兩種油脂水平下DHA添加對(duì)鯉生長(zhǎng)及脂質(zhì)代謝的影響

周繼術(shù) 曹艷姿 吉 紅 于海波

(西北農(nóng)林科技大學(xué)動(dòng)物科技學(xué)院水產(chǎn)科學(xué)系, 楊凌 712100)

二十二碳六烯酸(Docosahexaenoic acid, DHA)主要來(lái)源于海水魚(yú)與藻類[1], 屬于n-3系列的高不飽和脂肪酸(Highly unsaturated fatty acid, HUFA), 具有抗癌[2—4]、抗氧化[5—7]、防治心血管疾病[8]、提高免疫力[9,10]、調(diào)節(jié)脂代謝[11—13]等功能, 對(duì)大腦及神經(jīng)系統(tǒng)發(fā)育[14,15]與視覺(jué)功能[16]密切相關(guān)。在水產(chǎn)動(dòng)物上, DHA是海水魚(yú)尤其是海水仔稚魚(yú)正常生長(zhǎng)發(fā)育的必需脂肪酸之一[17], 對(duì)黑鯛[18—20]、舌鰨仔魚(yú)[21]等海水魚(yú)脂質(zhì)代謝也具有重要的調(diào)控作用, 而溫水性魚(yú)類如草魚(yú)、鯉等具有將18:3n-3合成為DHA的能力, 一般認(rèn)為DHA并非溫水性魚(yú)類的必需脂肪酸, 然而DHA對(duì)魚(yú)類生長(zhǎng)、脂代謝、細(xì)胞膜的流動(dòng)性以及免疫功能顯示出重要的調(diào)控作用[22—27]。但是高濃度HUFA的添加會(huì)給機(jī)體帶來(lái)過(guò)高的過(guò)氧化壓力[28—30], 從而影響草魚(yú)組織脂質(zhì)含量、抑制鯉及草魚(yú)脂質(zhì)沉積[31—33], 還可能造成生長(zhǎng)減緩、飼料轉(zhuǎn)化率降低等負(fù)面影響[34]。高濃度HUFA的添加對(duì)魚(yú)類造成的負(fù)面效應(yīng)是否和飼料中其他脂肪酸或脂肪總量水平相關(guān), 尚未見(jiàn)報(bào)道。

為了進(jìn)一步了解不同油脂水平下DHA的添加對(duì)淡水魚(yú)類的營(yíng)養(yǎng)生理作用, 本試驗(yàn)以鯉(Cyprinus carpioL.)為對(duì)象, 在6%與12%兩個(gè)油脂水平下, 探討了3%添加水平下DHA對(duì)鯉生長(zhǎng)、生物學(xué)性狀、體成份、血清理化性狀及組織結(jié)構(gòu)的影響, 旨在為研究淡水魚(yú)類脂質(zhì)營(yíng)養(yǎng)及其代謝調(diào)控提供基本資料。

1 材料與方法

1.1 試驗(yàn)飼料

以酪蛋白、糊精、纖維素、大豆油以及復(fù)合魚(yú)用預(yù)混料等為飼料原料, 采用Lovell[35]試驗(yàn)飼料配方配制鯉飼料。在6%與12%兩個(gè)油脂水平下, 以DHA制品(無(wú)錫訊達(dá)化學(xué)品廠, 江蘇, 無(wú)錫)分別替代3%的大豆油, 制成基礎(chǔ)組、基礎(chǔ)-DHA組、高脂組、高脂-DHA組4種等氮精制試驗(yàn)飼料。將各原料均粉碎過(guò)60目篩, 將原料按比例混勻后壓制成直徑為2 mm長(zhǎng)3 mm顆粒, 于冰箱冷藏保存?zhèn)溆?。DHA制品的脂肪酸組成見(jiàn)表 1, 試驗(yàn)飼料配方及營(yíng)養(yǎng)成分見(jiàn)表 2。

1.2 試驗(yàn)魚(yú)

試驗(yàn)鯉購(gòu)自陜西省周至縣某漁場(chǎng)。先挑選健康且個(gè)體相近的鯉馴養(yǎng)于容積為160 L的循環(huán)水養(yǎng)殖系統(tǒng)中, 經(jīng)過(guò)7d馴養(yǎng)后, 選出體質(zhì)健康的試驗(yàn)魚(yú)360尾(14.81±0.13) g, 隨機(jī)分成4個(gè)組, 每組3個(gè)重復(fù),每個(gè)重復(fù)30尾魚(yú), 飼養(yǎng)于12個(gè)循環(huán)水養(yǎng)殖缸中。

1.3 飼養(yǎng)管理

將4組試驗(yàn)飼料分別投喂4組試驗(yàn)魚(yú), 投飼率3.5%, 每天4次(8:30、11:30、14:30和17:30), 每次持續(xù)投喂時(shí)間為1h, 每次投食后清除殘餌和糞便。水源為經(jīng)曝氣的自來(lái)水, 每天記錄水溫及試驗(yàn)魚(yú)攝食等情況。試驗(yàn)期間水溫(27±2)℃, 水流速度為20—30 L/h, DO＞6 mg/L, pH 7.5—8.0。飼養(yǎng)中期稱重一次以調(diào)整投飼量, 飼養(yǎng)試驗(yàn)為74d。在飼養(yǎng)試驗(yàn)結(jié)束后, 對(duì)所有試驗(yàn)魚(yú)稱重并測(cè)量體長(zhǎng)和取樣。

1.4 樣品采集及分析

在飼養(yǎng)結(jié)束后, 將試驗(yàn)魚(yú)用濃度為100 mg/kg MS-222麻醉后, 隨機(jī)取9尾魚(yú)采血。將所有魚(yú)稱其體重并測(cè)量其體長(zhǎng), 再將魚(yú)進(jìn)行解剖后, 分離其內(nèi)臟、肝胰臟、脾臟、腸、腹腔脂肪、單側(cè)肌肉并稱重與量取魚(yú)腸長(zhǎng)。將分離的肌肉與肝胰臟于-20℃保存?zhèn)溆谩?/p>

鯉生長(zhǎng)指標(biāo)的測(cè)定試驗(yàn)結(jié)束后, 稱取所有試驗(yàn)魚(yú)體重, 其生長(zhǎng)指標(biāo)測(cè)定如下:

存活率(%)=末尾數(shù)/始尾數(shù)×100;

絕對(duì)生長(zhǎng)率(g/d)=(末尾均重-始尾均重)/飼養(yǎng)時(shí)間(d);

紅琴平時(shí)很少叫風(fēng)影的名字，開(kāi)口閉口的總喚他和尚，林燕常常拿著風(fēng)影敲過(guò)的木魚(yú)，坐在門(mén)檻上敲。他的眼神郁郁的，在懷想出家時(shí)的生活。風(fēng)影又體味著塵世的樂(lè)趣，特別是在紅琴身上，他體驗(yàn)著那種女人特有的味道，陽(yáng)光的溫暖，雨絲的纏綿，蘭花的馨香。對(duì)于林燕，村子里的好事者有各種猜測(cè)，他們用各自的縝密的邏輯推理得出了一個(gè)又一個(gè)結(jié)論，有人說(shuō)是紅琴落洞時(shí)山神在她身上播的種，也有人說(shuō)是風(fēng)影這個(gè)花和尚早就動(dòng)了花心，和尚也是人，是男人都會(huì)想做那點(diǎn)事兒。嘴巴長(zhǎng)在別人的臉上，人家要嚼舌頭風(fēng)影有什么辦法。

相對(duì)生長(zhǎng)率(%/d)=(末尾均重-始尾均重)/始尾均重×100;

飼料系數(shù)=飼料投喂量(g)/魚(yú)體增重量(g)。

鯉生物學(xué)性狀的測(cè)定分離魚(yú)體內(nèi)臟, 稱取魚(yú)體空殼、肝胰臟、脾臟、腸、腹腔脂肪、單側(cè)肌肉等的重量并測(cè)量魚(yú)體腸長(zhǎng)。鯉生物學(xué)性狀的測(cè)定指標(biāo)如下:

肥滿度(g/cm3%)=魚(yú)體重(g)/體長(zhǎng)(cm)3×100;

空殼比率(%)=空殼重(g)/體重(g)×100;

含肉率(%)=肌肉重(g)/體重(g)×100;

表 1 試驗(yàn)油脂脂肪酸組成Tab. 1 Fatty acids composition of dietary oils (%)

表 2 半純化試驗(yàn)飼料配方及營(yíng)養(yǎng)成分組成Tab. 2 Ingredient and proximate composition of the semi-purified experimental diet

肝胰臟指數(shù)(%)=肝胰臟重(g)/體重(g)×100;

腹脂指數(shù)(%)=腹腔脂肪重(g)/體重(g)×100;

腸重比(%)=腸重(g)/體重(g)×100;

脾臟指數(shù)(%)=脾臟重(g)/體重(g)×100。

飼料與肌肉及肝胰臟常規(guī)成分的測(cè)定飼料、肝胰臟與肌肉常規(guī)成分的測(cè)定根據(jù)AOAC[37]法, 分別用索氏抽提法測(cè)定肌肉及肝胰臟的粗脂肪含量, 用半微量凱氏定氮法測(cè)定粗蛋白含量。

血清樣品制備和指標(biāo)測(cè)定飼養(yǎng)試驗(yàn)結(jié)束后每箱隨機(jī)取鯉9尾, 于其尾靜脈處采血, 3000 r/min離心10min, 制備血清。血清谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)、總蛋白(TP)、白蛋白(ALB)、球蛋白(GLO)、總膽固醇(Chol)、甘油三脂(TG)、葡萄糖(GLU)等指標(biāo)由生化試劑盒(中生北控生物科技有限公司, 北京, 中國(guó))及全自動(dòng)生化分析儀(島津ILAB600)條件下進(jìn)行測(cè)定。

肝細(xì)胞及腹腔脂肪細(xì)胞觀察在飼養(yǎng)及饑餓試驗(yàn)結(jié)束后, 每個(gè)處理隨機(jī)取3尾魚(yú)解剖, 分離腸道、肝胰臟、腹腔脂肪組織立即于波恩氏液中固定。24h后自來(lái)水洗滌, 不同濃度梯度的酒精脫水,二甲苯透明, 石蠟包埋, 連續(xù)切片, HE染色, 中性樹(shù)膠封片。光學(xué)顯微鏡下進(jìn)行組織學(xué)觀察, 并利用顯微鏡的照相系統(tǒng)進(jìn)行肝細(xì)胞直徑及腹腔脂肪細(xì)胞的長(zhǎng)軸與短軸距離的測(cè)量。將脂肪細(xì)胞長(zhǎng)軸與短軸距離相乘, 計(jì)算脂肪細(xì)胞的面積。

1.5 數(shù)據(jù)分析

所得數(shù)據(jù)采用平均數(shù)±標(biāo)準(zhǔn)差(Mean±SD)表示。用SPSS17.0軟件(Chicago, IL, USA)對(duì)試驗(yàn)數(shù)據(jù)進(jìn)行單因素方差分析(One-way ANOVA)以及Duncan’s多重比較檢驗(yàn)組間差異,P＜0.05為差異顯著。

2 結(jié)果

2.1 DHA對(duì)鯉生長(zhǎng)的影響

在6%油脂水平下, 基礎(chǔ)-DHA組末尾均重(43.7±0.8) g、絕對(duì)生長(zhǎng)率(0.40±0.01) g/d與相對(duì)生長(zhǎng)率(2.67±0.03)%/d均顯著低于基礎(chǔ)組(P＜0.05)。在12%油脂水平下, 高脂-DHA組絕對(duì)生長(zhǎng)率(0.45±0.01) g/d顯著低于高脂組(0.49±0.01) g/d, (P＜0.05),而末尾均重與相對(duì)生長(zhǎng)率在兩高脂組間均無(wú)顯著差異(P＞0.05)?；A(chǔ)-DHA組的飼料系數(shù)顯著高于其他各組(P＜0.05), 而該飼料系數(shù)在其他三組間均無(wú)顯著差異。飼料的攝食量(3972—4212 g)與鯉的存活率(93%—99%)在各組間均無(wú)顯著差異(P＞0.05)。

2.2 DHA對(duì)鯉生物學(xué)性狀的影響

由表 4可以看出: 基礎(chǔ)-DHA組魚(yú)體肥滿度最低且顯著低于高脂-DHA組(P＜0.05)。基礎(chǔ)-DHA組腹脂指數(shù)為(0.37±0.05)%, 顯著低于基礎(chǔ)組(0.52±0.04)%、高脂組(0.70±0.05)%及高脂-DHA組(0.67±0.06)%的腹脂指數(shù)(P＜0.05)。同時(shí), 12%水平下的高脂組與高脂-DHA組的腹脂指數(shù)均高于6%水平下的基礎(chǔ)組與基礎(chǔ)-DHA組(P＜0.05)。空殼比率、含肉率、比腸長(zhǎng)與脾臟指數(shù)等在各組間均無(wú)顯著差異(P＞0.05)。

2.3 DHA對(duì)鯉體成分的影響

從表 5可以看出, 在12%脂肪水平下鯉肌肉與肝胰及粗脂肪含量均分別顯著高于6%脂肪水平組。在6%與12%脂肪水平下, DHA添加組的鯉肌肉粗脂肪含量[(1.82±0.07)%、(3.03±0.09)%]均分別顯著低于無(wú)DHA組[(2.58±0.07)%、(3.51±0.11)%;P＜0.05)]。鯉肌肉粗蛋白含量在各組間均無(wú)顯著差異(P＞0.05)。

表 3 DHA對(duì)鯉生長(zhǎng)的影響Tab. 3 Effect of dietary DHA fortification on growth performance of common carp

表 4 DHA對(duì)鯉生物學(xué)性狀的影響Tab. 4 Effect of dietary DHA fortification on biological parameters (n=13)

表 5 DHA對(duì)鯉肌肉及肝胰臟粗脂肪與粗蛋白含量的影響Tab. 5 Effect of dietary DHA fortification on crude lipid and crude protein of muscle and hepstosomatese (n=3)

2.4 DHA對(duì)鯉血清生化組成的影響

從表 6可以看出, 高脂-DHA組谷丙轉(zhuǎn)氨酶為(126.9±31.2) U/L, 該值顯著高于其他各組(58.9—70.2 U/L,P＜0.05), 同時(shí)該組的總蛋白與球蛋白等均達(dá)到各組中的最高值, 分別為(33.98±1.02) g/L與(23.66±0.89) g/L。分別在6%與12%油脂水平下,DHA的添加均顯著升高了鯉血清膽固醇含量 (P＜0.05), 而DHA的添加有降低血清甘油三酯含量的趨勢(shì)。血清谷草轉(zhuǎn)氨酶、白蛋白、白球比與葡萄糖等指血清生化指標(biāo)在各組間均無(wú)顯著差異(P＞0.05)。

2.5 DHA對(duì)鯉肝胰臟與腹脂組織的細(xì)胞大小的影響

從表 7可以看出, 6%脂肪水平下, 基礎(chǔ)-DHA組腹腔脂肪細(xì)胞面積為(67.5±7.0)×103μm2, 顯著低于基礎(chǔ)組; 當(dāng)飼料脂肪水平上升至12%水平時(shí), 兩組間魚(yú)體腹腔脂肪細(xì)胞面積無(wú)顯著差異(P＜0.05)。

3 討論

3.1 DHA對(duì)鯉生長(zhǎng)的影響

在黑鯛這樣海水魚(yú)的研究中發(fā)現(xiàn), 飼料中添加1.5%DHA, 黑鯛生長(zhǎng)及生物學(xué)指標(biāo)均沒(méi)有顯著變化, 但飼料效率顯著增高[20]。DHA并非草魚(yú)、鯉等溫水性魚(yú)類的必需脂肪酸, 本研究結(jié)果顯示, 3%DHA制品的添加不但并未提高鯉的生長(zhǎng)與飼料效率, 反而對(duì)其有明顯的抑制作用(表 3), 這與Du等[30]在草魚(yú)的研究中發(fā)現(xiàn), 隨飼料中魚(yú)油添加量的增加, 草魚(yú)攝食率及表觀生長(zhǎng)率下降相一致, 也與Takeuchi等[34]關(guān)于虹鱒飼料中添加過(guò)量n-3HUFA, 導(dǎo)致魚(yú)體生長(zhǎng)下降、飼料轉(zhuǎn)化效率降低的研究結(jié)果相一致,也與鯉、草魚(yú)[31—33]的研究結(jié)果相一致。

DHA的高不飽和性使其易受氧或其他自由基的攻擊, 從而產(chǎn)生有害的過(guò)氧化產(chǎn)物, 這些過(guò)氧化產(chǎn)物的產(chǎn)生會(huì)損傷體抗氧化能力[25,37—39]。然而, 也有研究發(fā)現(xiàn), DHA能夠增強(qiáng)機(jī)體抗氧化酶活性以及促進(jìn)自由基的清除[6,8,40—42]。因此, DHA對(duì)機(jī)體抗氧化能力的影響尚不明確。本研究結(jié)果顯示, 3%DHA制品的添加不但未能提高鯉的生長(zhǎng)與飼料效率, 反而對(duì)其有明顯的抑制作用(表 3), 雖然本次試驗(yàn)沒(méi)能檢測(cè)谷胱甘肽過(guò)氧化物酶、超氧化物岐化酶等抗氧化指標(biāo), 然而結(jié)合本次試驗(yàn)結(jié)果, 筆者推測(cè),DHA添加組的鯉較差的生長(zhǎng)效果可能與DHA的添加與食入有關(guān); 易于氧化的DHA在沒(méi)有額外的抗氧化物質(zhì), 如維生素E的供給, 會(huì)給鯉機(jī)體帶來(lái)過(guò)高的過(guò)氧化壓力, 從而帶給鯉較高的氧化應(yīng)激, 進(jìn)而影響鯉的生長(zhǎng), 降低鯉的飼料轉(zhuǎn)化效率, 而這一結(jié)果在鯉的以往研究[34]及哺乳動(dòng)物[43]中均有類似發(fā)現(xiàn)。

3.2 DHA對(duì)鯉脂質(zhì)代謝的影響

在本試驗(yàn)的研究結(jié)果中發(fā)現(xiàn), DHA的添加, 魚(yú)體肥滿度、腹脂指數(shù)、腹腔脂肪細(xì)胞面積與肌肉粗脂肪含量均低于同一脂肪水平下的無(wú)DHA的添加組(表 4—7)。本研究與Om等[18,19]的研究結(jié)果:飼料中DHA降低了黑鯛腹腔脂肪細(xì)胞脂質(zhì)蓄積和細(xì)胞直徑大小相一致。

表 6 DHA對(duì)鯉血清生理生化指標(biāo)的影響Tab. 6 Effect of dietary DHA fortification on serum biochemical index (n=3)

表 7 DHA對(duì)鯉肝胰臟與腹脂組織的細(xì)胞大小的影響Tab. 7 Effect of dietary DHA fortification on cell area of hetopancreas and adipose tissues (n=3)

DHA等多不飽和脂肪酸(PUFA)是動(dòng)物生命活動(dòng)的重要能量來(lái)源, 也是細(xì)胞膜的重要組成成分,在細(xì)胞表面信號(hào)傳導(dǎo)以及細(xì)胞生化代謝過(guò)程中起著重要作用[44]。對(duì)嚙齒動(dòng)物的研究表明, n-3PUFA能降低肥胖[45]。進(jìn)一步的研究發(fā)現(xiàn), PUFA能與鼠等哺乳動(dòng)物中的肝臟過(guò)氧化物酶增殖物激活受體(PPARs)、固醇調(diào)節(jié)元件結(jié)合蛋白(SREBP)、X受體 (LXRs)等核受體特異性地結(jié)合, 使PUFA調(diào)節(jié)相關(guān)酶的轉(zhuǎn)錄基因, 從而減少脂肪合成和體脂沉積,促進(jìn)了脂肪降解[44,46,47]。研究表明, 與陸生動(dòng)物一樣, PUFA能從分子水平上激活大西洋鮭PPAR受體, 調(diào)節(jié)相關(guān)酶的轉(zhuǎn)錄基因, 抑制脂合成酶的活性,促進(jìn)脂肪降解, 最終減少體脂沉積[48—50]。因此, PUFA等長(zhǎng)鏈脂肪酸是一種調(diào)控能量代謝的物質(zhì)[51], 它通過(guò)作用于PPARs、SREBP等核受體實(shí)現(xiàn)對(duì)細(xì)胞脂質(zhì)代謝的調(diào)控[52]。本研究室對(duì)水產(chǎn)動(dòng)物尤其是對(duì)草魚(yú)的長(zhǎng)期研究也發(fā)現(xiàn), DHA等PUFA對(duì)草魚(yú)脂肪細(xì)胞發(fā)育及某些脂質(zhì)氧化代謝基因, 如三酰甘油酯酶(ATGL)、過(guò)氧化物酶體增殖受體α激活因子1(PGC-1α)、過(guò)氧化物酶體增殖物激活受體(PPARs)等有顯著影響[33,53—55], 從而促進(jìn)了脂質(zhì)的氧化。雖然本次試驗(yàn)并未檢測(cè)以上脂質(zhì)代謝基因, 但本研究結(jié)果顯示, DHA的添加降低了魚(yú)體肥滿度、腹脂指數(shù)、腹腔脂肪細(xì)胞面積與肌肉粗脂肪含量, 為此,筆者推測(cè), 這一結(jié)果可能與DHA促進(jìn)了鯉脂質(zhì)及脂肪酸的氧化代謝有關(guān), 同時(shí), 本試驗(yàn)結(jié)果與DHA的添加影響草魚(yú)組織脂質(zhì)含量、抑制鯉及草魚(yú)脂質(zhì)沉積的研究結(jié)果[31—33]相一致。

在塞內(nèi)加爾舌鰨(Solea senegalensis)的研究發(fā)現(xiàn), 提高DHA及飼料油脂水平, 將提高其生長(zhǎng)率, 反之則降低其生長(zhǎng)率[56]。本研究發(fā)現(xiàn), 隨飼料脂肪水平的提高, DHA的添加對(duì)鯉生長(zhǎng)、肥滿度、腹脂指數(shù)、腹腔脂肪細(xì)胞面積、肌肉粗脂肪含量與血清膽固醇含量等的抑制作用均有減輕的趨勢(shì)(表3—7), 顯示出脂肪水平對(duì)DHA添加所造成的損傷的補(bǔ)償作用。

在本實(shí)驗(yàn)中添加DHA使鯉血清膽固醇含量并未顯著降低(表 6), 這與飼料PUFA有降低哺乳動(dòng)物血漿膽固醇含量的研究結(jié)果作用[57,58]不一致。HUFA對(duì)魚(yú)類膽固醇代謝的影響與哺乳動(dòng)物不同的原因還有待進(jìn)一步研究。鯉血清甘油三酯含量隨DHA添加有降低趨勢(shì)(表 6), 這在鼠[59]上也有類似的研究結(jié)果。研究表明, DHA對(duì)鯉脂質(zhì)代謝具有調(diào)控作用, DHA作為鯉的非必需脂肪酸, 其利用能力及其作用機(jī)制可能與哺乳動(dòng)物不同, 其相關(guān)機(jī)制還需進(jìn)一步研究。

4 總結(jié)

3%DHA制品的添加不但并未提高鯉的生長(zhǎng)與飼料效率, 反而對(duì)其有明顯的抑制作用。與此同時(shí),DHA的添加對(duì)魚(yú)體肥滿度、腹脂指數(shù)、腹腔脂肪細(xì)胞面積與肌肉粗脂肪含量均有抑制作用, 這可能與DHA的添加量可能超過(guò)了鯉的營(yíng)養(yǎng)需求量, 從而對(duì)魚(yú)體形成了的過(guò)氧化壓力, 進(jìn)而影響了魚(yú)機(jī)的生長(zhǎng)與健康有關(guān)。隨飼料脂肪水平的提高, DHA的添加對(duì)鯉生長(zhǎng)、肥滿度、腹脂指數(shù)、腹腔脂肪細(xì)胞面積、肌肉粗脂肪含量等所形成的抑制作用均有減輕趨勢(shì), 顯示出提高飼料脂肪水平可在一定程度下補(bǔ)償DHA添加所造成的機(jī)體損傷。

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