沈钰博 王际英 李宝山 刘财礼 王晓艳 黄炳山 王世信 孙永智
许氏平鲉幼鱼对饲料中精氨酸需求量的研究*
沈钰博1,2王际英2①李宝山2刘财礼1,2王晓艳2黄炳山2王世信2孙永智2
(1. 上海海洋大学 水产科学国家级实验教学示范中心 农业农村部鱼类营养与环境生态研究中心 水产动物遗传育种中心上海市协同创新中心 上海 201306;2. 山东省海洋资源与环境研究院 山东省海洋生态修复重点实验室 山东 烟台 264006)
本实验旨在研究饲料中不同精氨酸含量对许氏平鲉()幼鱼生长、体组成、血清生化指标及肝脏相关酶活力的影响,以确定其对精氨酸的最适需求量。在基础饲料中添加晶体L-精氨酸配制精氨酸含量分别为1.39%、1.83%、2.34%、2.80%、3.39%和4.08%的6组等氮等脂的实验饲料(D1、D2、D3、D4、D5和D6)。饲喂初始体重为(12.03±0.03) g的许氏平鲉幼鱼56 d。结果显示,随着饲料中精氨酸含量的升高,实验鱼的增重率(WGR)、特定生长率(SGR)和蛋白质效率(PER)均先升高后降低,在D3组达到最高值,显著高于D1、D2、D5和D6组(<0.05);饲料系数(FCR)先降低后升高,在D3组达到最低值,显著低于D1、D2、D5和D6组(<0.05);D3组肌肉粗蛋白含量最高且显著高于其他组(<0.05),肌肉水分、粗脂肪、粗灰分和全鱼水分、粗蛋白、粗脂肪和粗灰分无显著性差异;全鱼和肌肉总必需氨基酸(ΣEAA)和总氨基酸(ΣAA)含量均先升高后降低;血清谷丙转氨酶(ALT)活力先降低后升高,D3组显著小于其他组(<0.05),一氧化氮(NO)含量先升高后降低,D3和D4组显著高于其他组(<0.05),尿素氮(BUN)含量先升高后趋于平缓;肝脏超氧化物歧化酶(SOD)、溶菌酶(LZM)、总一氧化氮合酶(TNOS)和诱导型一氧化氮合酶(iNOS)活力均先升高后降低,丙二醛(MDA)含量先降低后升高,D2~D4组显著低于D1、D5和D6组(<0.05)。在本研究条件下,以WGR为评价指标,经一元二次回归分析得出,许氏平鲉幼鱼[(12.03±0.03) g]饲料中精氨酸适宜需求量为2.78% (占5.56%饲料粗蛋白)。
许氏平鲉幼鱼;精氨酸;需求量;生长
许氏平鲉),隶属于鲉形目(Scorpaeniformes)、鲉科(Scorpaenidae)、平鲉属(),又称黑鲪、黑头、黑寨等,属冷温性近海底层肉食性鱼类,在我国北部沿海、日本、朝鲜及 俄罗斯等区域均有分布(朱龙等, 1999)。因其适应性强、易饲养、肉质鲜美等优点和多栖息于礁岩海藻丛生区域,喜集群觅食等生物学特性,是网箱养殖和海洋牧场增殖的理想海水鱼种之一。目前,对许氏平鲉必需氨基酸的研究仅限于赖氨酸(严全根等, 2006)和蛋氨酸(Yan, 2007)。本研究通过在饲料中添加不同含量的精氨酸,研究其对许氏平鲉幼鱼生长、体成分、血清相关生化指标及肝脏相关酶活力影响,确定许氏平鲉幼鱼对精氨酸的最适需求量,为其配合饲料的开发提供参考。
本实验以鱼粉、玉米蛋白粉为主要蛋白源,以鱼油为主要脂肪源,设计粗蛋白约为50%,粗脂肪约为10%的基础饲料配方(Kim,2001; Lee, 2002)。添加混合晶体氨基酸使饲料的氨基酸组成比例与许氏平鲉幼鱼肌肉氨基酸组成比例一致(精氨酸除外)。在基础饲料中分别添加0、0.6%、1.2%、1.8%、2.4%和3.0%的L-精氨酸(纯度为98%,购自上海麦克林生化科技有限公司),配制6组精氨酸含量分别为1.39%、1.83%、2.34%、2.80%、3.39%和4.08%的实验饲料(表1),分别命名为D1、D2、D3、D4、D5和D6组。实验饲料氨基酸组成见表2。
各固体原料经粉碎过80目标准筛,按配比称重后,用逐级扩大法混合均匀,添加鱼油和适宜的蒸馏水,再次混合均匀,用小型饲料挤压机制成粒径为 2 mm和4 mm的颗粒饲料,60℃烘干,用塑料袋密封后于–20℃冰箱保存备用。
实验所用许氏平鲉幼鱼购自山东荣成裕源祥水产有限公司。正式实验前,挑选体质健壮的许氏平鲉幼鱼暂养于蓝色圆形养殖桶(桶高为80 cm,直径为70 cm,水深为50 cm),投喂D1组饲料驯化,14 d后挑选540尾规格均匀的许氏平鲉幼鱼[(12.03± 0.03) g],随机分配至18个桶,每桶30尾。正式养殖实验于2019年11月15日—2020年1月11日在山东省海洋资源与环境研究院室内循环水养殖系统进行。养殖期间每天定时(08:00和16:00)定量投喂2次,初始投喂量为鱼体重的1%,并按摄食情况及时调整投喂量,每日记录死鱼数量和重量。养殖期间确保水质符合以下条件:水温为16℃~18℃,溶氧(DO)> 8.0 mg/L,pH为7.5~8.2,盐度为27~28,氨氮和亚硝态氮<0.05 mg/L。
养殖实验结束后,禁食24 h,记录每桶鱼的数量并称总重,每桶随机取6尾用于鱼体常规成分测定,另取8尾测量体重及体长后,尾静脉取血,解剖分离内脏,分别称量内脏团和肝胰脏的重量。全鱼置于 –20℃保存,背肌、肝胰脏及血清样品置于–80℃保存,待测。
实验饲料及样品中水分采用105℃恒重法测定(GB/T 6435-2014),粗蛋白采用凯氏定氮法测定(GB/T 6432-2018),粗脂肪采用索氏抽提法测定(GB/T 6433-2006),粗灰分采用550℃失重法测定(GB/T 6438-2007),能量采用燃烧法测定(IKA, C6000, 德国),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, 日本)测定。
血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、白蛋白(Alb)、NO、尿素氮(BUN)、肝脏超氧化物歧化酶(SOD)、丙二醛(MDA)、溶菌酶(LZM)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、总抗氧化物酶(T-AOC)、碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、一氧化氮合酶(TNOS)和诱导型一氧化氮合酶(iNOS)均采用南京建成生物工程研究所试剂盒测定。血清和肝脏总蛋白(TP)含量采用考马斯亮蓝法测定。测定方法及酶活力单位参照试剂盒说明书。
表1 饲料配方及营养组成(%风干基础)
Tab.1 Formulation and nutritional composition of the experimental diets (% air-dry basis)
注:a: 混合氨基酸(g/kg饲料):天冬氨酸21.48 g;苏氨酸8.09 g;丝氨酸5.18 g;谷氨酸19.08 g;甘氨酸17.48 g;丙氨酸8.37 g;半胱氨酸5.75 g;缬氨酸6.36 g;蛋氨酸3.82 g;异亮氨酸4.90 g;亮氨酸13.96 g;酪氨酸5.07 g;苯丙氨酸7.64 g;赖氨酸19.87 g;组氨酸2.95 g
b: 维生素预混料(g/kg饲料):维生素A乙酸酯0.73 g;维生素B120.003 g;维生素C 121.2 g;维生素D30.003 g;DL维生素E生育酚乙酸酯18.8 g;维生素K 1.8 g;盐酸硫胺素2.7 g;盐酸吡哆醇1.8 g;烟酸27.8 g;叶酸(98%)0.68 g;核黄素9.1 g;泛酸钙12.7 g;肌醇181.8 g;生物素0.27 g
c: 矿物质预混料(g/kg饲料):硫酸镁80 g;磷酸二氢钠370 g;氯化钾130 g;柠檬酸铁40 g;硫酸锌20 g;氯化亚铜0.2 g;氯化铝0.15 g;碘化钾0.15 g;亚硒酸钠0.01 g;蛋氨酸锰2.0 g;氯化钴1.0 g
Note: a: Amino acid mixture (g/kg diet): Aspartic acid 21.48 g; threonine 8.09 g; serine 5.18 g; glutamic acid 19.08 g; glycine 17.48 g; alanine 8.37 g; cysteine 5.75 g; valine 6.36 g; methionine 3.82 g; isoleucine 4.90 g; leucine 13.96 g; tyrosine 5.07 g; phenylalanine 7.64 g; lysine 19.87 g; histidine 2.95 g
b: Vitamin premix (g/kg diet): Vitamin A acetate 0.73 g; vitamin B120.003 g; vitamin C 121.2 g; vitamin D30.003 g; DL vitamin E tocopherol 18.8 g; vitamin K 1.8 g, thiamine hydrochloride 2.7 g; pyridoxine hydrochloride 1.8 g; niacin acid 27.8 g; folic acid (98%) 0.68 g; riboflavin 9.1 g; calcium pantothenate 12.7 g; inositol 181.8 g; biotin 0.27 g
c: Mineral premix (g/kg diet): Magnesium sulfate 80 g; sodium dihydrogen phosphate 370 g; potassium chloride 130 g; ferric citrate 40 g; zinc sulfate 20 g; cuprous chloride 0.2 g; aluminum chloride 0.15 g; potassium iodide 0.15 g; sodium selenite 0.01 g; manganese methionine 2.0 g; cobalt chloride 1.0 g
增重率(weight gain rate, WGR, %)=(鱼体末重–鱼体初重)/鱼体初重×100;
特定生长率(specific growth rate, SGR, %/d)=(ln鱼体末重–ln鱼体初重)/养殖周期×100;
饲料系数(feed conversion ratio, FCR)=摄食饲料量/(鱼体末重–鱼体初重);
蛋白质效率(protein efficiency ratio, PER, %)=(鱼体末重–鱼体初重)/(摄食饲料量×饲料中粗蛋白的含量)×100;
摄食率(daily feed intake, DFI, %/d)=摄食饲料量/ [(鱼体初重+鱼体末重)/2×养殖天数]×100;
脏体比(viscerosomtic index, VSI, %)=内脏团重/鱼体末重×100;
肝体比(hepatosomatic index, HSI, %)=肝胰脏重/鱼体末重×100;
表2 实验饲料氨基酸组成(%干饲料)
Tab.2 Amino acid profiles of the experimental diets (% dry diet)
肥满度(condition factor, CF)=鱼体末重/体长3×100;
存活率(survival rate, SR,%)=终末鱼尾数/初始鱼尾数×100。
使用SPSS 18.0统计软件进行单因素方差分析(one-way ANOVA),差异显著(<0.05)时用Duncan’s检验进行多重比较分析,数据用平均值±标准差(Mean±SD)表示,采用一元二次回归分析,确定许氏平鲉幼鱼对饲料中精氨酸的最适需求量。
如表3所示,随着精氨酸含量的升高,实验鱼WGR、SGR均先升高后降低,在D3组出现最大值且显著高于其他组(<0.05)。FCR先降低后升高,D3组显著低于其他组(<0.05)。PER先升高后平稳,D3~D6组显著高于D1组(<0.05)。VSI和HSI无显著性差异(>0.05),CF先升高后降低,D3~D4组显著高于D1组(<0.05),各组成活率无显著差异(>0.05)。
表3 精氨酸对许氏平鲉幼鱼生长和形体指标的影响(=3, 平均值±标准差)
Tab.3 Effects of dietary arginine on growth performance and body indices of juvenile S. schlegelii (n=3, Mean±SD)
注:同行无字母或数据肩标相同字母表示差异不显著(>0.05),不同小写字母表示差异显著(<0.05)。下同
Note: In the same row, values with no letter or the same letter superscripts mean no significant difference (>0.05), while with different small letter superscripts mean significant difference (<0.05). The same as below
以WGR为评价指标,经一元二次回归性分析,许氏平鲉幼鱼对饲料中精氨酸的最适需求量为2.78%饲料(5.56%饲料蛋白质)(图1)。
图1 许氏平鲉幼鱼增重率与饲料精氨酸含量的相关性分析
如表4所示,饲料中精氨酸含量的对全鱼水分、粗蛋白、粗脂肪和粗灰分含量均无显著性影响(>0.05)。肌肉粗蛋白含量先升高后降低,D3组显著高于其他组(<0.05)。肌肉水分、粗脂肪和粗灰分含量均无显著性差异(>0.05)。
如表5所示,随着饲料中精氨酸含量的增加,全鱼缬氨酸(Val)、蛋氨酸(Met)、亮氨酸(Leu),异亮氨酸(Ile)、赖氨酸(Lys)、天冬氨酸(Asp)、总必需氨基酸(ΣEAA)和总氨基酸(ΣAA)均先升高后降低,精氨酸(Arg)含量先升高后平稳,其他氨基酸含量不受饲料精氨酸水平的影响。
如表6所示,随着饲料精氨酸含量的增加,肌肉蛋氨酸(Met)、精氨酸(Arg)、总必需氨基酸(ΣEAA)和总氨基酸(ΣAA)含量先升高后降低,赖氨酸(Lys)、亮氨酸(Leu)、谷氨酸(Glu)和天冬氨酸(Asp)含量先升高后平稳,其他氨基酸含量不受饲料精氨酸含量的影响。
表4 饲料中精氨酸水平对许氏平鲉幼鱼常规成分的影响(=3, 平均值±标准差,%湿重)
Tab.4 Effect of dietary arginine on the conventional compositions of juvenile S. schlegelii (n=3, Mean±SD,% wet weight)
如表7所示,随着饲料精氨酸含量的增加,血清谷丙转氨酶(ALT) (图2)活力先降低后升高,D3组显著小于其他组(<0.05);谷草转氨酶(AST)活力各组间无显著性差异;白蛋白(Alb)(图3)和NO(图4)含量先升高后降低;尿素氮(BUN)(图5)含量先升高后趋于平缓,D3~D6组显著高于D1组(<0.05)。
如表8所示,随着饲料精氨酸含量的增加,肝脏超氧化物歧化酶(SOD)(图6)、溶菌酶(LZM)、谷草转氨酶(AST)、总抗氧化物酶(T-AOC)、碱性磷酸酶(AKP)、总一氧化氮合酶(TNOS)(图8)和诱导型一氧化氮合酶(iNOS)(图9)活力均先升高后降低。丙二醛(MDA)(图7)含量先降低后升高,D2~D4组显著低于D1、D5和D6组(<0.05)。
表5 饲料中精氨酸水平对许氏平鲉幼鱼全鱼氨基酸组成的影响(=3, 平均值±标准差)
Tab.5 Effect of dietary arginine on amino acid profiles of whole fish of juvenile S. schlegelii (n=3, Mean±SD)
表6 饲料中精氨酸水平对许氏平鲉幼鱼肌肉氨基酸组成的影响(=3, 平均值±标准差)
Tab.6 Effect of dietary arginine on amino acid profiles of dorsal muscle of juvenile S. schlegelii (n=3, Mean±SD)
表7 饲料精氨酸对许氏平鲉幼鱼血清生化指标的影响(=6, 平均值±标准差)
Tab.7 Effects of dietary arginine on serum biochemical parameters of juvenile S.schlegelii (n=6, Mean±SD)
图2 血清谷丙转氨酶活力
图3 血清白蛋白含量
图4 血清一氧化氮含量
图5 血清尿素氮含量
表8 饲料精氨酸对许氏平鲉幼鱼肝脏相关酶活力的影响(=6, 平均值±标准差)
Tab.8 Effects of dietary arginine on liver enzyme activities of juvenile S.schlegelii(n=6, Mean±SD)
图6 肝脏超氧化物歧化酶活力
图7 肝脏丙二醛含量
图8 肝脏一氧化氮合酶活力
图9 肝脏诱导型一氧化氮合酶活力
本实验条件下,以增重率为评价指标,许氏平鲉幼鱼对精氨酸的最适需求量为2.78%饲料(5.56%饲料蛋白)。这一结果与斜带石斑鱼(6.07%饲料蛋白)、卵形鲳鲹(6.32%~6.35%饲料蛋白)和牙鲆(6.25%饲料蛋白)的研究结果相近(Lin, 2015; 韩凤禄等, 2016; Han, 2013),高于真鲷()(4.74%饲料蛋白)、遮目鱼(5.25%饲料蛋白)、银大马哈鱼() (4.90%饲料蛋白) (Klein, 1970; Fournier, 2003; Rahimnejad, 2014),低于黑鲷) (7.74%饲料蛋白)、青石斑鱼() (6.5%饲料蛋白) (Zhou, 2010、2012)。不同鱼种对精氨酸的需求量存在差异可能与鱼种、饲料蛋白源、饲料蛋白水平、实验环境、饲养方式和评估标准等有关。
本研究中,饲料精氨酸水平对全鱼粗蛋白、粗脂肪、粗灰分和水分无显著影响。与斜带石斑鱼,大口黑鲈(周恒永, 2011)、红鳍东方鲀() (张庆功, 2019)的研究结果一致。随着饲料精氨酸含量的增加,实验鱼肌肉粗蛋白含量先升高后降低,与吉富罗非鱼() (武文一, 2016)的研究结果一致。精氨酸提高肌肉蛋白质含量的原因可能是由于精氨酸代谢产生的谷氨酰胺和NO可激活肌肉中雷帕霉素靶蛋白(mTOR)信号途径,mTOR激活后会促进磷酸化核糖体S6蛋白激酶(p70s6激酶)和真核生物启动子4E-结合蛋白1 (eIF4E-BP1)磷酸化,形成用于多肽合成的激活启动复合物,促进蛋白质合成(Pervin, 2007)。韩凤禄等(2016)研究发现,随着饲料精氨酸含量的增加,斜带石斑鱼幼鱼肌肉粗蛋白和粗灰分含量先升高后趋于平缓,粗脂肪含量呈下降趋势。产生差异的原因可能与饲料原料组成和鱼种不同有关。研究证实,随着饲料精氨酸含量的升高,全鱼蛋白质的合成效率和沉积率也会显著提高,在精氨酸添加量超过其最适需求量之后,蛋白质合成效率和沉积率会显著下降,这是因为过量的精氨酸导致其他氨基酸的降解加快,而降低蛋白质合成和沉积,但在军曹鱼()中发现,当饲料精氨酸含量超过其最适需求量,蛋白质合成和沉积未受到显著影响(赵红霞等, 2007),这可能是鱼种及其规格不同导致的差异。
鱼体对于蛋白质的沉积是通过外源氨基酸的供给来合成的(张庆功等, 2019)。研究发现,黑鲷和大口黑鲈肌肉必需氨基酸含量随着饲料精氨酸摄入的增多而上升(Zhou, 2010; 周恒永, 2011)。本研究中,许氏平鲉幼鱼全鱼和肌肉的必需氨基酸含量随着饲料精氨酸含量的增加先升高后降低。当精氨酸达到其最适需求量时,饲料中各氨基酸含量达到平衡,此时,鱼体吸收的氨基酸以最大的效率进行机体蛋白的合成。摄入精氨酸过量后,氨基酸平衡被打破,多余的氨基酸被氧化分解,同时耗能,使得全鱼和肌肉的必需氨基酸含量下降。这种趋势与增重率和蛋白质效率一致。
溶菌酶是重要的抗菌物质,能溶解细菌细胞壁,破坏细菌肽聚糖支架,激活补体旁路和吞噬细胞活性,参与非特异性免疫防御,常用来作为鱼类非特异性免疫能力的评价指标(Zhu, 2013)。不同种类的鱼其适宜的精氨酸需求量存在差异,过高水平的精氨酸会降低鱼类血清中溶菌酶活力(Chen, 2015)。本研究中,肝脏溶菌酶活力受到精氨酸水平的显著影响,呈现先升高后降低的趋势,与卵形鲳鲹和大口黑鲈的研究结果相同(周恒永, 2011; 谭小红, 2015),表明适量的精氨酸可显著提高鱼类的非特异性免疫能力。关于精氨酸影响溶菌酶活力的机制已有相关报道,但精氨酸调控溶菌酶基因表达的机制有待研究。
饲料中适宜含量的精氨酸能显著提升许氏平鲉幼鱼的生长性能,提高蛋白质沉积,降低饲料系数,增强氨基酸代谢及非特异性免疫能力。以增重率为评价指标,许氏平鲉幼鱼[(12.03±0.03) g]精氨酸的适宜需求量为2.78%(占5.56%饲料粗蛋白)。
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Dietary Arginine Requirement of Juvenile Rockfish ()
SHEN Yubo1,2, WANG Jiying2①, LI Baoshan2, LIU Caili1,2, WANG Xiaoyan2, HUANG Bingshan2, WANG Shixin2, SUN Yongzhi2
(1.National Demonstration Center for Experimental Fisheries Science Education, Centre for Research on Environmental Ecology and Fish Nutrition (CREEFN) of Ministry of Agriculture and Rural Affairs, Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai Ocean University, Shanghai 201306, China; 2. Shandong Key Laboratory of Marine Ecological Restoration, Shandong Marine Resource and Environment Research Institute, Yantai, Shandong 264006, China)
An eight week experiment was conducted to investigate the effects of dietary arginine on the growth performance, body composition, serum biochemical parameters, and liver enzyme activities of juvenile Korean rockfish () and to determine the dietary arginine requirement. Six isonitrogenous and isolipidic diets were formulated to contain graded dietary arginine (1.5%, 1.0%, 2.0%, 2.5%, 3.5%, and 4.5% dry diet). The crystalline amino acid mixture was supplemented in the test diets to simulate the amino acid profile of the juvenilemuscle protein, except for arginine. The initial average body weight of the juvenilewas (12.03±0.03) g. The results showed that, with increasing dietary arginine, the weight gain rate (WGR), specific growth rate, and protein efficiency ratio significantly increased at first and then decreased; these were significantly higher in the 2.34% and 2.80% arginine diets than the 1.39% arginine diet, but the feed conversion ratio showed the opposite trend. The crude protein content of the muscle was significantly affected by dietary arginine levels and was significantly higher in the 2.34% arginine diet than the other groups. The concentrations of both total essential amino acids and total amino acids first increased and then decreased in the whole fish and muscle tissues. The levels of albumin and nitrogen oxide in the serum significantly increased first and then decreased (<0.05), whereas the serum blood urea nitrogen content first increased and then plateaued. However, the glutamic pyruvic transaminase in the serum significantly decreased at first and then increased, and was significantly lower in the 2.34% arginine diet than in other groups. The activities of superoxide dismutase, lysozyme, aspartate aminotransferase, total antioxidant enzymes, alkaline phosphatase, total nitric oxide synthase, and inducible nitric oxide synthase in the liver first significantly increased and then decreased, whereas the content of malonaldehyde first significantly decreased and then increased. With WGR as an evaluation index, the optimal arginine requirement of juvenilewas 2.78% (5.56% dietary protein).
Juvenile Korean rockfish (); Arginine; Requirement; Growth
S963
A
2095-9869(2022)03-0033-12
10.19663/j.issn2095-9869.20210114003
http://www.yykxjz.cn/
沈钰博, 王际英, 李宝山, 刘财礼, 王晓艳, 黄炳山, 王世信, 孙永智. 许氏平鲉幼鱼对饲料中精氨酸需求量的研究. 渔业科学进展, 2021, 42(3): 33–44
SHEN Y B, WANG J Y, LI B S, LIU C L, WANG X Y, HUANG B S, WANG S X, SUN Y Z. Dietary arginine requirement of juvenile rockfish (). Progress in Fishery Sciences, 2021, 42(3): 33–44
WANG Jiying, E-mail: ytwjy@126.com
* 山东省自然科学基金(ZR2020QC206)和烟台市科技计划(2018ZHGY066)共同资助 [This work was supported by Natural Science Foundation of Shandong Province (ZR2020QC206), and Science and Technology Development Project of Yantai (2018ZHGY066)]. 沈钰博,E-mail: shenyubo0121@163.com
王际英,研究员,E-mail: ytwjy@126.com
2021-01-14,
2021-03-30
(编辑 陈 辉)