飼料蛋氨酸對斜帶石斑魚生長性能、抗氧化及糖異生相關酶活性的影響

遲淑艷 王學武 譚北平 楊奇慧 董曉慧 劉泓宇 章 雙

(廣東海洋大學水產學院, 湛江 524088)

飼料蛋氨酸對斜帶石斑魚生長性能、抗氧化及糖異生相關酶活性的影響

遲淑艷 王學武 譚北平 楊奇慧 董曉慧 劉泓宇 章 雙

(廣東海洋大學水產學院, 湛江 524088)

實驗通過評價斜帶石斑魚幼魚生長性能、血清指標和相關酶活性的變化, 探討斜帶石斑魚獲得最大生長的飼料蛋氨酸(Met)水平與Met代謝關鍵酶活性和氧化損傷的關系。添加DL-Met使實驗飼料中Met的含量分別為0.71%、0.98%、1.26%、1.57%、1.86%和2.18%(Diet1-Diet6), 配制6組等氮等脂的飼料。選擇健康實驗魚[初重(9.75±0.05) g]隨機分為6組, 每天分別于8: 00和17: 00投喂實驗飼料, 養殖8周。結果表明, Diet3組魚體增重率和特定生長率顯著高于Diet1和Diet6組(P＜0.05); Diet4斜帶石斑魚幼魚的肥滿度顯著高于Diet1、Diet5和Diet6 (P＜0.05); Diet2和Diet3組血清總蛋白含量顯著高于Diet5組(P＜0.05), Diet3組幼魚血糖含量顯著低于Diet1組和Diet2組(P＜0.05), 血清總膽固醇含量在Diet3組逐漸降低, Diet4—6組顯著低于 Diet2組(P＜0.05); Diet3組肝臟超氧化物歧化酶(SOD)和過氧化氫酶(CAT)活性最高, 顯著高于其他各組(P＜0.05), Diet 4組肝臟磷酸烯醇式丙酮酸羧激酶(PEPCK)活性與Diet 3相比差異不顯著, 但是顯著低于其余各組(P＜0.05)。綜合以上結果, 以特定生長率為判據, 經二次曲線模型擬合可得斜帶石斑魚幼魚若獲得最大特定生長率, 其飼料中 Met的最適含量為 1.42%(占飼料蛋白 3.16%)。在該水平下, 魚體血糖、血清總膽固醇含量和PEPCK活性較低, 有利于改善魚體對能量的利用; SOD和CAT活性升高有利于改善魚體的氧化損傷。

蛋氨酸; 生長性能; 血清指標; 磷酸烯醇式丙酮酸羧激酶; 斜帶石斑魚

對哺乳動物的研究表明, 低蛋氨酸(Methionine restriction, MR)飲食可減少生物體線粒體活性氧的產生, 從而降低氧化損傷, 增加脂肪氧化進而減少脂肪沉積, 改善葡萄糖和胰島素的平衡[1—4]。蛋氨酸(Met)是魚類正常生長及其基礎代謝所必需, 海水養殖魚類飼料蛋氨酸適宜含量為0.8%—1.7%[5—9]。近期有研究表明, 蛋氨酸缺乏會導致大西洋鮭(Salmo salar)肝體比增大, 肝臟脂肪酸合成酶活性增加, 脂肪酸比例發生變化, 甘油三酯累積[10]。那么, 滿足魚類最大生長和降低生物體氧化損傷所需的蛋氨酸量是否一致呢？

本實驗通過研究斜帶石斑魚(Epinephelus coioides)生長性能、血清學指標和相關酶活性, 探討在減少魚粉用量的條件下, 飼料中不同蛋氨酸含量對斜帶石斑魚生長相關指標的影響, 在當下蛋氨酸與魚粉價格同時上漲的時期, 為飼料配方的合理配制提供參考。

1 材料與方法

1.1 實驗飼料配方及制作

以魚粉、豆粕和玉米蛋白粉為主要蛋白源, 豆油、魚油和大豆磷脂為脂肪源, 通過添加 DL-Met使實驗飼料中 Met的含量分別為 0.71%、0.98%、1.26%、1.57%、1.86%和2.18% (Diet1—Diet6)。除Met外, 添加晶體氨基酸使飼料中必需氨基酸組成與斜帶石斑魚肌肉氨基酸組成一致。隨Met含量增加, 通過改變非必需氨基酸(天冬氨酸︰甘氨酸)相應含量, 調節飼料至等氮(表1)。

表1 實驗飼料配方(%干重)Tab. 1 The formula of the experimental diets (% dry weight)

飼料原料經粉碎后過60目篩, 按配方稱重, 逐級混合均勻, 用雙螺桿擠壓機(華南理工大學科技實業總廠, F-75)加工制粒成3.0 mm的顆粒狀飼料, 晾干后于–20℃冰箱中儲存備用。

1.2 實驗用魚及飼養管理

實驗魚購自雷州石斑魚苗場, 水泥池中暫養兩周, 期間投喂商業飼料(中山統一飼料有限公司, 粗蛋白 46%), 使其逐漸適應人工配合飼料和飼養環境。養殖實驗在廣東海洋大學東海島生物研究基地室內海水魚養殖系統中進行。在實驗開始時, 隨機挑選規格一致、健康的斜帶石斑魚[初重(9.75± 0.05) g]于玻璃鋼桶(500 L)中。實驗設6個處理, 每個處理 3個重復, 每個重復 30尾魚。每天分別于8: 00和17: 00投喂直徑3.0 mm 的實驗飼料, 初始投喂量為3%體重, 并根據攝食情況適當調整。實驗期間水溫 28.5—31.5 , ℃ 鹽度 26—32, 溶解氧≥7 mg/L以上, pH 7.6—8.0, 氨氮濃度0.03 mg/L以下,實驗持續8周。

表2 實驗飼料氨基酸組成(%干重)Tab. 2 The amino acid composition of the experimental diets (% dry weight)

1.3 樣本采集及分析

養殖實驗結束, 饑餓24h后稱重, 計算增重率、蛋白質效率、特定生長率和飼料效率。每重復隨機取3尾魚測體長、體重后備測全魚粗蛋白、粗脂肪、粗灰分和水分; 另取3尾魚尾靜脈取血, 4℃保存用于檢測血液生化指標; 取血后解剖, 取肝臟和背肌迅速于液氮中保存, 后置于–80℃冷凍保存, 用于肝臟酶活力和肌肉氨基酸含量測定。

對飼料和全魚樣品進行常規養分分析[11], 水分測定采用 105℃烘干恒重法, 粗蛋白質采用凱氏定氮法(Kjeltec 8400凱氏定氮儀), 粗脂肪索式抽提法,粗灰分采用550℃馬弗爐灼燒法。將樣本(實驗原料、飼料和魚體肌肉)經由冷凍干燥處理后, 按照GB/T18246-2000進行酸水解和樣本處理, 經氨基酸自動分析儀測定氨基酸含量(國家糧食局成都糧油食品飼料質量監督檢驗測試中心, 日立L8900)。

血清葡萄糖(GLU)、總蛋白(TP)、膽固醇(CHOL)、血清甘油三酯(TG)采用全自動生化分析儀(日立7600-110型)測定。

肝臟超氧化物歧化酶(SOD)、過氧化氫酶(CAT)和勻漿液蛋白含量采用南京建成生物工程研究所試劑盒測定, 磷酸烯醇式丙酮酸羧激酶(PEPCK)采用武漢華美生物公司試劑盒測定。

1.4 計算方法

增重率(Weight gain, WG, %)=(末均重–初均重)/初均重×100;

特定生長率(Special growth rate, SGR, %/d)=(ln末均重–ln初均重)/飼養天數×100;

蛋白質效率(Protein efficiency ratio, PER, %)=(終末體重–初始體重)/(飼料攝食量×蛋白質含量)×100;

攝食率(Feed intake, FI, %) = l00×采食干飼料重(g) / [(實驗末均重+實驗初均重)/2)×天數];

成活率(Survival rate, SR, %)=末尾數/初尾數×100;

飼料系數(Feed coefficient rate, FCR)=攝食飼料干重/(末重–初重);

肥滿度(Condition factor, CF, %)=體重(g)/體長(cm)3×100。

1.5 統計分析

采用SPSS Version 17.0統計軟件對數據進行單因素方差分析(One-way ANOVA), 如有顯著性差異(P＜0.05), 則進行 Duncan’s多重比較。實驗數據用“平均數±標準差”表示。

2 結果

2.1 飼料蛋氨酸水平對斜帶石斑魚的生長性能和飼料利用效率的影響

由表3 可見, 飼料中Met水平對斜帶石斑魚成活率、蛋白質效率飼料系數和攝食率未產生顯著影響(P＞0.05)。隨著飼料Met水平的升高, 斜帶石斑魚的增重率和特定生長率均呈先升高, 后下降的趨勢, Diet3組魚體WG和SGR顯著高于Diet1和Diet6組(P＜0.05)。參考特定生長率數據, 經二次曲線模型擬合可得出方程y = –0.1445x2+0.4092x+2.03(R2=0.8451),即斜帶石斑魚幼魚若獲得最大特定生長率, 其飼料中Met的最適含量為1.42% (占飼料蛋白3.16%) (圖1)。

2.2 飼料蛋氨酸水平對斜帶石斑魚體成分和肥滿度的影響表4的結果表明, 飼料Met水平對斜帶石斑魚幼魚全魚體成分影響不顯著(P＞0.05)。Diet4斜帶石斑魚幼魚的肥滿度顯著高于 Diet1、Diet5和 Diet6 (P＜0.05)。飼料 Met含量對斜帶石斑魚幼魚肌肉必需氨基酸含量影響不顯著(P＞0.05)(表5)。

2.3 飼料蛋氨酸水平對斜帶石斑魚血清相關指標的影響

Diet3組幼魚血糖含量顯著低于 Diet1組和Diet2組(P＜0.05), 隨飼料 Met含量的升高, 當高于1.26% (Diet3)時血糖變化趨于穩定 (P＞0.05)。Diet2 和 Diet3組血清總蛋白含量顯著高于 Diet5組(P＜0.05), Diet2組總膽固醇含量顯著高于 Diet4、Diet5和Diet6組, 隨Met含量增加, 總膽固醇含量下降, Diet6組最低, 與Diet4和Diet5組相比差異不顯著(P＞0.05); 飼料 Met含量對幼魚血清甘油三酯含量未產生顯著影響(P＞0.05)。

表3 蛋氨酸對斜帶石斑魚的生長性能和飼料利用率的影響Tab. 3 The effects of dietary methionine on the growth performance and feed utilization of juvenile groupers

圖1 飼料蛋氨酸含量與斜帶石斑魚幼魚SGR的二次曲線模型Fig. 1 The relationship between dietary methionine and special growth rate of juvenile grouper

表4 蛋氨酸對斜帶石斑魚全魚常規成分(%干重)和肥滿度的影響Tab. 4 The body composition (% dry weight) and condition factor of juvenile grouper

表5 蛋氨酸對斜帶石斑魚幼魚肌肉必需氨基酸組成的影響(%干重)Tab. 5 The effects of dietary methionine on EAA composition in the muscles of juvenile groupers (% dry weight)

表6 蛋氨酸水平對斜帶石斑魚幼魚血清相關指標的影響Tab. 6 The effects of dietary methionine on the serum index of juvenile groupers

2.4 飼料蛋氨酸含量對斜帶石斑魚肝臟酶活力的影響

表7顯示Diet3組肝臟SOD 和CAT活性最高,其中 SOD活性與 Diet4組相比差異不顯著, 但是顯著高于其他各組(P＜0.05); CAT 活性則顯著高于其余各組(P＜0.05)。Diet4組肝臟PEPCK活性與Diet3相比差異不顯著, 但是顯著低于其余各組(P＜0.05)。

表7 飼料中蛋氨酸含量對斜帶石斑魚肝臟酶活性的影響Tab. 7 The effects of dietary methionine in diet on enzyme activities in the liver of juvenile groupers

3 討論

3.1 飼料蛋氨酸水平對斜帶石斑魚的生長性能和飼料利用效率的影響

本實驗以SGR為依據時, 經二次曲線模型擬合得出斜帶石斑魚幼魚(9.75 g)獲得最大 SGR所需飼料蛋氨酸量為1.42%(占飼料蛋白3.16%), 隨Met水平進一步升高, 魚體增重下降, 這與黑鯛(Sparus macrocephalus)[5]、建鯉(Cyprinus carpiovar Var. Jian)[6]、大黃魚(Pseudosciaena crocea R.)[8]和歐洲鱸(Dicentrarchus labrax)[9]等研究結果一致。然而, Luo等[7]參考全卵蛋白的氨基酸組成配制飼料, 得出斜帶石斑魚(13.25 g)獲得最大增重所需Met量為1.31% (胱氨酸含量0.26%), 并且當Met超過該水平后對魚體增重影響不顯著, 而本實驗以斜帶石斑魚肌肉氨基酸組成為模式, 并且初重低于前者, 可能導致 Met需要量上的差異。在本實驗中WG在Diet3 (1.26%)顯著升高, PER有升高趨勢但是未達顯著性差異, FCR和 FI均有下降趨勢但是同樣未達顯著性差異;王和偉的研究表明飼料中添加牛磺酸對斜帶石斑魚WG、PER和飼料效率要顯著高于未添加組, 但是隨添加水平升高各指標未出現顯著性差異[12]; 植物蛋白源替代魚粉后飼料中添加少量 Met, 可以顯著提高大菱鲆(Scophthalmus maximus L.)WG, 但是對PER和FI沒有顯著影響[13], 和本研究結果相近。研究表明斜帶石斑魚[7]、虹鱒(Salmo gairdneri)[14]和卵形鯧 鲹(Trachinotus ovatus L.)[15]肥滿度受飼料 Met水平的影響顯著, 且隨著Met水平的升高呈先升高,后趨于穩定的趨勢。本實驗飼料Met含量超過1.57% 時, 魚體肥滿度下降, 與0.71%組的狀態一致, 這種變化與其增重的變化趨勢一致。

3.2 飼料蛋氨酸水平對斜帶石斑魚血清相關指標的影響

魚類血清相關指標的變化與飼料中營養水平息息相關[16]。 許氏鲆 鲉(Sebastes schlegeli)[17]和黑鯛[5]的實驗表明, Met水平對血清甘油三酯均未產生顯著性影響, 然而, 許氏鲆 鲉分別攝食最低Met (0.58%)和最高Met (3.08%)的飼料, 血清總膽固醇含量均為最高, 隨著Met水平的升高基本呈現“W”的變化趨勢[17]; 飼料Met含量為0.75%組的黑鯛血清總膽固醇顯著高于1.09%—2.35%組, 血糖含量以最低Met (0.75%)和最高Met (2.35%)組顯著高于其余各組,獲得最大SGR其Met的最適需要量為1.71%(胱氨酸含量0.31%)[5]。Luo等[7]得出斜帶石斑魚血漿葡萄糖和總甘油三酯含量隨著飼料Met含量升高而升高, 在1.34%組升至最高而后顯著下降, 1.34%組血漿膽固醇含量顯著高于其余各組。然而, 本實驗中, 魚體獲得最大SGR時飼料Met含量在Diet3 (1.26%)和Diet4 (1.57%)之間, 血糖和總膽固醇含量也在Met 1.26%—1.57%水平間達最低, 之后趨于平穩。這是否說明這樣的飼料Met含量可以促使魚體更好地利用碳水化合物和脂類作為能量來滿足生長需要呢？MR飲食在哺乳動物的研究中雖然已展現出較多優勢, 但是其調控能量和新陳代謝的機制尚不清晰。Craig 和Moon[18]觀察到有趣的現象, 虹鱒攝食中(12%)和高(22%)碳水化合物-MR飼料6h后, 葡萄糖被快速吸收, 肉食性魚類的葡萄糖不耐受現象消失。隨后, Carig等[19]分別對虹鱒的肝細胞進行Met和無Met培養48h的實驗, 并用葡萄糖進行干預, 結果發現無Met的肝細胞對葡萄糖的吸收是增加的, 有可能是線粒體解偶聯增加, 導致細胞ATP下降, 促進膜上Na-葡萄糖轉運蛋白2(SGLT2)表達增加, 同時發現解偶聯蛋白2α(UCP2α)在轉錄水平上有所升高。

3.3 飼料蛋氨酸含量對斜帶石斑魚肝臟酶活力的影響

SOD和CAT是體內清除過氧化物的關鍵酶, 二者具有協同作用, 與生物體的健康狀況密切相關。李亮等[20]及馬春桃等[21]在對小鼠的研究中發現, 飼料中添加高于1%的Met可顯著降低血液中SOD活性。不同形式Met均能增強肉種雞機體的抗氧化功能,顯著提高肝臟、腎臟組織谷胱甘肽過氧化物酶、SOD活性和降低丙二醛含量[22]。Met可顯著提高生長中期草魚(Ctenopharyngodon idellus)肝臟SOD和CAT活性[23], 大菱鲆獲得最大SGR時, DL-Met適宜含量為1.58%, 肝臟TBARs含量先升高隨后下降, 提示Met含量升高對肝臟脂質過氧化物的生成有影響[24]。在本實驗中, 隨著Met水平的增高, SOD和CAT活力均呈現先升高后降低的趨勢, 表明適宜的Met含量可以有效提高抗氧化酶的活性, 有效清除過氧化物,改善生長。

PEPCK是糖異生途徑的關鍵酶, 它可催化草酰乙酸轉化為磷酸烯醇式丙酮酸, 繼而生成葡萄糖。當葡萄糖含量不足時PEPCK的活力會升高, 而當體內葡糖含量足夠時, 該酶活力降低[25]。飼料糖源和糖水平均能顯著影響大菱鲆PEPCK活性[26], 飼料添鲌加碳水化合物相比于無糖組能夠顯著降低翹嘴紅(Eryghroculter ilishaeformis Bleeker) PEPCK活性[27]。然而, 對河鱸(Perca fkuviatilis Linnaeus)[28]、大西洋鮭[29]和虹鱒[30]的研究表明, PEPCK的mRNA水平不受日糧中碳水化合物的影響。氨基酸對PEPCK活性的研究較少, 氨基酸單獨作用能夠誘導糖異生、糖酵解和氨基酸分解。Met可以轉化為三羧酸循環的中間產物琥珀酰輔酶A參加糖異生途徑。與不添加Met相比, 大鼠攝食含有Met的飼料, 肝臟PEPCK活性顯著降低[31]。在懷孕母豬飼糧中添加甜菜堿, 會通過提高新生仔豬肝臟Met代謝、后續DNA和組氨酸甲基化以及miRNA介導的轉錄后機制, 進而影響仔豬肝臟糖異生基因的表達。與無甜菜堿組仔豬相比,甜菜堿組仔豬肝臟PEPCK活性、PEPCK2(啟動子低甲基化)在mRNA水平和PEPCK蛋白水平的表達均顯著增加, 而PEPCK1在mRNA水平卻顯著下降(啟動子高甲基化)[32]。通過監測大西洋鮭稚魚肝臟β-同型半胱氨酸甲基轉移酶、S-腺苷高半胱氨酸水解酶和胱硫醚β-合酶的mRNA表達, 發現將高Met飼料和低Met飼料交替飼喂大西洋鮭稚魚, 可以更好地改善肝臟Met的利用效率[33]。與不加Met的處理組相比, Met顯著降低了虹鱒肝細胞葡萄糖-6-磷酸酶、葡萄糖激酶和絲氨酸脫水酶的表達, 但是對PEPCK沒有顯著影響[34], 沒有體現出Met對PEPCK的調控, 這是否與Met的濃度有關(2 mmol/L)？在本實驗中斜帶石斑魚PEPCK活性隨著飼料Met含量的升高呈先降低后升高的趨勢, 表明獲得最大SGR時, PEPCK的活性是較低的, 提示在該Met水平下, 糖異生作用不強, 當飼料Met水平低于或高于這個水平時,機體PEPCK活性上升糖異生途徑加強。

本實驗研究結果表明, 以 SGR為判據, 當飼料胱氨酸含量為0.46%, 斜帶石斑魚幼魚獲得最大SGR 時, 飼料Met的含量為1.42%(占飼料蛋白3.16%)。在該水平下, 魚體血糖、血清總膽固醇含量和PEPCK 活性較低有利于改善魚體對能量的利用, SOD和CAT活性升高有利于改善魚體的氧化損傷。

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EFFECTS OF DIETARY METHIONINE ON THE GROWTH PERFORMANCE,
ANTI-OXIDATION AND ACIVITIES OF GLUCONEOGENESIS-RELATED ENZYME IN JUVENILE GROUPERS, EPINEPHELUS COIOIDES

CHI Shu-Yan, WANG Xue-Wu, TAN Bei-Ping, YANG Qi-Hui, DONG Xiao-Hui, LIU Hong-Yu
and ZHANG Shuang
(Laboratory of Aquatic Economic Animal Nutrition and Feed, College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China)

In this study, we investigated the effects of dietary methionine (Met) on the growth performance, the serum index, the activities of key enzymes in Met metabolism, and the oxidation of juvenile groupers (Epinephelus coioides). Six iso-nitrogen and iso-lipid diets were prepared with DL-Met supplement at different concentrations, 0.71%, 0.98%, 1.26%, 1.57%, 1.86%, and 2.18% (Diet1-Diet6). Each treatment was randomly assigned to a triplicate of 30 fish [initial weight (9.75±0.05) g] per aquarium. Fish were fed at 8: 00 and 17: 00 every day and were maintained in a flow- through aquaria for eight weeks. The results showed that the weight gain and the specific growth rate of the Diet3 group were significantly higher than those of the Diet1 and Diet6 groups (P＜0.05). The condition factor of the Diet4 group was significantly higher than that of the Diet1, Diet5 and Diet6 groups (P＜0.05). The levels of serum total proteins of the Diet2 and Diet3 groups were significantly higher than that of the Diet5 group (P＜0.05). The level of blood glucose of the Diet3 group was significantly lower than that of the Diet1 and Diet2 groups (P＜0.05). The level of serum total cholesterol was gradually reduced in the Diet3 group, and it was significantly decreased in the Diet4 and Diet6 groups compared to the Diet2 group (P＜0.05). The activities of superoxide dismutase (SOD) and catalase (CAT) in the liver of the Diet3 group were the highest (P＜0.05). There was no significant difference in the activity of phosphoenolpyruvate carboxylase kinase (PEPCK) between the Diet3 and the Diet4 groups, but it was significantly lower than that in the other groups (P＜0.05). Quadratic regression analysis of the specific growth rate corresponding to the level of dietary methionine indicated that the optimal concentration of dietary methionine for the growth of juvenile groupers was 1.42% of dry diet in the presence of 0.46% cystine (corresponding to 3.16% of dietary protein on a dry weight basis). At this level, the blood glucose, the serum total cholesterol and the activity of PEPCK could be relatively low, which would help improve the energy efficiency in fish metabolism. Also the increased activities of SOD and CAT may help protect the body from oxidative damages.

Methionine; Growth performance; Serum index; PEPCK; Epinephelus coioides

S965.3

A

1000-3207(2015)04-0645-08

10.7541/2015.86

2014-11-27;

2015-03-05

國家自然科學基金(31402310); 公益性行業(農業)科研專項(201003020); 廣東省科技創新項目(2013KJCX0097); 廣東省高等學校科技創新重點項目(粵財教2011-473)資助

遲淑艷(1977—), 女, 內蒙古赤峰人; 博士; 主要從事水產動物營養與飼料學研究。E-mail: chishuyan77@163.com

譚北平(1967—), 男, 湖北巴東人; 博士; 主要從事水產動物營養與飼料學研究。E-mail: bptan@126.com