氧化魚油和維生素E對(duì)大黃魚SOD和CA T酶活性的影響

唐 筱,王 珺,2,徐后國,艾慶輝,左然濤

(1.中國海洋大學(xué),山東青島266003;2.廣東省微生物研究所,廣東廣州,510070)

氧化魚油和維生素E對(duì)大黃魚SOD和CA T酶活性的影響

唐 筱1,王 珺1,2,徐后國,艾慶輝＊＊,左然濤

(1.中國海洋大學(xué),山東青島266003;2.廣東省微生物研究所,廣東廣州,510070)

實(shí)驗(yàn)設(shè)計(jì)了8種飼料,其中飼料1采用新鮮魚油(過氧化值POV=1.72 meq·kg-1),作為對(duì)照組,飼料2,3和4采用氧化魚油組(POV分別為28.29,62.79和104.21 meq·kg-1),飼料1～4中均未補(bǔ)充維生素E。在飼料1～4中分別補(bǔ)充600 IU·kg-1維生素E制作成飼料5～8。用實(shí)驗(yàn)飼料飼喂大黃魚幼魚(初體質(zhì)量7.82±0.68 g)10周。實(shí)驗(yàn)結(jié)果顯示,氧化魚油使大黃魚血清抗氧化酶超氧化物歧化酶(SOD)和過氧化氫酶(CAT)活性顯著升高(P<0.05);補(bǔ)充維生素E使氧化魚油組的SOD和CAT活性降低。結(jié)果顯示,維生素E對(duì)氧化魚油對(duì)大黃魚的負(fù)面影響具有一定的補(bǔ)償作用。

大黃魚;氧化魚油;維生素E;SOD;CAT

脂肪是肉食性魚類的最主要的能量來源,魚油是很多肉食性魚類飼料中必需的原料成分[1]。但是在飼料加工、處理和儲(chǔ)藏過程中,脂肪很容易被氧化[2-3]。氧化了的脂肪對(duì)魚類的存活、生長和免疫系統(tǒng)均產(chǎn)生不利的影響[4-8]。脂肪氧化會(huì)導(dǎo)致細(xì)胞膜損傷,尤其是對(duì)富含不飽和脂肪酸的細(xì)胞器具有很大的危害[5]。

維生素E是具有α-生育酚生物活性的所有分子的統(tǒng)稱,維生素E作為脂溶性生物組織抗氧化劑,具有保護(hù)含磷脂的生物膜的作用,它能通過抑制脂質(zhì)過氧化從而避免細(xì)胞的最終破裂。Lewis-McCrea等對(duì)大西洋鳙鰈(Hippoglossus hippoglossusL.)的研究證明維生素E可降低氧化魚油對(duì)魚體的損害[9]。

有研究顯示氧化魚油對(duì)日本牙鲆[10]、雜交羅非魚[11]、大西洋鮭[12]和大西洋鳙鰈[9,13]生長和抗氧化酶活性均會(huì)產(chǎn)生影響。大黃魚(Pseudosciaena croceaR.)屬鱸形目,石首魚科,黃魚屬,也稱黃魚、大黃花、大鮮,是我國重要的養(yǎng)殖品種。大黃魚屬肉食性魚類,其飼料中脂肪尤其是富含不飽和脂肪酸的魚油的含量較高,在濕熱環(huán)境下極易發(fā)生氧化反應(yīng)。有關(guān)大黃魚營養(yǎng)生理的研究己有相關(guān)報(bào)道[14-17],而氧化魚油對(duì)大黃魚抗氧化酶活性的影響卻未見報(bào)道。體內(nèi)抗氧化酶的活力可以反映機(jī)體氧化—抗氧化狀態(tài),從側(cè)面體現(xiàn)養(yǎng)殖動(dòng)物的健康狀態(tài)。本實(shí)驗(yàn)對(duì)不同氧化程度的魚油對(duì)大黃魚抗氧化酶活性的影響以及維生素E的補(bǔ)償作用進(jìn)行了研究。

1 材料與方法

1.1 實(shí)驗(yàn)飼料

實(shí)驗(yàn)設(shè)計(jì)制作了8種飼料。其中飼料1為新鮮魚油(過氧化值POV=1.72 meq·kg-1),作為對(duì)照組,飼料2,3和4為氧化魚油組(POV分別為28.29,62.79和104.21 meq·kg-1),飼料1～4中均未補(bǔ)充維生素E。在飼料1～4中分別補(bǔ)充600 IU·kg-1維生素E制作成飼料5～8(見表1)。實(shí)驗(yàn)采用未添加抗氧化劑的鯡魚魚油作為主要的脂肪源。

表1 不同氧化程度魚油和維生素E實(shí)驗(yàn)設(shè)計(jì)Table 1 Experimental design

氧化魚油制作方法參考Koshio等方法[18],過氧化值采用國標(biāo)法(GB/T 5538-2005)測定。實(shí)驗(yàn)飼料配方及營養(yǎng)組見表2。飼料制作時(shí),所有原料粉碎后過220μm篩網(wǎng),各原料采用逐級(jí)放大法混合均勻,然后加入適量的水揉勻,經(jīng)雙螺桿擠條機(jī)(華南理工大學(xué)研制的F-III(26)型)加工成適宜2種粒徑大小的顆粒狀飼料(1.5 mm×3.0 mm和2.5 mm×5.0 mm),比例1∶2。飼料在38℃下,鼓風(fēng)干燥箱中烘12 h,裝入塑料密封袋中保存?zhèn)溆谩?/p>

表2 實(shí)驗(yàn)基礎(chǔ)飼料配方及營養(yǎng)組成(%干物質(zhì))Table 2 Formulation and proximate composition of the experimental diets(%dry matter)

1.2 實(shí)驗(yàn)過程

養(yǎng)殖實(shí)驗(yàn)在浙江省寧波市象山港海水網(wǎng)箱養(yǎng)殖區(qū)進(jìn)行。實(shí)驗(yàn)所用大黃魚選用當(dāng)年人工孵化的同一批魚苗,實(shí)驗(yàn)開始前在網(wǎng)箱(3.0 m×3.0 m×3.0 m)中暫養(yǎng)2周,暫養(yǎng)期間投喂對(duì)照組飼料,使之逐漸適應(yīng)實(shí)驗(yàn)飼料、投喂時(shí)間和養(yǎng)殖環(huán)境。

實(shí)驗(yàn)魚經(jīng)2周的馴化后,禁食24 h,以丁香酚(1∶10 000,上海試劑廠,中國上海)麻醉,挑選出體格健壯、規(guī)格一致的大黃魚(初始體質(zhì)量為(7.82±0.68) g),作為實(shí)驗(yàn)用魚。實(shí)驗(yàn)魚隨機(jī)養(yǎng)殖在24個(gè)海水網(wǎng)箱(1.0 m×1.0 m×1.5 m)中,每個(gè)網(wǎng)箱放養(yǎng)60尾實(shí)驗(yàn)魚。將網(wǎng)箱隨機(jī)分成8組,分別投喂8種飼料。每天投喂2次(04··30和16··30),飽食投喂(以實(shí)驗(yàn)魚不再爭搶飼料為準(zhǔn))。實(shí)驗(yàn)期間1～4周飼喂小粒徑飼料,5～10周飼喂大粒徑飼料。實(shí)驗(yàn)期間水溫為26.5～30.5℃,鹽度約為28,溶解氧約為7 mg·L-1。飼養(yǎng)實(shí)驗(yàn)為期10周。

1.3 樣品采集和分析

養(yǎng)殖實(shí)驗(yàn)結(jié)束后,實(shí)驗(yàn)魚禁食24 h,每網(wǎng)箱隨機(jī)取6尾魚,經(jīng)丁香酚麻醉后,經(jīng)尾靜脈抽血以分離出血清,測定酶活。

超氧化物歧化酶(SOD)和過氧化氫酶(CAT)活性采用試劑盒法測定。SOD活性用黃嘌呤氧化酶法測定,其活性單位定義為:在37℃條件下,每毫克組織蛋白在1 mL反應(yīng)液中SOD抑制率達(dá)50%時(shí)所對(duì)應(yīng)的SOD量為1個(gè)SOD活力單位。CAT活性單位定義為:每克組織蛋白中過氧化氫酶(CAT)每秒鐘分解吸光度為0.50～0.55的底物中的過氧化氫相對(duì)量為1個(gè)過氧化氫酶的活力單位。

采用Statistica 6.0統(tǒng)計(jì)軟件對(duì)所得數(shù)據(jù)進(jìn)行單因素方差分析(ANOVA),若差異達(dá)到顯著,進(jìn)行Duncan多重比較,顯著性水平為P<0.05。

2 實(shí)驗(yàn)結(jié)果

3 討論

表3 不同氧化程度的魚油和維生素E對(duì)大黃魚血清超氧化物歧化酶(SOD)、過氧化氫酶(CAT)活性的影響Table 3 Superoxide dismutase(SOD)and catalase(CAT)of large yellow croaker fed diets containing different levels of oxidized fish oil and vitamin E for 10 weeks1

氧化魚油和維生素E對(duì)大黃魚血清超氧化物歧化酶(SOD)和過氧化氫酶(CAT)活性均具有顯著影響(P<0.05)。未補(bǔ)充維生素E時(shí),大黃魚血清SOD和CAT活性均隨魚油過氧化值的升高而顯著升高(P< 0.05)。補(bǔ)充維生素E后,新鮮魚油組大黃魚血清SOD和CA T的活性升高,而氧化魚油組大黃魚血清SOD和CAT活性均顯著降低(P<0.05)(見表3)。

動(dòng)物體清除體內(nèi)過多自由基的防御系統(tǒng)由非酶系統(tǒng)和抗氧化酶系統(tǒng)共同構(gòu)成。非酶系統(tǒng)主要包括維生素E、維生素C、β-胡蘿卜素、谷胱甘肽、巰基類化合物及微量元素如硒、銅、鋅等,主要功能是抗氧化損傷;抗氧化酶系統(tǒng)主要包括超氧化物歧化酶(清除超氧陰離子)、過氧化氫酶(清除過氧化氫)、谷胱甘肽過氧化物酶及谷胱甘肽-硫-轉(zhuǎn)移酶(清除脂質(zhì)氫過氧化物)及谷胱甘肽還原酶(維持體內(nèi)還原性谷胱甘肽的水平)等,其主要功能是清除體內(nèi)自由基[3]。維生素E和抗氧化酶共同組成機(jī)體抗氧化防御系統(tǒng),機(jī)體維生素E的含量可直接影響氧化油脂對(duì)抗氧化酶的作用[11,19]。本實(shí)驗(yàn)的結(jié)果顯示,氧化魚油使大黃魚血清中超氧化物岐化酶(SOD)和過氧化氫酶(CAT)活性升高,補(bǔ)充高濃度的維生素E則降低了這2種抗氧化酶的活性。這與Mourente等的研究結(jié)果相一致,他們發(fā)現(xiàn)氧化魚油使海鯛(S parus aurata)的SOD和CAT活性升高,而添加維生素E則降低了酶的活性[3]。Fontagné等也發(fā)現(xiàn)氧化魚油使西伯利亞鱘魚(Acipenser baeri)的SOD活性升高[19]。氧化魚油使抗氧化酶的活性提高的原因可能是攝食氧化了的魚油后細(xì)胞中氧自由基等生物活性物質(zhì)的濃度升高,增加了抗氧化酶反應(yīng)底物的濃度,進(jìn)而使提高SOD和CA T的活性。但任澤林等對(duì)鯉魚的研究中則發(fā)現(xiàn)氧化魚油降低了鯉魚肝胰臟中SOD與CA T活性[21]。不同的結(jié)果可能是由于養(yǎng)殖條件、飼料營養(yǎng)組成、魚油氧化程度不同以及動(dòng)物對(duì)魚油氧化耐受力的種屬差異所造成的[3,20]。補(bǔ)充維生素E使攝食含氧化魚油的魚體血清的抗氧化酶SOD和CAT活性降低,可能是由于添加維生素E降低了氧自由基等抗氧化酶反應(yīng)底物的濃度。可見,在飼料中補(bǔ)充維生素E在一定程度上可以減輕或補(bǔ)償氧化魚油所產(chǎn)生的負(fù)面影響。根據(jù)實(shí)驗(yàn)結(jié)果,在確定飼料中維生素E的適宜添加量時(shí),應(yīng)考慮飼料中脂類的氧化情況。

致謝:作者衷心感謝吳敏、王兆琦和史宣在實(shí)驗(yàn)分析和文章寫作過程中給予的充分幫助。

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Abstract: The effects of oxidized fish oil and vitamin E supplementation on antioxydant capacity of large yellow croaker(Pseudosciaena croceaR.)were investigated.Eight experimental diets,which differed in the oxidation level of the fish oil and vitamin E supplementation included:Diet 1(control,fresh fish oil (peroxide value(POV)=1.72 meq·kg-1);Diet 2,3 and 4 oxidized fish oil(POV=28.29,62.79 and 104.21 meq·kg-1,respectively).Diets 5～8 were formulated by supplementing vitamin E(600 IU· kg-1),in the form ofα-tocopheryl acetate,to diets 1～4,respectively.Experimental diets were fed to large yellow croaker(initial body weight(7.82±0.48)g)for 10 weeks.Significant difference in SOD and CAT were found among dietary treatments(P<0.05).The enzymatic activities were significantly increased with the increasing of POV(P<0.05),and decreased by the vitamin E supplementation.The results indicated that the negative effect of fish oil oxidization could be alleviated or compensated to some extent.The effect of vitamin E on croaker was influenced by the oxidization of fish oil,and this should be considered when evaluating the optimal dietary vitamin E concentration.

Key words: large yellow croaker(Pseudosciaena croceaR.);oxidized fish oil;vitamin E;SOD;CAT

責(zé)任編輯 于 衛(wèi)

Effects of Oxidized Fish Oil and Vitamin E Supplementation on Activities of SOD and CAT in Large Yellow Croaker(Pseudosciaena crocea R.)

TANG Xiao1,WAN GJun1,2,XU Hou-Guo,AI Qing-Hui,ZUO Ran-Tao1
(1.Ocean University of China,Qingdao 266003,China;2.Guangdong Institute of Microbiology,Guangzhou 510070,China)

S963

A

1672-5174(2010)09Ⅱ-055-05

國家“十一五”科技支撐計(jì)劃(2006BAD03BO3);中國海洋大學(xué)大學(xué)生科研訓(xùn)練計(jì)劃(OUC-SRDP,0912010616)資助

2010-07-10;

2010-09-01

唐 筱(1988-),女,本科生。E-mail:erica_tx@hotmail.com

E-mail:qhai@ouc.edu.cn