氧化魚粉對肉仔雞生長性能、消化道結(jié)構(gòu)和功能的影響

廖瑞波　閆海潔　張　姝　劉國華　常文環(huán)　劉　偉　吝常華　黃向陽　蔡輝益

(中國農(nóng)業(yè)科學(xué)院飼料研究所，農(nóng)業(yè)部飼料生物技術(shù)重點(diǎn)開放實(shí)驗(yàn)室，北京100081)

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氧化魚粉對肉仔雞生長性能、消化道結(jié)構(gòu)和功能的影響

廖瑞波閆海潔*張姝劉國華常文環(huán)劉偉吝常華黃向陽蔡輝益**

(中國農(nóng)業(yè)科學(xué)院飼料研究所，農(nóng)業(yè)部飼料生物技術(shù)重點(diǎn)開放實(shí)驗(yàn)室，北京100081)

本試驗(yàn)旨在探索氧化的魚粉蛋白質(zhì)對肉仔雞生長性能、消化道結(jié)構(gòu)和功能的影響。選用健康、體重相近的1日齡肉公雞180只，隨機(jī)分為3個組，每組6個重復(fù)，每個重復(fù)10只雞。3個組分別飼喂對照飼糧(CON組)、含2%正常魚粉飼糧(FM組)和含2%氧化魚粉飼糧(OFM組)，試驗(yàn)期21 d。結(jié)果表明：1)與CON組與FM組相比，OFM組顯著降低肉仔雞體重和平均日增重(P<0.05)，顯著提高肉仔雞料重比(P<0.05)；2)與CON組與FM組相比，OFM組肉仔雞出現(xiàn)持續(xù)的腹瀉癥狀，且14和21日齡肉仔雞糞便pH顯著降低(P<0.05)，糞便中水分含量顯著提高(P<0.05)；3)對于21日齡肉仔雞回腸的絨毛高度、隱窩深度和絨毛高度/隱窩深度，3個組間無顯著差異(P>0.05)；4)肉仔雞嗉囊中谷胱甘肽、氧化型谷胱甘肽、脂質(zhì)過氧化物和丙二醛含量和谷胱甘肽/氧化型谷胱甘肽值在3個組間無顯著差異(P>0.05)，而OFM組與CON組和FM組相比顯著提高了肉仔雞回腸中脂質(zhì)過氧化物和丙二醛含量(P<0.05)。結(jié)果提示，氧化魚粉對肉仔雞回腸形態(tài)結(jié)構(gòu)沒有顯著影響，但顯著破壞了回腸的氧化還原平衡狀態(tài)，造成肉仔雞腹瀉，影響消化道的功能，顯著降低早期肉仔雞的生長性能。

肉仔雞；氧化魚粉；消化道；生長性能

魚粉在加工和儲藏期間發(fā)生的抗氧化和促氧化反應(yīng)對魚粉品質(zhì)有重要影響[1]。魚粉中的脂質(zhì)氧化造成的高溫對蛋白質(zhì)品質(zhì)有毒害作用[2]，但脂質(zhì)氧化本身對魚粉蛋白質(zhì)品質(zhì)影響方面的研究較少[3]。脂質(zhì)過氧化物(LPO)，如羥基壬烯酸、丙二醛(MDA)和丙烯醛可以與賴氨酸、組氨酸和半胱氨酸殘基反應(yīng)[4-6]，更重要的是氧化反應(yīng)是一系列鏈?zhǔn)椒磻?yīng)，反應(yīng)產(chǎn)生的自由基可能直接攻擊蛋白質(zhì)。而蛋白質(zhì)氧化定義為由自由基直接誘導(dǎo)的或由氧化反應(yīng)次級產(chǎn)物誘導(dǎo)的蛋白質(zhì)共價修飾[7]。

魚粉富含不飽和脂肪酸，容易發(fā)生氧化性酸敗，脂肪的酸敗加速了蛋白質(zhì)的變質(zhì)[8]。低脂肪魚和高脂肪魚在溫度為40～115 ℃下加熱20 min，蛋白質(zhì)中自由巰基(—SH)含量線性降低，同時二硫鍵(S—S)含量增加[9]。魚粉即便是在低溫儲藏(低于0 ℃)條件下，也出現(xiàn)巰基含量降低的情況[10]。雖然如此，巰基氧化轉(zhuǎn)化為二硫鍵需要溫度高于50 ℃，95 ℃時反應(yīng)迅速，在20 min后達(dá)到平衡狀態(tài)[9]。蛋白質(zhì)內(nèi)部的巰基轉(zhuǎn)換為二硫鍵形成交聯(lián)會產(chǎn)生不溶性的蛋白質(zhì)膠體結(jié)構(gòu)[11]，而蛋白質(zhì)的生化結(jié)構(gòu)[自由巰基含量、巰基/(巰基+二硫鍵)值]的改變會造成飼糧中蛋白質(zhì)消化率的改變[12]。Tang等[13]報(bào)道，氧化的大豆蛋白產(chǎn)生自由基，會降低大鼠消化道的抗氧化狀態(tài)，導(dǎo)致氧化還原狀態(tài)失衡。Wu等[14]報(bào)道，氧化的大豆蛋白降低肉仔雞前段消化道中多種酶的活性。故推測氧化的飼料蛋白質(zhì)可能引發(fā)肉仔雞消化道功能改變，影響生長性能。而魚粉富含不飽和脂肪酸，在加工和儲藏過程中易發(fā)生氧化性酸敗[1]，加速魚粉蛋白質(zhì)的變質(zhì)[2]。Timm-Heinrich等[10]也認(rèn)為魚粉中的油脂可能會誘導(dǎo)蛋白質(zhì)氧化，發(fā)生共價修飾[7]。因此，本試驗(yàn)旨在通過研究氧化魚粉對肉仔雞生長性能、消化道結(jié)構(gòu)和功能的影響，探究氧化魚粉對肉仔雞不利影響的機(jī)理，為生產(chǎn)實(shí)踐中緩解這種不利影響提供理論基礎(chǔ)。

1　材料與方法

1.1試驗(yàn)材料

本研究以國產(chǎn)魚粉為基礎(chǔ)試驗(yàn)材料，參照Lagrain等[15]報(bào)道的方法，按每20 g魚粉放入100 mL含有8 mL/L雙氧水的蒸餾水的比例，95 ℃加熱1 h，之后自然晾曬風(fēng)干粉碎制成。試驗(yàn)中使用的正常魚粉和氧化魚粉為相同種類魚粉。

1.2試驗(yàn)設(shè)計(jì)

選用健康體重相近[(37.47±0.77) g]1日齡愛拔益加肉公雞(北京華都肉雞公司提供)180只，采用單因素完全隨機(jī)設(shè)計(jì)，將試驗(yàn)肉仔雞分為3個組，每組6個重復(fù)，每個重復(fù)10只雞。對照(CON)組、魚粉(FM)組和氧化魚粉(OFM)組分別飼喂對照飼糧、含2%正常魚粉的飼糧和含2%氧化魚粉的飼糧。飼糧營養(yǎng)水平參照Aviagen公司品種推薦標(biāo)準(zhǔn)，飼糧組成及營養(yǎng)水平見表1。

表1　飼糧組成及營養(yǎng)水平(風(fēng)干基礎(chǔ))

1)預(yù)混料為千克飼糧提供 The premix provided the following per kg of diets：VA 12 500 IU，VD33 500 IU，VE 25 mg，VB13.5 mg，VB28.5 mg，VB65 mg，VB120.03 mg，VK32.5 mg，煙酸 nicotinic acid 30 mg，D-泛酸D-pantothenic acid 15 mg，葉酸 folic acid 1.0 mg，生物素 biotin 0.1 mg，Zn (as zinc sulfate) 110 mg，Mn (as manganese sulfate) 110 mg，F(xiàn)e (as ferrous sulfate) 80 mg，Cu (as copper sulfate) 8 mg，I (as potassium iodide) 0.35 mg，Se (as sodium selenite) 0.15 mg。

2)粗蛋白質(zhì)為測定值，其余為計(jì)算值。Crude protein was a measured value, while the others were calculated values.

1.3飼養(yǎng)管理

試驗(yàn)期為21 d，所有肉雞自由采食和飲水，采用層籠飼養(yǎng)，光照制度采用1 h(黑暗)/23 h(燈光)。人工控溫，育雛期前4 d舍內(nèi)溫度控制在33 ℃左右，以后每周降低2 ℃，直至22 ℃。7日齡免疫新支二聯(lián)苗(滴鼻點(diǎn)眼)，14日齡免疫法氏囊疫苗(飲水)。隨時觀察、記錄雞只的采食和健康狀況。

1.4樣品采集和指標(biāo)測定

1.4.1魚粉中自由巰基含量

巰基含量測定采用Ellman法，具體操作參照Beveridge等[16]、Buttkus[17]報(bào)道的方法。

1.4.2生長性能

試驗(yàn)期間每日記錄雞只健康狀況。21日齡07:00以重復(fù)為單位稱量試驗(yàn)雞的空腹重。根據(jù)試驗(yàn)記錄，以重復(fù)為單位計(jì)算體重、平均日增重(ADG)、平均日采食量(ADFI)和料重比(F/G)。

1.4.3糞便pH和水分含量

收集14和21日齡新鮮糞便，混勻，測定pH(Testo 206便攜式pH計(jì))和水分含量。

1.4.4回腸形態(tài)，嗉囊和回腸氧化狀態(tài)指標(biāo)

21日齡時每個重復(fù)按照平均體重選取1只雞，電擊處死，打開腹腔，取2段2 cm左右的回腸和3 cm2的嗉囊樣品。

一段回腸組織用生理鹽水沖洗腸道，浸入4%的甲醛溶液固定，固定后做石蠟切片。將已固定好的組織進(jìn)行修整、脫水、包埋、切片和蘇木精-伊紅(HE)染色制成切片，100×光鏡下用顯微測微尺測定絨毛高度、隱窩深度，并計(jì)算絨毛高度/隱窩深度(V/C)值。

嗉囊和另外一段回腸組織稱重、研磨制成組織勻漿液。利用紫外分光光度計(jì)(賽默飛爾科技公司，Nano Drop 2000)測定組織中總蛋白用于生化指標(biāo)分析。組織中谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量分別使用南京建成生物工程研究所A061和A061-1試劑盒測定，方法參考Tietze[18]，兩者的差值為還原型谷胱甘肽；MDA含量使用南京建成生物工程研究所A003-1試劑盒測定，方法為硫代巴比妥酸(TBA)法，參考Todorova等[19]；LPO含量采用TBA法，使用南京建成生物工程研究所A106試劑盒測定。

1.5數(shù)據(jù)統(tǒng)計(jì)與分析

所有試驗(yàn)數(shù)據(jù)使用Excel 2007進(jìn)行整理，使用SPSS 19.0統(tǒng)計(jì)軟件進(jìn)行單因素方差分析(one-way ANOVA)，組間平均值采用LSD多重比較進(jìn)行差異顯著性檢驗(yàn)。數(shù)據(jù)采用平均值±標(biāo)準(zhǔn)誤來表示，以P<0.05為差異顯著。

2　結(jié)　果

2.1魚粉中自由巰基含量

經(jīng)測定，正常魚粉中自由巰基含量為31.11 μmol/g，使用8 mL/L雙氧水加工制成的氧化魚粉中自由巰基含量為13.32 μmol/g，自由巰基含量降低了57.18%。

2.2氧化魚粉對肉仔雞生長性能的影響

氧化魚粉對肉仔雞生長性能的影響見表2。對1～21日齡肉仔雞而言，3個組間ADFI無顯著差異(P>0.05)。CON和FM 2個組間肉仔雞體重、ADG和F/G無顯著差異(P>0.05)。與CON組和FM組相比，OFM組的肉仔雞體重、ADG顯著降低(P<0.05)，而F/G顯著提高(P<0.05)。

表2　氧化魚粉對肉仔雞生長性能的影響

同列數(shù)據(jù)肩標(biāo)相同字母或無字母表示差異不顯著(P>0.05)，不同字母表示差異顯著(P<0.05)。下表同。

In the same column, values with the same or no letter superscripts mean no significant difference (P>0.05), while with different letter superscripts mean significant difference (P<0.05). The same as below.

2.3氧化魚粉對肉仔雞糞便pH和水分含量的影響

氧化魚粉對肉仔雞糞便pH和水分含量的影響見表3。與CON組和FM組相比，OFM組的糞便pH顯著降低(P<0.05)，水分含量顯著提高(P<0.05)。且飼喂含氧化魚粉的飼糧后，14和21日齡肉仔雞糞便的水分含量分別為77.88%和75.93%。

表3　氧化魚粉對肉仔雞糞便pH和水分含量的影響

2.4氧化魚粉對肉仔雞回腸形態(tài)的影響

如表4所示，對21日齡肉仔雞回腸絨毛高度，隱窩深度和V/C值而言，3個組間無顯著差異(P>0.05)。

表4　氧化魚粉對肉仔雞回腸形態(tài)的影響

2.5氧化魚粉對嗉囊、回腸氧化狀態(tài)的影響

如表5所示，與CON組和FM組相比，OFM組未顯著影響21日齡肉仔雞嗉囊中的GSH、GSSG含量和GSH/GSSG值以及LPO和MDA含量(P>0.05)。與其他2種飼糧相比，飼喂正常魚粉飼糧肉仔雞回腸中GSH含量顯著提高(P<0.05)。與CON組和FM組相比，OFM組肉仔雞回腸中LPO和MDA含量顯著提高(P<0.05)。

表5　氧化魚粉對肉仔雞嗉囊和回腸氧化狀態(tài)的影響

3　討　論

3.1魚粉中自由巰基含量

自由巰基轉(zhuǎn)換成二硫鍵是蛋白質(zhì)氧化過程中的顯著變化，可以作為檢測蛋白質(zhì)氧化的指標(biāo)。同時Ellman法是測定蛋白質(zhì)中自由巰基含量廣泛采用的方法，因此本研究采用Ellman法測定氧化前后魚粉中自由巰基含量。研究表明，即便是低溫儲藏(低于0 ℃)，魚粉也會出現(xiàn)巰基含量降低的情況[10]。低脂肪魚和高脂肪魚在溫度為40～115 ℃下加熱20 min，蛋白質(zhì)中自由巰基含量線性降低，二硫鍵含量增加[9]。自由巰基含量可以用于評判飼料或原料質(zhì)量的高低。Sunde等[20]報(bào)道高品質(zhì)的飼料蛋白質(zhì)中自由巰基含量顯著高于低品質(zhì)飼料中相應(yīng)的含量。Aslaksen等[21]報(bào)道，魚粉飼糧中二硫鍵含量在23.3～32.7 nmol/mg prot。而Timm-Heinrich等[10]發(fā)現(xiàn)初始魚粉中巰基含量在16.5～17.3 μmol/g，低溫條件下存儲12 d后巰基含量為12.9～14.9 μmol/g。而本試驗(yàn)中正常魚粉中巰基含量為31.11 μmol/g，氧化后自由巰基含量為13.32 μmol/g，與前人研究結(jié)果差別可能是由于用于加工魚粉的魚種類的差異。此外，自由巰基的氧化僅是蛋白質(zhì)中氨基酸側(cè)鏈氧化中的一種，除側(cè)鏈氧化外，蛋白質(zhì)氧化也可能造成蛋白質(zhì)骨架的氧化或蛋白質(zhì)碎片化[22]。

3.2氧化魚粉對肉仔雞生長性能的影響

蛋白質(zhì)的氧化修飾誘導(dǎo)蛋白質(zhì)一系列結(jié)構(gòu)性改變[23]，會降低蛋白的品質(zhì)和消化率[24-26]，造成其營養(yǎng)價值的損失[27]以及生物利用率的降低[28]，這些因素均是肉仔雞生長性能降低的原因。Laohabanjong等[29]發(fā)現(xiàn)與高度酸敗魚粉[硫代巴比妥酸反應(yīng)物(TBARS)=62.31 mg/kg]相比，中度酸敗魚粉(TBARS=22.52 mg/kg)顯著降低黑虎蝦最終體重，日增重和生長速率最低。該結(jié)果暗示影響生長性能重要的因素可能并非脂質(zhì)過氧化，而可能是脂質(zhì)過氧化造成的蛋白質(zhì)氧化。本研究中飼喂氧化魚粉的肉仔雞體重和ADG顯著降低，F(xiàn)/G顯著提高。Wu等[14]發(fā)現(xiàn)經(jīng)過高溫烘烤的分離大豆蛋白，其巰基含量顯著降低，而飼喂烘烤的大豆蛋白造成肉仔雞平均體重顯著降低，這與本試驗(yàn)結(jié)果相一致。

3.3氧化魚粉對肉仔雞糞便pH和水分含量的影響

醫(yī)學(xué)研究表明，糞便的外觀和一致性代表了一個重要的癥狀學(xué)線索[30]，可以用于疾病的診斷。參照Ogunji等[31]報(bào)道的糞便視覺評分，氧化魚粉組糞便疏松，有少量圓錐形糞便；該組糞便水分含量高于75%，盡管未觀察到游離水的存在，但該組肉仔雞表現(xiàn)出持續(xù)的腹瀉癥狀。而Engberg等[32]用氧化的植物油(9%的菜籽油，2%的大豆油，均為156 mEq O2/kg)飼喂肉仔雞，并未觀察到腹瀉癥狀。Yuan等[33]用氧化魚油(786.5 mEq O2/kg)飼喂斷奶仔豬，也未報(bào)道動物出現(xiàn)腹瀉癥狀。因此，氧化魚粉是誘導(dǎo)肉仔雞腹瀉的因素。而腸道完整性受損傷也會造成水分重吸收量的降低，進(jìn)一步造成腹瀉。

本研究中，OFM組肉仔雞糞便pH顯著降低。微生物的代謝產(chǎn)物(乳酸、乙酸、丙酸和丁酸)是降低腸道黏膜pH[34]和糞便pH[35]的主要原因。通常認(rèn)為中度酸性的糞便是正常的，而糞便過度酸性可能暗示消化道的某些病變[36]。斷奶仔豬的研究表明，糞便的pH與腹瀉有關(guān)聯(lián)[37]，并通常將糞便pH的降低作為滲透性腹瀉的診斷指標(biāo)[38]。家禽方面，尚無類似報(bào)道，因此無法依據(jù)糞便pH判定腹瀉類型。但糞便表觀狀態(tài)、水分含量、pH的結(jié)果表明氧化魚粉造成肉仔雞消化道的功能障礙。

3.4氧化魚粉對肉仔雞回腸形態(tài)的影響

諸多研究將飼糧成分對腸道形態(tài)(絨毛高度、隱窩深度和V/C值)的影響作為評價動物健康的一項(xiàng)指標(biāo)[39-42]，甚至有研究表明消化道局部或系統(tǒng)性炎癥反應(yīng)與小腸絨毛高度有關(guān)聯(lián)[43-44]。盡管2%的氧化魚粉顯著影響前期肉仔雞的生長性能，但回腸形態(tài)結(jié)構(gòu)并未受到氧化魚粉的影響。前人研究報(bào)道認(rèn)為，蛋白質(zhì)氧化對消化道結(jié)構(gòu)有損傷作用。如Chen等[26]報(bào)道，100 ℃加熱8 h的大豆蛋白顯著降低前段消化道和回腸的相對重量，氧化的蛋白質(zhì)可能會對肉仔雞消化道和器官的發(fā)育有負(fù)面影響；Xie等[45]報(bào)道蛋白質(zhì)氧化的終極產(chǎn)物誘導(dǎo)炎性腸病(IBD)病人的腸道組織損傷。

3.5氧化魚粉對嗉囊、回腸氧化狀態(tài)的影響

消化道指胃腸道，包括前段的消化器官和后段的腸道。嗉囊是肉雞體內(nèi)微生物活動重要的器官之一[46-47]，同時在肉雞采食3 h后，約1/2的食物會停留在嗉囊中[48]。因此除回腸外，本試驗(yàn)也觀察氧化魚粉對嗉囊的影響，研究結(jié)果顯示氧化魚粉對嗉囊中氧化狀態(tài)無顯著影響，而顯著提高前期肉仔雞回腸的中LPO和MDA的含量。

對肉仔雞而言，消化道的抗氧化能力在維持消化道正常功能方面也發(fā)揮著重要作用[49]。本研究結(jié)果表明，氧化魚粉誘導(dǎo)的消化道應(yīng)激狀態(tài)，破壞了肉仔雞消化道中氧化還原平衡。這與前人使用氧化的飼料原料得到的研究結(jié)果一致，Tang等[13]報(bào)道氧化的大豆蛋白造成大鼠消化道中氧化平衡狀態(tài)破壞，降低抗氧化能力；Yuan等[33]報(bào)道，氧化魚油誘導(dǎo)斷奶仔豬氧化應(yīng)激，造成血清和肝臟中抗氧化酶活性降低，MDA含量提高。Ferretti等[50]報(bào)道，進(jìn)入消化道上皮細(xì)胞的面筋蛋白抗原片段通過激活核轉(zhuǎn)錄因子-κB(NF-κB)，誘導(dǎo)促炎癥細(xì)胞因子和環(huán)氧化酶2(COX2)、誘導(dǎo)型一氧化氮合酶(iNOS)的轉(zhuǎn)錄，造成前列腺素和一氧化氮(NO)代謝產(chǎn)物大量產(chǎn)生，最終導(dǎo)致上皮細(xì)胞的氧化應(yīng)激。Xie等[45]也報(bào)道蛋白質(zhì)氧化的終極產(chǎn)物通過氧化介導(dǎo)的通路誘導(dǎo)人消化道上皮細(xì)胞死亡。結(jié)合回腸形態(tài)觀察的結(jié)果，2%的氧化魚粉僅改變了肉仔雞回腸中的氧化平衡狀態(tài)，并未造成回腸上皮細(xì)胞的損傷。

4　結(jié)　論

氧化魚粉對肉仔雞回腸形態(tài)結(jié)構(gòu)沒有顯著影響，但顯著破壞了回腸的氧化還原平衡狀態(tài)，造成肉仔雞腹瀉，影響消化道的功能，顯著降低早期肉仔雞的生長性能。

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*Contributed equally

**Corresponding author, professor, E-mail: caihuiyi@caas.cn

(責(zé)任編輯田艷明)

Effects of Oxidized Fish Meal on Growth Performance, Gut Structure and Functions of Broilers

LIAO RuiboYAN Haijie*ZHANG ShuLIU GuohuaCHANG WenhuanLIU WeiLIN ChanghuaHUANG XiangyangCAI Huiyi**

(Key Laboratory of Feed Biotechnology of Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China)

This experiment was conducted to study the effects of oxidized fish meal on the growth performance, gut structure and functions of broilers. A total of 180 male broilers with good health and similar weight were divided into 3 groups with 6 replicates of 10 birds each. The chicks were fed control diet (CON group), containing 2% normal fish meal diet (FM group) and 2% oxidized fish meal diet (OFM group), respectively. The experiment lasted for 21 d. The results were demonstrated that: compared with CON group and FM group, 1) OFM group significantly reduced the body weight and average daily gain (P<0.05), significantly increased the ratio of feed to gain of broilers (P<0.05). 2) OFM group resulted in constantly diarrhea of broilers, significantly decreased the fecal pH (P<0.05), and significantly increased the moisture content of faeces of broilers at 14- and 21-day-old (P<0.05). 3) The villus height, crypt depth and ratio of villus height to crypt depth of ileum of 21-day-old broilers did not significantly differ across three groups (P>0.05). 4) There were no significant differences for contents of glutathione (GSH), oxidized glutathione (GSSG), lipid peroxide (LPO), malondialdehyde (MDA) and ratio of GSH to GSSG in crop among there groups (P>0.05), however, the contents of LPO and MDA were significantly increased in the OFM group (P<0.05). In conclusion, oxidized fish meal can not only induce the imbalance of redox states in ileum, but also induce the diarrhea and influence the gut functions, and significantly decrease the growth performance of broilers in early stage, however there is no significant effects on the ileum morphology.[ChineseJournalofAnimalNutrition, 2016, 28(10)：3084-3092]

broilers; oxidized fish meal; gut; growth performance

10.3969/j.issn.1006-267x.2016.10.010

2016-04-05

國家肉雞產(chǎn)業(yè)技術(shù)體系(CARS-42)

廖瑞波(1986—)，男，河南洛陽人，博士研究生，家禽營養(yǎng)與飼料科學(xué)專業(yè)。E-mail： liao231@163.com

S831.5

A

1006-267X(2016)10-3084-09

*同等貢獻(xiàn)作者

**通信作者：蔡輝益，研究員，博士生導(dǎo)師，E-mail： caihuiyi@caas.cn