 2 2 2 2 2 混合氨基酸Amino acid mix2 1.75 1.75 1.75 1.75 1.75 1.75 复合添加剂Composite additive3 0.1 0.1 0.1 0.1 0.1 0.1 预混料Premix4 0.5 0.5 0.5 0.5 0.5 0.5 胆碱Choline chloride 0.5 0.5 0.5 0.5 0.5 0.5 总和Sum 100 100 100 100 100 100 营养水平 Nutrient levels5 水分Moisture 7.14 7.75 6.50 6.80c 6.81 6.29 粗蛋白质Crude protein 51.46 51.21 51.38 51.37 51.46 51.16 粗脂肪Crude fat 7.69 7.51 7.60 7.48 7.41 7.35 粗灰分Crude ash 9.71 9.62 9.72 9.67 9.71 9.68 赖氨酸Lysine 2.31 2.61 2.91 3.51 4.71 7.11 总能 Gross energy (MJ/kg)6 17.45 17.45 17.45 17.45 17.45 17.45 注: 1复合蛋白粉由蚯蚓粉:谷朊粉1﹕2组成 The composite protein is composed of earthworm powder: millet powder 1﹕2; 2混合氨基酸为1.10%苏氨酸、0.55%蛋氨酸、0.10%色氨酸 The mixed amino acids are 1.10% threonine, 0.55% methionine and 0.10% tryptophan; 3复合添加剂由植酸酶、复合酶、蛋白酶、维生素C组成 The coomposite additive is composed of phytase, compound enzyme, protease and vitamin C; 4预混料为每千克饲料提供 The premix provided the following per kg of the diet: VA 6000 IU, VD3 2500.0 IU, VE 200.0 mg, VK3 10.0 mg, VB1 25.0 mg, VB2 45.0 mg, 烟酸 nicotinic acid 200.0 mg, VB6 20.0 mg, 泛酸钙 Ca-pantothenate acid 60.0 mg, 叶酸 folic acid 10.0 mg, VB12 0.1 mg, 生物素 biotin 1.5 mg, 肌醇 inositol 200.0 mg, NaSeO3·5H2O 0.3 mg, CoCZ·6H2O 0.4 mg, KI 0.8 mg, CuSO4·5H2O 10.0 mg, MnSO4·4H2O 20.0 mg, ZnSO4·H2O 50.0 mg, FeSO4·7H2O 150.0 mg, MgSO4·7H2O 500.0 mg, NaCl 1000.0 mg; 5营养水平为实测值 Nutritional level is measured value; 6为计算值, 蛋白质以23.64 MJ/kg, 脂肪以39.50 MJ/kg, 淀粉以17.15 MJ/kg计算 The calculated value, protein is 23.64 MJ/kg, fat is 39.50 MJ/kg, and starch is 17.15 MJ/kg 表 2 试验饲料氨基酸组成(干物质基础)Table 2. Amino acid composition of experimental diets (DM basis)项目Item (%) 饲料赖氨酸水平 Dietary lysine level (%) 2.31 2.61 2.91 3.51 4.71 7.11 天冬氨酸Asp 3.18 3.18 3.18 3.18 3.18 3.18 苏氨酸Thr 2.40 2.40 2.40 2.40 2.40 2.40 丝氨酸Ser 1.63 1.63 1.63 1.63 1.63 1.63 谷氨酸Glu 7.39 7.39 7.39 7.39 7.39 7.39 脯氨酸Pro 2.17 2.17 2.17 2.17 2.17 2.17 甘氨酸Gly 1.91 1.91 1.91 1.91 1.91 1.91 丙氨酸Ala 6.72 6.42 6.12 5.52 4.32 1.92 胱氨酸Cys 0.49 0.49 0.49 0.49 0.49 0.49 缬氨酸Val 1.79 1.79 1.79 1.79 1.79 1.79 蛋氨酸Met 1.27 1.27 1.27 1.27 1.27 1.27 异亮氨酸Ile 1.54 1.54 1.54 1.54 1.54 1.54 亮氨酸Leu 2.95 2.95 2.95 2.95 2.95 2.95 酪氨酸Tyr 1.13 1.13 1.13 1.13 1.13 1.13 苯丙氨酸Phe 1.74 1.74 1.74 1.74 1.74 1.74 组氨酸His 0.99 0.99 0.99 0.99 0.99 0.99 赖氨酸Lys 2.31 2.61 2.91 3.51 4.71 7.11 精氨酸Arg 2.28 2.28 2.28 2.28 2.28 2.28 色氨酸Trp 0.50 0.50 0.50 0.50 0.50 0.50 总氨基酸TAA 42.39 42.39 42.39 42.39 42.39 42.39 1.2 养殖管理
试验所用幼鳝来自江西农业大学黄鳝繁养殖基地, 随机将720条体质健壮、无病无伤的人工繁养殖幼鳝[体质量(7.64±0.04) g]分为6组, 每组4个重复, 每个重复30尾。养殖试验于江西农业大学水产养殖基地进行, 养殖箱规格为86 cm×62 cm×61 cm, 水深为20—25 cm, 水葫芦覆盖水面2/3以上。养殖时间在2023年6—9月, 周期为90d。试验期间每天17: 00进行饱食投喂, 水温在25.2—34.8℃缓慢变化(高于32℃的天数占养殖天数35.56%), pH6.5—7.3, 氨氮浓度<2.5 mg/L, 亚硝酸盐浓度<0.15 mg/L, 溶解氧>6 mg/L。3—4天换水1次, 每次换水将箱内水换完, 补充曝气自来水, 每两次换水测定1次水质。
1.3 样品采集
试验开始前挑选8尾7.64 g左右幼鳝用作初始营养成分测定, 养殖试验结束后禁食24h, 对每个养殖箱进行计数称重, 计算各组末均重(FBW)、增重率(WGR)、特定生长率(SGR)、饲料系数(FCR)、成活率(SR)等指标。每箱随机取4尾均重左右幼鳝, 准确称重后测量体长, 断尾取血后于冰盘上解剖, 分离并记录内脏、肝脏、脾脏、肠道、胴体重, 计算形体参数。准确称取部分肝脏、肠道组织, 液氮速冻后转移至–80℃冰箱保存待测, 收集剩余幼鳝胴体、肝脏测定营养成分。血液于4℃静置24h, 吸取上清液保存于–80℃待测。每箱随机取1尾均重左右幼鳝用作全鱼营养成分测定。
1.4 生长性能及形体参数指标计算
增重率(Weight gain rate, WGR, %)=(Wt–W0)/W0×100
特定生长率(Specific growth rate, SGR, %/d)=(LnWt–LnW0)/t×100
饲料系数(Feed conversion ratio, FCR)=F/(Wt–W0)
存活率(Survival rate, SR, %)=Nt/N0×100
蛋白质效率(Protein efficiency ratio, PER, %)=(Wt–W0)/(F×CP)×100
蛋白质沉积率(Protein retention rate, PRR, %)=(Wt×CPt–W0×CP0)/(F×CP)×100
脏体指数(Viscerasomatic index, VSI)=(VW/W)×100
肝体指数(Hepatosomatic index, HSI)=(HW/W)×100
肠体指数(Intestosomatic index, ISI)=(IW/W)×100
脾体指数(Splenic idex, SI)=(SW/W)×100
肥满度(Condition factor, CF, g/cm3)=W/L3
胴体率(Carcass percentage, CP, %)=(CW/W)×100
式中, W0、Wt分别代表试验黄鳝初均重和末均重(g); t代表养殖天数(d); F代表平均摄食量(g); CP代表饲料粗蛋白质水平; CP0、CPt分别代表试验前后黄鳝全鱼粗蛋白质含量; Nt、N0分别代表试验结束和开始时幼鳝尾数。VW、HW、SW、IW、CW、W分别代表取样黄鳝内脏、肝脏、脾脏、肠道、胴体及全鱼重量(g); L代表取样黄鳝体长(cm)。
1.5 血清生化指标测定
按照南京建成生物工程研究所试剂说明书测定血清白蛋白(ALB)、总蛋白(TP)、血氨(Sa)、谷丙转氨酶(GPT)、谷草转氨酶(GOT)、溶菌酶(LZM)、碱性磷酸酶(AKP); 肝脏GPT和GOT; 肝胰脏及前肠蛋白酶、脂肪酶、淀粉酶活性。
1.6 营养成分测定及氨基酸营养价值评定
饲料及鱼体常规营养成分和氨基酸含量均采用同样的测定方法: 水分含量测定参考GB/T 6435—2014; 粗蛋白质参考GB/T 6432—1994; 粗脂肪参考GB/T 6433—2006; 粗灰分参考GB/T 6438—2007。取2.31%、3.51%和7.11%水平肌肉冷冻干燥后参考GB/T 18246—2019测定氨基酸含量, 所用仪器为日立L8900氨基酸分析仪。
1.7 实时荧光定量PCR
选择2.31%、3.51%和7.11%水平幼鳝测定肝脏中的基因相对表达量, 相关基因引物序列见表 3, 从肝脏中提取总RNA, 通过1.0%琼脂糖凝胶电泳检测其完整性, epoch biotek测定样品RNA纯度和浓度, 以SynScript®Ⅲ RT SuperMix for qPCR (擎科)进行逆转录扩增。实时荧光定量检测使用ABI, QuantStudio stepone Plus, 反应体系为20 μL, 包含ArtiCanCEO SYBR qPCR Mix10 μL, cDNA 1 μL, 10 μmol/L Primer F及Primer R 0.8 μL, 7.4 μL ddH2O。程序为95℃ 5min, 95℃ 15s, 60℃ 20s, 72℃ 20s, 40个循环。以18S为内参基因, 采用2–ΔΔCt法计算目的基因相对表达量。
表 3 实时荧光定量PCR引物序列Table 3. The primers sequence of quantitative RT-PCR引物名称
Gene name引物序列
Primer sequence (5′—3′)产物大小
Product
length
(bp)GenBank
登录号
Accession No.mTOR-F CGTTCCGACTGACGAGGATG 152 XM_020612477.1 mTOR-R CAGCGGGTCATAGACGAAGG S6K1-F TGAAGAACTCCCAACCCACAC 101 XM_020602184.1 S6K1-R GAGTCTCTACAGGGAGGTGGA eIF4E-BP2-F AATGCGTCAAAGGGTCACTCA 132 XM_020603221.1 eIF4E-BP2-R GCCCTCACTGCCAATCCAA IGF-1-F ACCCTGACTCCGACGACAA 140 XM_020613661.1 IGF-1-R CGTGACCGTCGTGTACTGG 18S-F GTGAAATTCTTGGACCGGCG 145 XM_020605951.1 18S-R GGATCGCTAGTTGGCATCGT 1.8 数据处理与分析
试验结果采用平均值±标准误(mean±SE)表示, 用Spss 26.0软件进行单因素方差分析(One-way ANOVA), 再用Duncan氏法进行多重比较, 显著水平为P<0.05, 折线拟合采用Excel 2016。
2. 结果
2.1 饲料赖氨酸水平对幼鳝生长性能的影响
由表 4可知, 各组幼鳝FBW、WGR、SGR、PER和PRR随饲料赖氨酸水平的升高呈先升高再下降的趋势, FCR呈相反趋势; 3.51%水平FBW、WGR、SGR显著高于2.31%—2.91%水平(P<0.05), 与4.71%—7.11%水平无显著差异(P>0.05); FCR显著小于2.31%—2.61%及4.71%—711%水平(P<0.05), 与2.91%水平无显著差异(P>0.05); PER、PRR显著高于其余水平(P>0.05)。饲料赖氨酸水平对幼鳝SR无显著影响(P>0.05), 各组均保持在93%以上。以WGR、SGR、FCR、PRR和PER为评价指标, 通过折线回归(图 1)得出幼鳝(7.64±0.04) g的适宜赖氨酸需求量为3.38%—3.55% (占饲料蛋白质6.57%—6.90%)。
表 4 饲料赖氨酸水平对幼鳝生长性能的影响Table 4. Effects of dietary lysine levels on growth performance of juvenile Monopterus albus项目Item 饲料赖氨酸水平 Dietary lysine level (%) 2.31 2.61 2.91 3.51 4.71 7.11 初均重IBW (g) 7.61±0.02a 7.64±0.02a 7.64±0.02a 7.63±0.02a 7.64±0.02a 7.62±0.02a 末均重FBW (g) 27.71±0.99c 28.69±0.37c 30.20±1.25bc 34.76±1.74a 33.23±1.79ab 30.91±0.92abc 增重率WGR (%) 264.10±13.69c 275.79±5.14c 295.65±17.40bc 355.52±22.83a 335.05±23.28ab 305.58±12.00abc 特定生长率SGR (%/d) 1.43±0.04c 1.47±0.02c 1.52±0.05bc 1.68±0.06a 1.62±0.06ab 1.55±0.03abc 饲料系数FCR 1.86±0.08a 1.77±0.06a 1.73±0.07ab 1.53±0.10b 1.76±0.08a 1.83±0.02a 成活率SR (%) 96.67±1.92a 95.00±1.67a 96.67±3.33a 96.67±1.92a 93.33±0.00a 97.50±0.83a 蛋白质效率PER (%) 114.35±3.99b 123.64±4.22b 124.02±3.69b 142.68±8.74a 124.21±5.62b 116.03±1.42b 蛋白质沉积率PRR (%) 18.70±0.66b 20.81±0.71b 20.89±0.62b 25.13±1.53a 21.24±0.96b 19.39±0.24b 注: 表中同一行右上角相同字母表示差异不显著(P>0.05), 不同字母表示差异显著(P<0.05); 下同Note: In the same row, values with no letter or the same superscript letter mean no significant difference (P>0.05), while with different small superscript letter mean significant difference (P<0.05); the same applies below ![]()
图 1 饲料赖氨酸水平与幼鳝WGR (a)、SGR (b)、FCR (c)、PRR (d)、PER (e)间的关系Figure 1. The relationship between dietary lysine levels and WGR (a), SGR (b), FCR (c), PRR (d), and PER (e) of juvenile Monopterus albus2.2 饲料赖氨酸水平对幼鳝形体参数的影响
由表 5可知, 饲料赖氨酸水平对幼鳝HSI、ISI、SI和CF无显著影响(P>0.05)。随饲料赖氨酸水平的升高, 幼鳝VSI呈下降再上升趋势, 4.71%水平显著小于2.31%—2.91% (P<0.05), 与3.51%及7.11%水平差异不显著(P>0.05); CP呈先升高后下降趋势, 4.71%水平显著大于2.30%—2.91% (P<0.05), 与3.51%及7.11%水平差异不显著(P>0.05)。
表 5 饲料赖氨酸水平对幼鳝形体参数的影响Table 5. Effects of dietary lysine levels on body parameters of juvenile Monopterus albus项目Item 饲料赖氨酸水平 Dietary lysine level (%) 2.31 2.61 2.91 3.51 4.71 7.11 脏体指数VSI 6.63±0.26a 6.55±0.25a 6.35±0.27ab 5.96±0.19abc 5.38±0.25c 5.75±0.30bc 肝体指数HSI 3.57±0.31a 3.72±0.28a 3.72±0.20a 3.56±0.17a 3.11±0.25a 3.33±0.21a 肠体指数ISI 1.19±0.05a 1.23±0.06a 1.11±0.06a 1.10±0.05a 1.13±0.04a 1.11±0.05a 脾体指数SI 0.23±0.05a 0.23±0.08a 0.17±0.02a 0.19±0.03a 0.17±0.03a 0.15±0.02a 胴体率CP (%) 85.87±0.33c 86.60±0.35bc 86.95±0.47bc 87.34±0.40ab 88.41±0.44a 87.51±0.66ab 肥满度CF (g/cm3) 0.21±0.01a 0.21±0.01a 0.22±0.01a 0.22±0.01a 0.22±0.01a 0.22±0.01a 2.3 饲料赖氨酸水平对幼鳝体成分的影响
由表 6可知, 随饲料赖氨酸水平的升高, 幼鳝肌肉、全鱼水分呈先下降后上升趋势, 但无显著差异(P>0.05)。饲料赖氨酸水平对肌肉、肝脏灰分无显著影响(P>0.05), 但4.11%—7.11%水平全鱼灰分显著低于2.31%与2.91%水平(P<0.05)。随饲料赖氨酸水平的升高, 幼鳝肌肉、全鱼粗脂肪呈先上升后下降趋势, 3.51%水平肌肉粗脂肪显著高于2.31%—2.61%及4.71%—7.11%水平, 4.71%水平全鱼粗脂肪显著高于其余水平(P<0.05); 肝脏粗脂肪含量呈上升趋势, 7.11%水平显著高于2.31%—2.91%水平(P<0.05)。随着饲料赖氨酸水平的升高, 幼鳝肝脏、全鱼粗蛋白质水平呈先升高后下降趋势, 4.71%—7.11%水平肝脏粗蛋白质显著高于2.31%—3.51%水平(P<0.05), 3.51%水平全鱼粗蛋白质显著高于其余水平(P<0.05); 肌肉粗蛋白质呈先升高后不变趋势, 4.71%—7.11%水平显著高于2.31%—2.91%水平(P<0.05), 与3.51%水平差异不显著(P>0.05)。
表 6 饲料赖氨酸水平对幼鳝肌肉、肝脏、全鱼营养成分的影响Table 6. Effects of dietary lysine levels on nutrient composition of muscle, liver, and whole body of juvenile Monopterus albus项目Item (%) 饲料赖氨酸水平Dietary lysine level (%) 2.31 2.61 2.91 3.51 4.71 7.11 肌肉Muscle 水分Moisture 73.66±0.64a 72.83±0.43a 71.74±0.70a 71.29±0.81a 72.75±1.27a 72.61±0.86a 粗灰分Crude ash 1.09±0.02a 1.06±0.02a 1.02±0.03a 1.03±0.03a 1.09±0.01a 1.06±0.04a 粗脂肪Crude lipid 5.11±0.22c 5.67±0.21c 7.77±0.07a 8.08±0.16a 6.63±0.11b 6.41±0.23b 粗蛋白Crude protein 19.50±0.07c 19.94±0.09bc 19.87±0.27bc 20.26±0.08ab 20.57±0.09a 20.65±0.01a 肝脏Liver 水分Moisture 70.24±0.34a 69.31±0.29a 69.77±0.20a 69.06±0.77a 70.21±0.56a 69.23±0.30a 粗灰分Crude ash 0.90±0.02a 0.82±0.01a 0.74±0.01a 0.71±0.01a 0.80±0.18a 0.81±0.03a 粗脂肪Crude lipid 4.00±0.52c 4.59±0.15bc 4.77±0.09bc 5.32±0.10ab 5.35±0.06ab 5.71±0.26a 粗蛋白Crude protein 7.61±0.21b 7.69±0.08b 7.56±0.15b 7.85±0.06b 9.10±0.66a 8.98±0.26a 全鱼Whole body 水分Moisture 71.83±0.67a 70.62±0.44a 71.15±0.30a 70.66±0.62a 70.50±0.50a 71.59±1.04a 粗灰分Crude ash 2.40±0.03a 2.10±0.02cd 2.25±0.03b 2.14±0.01c 2.05±0.01d 2.07±0.04cd 粗脂肪Crude lipid 8.91±0.19c 9.29±0.36bc 8.88±0.13c 9.82±0.23b 10.70±0.42a 9.51±0.06bc 粗蛋白Crude protein 16.55±0.21c 16.89±0.04bc 16.90±0.07bc 17.49±0.04a 17.09±0.16b 16.87±0.06bc 2.4 饲料赖氨酸水平对幼鳝肌肉氨基酸含量的影响
由表 7可知, 随饲料赖氨酸水平的升高, 幼鳝肌肉蛋氨酸、酪氨酸、赖氨酸、色氨酸含量呈升高趋势, 均在7.11%水平取得最高值且显著高于2.31%水平(P<0.05), 其余氨基酸呈先下降后上升趋势, 除精氨酸在2.31%水平有最大值, 其余均在7.11%水平取得最高值。随饲料赖氨酸水平的升高, 幼鳝肌肉总氨基酸、必需氨基酸、非必需氨基酸、半必需氨基酸均呈先下降后上升趋势。在7.11%水平取得最高值且显著高于3.51%水平(P<0.05)。EAA/TAA为39.08%—39.88%, EAA/NEAA为75.08%—77.88%, 均在3.51%水平取得最大值且显著大于2.31%和7.11%水平(P<0.05)。
表 7 饲料赖氨酸水平对幼鳝肌肉氨基酸含量的影响Table 7. Effect of dietary lysine levels on amino acid content in muscle of juvenile Monopterus albus项目 Item (%) 饲料赖氨酸水平Dietary lysine level (%) 2.31 3.51 7.11 天冬氨酸Asp 6.70±0.04a 6.48±0.04b 6.76±0.01a 苏氨酸Thr 3.14±0.20ab 3.10±0.02b 3.20±0.01a 丝氨酸Ser 2.77±0.01a 2.68±0.02b 2.79±0.01a 谷氨酸Glu 11.18±0.04a 10.87±0.07b 11.35±0.01a 脯氨酸Pro 2.91±0.02a 2.68±0.03b 2.81±0.01a 甘氨酸Gly 4.38±0.04a 3.79±0.07c 4.03±0.02b 丙氨酸Ala 4.49±0.01a 4.20±0.04b 4.38±0.01a 胱氨酸Cys 0.75±0.01b 0.74±0.01b 0.79±0.01a 缬氨酸Val 3.33±0.01ab 3.27±0.02b 3.36±0.01a 蛋氨酸Met 1.94±0.01b 1.99±0.02b 2.12±0.02a 异亮氨酸Ile 3.19±0.02ab 3.17±0.02b 3.26±0.01a 亮氨酸Leu 5.35±0.01a 5.22±0.04b 5.36±0.01a 酪氨酸Tyr 2.15±0.02b 2.25±0.01a 2.30±0.01a 苯丙氨酸Phe 2.85±0.03a 2.75±0.02b 2.85±0.01a 组氨酸His 1.62±0.01a 1.61±0.01a 1.63±0.01a 赖氨酸Lys 5.97±0.02b 5.95±0.04b 6.15±0.01a 精氨酸Arg 4.41±0.01a 4.25±0.04b 4.38±0.02a 色氨酸Trp 0.76±0.01b 0.78±0.01a 0.79±0.01a 总氨基酸TAA 67.87±0.06a 65.82±0.51b 68.32±0.07a 必需氨基酸EAA 26.52±0.04b 26.25±0.19b 27.09±0.02a 非必需氨基酸NEAA 35.32±0.01a 33.71±0.28b 35.21±0.04a 半必需氨基酸HEAA 6.03±0.02a 5.87±0.05b 6.01±0.02a EAA/TAA 39.08±0.03c 39.88±0.02a 39.66±0.01b EAA/NEAA 75.08±0.11c 77.88±0.07a 76.94±0.03b 注: 必需氨基酸、半必需氨基酸均以成人所需为参考标准计算Note: Essential amino acids and semi-essential amino acids are calculated with adult requirements as reference standards 2.5 饲料赖氨酸水平对幼鳝血清、肝脏生化指标的影响
由表 8可知, 随饲料赖氨酸水平的升高, 幼鳝血清TP、ALB和AKP均呈升高趋势, 7.11%水平血清TP和ALB显著高于2.31%水平(P<0.05), AKP显著高于2.31%—2.91%水平(P<0.05), 与其余水平差异不显著(P>0.05)。随饲料赖氨酸水平的升高, 幼鳝血清LZM呈先上升后下降趋势, 血清GOT、GPT活性及Sa呈先下降后上升趋势; 其中4.71%水平LZM活性显著高于2.31%—2.91%水平(P<0.05), 与其余水平差异不显著(P>0.05); 3.51%水平Sa显著低于其余水平(P<0.05); 2.91%—3.51%水平血清GOT显著低于2.31%水平(P<0.05), 与其余水平差异不显著(P>0.05)。随饲料赖氨酸水平的升高, 幼鳝肝脏GOT呈升高趋势、肝脏GPT呈先上升再下降趋势, 7.11%水平肝脏GOT显著高于2.31水平(P<0.05), 与其余水平差异不显著(P>0.05)。
表 8 饲料赖氨酸水平对幼鳝血清、肝脏生化指标的影响Table 8. Effects of dietary lysine levels on serum and liver biochemical indexes of juvenile Monopterus albus项目Item 饲料赖氨酸水平Dietary lysine level (%) 2.31 2.61 2.91 3.51 4.71 7.11 血清Serum 总蛋白TP (g/L) 34.63±3.85b 39.74±4.81ab 46.06±4.27ab 45.84±4.37ab 47.72±5.51ab 50.18±4.56a 白蛋白ALB (g/L) 8.51±0.80b 9.85±0.37ab 10.54±0.87ab 10.61±0.52ab 10.75±0.54a 11.97±0.87a 碱性磷酸酶AKP
(金氏单位/100 mL)0.56±0.10c 0.88±0.22bc 0.92±0.28bc 1.54±0.07abc 1.90±0.67ab 2.22±0.36a 血氨Sa (μmol/L) 136.42±7.77ab 124.05±5.75b 123.63±1.66b 108.78±0.71c 128.72±5.94ab 143.02±4.39a 溶菌酶活性LZM (μg/mL) 1281.20±142.67c 1488.15±129.98bc 1510.90±164.92bc 1879.94±174.98abc 2149.13±288.63a 2018.96±242.23ab 谷草转氨酶GOT (U/L) 6.30±1.06a 4.48±0.33ab 2.90±0.60b 3.01±0.78b 4.29±0.96ab 4.68±0.58ab 谷丙转氨酶GPT (U/L) 1.99±0.35a 1.49±0.33a 1.45±0.46a 1.48±0.22a 2.19±0.32a 2.23±0.89a 肝脏Liver 谷草转氨酶GOT (U/g prot) 12.17±0.85b 15.93±2.28ab 18.66±1.65a 18.83±1.56a 18.86±1.59a 20.24±2.52a 谷丙转氨酶GPT (U/g prot) 15.31±1.38a 18.09±1.75a 19.76±0.82a 20.22±0.87a 19.10±0.58a 18.68±2.15a 2.6 饲料赖氨酸水平对幼鳝消化酶活性的影响
由表 9可知, 饲料赖氨酸水平对幼鳝肝胰脏、前肠淀粉酶活性均无显著影响(P>0.05)。随饲料赖氨酸水平的升高, 幼鳝肝胰脏、前肠蛋白酶活性呈先升高后降低趋势, 2.91%—3.51%水平肝胰脏蛋白酶活性显著高于2.31%—2.61%水平(P<0.05), 与其余水平差异不显著(P>0.05); 3.51%水平前肠蛋白酶活性显著高于7.11%水平(P<0.05), 与其余水平差异不显著(P>0.05)。随饲料赖氨酸水平的升高, 幼鳝前肠脂肪酶活性呈先上升后下降趋势, 3.59%水平显著高于其余水平(P<0.05); 肝胰脏脂肪酶活性呈先上升后下降趋势但各水平间差异不显著(P>0.05)。
表 9 饲料赖氨酸水平对幼鳝肝胰脏、前肠酶活性的影响Table 9. Effects of dietary lysine levels on digestive enzyme activities in hepatopancreas and foregut of juvenile Monopterus albus项目Item 饲料赖氨酸水平Dietary lysine level (%) 2.31 2.61 2.91 3.51 4.71 7.11 肝胰脏Hepatopancreas 脂肪酶LPS (U/g prot) 1.46±0.15a 1.65±0.27a 1.61±0.20a 1.71±0.18a 1.86±0.19a 1.69±0.08a 蛋白酶TPS (U/mg prot) 75.79±10.59b 82.77±3.11b 108.17±4.04a 107.80±8.88a 96.57±4.11ab 94.35±6.55ab 淀粉酶AMS (U/mg prot) 0.59±0.29a 0.46±0.24a 0.61±0.25a 0.81±0.11a 0.62±0.14a 0.84±0.23a 前肠Foregut 脂肪酶LPS (U/g prot) 3.49±1.85b 6.13±0.37b 6.79±1.05b 10.21±0.59a 6.64±1.28b 6.26±0.53b 蛋白酶TPS (U/mg prot) 103299.60±
16827.24ab106732.50±
11655.16ab103911.05±
20000.41ab120568.67±
7039.39a83889.73±
11920.01ab71762.37±
3815.82b淀粉酶AMS (U/mg prot) 1.17±0.26a 0.99±0.09a 1.31±0.40a 1.48±0.43a 1.19±0.09a 1.05±0.10a 2.7 饲料赖氨酸水平对幼鳝肝脏蛋白质代谢相关基因相对表达量的影响
由图 2可知, 3.51%水平幼鳝肝脏mTOR、S6K1及IGF-1的相对表达量显著高于2.31%与7.11%水平(P<0.05); 2.31%水平eIF4E-BP2的相对表达量显著高于3.51%与7.11%水平(P<0.05)。
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图 2 饲料赖氨酸水平对幼鳝肝脏mTOR、S6K1、eIF4E-BP2和IGF-1相对表达量的影响Figure 2. Effects of dietary lysine levels on the relative expression of mTOR, S6K1, eIF4E-BP2, and IGF-1 in the liver of juvenile Monopterus albus3. 讨论
3.1 饲料赖氨酸水平对幼鳝生长性能的影响
赖氨酸作为鱼类的必需氨基酸, 对鱼类的生长发育有重要作用。在本试验中, 随饲料赖氨酸水平的升高, 幼鳝的生长性能呈先升高后下降趋势, 在3.51%水平生长最佳。说明幼鳝能够较好地利用外源添加的晶体DL-赖氨酸, 但缺乏或过量的赖氨酸均会破坏饲料的氨基酸平衡, 影响其生长和饲料利用。在红鳍东方鲀(Takifugu rubripes)[7]幼鱼、胭脂鱼(Myxocyprinus asiaticus)[25]幼鱼的研究中也有类似的结果。雷帕霉素靶蛋白(mTOR)可调节鱼体生长和蛋白质翻译, 对营养素敏感的mTORC1被激活后, 下游作用因子S6K1被磷酸化, 并抑制eIF4E-BP2的表达, 激活蛋白质翻译[12]。氨基酸被认为是鱼类GH/IGF生长轴的调节因子, IGF信号在哺乳动物生长发育中发挥重要作用[26]。在本试验中, 与2.31%水平相比, 3.51%水平上调了肝脏mTOR、S6K1及IGF-1的相对表达量, 降低了eIF4E-BP2的相对表达量。这说明适宜的赖氨酸水平激活了mTOR和GH/IGF信号通路, 促进幼鳝生长和蛋白质合成, 在异育银鲫(Carassius auratus gibelio)[27]和鳃棘鲈(Plectropomus leopardus)[9]的研究中也有相似的结果。与其他黄鳝上的研究相比, 本试验中各组PRR和PER均偏低[28], 而FCR整体偏高[24], 这可能是由于养殖过程中有部分时间段温度过高(高于32℃的天数占养殖天数的35.56%), 使得黄鳝处于应激状态, 食欲减退导致的。因而今后有必要研究在高温下黄鳝的消化情况以探究适宜的投喂策略。以WGR、SGR、FCR、PRR、PER为评价指标, 通过折线回归得出幼鳝的适宜赖氨酸需求量为3.38%—3.53% (6.57%—6.90%), 以占饲料蛋白质水平为标准, 此结果高于草鱼幼鱼的2.07% (5.44%)[16], 鳃棘鲈的2.60%—2.97% (4.91%—5.60%)[9], 日本鲈(Lateolabrax japonicus)的2.49%—2.61% (5.80%—6.07%)[29], 但低于鳡(Elopichthys bambusa)幼鱼的3.40% (7.39%)[30], 与大黄鱼仔鱼的3.34%—3.37% (6.49%—6.55%)[14]相近。
研究发现, 以生长为评价指标大西洋鲑(Salmo salar L.)[31]、黑鲷(Acanthopagrus schlegelii)[32]等鱼类中存在赖氨酸与精氨酸的拮抗作用, 在大西洋鲑[33]的研究发现这可能是因为精氨酸与赖氨酸均用刷状缘膜为载体, 其吸收在中肠存在竞争抑制现象, 且二者同为碱性氨基酸, 在吸收后的代谢过程也存在竞争抑制[34]。但在虹鳟(Oncorhynchus mykiss)[35]、杂交条纹鲈(Moronesaxatilis×M. chrysops)[36]的研究中并未发现拮抗作用。也有部分研究者认为二者的拮抗可能并不表现在生长性能上, Dong等[10]发现饲料赖氨酸过量使得罗氏沼虾(Macrobrachium rosenbergii)血清精氨酸含量下降, 沈勇等[34]发现随饲料精氨酸/赖氨酸比值的增大, 全雄黄颡鱼全鱼大部分氨基酸的沉积率呈先上升后降低的趋势。在本研究中, 过量的赖氨酸导致幼鳝生长性能下降, 2.31%水平幼鳝肌肉精氨酸含量最高, 但后续并未与饲料赖氨酸水平呈反比变化。Huo等[37]发现过量的精氨酸对黄鳝的生长无影响, 但降低了其免疫能力。因此, 对于黄鳝是否存在赖氨酸与精氨酸的拮抗还需进一步的研究。
3.2 饲料赖氨酸水平对幼鳝形体参数的影响
黄鳝的胴体是人们主要的食用部分。本试验中, 4.71%水平VSI显著低于2.30%—2.91%水平, CP则显著高于2.30%—2.91%水平, 说明适宜的赖氨酸水平能够增加黄鳝的胴体率。在草鱼[38]上的研究也发现赖氨酸甲酯能够降低草鱼的VSI和HSI, 提高CP。在军曹鱼幼鱼[17]上也发现其VSI随饲料赖氨酸水平的升高呈先下降后上升趋势, 且赖氨酸缺乏组VSI显著升高。
3.3 饲料赖氨酸水平对幼鳝体成分的影响
本试验中, 随饲料赖氨酸水平的升高, 幼鳝肌肉粗蛋白质含量呈先上升后稳定的趋势, 全鱼、肝脏粗蛋白质含量呈先上升后下降趋势, 这与PRR的结果也一致。在六丝马鲅(Polydactylus sexfilis)[39]幼鱼、大西洋鳕(Atlantic cod)[40]上也有相同的结果。这可能是因为在赖氨酸缺乏时加入赖氨酸能够改善饲料氨基酸平衡, 促进鱼体对饲料的吸收利用从而增强机体蛋白质合成, 而当赖氨酸过量时, 又使得饲料氨基酸平衡被破坏, 使得多余氨基酸参与氧化分解供能产生尿素和氨排出体外; 过量的赖氨酸也可能影响精氨酸的吸收利用, 使得机体蛋白质沉积减少。赖氨酸能够通过合成肉碱来促进长链脂肪酸转移到线粒体中, 从而在脂质β-氧化中发挥重要作用[41], 已有不少研究发现赖氨酸能够促进脂解从而减少鱼体脂质沉积[42]。在本试验中, 随饲料赖氨酸水平的升高, 幼鳝肌肉、全鱼粗脂肪含量呈先上升后下降趋势, 肝脏粗脂肪含量呈上升趋势。在乌苏里拟鲿[13]幼鱼上也发现肌肉粗脂肪随赖氨酸水平的升高先升高后下降, 在团头鲂(Megalobrama amblycephala)[43]上的研究则发现全鱼粗脂肪随赖氨酸水平的升高先稳定后升高。这可能是因为适宜赖氨酸水平提高了幼鳝脂肪酶活性, 从而提高了幼鳝对脂质的消化吸收能力, 随着赖氨酸的进一步升高, 肉碱合成增多, 使得脂肪酸通过线粒体膜的转运增多, 减少肌肉脂质沉积[40]。此前的许多研究都表明, 饲料赖氨酸水平对鱼体灰分无显著影响[12, 42]。在本试验中, 随饲料赖氨酸水平的升高, 幼鳝全鱼粗灰分呈下降趋势, 在半滑舌鳎(Cynoglossus semilaevis Günther)[44]、虹鳟[45]的研究中也发现了相同的结果, 具体原因还需进一步分析。
3.4 饲料赖氨酸水平对幼鳝肌肉氨基酸含量的影响
蛋白质的营养价值取决于其含氨基酸的含量及比例。本试验中, 随饲料赖氨酸水平的升高, 除精氨酸在2.31%水平有最大值, 其余氨基酸均在7.11%水平取得最大值。幼鳝肌肉总氨基酸、必需氨基酸、非必需氨基酸呈先下降后上升趋势, 均在7.11%水平取得最大值。这说明满足营养物质沉积所需的赖氨酸要比满足生长高[46]。氨基酸比例也是评价蛋白质的重要条件之一, 本试验中各组幼鳝肌肉EAA/TAA为39.08%—39.88%, EAA/NEAA为75.08%—77.88%, 且均在3.51%水平取得最大值, 说明适宜赖氨酸水平能够提高幼鳝肌肉必需氨基酸的比例。
3.5 饲料赖氨酸水平对幼鳝血清、肝脏生化指标的影响
血清生化指标常用于鱼类生理学诊断以确定其营养代谢和健康状况[47]。血清TP具有维持血浆胶体渗透压, 运输多种代谢物质的功能, 血清ALB是血浆中主要的蛋白载体, 二者也被认为具有增强免疫反应的作用[27]; 超过90%的血清TP以及所有的血清ALB是由肝脏合成, 因而血清TP和ALB可以用来反映肝脏合成功能。氨是鱼类氨基酸分解代谢的主要产物, 鱼体内肝脏代谢产生的氨, 在血液中逐渐累积, 最后由鳃排出体外[48]。因此血氨(Sa)含量可以反映蛋白质的分解和合成情况。在本试验中, 随饲料赖氨酸水平的升高, 幼鳝血清TP、ALB呈先上升后趋于稳定的趋势, 均在7.11%水平取得最大值; Sa呈先下降后上升趋势, 3.51%水平有最低值。在斜带石斑鱼(Epinephelus coioides)[49]幼鱼上也有相同的趋势。这说明赖氨酸缺乏使得幼鳝体内氨基酸分解代谢旺盛, 随着赖氨酸水平的升高, 合成代谢增强, 分解代谢减弱; 赖氨酸过量时, 分解和合成代谢的综合作用造成了血清TP、ALB呈稳定趋势, Sa呈上升趋势。
谷丙转氨酶(GPT)主要分布于肝脏, 谷草转氨酶(GOT)主要分布于心肌, 其次在肝脏、骨骼肌等组织, 二者在鱼体的氨基酸代谢方面发挥重要作用[50]。当相应细胞受损时, 细胞膜通透性增加, 胞浆内的GOT、GPT释放入血, 使得其在血清中活性升高, 故常用来评价机体氨基酸代谢和肝功能情况。本试验中, 血清GOT、GPT随饲料赖氨酸水平的升高呈先下降再上升趋势, 血清GOT在2.91%水平取得最小值。肝脏GOT随饲料赖氨酸水平的升高呈先上升后稳定的趋势, 在7.11%水平取得最大值, 肝脏GPT呈先上升后下降的趋势, 在3.15%水平取得最大值且与其余水平差异不显著。在许氏平鲉(Sebastes schlegeli)[51]幼鱼、鳡[30]幼鱼的研究中也有相似的结果。说明适宜赖氨酸水平能够增强幼鳝肝脏转氨酶活性, 增强其对氨基酸的吸收利用; 赖氨酸缺乏不仅会影响幼鳝氨基酸代谢, 减少蛋白质沉积, 还可能对肝脏细胞造成损害。
碱性磷酸酶(AKP)能直接参与生物体磷酸基团的转移和代谢过程, 与脊椎动物的骨化和蛋白质分泌有关, 在鱼类对营养物质的吸收利用中发挥重要作用, 是水生动物的生长调控酶类之一[52]。在本试验中, 幼鳝血清AKP随饲料赖氨酸水平的升高而升高, 在鳡[30]幼鱼的研究中发现血清AKP随饲料赖氨酸水平的升高呈先升高后下降趋势; 胡凯等[53]也发现适宜水平的80%赖氨酸硫酸盐能显著提高生长中期草鱼前、中、后肠AKP活性。这说明适宜赖氨酸能够促进幼鳝对营养物质的吸收, 过量赖氨酸并未降低血清AKP可能是因为不同鱼类对赖氨酸过量的耐受度不同。溶菌酶(LZM)是白细胞分泌的一种分解酶, 也存在于各种组织(如血液)和分泌物(如黏液)中, 是鱼类非特异性免疫的重要组成部分。本试验中, 幼鳝血清LZM随饲料赖氨酸水平的升高呈先上升后趋于稳定的趋势, 在4.71%水平最高。Wu等[18]的研究也发现适宜赖氨酸水平显著提高了拟穴青蟹幼蟹LZM基因的转录水平。说明适宜赖氨酸水平能显著提高幼鳝非特异性免疫能力, 维持机体健康。
3.6 饲料赖氨酸水平对幼鳝肝脏、前肠消化酶活性的影响
消化酶主要包括蛋白酶、脂肪酶、淀粉酶, 能够把大分子蛋白质、脂肪和淀粉降解为肽、氨基酸, 脂肪酸, 甘油和寡糖等小分子物质, 有利于营养物质消化吸收, 其活性对机体生长发育极为重要。本试验中, 随饲料赖氨酸水平的升高, 幼鳝肝胰脏、前肠蛋白酶活性呈先升高后下降趋势, 分别在2.91%和3.51%水平取得最大值。Liu等[54]在海参(Apostichopus japonicus)上的研究也发现了相同的结果, 这可能是因为赖氨酸能够刺激鱼类胰腺释放胰蛋白酶原, 从而影响胰蛋白酶的合成和分泌[55]; 也可能是赖氨酸直接增加了机体胰蛋白酶的合成[56]。随饲料赖氨酸水平的升高, 幼鳝前肠脂肪酶酶活性呈先升高后下降趋势, 在3.59%水平取得最大值, 脂肪酶活性的变化能够解释幼鳝脂肪沉积的结果。在鳃棘鲈[9]幼鱼、亚成年草鱼[57]上也有相同的结果。这些结果说明适宜赖氨酸水平可通过提高幼鳝肝胰脏、前肠消化酶活性来增强其对营养物质的消化吸收, 从而促进生长。
4. 结论
综上, 适宜赖氨酸水平能提高幼鳝消化能力及非特异性免疫能力, 通过促进GH/IGF及mTOR通路相关基因表达增强机体蛋白质沉积, 从而提高幼鳝生长性能。以WGR、SGR、FCR、PRR和PER为评价指标, 综合血清、肝脏的生化指标, 黄鳝幼鳝[(7.64±0.04) g]的适宜赖氨酸需求量为3.38%—3.55% (占饲料蛋白质6.57%—6.90%)。
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图 1 饲料赖氨酸水平与幼鳝WGR (a)、SGR (b)、FCR (c)、PRR (d)、PER (e)间的关系
Figure 1. The relationship between dietary lysine levels and WGR (a), SGR (b), FCR (c), PRR (d), and PER (e) of juvenile Monopterus albus
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图 2 饲料赖氨酸水平对幼鳝肝脏mTOR、S6K1、eIF4E-BP2和IGF-1相对表达量的影响
Figure 2. Effects of dietary lysine levels on the relative expression of mTOR, S6K1, eIF4E-BP2, and IGF-1 in the liver of juvenile Monopterus albus
表 1 试验饲料组成及营养水平(干物质基础)
Table 1 Composition and nutritional level of the experimental diets (DM basis)
原料Ingredient (%) 饲料赖氨酸水平
Dietary lysine level (%)2.31 2.61 2.91 3.51 4.71 7.11 鱼粉Fish meal 30 30 30 30 30 30 复合蛋白粉Compound protein1 6 6 6 6 6 6 豆粕Soybean meal 16 16 16 16 16 16 玉米蛋白粉Corn protein powder 6 6 6 6 6 6 棉籽蛋白Cottonseed protein 6 6 6 6 6 6 小麦粉Wheat meal 20 20 20 20 20 20 碎米粉Crushed rice flour 2 2 2 2 2 2 豆油Soybean oil 4.35 4.35 4.35 4.35 4.35 4.35 DL-赖氨酸Lysine 0 0.3 0.6 1.2 2.4 4.8 L-丙氨酸Alanine 4.8 4.5 4.2 3.6 2.4 0 磷酸二氢钙Ca(H2PO4) 2 2 2 2 2 2 混合氨基酸Amino acid mix2 1.75 1.75 1.75 1.75 1.75 1.75 复合添加剂Composite additive3 0.1 0.1 0.1 0.1 0.1 0.1 预混料Premix4 0.5 0.5 0.5 0.5 0.5 0.5 胆碱Choline chloride 0.5 0.5 0.5 0.5 0.5 0.5 总和Sum 100 100 100 100 100 100 营养水平 Nutrient levels5 水分Moisture 7.14 7.75 6.50 6.80c 6.81 6.29 粗蛋白质Crude protein 51.46 51.21 51.38 51.37 51.46 51.16 粗脂肪Crude fat 7.69 7.51 7.60 7.48 7.41 7.35 粗灰分Crude ash 9.71 9.62 9.72 9.67 9.71 9.68 赖氨酸Lysine 2.31 2.61 2.91 3.51 4.71 7.11 总能 Gross energy (MJ/kg)6 17.45 17.45 17.45 17.45 17.45 17.45 注: 1复合蛋白粉由蚯蚓粉:谷朊粉1﹕2组成 The composite protein is composed of earthworm powder: millet powder 1﹕2; 2混合氨基酸为1.10%苏氨酸、0.55%蛋氨酸、0.10%色氨酸 The mixed amino acids are 1.10% threonine, 0.55% methionine and 0.10% tryptophan; 3复合添加剂由植酸酶、复合酶、蛋白酶、维生素C组成 The coomposite additive is composed of phytase, compound enzyme, protease and vitamin C; 4预混料为每千克饲料提供 The premix provided the following per kg of the diet: VA 6000 IU, VD3 2500.0 IU, VE 200.0 mg, VK3 10.0 mg, VB1 25.0 mg, VB2 45.0 mg, 烟酸 nicotinic acid 200.0 mg, VB6 20.0 mg, 泛酸钙 Ca-pantothenate acid 60.0 mg, 叶酸 folic acid 10.0 mg, VB12 0.1 mg, 生物素 biotin 1.5 mg, 肌醇 inositol 200.0 mg, NaSeO3·5H2O 0.3 mg, CoCZ·6H2O 0.4 mg, KI 0.8 mg, CuSO4·5H2O 10.0 mg, MnSO4·4H2O 20.0 mg, ZnSO4·H2O 50.0 mg, FeSO4·7H2O 150.0 mg, MgSO4·7H2O 500.0 mg, NaCl 1000.0 mg; 5营养水平为实测值 Nutritional level is measured value; 6为计算值, 蛋白质以23.64 MJ/kg, 脂肪以39.50 MJ/kg, 淀粉以17.15 MJ/kg计算 The calculated value, protein is 23.64 MJ/kg, fat is 39.50 MJ/kg, and starch is 17.15 MJ/kg 
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表 2 试验饲料氨基酸组成(干物质基础)
Table 2 Amino acid composition of experimental diets (DM basis)
项目Item (%) 饲料赖氨酸水平 Dietary lysine level (%) 2.31 2.61 2.91 3.51 4.71 7.11 天冬氨酸Asp 3.18 3.18 3.18 3.18 3.18 3.18 苏氨酸Thr 2.40 2.40 2.40 2.40 2.40 2.40 丝氨酸Ser 1.63 1.63 1.63 1.63 1.63 1.63 谷氨酸Glu 7.39 7.39 7.39 7.39 7.39 7.39 脯氨酸Pro 2.17 2.17 2.17 2.17 2.17 2.17 甘氨酸Gly 1.91 1.91 1.91 1.91 1.91 1.91 丙氨酸Ala 6.72 6.42 6.12 5.52 4.32 1.92 胱氨酸Cys 0.49 0.49 0.49 0.49 0.49 0.49 缬氨酸Val 1.79 1.79 1.79 1.79 1.79 1.79 蛋氨酸Met 1.27 1.27 1.27 1.27 1.27 1.27 异亮氨酸Ile 1.54 1.54 1.54 1.54 1.54 1.54 亮氨酸Leu 2.95 2.95 2.95 2.95 2.95 2.95 酪氨酸Tyr 1.13 1.13 1.13 1.13 1.13 1.13 苯丙氨酸Phe 1.74 1.74 1.74 1.74 1.74 1.74 组氨酸His 0.99 0.99 0.99 0.99 0.99 0.99 赖氨酸Lys 2.31 2.61 2.91 3.51 4.71 7.11 精氨酸Arg 2.28 2.28 2.28 2.28 2.28 2.28 色氨酸Trp 0.50 0.50 0.50 0.50 0.50 0.50 总氨基酸TAA 42.39 42.39 42.39 42.39 42.39 42.39
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表 3 实时荧光定量PCR引物序列
Table 3 The primers sequence of quantitative RT-PCR
引物名称
Gene name引物序列
Primer sequence (5′—3′)产物大小
Product
length
(bp)GenBank
登录号
Accession No.mTOR-F CGTTCCGACTGACGAGGATG 152 XM_020612477.1 mTOR-R CAGCGGGTCATAGACGAAGG S6K1-F TGAAGAACTCCCAACCCACAC 101 XM_020602184.1 S6K1-R GAGTCTCTACAGGGAGGTGGA eIF4E-BP2-F AATGCGTCAAAGGGTCACTCA 132 XM_020603221.1 eIF4E-BP2-R GCCCTCACTGCCAATCCAA IGF-1-F ACCCTGACTCCGACGACAA 140 XM_020613661.1 IGF-1-R CGTGACCGTCGTGTACTGG 18S-F GTGAAATTCTTGGACCGGCG 145 XM_020605951.1 18S-R GGATCGCTAGTTGGCATCGT
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表 4 饲料赖氨酸水平对幼鳝生长性能的影响
Table 4 Effects of dietary lysine levels on growth performance of juvenile Monopterus albus
项目Item 饲料赖氨酸水平 Dietary lysine level (%) 2.31 2.61 2.91 3.51 4.71 7.11 初均重IBW (g) 7.61±0.02a 7.64±0.02a 7.64±0.02a 7.63±0.02a 7.64±0.02a 7.62±0.02a 末均重FBW (g) 27.71±0.99c 28.69±0.37c 30.20±1.25bc 34.76±1.74a 33.23±1.79ab 30.91±0.92abc 增重率WGR (%) 264.10±13.69c 275.79±5.14c 295.65±17.40bc 355.52±22.83a 335.05±23.28ab 305.58±12.00abc 特定生长率SGR (%/d) 1.43±0.04c 1.47±0.02c 1.52±0.05bc 1.68±0.06a 1.62±0.06ab 1.55±0.03abc 饲料系数FCR 1.86±0.08a 1.77±0.06a 1.73±0.07ab 1.53±0.10b 1.76±0.08a 1.83±0.02a 成活率SR (%) 96.67±1.92a 95.00±1.67a 96.67±3.33a 96.67±1.92a 93.33±0.00a 97.50±0.83a 蛋白质效率PER (%) 114.35±3.99b 123.64±4.22b 124.02±3.69b 142.68±8.74a 124.21±5.62b 116.03±1.42b 蛋白质沉积率PRR (%) 18.70±0.66b 20.81±0.71b 20.89±0.62b 25.13±1.53a 21.24±0.96b 19.39±0.24b 注: 表中同一行右上角相同字母表示差异不显著(P>0.05), 不同字母表示差异显著(P<0.05); 下同Note: In the same row, values with no letter or the same superscript letter mean no significant difference (P>0.05), while with different small superscript letter mean significant difference (P<0.05); the same applies below
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表 5 饲料赖氨酸水平对幼鳝形体参数的影响
Table 5 Effects of dietary lysine levels on body parameters of juvenile Monopterus albus
项目Item 饲料赖氨酸水平 Dietary lysine level (%) 2.31 2.61 2.91 3.51 4.71 7.11 脏体指数VSI 6.63±0.26a 6.55±0.25a 6.35±0.27ab 5.96±0.19abc 5.38±0.25c 5.75±0.30bc 肝体指数HSI 3.57±0.31a 3.72±0.28a 3.72±0.20a 3.56±0.17a 3.11±0.25a 3.33±0.21a 肠体指数ISI 1.19±0.05a 1.23±0.06a 1.11±0.06a 1.10±0.05a 1.13±0.04a 1.11±0.05a 脾体指数SI 0.23±0.05a 0.23±0.08a 0.17±0.02a 0.19±0.03a 0.17±0.03a 0.15±0.02a 胴体率CP (%) 85.87±0.33c 86.60±0.35bc 86.95±0.47bc 87.34±0.40ab 88.41±0.44a 87.51±0.66ab 肥满度CF (g/cm3) 0.21±0.01a 0.21±0.01a 0.22±0.01a 0.22±0.01a 0.22±0.01a 0.22±0.01a
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表 6 饲料赖氨酸水平对幼鳝肌肉、肝脏、全鱼营养成分的影响
Table 6 Effects of dietary lysine levels on nutrient composition of muscle, liver, and whole body of juvenile Monopterus albus
项目Item (%) 饲料赖氨酸水平Dietary lysine level (%) 2.31 2.61 2.91 3.51 4.71 7.11 肌肉Muscle 水分Moisture 73.66±0.64a 72.83±0.43a 71.74±0.70a 71.29±0.81a 72.75±1.27a 72.61±0.86a 粗灰分Crude ash 1.09±0.02a 1.06±0.02a 1.02±0.03a 1.03±0.03a 1.09±0.01a 1.06±0.04a 粗脂肪Crude lipid 5.11±0.22c 5.67±0.21c 7.77±0.07a 8.08±0.16a 6.63±0.11b 6.41±0.23b 粗蛋白Crude protein 19.50±0.07c 19.94±0.09bc 19.87±0.27bc 20.26±0.08ab 20.57±0.09a 20.65±0.01a 肝脏Liver 水分Moisture 70.24±0.34a 69.31±0.29a 69.77±0.20a 69.06±0.77a 70.21±0.56a 69.23±0.30a 粗灰分Crude ash 0.90±0.02a 0.82±0.01a 0.74±0.01a 0.71±0.01a 0.80±0.18a 0.81±0.03a 粗脂肪Crude lipid 4.00±0.52c 4.59±0.15bc 4.77±0.09bc 5.32±0.10ab 5.35±0.06ab 5.71±0.26a 粗蛋白Crude protein 7.61±0.21b 7.69±0.08b 7.56±0.15b 7.85±0.06b 9.10±0.66a 8.98±0.26a 全鱼Whole body 水分Moisture 71.83±0.67a 70.62±0.44a 71.15±0.30a 70.66±0.62a 70.50±0.50a 71.59±1.04a 粗灰分Crude ash 2.40±0.03a 2.10±0.02cd 2.25±0.03b 2.14±0.01c 2.05±0.01d 2.07±0.04cd 粗脂肪Crude lipid 8.91±0.19c 9.29±0.36bc 8.88±0.13c 9.82±0.23b 10.70±0.42a 9.51±0.06bc 粗蛋白Crude protein 16.55±0.21c 16.89±0.04bc 16.90±0.07bc 17.49±0.04a 17.09±0.16b 16.87±0.06bc
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表 7 饲料赖氨酸水平对幼鳝肌肉氨基酸含量的影响
Table 7 Effect of dietary lysine levels on amino acid content in muscle of juvenile Monopterus albus
项目 Item (%) 饲料赖氨酸水平Dietary lysine level (%) 2.31 3.51 7.11 天冬氨酸Asp 6.70±0.04a 6.48±0.04b 6.76±0.01a 苏氨酸Thr 3.14±0.20ab 3.10±0.02b 3.20±0.01a 丝氨酸Ser 2.77±0.01a 2.68±0.02b 2.79±0.01a 谷氨酸Glu 11.18±0.04a 10.87±0.07b 11.35±0.01a 脯氨酸Pro 2.91±0.02a 2.68±0.03b 2.81±0.01a 甘氨酸Gly 4.38±0.04a 3.79±0.07c 4.03±0.02b 丙氨酸Ala 4.49±0.01a 4.20±0.04b 4.38±0.01a 胱氨酸Cys 0.75±0.01b 0.74±0.01b 0.79±0.01a 缬氨酸Val 3.33±0.01ab 3.27±0.02b 3.36±0.01a 蛋氨酸Met 1.94±0.01b 1.99±0.02b 2.12±0.02a 异亮氨酸Ile 3.19±0.02ab 3.17±0.02b 3.26±0.01a 亮氨酸Leu 5.35±0.01a 5.22±0.04b 5.36±0.01a 酪氨酸Tyr 2.15±0.02b 2.25±0.01a 2.30±0.01a 苯丙氨酸Phe 2.85±0.03a 2.75±0.02b 2.85±0.01a 组氨酸His 1.62±0.01a 1.61±0.01a 1.63±0.01a 赖氨酸Lys 5.97±0.02b 5.95±0.04b 6.15±0.01a 精氨酸Arg 4.41±0.01a 4.25±0.04b 4.38±0.02a 色氨酸Trp 0.76±0.01b 0.78±0.01a 0.79±0.01a 总氨基酸TAA 67.87±0.06a 65.82±0.51b 68.32±0.07a 必需氨基酸EAA 26.52±0.04b 26.25±0.19b 27.09±0.02a 非必需氨基酸NEAA 35.32±0.01a 33.71±0.28b 35.21±0.04a 半必需氨基酸HEAA 6.03±0.02a 5.87±0.05b 6.01±0.02a EAA/TAA 39.08±0.03c 39.88±0.02a 39.66±0.01b EAA/NEAA 75.08±0.11c 77.88±0.07a 76.94±0.03b 注: 必需氨基酸、半必需氨基酸均以成人所需为参考标准计算Note: Essential amino acids and semi-essential amino acids are calculated with adult requirements as reference standards
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表 8 饲料赖氨酸水平对幼鳝血清、肝脏生化指标的影响
Table 8 Effects of dietary lysine levels on serum and liver biochemical indexes of juvenile Monopterus albus
项目Item 饲料赖氨酸水平Dietary lysine level (%) 2.31 2.61 2.91 3.51 4.71 7.11 血清Serum 总蛋白TP (g/L) 34.63±3.85b 39.74±4.81ab 46.06±4.27ab 45.84±4.37ab 47.72±5.51ab 50.18±4.56a 白蛋白ALB (g/L) 8.51±0.80b 9.85±0.37ab 10.54±0.87ab 10.61±0.52ab 10.75±0.54a 11.97±0.87a 碱性磷酸酶AKP
(金氏单位/100 mL)0.56±0.10c 0.88±0.22bc 0.92±0.28bc 1.54±0.07abc 1.90±0.67ab 2.22±0.36a 血氨Sa (μmol/L) 136.42±7.77ab 124.05±5.75b 123.63±1.66b 108.78±0.71c 128.72±5.94ab 143.02±4.39a 溶菌酶活性LZM (μg/mL) 1281.20±142.67c 1488.15±129.98bc 1510.90±164.92bc 1879.94±174.98abc 2149.13±288.63a 2018.96±242.23ab 谷草转氨酶GOT (U/L) 6.30±1.06a 4.48±0.33ab 2.90±0.60b 3.01±0.78b 4.29±0.96ab 4.68±0.58ab 谷丙转氨酶GPT (U/L) 1.99±0.35a 1.49±0.33a 1.45±0.46a 1.48±0.22a 2.19±0.32a 2.23±0.89a 肝脏Liver 谷草转氨酶GOT (U/g prot) 12.17±0.85b 15.93±2.28ab 18.66±1.65a 18.83±1.56a 18.86±1.59a 20.24±2.52a 谷丙转氨酶GPT (U/g prot) 15.31±1.38a 18.09±1.75a 19.76±0.82a 20.22±0.87a 19.10±0.58a 18.68±2.15a
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表 9 饲料赖氨酸水平对幼鳝肝胰脏、前肠酶活性的影响
Table 9 Effects of dietary lysine levels on digestive enzyme activities in hepatopancreas and foregut of juvenile Monopterus albus
项目Item 饲料赖氨酸水平Dietary lysine level (%) 2.31 2.61 2.91 3.51 4.71 7.11 肝胰脏Hepatopancreas 脂肪酶LPS (U/g prot) 1.46±0.15a 1.65±0.27a 1.61±0.20a 1.71±0.18a 1.86±0.19a 1.69±0.08a 蛋白酶TPS (U/mg prot) 75.79±10.59b 82.77±3.11b 108.17±4.04a 107.80±8.88a 96.57±4.11ab 94.35±6.55ab 淀粉酶AMS (U/mg prot) 0.59±0.29a 0.46±0.24a 0.61±0.25a 0.81±0.11a 0.62±0.14a 0.84±0.23a 前肠Foregut 脂肪酶LPS (U/g prot) 3.49±1.85b 6.13±0.37b 6.79±1.05b 10.21±0.59a 6.64±1.28b 6.26±0.53b 蛋白酶TPS (U/mg prot) 103299.60±
16827.24ab106732.50±
11655.16ab103911.05±
20000.41ab120568.67±
7039.39a83889.73±
11920.01ab71762.37±
3815.82b淀粉酶AMS (U/mg prot) 1.17±0.26a 0.99±0.09a 1.31±0.40a 1.48±0.43a 1.19±0.09a 1.05±0.10a
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