高 進(jìn),艾慶輝,麥康森

(中國海洋大學(xué)海水養(yǎng)殖教育部重點(diǎn)實(shí)驗(yàn)室;水產(chǎn)學(xué)院,山東青島266003)

微顆粒飼料中添加谷氨酰胺對大黃魚稚魚生長、存活及消化酶活力的影響

高 進(jìn),艾慶輝＊＊,麥康森

(中國海洋大學(xué)海水養(yǎng)殖教育部重點(diǎn)實(shí)驗(yàn)室;水產(chǎn)學(xué)院,山東青島266003)

以初始體質(zhì)量為(2.75±0.31)mg的大黃魚(Pseudosciaena crocea)稚魚(15日齡)為實(shí)驗(yàn)對象,在基礎(chǔ)飼料中分別添加0.00%(對照組)、0.60%、1.20%和1.80%的谷氨酰胺,并用甘氨酸調(diào)節(jié)總蛋白質(zhì)水平一致,制作而成4種粗蛋白58%左右、粗脂肪16%左右的實(shí)驗(yàn)微顆粒飼料,在室內(nèi)養(yǎng)殖系統(tǒng)中投喂大黃魚稚魚30 d,研究飼料谷氨酰胺對大黃魚稚魚生長、存活及消化酶活力的影響。實(shí)驗(yàn)結(jié)果表明,大黃魚稚魚的特定生長率和存活率隨飼料中谷氨酰胺含量的升高有上升的趨勢,但差異未達(dá)到顯著水平(P>0.05)。飼料中谷氨酰胺的添加未對大黃魚稚魚腸段、胰段胰蛋白酶活力和淀粉酶活力,腸段氨基肽酶活力和堿性磷酸酶活力產(chǎn)生顯著的影響(P>0.05)。飼料中添加谷氨酰胺未能對大黃魚稚魚的生長、存活和消化酶活力產(chǎn)生顯著的影響,可能與基礎(chǔ)飼料中魚粉、魚肉水解蛋白等蛋白源所含有的谷氨酰胺已經(jīng)達(dá)到或超過稚魚吸收、利用谷氨酰胺的閾值,額外添加的谷氨酰胺不能被吸收利用而未能表現(xiàn)出促進(jìn)效果。本實(shí)驗(yàn)條件下,大黃魚稚魚基礎(chǔ)飼料中無需額外添加谷氨酰胺即可滿足稚魚正常生長發(fā)育的需要。

大黃魚稚魚;谷氨酰胺;生長;存活;消化酶活力

谷氨酰胺(Glutamine)是動物血漿中豐富的游離氨基酸,是動物體內(nèi)蛋白質(zhì)、核苷酸和氨基糖合成的重要前體,也是快速分裂的細(xì)胞包括活化淋巴細(xì)胞和腸上皮細(xì)胞重要的能量供體[1-3]。在病理狀態(tài)下,補(bǔ)充谷氨酰胺可以促進(jìn)病變或損傷組織(如肝臟或者腸道)的修復(fù)和加速患者的恢復(fù)[4-5]。

研究發(fā)現(xiàn),在日糧中添加谷氨酰胺可以提高人、鼠、豬腸道的重量,促進(jìn)腸絨毛和腸黏膜的發(fā)育,提高腸道蛋白質(zhì)含量、DNA含量及消化酶活力[6-10]。Lin和Zhou[11]發(fā)現(xiàn)飼料中添加谷氨酰胺可以增加建鯉幼魚體增重、攝食量、腸道重量、腸絨毛高度、堿性磷酸酶活力和鈉鉀ATP酶活力。

大黃魚(Pseudosciaena crocea,Richardson)隸屬鱸形目、石首魚科、黃魚屬,是集群暖溫性近海洄游魚類,是我國重要的經(jīng)濟(jì)海水魚類。自從1980年代,大黃魚人工育苗成功后,大黃魚的人工養(yǎng)殖發(fā)展迅速,產(chǎn)生了巨大的經(jīng)濟(jì)和社會效益。稚魚對營養(yǎng)物質(zhì)消化吸收的能力較低且稚魚餌料中營養(yǎng)成分不完全,從而導(dǎo)致部分稚魚因營養(yǎng)缺乏而死亡。目前,大黃魚稚魚營養(yǎng)生理的研究已有相關(guān)報(bào)道[12-17]。本實(shí)驗(yàn)擬以大黃魚稚魚為研究對象,在微顆粒飼料中添加不同含量的谷氨酰胺以探討其對大黃魚稚魚生長、存活及消化酶活力的影響。

1 實(shí)驗(yàn)材料與方法

1.1 實(shí)驗(yàn)魚苗及養(yǎng)殖系統(tǒng)

選用初始體質(zhì)量(2.75±0.31)mg的15日齡大黃魚稚魚(購買于福建省寧德市水產(chǎn)技術(shù)推廣站育苗場)作為實(shí)驗(yàn)對象,進(jìn)行30 d的攝食生長實(shí)驗(yàn)。實(shí)驗(yàn)共設(shè)4個處理,每個處理設(shè)3個重復(fù)。實(shí)驗(yàn)系統(tǒng)由12只藍(lán)色塑料方桶(70 cm×60 cm×50 cm,有效容積200 L)組成,每桶放魚苗3 000尾。實(shí)驗(yàn)用桶均懸掛于一充滿水的水泥池中,實(shí)驗(yàn)用水為經(jīng)雙層沙濾的新鮮海水,水溫21～23℃,鹽度25～28,p H=7.8～8.2,溶氧量在6 mg/L以上,光周期為14 L∶10 D,水面光強(qiáng)在1 000 lx以下。換水量隨日齡增長遞增,每天50%～200%。每天吸底、換水2次。每天投喂8次,養(yǎng)殖過程均采用飽食投喂。

1.2 實(shí)驗(yàn)飼料

以低溫干燥白魚粉、低溫干燥蝦粉、魷魚粉、魚肉水解蛋白和酵母浸出粉為蛋白源;魚油、精制魚油及大豆卵磷脂為主要脂肪源;同時(shí)添加維生素混合物、礦物質(zhì)混合物、誘食劑等配制成基礎(chǔ)飼料。以甘氨酸為粗蛋白質(zhì)水平調(diào)節(jié)組分(甘氨酸不具有谷氨酰胺具有的功能[18]),在基礎(chǔ)飼料中添加0.00%,0.60%,1.20%及1.80%的谷氨酰胺。固態(tài)飼料原料粉碎后,過140目篩,充分混合,然后添加魚油等脂肪源充分混合,加水混合后用雙螺桿制粒機(jī)制粒,50℃烘干,然后破碎、過篩、分級而成粗蛋白58%左右、粗脂肪16%左右、含4個梯度谷氨酰胺的實(shí)驗(yàn)微顆粒飼料(見表1)。

表1 實(shí)驗(yàn)微顆粒飼料配方及營養(yǎng)組成(%干物質(zhì))Table 1 Formulation and proximate chemical composition of the experimental diets

1.3 取樣

實(shí)驗(yàn)開始時(shí),從育苗池中隨機(jī)取稚魚3組,每組300尾,用吸水紙吸干體表水分,然后稱質(zhì)量,取其平均值為初始體質(zhì)量。實(shí)驗(yàn)結(jié)束時(shí),饑餓稚魚24 h,通過點(diǎn)數(shù)確定存活尾數(shù),每桶隨機(jī)取樣30尾稱質(zhì)量。其余稚魚樣品液氮封存,然后-70℃保存,以進(jìn)行消化酶活力分析。

1.4 飼料樣品常規(guī)分析

飼料樣品均在105℃烘干至恒重后依照AOAC[19]方法進(jìn)行組成分析。采用凱氏定氮法測定樣品的總氮含量,將測定結(jié)果乘以6.25得粗蛋白含量;采用索氏抽提法,以乙醚為抽提劑測定粗脂肪含量;將樣品在電爐上炭化后,在馬福爐中灼燒(550℃)8 h后測得樣品灰分含量。

1.5 消化酶活力分析

依照Zambonino和Cahu[20]的方法,在冰上解剖大黃魚稚魚,將稚魚消化道分為腸段和胰段,取0.1 g腸段或胰段以0℃蒸餾水勻漿,3 300 g離心3 min,取上清液進(jìn)行酶活力分析。胰蛋白酶活力的分析按照Holm等[21]的方法,用Nα-Benzoyl-DL-arginine-p-nitroanilide BAPNA(Sigma B-4875)作底物。淀粉酶活力參照Métais和Bieth[22]的方法測定,用碘溶液顯示未水解的淀粉(Sigma S-9765)。腸道總堿性磷酸酶活力的分析參照Bessey等[23]的方法,用p-Nitrophenylphosphate(PNPP,Merck 106850)作底物。腸道總亮氨酸氨肽酶活力的分析參照Maroux[24]方法,用Leucine-p-nitroanilide(Sigma L-9125)作底物。蛋白質(zhì)濃度依照Bradford[25]的方法測定,酶活力用比活力(mU/mg Pro或U/mg Pro)表示。

1.6 計(jì)算公式和統(tǒng)計(jì)方法

存活率(Survival,%)=終末尾數(shù)/初始尾數(shù)×100;

特定生長率(Specific Growth Rate,%/d)=

(Ln終末體質(zhì)量-Ln初始體質(zhì)量)/養(yǎng)殖天數(shù)× 100;

實(shí)驗(yàn)所得數(shù)據(jù)用平均值±標(biāo)準(zhǔn)差(n=3)表示,實(shí)驗(yàn)數(shù)據(jù)用SPSS 15.0 for windows軟件進(jìn)行單因子方差(ANOVA)分析,當(dāng)差異顯著(P<0.05)時(shí),再進(jìn)行Tukey多重比較(Tukey HSD test)。

2 實(shí)驗(yàn)結(jié)果

2.1 谷氨酰胺對大黃魚稚魚生長和存活的影響

飼料中添加不同含量的谷氨酰胺在一定程度上促進(jìn)了大黃魚稚魚的生長,稚魚的特定生長率在每天10.40%～10.80%之間,但差異未達(dá)到顯著水平(P> 0.05)。

稚魚的存活率在26%～29%之間,隨飼料谷氨酰胺添加量的增加有上升的趨勢,但各處理間差異未達(dá)到顯著水平(P>0.05)(見表2)。

表2 飼料中添加谷氨酰胺對大黃魚稚魚生長、存活的影響①數(shù)據(jù)為3個重復(fù)的平均值±標(biāo)準(zhǔn)差Values are expressed as means± S.D.(n=3);②BW:body weight;③SGR:specific growth rate.Table 2 Specific growth rate(SGR),survival of large yellow croaker larvae fed the experimental micro-diet for 30 d

2.2 谷氨酰胺對大黃魚稚魚消化酶活力的影響

飼料中添加梯度含量的谷氨酰胺未對大黃魚稚魚腸段、胰段胰蛋白酶活力和淀粉酶活力產(chǎn)生顯著的影響(P>0.05)(見表3)。胰段胰蛋白酶活力有隨著谷氨酰胺添加量上升而升高的趨勢(58.84～65.39 mU/ mg Pro),腸段胰蛋白酶活力有隨著飼料谷氨酰胺添加量的上升有下降的趨勢(39.26～28.40 mU/mg Pro) (見表3)。

表3 飼料中添加谷氨酰胺對大黃魚稚魚腸段及胰段淀粉酶活力和胰蛋白酶活力的影響①數(shù)據(jù)為3個重復(fù)的平均值±標(biāo)準(zhǔn)差Values are expressed as means± S.D.(n=3);②BW:body weight;③SGR:specific growth rate.Table 3 Activities of trypsin and amylase in pancreatic or intestinal segments of large yellow croaker larvae fed the experimental micro-diet for 30 d

飼料中添加谷氨酰胺未對大黃魚稚魚腸道氨基肽酶和堿性磷酸酶活力產(chǎn)生顯著的差異(P>0.05)(見表4)。堿性磷酸酶活力在123.00～141.00 mU/mg Pro之間,氨基肽酶活力在17.00～23.00 mU/mg Pro之間,堿性磷酸酶和氨基肽酶活力均未表現(xiàn)出規(guī)律的變化趨勢。

表4 飼料中添加谷氨酰胺對大黃魚稚魚腸道堿性磷酸酶及氨基肽酶活力的影響①Table 4 Activities of aminopeptidase N(AN)and alkaline phosphatase(AP)of total intestinal segments of large yellow croaker larvae fed the experimental micro-diet for 30 d

3 討論

本實(shí)驗(yàn)發(fā)現(xiàn),在基礎(chǔ)飼料中添加谷氨酰胺未對大黃魚稚魚的生長、存活及消化酶活力

產(chǎn)生顯著影響,但有提高稚魚生長和存活的趨勢。楊奇慧[26]在羅非魚幼魚中得到了和本實(shí)驗(yàn)相似的研究結(jié)果,飼料中添加谷氨酰胺對雜交羅非魚的增重率、特異生長率、成活率及飼料系數(shù)均無顯著影響(P> 0.05)。然而,與本實(shí)驗(yàn)不同,以往研究發(fā)現(xiàn)飼料中添加谷氨酰胺可以促進(jìn)陸生動物腸道發(fā)育和提高消化酶活力[6,8-9,27-28]。在魚類中也觀察到了和本實(shí)驗(yàn)結(jié)果相反的實(shí)驗(yàn)結(jié)果,在飼料中添加谷氨酰胺可以促進(jìn)建鯉幼魚的生長和攝食,且魚腸道蛋白酶和脂肪酶活力隨飼料谷氨酰胺添加量上升而提高[11]。建鯉腸道消化酶活力的提高可能與谷氨酰胺提高了幼魚肝胰臟的發(fā)育有關(guān),攝食谷氨酰胺飼料組建鯉的肝指數(shù)和肝胰臟蛋白質(zhì)含量均高于對照組[11]。

本實(shí)驗(yàn)未觀察到谷氨酰胺的促生長等作用效果,可能因?yàn)楣劝滨０穼游锏淖饔眯Ч嬖趧┝肯嚓P(guān)性。體外實(shí)驗(yàn)發(fā)現(xiàn),0.5 mmol/L谷氨酰胺誘發(fā)了鼠雜交瘤細(xì)胞核糖核苷酸的合成,但是在0.5～9 mmol/L谷氨酰胺水平內(nèi)無顯著差異[29]。過氧化氫氧化壓力培養(yǎng)基中添加4 mmol/L谷氨酰胺即可為腸上皮細(xì)胞提供足夠的保護(hù),谷氨酰胺濃度的提高未產(chǎn)生更好的保護(hù)效果[30]。Lin和Zhou[11]研究發(fā)現(xiàn)飼料中添加谷氨酰胺顯著提高了建鯉幼魚體增重、攝食量、腸重量、腸絨毛高度和消化酶活力,各參數(shù)隨飼料谷氨酰胺添加量的提高有先上升然后穩(wěn)定的趨勢。較高含量的谷氨酰胺未能發(fā)揮更好的作用可能因?yàn)榧?xì)胞或者動物利用高濃度谷氨酰胺能力有限[29]。動物不能充分利用高含量的谷氨酰胺可能與谷氨酰胺運(yùn)輸能力有限和谷氨酰胺代謝酶活力低且反應(yīng)滯后有關(guān)[31-34]。本實(shí)驗(yàn)中,為滿足稚魚的營養(yǎng)需求基礎(chǔ)飼料中添加了76%的優(yōu)質(zhì)蛋白源,其中包括48%的低溫干燥魚粉和3%的魚肉水解蛋白,這些蛋白源中可能已經(jīng)含有達(dá)到或超過稚魚利用能力閾值的谷氨酰胺,所以額外添加的谷氨酰胺未被充分利用而未表現(xiàn)出作用效果。

動物對谷氨酰胺的需要還具有種屬差異性。谷氨酰胺是哺乳動物淋巴細(xì)胞培養(yǎng)基必需的,是哺乳動物白細(xì)胞進(jìn)行有絲分裂的必需物質(zhì)[35-36]。但是谷氨酰胺不是魚類細(xì)胞增殖必需的,甚至可能抑制魚類細(xì)胞的增殖[37]。Ganassin等[38]發(fā)現(xiàn),在培養(yǎng)基中添加谷氨酰胺能抑制虹鱒白血球凝集素誘發(fā)的增殖,此外研究發(fā)現(xiàn)石斑魚也不需要谷氨酰胺對有絲分裂反應(yīng)[39]。而Rosenberg-Wiser和Avtalion[40]發(fā)現(xiàn)8 mmol/L谷氨酰胺促進(jìn)了鯉白細(xì)胞植物凝集素誘發(fā)的增殖。奇努克鮭魚胚胎細(xì)胞系CHSE對谷氨酰胺表現(xiàn)出條件需要特性,在基本培養(yǎng)基MEM中谷氨酰胺促進(jìn)了胚胎細(xì)胞的增殖,而在L-15培養(yǎng)基中,谷氨酰胺抑制了細(xì)胞的生長[37]。目前,谷氨酰胺的作用效果在哺乳動物中已被廣泛的證實(shí),但是在魚類研究較少,其對魚類細(xì)胞增殖、腸道發(fā)育及魚類生長等的作用效果和作用機(jī)制仍需要深入研究。

本實(shí)驗(yàn)條件下,在飼料中添加谷氨酰胺未對大黃魚稚魚的生長、存活和消化酶活力產(chǎn)生顯著的影響。表明,本基礎(chǔ)飼料配方足以滿足大黃魚稚魚正常生長發(fā)育的營養(yǎng)需要,無需額外補(bǔ)充谷氨酰胺。

致謝:本實(shí)驗(yàn)的養(yǎng)殖和分析過程中得到了謝奉軍、程鎮(zhèn)燕和李慶飛的大力協(xié)助,在此表示感謝。

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Abstract: Four experimental micro-diets supplemented with 0.00%(control),0.60%,1.20%and 1.80%glutamine were fed to large yellow croaker larvae(15days post hatch,initial body weight 2.75± 0.31 mg)for 30 days to investigate the effects of glutamine on growth,survival and activities of selected digestive enzyme of larvae.Glycine was used to adjust the level of crude protein to be about 58%,and crude lipid were about 16%in the diets.The results showed that specific growth rate and survival of large yellow croaker larvae increased with the supplementation of glutamine,but no significant difference was observed among dietary treatments(P>0.05).There were no significant differences in activities of amylase and trypsin in intestinal or pancreatic segments,alkaline phosphatase and aminopeptidase N in intestinal segments among fish fed different diets(P>0.05).In this experiment,dietary glutamine didn’t show significant influence on growth,survival and activities of digestive enzyme of large yellow croaker larvae. This may be related to the content of glutamine contained in fish meal,hydrolysated fish meal and other protein sources in the diets,which perhaps had satisfied or exceeded the limit that the larvae could absorb and utilize.So additional glutamine supplementation could not be absorbed and utilized to perform the effects.The results showed that the basal diet could satisfy the nutrition requirement of the larvae,and no more additional glutamine was required by large yellow croaker larvae.

Key words: large yellow croaker larvae;Glutamine;growth;survival;activities of digestive enzymes

責(zé)任編輯 于 衛(wèi)

Effects of Dietary Glutamine on Growth,Survival and Activities of Selected Digested Enzymes of Large Yellow Croaker(Pseudosciaena crocea)Larvae

GAO Jin,AI Qing-Hui,MAI Kang-Sen
(The Key Laboratory of Mariculture,Ministry of Education;College of Fishery,Ocean University of China,Qingdao 266003,China)

S963.73+1

A

1672-5174(2010)09Ⅱ-049-06

國家自然科學(xué)基金項(xiàng)目(30400335)資助

2010-04-08;

2010-06-23

高 進(jìn)(1985-),男,碩士生;主要從事水產(chǎn)動物營養(yǎng)生理研究。E-mail:jingao851227@163.com

E-mail:qhai@ouc.edu.cn