王雅平 李寶山 王際英 王成強(qiáng) 王曉艷 王麗麗 王世信 孫永智 郝甜甜
大菱鲆幼魚對(duì)飼料中泛酸最適需求量的研究*
王雅平1,2李寶山2王際英2①王成強(qiáng)2王曉艷2王麗麗1,2王世信2孫永智2郝甜甜2
(1. 上海海洋大學(xué) 水產(chǎn)科學(xué)國(guó)家級(jí)實(shí)驗(yàn)教學(xué)示范中心 農(nóng)業(yè)農(nóng)村部魚類營(yíng)養(yǎng)與環(huán)境生態(tài)研究中心 水產(chǎn)動(dòng)物遺傳育種中心上海市協(xié)同創(chuàng)新中心 上海 201306;2. 山東省海洋資源與環(huán)境研究院 山東省海洋生態(tài)修復(fù)重點(diǎn)實(shí)驗(yàn)室 煙臺(tái) 264006)
為研究大菱鲆()幼魚對(duì)泛酸的最適需求量,在基礎(chǔ)配方中添加不同梯度的泛酸鈣,制成6組泛酸含量分別為6.24、10.64、15.02、23.81、41.40和76.57 mg/kg等氮等能實(shí)驗(yàn)飼料,投喂初始體重為(24.73±0.10) g的大菱鲆幼魚80 d。結(jié)果顯示:1)泛酸對(duì)幼魚成活率(SR)無(wú)顯著影響(>0.05),10.64~76.57 mg/kg飼料組增重率(WGR)和特定生長(zhǎng)率(SGR)顯著提高(< 0.05);泛酸含量超過(guò)23.81 mg/kg時(shí),肝體比(HSI)顯著降低(<0.05);2)隨著飼料中泛酸含量的提高,全魚粗蛋白、粗脂肪和肌肉粗蛋白均呈先升后降趨勢(shì),肝臟脂肪含量顯著下降(<0.05);3)腸道消化酶、Na+, K+-ATPase和肝臟膽堿酯酶(ChE)活力隨泛酸添加量的增加呈先上升后下降趨勢(shì),10.64~76.57 mg/kg飼料組腸道肌酸激酶(CK)活力顯著提高(<0.05);4)血清、肝臟中過(guò)氧化氫酶(CAT)、超氧化物歧化酶(SOD)活力均呈先升后降趨勢(shì);6.24 mg/kg飼料組血清丙二醛(MDA)含量顯著高于其他5組(<0.05);5)隨泛酸含量的增加,肝臟脂肪合成酶(FAS)基因表達(dá)量先升后降,脂蛋白酯酶(LPL)基因表達(dá)量顯著上升(<0.05)。研究表明,飼料中添加適宜的泛酸能顯著增強(qiáng)大菱鲆幼魚腸道消化、吸收能力和機(jī)體抗氧化能力,并可提高脂肪代謝相關(guān)基因的表達(dá)水平,從而提高其生長(zhǎng)性能。以增重率為判據(jù),經(jīng)折線模型擬合得出,初體重為24.73 g的大菱鲆幼魚對(duì)泛酸的需求量為16.08 mg/kg飼料。
大菱鲆;泛酸;生長(zhǎng)性能;需求
泛酸(Pantothenic acid)又稱維生素B5,是生物體必需的一種水溶性維生素(楊延輝等, 2008)。泛酸在生物體內(nèi)主要以輔酶A(CoA)和?;d體蛋白(Acyl carrierprotein, ACP) 2種活性形式存在,其主要代謝功能包括脂肪酸、膽固醇和類固醇的合成,參與能量代謝,合成抗體和乙?;憠A(Jobling, 2012; Zhu, 2004);泛酸還具有抗脂質(zhì)過(guò)氧化作用,當(dāng)泛酸不足時(shí),過(guò)氧化物酶體脂肪酸β-氧化受到抑制,從而增加機(jī)體氧化損傷(Chen, 2015);除此之外,泛酸不足還會(huì)引起魚體生長(zhǎng)遲緩、厭食、棒狀鰓、鰭條出血等器官病變,嚴(yán)重缺乏時(shí)還會(huì)導(dǎo)致魚體死亡(Jobling, 2012)。目前,國(guó)內(nèi)外對(duì)魚類泛酸需求的研究較多,如黑帶石斑魚() (Lin, 2012)的泛酸需求量為11.0 mg/kg,寶石鱸() (宋理平, 2009)為12.40 mg/kg,大黃魚()(張春曉, 2006)為11.20 mg/kg,真鯛() (Yano, 1988)為10 mg/kg,雜交條紋狼鱸(×) (Raggi,2016)為18.8 mg/kg。
大菱鲆(L)屬鰈形目(Pleuronectiformes),鲆科(Bothidae),菱鲆屬,俗稱“多寶魚”,是名貴的低溫經(jīng)濟(jì)魚類。自20世紀(jì)90年代引種到我國(guó)以來(lái),已經(jīng)在我國(guó)北方沿海地區(qū)大規(guī)模養(yǎng)殖(彭墨等, 2014; 陳慕雁等, 2005)。目前,關(guān)于大菱鲆維生素的研究主要集中在脂溶性維生素(?;赖? 2014),而在水溶性維生素方面研究較少(謝全森等, 2008)。泛酸是生物體必需的營(yíng)養(yǎng)素,但在大菱鲆中尚未見對(duì)泛酸需求量的相關(guān)報(bào)道。因此,本研究以大菱鲆為研究對(duì)象,通過(guò)研究飼料中添加不同梯度的泛酸對(duì)其生長(zhǎng)、體組成、飼料利用、抗氧化性能、脂肪代謝的影響,確定大菱鲆對(duì)飼料泛酸的最適需求量,為泛酸在大菱鲆的飼料研究與應(yīng)用提供基礎(chǔ)數(shù)據(jù)。
以魚粉、酪蛋白為主要蛋白源,魚油為主要脂肪源,補(bǔ)充礦物質(zhì)和維生素預(yù)混料(不含泛酸),配制成6組等氮等能的基礎(chǔ)飼料(表1)。每千克基礎(chǔ)飼料中分別添加0、5、10、20、40和80 mg的D-泛酸鈣(購(gòu)自盛瑞源生物科技公司,純度>96%),命名為P1、P2、P3、P4、P5、P6組,P1為對(duì)照組。按實(shí)驗(yàn)配方將所需原料粉碎過(guò)80目篩,按比例稱重混勻,加入魚油及適量水再次混勻,經(jīng)螺旋擠壓機(jī)加工成粒徑為3 mm的飼料顆粒,60℃烘干后放置-20℃冰箱備用。利用高效液相層析色譜(HPLC)測(cè)得實(shí)驗(yàn)飼料中泛酸的含量分別為6.24、10.64、15.02、23.81、41.40和76.57 mg/kg。
實(shí)驗(yàn)所用大菱鲆幼魚購(gòu)自蓬萊宗哲養(yǎng)殖有限公司。養(yǎng)殖實(shí)驗(yàn)于2017年11月11日~2018年2月4日在山東省海洋資源與環(huán)境研究院室內(nèi)循環(huán)水養(yǎng)殖系統(tǒng)進(jìn)行。正式實(shí)驗(yàn)前挑選體質(zhì)健壯的大菱鲆幼魚暫養(yǎng)于圓形養(yǎng)殖桶(桶高80 cm,直徑70 cm,水深50 cm)中,投喂P1組飼料馴化,14 d后挑選規(guī)格均勻的大菱鲆幼魚[(24.73±0.10) g] 540尾,隨機(jī)分配至18個(gè)養(yǎng)殖桶中,每桶30尾魚,每種實(shí)驗(yàn)飼料隨機(jī)投喂3個(gè)養(yǎng)殖桶的實(shí)驗(yàn)魚。養(yǎng)殖實(shí)驗(yàn)進(jìn)行80 d,每天投喂2次(08:00,16:00),初始日投喂量占體重的1.5%左右,并根據(jù)攝食情況及時(shí)調(diào)整投喂量。投喂30 min后排殘餌,數(shù)顆粒,記錄殘餌數(shù)量。養(yǎng)殖期間,確保水質(zhì)符合以下條件:水溫(17.0±0.5)℃,鹽度28~32,pH 7.6~ 8.2,溶氧>7.0 mg/L,氨氮濃度和亞硝酸<0.1 mg/L。
表1 實(shí)驗(yàn)飼料基礎(chǔ)配方及營(yíng)養(yǎng)成分(干物質(zhì)%)
Tab.1 Formulation and nutrient composition of the basal experimental diets (dry matter %)
注: a. 礦物質(zhì)預(yù)混料配方參考王際英等(2014). b. 維生素預(yù)混料(mg/kg飼料): 硫胺素,15 mg;核黃素,15 mg;煙酸,100 mg;吡哆醇,20 mg;氰鈷胺,4 mg;生物素,1 mg;肌醇,200 mg;葉酸,5 mg;氯化膽堿,1000 mg;抗壞血酸,240 mg;維生素A,20 mg;維生素D,8 mg;維生素E,150 mg;維生素K,10 mg
Note: a. Same contents of mineral premix as reference Wang(2014). b. Vitamin mixture (mg/kg diet): Thiamin, 15 mg; Riboflavin, 15 mg; Niacin acid, 100 mg; Pyridoxine, 20 mg; Cyanocobalamin, 4 mg; Biotin, 1 mg; Inositol, 200 mg; Folic acid, 5 mg; Choline chloride, 1000 mg; Ascorbic acid, 240 mg; Retinol acetate, 20 mg; Cholecalciferol, 8 mg; Alpha-tocopherol, 150 mg; Vitamin K, 10 mg
全魚、背肌、肝臟、腸道及血清取樣方法參考 梅琳等(2015)。
同時(shí),每桶中隨機(jī)取3尾實(shí)驗(yàn)魚,無(wú)菌條件下解剖,取肝尖置于2 ml無(wú)RNase離心管中并迅速于液氮中速凍,–80℃超低溫冰箱中保存,用于組織中相關(guān)基因的熒光定量分析。
1.4.1 生長(zhǎng)指標(biāo)計(jì)算
增重率(Weight gain rate WGR, %)=(魚體末重–魚體初重)/魚體初重×100;
特定生長(zhǎng)率(Specific growth rate, SGR, %/d) =(ln魚體末重–ln魚體初重)/養(yǎng)殖周期×100;
飼料系數(shù)(Feed conversion ratio, FCR)=攝食量/體增重×100;
攝食率 (Feed intake, FI, %/d)=攝食量/[(魚體初重量+魚體末重量)/2×養(yǎng)殖周期]×100
蛋白質(zhì)效率(Protein efficiency ratio, PER)=(魚體末重–魚體初重)/蛋白質(zhì)總攝入量;
臟體比(Viscerosomtic index, VSI)=內(nèi)臟團(tuán)濕重/魚體末重×100;
肝體比(Hepatosomatic index, HSI, %)=肝臟濕重/魚體末重×100;
肥滿度(Condition factor, CF,)=魚體末重/體長(zhǎng)3× 100;
存活率(Survival rate, SR, %)=終末魚尾數(shù)/初始魚尾數(shù)×100。
1.4.2 體成分測(cè)定 實(shí)驗(yàn)飼料及體壁中水分用105℃恒重法測(cè)定(GB/T6435-2006),粗蛋白用凱氏定氮法測(cè)定(GB/T6432-2006),粗脂肪用索氏抽提法測(cè)定(GB/T6433-2006),粗灰分用550℃失重法測(cè)定(GB/T 6438-2007),能量用燃燒法測(cè)定(IKA, C6000, 德國(guó))。
1.4.3 酶活測(cè)定 腸道淀粉酶(Amylase)、脂肪酶(Lipase)、胰蛋白酶(Trypsin)、Na+,K+-ATPase、肌酸激酶(Creatine Kinase,CK)活性、肝臟中膽堿酯酶(Acetylcholinesterase,AChE),血清和肝臟中超氧化物歧化酶(Superoxide dismutase,SOD)、過(guò)氧化氫酶(Catalase, CAT)活力及丙二醛(Malondialdehyde, MDA)含量均采用南京建成生物工程研究所試劑盒測(cè)定。酶活力單位參照試劑盒說(shuō)明書。
1.4.4 肝臟脂代謝相關(guān)基因差異表達(dá)分析 按照TaKaRa公司的RNAiso Plus試劑盒說(shuō)明書提取肝臟總RNA,通過(guò)電泳檢測(cè)RNA的完整性,然后檢測(cè)其純度。最后,采用PrimeScript? RT reagent Kit with gDNA Eraser反轉(zhuǎn)錄試劑盒合成cDNA,并置于–20℃待用。
使用Primer 5.0設(shè)計(jì)引物,同時(shí)設(shè)計(jì)引物作為內(nèi)參,所有引物均由生工生物技術(shù)(上海)股份有限公司合成,引物序列參見表2。使用TaKaRa公司生產(chǎn)的SYBR? Premix ExTMⅡ試劑盒進(jìn)行熒光定量PCR,具體步驟參見說(shuō)明書。PCR程序?yàn)椋?5℃、30 s,1個(gè)循環(huán);95℃、5 s,57℃、30 s,72℃、60 s共40個(gè)循環(huán):95℃熔解曲線檢測(cè)反應(yīng)特異性。在本實(shí)驗(yàn)中,F(xiàn)AS、LPL、-的擴(kuò)增效率分別是0.986、0.980、0.991。每個(gè)復(fù)孔設(shè)置參照基因,根據(jù)2-ΔΔCt計(jì)算法進(jìn)行相對(duì)定量后,分析肝臟中FAS和LPL的相對(duì)表達(dá)量。
采用SPSS18.0軟件進(jìn)行單因素方差分析(One-way ANOVA),差異顯著(<0.05)時(shí)用Duncan’s檢驗(yàn)進(jìn)行多重比較分析。統(tǒng)計(jì)數(shù)據(jù)以平均值±標(biāo)準(zhǔn)差(Means± SD,=3)的形式表示。采用折線模型擬合得出大菱鲆幼魚對(duì)飼料中泛酸基于增重率的最適需求量。
由表3可見,添加泛酸對(duì)大菱鲆幼魚的存活率(SR)無(wú)顯著影響(>0.05),均在98%以上。隨飼料泛酸含量的增加,大菱鲆幼魚的增重率(WGR)和特定生長(zhǎng)率(SGR)均呈先上升后下降趨勢(shì),P3、P4組顯著高于其他組(<0.05),各添加組均顯著高于對(duì)照組(<0.05)。各添加組的飼料系數(shù)(FCR)和攝食率(FI)呈先降后升趨勢(shì),且均顯著低于對(duì)照組(>0.05)。添加組蛋白質(zhì)效率(PER)顯著上升(<0.05),在P3組達(dá)到最大。添加泛酸對(duì)臟體比(VSI)和肥滿度(CF)無(wú)顯著影響(>0.05),肝體比(HSI)呈先下降后上升趨勢(shì),P4和P5組顯著低于其他各組(<0.05)。
表2 定量PCR引物序列
Tab.2 Nucleotide sequences of the primers used to assay gene expression by real-time PCR
表3 飼料泛酸含量對(duì)大菱鲆幼魚生長(zhǎng)性能、飼料利用及形體指標(biāo)的影響
Tab.3 Effects of dietary pantothenic acid on growth performance, feed utilization and body indices of juvenile S. maximus
注: 同一行肩標(biāo)不同表示不同組間差異顯著(<0.05),下表同
Note: Values within the same row with different letters are significantly different (<0.05). The same as the following
采用折線模型分析方法,擬合增重率與飼料中泛酸含量的關(guān)系:初始體重為(24.73±0.10) g的大菱鲆幼魚飼料中泛酸的最適含量為16.08 mg/kg (圖1)。
圖1 大菱鲆幼魚增重率與飼料泛酸含量的折線回歸分析
飼料中添加泛酸對(duì)大菱鲆幼魚體組成的影響見表4。由表4可知,全魚體組成中,水分含量在77.59%~ 79.51%之間,且各組之間無(wú)顯著差異(>0.05);粗脂肪含量先上升后下降,P3組達(dá)到最大值;隨著泛酸含量的增加,粗蛋白和粗灰分顯著上升,最終呈平穩(wěn)趨勢(shì)。肌肉體組成中,水分含量無(wú)顯著差異(>0.05);P3和P4組粗蛋白含量顯著高于其他組(<0.05);粗脂肪及粗灰分含量差異不顯著(>0.05);肝臟粗脂肪含量隨泛酸的增加顯著下降(<0.05)。
隨著飼料泛酸含量的增加,大菱鲆幼魚淀粉酶、脂肪酶和Na+,K+-ATPase活力呈先上升后下降趨勢(shì),P4組脂肪酶顯著高于其他各組(<0.05),淀粉酶和Na+,K+-ATPase活力在P5組達(dá)到最大值。P5組胰蛋白酶活力顯著高于其他組(<0.05)。各添加組肌酸激酶(CK)和膽堿酯酶(ChE)活力顯著高于對(duì)照組(<0.05) (表5)。
隨著飼料中泛酸含量的增加,肝臟中過(guò)氧化氫酶(CAT)和超氧化物歧化酶(SOD)活力均呈先上升后下降趨勢(shì),P3-P6組丙二醛(MDA)含量顯著低于對(duì)照組(<0.05)。血清中過(guò)氧化氫酶(CAT)活力隨飼料泛酸含量的升高呈先升后降趨勢(shì),P4組達(dá)到最高值(< 0.05)。P2~P4組的超氧化物歧化酶(SOD)活力顯著高于對(duì)照組(<0.05),并在P4組達(dá)到最大值。血清中丙二醛(MDA)含量隨泛酸添加量的增加顯著下降(< 0.05),P6組含量最低(表6)。
表4 飼料中泛酸水平對(duì)大菱鲆幼魚常規(guī)成分的影響(%濕重)
Tab.4 Effects of dietary pantothenic acid levels on proximate composition of juvenile S. maximus (% wet weight)
表5 泛酸對(duì)大菱鲆幼魚腸道消化相關(guān)酶活的影響
Tab.5 Effects of pantothenic acid on the activities of intestinal digestive physiology and liver physiological enzymes of juvenile S. maximus
表6 泛酸對(duì)大菱鲆幼魚肝臟和血清抗氧化能力的影響
Tab.6 Effects of dietary pantothenic acid on liver and serum antioxidant capacity of juvenile S.maximus
研究結(jié)果顯示,大菱鲆肝臟脂肪合成酶(FAS)mRNA表達(dá)量隨泛酸含量的增加呈先升后降趨勢(shì),P3、P4組顯著高于P1、P6組(<0.05) (圖2);脂蛋白酯酶(LPL)mRNA表達(dá)量隨泛酸增加呈上升趨勢(shì),且P4到P6組顯著高于其他組(<0.05) (圖3)。
圖2 肝臟脂肪合成酶基因相對(duì)表達(dá)量
標(biāo)有相同上標(biāo)字母的數(shù)值表示差異不顯著(>0.05),下同
Values with the same letters are not significantly different at>0.05. The same as belwo
圖3 肝臟脂蛋白酯酶基因相對(duì)表達(dá)量
體蛋白,體脂肪和體灰分沉積會(huì)造成水生動(dòng)物的增重,飼料中主要營(yíng)養(yǎng)物質(zhì)的利用效率對(duì)水生動(dòng)物生長(zhǎng)有重要影響(Mommsen, 2001)。飼料泛酸水平對(duì)不同魚種的體組成影響存在差異。研究表明,飼料泛酸顯著影響翠鱧魚體粗蛋白和粗灰分含量(Zehra, 2018),湖鱒脂肪含量隨著飼料泛酸水平增加而增加(Poston, 1982)。本研究中,飼料中添加泛酸對(duì)全魚和背肌水分無(wú)顯著影響,當(dāng)泛酸含量從6.24 mg/kg增加到41.40 mg/kg時(shí),實(shí)驗(yàn)魚體粗蛋白和粗脂肪積累增多,泛酸含量繼續(xù)增加時(shí),蛋白沉積受到抑制,與在草魚(劉安龍等, 2007)、吉富羅非魚(黃鳳等, 2014)的研究結(jié)果類似。其促進(jìn)蛋白和脂肪沉積的原因是:第一,泛酸以輔酶A的形式在蛋白質(zhì)的合成和轉(zhuǎn)運(yùn)中發(fā)揮重要作用(Wen, 2009);第二,泛酸作為?;d體蛋白的組成之一,調(diào)節(jié)脂肪的代謝(田娟等, 2009);第三,從本研究結(jié)果可知,泛酸可顯著提高腸消化和吸收酶的活性,從而提高大菱鲆幼魚對(duì)蛋白質(zhì)、脂肪的消化吸收能力。P2~P5組全魚粗灰分含量與P1組差異顯著,這與吉富羅非魚的研究結(jié)果一致(黃鳳等, 2014),可能是泛酸提高了腸道對(duì)鈣磷的吸收,促進(jìn)了其在體內(nèi)的蓄積(張璐等, 2016)。本研究結(jié)果顯示,適宜的泛酸添加水平對(duì)大菱鲆背肌的粗脂肪和粗灰分的蓄積有一定的促進(jìn)作用,但無(wú)顯著差異。膽固醇和甘油三酯主要在肝臟內(nèi)合成,P1組肝臟脂肪含量顯著增加,可能是因?yàn)榉核岵蛔阌绊懙街舅嵫趸?,造成多余脂肪在肝臟中沉積(黃鳳等, 2014)。該結(jié)果與石斑魚的研究結(jié)果類似(Lin, 2012)。
化學(xué)性消化是水生動(dòng)物的主要消化方式,而消化酶在其中發(fā)揮重要作用。腸上皮營(yíng)養(yǎng)物質(zhì)消化是消化酶催化ATP結(jié)合的過(guò)程,其活力可直接揭示魚類的消化能力(Qian, 2015)。本研究中,攝食缺乏泛酸的飼料降低了蛋白酶、淀粉酶和脂肪酶活力,表明泛酸缺乏導(dǎo)致魚體腸道消化功能受阻。隨著泛酸含量從6.24 mg/kg增加到41.40 mg/kg,腸道消化酶活力顯著增加,表明泛酸是維持魚類腸道消化功能的重要營(yíng)養(yǎng)物質(zhì),這可能是因?yàn)槟c道功能與組織CoA的含量成正比,泛酸作為CoA的組成成分,參與蛋白質(zhì)和脂質(zhì)的代謝和轉(zhuǎn)運(yùn),提高消化酶活性,增強(qiáng)蛋白質(zhì)等營(yíng)養(yǎng)物質(zhì)在體內(nèi)的消化利用率(Qian, 2015; Feng, 2013)。Na+-K+泵主要由腸上皮刷狀緣細(xì)胞表面存在的ATP酶構(gòu)成,其活性可以間接反映腸黏膜的吸收功能(Almansa, 2001)。肌酸激酶參與細(xì)胞內(nèi)能量轉(zhuǎn)運(yùn)和ATP再生(Tang, 2009)。本研究中,泛酸添加量超過(guò)10.64 mg/kg時(shí),Na+,K+-ATP酶和CK活力顯著上升。說(shuō)明適宜水平的泛酸可以通過(guò)提高腸道Na+,K+-ATP酶活性提高ATP水解能力,并使CK的活力增強(qiáng),為氨基酸、葡萄糖等營(yíng)養(yǎng)物質(zhì)在腸內(nèi)的轉(zhuǎn)運(yùn)提供能量,從而促進(jìn)營(yíng)養(yǎng)物質(zhì)在腸道的消化和吸收。這與文澤平(2008)對(duì)建鯉的研究結(jié)果相一致。泛酸在乙酰膽堿的合成中承擔(dān)重要作用,而膽堿酯酶能迅速破壞組織內(nèi)的乙酰膽堿。添加組ChE活力顯著高于P1組。可能是脂肪代謝的增加導(dǎo)致肝臟中?;鵆oA增多,繼而產(chǎn)生大量的乙酰膽堿,過(guò)多的底物誘導(dǎo)ChE活力增強(qiáng)(杜金梁等, 2017)。
體內(nèi)抗氧化酶(SOD-CAT)系統(tǒng)可以清除體內(nèi)自由基,對(duì)維持機(jī)體正常代謝和生理功能有重要作用。SOD-CAT系統(tǒng)代表機(jī)體抵抗氧化應(yīng)激和增加抗自由基酶活性的第一道防線(Liu, 2007)。SOD-CAT系統(tǒng)代表機(jī)體抵抗氧化應(yīng)激與增加抗自由基酶活性的第一道防線(Winston, 1991),本研究結(jié)果顯示,飼料中添加一定量的泛酸后,能顯著提高肝臟和血清中的CAT活性,與對(duì)團(tuán)頭魴的報(bào)道一致(Qian, 2015)。本研究中,肝臟和血清SOD活性均在對(duì)照組中最低,當(dāng)飼料泛酸添加量超過(guò)23.81 mg/kg時(shí),二者表現(xiàn)出不同的變化趨勢(shì):血清中的SOD活性呈下降趨勢(shì),肝臟中的SOD活力繼續(xù)上升。說(shuō)明飼料中過(guò)多的泛酸降低了血清的抗氧化能力。凡納濱對(duì)蝦() (袁野等, 2016)、團(tuán)頭魴(Qian, 2015)的研究中也有類似報(bào)道。脂質(zhì)過(guò)氧化產(chǎn)生的MDA含量可以反映機(jī)體受自由基攻擊程度,MDA從膜上產(chǎn)生的位置釋放后,可與蛋白質(zhì)、核酸反應(yīng),進(jìn)而抑制蛋白質(zhì)的合成(李涌泉等, 2008)。本研究結(jié)果顯示,添加量超過(guò)10.64 mg/kg時(shí),肝臟MDA含量顯著下降,而泛酸水平超過(guò)6.24 mg/kg時(shí),血清中MDA含量顯著下降。這一結(jié)果與在凡納濱對(duì)蝦(袁野等, 2016)、鯉魚(Li, 2015)上的研究一致。Ayyat等(2011)的研究發(fā)現(xiàn),CoA通過(guò)增強(qiáng)脂肪酸的代謝來(lái)清除脂質(zhì)過(guò)氧化物。表明適宜水平的泛酸可以抵抗脂質(zhì)過(guò)氧化和保護(hù)組織。
魚體脂肪沉積主要是受體內(nèi)脂肪合成、降解和轉(zhuǎn)運(yùn)共同調(diào)控的結(jié)果。因此,測(cè)定魚類肝臟中脂肪代謝相關(guān)基因的表達(dá)量,可以有效地反映魚體脂肪沉積情況(徐超等, 2016)。FAS是脂肪酸從頭合成的限速酶,對(duì)體內(nèi)脂肪從頭合成發(fā)揮主導(dǎo)作用(Guo, 2007)。本研究中,隨著泛酸含量的增加,F(xiàn)AS表達(dá)量呈先升后降趨勢(shì),這表明適量泛酸能夠提高肝臟中FAS的表達(dá)水平;這也暗示適量的泛酸可能促進(jìn)機(jī)體脂肪的合成水平,類似的結(jié)果在團(tuán)頭魴中也有報(bào)道(Qian, 2015)。LPL促進(jìn)組織對(duì)脂肪酸的攝取,進(jìn)而增強(qiáng)對(duì)肝臟脂肪酸的β-氧化能力(朱定貴等, 2011)。本研究結(jié)果顯示,相比對(duì)照組,P4~P6組的LPL表達(dá)量顯著升高,同時(shí),魚體肝臟粗脂肪含量減少,產(chǎn)生這一結(jié)果的原因可能是由于LPL表達(dá)量的增加,提高了脂肪的氧化代謝速率,同時(shí)FAS表達(dá)量顯著降低,使得肝臟脂肪合成量下降。這一研究結(jié)果與瓦氏黃顙魚()的研究結(jié)果一致(覃川杰等, 2015)。
綜上所述,泛酸是大菱鲆幼魚維持正常生長(zhǎng)和生理所必需的維生素。飼料中適宜水平的泛酸可以增強(qiáng)機(jī)體消化吸收和清除自由基的能力,并能影響肝臟中脂肪代謝相關(guān)基因的表達(dá)水平。以增重率為評(píng)價(jià)指標(biāo),24.73 g大菱鲆幼魚最適泛酸需求量為16.08 mg/kg飼料。
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Study on the Optimum Dietary Pantothenic Requirement of Juvenile Turbot (L.)
WANG Yaping1,2, LI Baoshan2, WANG Jiying①, WANG Chengqiang2, WANG Xiaoyan2, WANG Lili1,2, WANG Shixin2, SUN Yongzhi2, HAO Tiantian2
(1. National Demonstration Center for Experimental Fisheries Science Education, Centre for Research on Environmental Ecology and Fish Nutrion (CREEFN) of the Ministry of Agriculture and Rural Affairs Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai Ocean University, Shanghai 201306; 2. Shandong Key Laboratory of Marine Ecological Restoration, Shandong Marine Resource and Environment Research Institute, Yantai 264006)
An 80-day feeding trial was conducted to investigate the pantothenic acid dietary requirements of juvenile turbot. Six isoenergetic and isonitrogenous diets were formulated using fish meal and casein as a protein source. The basal diet was supplemented with calcium D-pantothenic acid at 6.24, 10.64, 15.02, 23.81, 41.40, or 76.57 mg/kg and fed to juveniles weighing (24.73±0.10) g. The results were as follows: 1) No significant differences in juvenile turbot survival rate (SR) were found between the dietary treatments (>0.05) and the weight growth rate (WGR) and specific growth rate (SGR) increased as pantothenic acid levels increased from 10.64 to 76.57 mg/kg (<0.05). For dietary pantothenic acid content greater than 23.81 mg/kg, the hepatosomatic index decreased significantly (<0.05). 2) The crude protein and lipid levels of the whole body and crude protein levels of the muscle initially increased and then decreased with increasing dietary pantothenic acid levels, whereas the crude lipid content of the liver decreased (<0.05). 3) The activities of the intestinal digestive enzymes Na+, K+-ATPase and hepatic cholinesterase (ChE) initially increased and then decreased with increasing dietary pantothenic acid levels, whereas intestinal creatine kinase (CK) activity increased significantly from 10.64 to 76.57 mg/kg (<0.05). 4) Hepatic and serum catalase (CAT) and superoxide dismutase (SOD) activities were significantly lower in the control group than in the groups with calcium pantothenic acid-enriched diets (<0.05). Turbot fed a 6.24 mg/kg pantothenic acid diet had a higher serum malondialdehyde content than those fed other diets (<0.05). 5) Fatty acid synthetase (FAS) expression increased and then decreased with increasing levels of pantothenic acid. Lipoprotein lipase (LPL) expression was significantly up-regulated in the liver with increasing levels of pantothenic acid (<0.05). In conclusion, appropriate levels of dietary pantothenic acid significantly improved intestinal digestion and absorption capacity, thus, improving nonspecific immunity and the expression of fat-related genes, and consequently, the growth and body composition of juvenile turbot. Based on broken-line regression analysis of WGR, the optimum dietary pantothenic acid requirement of juvenile turbot with a body weight of 24.73 g was 16.08 mg/kg.
Juvenile turbot; Pantothenic acid; Growth performance; Requirement
S963
A
2095-9869(2019)06-0066-10
10.19663/j.issn2095-9869.20180821001
http://www.yykxjz.cn/
王雅平, 李寶山, 王際英, 王成強(qiáng), 王曉艷, 王麗麗, 王世信, 孫永智, 郝甜甜. 大菱鲆幼魚對(duì)飼料中泛酸最適需求量的研究. 漁業(yè)科學(xué)進(jìn)展, 2019, 40(6): 66–75
Wang YP, Li BS, Wang JY, Wang CQ, Wang XY, Wang LL, Wang SX, Sun YZ, Hao TT. Study on the optimum dietary pantothenic requirement of juvenile turbot (L.). Progress in Fishery Sciences, 2019, 40(6): 66–75
* 山東省重點(diǎn)研發(fā)計(jì)劃(2016GSF115005)和煙臺(tái)市科技計(jì)劃(2018ZHGY066)共同資助 [This work was supported by Shandong Key Research and Development Plan (2016GSF115005), and Science and Technology Development Project of Yantai (2018ZHGY066)]. 王雅平,E-mail: m15552437686@163.com
王際英,研究員,E-mail: ytwjy@126.com
2018-08-21,
2018-09-21
WANG Jiying, E-mail: ytwjy@126.com
(編輯 江潤(rùn)林)