鄭琛，李發(fā)弟，李飛，周巨旺，段鵬偉，劉繪匯，樊海苗，朱威力，劉婷

代乳粉添加單寧酸對(duì)7—28日齡湖羊羔羊胃腸道發(fā)育的影響

鄭琛1，李發(fā)弟2,3，李飛2，周巨旺1，段鵬偉1，劉繪匯1，樊海苗1，朱威力1，劉婷1

（1甘肅農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院，蘭州 730070；2蘭州大學(xué)草地農(nóng)業(yè)生態(tài)系統(tǒng)國(guó)家重點(diǎn)實(shí)驗(yàn)室/蘭州大學(xué)農(nóng)業(yè)農(nóng)村部草牧業(yè)創(chuàng)新重點(diǎn)實(shí)驗(yàn)室/ 蘭州大學(xué)草地農(nóng)業(yè)科技學(xué)院，蘭州 730020；3甘肅省肉羊繁育生物技術(shù)工程實(shí)驗(yàn)室，甘肅民勤 733300）

【】觀察代乳粉中添加單寧酸對(duì)7—28日齡湖羊羔羊胃腸道生長(zhǎng)發(fā)育的影響。選擇同質(zhì)性好的7日齡湖羊公羔（雙羔）30只，隨機(jī)分為2組，每組15只，每只1個(gè)重復(fù)，分別飼喂對(duì)照代乳粉或單寧酸含量為0.2%的代乳粉，試驗(yàn)期21 d。羔羊28日齡時(shí)飼養(yǎng)試驗(yàn)結(jié)束，每組隨機(jī)選擇8只羔羊進(jìn)行屠宰，分離胃室和腸段，分別稱量各胃室和各腸段凈質(zhì)量以及含內(nèi)容物質(zhì)量，并量取各腸段的長(zhǎng)度，計(jì)算各胃室和各腸段的相對(duì)質(zhì)量和相對(duì)長(zhǎng)度及內(nèi)容物分布。腸道組織形態(tài)學(xué)和上皮細(xì)胞凋亡率用皺胃胃底腺區(qū)及十二指腸、空腸和回腸中段的組織樣品測(cè)定。腸道屏障功能相關(guān)的閉鎖蛋白（occludin）、閉鎖小帶1（zonula occludens-1，ZO-1）和緊密連接蛋白1（claudin 1）mRNA表達(dá)量用采集的十二指腸、空腸和回腸黏膜測(cè)定。除十二指腸指數(shù)（%活體質(zhì)量，=0.012）和相對(duì)質(zhì)量（%全腸質(zhì)量，=0.034；%全胃腸質(zhì)量，=0.017）、空腸和結(jié)腸相對(duì)長(zhǎng)度（%全腸長(zhǎng)度，=0.030，=0.004）及結(jié)腸內(nèi)容物分布（%活體質(zhì)量，=0.039）外，單寧酸對(duì)羔羊胃腸指數(shù)（%活體質(zhì)量）、胃腸相對(duì)質(zhì)量（%全胃質(zhì)量、%全腸質(zhì)量和%全胃腸質(zhì)量）、腸道相對(duì)長(zhǎng)度（%全腸長(zhǎng)度）、內(nèi)容物相對(duì)質(zhì)量（%活體質(zhì)量）、胃腸內(nèi)容物相對(duì)總胃/腸內(nèi)容物及總胃腸內(nèi)容物相對(duì)質(zhì)量（%總胃內(nèi)容物、總腸內(nèi)容物、總胃腸內(nèi)容物）以及小腸claudin 1蛋白mRNA表達(dá)量均沒(méi)有產(chǎn)生顯著影響（＞0.05），但顯著提高羔羊十二指腸肌層厚度并降低絨毛寬度（=0.013，=0.001），顯著上調(diào)空腸ZO-1蛋白mRNA表達(dá)量（=0.003），此外，單寧酸有上調(diào)羔羊空腸occludin蛋白mRNA表達(dá)量并降低空腸絨毛寬度和隱窩深度以及空腸和回腸上皮細(xì)胞凋亡率的趨勢(shì)（=0.077，=0.073，=0.062，=0.097，=0.052）。單寧酸顯著降低7—28日齡湖羊羔羊十二指腸相對(duì)質(zhì)量和空腸相對(duì)長(zhǎng)度，但可通過(guò)提高十二指腸肌層厚度、上調(diào)空腸ZO-1蛋白和occludin蛋白mRNA表達(dá)量并降低空腸隱窩深度及空腸和回腸上皮細(xì)胞凋亡率來(lái)改善腸屏障功能。

單寧酸；羔羊；代乳粉；胃腸道；發(fā)育

0 引言

【研究意義】胃腸道是哺乳動(dòng)物最重要的消化器官，承擔(dān)著為機(jī)體供給養(yǎng)分的重任，同時(shí)也是機(jī)體最大的免疫器官，起到保障機(jī)體健康的作用[1]。幼齡哺乳動(dòng)物出生前，胃腸道的發(fā)育與內(nèi)部信號(hào)分子有關(guān)[2]，出生后則取決于胃腸道內(nèi)養(yǎng)分及各種生物活性因子[3]。因此，研究幼齡動(dòng)物胃腸道的發(fā)育規(guī)律及調(diào)控效果，對(duì)提高動(dòng)物養(yǎng)分消化利用率有重要意義?！厩叭搜芯窟M(jìn)展】單寧酸是一種植物次級(jí)代謝物，廣泛存在于豆科植物和高粱、五倍子、石榴等[4]，可溶于水和乙醇，化學(xué)結(jié)構(gòu)較為復(fù)雜[5]，而其復(fù)雜的多元酚羥基結(jié)構(gòu)，決定了單寧酸具有抗瀉、抗菌、抗氧化和抗病毒等生物學(xué)功能[6]。目前，歐盟已批準(zhǔn)單寧酸可作為飼料添加劑，在豬、肉雞和奶牛的養(yǎng)殖中已開(kāi)始使用[7]。REZAR等[8]發(fā)現(xiàn)肉仔雞飼糧中添加0.2%栗木單寧酸能顯著增加糞干物質(zhì)含量，減少腹瀉的發(fā)生。隋慧等[9]和孫展英等[10]也均發(fā)現(xiàn)，飼糧中添加0.05 %和0.1%單寧酸可降低仔豬腹瀉率。GIRARD等[11]也指出，在產(chǎn)腸毒素大腸桿菌誘發(fā)腹瀉的斷奶仔豬模型中，添加1%栗木單寧提取物（54%水解單寧）可顯著降低腹瀉率。此外，單寧酸也在抑制產(chǎn)氣莢膜梭菌[12]、大腸桿菌[13]和金黃色葡萄球菌[14]等病原菌、提高總抗氧化活力和抑制丙二醛[15-16]、抑制甲型流感病毒受體結(jié)合[17]和丙型肝炎病毒進(jìn)入細(xì)胞[18]、以及通過(guò)損傷寄生蟲(chóng)表皮蛋白而抑制寄生蟲(chóng)[19]等方面發(fā)揮著重要作用?！颈狙芯壳腥朦c(diǎn)】通常情況下，腸黏膜的屏障作用和養(yǎng)分吸收作用與上皮細(xì)胞的抗氧化活性密切相關(guān)，因此，很多學(xué)者認(rèn)為具有抗氧化活性的植物提取物或次級(jí)代謝物，如單寧酸等，可通過(guò)改善腸黏膜上皮細(xì)胞組織形態(tài)而提高養(yǎng)分消化利用率[20]。JAMROZ等[21]報(bào)道，飼糧添加0.025%和0.05% 甜栗水解單寧對(duì)肉仔雞腸道無(wú)顯著影響，而添加量為0.1%時(shí)對(duì)空腸上皮細(xì)胞有負(fù)面作用。但LIU等[22]報(bào)道，0.2%栗木單寧可提高熱應(yīng)激肉雞空腸黏膜絨毛高度，ZHAO等[23]指出，飼糧中添加0.1%單寧可提高湖羊空腸和回腸黏膜絨毛高度和絨毛高度/隱窩深度值（V/C），劉洋等[24]也指出，0.05%栗木水解單寧可修復(fù)肉雞腸道損傷。鑒于此，本研究提出假設(shè)，適宜劑量的單寧酸可通過(guò)改善羔羊胃腸道內(nèi)環(huán)境而影響胃腸道生長(zhǎng)發(fā)育?！緮M解決的關(guān)鍵問(wèn)題】因此，本試驗(yàn)選擇7日齡湖羊公雙羔作為試驗(yàn)對(duì)象，研究代乳粉中添加單寧酸對(duì)胃腸道生長(zhǎng)發(fā)育的影響，為羔羊生產(chǎn)提供基礎(chǔ)數(shù)據(jù)。

1 材料與方法

1.1 試驗(yàn)設(shè)計(jì)及動(dòng)物

試驗(yàn)選取同質(zhì)性好的7日齡湖羊公羔（雙羔）30只（甘肅金昌中天羊業(yè)有限公司），采用對(duì)照試驗(yàn)設(shè)計(jì)，羔羊隨機(jī)分入2個(gè)處理組（對(duì)照組和0.2%單寧酸組），每組15只，每只1個(gè)重復(fù)，保證羔羊7日齡體重組間無(wú)差異（對(duì)照組（4.09 ± 0.66）kg，單寧酸組（3.89 ± 0.62）kg）。試驗(yàn)期21 d，羔羊28日齡結(jié)束試驗(yàn)，并從每組隨機(jī)選擇8只羔羊進(jìn)行屠宰。

1.2 羔羊飼養(yǎng)管理

羔羊在1—3日齡時(shí)吸吮初乳，4日齡時(shí)與母羊分離，開(kāi)始用奶瓶訓(xùn)飼代乳粉，7日齡晨飼前空腹稱重并分組。8日齡開(kāi)始，對(duì)照組羔羊飼喂羔羊?qū)Ｓ么榉郏ū本┚珳?zhǔn)動(dòng)物營(yíng)養(yǎng)研究中心，養(yǎng)分濃度見(jiàn)表1），單寧酸組羔羊則飼喂在對(duì)照組代乳粉基礎(chǔ)上添加0.2%單寧酸（MACKLIN?，上海麥克林生化科技有限公司，AR，純度98%）的代乳粉。飼喂時(shí)，用溫水溶解代乳粉，比例為5﹕1，每天分別在6：00、12：00、18：00和24：00飼喂4次，喂量為羔羊體重的2 %。羔羊單籠飼養(yǎng)，自由飲水。

1.3 屠宰和指標(biāo)測(cè)定

屠宰前羔羊不禁水禁食。羔羊28日齡時(shí)，先稱量宰前活重，然后切斷頸靜脈放血致死。迅速打開(kāi)羔羊腹腔，按馬仲華[25]的方法分出4個(gè)胃室和6段腸段，結(jié)扎各胃室和腸段的連接處并分離。稱量各胃室和各腸段含內(nèi)容物的質(zhì)量后，排空內(nèi)容物并稱量各胃室和各腸段的凈質(zhì)量，計(jì)算各胃室和各腸段的相對(duì)質(zhì)量及其內(nèi)容物分布情況。同時(shí)量取各腸段的長(zhǎng)度以計(jì)算相對(duì)長(zhǎng)度。

1.4 組織樣品采集及形態(tài)學(xué)和上皮細(xì)胞凋亡率測(cè)定

確定采樣部位，用手術(shù)剪從皺胃胃底腺區(qū)以及十二指腸、空腸和回腸中部采集約1 cm2組織塊，轉(zhuǎn)移至10 mL帶蓋離心管中，加入10 mL多聚甲醛以固定樣品。然后將樣品送至成都里來(lái)生物科技有限公司，用蘇木精-伊紅染色法進(jìn)行形態(tài)學(xué)測(cè)定，用TUNEL法測(cè)定上皮細(xì)胞凋亡率。

1.5 小腸黏膜緊密連接蛋白mRNA表達(dá)量測(cè)定

截取十二指腸、空腸和回腸中段組織樣品，用載玻片刮取黏膜后轉(zhuǎn)入2 mL凍存管并迅速轉(zhuǎn)入液氮罐冷凍，帶回實(shí)驗(yàn)室后轉(zhuǎn)入-80℃冰箱冷凍保存，用于測(cè)定小腸黏膜閉鎖蛋白（occludin）、閉鎖小帶1（zonula occludens-1，ZO-1）和緊密連接蛋白1（claudin 1）mRNA表達(dá)量。提取十二指腸、空腸和回腸黏膜中的總RNA，然后將各樣品RNA反轉(zhuǎn)錄為cDNA。Oligo 7.0設(shè)計(jì)引物（occludin引物長(zhǎng)度93 bp，GenBank號(hào)NC_040267；ZO-1和claudin 1引物長(zhǎng)度分別為163 bp和216 bp，參考LIU等[26]），以β-Actin為內(nèi)參基因（長(zhǎng)度97 bp，GenBank號(hào)NC_040362）。RT-PCR使用20 μL擴(kuò)增體系：10 μL 2×Biogold qPCR SuperMix（2×Biogold qPCR Mixture，浙江博而金科技股份有限公司），0.4 μL上下游引物，1 μL cDNA，8.2 μL ddH2O。反應(yīng)條件為：95℃預(yù)變性3 min，95℃變性10 s，60℃退火20 s，72℃延伸10 s，40個(gè)循環(huán)，72℃延伸10 min。目的基因的相對(duì)表達(dá)量用2-ΔΔCt法計(jì)算。

表1 羔羊代乳粉養(yǎng)分濃度（風(fēng)干基礎(chǔ)，%）

營(yíng)養(yǎng)水平均為實(shí)測(cè)值 The nutrient levels are measured values

1.6 數(shù)據(jù)統(tǒng)計(jì)分析

試驗(yàn)數(shù)據(jù)用SPSS 22.0統(tǒng)計(jì)分析軟件對(duì)數(shù)據(jù)進(jìn)行獨(dú)立樣本t檢驗(yàn)，以≤0.05表示為差異顯著，以0.05＜≤0.10表示差異具有顯著趨勢(shì)。

2 結(jié)果

2.1 單寧酸對(duì)羔羊胃腸道相對(duì)質(zhì)量和相對(duì)長(zhǎng)度的影響

從表2可以看出，單寧酸顯著降低羔羊十二指腸相對(duì)質(zhì)量（%活體質(zhì)量，=0.012；全腸質(zhì)量，=0.034；%全胃腸質(zhì)量，=0.017），并有降低羔羊盲腸相對(duì)質(zhì)量的趨勢(shì)（%活體質(zhì)量，=0.072），對(duì)羔羊胃室和其他腸段指數(shù)沒(méi)有產(chǎn)生顯著影響（＞0.05）。

從表3可以看出，單寧酸顯著降低羔羊空腸相對(duì)長(zhǎng)度并顯著增加結(jié)腸相對(duì)長(zhǎng)度（%全腸長(zhǎng)度，=0.030，=0.004），對(duì)羔羊其他腸段相對(duì)長(zhǎng)度無(wú)顯著影響（＞0.05）。

2.2 單寧酸對(duì)羔羊胃腸道內(nèi)容物分布的影響

從表4可以看出，單寧酸顯著降低羔羊結(jié)腸內(nèi)容物相對(duì)活體質(zhì)量（%活體質(zhì)量，=0.039），并有降低皺胃內(nèi)容物相對(duì)活體質(zhì)量的趨勢(shì)（%活體質(zhì)量，=0.053），對(duì)羔羊胃室及其他腸段內(nèi)容物相對(duì)質(zhì)量（%總胃內(nèi)容物、%總腸內(nèi)容物和%總胃腸內(nèi)容物）沒(méi)有產(chǎn)生顯著影響（＞0.05）。

表2 單寧酸對(duì)羔羊胃腸相對(duì)質(zhì)量的影響

同行數(shù)據(jù)后所標(biāo)字母相異表示差異顯著（＜0.05），所標(biāo)字母相同表示差異不顯著（＞0.05）。下同

Different letters in the same row means significant difference between the treatments (＜0.05), same letter in the same row means not significant difference between treatments (＞0.05). The same as below

表3 單寧酸對(duì)羔羊腸道相對(duì)長(zhǎng)度的影響

表4 單寧酸對(duì)羔羊胃腸內(nèi)容物相對(duì)質(zhì)量的影響

2.3 單寧酸對(duì)羔羊皺胃和小腸組織形態(tài)的影響

從表5可以看出，單寧酸顯著提高羔羊十二指腸肌層厚度并顯著降低絨毛寬度（=0.013，=0.001），此外，單寧酸有降低空腸絨毛寬度和隱窩深度的趨勢(shì)（=0.073，=0.062）。單寧酸對(duì)羔羊皺胃和小腸其他形態(tài)學(xué)指標(biāo)未產(chǎn)生顯著影響（＞0.05）。

從表6可以看出，單寧酸顯著上調(diào)羔羊空腸ZO-1蛋白mRNA表達(dá)量（=0.003），且有上調(diào)空腸occludin蛋白mRNA表達(dá)量及降低空腸和回腸上皮細(xì)胞凋亡率的趨勢(shì)（=0.077，=0.097，=0.052）。單寧酸對(duì)羔羊十二指腸ZO-1蛋白和claudin 1蛋白mRNA表達(dá)量及上皮細(xì)胞凋亡率、空腸claudin 1蛋白mRNA表達(dá)量以及回腸occludin蛋白、ZO-1蛋白和claudin 1蛋白mRNA表達(dá)量均沒(méi)有產(chǎn)生顯著影響（＞0.05）。

3 討論

3.1 單寧酸對(duì)羔羊胃腸道相對(duì)質(zhì)量的影響

反芻動(dòng)物出生時(shí)皺胃占總?cè)莘e的60%，瘤網(wǎng)胃占35%，而隨著生長(zhǎng)發(fā)育，前胃迅速發(fā)育，成年時(shí)瘤胃占整個(gè)胃重的80%，皺胃僅占8%[27]；同樣，新生反芻動(dòng)物腸管占消化道的70%—80%，而隨日齡增長(zhǎng)和飼糧變化，腸道比例逐漸下降，胃的比例大大上升，成年時(shí)腸管占消化道的比例降為30%—50%[28]。初乳、乳源性促生長(zhǎng)因子、營(yíng)養(yǎng)水平、斷奶等均會(huì)影響幼齡反芻動(dòng)物胃腸道發(fā)育，飼糧也是影響胃腸道發(fā)育的重要因子[29]。本次試驗(yàn)中，單寧酸顯著降低羔羊十二指腸相對(duì)質(zhì)量，對(duì)其他胃室和腸段均未產(chǎn)生顯著影響，這是因?yàn)閱螌幩崤c蛋白質(zhì)可結(jié)合為緊密的絡(luò)合物[30]，雖然皺胃中的強(qiáng)酸可分解該絡(luò)合物，但羔羊皺胃功能發(fā)育不完善，pH較高，不能有效分解單寧酸和蛋白質(zhì)的絡(luò)合物，導(dǎo)致流入十二指腸的可消化蛋白質(zhì)數(shù)量減少，對(duì)十二指腸的刺激作用減弱[1]。此外，在試驗(yàn)全期，羔羊均飼喂液態(tài)代乳粉，胃腸道缺乏有效的物理刺激，導(dǎo)致羔羊胃腸道發(fā)育延滯，尤其是瘤胃[28-29,31]。

3.2 單寧酸對(duì)羔羊胃腸道內(nèi)容物分布的影響

本次試驗(yàn)中，除結(jié)腸內(nèi)容物相對(duì)活體質(zhì)量（%活體質(zhì)量）外，單寧酸對(duì)羔羊胃室和腸道各段的內(nèi)容物分布并未產(chǎn)生顯著的影響，這是因?yàn)?，胃腸道內(nèi)容物的分布主要由飼糧的物理形態(tài)及胃腸道的蠕動(dòng)所決定[28]。本試驗(yàn)全期均給羔羊飼喂液態(tài)代乳粉，會(huì)縮短胃腸道中食糜的停留時(shí)間，而添加量為0.2%的單寧酸不足以改變飼糧物理特性，導(dǎo)致羔羊胃腸道內(nèi)容物分布無(wú)顯著差異[28]。此外，胃腸道的節(jié)律運(yùn)動(dòng)由機(jī)體神經(jīng)系統(tǒng)、內(nèi)分泌系統(tǒng)、食糜壓力差和物理化學(xué)性質(zhì)等多種因素調(diào)控[32]，單寧酸作為外源添加的少量植物次級(jí)代謝物，不能對(duì)羔羊胃腸道蠕動(dòng)和內(nèi)容物分布產(chǎn)生顯著影響。

3.3 單寧酸對(duì)羔羊皺胃和小腸組織形態(tài)的影響

幼齡反芻動(dòng)物由于瘤胃功能發(fā)育不完善，大量養(yǎng)分會(huì)進(jìn)入皺胃和小腸，皺胃和小腸也成為了養(yǎng)分最主要的消化吸收部位[33-35]，而養(yǎng)分的吸收取決于皺胃和小腸的組織形態(tài)。腸道肌層主要參與蠕動(dòng)，以及通過(guò)緊張性收縮維持腸道正常形態(tài)[36]；腸道絨毛與養(yǎng)分吸收有關(guān)，絨毛越高表明吸收表面積越大[37]；腸道隱窩內(nèi)的未分化細(xì)胞與腸絨毛形態(tài)和功能正常有關(guān)[38]；V/C值與腸黏膜上皮的成熟有關(guān)，比值越高表明成熟細(xì)胞所占比例越大，腸道吸收功能越強(qiáng)[36]。本次試驗(yàn)中，單寧酸顯著提高羔羊十二指腸肌層厚度并顯著降低絨毛寬度，且有提高羔羊回腸絨毛寬度和降低空腸絨毛寬度和隱窩深度的趨勢(shì)，這是因?yàn)槭改c可消化蛋白質(zhì)數(shù)量降低，腸絨毛的脫落減緩，導(dǎo)致絨毛高度較高而降低絨毛寬度，并使肌層厚度增加[23]。而食糜快速通過(guò)十二指腸后，較多的蛋白質(zhì)刺激空腸段絨毛脫落，導(dǎo)致空腸絨毛高度變低，寬度變窄，且由于空腸上皮細(xì)胞成熟度升高而使隱窩深度變淺[39]。但本試驗(yàn)中，采食含單寧酸代乳粉羔羊的十二指腸和空腸V/C值均高于對(duì)照組羔羊，表明單寧酸有助于提高羔羊十二指腸和空腸的養(yǎng)分吸收能力[23]。本試驗(yàn)中單寧酸顯著上調(diào)羔羊空腸ZO-1蛋白mRNA表達(dá)量且有上調(diào)十二指腸和空腸occludin蛋白mRNA蛋白表達(dá)量的趨勢(shì)，小腸上皮細(xì)胞凋亡率均低于對(duì)照組羔羊，也與羔羊小腸形態(tài)學(xué)結(jié)果相符合。腸道緊密連接蛋白是腸黏膜屏障中機(jī)械屏障的組成成分，對(duì)保障屏障正常功能、阻止病原微生物入侵、養(yǎng)分的選擇性通透等起重要作用[40]。幼齡哺乳動(dòng)物出生后，腸道開(kāi)始逐漸發(fā)育，上皮細(xì)胞凋亡和有絲分裂共同作用，使功能逐漸完善，而母乳或胃腸道中的胰高血糖素樣肽（glucagon-like peptide）、胰島素（insulin）、胰島素樣生長(zhǎng)因子（insulin- like growth factors，IGFs）、瘦素（leptin）、腫瘤壞死因子α（tumor necrosis factor α，TNF-α）、表皮生長(zhǎng)因子（epidermal growth factor，EGF）等，都會(huì)對(duì)上皮細(xì)胞的增殖、分化和凋亡產(chǎn)生重要作用[39]。通常情況下，腸道炎癥出現(xiàn)時(shí)，會(huì)使TNF-α等細(xì)胞因子的分泌量增加，而TNF-α也是誘導(dǎo)腸道上皮細(xì)胞凋亡的重要信號(hào)之一[39]。但單寧酸有多種抗炎活性[41-42]，可以抑制炎癥因子，從而保障腸道屏障功能并降低上皮細(xì)胞凋亡率，這也是本次試驗(yàn)中，采食含單寧酸代乳粉羔羊小腸緊密連接蛋白mRNA表達(dá)量上調(diào)且上皮細(xì)胞凋亡率降低的主要原因。Liu等[43]研究發(fā)現(xiàn)，炎癥小鼠注射從水柏枝（）中提取的水解單寧后，抗炎作用明顯。Hoffmann等[44]也報(bào)道，委陵菜（）提取物富含單寧，表現(xiàn)出很強(qiáng)的抗炎作用。此外，Liu[43]等研究發(fā)現(xiàn)，飼糧添加0.2%栗木單寧可上調(diào)熱應(yīng)激肉雞空腸黏膜ZO-1蛋白mRNA表達(dá)量，也與本試驗(yàn)中單寧酸改善羔羊腸道屏障功能的結(jié)果相似。

4 結(jié)論

7—28日齡湖羊羔羊代乳粉中添加0.2%單寧酸顯著降低十二指腸相對(duì)質(zhì)量和空腸相對(duì)長(zhǎng)度，顯著提高結(jié)腸相對(duì)長(zhǎng)度，對(duì)胃腸道其他部位的相對(duì)質(zhì)量和長(zhǎng)度無(wú)顯著影響。單寧酸顯著提高羔羊十二指腸肌層厚度、降低絨毛寬度并上調(diào)空腸ZO-1蛋白mRNA表達(dá)量，且有上調(diào)羔羊空腸occludin蛋白mRNA表達(dá)量并降低空腸絨毛寬度和隱窩深度以及空腸和回腸上皮細(xì)胞凋亡率的趨勢(shì)。表明單寧酸對(duì)7—28日齡湖羊羔羊胃腸道生長(zhǎng)的影響較為微弱，但在調(diào)節(jié)小腸組織形態(tài)并維持正常屏障功能方面發(fā)揮一定的作用。

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Effects of Tannic Acid Addition in Milk Replacer on Development of Gastrointestinal Tract of 7 to 28 Days Old Hu Lambs

ZHENG Chen1, LI FaDi2,3, LI Fei2, ZHOU JuWang1, DUAN PengWei1, LIU HuiHui1, FAN HaiMiao1, ZHU WeiLi1, LIU Ting1

(1College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070;2State Key Laboratory of Grassland Agro-ecosystems, Lanzhou University/Key laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs, Lanzhou University/College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020;3Engineering Laboratory of Mutton Sheep Breeding and Reproduction Biotechnology in Gansu Province, Minqin 733300, Gansu)

【】This study was conducted to investigate the effects of tannic acid supplementation to milk replacer on the development of gastrointestinal tract of 7 to 28 day-old Hu lambs. 【】Thirty 7 day-old Hu male lambs were chosen and divided into 2 groups, fifteen lambs in each group and each lamb as a duplication. Lambs were fed milk replacer with or without 0.2% tannic acid, respectively. The test lasted 21 days. Eight lambs were selected from each group randomly and slaughtered at 28 day-old. The weights of the compound stomach and the intestinal tract with and without content, and lengths of the intestinal tract were measured, then the relative quality and length were calculated. While the tissue samples from fundus gland region of the abomasum, the middle part of duodenum, jejunum and ileum were fixed in paraformaldehyde to analyse the histomorphology, and the apoptotic rate of intestinal epithelial cells as well. And the mRNA expression of occludin, ZO-1 and claudin 1 protein of duodenum, jejunum and ileum mucosa were measured.【】The results showed that except relative weights of duodenum (% body weight,=0.012; % intestinal tract weight,=0.034; % gastrointestinal tract weight,=0.017), relative length of jejunum and colon (% intestinal tract length,=0.030, P=0.004), and content of colon (% body weight,=0.039), the relative weights (% body weight, % stomach weight, % intestinal tract weight, and % gastrointestinal tract weight), relative lengths (% intestinal tract length), content of stomach and intestinal tract (% body weight, % stomach content weight, % intestinal tract content weight, and % gastrointestinal tract content weight), and mRNA expression of claudin 1 protein in intestinal tract of lambs were not affected by tannic acid (＞0.05). However, tannic acid elongated the muscular thickness of duodenum and decreased the villus width of lamb duodenum significantly (=0.013=0.001), and up-regulated mRNA expression of ZO-1 protein of jejunum (=0.003). And there was a tendency that tannic acid up-regulated mRNA expression of occludin protein of jejunum (=0.077), and decreased villus width and crypt depth of jejunum and the apoptotic rate of jejunum and ileum cells (=0.073,=0.062,=0.097,=0.052). 【】In conclusion, tannic acid decreased the relative weights of duodenum and relative length of jejunum of 7-28 day-old Hu lambs, but improved the barrier function of intestinal tract via elongating the muscular thickness of duodenum, up-regulating the mRNA expression of ZO-1 and occludin protein of jejunum, and decreasing the crypt depth of jejunum and the apoptotic rate of jejunum and ileum cells.

tannic acid; lamb; milk replacer; gastrointestinal tract; development

10.3864/j.issn.0578-1752.2019.21.019

2019-06-24；

甘肅農(nóng)業(yè)大學(xué)青年研究生指導(dǎo)教師扶持基金項(xiàng)目（GAU-QNDS-201603）

鄭琛，E-mail：zhengc@gsau.edu.cn。通信作者劉婷，E-mail：liuting@gsau.edu.cn

（責(zé)任編輯 林鑒非）