• ISSN 1000-0615
  • CN 31-1283/S
Volume 45 Issue 10
Oct.  2021
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Effects of Schizochytrium sp. oil combined with exogenous EPA on fatty acid composition, FAD and ELO gene expression and lipid metabolism of juvenile Ctenopharygodon idella

  • Corresponding author: JI Hong, jihong@nwsuaf.edu.cn
  • Received Date: 2020-09-25
    Accepted Date: 2021-01-31
    Available Online: 2021-09-10
  • The grass carp (Ctenopharyngodon idella) dietary Schizochytrium sp. stearin oil may show better growth performance than dietary fish oil by saving energy required for de novo synthesis of DHA, promoting lipolysis energy supply and inhibiting protein catabolism. The purpose of this study was to investigate whether DHA-rich Schizochytrium sp. stearin oil needs to be compatible with EPA and its possible mechanism. Equipped with five groups of iso-nitrogen and isoenergetic diets and fed grass carp (22.70 g±0.80 g) for 49 d. The 0.52% n-3 long-chain polyunsaturated fatty acid (LC PUFA) of the diets were provided with fish oil (F-O); Sc stearin oil (S-O); stearin oil (DHA)∶EPA =3∶2 (SE1-O); stearin oil (DHA)∶EPA = 1∶1(SE2-O); EPA (E-O), respectively. The results showed: 1) There was no difference in the weight gain rate (WG), specific growth rate (SGR) and feed conversion ratio (FCR) among all the groups; 2) The content of crude protein in muscle of S-O group was significantly higher than those of E-O group; 3) the content of DHA in muscle of S-O group was significantly higher than that of F-O group, SE1-O group and E-O group; 4) there was no significant difference in atherogenicity index among the groups, the thrombogenicity index of SE2-O group was significantly higher than that of F-O group, the hypocholesterolemic/ Hypercholesterolemic ratio in S-O group and SE2-O group was markedly lower than those in F-O group; 5) the adipose size of intraperitoneal fat in E-O group was notably higher than those in F-O group and SE1-O group, the adipose TAG lipase(ATGL)and carnitine palmitoyltransferase 1(CPT1) mRNA levels in E-O group were apparently down-regulated; in muscle, the fatty acid desaturase (FAD)mRNA level was markedly higher in E-O group than that in S-O group, the fatty acid elongase(ELO)mRNA level was obviously down-regulated in S-O group. Studies have shown that the use of Sc oil alone or in combination with EPA had no significant effect on growth, n-3LC PUFA content and lipid hydrolysis of adipose tissue of grass carp. When EPA was the sole source of n-3LC PUFA, the lipid hydrolysis of adipose tissue was reduced and the content of crude protein in muscle was reduced. High level of DHA in the diet would weaken the body's ability to synthesize LC PUFA. Compared to EPA, grass carp may need DHA more, and the Sc oil could be used alone in the feed of grass carp without combination with EPA.
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    [11] LIU Zhaojun, ZHAO Jie, BO Yunxuan, XIAO Lanying, SHI Kaili, SONG Xuehong. Expression patterns of IL-8 protein during bacterial LPS-induced inflammatory response in grass carp (Ctenopharyngodon idella). Journal of fisheries of china, 2017, 41(7): 1028-1035.  doi: 10.11964/jfc.20160210278
    [12] XIAO Tiaoyi, ZHOU Zhiyu, WANG Ronghua, LI Yaoguo, JIN Shengzhen, LI Wei, WANG Hongquan. The expression and transmission of immune factors between generations in maternal Ctenopharyngodon idella after immunization with GCRV attenuated vaccine. Journal of fisheries of china, 2017, 41(8): 1308-1318.  doi: 10.11964/jfc.20161210651
    [13] WANG Shentong, ZHANG Meng, SHEN Yubang, LI Jiale. Polymorphisms of the GH gene 3′ partial sequence and their associations with growth traits and muscle composition in juvenile grass carp (Ctenopharyngodon idella). Journal of fisheries of china, 2017, 41(9): 1329-1337.  doi: 10.11964/jfc.20160910546
    [14] DONG Chuanju, ZHANG Jiangfan, LI Shengjie, LV Hongzao, NIE Guoxing, LI Xuejun. Genome-wide identification, phylogeny, and expression of IL17 receptor genes in common carp (Cyprinus carpio) and grass carp (Ctenopharyngodon idella). Journal of fisheries of china, 2018, 42(11): 1693-1703.  doi: 10.11964/jfc.20180311198
    [15] LIU Xiaojuan, LUO Wei, WANG Chunfang, LI Dapeng, Dominique BUREAU. Establishment of bioenergy models to predict growth, feed requirement and waste output of grass carp (Ctenopharyngodon idella). Journal of fisheries of china, 2018, 42(6): 950-967.  doi: 10.11964/jfc.20170510828
    [16] LIN Ken, FENG Jingyun, YANG Huishi, CHEN Yongjun, CHEN Xiaochuan, LUO Hao, HUANG Wang, WANG Haitao, LUO Li. Effects of inositol supplementation to practical dietary on growth performance, lipid metabolism and antioxidant activity of Ctenopharyngodon idella. Journal of fisheries of china, 2018, 42(9): 1428-1437.  doi: 10.11964/jfc.20170910968
    [17] LU Ronghua, ZHANG Wenya, ZHANG Yuru, YANG Liping, WANG Junli, MENG Xiaolin, NIE Guoxing. Isolation and identification of hepatocellular exosomes and their effects on the expression of miR-122/33 and immune-related genes in grass carp(Ctenopharyngodon idella). Journal of fisheries of china, 2020, 44(1): 1-10.  doi: 10.11964/jfc.20181211575
    [18] WANG Shentong, SHEN Yubang, MENG Xinzhan, WANG Rongquan, LI Jiale. Genetic variability in wild and selected populations of Ctenopharyngodon idella using microsatellite markers. Journal of fisheries of china, 2018, 42(8): 1273-1284.  doi: 10.11964/jfc.20171010997
    [19] HU Kai, LI Shuang’an, FENG Lin, JIANG Weidan, WU Pei, LIU Yang, JIANG Jun, KUANG Shengyao, TANG Ling, ZHOU Xiaoqiu. Protective effect of myo-inositol on oxidative damage of head kidney and spleen in juvenile grass carp (Ctenopharyngodon idella) induced by Aeromonas hydrophila. Journal of fisheries of china, 2019, 43(10): 2256-2267.  doi: 10.11964/jfc.20190811930
    [20] CHEN Yanna, LU Ronghua, YANG Guokun, ZHANG Yuru, QIN Chaobin, JI Hong, NIE Guoxing. Effects of replacing soybean oil with black soldier fly (Hermetia illucens) larvae oil on the growth performance, antioxidant ability and intestinal microbiota of grass carp (Ctenopharyngodon idella). Journal of fisheries of china, 2019, 43(10): 2241-2255.  doi: 10.11964/jfc.20190911949
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Effects of Schizochytrium sp. oil combined with exogenous EPA on fatty acid composition, FAD and ELO gene expression and lipid metabolism of juvenile Ctenopharygodon idella

    Corresponding author: JI Hong, jihong@nwsuaf.edu.cn
  • College of Animal Science and Technology, Northwest A&F University, Yangling    712100, China

Abstract: The grass carp (Ctenopharyngodon idella) dietary Schizochytrium sp. stearin oil may show better growth performance than dietary fish oil by saving energy required for de novo synthesis of DHA, promoting lipolysis energy supply and inhibiting protein catabolism. The purpose of this study was to investigate whether DHA-rich Schizochytrium sp. stearin oil needs to be compatible with EPA and its possible mechanism. Equipped with five groups of iso-nitrogen and isoenergetic diets and fed grass carp (22.70 g±0.80 g) for 49 d. The 0.52% n-3 long-chain polyunsaturated fatty acid (LC PUFA) of the diets were provided with fish oil (F-O); Sc stearin oil (S-O); stearin oil (DHA)∶EPA =3∶2 (SE1-O); stearin oil (DHA)∶EPA = 1∶1(SE2-O); EPA (E-O), respectively. The results showed: 1) There was no difference in the weight gain rate (WG), specific growth rate (SGR) and feed conversion ratio (FCR) among all the groups; 2) The content of crude protein in muscle of S-O group was significantly higher than those of E-O group; 3) the content of DHA in muscle of S-O group was significantly higher than that of F-O group, SE1-O group and E-O group; 4) there was no significant difference in atherogenicity index among the groups, the thrombogenicity index of SE2-O group was significantly higher than that of F-O group, the hypocholesterolemic/ Hypercholesterolemic ratio in S-O group and SE2-O group was markedly lower than those in F-O group; 5) the adipose size of intraperitoneal fat in E-O group was notably higher than those in F-O group and SE1-O group, the adipose TAG lipase(ATGL)and carnitine palmitoyltransferase 1(CPT1) mRNA levels in E-O group were apparently down-regulated; in muscle, the fatty acid desaturase (FAD)mRNA level was markedly higher in E-O group than that in S-O group, the fatty acid elongase(ELO)mRNA level was obviously down-regulated in S-O group. Studies have shown that the use of Sc oil alone or in combination with EPA had no significant effect on growth, n-3LC PUFA content and lipid hydrolysis of adipose tissue of grass carp. When EPA was the sole source of n-3LC PUFA, the lipid hydrolysis of adipose tissue was reduced and the content of crude protein in muscle was reduced. High level of DHA in the diet would weaken the body's ability to synthesize LC PUFA. Compared to EPA, grass carp may need DHA more, and the Sc oil could be used alone in the feed of grass carp without combination with EPA.

  • 裂殖壶藻(Schizochytrium sp.) 属真菌门 (Eumycophyta) 卵菌纲 (Oomycetes) 水霉目(Saprolegniales)破囊壶菌科(Thraustochytriaceae) ,是一类单细胞、球形的海洋真菌,也称裂壶藻和裂殖壶菌[1]。裂殖壶藻含有大量的脂质, 约占细胞干质量的55%~75%,且多为不饱和脂肪酸,DHA占比49%[2-3]。裂殖壶藻约含水分1.49%、灰分9.79%、蛋白质15.49%、碳水化合物29.26%,含有丰富的Mg、Ca、Na、Fe等元素,还富含油脂、色素、角鲨烯等活性物质。裂殖壶藻的人工培养不受季节的影响,具有对环境无污染、异养方式下培养生长速度快、营养成分稳定等诸多优点[2]。从裂殖壶藻中提取精炼藻油的过程中,产生了硬脂和蜡脂等2种工业副产品。

    目前关于裂殖壶藻对鱼类生长及脂合成调控的研究主要集中在海水鱼中,而淡水鱼相关研究鲜有报道。裂殖壶藻中含有高水平的DHA,但缺乏EPA,有研究指出,以裂殖壶藻作为油脂源时,这2种脂肪酸的不平衡是造成水产动物生长受阻的一个潜在原因,例如,利用裂殖壶藻粉和隐甲藻(Crypthecodinium)替代饲料中的鱼油而不替代乌贼粉时,金头鲷(Sparus aurata)的成活率、生长性能和饲料利用率均未受到显著影响,然而在替代全部脂肪源时其成活率和生长则均呈下降的趋势[4];用裂殖壶藻粉替代50%的鱼油时没有影响海鲈(Lateolabrax japonicus)的生长,但完全替代鱼油时会阻碍海鲈生长[5]。DHA和EPA在调节鱼类生长性能、生存、脂质代谢、繁殖和免疫功能等方面具有重要的作用[6-7],已有大量研究表明DHA和EPA的比例对大口黑鲈(Micropterus salmoides)[8]、黄鳝(Epinephelus coioides)[9]、黑鲷(Acanthopagrus schlegelii)[10]和大西洋鲑 (Salmo salar)[11]等的生长和脂质代谢有重要作用,且最适DHA和EPA比例具有物种特异性[12]。在草鱼 (Ctenopharyngodon idella) 方面,研究表明,饲料DHA/EPA比率在4.93~0.21对草鱼幼鱼的生长、脂质蓄积和抗氧化状态没有显著性影响[13]

    草鱼是我国产量最高的淡水养殖品种,2019年产量达到5533083 t,占全国淡水养殖产量的18.36%[14]。当前,草鱼生产中大量投喂配合饲料,青饲料的投喂减少,高能量摄入的养殖模式虽然提高了草鱼的生长速度,但易造成营养性脂肪肝和脂质代谢紊乱的情况,影响了商品鱼的品质和养殖效果[15]。一般认为,草鱼幼鱼必需脂肪酸为1%亚油酸 LA和0.5%~1.0%亚麻酸α-LNA或者0.5% n-3 长链多不饱和脂肪酸(LC PUFA)[16]。吉红等[17]认为n-3 LC PUFA可抑制草鱼肝胰脏脂质过度沉积和脂合成酶活力及降低LPL表达丰度[18],对草鱼最适n-3 LC PUFA水平的研究表明,草鱼日粮中添加0.52% n-3 LC PUFA可促进草鱼生长,调控体脂在组织间分配,提高脂质利用能力[19]

    在鱼油鱼粉价格高昂且资源短缺的情况下,将富含LC PUFA的微藻工业副产品资源充分利用在水产饲料中显得极为重要。本实验室在以裂殖壶藻油替代鱼油的前期研究中发现,裂殖壶藻硬脂能促进草鱼的生长、促进脂质水解并调节脂质的再分配和利用等[20],本实验在此基础上,将裂殖壶藻硬脂配伍外源性EPA应用于草鱼日粮中,进一步探究裂殖壶藻油是否能在草鱼饲料中作为n-3 LC PUFA的唯一来源,是否有必要配伍EPA使用,并探究饲料中DHA和EPA对草鱼肌肉合成n-3 LC PUFA机制的影响。

    • 配置5组等氮等能日粮,主要油源有精炼鱼油(DHA∶EPA=2∶3;江苏省无锡市迅达有限责任公司),裂殖壶藻硬脂(下文简称为藻油;含39.72% DHA,8.9% DPA)由山东省临沂市友康生物科技有限公司提供,精炼EPA购自江苏省无锡市迅达有限责任公司。根据本实验室前期研究结果,设置日粮n-3 LC PUFA水平为0.52%[19],分别以鱼油(F-O);藻油(S-O);藻油DHA∶EPA=2∶3(SE1-O);藻油DHA∶EPA=1∶1(SE2-O);EPA(E-O)提供饲料中的0.52% n-3 LC PUFA,5组日粮n-3 LC PUFA的实测值均为0.50%。所有原料粉碎混匀,加入油搓匀后加入70%的水,用饸烙机制成直径2 mm的颗粒,放置于室温阴凉处通风干燥24 h,在−20 °C保存待用。饲料配方和常规成分如表1所示,饲料脂肪酸组成如表2所示。

      项目
      items
      0.50%n-3LC PUFA
      F-OS-OSE1-OSE2-OE-O
      原料 ingredients
        酪蛋白 casein 320.00 320.00 320.00 320.00 320.00
        明胶 gelatin 80.00 80.00 80.00 80.00 80.00
        糊精 dextrin 280.00 280.00 280.00 280.00 280.00
        微晶纤维素 cellulose 199.00 199.00 199.00 199.00 199.00
        猪油 lard oil 3.00 9.30 11.23 10.85 12.70
        大豆油 soybean oil 15.00 15.00 15.00 15.00 15.00
        亚麻油 linseed oil 15.00 15.00 15.00 15.00 15.00
        精炼鱼油 refined fish oil 17.00 0.00 0.00 0.00 0.00
        裂殖壶藻硬脂 stearin oil1 0.00 10.70 4.65 5.84 0.00
        精炼 EPA refined EPA 0.00 0.00 4.12 3.31 7.30
        羧甲基纤维素钠 carboxymethyl celluose 20.00 20.00 20.00 20.00 20.00
        混合无机盐 mineral mix2 40.00 40.00 40.00 40.00 40.00
        混合维生素 vitamin mix3 10.00 10.00 10.00 10.00 10.00
        BHT 1.00 1.00 1.00 1.00 1.00
        总计 total 1000 1000 1000 1000 1000
      常规成分 proximate composition
        粗蛋白质/(%, N%×6.25) crude protein 35.83 35.74 35.81 35.77 35.80
        粗脂肪/% crude lipid 4.85 4.86 4.85 4.84 4.86
        水分/% moisture 12.10 11.82 11.90 12.30 12.05
      注:1. 裂殖壶藻硬脂油含 39.72% DHA, 8.9%DPA(%总脂肪酸);2. 混合无机盐(毫克/100克饲料)含硫酸铝钾 0.159,碳酸钙 18.101,磷酸二氢钙 44.601,氯化钴 0.07,硫酸镁 5.216,硫酸锰 0.007,氯化钾 16.553,碘化钾 0.014,碳酸锌 0.192,磷酸二氢钠 13.605,硒酸钠 0.006,硫酸铜 0.075,柠檬酸铁 1.338;3. 混合维生素(毫克/100克饲料)含硫胺素5,核黄素5,维生素 A 25 00 IU,维生素 E 40 IU,维生素 D 3 24 00 IU,甲萘醌 4,盐酸吡哆醇 4,氰钴胺0.01,生物素 0.6,泛酸钙 10,叶酸 1.5,烟酸 20,肌醇 200
      Notes: 1. Stearin oil contained 39.72% DHA,8.9%DPA (% of total fatty acids); 2. The mineral mix contained (g/100 g of diet): KAl(SO4), 0.159; CaCO3, 18.101; CaH2PO4, 44.601; CoCl, 0.070; MgSO4, 5.216; MnSO4·H2O, 0.070; KCl, 16.553; KI, 0.014; ZnCO3,0.192; NaH2PO4, 13.605; Na2SeO3, 0.006; CuSO4·5H2O, 0.075; Ferric citrate, 1.338; 3. The vitamin mix contained (mg/100 g of diet): thiamine, 5; riboflavin, 5; vitamin A,2500 IU; vitamin E, 40; vitamin D3, 2400 IU; menadione, 4; pyridoxine HCl, 4;cyanocobalamin, 0.01; biotin, 0.6; calcium pantothenate, 10; folic acid, 1.5; niacin, 20;inositol, 200

      Table 1.  Formulation and proximate composition of the experimental diets (g/kg DM)

      脂肪酸
      fatty acids
      组别 group
      F-OS-OSE1-OSE2-OE-O
      C14:0 0.17 0.17 0.49 0.47 0.48
      C16:0 11.03 18.91 13.93 14.67 10.53
      C18:0 6.57 6.19 6.87 6.83 8.83
      SFA 17.78 25.27 21.29 21.96 19.84
      C16:1n-7 1.24 0.42 0.50 0.50 0.61
      C18:1n-9 24.74 19.34 23.58 23.43 25.33
      C20:1n-9 0.59 0.34 0.36 0.35 0.20
      MUFA 26.58 20.10 24.44 24.28 26.14
      C18:2n-6 22.67 22.54 22.49 22.51 22.41
      C20:4n-6 0.78 0.29 0.10 0.09 0.58
      n-6PUFA 23.45 22.83 22.59 22.61 22.99
      C18:3n-3 20.56 20.15 19.18 19.33 19.31
      C20:5n-3 6.71 0.21 6.34 5.35 11.03
      C22:5n-3 0.59 2.20 1.08 1.18 0.00
      C22:6n-3 4.32 9.23 4.18 5.29 0.68
      DHA+EPA 11.03 9.44 10.52 10.63 11.71
      DHA/EPA 0.64 43.95 0.66 0.99 0.06
      n-3PUFA 32.19 31.79 30.78 31.15 31.03
      n-3LC PUFA 11.63 11.64 11.59 11.82 11.71
      n-3/n-6 1.37 1.39 1.36 1.38 1.35
      注:SFA,饱和脂肪酸;MUFA,单不饱和脂肪酸;PUFA,多不饱和脂肪酸 ;LC-PUFA,长链多不饱和脂肪酸,下同
      Notes: SFA.saturated fatty acids;MUFA. monounsaturated fatty acids; PUFA. polyunsaturated fatty acids; LC-PUFA.long chain polyunsaturated fatty acids; the same below

      Table 2.  Fatty acid composition of the experimental diets (% of total fatty acids)

    • 草鱼幼鱼由西北农林科技大学安康水产试验示范站提供。草鱼在水泥池(4.75 m×1.65 m×0.80 m)中饲喂对照组饲料暂养28 d。饥饿24 h后,挑选规格相似、健康的216尾幼鱼(体质量22.70±0.80 g)随机分至循环水系统的缸(225 L,d=0.55 m,h=0.5 m),将15个缸随机分为5组,每组3个重复,每个重复12尾鱼,停食24 h正式开始试验,每组随机分配饲喂5组试验饲料49 d。试验期间每天饱食投喂3次分别为8:30,12:30,16:30。试验用水采用曝气后的井水,光周期为12 h/12 h。每日监测水质,并记录试验鱼死亡数量等。饲喂期间水质测定结果如下:水温(26.50±0.70) °C;溶解氧 (7.70±0.55) mg/L;pH 6.89±0.31;氨氮 (0.09±0.05) mg/L。

    • 样品采集过程严格按照西北农林科技大学动物管理委员会的要求执行,尊重动物福利与道德规范。采样前,草鱼饥饿24 h后使用MS-222 (50 mg/L)麻醉。每个缸的鱼都称重并测量长度。从每个缸取2尾鱼在−20 °C保存。取5尾鱼解剖并称重内脏、肾脏、肝脏、脾脏以及腹腔脂肪组织,并测量肠的长度。特定生长率(SGR)、增重率(WG)、饲料转化率(FCR)、内脏指数(VSI)、肥满度(CF)、肾指数(KI)、肝体比(HSI)、脾脏指数(SI)、腹腔脂肪指数(IPFI)以及肠体指数(RIW)根据以下的公式计算得出。然后肝脏、IPF、肠和肌肉储存在−20 °C,将其中2尾鱼的脂肪组织样品固定在4%多聚甲醛溶液中用于组织学观察。另取5尾鱼解剖后将肝脏、脂肪和肌肉保存在−80 °C用于组织基因表达测定。

      特定生长率(specific growth rate, SGR, %/d)=[ln(鱼末均重)−ln(鱼初均重)]/实验天数×100%;

      增重率(weight gain rate, WGR, %)=(末重−初重)/初重×100%;

      饲料转化率(feed conversion ratio, FCR, %)=投饲总量/体增重;

      内脏指数(viscerosomatic index, VSI, %) =内脏重/鱼体质量×100%;

      肥满度(condition factor, CF, g/cm3)=体质量/体长3×100;

      肾指数(kidney index, KI, %)=肾脏重/鱼体质量×100%;

      肝体比(hepatosomatic index, HSI, %)=肝脏重/鱼体质量×100%;

      脾脏指数(spleen index, SI, %)=脾脏重/鱼体质量×100%;

      腹腔脂肪指数(intraperitoneal fat index, IPFI, %)=腹腔脂肪量/鱼体质量×100%;

      肠体指数(relative intestine weight, RIW, %)=肠重/鱼体质量×100%。

    • 饲料、组织和全鱼的常规成分按照AOAC所述方法测定[21]。其中,水分用150 °C恒温干燥法测定,粗蛋白质(N%×6.25)含量使用凯氏定氮法测定,粗脂肪含量用索氏抽提法,粗灰分含量使用马弗炉550 °C灰化12 h测定。

    • 用氯仿-甲醇提取肌肉、肝脏和日粮中的脂质[22],脂肪酸甲酯是用0.4 mol/L KOH-甲醇制备[23],再加2 mL去离子水,待分层后提取上层溶液在气象色谱仪(安捷伦7820a,安捷伦科技,美国)上进行测定。每种脂肪酸与已知标品(47015-U, Sigma-Aldrich, Inc., St. Louis, USA)进行比对鉴别。实验结果按面积归一化法计算不同脂肪酸含量,以总脂肪酸的百分比的形式呈现。脂肪酸健康指数按照下述方法计算。

      动脉粥样硬化指数 (atherogenicity index, AI)和血栓形成指数 (thrombogenicity index, TI)是根据Ulbricht等[24]所述方法计算:

      AI= [C12: 0+(4×C14: 0) +C16: 0]/(n−3 PUFA+n−6 PUFA+MUFA);

      TI= (C14: 0+C16: 0+ C18: 0)/[(0.5×MUFA)+ (0.5×n−6 PUFA)+ (3×n−3 PUFA)+(n−3 PUFA/n−6 PUFA)]

      胆固醇血症指数 (hypocholesterolemic/hypercholesterolemic FA ratio, h/H)是根据Santos-Silva等[25]所述方法计算:

      h/H= (C18: 1n−9+C18: 2n−6+C20: 4n-6+C18: 3n−3+C20: 5n−3+C22: 5n−3+C22: 6n−3)/(C14: 0+C16: 0)

    • 石蜡切片的制作按照Liu等[26]所述的方法进行,并使用苏木精和伊红染色,脂肪组织石蜡切片由西安依科生物科技有限公司制作。观察组织学样品用正置显微镜(Leica biosystems,Germany)拍照。使用Photoshop (Adobe,San Jose,USA)对每个图像的平均脂肪细胞大小进行定量,计算每个组5个不重复的图像平均值[27]

    • 肝脏组织的甘油三酯含量采用南京建成生物工程研究所试剂盒检测。

    • RNA提取、浓度测定、反转录和RT-qPCR均按前述方法进行[28],采用CFX96实时定量PCR检测系统。qPCR反应体系为,初始激活步骤为95 °C 3 min,随后为38个循环的95 °C 15 s,60 °C 30 s,最后为65 °C 5 s。PCR反应后采用扩增曲线获得目的基因与管家基因的β-actin的CT值,计算它们之间CT值的差值记为△Ct,根据公式2−ΔΔCt计算各组目的基因的表达量[29-30]。基因引物序列见表3,由陕西省杨凌奥科鼎盛生物科技有限公司合成。

      基因
      genes
      登录序号
      accession number
      上游
      forward (5′-3′)
      下游
      reverse (5′-3′)
      PPARα FJ623265 CGCTGAGGTTCGGATATTT ACGTCACCTGGTCATTTAAG
      HSL HQ446238 TGGAACGTTACTGAGTCTGG AAGCGCACGTTGACTGG
      ATGL HQ845211 TCGTGCAAGCGTGTATATG GCTCGTACTGAGGCAAATTA
      CPT1 JF728839 GCATCCATGACACGTTTATTC GAAGTTTCTCTTCTCTCGTCTC
      FAD FJ641974 GCAGTTCTACGGTGTGTTT CAGTCTTGGTGTTTCTCATAGT
      ELO HQ637463 GCTTCTGCTGGACAACTAC GCGTCAGGAAGAGGTTATATG
      β-actin DQ211096 GACCTGACTGACTACCTCAT CGAAGTCAAGAGCCACATAG
      注:PPARα. 过氧化物酶体增殖活化受体;ATGL. 甘油三酯水解酶;CPT-1. 肉碱棕榈酰转移酶1;HSL. 激素敏感性脂肪酶;FAD. 脂肪酸去饱和酶;ELO. 脂肪酸延长酶
      Notes: PPARα. α peroxisome proliferator-activated receptor-α; ATGL. adipose TAG lipase; CPT-1. 1 carnitine palmitoyltransferase 1; HSL. hormone sensitive lipase; FAD. fatty acid desaturase; ELO. fatty acid elongase

      Table 3.  Primers used in real-time quantitative PCR.

    • 数据均采用平均数±标准差(mean±SD)表示,用SPSS 18.0软件(Chicago, IL, USA)对数据进行单因素方差分析、Duncan氏多重比较检验组间的差异。

    2.   结果
    • 各组间SGR、FCR、CF、HSI、SI、IPFI差异不显著(P>0.05)。S-O组的KI显著低于F-O组(P<0.05)。SE2-O组VSI显著低于F-O组(P<0.05)。S-O组及SE2-O组肠指数显著低于F-O组(P<0.05) (表4)。

      项目    
      items    
      组别 group
      F-OS-OSE1-OSE2-OE-O
      末重/g FBW 30.35±1.74 31.03±1.81 31.02±1.35 30.69±0.46 29.84±1.58
      增重率/% WG 37.97±7.88 41.06±8.23 41.02±6.16 39.51±2.10 35.63±7.17
      特定生长率/(%/d) SGR 0.65±0.12 0.70±0.12 0.70±0.09 0.68±0.03 0.62±0.11
      饲料转化率/% FCR 2.86±0.62 2.74±0.58 2.63±0.50 2.98±0.31 2.84±0.52
      肥满度/(g/cm3) CF 2.00±0.08 1.93±0.05 1.91±0.04 1.95±0.02 1.93±0.03
      肝体比/% HSI 3.11±0.12 3.27±0.16 3.08±0.24 3.05±0.11 2.96±0.31
      内脏指数/% VSI 16.79±0.49a 15.84±0.41ab 16.20±0.78ab 15.62±0.67b 16.01±0.51ab
      脾脏指数/% SI 0.12±0.01a 0.08±0.01b 0.11±0.02ab 0.09±0.01ab 0.11±0.02ab
      肾脏指数/% KI 0.31±0.02 0.28±0.04 0.28±0.01 0.27±0.03 0.31±0.03
      肠体指数/% RIW 3.06±0.42a 2.42±0.15b 2.64±0.36ab 2.49±0.09b 2.90±0.19ab
      脂肪指数/% IPFI 1.24±0.02 1.33±0.06 1.33±0.21 1.36±0.13 1.35±0.14
      注:数据表示为平均值±标准差。同行上标不同表示差异显著(P<0.05),下表同
      Notes: Values are means ± SD, of three replicates. Values in the same line with different superscript letters are significantly different(P<0.05), the same below

      Table 4.  Effects of Schizochytrium sp. oil combined with exogenous EPA on growth performance and biological parameters of grass carp

    • 全鱼水分、粗脂肪及灰分,肌肉中粗脂肪含量和肝脏甘油三酯含量在各组间无显著差异(P>0.05)。SE2-O组的肌肉水分含量显著低于F-O组、S-O组和E-O组(P<0.05)。E-O组肌肉粗蛋白显著低于S-O组(P<0.05)(表5)。

      项目    
      items    
      组别 group
      F-OS-OSE1-OSE2-OE-O
      肝脏 hepatopancreas
        甘油三酯/(mmol/g prot) triglyceride 0.30±0.05 0.25±0.03 0.31±0.05 0.30±0.07 0.30±0.06
      肌肉 muscle
        水分/% moisture 79.36±0.48a 79.40±0.27a 79.09±0.66ab 78.55±0.35b 79.41±0.87a
        粗蛋白质/% crude protein 20.78±0.28ab 21.13±0.29a 20.82±0.29ab 20.57±0.35ab 20.26±0.25b
        粗脂肪/% crude lipid 1.61±0.39 1.54±0.20 1.66±0.92 1.90±0.46 1.93±0.72
      全鱼 whole body
        水分/% moisture 75.37±1.30 75.52±0.85 74.02±0.42 75.10±1.21 72.68±1.14
        粗脂肪/% crude lipid 6.49±0.35 6.39±0.41 6.52±0.50 6.50±0.44 7.07±0.15
        灰分/% ash 3.15±0.31 2.93±0.37 2.65±0.24 2.90±0.11 3.04±0.20

      Table 5.  Effects of Schizochytrium sp. oil combined with exogenous EPA on proximate composition of grass carp

    • 在肌肉中,C18:2n-6, MUFA, n-6PUFA, n-3PUFA, n-3 LC PUFA及n-3/n-6在各组间均无显著差异(P>0.05),SE2-O组的SFA显著高于F-O组(P<0.05)。E-O组的EPA含量显著高于其他3组含藻油的组别(P<0.05)(表6)。S-O组DHA含量显著高于F-O组、SE1-O组和E-O组(P<0.05),SE2-O组和E-O组C18:3n-3显著低于F-O组(P<0.05)。在肝脏中,SFA在SE1-O组和E-O组显著高于其他组,而MUFA显著低于其他组(P<0.05)。S-O组的n-3PUFA显著高于其他组(P<0.05),C18:3n-3,n-3 LC PUFA在各组间无显著差异(P>0.05)(表7)。

      脂肪酸
      fatty acids
      肌肉 muscle
      F-OS-OSE1-OSE2-OE-O
      C14:0 1.47±0.68 1.76±0.26 1.58±0.29 1.32±0.73 1.79±0.29
      C16:0 24.15±1.53b 25.43±1.23ab 24.71±1.31ab 25.82±0.33a 24.42±0.64ab
      C18:0 9.02±1.00 10.30±0.69 9.35±1.09 9.68±0.74 9.89±0.76
      SFA 34.63±1.87b 37.48±1.76ab 35.65±2.39ab 36.81±1.40a 36.10±0.96ab
      C16:1n-7 5.49±0.68 5.00±0.65 5.61±0.65 6.01±0.45 5.10±0.74
      C18:1n-9 26.69±2.40ab 24.05±1.95b 27.32±2.70a 25.87±1.31ab 26.48±1.80ab
      C20:1n-9 0.08±0.07 0.14±0.03 0.09±0.12 0.07±0.07 0.06±0.08
      MUFA 32.26±3.11 29.20±2.53 33.02±3.39 31.95±1.57 31.65±2.40
      C18:2n-6 9.99±0.58 9.14±0.52 9.24±0.93 9.56±0.51 9.89±0.56
      C20:4n-6 1.09±0.30 1.04±0.56 0.74±0.41 1.11±0.22 1.08±0.35
      n-6PUFA 11.08±0.85 10.18±0.79 9.98±1.17 10.67±0.71 10.97±0.66
      C18:3n-3 3.37±0.44a 2.63±0.56abc 3.14±0.83ab 2.21±0.35c 2.45±0.37bc
      C20:5n-3 4.74±0.27ab 3.79±0.93b 4.24±1.27b 3.85±0.32b 5.57±0.97a
      C22:5n-3 2.25±0.47b 3.14±0.86a 2.55±0.48ab 2.69±0.16ab 2.21±0.27b
      C22:6n-3 11.66±1.18b 13.59±1.64a 11.42±1.50b 11.81±1.19ab 11.05±0.73b
      DHA+EPA 16.41±1.36 17.38±2.16 15.65±2.62 15.66±1.07 16.62±1.16
      DHA/EPA 2.46±0.20bc 3.73±0.95a 2.80±0.53bc 3.09±0.53ab 2.03±0.34c
      n-3PUFA 22.03±1.38 23.14±1.79 21.34±2.31 20.56±0.98 21.28±1.59
      n-3LC PUFA 18.66±1.82 20.52±2.35 18.20±3.09 18.35±1.22 18.83±1.37
      n-3/n-6 2.00±0.23 2.29±0.33 2.17±0.45 1.93±0.16 1.94±0.10

      Table 6.  Effects of Schizochytrium sp. oil combined with exogenous EPA on the fatty acid composition of muscle in grass carp (% of total fatty acids) %

      脂肪酸
      fatty acids
      肝脏 hepatopancreas
      F-OS-OSE1-OSE2-OE-O
      C14:0 2.35±0.10 2.12±0.31 2.33±0.26 2.43±0.14 2.31±0.31
      C16:0 25.93±1.29b 26.27±0.58b 28.80±0.59a 28.95±0.95a 27.64±0.07a
      C18:0 7.27±0.70bc 7.61±2.10bc 9.52±0.27b 5.84±1.36c 14.08±2.77a
      SFA 35.56±1.99c 36.20±2.22c 40.65±0.71b 37.22±0.77c 44.03±2.49a
      C16:1n-7 10.99±0.60 10.61±1.23 10.36±1.06 11.22±0.64 10.65±0.45
      C18:1n-9 45.07±0.83a 44.41±2.14a 41.13±1.15b 45.88±1.18a 39.33±1.71b
      C20:1n-9 1.19±0.41a 1.24±0.38a 1.05±0.27ab 0.62±0.06b 0.64±0.26b
      MUFA 57.25±0.47a 56.06±2.09a 52.53±0.70b 57.72±0.71a 50.61±1.71b
      C18:2n-6 3.32±1.06a 3.35±0.74a 2.81±0.64ab 1.63±0.12b 1.79±0.52b
      C20:4n-6 0.10±0.03 0.12±0.03 0.12±0.02 0.09±0.01 0.09±0.04
      n-6PUFA 3.42±1.04a 3.46±0.76a 2.93±0.66ab 1.72±0.11c 1.88±0.56bc
      C18:3n-3 1.90±0.33 1.85±0.31 2.05±0.29 1.71±0.47 2.01±0.07
      C20:5n-3 0.45±0.09a 0.24±0.07c 0.39±0.06ab 0.24±0.02c 0.30±0.08bc
      C22:5n-3 0.17±0.07b 0.42±0.18a 0.29±0.07ab 0.27±0.04ab 0.15±0.03b
      C22:6n-3 1.25±0.18ab 1.76±0.64a 1.41±0.27ab 1.13±0.18ab 1.02±0.27b
      DHA+EPA 1.70±0.25ab 2.00±0.70a 1.79±0.21ab 1.36±0.19ab 1.32±0.35b
      DHA/EPA 2.83±0.48 7.27±0.84 3.77±1.27 4.72±0.43 3.43±0.26
      n-3PUFA 3.77±0.64c 4.28±0.90a 4.14±0.50bc 3.34±0.41b 3.48±0.39bc
      n-3LC PUFA 1.87±0.31 2.07±0.44 2.08±0.27 1.63±0.23 1.47±0.38
      n-3/n-6 1.15±0.27b 1.27±0.31b 1.49±0.49ab 1.95±0.29a 1.94±0.49a

      Table 7.  Effects of Schizochytrium sp. oil combined with exogenous EPA on the fatty acid composition of hepatopancreas in grass carp (% of total fatty acids) %

    • 动脉粥样硬化指数AI在各组间无差异(P>0.05)。SE2-O组血栓形成指数TI显著高于F-O组(P<0.05),与其他组无差异(P>0.05)。胆固醇血脂指数h/H在S-O组和SE2-O组显著低于F-O组(P<0.05)(表8)。

      项目 itemsF-OS-OSE1-OSE2-OE-O
      动脉粥样硬化指数 AI 0.46±0.01 0.52±0.04 0.48±0.05 0.49±0.05 0.49±0.02
      血栓形成指数 TI 0.39±0.02b 0.41±0.02ab 0.41±0.02ab 0.43±0.02a 0.42±0.02ab
      胆固醇血症指数 h/H 2.34±0.12a 2.12±0.15b 2.24±0.20ab 2.11±0.11b 2.24±0.09ab
      注:AI,动脉粥样硬化指数;TI,血栓形成指数;h/H. 胆固醇血症指数
      Notes: AI. atherogenicity index; TI. thrombogenicity index; h/H. hypocholesterolemic/hypercholesterolemic FA ratio

      Table 8.  Effects of Schizochytrium sp. oil combined with exogenous EPA on health index of fatty acid in muscle of grass carp

    • 腹腔脂肪组织中脂肪细胞的大小如图1-a所示。F-O组脂肪细胞显著小于E-O组 (P<0.05),但与其他组没有差异(P>0.05)(图1-b)。

      Figure 1.  Effects of Schizochytrium sp. oil combined with exogenous EPA on the lipid accumulation of intraperitoneal fat in juvenile grass carp

    • 在腹腔脂肪组织中,与F-O和S-O相比,E-O组的ATGL和CPT1 mRNA水平显著下调(P<0.05);HSL和PPARα在各组间无显著性差异(P>0.05)(图2-a)。在肌肉组织中,E-O组FAD mRNA水平显著高于S-O组(P<0.05),ELO mRNA水平在S-O组显著下调(P<0.05)(图2-b)。

      Figure 2.  Effects of Schizochytrium sp. oil combined with exogenous EPA on the lipid-metabolism-related gene expression of the intraperitoneal fat (a) and FAD, ELO gene expression of muscle in juvenile grass carp (b)

    3.   讨论
    • 本研究中,生长性能在各组间无显著性差异,裂殖壶藻油可以完全替代鱼油单独作为草鱼日粮中的n-3 LC PUFA来源,无需外源添加EPA。在以微藻替代鱼油的研究发现,富含DHA的裂殖壶藻粉对星斑川鲽(Platichthys stellatus)幼鱼的生长显著优于裂殖壶藻粉+富含EPA的微拟球藻粉混合组和微拟球藻粉组,且与鱼油组的生长无显著差异[31]。裂殖壶藻能有效提高鱼类的生长性能可能与它含有丰富的DHA有关[32]。关于星斑川鲽DHA和EPA比例的研究中发现,DHA对星斑川鲽幼鱼生长性能的促进作用优于EPA[33]。Trushenski等[34]也认为DHA就可以满足军曹鱼(Rachycentron canadum)生长。还有研究表明,高DHA组的实验鱼生长最快,成活率最高[35-36]。有学者指出,DHA比 EPA 更能促进大部分鱼类的生长,过高的EPA含量和过低的DHA含量都会对仔稚鱼的神经功能产生消极的影响,DHA可能发挥着更为关键的作用[37,16]。本研究中E-O组的肌肉粗蛋白含量显著低于S-O组可能与上述原因相关。另外,由于日粮中仅以EPA为LC PUFA的来源时,减弱脂肪组织的水解能力和游离脂肪酸的β氧化能力,从而减弱了脂肪在组织间的分配能力和供能利用,导致E-O组肌肉组织的粗蛋白质含量显著降低。此外,脾脏是水产动物的主要免疫器官,脾指数可以反映免疫器官的生长发育状态[38],脾指数在S-O组显著低于F-O组,表明鱼油在促进脾等免疫器官生长发育方面比裂殖壶藻油更具有优势。S-O组及SE1-O组的肠指数显著低于F-O组,可能与鱼油的消化率低于微藻有关[39],推测鱼油可能加重了肠道的消化吸收负担。

    • 生物体对LC PUFA的需求量与其LC PUFA合成能力、食物及环境因子等因素有关[40-41]。淡水硬骨鱼类可以将C18PUFA转化成LC PUFA,而海水鱼类不具有该能力或该能力很弱[42]。研究表明草鱼机体可能具有比其他杂食性淡水鱼更强的LC PUFA合成能力[43]。脂肪酸去饱和酶(FAD)作为LC PUFA合成过程中的限速酶发挥作用,脂肪酸延长酶(ELO)则参与不饱和脂肪酸碳链的延长,FAD和ELO在LC PUFA的合成中起着关键作用,FAD和ELO的活性和底物特异性在不同鱼类之间的差异比较大[44-45]。富含C18PUFA的植物油促进LC PUFA合成关键酶的转录,而富含LC PUFA的鱼油则起抑制作用[46]。但调控LC PUFA合成的机制尚不清楚,推测与植物油缺乏鱼油富含的n-3 LC PUFA有关,减少了对LC PUFA合成以及相关酶基因的表达和酶活性的反馈抑制[47]

      在本研究中,FAD mRNA水平在E-O组显著高于S-O组,S-O组的ELO mRNA水平显著低于F-O组,SE2-O组和E-O组,表明在本实验中各组日粮含有等量的亚麻酸的情况下,日粮中等量但不同组成的n-3 LC PUFA会影响FAD和ELO的活性,推测仅以DHA为日粮中LC PUFA为来源会减弱FAD及ELO的活性,即日粮中高水平DHA可能会减弱机体合成LC PUFA的能力,这可能是因为S-O组日粮中富含DHA,自身不需要再合成大量DHA,因此对草鱼去饱和酶及延长酶活性形成了反馈抑制。同样,Thomassen等[48]以菜籽油和鱼油为对照组,菜籽油+EPA和菜籽油+EPA+DHA为处理组,得出大西洋鲑在LC PUFA合成过程中的去饱和及延长反应受DHA抑制,而不是EPA,Betancor等[49]在大西洋鲑的研究中也得出了同样的结论。在黄鳝的研究中表明高含量的DHA会抑制FAD基因的表达[9],在黑鲷的研究中也发现,随着DHA/EPA比例的升高,延长酶和去饱和酶下调[10]。在本研究中,随着日粮中EPA含量的增加,肌肉FAD和ELO表达水平也随之上调,这与肌肉中亚麻酸在日粮中含有EPA的组中有下降趋势的结果基本一致。这也解释了在E-O组的日粮中虽不含DHA,但肌肉和肝脏中却积累了高于EPA 2倍含量的DHA,这一现象的原因可能是机体通过激活FAD和ELO的活性从而由内源性途径合成了DHA。也有研究指出,亚麻酸在鱼体内用于β氧化以及合成LC PUFA[50-51],推测当亚麻酸合成LC PUFA的过程被抑制时,亚麻酸便倾向于β氧化。因此,草鱼体内亚麻酸的分配和去路有待进一步研究。从肌肉及肝脏各组脂肪酸组成的对比来看,推测草鱼有选择性保留具有调节膜流动性作用的DHA的机制,而EPA则易于被氧化供能,这在罗非鱼中也有相关报道[52-53],另一方面EPA可能作为底物转化为DHA,由此看来草鱼对DHA的需求量要高于EPA。在DHA/EPA比例的研究中也指出,DHA比EPA更加高效,鱼可选择性的消耗EPA而保留DHA[54]。另外,E-O组的肌肉EPA含量最高,这与E-O组的日粮中高含量的EPA来源有关,n-3 LC PUFA含量在肌肉及肝脏均无显著性差异,说明在藻油中添加EPA没有起到更进一步提高肌肉品质的作用。

    • 摄食大量的饱和脂肪酸(SFA)会使人体血液胆固醇含量上升,从而导致心血管疾病[55]。因此,为人类提供含优质脂肪酸的食物很关键。具有低比例的AI和TI,高比例的h/H的脂质更加适合人类食用[56]。SE2-O组TI显著高于F-O组,S-O组的h/H显著低于F-O组。S-O组的肌肉脂肪酸健康指数次于F-O组,添加EPA后,h/H在SE2-O组和E-O组虽有上升的趋势,但SE2-O组和E-O组的h/H及TI均与S-O组无显著差异。对AI, TI, h/H的分析说明,在藻油中添加EPA未能有效改善肌肉脂肪酸健康指数。肌肉脂肪酸组成反映日粮脂肪酸组成,分析日粮脂肪酸组成可知,添加EPA后,日粮中SFA比例随着C16:0比例下降,MUFA比例虽升高,但在肌肉中MUFA和n-3PUFA的含量没有得到改善,这可能是添加EPA后肌肉脂肪酸健康指数没有明显改善的原因,同时也与裂殖壶藻油本身含有高比例的SFA和低比例的MUFA有关,裂殖壶藻油中C16:0和DHA含量高,而其他种类的脂肪酸含量微乎其微,不如鱼油的脂肪酸种类丰富。本研究中的AI和TI的指数与鲤 (Cyprinus carpio)[57]和南方大口鲇(Silurus meridionalis)[58]等多种鱼类的指数较为接近,且远远优于乳制品和家畜动物脂质的相关指数[24],表明草鱼摄食含有藻油的日粮时,其肌肉脂质的质量与摄食鱼油的草鱼相近,对人体健康是有益。

    • 脂解由ATGL、HSL和MGL等一系列酶调控,在ATGL的催化下,甘油三酯转变成甘油二酯,HSL催化甘油二酯转变为单酰基甘油,MGL催化单酰基甘油生成甘油[59]。β氧化相关的酶类参与组织中脂肪酸的氧化分解供能过程, HSL与脂肪酸β氧化密切相关,是脂肪动员的主要限速酶,ATGL是脂质水解的限速酶[60],CPT1被认为是β氧化的限速酶[59]。研究表明黑鲷摄食含有EPA和DHA的日粮均能减小脂肪细胞大小[61]。在本研究中,E-O组的脂肪细胞大小显著高于F-O组和SE1-O组,这与脂肪水解基因ATGL在E-O组显著下调有关。在日粮中均含有0.52% n-3 LC PUFA的情况下,相比于其他各组,日粮中仅以EPA为LC PUFA的来源时,可能会减弱脂肪组织的水解能力和游离脂肪酸的β氧化能力,从而减弱脂肪在组织间的分配能力和供能利用,这也是E-O组肌肉组织的粗蛋白质含量显著降低的原因之一。此外,裂殖壶藻油配伍EPA对脂肪的水解能力没有产生影响。

    4.   结论
    • 综上所述,单独使用裂殖壶藻油和裂殖壶藻油配伍EPA对草鱼的生长性能、n-3 LC PUFA含量及脂肪组织的水解没有显著性影响。相对于EPA,草鱼可能更需要DHA。本实验结果认为,草鱼生产中可以单独使用裂殖壶藻油,而无需配伍EPA。

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