飼料中添加合成蝦青素對中華絨螯蟹成體雌蟹性腺發(fā)育、色澤和抗氧化能力的影響

麻 楠龍曉文趙 磊常國亮吳旭干,成永旭,

(1.上海海洋大學(xué)農(nóng)業(yè)部淡水水產(chǎn)種質(zhì)資源重點實驗室, 上海 201306; 2.上海海洋大學(xué), 上海市教委水產(chǎn)動物遺傳育種協(xié)同創(chuàng)新中心, 上海 201306; 3.淮陰師范學(xué)院, 江蘇省特色水產(chǎn)繁育工程實驗室, 淮安 223300)

飼料中添加合成蝦青素對中華絨螯蟹成體雌蟹性腺發(fā)育、色澤和抗氧化能力的影響

麻 楠1龍曉文1趙 磊1常國亮3吳旭干1,2成永旭1,2

(1.上海海洋大學(xué)農(nóng)業(yè)部淡水水產(chǎn)種質(zhì)資源重點實驗室, 上海 201306; 2.上海海洋大學(xué), 上海市教委水產(chǎn)動物遺傳育種協(xié)同創(chuàng)新中心, 上海 201306; 3.淮陰師范學(xué)院, 江蘇省特色水產(chǎn)繁育工程實驗室, 淮安 223300)

采用在基礎(chǔ)飼料中分別添加0、130和260 mg/kg的合成蝦青素配制成3種粗蛋白和粗脂肪含量分別為42%和16%的等氮等脂的中華絨螯蟹(Eriocheir sinensis)育肥飼料(分別記為飼料1、2和3), 以中華絨螯蟹商業(yè)育肥飼料作為飼料4, 分別投喂4組雌蟹(每個飼料組3個重復(fù)水槽, 每個水槽中12只蟹), 進行為期60d的室內(nèi)養(yǎng)殖實驗, 以探討添加合成蝦青素對中華絨螯蟹成體雌蟹性腺發(fā)育、色澤及抗氧化能力的影響。結(jié)果顯示: (1)實驗進行到30d和60d, 在飼料中添加蝦青素對雌體肝胰腺指數(shù)(HSI)和性腺指數(shù)(GSI)均無顯著影響(P>0.05)。(2)性腺、肝胰腺和頭胸甲中的總類胡蘿卜素含量和紅度值(a值)均以飼料3組最高, 性腺亮度值(L值)和黃度值(b值)以飼料1組最高(P<0.05); 飼料2組頭胸甲的b值最高, 飼料1組最低(P<0.05)。(3) 飼料1組中華絨螯蟹血清過氧化物酶(POD)活性顯著高于其他組, 飼料4組的過氧化氫酶(CAT)、谷胱甘肽還原酶(GR)、乳酸脫氫酶(LDH)、總抗氧化能力(T-AOC)、丙二醛(MDA)和乳酸(LD)含量最高(P<0.05); 肝胰腺中的SOD、T-AOC、GSH-Px和GR活性均以飼料1組最高, 飼料2組最低(P<0.05)。(4) 飼料2組血清酸性磷酸酶(ACP)活性和血藍蛋白(Hc)含量顯著高于其他組, 其余各組間差異不顯著。血清中的堿性磷酸酶(ALP)和γ-谷氨酰轉(zhuǎn)移酶(γ-GT)活性和一氧化氮(NO)含量均以飼料4組最高(P<0.05), 肝胰腺中的ACP、ALP和γ-GT活性以飼料1組最高。綜上, 在育肥飼料中添加合成蝦青素對成體雌蟹性腺發(fā)育無顯著影響, 但可顯著提高頭胸甲、肝胰腺和卵巢中的類胡蘿卜素總量、色澤和抗氧化能力, 建議雌蟹育肥飼料中合成蝦青素的含量為90 mg/kg左右。

合成蝦青素; 中華絨螯蟹; 雌體; 性腺發(fā)育; 色澤; 抗氧化能力

中華絨螯蟹(Eriocheir sinensis)(以下簡稱河蟹), 是我國重要的經(jīng)濟養(yǎng)殖蟹類之一。因其營養(yǎng)價值高和風(fēng)味獨特而深受廣大消費者的喜愛[1,2],具有較高的市場經(jīng)濟價值, 2015年全國河蟹養(yǎng)殖總產(chǎn)量高達8.23×108kg左右[3]。盡管我國河蟹養(yǎng)殖產(chǎn)量巨大, 但池塘養(yǎng)殖河蟹普遍存在煮熟后殼色、卵巢和肝胰腺顏色較淡的問題, 野生和湖泊增殖的河蟹通常殼色和卵巢顏色較紅[4,5]。色澤是影響消費者對甲殼動物感官評價的重要因素之一[4—9], 色澤較淡會直接影響到商品河蟹的市場價值[5]。因此,改善池塘養(yǎng)殖河蟹的色澤十分重要。

研究表明, 甲殼動物的體色與其體內(nèi)的類胡蘿卜素(尤其是蝦青素)含量密切相關(guān)[10—13]。甲殼動物自身無法從頭合成蝦青素等類胡蘿卜素, 但其可利用餌料中的外源類胡蘿卜素直接儲存或轉(zhuǎn)化后沉積于體內(nèi)[4,11,13—15], 因此水產(chǎn)甲殼類在很大程度上依賴于飼料中的類胡蘿卜素[14,15]。池塘養(yǎng)殖蝦蟹類顏色較淡可能與其餌料中類胡蘿卜素含量較低有關(guān)[4,11]。此外, 蝦青素等類胡蘿卜素在甲殼動物機體維生素A合成、抗脅迫和免疫應(yīng)答等多種生理過程中發(fā)揮重要作用[10,15—20]。近年來, 在蝦蟹類飼料中添加不同來源的類胡蘿卜素改善其體色、抗氧化能力和免疫力[10,11,18,21,22]等研究結(jié)果表明,蝦青素的效果優(yōu)于其他類胡蘿卜素[23—26]。蝦青素是甲殼動物體內(nèi)主要的類胡蘿卜素之一, 主要以蝦青素酯的形式存在[27—30]。在飼料中添加蝦青素對蝦類色澤、抗氧化能力和免疫力均具有一定的影響, 但不同種類和發(fā)育階段的適宜添加量存在較大差異[8,10,25,26,31]。在飼料中使用的蝦青素主要有2種來源, 合成蝦青素和雨生紅球藻粉來源的蝦青素與紅法夫酵母來源的天然蝦青素, 前者由于價格相對便宜、來源方便, 已成為飼用蝦青素的主要來源[22,30,32]。眾所周知, 肝胰腺和性腺是河蟹重要的可食部分, 其性腺發(fā)育狀況和色澤是評價其食用價值和經(jīng)濟價值的重要指標[2,4,33]。迄今為止, 尚未見在飼料中添加合成蝦青素含量對河蟹卵巢發(fā)育、色澤和抗氧化能力影響的報道。鑒于此, 探討合成蝦青素添加水平對成體雌蟹性腺發(fā)育、顏色和抗氧化能力的影響, 旨在為河蟹育肥飼料配制及品質(zhì)調(diào)控提供理論依據(jù)和實踐參考。

1 材料與方法

1.1 實驗飼料

以豆粕、菜籽粕和魚粉作為主要蛋白源, 以魚油、豬油、大豆油和菜籽油作為脂肪源配制基礎(chǔ)飼料(飼料1), 在基礎(chǔ)飼料中分別添加0.13%和0.26%的合成蝦青素(巴斯夫露康定紅, 蝦青素含量為10%), 分別記為飼料2和飼料3; 成蟹商業(yè)育肥飼料記為飼料4。在配制飼料前, 所有飼料原料均粉碎后過60目篩, 按照飼料配方將原料充分混合, 加30%蒸餾水混合后, 用膨化機制成沉性膨化飼料,粒徑4.5—5 mm, 長度10 mm左右, 所有飼料在實驗室風(fēng)干后用黑色密封袋包裝于–20℃冰箱中保存?zhèn)溆谩嶒烇暳吓浞郊盃I養(yǎng)成分見表 1。

1.2 實驗用蟹及養(yǎng)殖管理

實驗用蟹采自上海海洋大學(xué)崇明基地, 均為生殖蛻殼后的雌蟹, 體質(zhì)量(88.70±2.11) g, 殼長(54.13± 0.76) mm, 殼寬(57.52±0.51) mm, 殼高(30.08± 0.62) mm, 從中挑選144只附肢健全、體無外傷、活力較好的個體用于實驗, 雌體初始的平均性腺指數(shù)為0.34%左右。養(yǎng)殖實驗在室內(nèi)循環(huán)水養(yǎng)殖PE (Polyethylene)圓桶(直徑×高=Φ1080 mm× H1200 mm)中進行, 實驗采用漂白粉消毒曝氣后的河道水。實驗期間水深50 cm左右。每個飼料組設(shè)3個重復(fù)水槽, 每個水槽中隨機放入12只雌蟹, 在PE桶中暫養(yǎng)3—5d后開始正式實驗, 于2015年8月28日開始正式實驗。

實驗期間, 每日18:00左右投喂, 平均水溫高于20℃時, 按總體質(zhì)量的2%—3%投喂, 當(dāng)水溫為15—20℃, 則按照總體質(zhì)量的1.5%左右投喂, 根據(jù)水溫和攝食等情況靈活調(diào)整。投喂方式為均勻潑灑, 投喂后2—3h檢查殘餌情況, 并于次日上午9:00左右檢查殘餌和死亡情況, 做好相關(guān)記錄。在養(yǎng)殖過程中, 每隔2天吸1次殘餌和糞便, 每隔3天測定1次水質(zhì)指標, 根據(jù)水質(zhì)指標每2周左右適當(dāng)換水或加水, 養(yǎng)殖期間水質(zhì)指標要求為: pH 7.0—9.0, 平均溶氧>4 mg/L, 氨氮<0.5 mg/L, 亞硝酸鹽<0.15 mg/L,這些均在河蟹養(yǎng)殖的安全水質(zhì)指標范圍內(nèi), 養(yǎng)殖實驗為期60d。

1.3 樣品采集

正式實驗30d后停食1d, 從每個PE桶中隨機采樣3只雌蟹。用吸水紙擦干雌蟹體表水分并用電子天平稱重(精確度=0.01 g); 用1.0 mL無菌注射器從第三支步足基部抽取2.0 mL血淋巴樣品分裝于兩個1.5 mL離心管中, 于–40℃保存; 然后解剖取出肝胰腺和性腺, 準確稱重后分別計算肝胰腺指數(shù)(Hepatosomatic index, HSI=肝胰腺質(zhì)量/體質(zhì)量× 100%)和性腺指數(shù)(Gonadosomatic index, GSI=卵巢質(zhì)量/體質(zhì)量×100%), 肝胰腺和性腺樣品稱重后于–40℃冰箱中保存?zhèn)溆?。實?0d后停食1d, 從每個PE桶中隨機采樣3只雌蟹稱重, 解剖采集頭胸甲、性腺和肝胰腺, 并計算HSI和GSI, 樣品于–40℃冰箱中保存。

1.4 常規(guī)成分、類胡蘿卜素含量及L*a*b值測定

按照AOAC[34]的標準方法測定飼料水分(105℃烘干法)、粗蛋白(凱氏定氮法)和粗灰分(550℃灼燒法); 按照Folch等[35]的方法, 采用氯仿鯰甲醇(v/v=2鯰1)提取總脂并測定含量。參考Johnston等[36]的方法提取樣品中的類胡蘿卜素, 用分光光度計(型號: T6新世紀, 北京普析通用儀器有限責(zé)任公司)在波長470 nm處測定其吸光度值, 并根據(jù)蝦青素標準曲線(y=4.2327x–0.0108, R2=0.9996)計算樣品中總類胡蘿卜素含量。參考Tume等[8]的方法, 采用液相色譜法測定蝦青素含量, 色譜柱為Luna 3u Silica (150 mm×4.60 mm, phenomenex, USA), 流動相為正己烷和丙酮(83鯰17, v/v), 流速1.0 mL/min, 檢測波長為478 nm, 進樣量為20 μL。肝胰腺、卵巢和頭胸甲充分冷凍干燥后, 采用色差儀(型號: CR-400, 日本柯尼卡美能達公司)在D65光源下測定其表面L*a*b值[L代表亮度值, 從0(黑)到100(白); a代表紅綠軸的顏色飽和度, 其中–a為綠, +a為紅; b代表藍黃軸的顏色飽和度, 其中–b為藍, +b為黃]。由于頭胸甲有些區(qū)域不太平整, 且不同位置的顏色有一定差別, 因此選擇每只河蟹頭胸甲上相對平整的6個測量點進行測量(圖 1), 取其平均值作為該個體的測量值。

表 1 實驗飼料配方及營養(yǎng)成分(%干重)Tab.1 Formulation and proximate composition of experimental diet (% dry weight)

1.5 樣品制備、抗氧化及免疫指標的測定

血淋巴解凍后用微型勻漿器勻漿30s后, 在4℃, 12000 r/min條件下離心20min, 取出上清液(血清)待測。稱取0.2 g左右的肝胰腺, 加入1 mL (w鯰v=1鯰5)預(yù)冷的生理鹽水后用微型勻漿器(型號: T10B, 德國IKA公司生產(chǎn))勻漿30s后, 在4℃, 12000 r/min條件下離心20min, 取中間清液再次離心后, 取中間清液用于后續(xù)分析。采用南京建成生物工程研究所生產(chǎn)的試劑盒測定超氧化物歧化酶(SOD)、過氧化氫酶(CAT)、過氧化物酶(POD)、谷胱甘肽過氧化物酶(GSH-Px)、谷胱甘肽還原酶(GR)、總抗氧化能力(T-AOC)、乳酸脫氫酶(LDH)、乳酸(LD)、一氧化氮(NO)、丙二醛(MDA)和酸性磷酸酶(ACP); 根據(jù)陳彥良等[37]的方法測定血清中酚氧化酶活性(PO); 使用全自動生化分析儀(型號: BS-200, 深圳邁瑞生物醫(yī)療電子股份有限公司)及其配套試劑盒測定堿性磷酸酶(ALP)和γ-谷氨酰轉(zhuǎn)肽酶(γ-GT)。血藍蛋白(Hc)測定參考Nickerson[38]的方法, 用Tris-Ca緩沖液(50 mmol/L Tris-HCl +10 mmol/L CaCl2, pH=8.0)將血清稀釋70倍后, 在335 nm波長下比色測定OD值, 血藍蛋白含量(mg/mL)=3.717×A335×稀釋倍數(shù)。

圖 1 成體雌蟹頭胸甲L*a*b值測定位點Fig.1 Measure points of carapace L*a*b values of adult female E.sinensis

1.6 數(shù)據(jù)處理與分析

采用SPSS 16.0軟件統(tǒng)計分析實驗數(shù)據(jù), 所有數(shù)據(jù)均以平均值±標準誤表示。采用Levene法對所有數(shù)據(jù)進行方差齊性檢驗, 當(dāng)不滿足齊性方差時對百分比數(shù)據(jù)進行反正弦或平方根處理。采用ANOVA對實驗數(shù)據(jù)進行方差分析, 用Duncan法進行多重比較, 取P<0.05為差異顯著。

2 結(jié)果

2.1 飼料中蝦青素添加水平對成體雌蟹肝胰腺指數(shù)和性腺指數(shù)的影響

由圖 2可知, 經(jīng)過30d育肥后, 各組雌蟹肝胰腺指數(shù)(HSI)和性腺指數(shù)(GSI)均無顯著差異(P>0.05), HSI在10.25%—11.16%, 其中以飼料3組最高, 飼料4組最低, 各組GSI在3.64%—4.27%。經(jīng)過60d育肥后, 各飼料組HSI和GSI均無顯著差異(P>0.05), HSI在8.00%—8.85%, GSI在8.25%—8.63%, 與第30天相比, HSI略有下降, GSI明顯升高。

圖 2 飼料中蝦青素添加水平對成體雌蟹肝胰腺(a)和性腺(b)發(fā)育的影響Fig.2 Effects of dietary supplementation of astaxanthin on hepatopancreas (a) and (b) gonadal development of adult female E.sinensis

2.2 飼料蝦青素添加水平對成體雌蟹色澤指標的影響

各組雌蟹色澤指標見表 2。飼料3組性腺中的總類胡蘿卜素含量和a值顯著高于飼料1和飼料4組(P<0.05), 與飼料2組差異不顯著(P>0.05), 而L值和b值均以飼料1組最高, 飼料3組最低(P<0.05)。飼料3組肝胰腺中總類胡蘿卜素含量和a值最高, 這2個指標分別在飼料4和飼料1組最低(P<0.05), 各組肝胰腺L值和b值均無顯著差異(P>0.05)。就頭胸甲而言, 飼料3組總類胡蘿卜素含量顯著高于飼料1和飼料4組(P<0.05), 與飼料2組無顯著差異(P>0.05); 頭胸甲a值以飼料2組最高, 飼料1組最低, 而b值以飼料3組最高, 飼料1組最低(P<0.05)。

2.3 飼料中蝦青素添加水平對成體雌蟹抗氧化指標的影響

雌蟹血清中抗氧化指標見表 3。各組血清超氧化物歧化酶(SOD)和谷胱甘肽過氧化酶(GSH-Px)活性均無顯著差異(P>0.05), 飼料1組過氧化物酶(POD)活性顯著高于其他組(P<0.05), 其余3組間差異不顯著(P>0.05)。血清過氧化氫酶(CAT)、谷胱甘肽還原酶(GR)、乳酸脫氫酶(LDH)、總抗氧化能力(T-AOC)、丙二醛(MDA)和乳酸(LD)含量均以飼料4組最高(P<0.05)。由表 4可知, 肝胰腺中的SOD、T-AOC、GSH-Px和GR活性均以飼料1組最高, 大部分以飼料2組最低(P<0.05), 而各組POD、MDA和LD均無顯著差異。

2.4 飼料中蝦青素添加水平對成體雌蟹免疫指標的影響

表 2 飼料中蝦青素添加水平對成體雌蟹類胡蘿卜素含量和L*a*b值的影響Tab.2 Effects of dietary supplementation of astaxanthin on tissue carotenoid content and L*a*b value of adult female E.sinensis

表 3 飼料中蝦青素添加水平對成體雌蟹血清中抗氧化指標的影響Tab.3 Effects of dietary supplementation of astaxanthin on serum antioxidant indices of adult female E.sinensis

各組雌蟹免疫指標如表 5所示。飼料2組血清酸性磷酸酶(ACP)活性和血藍蛋白(Hc)含量顯著高于其他組(P<0.05), 其余各組間差異不顯著, 而堿性磷酸酶(ALP)、γ-谷氨酰轉(zhuǎn)移酶(γ-GT)活性和一氧化氮(NO)含量均以飼料4組最高(P<0.05); 各組血酚氧化酶(PO)活性較為接近, 在16.44—19.04 U/mgprot之間, 但均無顯著差異。就肝胰腺而言, 飼料1組ACP、ALP和γ-GT活性均顯著高于其他組(P<0.05), 而各組肝胰腺NO含量差異不顯著(P>0.05)。

3 討論

3.1 飼料中蝦青素添加水平對成體雌蟹性腺發(fā)育的影響

性腺和肝胰腺是河蟹主要的可食部分, 性腺指數(shù)(GSI)和肝胰腺指數(shù)(HSI)是評價蟹類食用價值的重要指標[2,33]。本研究結(jié)果表明, 飼料中添加不同含量的合成蝦青素對雌蟹GSI和HSI均無顯著影響,這與郭春雨[39]對河蟹一齡扣蟹的研究結(jié)果不同。出現(xiàn)這種差異的可能原因為: (1)研究對象所處的階段不同, 初始平均體質(zhì)量為27 g左右, 未完成生殖蛻殼的一齡雌體扣蟹實驗過程中需要經(jīng)過生殖蛻殼后才可能進行卵巢發(fā)育, 在飼料中添加了蝦青素可能促進了其蛻殼速度, 從而促進其性腺發(fā)育; 在本研究中實驗對象為初始平均體質(zhì)量為90 g左右, 完成生殖蛻殼的一齡成熟雌蟹, 實驗過程不需要再進行生殖蛻殼。(2)前者采用雨生紅球藻粉中提取的天然蝦青素, 主要為全反式左旋蝦青素[40—42], 可能具有較高的生物利用率; 在本研究中采用的合成蝦青素, 包含多種同分異構(gòu)體的蝦青素, 全反式左旋蝦青素僅占合成蝦青素中含量的25%左右[43,44], 今后需要進一步研究不同結(jié)構(gòu)異構(gòu)體和來源的蝦青素對河蟹不同生長發(fā)育階段的影響, 以全面評估不同蝦青素的生物學(xué)功效。研究表明, 蝦青素在水生動物性腺發(fā)育過程中主要起到抗氧化和防止光對卵巢的氧化損傷的作用, 對斑節(jié)對蝦(Penaeus monodon)、虹鱒(Oncorhynchus mykiss)、大西洋鱈(Gadus morhua)等水生動物的性腺發(fā)育和生殖量并無直接影響[45—47]。甲殼動物卵巢的發(fā)育受到激素、神經(jīng)遞質(zhì)、營養(yǎng)條件和水溫、鹽度和光照等共同影響[33], 在本實驗條件下, 在飼料中添加合成蝦青素對雌蟹性腺發(fā)育并無顯著影響。河蟹育肥過程中GSI明顯升高, HSI顯著下降, 這是卵巢快速發(fā)育過程中, 肝胰腺中的部分營養(yǎng)素轉(zhuǎn)移到卵巢中了, 從而導(dǎo)致肝胰腺指數(shù)下降[33,48,49]。

表 4 飼料中蝦青素添加水平對成體雌蟹肝胰腺中抗氧化指標的影響Tab.4 Effects of dietary supplementation of astaxanthin on hepatopancreatic antioxidant indices of adult female E.sinensis

表 5 飼料中蝦青素添加水平對成體雌蟹免疫指標的影響Tab.5 Effects of dietary supplementation of astaxanthin on immune indices of adult female E.sinensis

3.2 飼料中蝦青素添加水平對成體雌蟹色澤的影響

甲殼動物的體色與其體內(nèi)類胡蘿卜素的含量和組成密切相關(guān)[10,50,51]。盡管甲殼動物自身無法從頭合成蝦青素, 但可利用餌料中外源類胡蘿卜素轉(zhuǎn)化成蝦青素或直接將餌料中其他類胡蘿卜素和蝦青素貯存于體內(nèi)[13—15], 飼料中蝦青素對甲殼動物的色澤影響較大[11,24]。在本研究中, 添加蝦青素飼料組的雌蟹性腺、肝胰腺和頭胸甲中的類胡蘿卜素總量顯著高于對照組和商業(yè)飼料組, 且總類胡蘿卜素含量以卵巢最高, 這與飼料中補充蝦青素可顯著提高日本對蝦(Penaeus japonicus)和羅非魚(O.mossambicus × O.niloticus)體內(nèi)類蘿卜素總量的研究結(jié)果相似[52,53], 說明雌蟹可有效利用飼料中添加的合成蝦青素并沉積于體內(nèi)。此外, L*a*b值是評價水生動物色澤的重要指標, 其大小與組織中的類胡蘿卜素, 尤其是蝦青素含量有關(guān)[32,54,55]。本研究發(fā)現(xiàn)雌蟹性腺、肝胰腺和頭胸甲的紅度(a值)及頭胸甲黃度(b值)隨飼料中蝦青素含量的升高而明顯上升, 這與在銀大馬哈魚(Oncorhynchus kisutch)和帝王蟹(Paralithodes camtschaticus)上的研究結(jié)果相類似[21,32]。與之相反的是, 添加蝦青素飼料組雌蟹性腺的L值和b值顯著低于對照組和商業(yè)飼料組, 但各組頭胸甲和肝胰腺L值無顯著差異。虹鱒(Oncorhynchus mykiss)和大馬哈魚(Salmo salar L.)飼料中添加合成蝦青素(主要為反式蝦青素)的研究發(fā)現(xiàn),肌肉L值隨其類胡蘿卜素含量的升高呈線性下降趨勢[32,56]。我們推測, 可能是由于本研究雌蟹性腺中所積累的類胡蘿卜素含量隨飼料蝦青素水平的上升而升高, 且要高于其他組織, 因此L值隨類胡蘿卜素含量的升高而降低[32,56], 因此實驗說明了飼料中添加適宜的合成蝦青素可顯著提高雌蟹組織紅度,但攝食高含量蝦青素則會降低卵巢亮度。

3.3 飼料中不同蝦青素添加水平對成體雌蟹抗氧化及免疫性能的影響

甲殼動物的抗氧化防御系統(tǒng)由酶促和非酶促2個體系組成, 酶促體系包括SOD、CAT、GSHPx等抗氧化酶, 非酶促體系包括維生素、類胡蘿卜素、氨基酸和金屬蛋白等, 在2個體系的相互協(xié)同作用下, 使機體免受自由基的侵害[57—59]。在本研究中, 雌蟹血清和肝胰腺中的大部分抗氧化酶活性均以飼料1和飼料4組最高, 添加蝦青素的飼料組相對較低, 這與裴素蕊等[57]對凡納濱對蝦(Litopenaeus vannamei)的研究結(jié)果較為相似, 可能是由于雌蟹攝食蝦青素后作為非酶體系成分參與體內(nèi)的抗氧化過程, 清除體內(nèi)過多的氧自由基[60], 從而減少了抗氧化酶的反應(yīng)底物, 最終導(dǎo)致其活性的降低[57,61]。飼料添加蝦青素可以降低河蟹肝胰腺和血清中的MDA含量, 這說明蝦青素飼料組雌蟹機體所受氧化脅迫相對較低, 因此, 在飼料中添加合成蝦青素可減少雌蟹機體的氧化損傷。

ACP和ALP作為2種重要的磷酸酶, 對機體的代謝和免疫都具有重要的作用[62]。在本研究中添加蝦青素組血清中ACP活性高于對照組和商業(yè)飼料組, 與崔培等[63]對錦鯉(Cyprinus carpio haematopterus)的研究結(jié)果較為相似。ALP活性以飼料4組最高, 結(jié)合飼料4組雌蟹血清中較高的γ-GT活性說明了飼料中添加合成蝦青素降低了雌蟹的應(yīng)激水平, 因此血清免疫酶活性保持在較低的水平, 對照組和商業(yè)飼料組的雌蟹對外源蝦青素的攝取較少, 因此通過激活免疫相關(guān)酶來增強機體的免疫系統(tǒng)功能[21]。此外, 酚氧化酶(PO)被認為是甲殼動物對病原體識別和防御的重要酶類之一, 是評價甲殼動物免疫抗病性能的重要指標[64—66]。在本實驗中,各組間PO差異不顯著, 這與Supamattaya等[10]的研究結(jié)果相吻合, 可能是未引起相關(guān)的免疫應(yīng)答。血藍蛋白(Hc)主要存在于甲殼動物的血淋巴中, 發(fā)揮著氧氣運輸和免疫防御的功能[67]。在本研究中, 飼料2組血清Hc含量顯著高于其他組, 但隨著蝦青素添加水平的升高, 血淋巴中的Hc含量顯著降低, 這暗示飼料中適宜濃度的蝦青素可提高雌蟹的免疫力, 但吸收過高的飼料蝦青素可能會增加體內(nèi)維生素A的合成[45,68,69], 從而對機體產(chǎn)生毒性作用。類似的現(xiàn)象已在鼠、兔和人類中有所發(fā)現(xiàn)[70—73]。

綜上所述, 在飼料中添加合成蝦青素對中華絨螯蟹成體雌蟹性腺發(fā)育無顯著影響, 但可提高性腺、肝胰腺和頭胸甲中的類胡蘿卜素總量, 改善色澤, 同時提高機體抗氧化能力及免疫性能。綜合考慮成本、著色效果、抗氧化能力和免疫力等因素,建議河蟹雌蟹育肥飼料中合成蝦青素含量為90 mg/ kg左右。

致謝:

上海海洋大學(xué)甲殼動物營養(yǎng)繁殖研究室吳仁福同學(xué)協(xié)助進行類胡蘿卜素測定, 劉澤華同學(xué)參與部分采樣工作, 中國科學(xué)院海洋研究所蘇芳同學(xué)幫助進行蝦青素測定, 在此表示感謝。

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EFFECTS OF DIETARY SUPPLEMENTATION OF SYNTHETIC ASTAXANTHIN ON OVARIAN DEVELOPMENT, COLORATION AND ANTIOXIDANT CAPACITY OF ADULT FEMALE CHINESE MITTEN CRAB, ERIOCHEIR SINENSIS

MA Nan1, LONG Xiao-Wen1, ZHAO Lei1, CHANG Guo-Liang3, WU Xu-Gan1,2and CHENG Yong-Xu1,2
(1.Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai
201306, China; 2.Collaborative Innovation Center of Aquatic Animal Breeding Center Certificated by Shanghai Municipal Education Commission, Shanghai Ocean University, Shanghai 201306, China; 3.Jiangsu Engineering Laboratory for Characteristic Aquatic Species Breeding, Huaiyin Normal University, Huai’an 223300, China)

To investigate the effects of dietary supplementation of synthetic astaxanthin on ovarian development, coloration and antioxidant capacity of adult female Chinese mitten crab (Eriocheir sinensis), three isonitrogenous and isolipidic fattening diets with 42% crude protein and 16% crude lipid were formulated by adding 0, 130 and 260 mg/kg synthetic astaxanthin (named as Diet 1, Diet 2, and Diet 3, respectively), and commercial fattening diet was used (named as Diet 4).Each diet was fed to triplicate tanks with 12 female crabs per tank for 60-day fattening.The results showed that dietary supplementation of synthetic astaxanthin had no significant effect on the hepatosomatic index (HSI) and gonadosomatic index (GSI) of adult female E.sinensis (P>0.05).Diet 3 treatment had the highest contents of carotenoid and redness value (a), and Diet 1 group had the highest lightness value (L) and yellow value (b) in the ovaries.The highest and the lowest b value of carapace were found in Diet 2 and Diet 1 treatments, respectively (P<0.05).Diet 1 group had the highest activity of peroxidase (POD) in the serum.The activities of catalase (CAT), glutathione reductase (GR), lactic dehydrogenase (LDH), total antioxidant capacity (T-AOC) and the contents of malondialdehyde (MDA) and lactic acid (LD) in serum from Diet 4 group were significantly higher than those of other groups (P<0.05).The highest and lowest activities of superoxide dismutase (SOD), T-AOC, glutathione peroxidase (GSH-Px) and GR in hepatopancreas were found in Diet 1 treatment and Diet 2 treatment, respectively (P<0.05).The activity of acid phosphatase (ACP) and hemocyanin (Hc) content in the serum from Diet 2 treatment were signifi-cantly higher than those of other treatments (P<0.05).The highest activities of alkaline phosphatase (ALP) and γ-glutamyl transpeptidase (γ-GT), and nitric oxide (NO) content were found in Diet 4 treatment.The highest activities of ACP, ALP and γ-GT in the hepatopancreas were shown in Diet 1 group.In conclusion, dietary supplementation of synthetic astaxanthin did not significantly affect the ovarian development of adult female crabs, but could enhance content of carotenoid, coloration and antioxidant capacity.These results suggest that the appropriate content of synthetic astaxanthin in the fattening diet is 90 mg/kg for female E.sinensis.

Synthetic astaxanthin; Eriocheir sinensis; Female; Ovarian development; Coloration; Antioxidant capacity

S968.25

A

1000-3207(2017)04-0755-11

10.7541/2017.94

2016-08-18;

2017-02-17

科技部港澳臺科技合作專項項目(2014DFT30270); 上海市科學(xué)技術(shù)委員會科研項(13320502100, 16DZ2281200); 上海高校水產(chǎn)學(xué)一流學(xué)科建設(shè)項目(2012-62-0908); 深圳澳華農(nóng)牧科技有限公司委托項目(D-8006-15-0054); 江蘇省科技廳政策引導(dǎo)類產(chǎn)學(xué)研合作項目(BY2016062)資助 [Supported by the Hongkong, Macao and Taiwan Science and Technology Cooperation Projects (2014DFT30270); the Projects (13320502100 and 16DZ2281200) from Shanghai Municipal Science and Technology Commission; the Shanghai Universities First-class Disciplines Project of Fisheries (No.2012-62-0908) from Shanghai Municipal Education Committee; Technology Commission and the R&D Project (D-8006-15-0054) from Shenzhen Alpha Feed Co.Ltd and Jiangsu Department of Science and Technology Policy Guidance Research Cooperation Project (BY2016062)]

麻楠(1991—), 女, 山東濱州人; 在讀研究生; 研究方向為水產(chǎn)動物營養(yǎng)與生理。E-mail: 1058974664@qq.com

吳旭干, E-mail: xgwu@shou.edu.cn