活性酵母對(duì)脂多糖應(yīng)激黃羽肉雞腸道健康的影響

潘迪子 李國(guó)軍 胡貴麗 王玉詩(shī) 張 博 賀 喜*

(1.湖南農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院，飼料安全與高效利用教育部工程研究中心，湖南畜禽安全生產(chǎn)協(xié)同創(chuàng)新中心，長(zhǎng)沙410128；2.法國(guó)樂(lè)斯福工業(yè)公司，上海200030)

活性酵母對(duì)脂多糖應(yīng)激黃羽肉雞腸道健康的影響

潘迪子1李國(guó)軍1胡貴麗1王玉詩(shī)1張 博2賀 喜1*

(1.湖南農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院，飼料安全與高效利用教育部工程研究中心，湖南畜禽安全生產(chǎn)協(xié)同創(chuàng)新中心，長(zhǎng)沙410128；2.法國(guó)樂(lè)斯福工業(yè)公司，上海200030)

本試驗(yàn)旨在研究活性酵母對(duì)脂多糖(LPS)應(yīng)激黃羽肉雞腸道健康的影響。選取480羽1日齡黃羽肉公雞，隨機(jī)分成6個(gè)組，分別為抗生素組(基礎(chǔ)飼糧+0.025‰抗生素)、抗生素+LPS組(基礎(chǔ)飼糧+0.025‰抗生素，注射LPS)、0.05%活性酵母組(基礎(chǔ)飼糧+0.05%活性酵母)、0.05%活性酵母+LPS組(基礎(chǔ)飼糧+0.05%活性酵母，注射LPS)、0.50%活性酵母組(基礎(chǔ)飼糧+0.50%活性酵母)和0.50%活性酵母+LPS組(基礎(chǔ)飼糧+0.50%活性酵母，注射LPS)，每組8個(gè)重復(fù)，每個(gè)重復(fù)10只雞。試驗(yàn)期56 d?？股?LPS組、0.05%活性酵母+LPS組和0.50%活性酵母+LPS組的試驗(yàn)雞于21、23、25和27日齡每只雞肌肉注射2 mL 0.2 mg/mL LPS，其余試驗(yàn)雞肌肉注射等量生理鹽水。于21和27日齡注射LPS或生理鹽水后2、4、6、8、10、12和24 h測(cè)量試驗(yàn)雞的直腸溫度，并檢測(cè)27和56日齡試驗(yàn)雞的腸道細(xì)胞凋亡指數(shù)以及27、35和56日齡試驗(yàn)雞的腸道食糜微生物數(shù)量和腸道形態(tài)結(jié)構(gòu)。結(jié)果表明：1)與注射生理鹽水相比，LPS刺激顯著提高了21日齡注射后2 h和27日齡注射后2和4 h黃羽肉雞的直腸溫度(P<0.05)，顯著降低了21日齡注射后12 h和27日齡注射后8 h黃羽肉雞的直腸溫度(P<0.05)；飼糧中添加活性酵母對(duì)黃羽肉雞的直腸溫度無(wú)顯著影響(P>0.05)；飼糧中添加活性酵母與肌肉注射LPS對(duì)黃羽肉雞的直腸溫度無(wú)顯著交互作用(P>0.05)。2)與注射生理鹽水相比，LPS應(yīng)激顯著提高了27日齡黃羽肉雞的十二指腸和回腸細(xì)胞凋亡指數(shù)(P<0.05)；飼糧中添加活性酵母對(duì)黃羽肉雞的腸道細(xì)胞凋亡指數(shù)無(wú)顯著影響(P>0.05)；飼糧中添加活性酵母與肌肉注射LPS對(duì)黃羽肉雞的腸道細(xì)胞凋亡指數(shù)無(wú)顯著交互作用(P>0.05)。3)與抗生素相比，飼糧中添加0.05%和0.50%的活性酵母顯著提高了27、35和56日齡黃羽肉雞回腸食糜酵母菌數(shù)量(P<0.05)；與注射生理鹽水相比，LPS應(yīng)激對(duì)黃羽肉雞回腸和盲腸食糜微生物數(shù)量無(wú)顯著影響(P>0.05)；飼糧中添加活性酵母與肌肉注射LPS對(duì)黃羽肉雞腸道食糜微生物數(shù)量無(wú)顯著交互作用(P>0.05)。4)與抗生素相比，飼糧中添加0.50%活性酵母顯著提高了35日齡黃羽肉雞的空腸絨毛高度(P<0.05)；與注射生理鹽水相比，LPS應(yīng)激顯著提高了56日齡黃羽肉雞的十二指腸隱窩深度(P<0.05)；飼糧中添加活性酵母與肌肉注射LPS對(duì)黃羽肉雞的腸道形態(tài)結(jié)構(gòu)無(wú)顯著交互作用(P>0.05)。綜上所述，LPS能成功誘導(dǎo)黃羽肉雞的免疫應(yīng)激反應(yīng)，飼糧中添加活性酵母能夠提高黃羽肉雞腸道絨毛高度和食糜中酵母菌數(shù)量，改善腸道黏膜及菌群結(jié)構(gòu)，但肌肉注射LPS與飼糧中添加活性酵母無(wú)顯著交互作用。

活性酵母；黃羽肉雞；脂多糖；腸道功能

現(xiàn)代集約化的畜牧生產(chǎn)環(huán)境導(dǎo)致有害微生物、內(nèi)毒素和飼糧中的抗原分子極易引起動(dòng)物機(jī)體的免疫應(yīng)激反應(yīng)，而動(dòng)物長(zhǎng)期處于免疫應(yīng)激狀態(tài)下，會(huì)導(dǎo)致腸道生長(zhǎng)受阻，易激發(fā)腸炎，破壞腸道屏障功能[1-2]，進(jìn)而影響生長(zhǎng)，給養(yǎng)殖業(yè)造成巨大的經(jīng)濟(jì)損失[3-5]?；钚越湍甘且环N單細(xì)胞真菌，屬于兼性厭氧菌，是由鮮酵母經(jīng)壓榨干燥脫水而得到的干酵母制品，能耐受胃內(nèi)的酸性環(huán)境，保持代謝活性。Yang等[5]研究表明，飼糧中添加活性酵母能夠顯著緩解脂多糖(lipopolysaccharide, LPS)誘導(dǎo)的白羽肉仔雞的炎癥反應(yīng)，但未從家禽腸道健康角度進(jìn)行研究探討。也有研究表明，飼糧中添加酵母能夠增加肉雞腸道中乳酸菌的數(shù)量、減少沙門氏菌數(shù)量，還能產(chǎn)生大量的氨基酸和B族維生素，促進(jìn)腸道的消化與吸收功能，保證腸道健康[6]。鑒于維持畜禽腸道健康的重要性，本研究以黃羽肉雞為試驗(yàn)對(duì)象，探討活性酵母對(duì)LPS應(yīng)激黃羽肉雞腸道健康的影響，為酵母菌制劑更好地在畜禽養(yǎng)殖中應(yīng)用提供理論依據(jù)。

1 材料與方法

1.1 試驗(yàn)材料

活性酵母的菌株為啤酒酵母，經(jīng)檢測(cè)活性為1.2×1010CFU/g，干物質(zhì)含量為93.43%，粗蛋白質(zhì)含量為45.76%，由法國(guó)樂(lè)斯福工業(yè)有限公司提供；抗生素為硫酸抗敵素(STY1506023)，硫酸黏菌素含量為10%，購(gòu)自麗珠集團(tuán)福州福興醫(yī)藥有限公司；大腸桿菌(Escherichiacoli)LPS，血清型為O127∶B8，購(gòu)自美國(guó)Sigma公司，現(xiàn)用現(xiàn)配；滅菌生理鹽水注射液濃度為500 μg/mL。

1.2 試驗(yàn)動(dòng)物與試驗(yàn)設(shè)計(jì)

選取480羽1日齡新廣黃K99黃羽肉公雞，隨機(jī)分成6個(gè)組，分別為抗生素組(基礎(chǔ)飼糧+0.025‰抗生素)、抗生素+LPS組(基礎(chǔ)飼糧+0.025‰抗生素，注射LPS)、0.05%活性酵母組(基礎(chǔ)飼糧+0.05%活性酵母)、0.05%活性酵母+LPS組(基礎(chǔ)飼糧+0.05%活性酵母，注射LPS)、0.50%活性酵母組(基礎(chǔ)飼糧+0.50%活性酵母)和0.50%活性酵母+LPS組(基礎(chǔ)飼糧+0.50%活性酵母，注射LPS)，每組8個(gè)重復(fù)，每個(gè)重復(fù)10只雞，各組試驗(yàn)雞初始體重?zé)o顯著差異(P>0.05)。抗生素+LPS組、0.05%活性酵母+LPS組和0.50%活性酵母+LPS組的試驗(yàn)雞于21、23、25和27日齡08:00—09:00每只雞肌肉注射2 mL 0.2 mg/mL LPS(LPS溶解在蒸餾水中)，其余試驗(yàn)雞肌肉注射等量生理鹽水。于21和27日齡注射LPS或生理鹽水后2、4、6、8、10、12和24 h，每重復(fù)取1只雞，用玻璃棒水銀柱式獸用體溫計(jì)插入試驗(yàn)雞直腸約3 cm處，5 min后讀數(shù)，用于直腸溫度測(cè)定。常規(guī)飼養(yǎng)管理。試驗(yàn)期56 d。

1.3 試驗(yàn)飼糧

基礎(chǔ)飼糧參照NRC(1994)和《雞飼養(yǎng)標(biāo)準(zhǔn)》(NY/T 33—2004)推薦的營(yíng)養(yǎng)水平配制，基礎(chǔ)飼糧組成及營(yíng)養(yǎng)水平見表1。

表1 基礎(chǔ)飼糧組成及營(yíng)養(yǎng)水平(風(fēng)干基礎(chǔ))Table 1 Composition and nutrient levels of basal diets (air-dry basis) %

續(xù)表1項(xiàng)目Items含量Content1～28日齡Agedfrom1to28days29～56日齡Agedfrom29to56days營(yíng)養(yǎng)水平Nutrientlevels2)代謝能ME/(MJ/kg)12.1312.55粗蛋白質(zhì)CP21.0019.00有效磷AP0.450.40鈣Ca1.000.90賴氨酸Lys1.151.00蛋氨酸Met0.500.40

1)預(yù)混料為每千克飼糧提供 Premix provided the following per kg of diets：VA 9 500 IU，VD362.5 μg，VK32.65 mg，VB12 mg，VB26 mg，VB120.025 mg，VE 30 IU，生物素 biotin 0.032 5 mg，葉酸 folic acid 1.25 mg，泛酸 pantothenic acid 12 mg，煙酸 nicotinic acid 50 mg，Cu (as copper sulfate) 8 mg，Zn (as zinc sulfate) 75 mg，F(xiàn)e (as ferrous sulfate) 80 mg，Mn (as manganese sulfate) 100 mg，Se (as sodium selenite) 0.15 mg，I (as potassium iodide) 0.35 mg。

2)營(yíng)養(yǎng)水平為計(jì)算值。Nutrient levels were calculated values.

1.4 測(cè)定指標(biāo)與方法

1.4.1 腸道食糜微生物數(shù)量

于試驗(yàn)雞27、35和56日齡時(shí)，每重復(fù)取1只雞屠宰，取回腸的后1/2段及盲腸中食糜，用生理鹽水沖食糜于鋁盒中，再將處理好的樣品放置于-20 ℃冰箱保存。采用平板涂布法測(cè)定回腸和盲腸食糜中微生物數(shù)量，酵母菌采用馬鈴薯葡萄糖瓊脂(PDA)培養(yǎng)基有氧培養(yǎng)72 h進(jìn)行計(jì)數(shù)，大腸桿菌采用麥康凱培養(yǎng)基有氧培養(yǎng)24 h進(jìn)行計(jì)數(shù)，乳酸桿菌和雙歧桿菌分別用乳酸細(xì)菌(MRS)培養(yǎng)基和溶菌肉湯(BL)培養(yǎng)基厭氧培養(yǎng)48 h進(jìn)行計(jì)數(shù)。

1.4.2 腸道形態(tài)結(jié)構(gòu)與腸道細(xì)胞凋亡指數(shù)

取27、35和56日齡試驗(yàn)雞的十二指腸、空腸和回腸中段1 cm腸段，小心去除腸道內(nèi)食糜后，固定于4%多聚甲醛溶液中，再將固定好的腸道組織經(jīng)脫水→透明→浸蠟→包埋→修塊→切片→展開→貼片等一系列處理后，用蘇木精-伊紅(HE)染色制成組織切片，在顯微鏡下測(cè)量每個(gè)組織切片上10根最長(zhǎng)絨毛的高度，以其均值作為相應(yīng)的絨毛高度(VH)和隱窩深度(CD)，并計(jì)算絨毛高度/隱窩深度(VH/CD)。腸道細(xì)胞凋亡采用Tunel原位凋亡檢測(cè)法，其原理是細(xì)胞凋亡的發(fā)生使內(nèi)源性核酸酶激活，DNA的一條鏈出現(xiàn)缺口，產(chǎn)生一系列3′-OH末端，在脫氧核糖核甘酸末端轉(zhuǎn)移酶作用下，用生物素-dUTP標(biāo)記組織細(xì)胞原位DNA切口。將包埋的腸道組織經(jīng)脫蠟脫水→酶解→標(biāo)記→信號(hào)轉(zhuǎn)化和分析→復(fù)染等處理后，計(jì)算21和56日齡試驗(yàn)雞的腸道細(xì)胞凋亡指數(shù)。

1.5 統(tǒng)計(jì)分析

采用SPSS 17.0統(tǒng)計(jì)軟件中的雙因素方差分析(two-way ANOVA)分析活性酵母和LPS 2個(gè)主效應(yīng)，用GLM程序分析它們之間的互作關(guān)系。差異顯著時(shí)用Duncan氏法進(jìn)行多重比較，以P<0.05作為差異顯著性的判斷標(biāo)準(zhǔn)。其中活性酵母和LPS為全模型的2個(gè)固定效應(yīng)與互作效應(yīng)，交互作用顯著時(shí)，采用單因素方差分析(one-way ANOVA)進(jìn)行分析。

2 結(jié) 果

2.1 活性酵母對(duì)LPS應(yīng)激黃羽肉雞直腸溫度的影響

由表2可知，與注射生理鹽水相比，LPS刺激顯著提高了21日齡注射后2 h和27日齡注射后2和4 h黃羽肉雞的直腸溫度(P<0.05)，顯著降低了21日齡注射后12 h和27日齡注射后8 h黃羽肉雞的直腸溫度(P<0.05)；飼糧中添加活性酵母對(duì)黃羽肉雞的直腸溫度無(wú)顯著影響(P>0.05)；飼糧中添加活性酵母與肌肉注射LPS對(duì)黃羽肉雞的直腸溫度無(wú)顯著交互作用(P>0.05)。

表2 活性酵母對(duì)LPS應(yīng)激黃羽肉雞直腸溫度的影響Table 2 Effects of active yeast on the rectal temperature of yellow-feathered broilers challenged by LPS ℃

同列數(shù)據(jù)肩標(biāo)相同或無(wú)字母表示差異不顯著(P>0.05)，不同小寫字母表示差異顯著(P<0.05)，不同大寫字母表示差異極顯著(P<0.01)。下表同。

In the same column, values with the same or no letter superscripts mean no significant difference (P>0.05), and with different small letter superscripts mean significant difference (P<0.05), while with different large letter superscripts mean significant difference (P<0.01). The same as below.

2.2 活性酵母對(duì)LPS應(yīng)激黃羽肉雞腸道細(xì)胞凋亡指數(shù)的影響

由表3可知，與注射生理鹽水相比，LPS應(yīng)激顯著提高了27日齡黃羽肉雞的十二指腸和回腸細(xì)胞凋亡指數(shù)(P<0.05)；LPS應(yīng)激對(duì)56日齡黃羽肉雞的十二指腸、空腸和回腸細(xì)胞凋亡指數(shù)均無(wú)顯著影響(P>0.05)。飼糧中添加活性酵母對(duì)黃羽肉雞的腸道細(xì)胞凋亡指數(shù)無(wú)顯著影響(P>0.05)。飼糧中添加活性酵母與肌肉注射LPS對(duì)黃羽肉雞的腸道細(xì)胞凋亡指數(shù)無(wú)顯著交互作用(P>0.05)。

表3 活性酵母對(duì)LPS應(yīng)激黃羽肉雞腸道細(xì)胞凋亡指數(shù)的影響Table 3 Effects of active yeast on the apoptotic index of intestinal cell of yellow-feathered broilers challenged by LPS

2.3 活性酵母對(duì)LPS應(yīng)激黃羽肉雞腸道食糜微生物數(shù)量的影響

由表4、5、6可知，與抗生素相比，飼糧中添加0.05%和0.50%的活性酵母顯著提高了27、35和56日齡黃羽肉雞的回腸食糜酵母菌數(shù)量(P<0.05)；與注射生理鹽水相比，LPS應(yīng)激對(duì)黃羽肉雞的回腸和盲腸食糜酵母菌、乳酸桿菌、雙歧桿菌、大腸桿菌數(shù)量無(wú)顯著影響(P>0.05)；飼糧中添加活性酵母與肌肉注射LPS對(duì)黃羽肉雞的腸道食糜微生物數(shù)量無(wú)顯著交互作用(P>0.05)。

2.4 活性酵母對(duì)LPS應(yīng)激黃羽肉雞腸道形態(tài)結(jié)構(gòu)的影響

由表7、8、9可知，與抗生素相比，飼糧中添加0.50%活性酵母顯著提高了35日齡黃羽肉雞的空腸絨毛高度(P<0.05)；與注射生理鹽水相比，LPS應(yīng)激顯著提高了56日齡黃羽肉雞的十二指腸隱窩深度(P<0.05)；飼糧中添加活性酵母與肌肉注射LPS對(duì)黃羽肉雞的十二指腸、空腸和回腸絨毛高度、隱窩深度和絨毛高度/隱窩深度無(wú)顯著交互作用(P>0.05)。

表4 活性酵母對(duì)LPS應(yīng)激黃羽肉雞27日齡腸道食糜微生物數(shù)量的影響Table 4 Effects of active yeast on microflora number of intestinal chyme of yellow-feathered broilers aged at 27 days challenged by LPS lg(CFU/g)

表5 活性酵母對(duì)LPS應(yīng)激黃羽肉雞35日齡腸道食糜微生物數(shù)量的影響Table 5 Effects of active yeast on microflora number of intestinal chyme of yellow-feathered broilers aged at 35 days challenged by LPS lg(CFU/g)

續(xù)表5組別Groups回腸Ileum酵母菌Saccharo-mycetes乳酸桿菌Lacto-bacillus雙歧桿菌Bifido-bacterium大腸桿菌Escherichiacoli盲腸Cecum酵母菌Saccharo-mycetes乳酸桿菌Lacto-bacillus雙歧桿菌Bifido-bacterium大腸桿菌Escherichiacoli抗生素+LPS組Antibiotic+LPSgroup3.475.636.294.653.895.975.895.670.05%活性酵母組0.05%activeyeastgroup4.515.206.604.794.495.825.975.510.05%活性酵母+LPS組0.05%activeyeast+LPSgroup4.595.526.164.674.475.446.044.910.50%活性酵母組0.50%activeyeastgroup4.715.546.324.614.565.525.785.240.50%活性酵母+LPS組0.50%activeyeast+LPSgroup4.615.396.684.914.525.756.235.14SEM0.090.090.090.060.090.090.110.11主效應(yīng)Maineffect活性酵母Activeyeast抗生素Antibiotic3.56b5.536.284.744.135.595.885.690.05%4.55a5.366.384.734.485.606.005.210.50%4.66a5.466.504.754.545.646.015.19脂多糖LPS-4.315.396.404.744.475.505.885.48+4.225.516.384.734.295.716.055.22P值P-value活性酵母Activeyeast<0.0010.7370.6330.9740.1410.9930.8910.143脂多糖LPS0.4300.4950.9210.9690.3290.2780.4590.276活性酵母×脂多糖Activeyeast×LPS0.4510.5290.2250.2070.5120.0530.7240.526

表6 活性酵母對(duì)LPS應(yīng)激黃羽肉雞56日齡腸道食糜微生物數(shù)量的影響Table 6 Effects of active yeast on microflora number of intestinal chyme of yellow-feathered broilers aged at 56 days challenged by LPS lg(CFU/g)

續(xù)表6組別Groups回腸Ileum酵母菌Saccharo-mycetes乳酸桿菌Lacto-bacillus雙歧桿菌Bifido-bacterium大腸桿菌Escherichiacoli盲腸Cecum酵母菌Saccharo-mycetes乳酸桿菌Lacto-bacillus雙歧桿菌Bifido-bacterium大腸桿菌Escherichiacoli0.05%活性酵母+LPS組0.05%activeyeast+LPSgroup4.435.366.404.674.306.286.115.510.50%活性酵母組0.50%activeyeastgroup4.345.656.384.774.066.436.575.740.50%活性酵母+LPS組0.50%activeyeast+LPSgroup4.165.706.114.704.456.436.205.70SEM0.110.060.080.100.100.080.090.09主效應(yīng)Maineffect活性酵母Activeyeast抗生素Antibiotic3.27b5.546.264.664.256.156.195.650.05%4.45a5.446.464.404.386.346.195.490.50%4.25a5.686.254.734.266.436.385.72脂多糖LPS-4.105.556.394.434.356.306.385.59+3.875.556.254.764.236.316.135.65P值P-value活性酵母Activeyeast<0.0010.2720.4780.3060.8520.4210.6310.607脂多糖LPS0.1560.9990.3970.0780.5770.9510.1820.731活性酵母×脂多糖Activeyeast×LPS0.5290.6690.8000.3090.1780.8420.8950.874

表7 活性酵母對(duì)LPS應(yīng)激黃羽肉雞27日齡腸道形態(tài)結(jié)構(gòu)的影響Table 7 Effects of active yeast on the intestinal morphology of yellow-feathered broilers aged at 27 days challenge by LPS

續(xù)表7組別Groups十二指腸Duodenum絨毛高度VH/μm隱窩深度CD/μm絨毛高度/隱窩深度VH/CD空腸Jejunum絨毛高度VH/μm隱窩深度CD/μm絨毛高度/隱窩深度VH/CD回腸Ileum絨毛高度VHμm隱窩深度CD/μm絨毛高度/隱窩深度VH/CD0.05%活性酵母+LPS組0.05%activeyeast+LPSgroup403.4288.01.5280.0211.41.4228.7147.31.50.50%活性酵母組0.50%activeyeastgroup264.3192.01.5322.4233.01.4237.7170.11.40.50%活性酵母+LPS組0.50%activeyeast+LPSgroup404.1296.41.3294.6225.31.2206.1137.01.4SEM16.013.60.08.45.40.05.95.50.0活性酵母Activeyeast抗生素Antibiotic390.6297.31.3263.9208.71.3212.8147.61.60.05%397.6288.91.4289.1216.91.4238.1161.91.50.50%324.2236.71.4308.5229.11.3221.9155.11.4脂多糖LPS-337.9251.71.4290.0220.91.3232.3161.21.6+402.2295.21.3284.3215.51.3216.2148.11.4P值P-value活性酵母Activeyeast0.1480.1910.0960.0960.3280.7210.2070.5550.346脂多糖LPS0.0600.1580.4680.7250.6290.8870.1720.2530.193活性酵母×脂多糖Activeyeast×LPS0.1260.1750.5460.3290.8720.4740.4880.0750.215

表8 活性酵母對(duì)LPS應(yīng)激黃羽肉雞35日齡腸道形態(tài)結(jié)構(gòu)的影響Table 8 Effects of active yeast on the intestinal morphology of yellow-feathered broilers aged at 35 days challenged by LPS

續(xù)表8組別Groups十二指腸Duodenum絨毛高度VH/μm隱窩深度CD/μm絨毛高度/隱窩深度VH/CD空腸Jejunum絨毛高度VH/μm隱窩深度CD/μm絨毛高度/隱窩深度VH/CD回腸Ileum絨毛高度VH/μm隱窩深度CD/μm絨毛高度/隱窩深度VH/CD0.05%活性酵母+LPS組0.05%activeyeast+LPSgroup405.4328.61.2302.3231.91.3207.7142.71.50.50%活性酵母組0.50%activeyeastgroup378.6304.01.3363.0234.51.6219.4160.81.50.50%活性酵母+LPS組0.50%activeyeast+LPSgroup366.2269.81.3302.5215.21.4227.0135.71.7SEM7.78.30.010.79.80.03.77.30.1活性酵母Activeyeast抗生素Antibiotic380.5278.21.3266.5b209.41.3217.2146.61.60.05%394.3322.21.3286.5ab221.11.3212.9159.21.40.50%372.4285.31.3332.8a224.81.5223.6149.41.6脂多糖LPS-382.8296.01.3306.7216.61.5214.3156.31.6+382.2295.61.3284.8220.31.3221.9147.01.5P值P-value活性酵母Activeyeast0.5260.0910.3440.0190.8200.0600.5030.7440.713脂多糖LPS0.9410.8490.7020.2080.8590.3610.4510.5010.860活性酵母×脂多糖Activeyeast×LPS0.5880.3690.8180.1160.7170.3100.3520.1930.505

表9 活性酵母對(duì)LPS應(yīng)激黃羽肉雞56日齡腸道形態(tài)結(jié)構(gòu)的影響Table 9 Effects of active yeast on the intestinal morphology of yellow-feathered broilers aged at 56 days challenged by LPS

續(xù)表9組別Groups十二指腸Duodenum絨毛高度VH/μm隱窩深度CD/μm絨毛高度/隱窩深度VH/CD空腸Jejunum絨毛高度VH/μm隱窩深度CD/μm絨毛高度/隱窩深度VH/CD回腸Ileum絨毛高度VH/μm隱窩深度CD/μm絨毛高度/隱窩深度VH/CD0.05%活性酵母+LPS組0.05%activeyeast+LPSgroup1319.8202.66.61008.6200.34.7910.3174.25.20.50%活性酵母組0.50%activeyeastgroup1312.2186.27.01081.2198.85.4854.4177.15.40.50%活性酵母+LPS組0.50%activeyeast+LPSgroup1217.8219.85.71014.0201.45.0830.6217.14.2SEM21.95.30.232.03.50.220.56.80.2活性酵母Activeyeast抗生素Antibiotic1219.0198.86.31164.7201.55.8918.6171.15.40.05%1251.4199.66.31096.0212.35.1953.2189.85.10.50%1265.0204.66.31047.6190.65.2840.1201.14.7脂多糖LPS-1230.9188.0b6.61140.9207.95.6948.9188.15.3+1260.8211.9a6.11062.0202.15.2874.5186.74.8P值P-value活性酵母Activeyeast0.6130.8330.9990.2540.2590.1700.0690.3120.267脂多糖LPS0.4180.0210.1410.1520.5080.1930.0980.9190.172活性酵母×脂多糖Activeyeast×LPS0.0850.4110.1080.4570.1390.9130.7480.1040.117

3 討 論

3.1 活性酵母對(duì)LPS應(yīng)激黃羽肉雞直腸溫度的影響

LPS作為細(xì)菌的一種內(nèi)毒素，進(jìn)入動(dòng)物體后能夠刺激產(chǎn)生內(nèi)生致熱原細(xì)胞，使其產(chǎn)生和釋放內(nèi)生致熱原，進(jìn)而影響體溫調(diào)節(jié)中樞，最終引起體溫升高[7]。當(dāng)動(dòng)物機(jī)體接收到應(yīng)激源刺激后，感受器會(huì)發(fā)出信號(hào)，通過(guò)神經(jīng)傳遞到低級(jí)中樞，再向上傳遞到下丘腦；下丘腦分泌腎上腺皮質(zhì)激素釋放激素(CRH)，CRH經(jīng)垂體門脈系統(tǒng)到達(dá)垂體前葉，刺激分泌促腎上腺皮質(zhì)激素(ACTH)；ACTH進(jìn)入血液循環(huán)系統(tǒng)后，最終腎上腺皮質(zhì)分泌糖皮質(zhì)激素來(lái)調(diào)節(jié)應(yīng)激反應(yīng)[8]。肌肉注射LPS后2 h黃羽肉雞的直腸溫度顯著升高，說(shuō)明注射LPS成功引發(fā)了黃羽肉雞的免疫應(yīng)激反應(yīng)；21日齡注射LPS后4 h黃羽肉雞的直腸溫度已恢復(fù)至正常水平，且在注射LPS后12 h出現(xiàn)了溫度的逆轉(zhuǎn)性生理調(diào)節(jié)反應(yīng)，說(shuō)明LPS的應(yīng)激效果已經(jīng)消除；而27日齡注射LPS后6 h黃羽肉雞的直腸溫度恢復(fù)至正常水平，注射LPS后8 h就出現(xiàn)了溫度的逆轉(zhuǎn)性生理調(diào)節(jié)反應(yīng)，這可能是連續(xù)注射LPS導(dǎo)致黃羽肉雞從急性應(yīng)激轉(zhuǎn)向了慢性應(yīng)激，加快了對(duì)LPS抗原的清除和機(jī)體的調(diào)節(jié)反應(yīng)時(shí)間軸。Liu等[9]研究發(fā)現(xiàn)，每千克體重注射500 μg LPS后8 h雞的體溫達(dá)到最高，12 h后恢復(fù)正常。雖然在反應(yīng)時(shí)間上與本試驗(yàn)有一定的差異，但都能達(dá)到體溫升高的效果，LPS應(yīng)激對(duì)體溫的調(diào)節(jié)機(jī)制還有待進(jìn)一步探究。另外，活性酵母在LPS的免疫應(yīng)激過(guò)程中對(duì)黃羽肉雞的直腸溫度無(wú)顯著影響，說(shuō)明活性酵母只是作為一種飼料添加劑被肉雞采食而不會(huì)刺激機(jī)體引起致熱反應(yīng)。

3.2 活性酵母對(duì)LPS應(yīng)激黃羽肉雞腸道細(xì)胞凋亡的影響

細(xì)胞凋亡是機(jī)體細(xì)胞在一定的病理或生理?xiàng)l件下由多種基因嚴(yán)格控制的細(xì)胞自主性死亡，是在細(xì)胞受到外界信號(hào)刺激后引發(fā)的一系列控制開關(guān)的開啟或關(guān)閉的復(fù)雜生理過(guò)程。LPS是革蘭氏陰性菌細(xì)胞壁外膜的主要成分，可引起機(jī)體的毒性病理活動(dòng)，促進(jìn)細(xì)胞因子的釋放，誘導(dǎo)細(xì)胞異常增殖，從而導(dǎo)致細(xì)胞凋亡。本試驗(yàn)采用Tunel原位凋亡檢測(cè)法標(biāo)記腸道細(xì)胞DNA裂解片段末端，進(jìn)行腸道細(xì)胞凋亡評(píng)估。試驗(yàn)結(jié)果表明，LPS應(yīng)激顯著提高了27日齡黃羽肉雞的十二指腸和回腸細(xì)胞凋亡指數(shù)，證明試驗(yàn)成功建立了應(yīng)激模型，LPS應(yīng)激造成了腸道損傷。Williams等[10]研究顯示，在小鼠腹部按每千克體重注射LPS≥0.125 mg/kg能導(dǎo)致腸上皮細(xì)胞快速凋亡。本試驗(yàn)結(jié)果也表明，飼糧中添加活性酵母對(duì)黃羽肉雞的腸道細(xì)胞凋亡指數(shù)無(wú)顯著影響，可能是活性酵母維持了有益的腸道微生態(tài)環(huán)境，增強(qiáng)了腸道黏膜屏障的免疫功能[11]。

3.3 活性酵母對(duì)LPS應(yīng)激黃羽肉雞腸道食糜微生物數(shù)量的影響

健康動(dòng)物的腸道寄生著大量的菌群，穩(wěn)定的微生態(tài)環(huán)境能協(xié)助動(dòng)物產(chǎn)生免疫反應(yīng)，對(duì)外襲菌群起著生物屏障作用。大腸桿菌、乳酸桿菌和雙歧桿菌是腸道內(nèi)的3種數(shù)量較多的正常菌群。本試驗(yàn)結(jié)果表明，飼糧中添加0.05%和0.50%的活性酵母顯著提高了27、35和56日齡黃羽肉雞的回腸食糜酵母菌數(shù)量，而對(duì)回腸和盲腸食糜乳酸桿菌、雙歧桿菌和大腸桿菌數(shù)量無(wú)顯著影響。陳生龍[12]報(bào)道，斷奶仔豬飼糧中添加2×107CFU/g的活酵母，可提高腸道內(nèi)酵母菌的數(shù)量，并對(duì)腸道微生物菌群結(jié)構(gòu)具有一定的改善作用，與本試驗(yàn)結(jié)果相一致。本試驗(yàn)結(jié)果也表明，LPS應(yīng)激有增加56日齡黃羽肉雞回腸食糜大腸桿菌數(shù)量的趨勢(shì)，這與馮焱等[13]的研究報(bào)道相似。胡友軍等[14]的研究結(jié)果表明，早期斷奶仔豬飼糧中添加活性酵母可顯著降低腸道內(nèi)容物中大腸桿菌數(shù)量。

3.4 活性酵母對(duì)LPS應(yīng)激黃羽肉雞腸道形態(tài)結(jié)構(gòu)的影響

動(dòng)物腸道的絨毛高度、隱窩深度和絨毛高度/隱窩深度可反映其發(fā)育狀況和腸道屏障功能[15-16]，絨毛高度和隱窩深度的變化也是引起腸道功能和吸收機(jī)能改變的主要原因[17-18]。本試驗(yàn)結(jié)果表明，LPS應(yīng)激可顯著提高56日齡黃羽肉雞的十二指腸隱窩深度。趙珂立等[19]研究報(bào)道，給小鼠按每千克體重腹腔注射0.8 mg/kg LPS，腸道隱窩深度顯著增加，與本試驗(yàn)結(jié)果相一致。范偉等[20]研究顯示，給仔豬按每千克體重注射100 μg/kg LPS，十二指腸、空腸和回腸的隱窩深度均顯著增加，表明LPS應(yīng)激可造成腸道損傷。本試驗(yàn)結(jié)果發(fā)現(xiàn)，飼糧中添加0.50%活性酵母可顯著提高35日齡黃羽肉雞的空腸絨毛高度，并有提高絨毛高度/隱窩深度的趨勢(shì)，表明活性酵母可促進(jìn)腸道絨毛的發(fā)育，改善腸道形態(tài)結(jié)構(gòu)。張愛武等[21]研究結(jié)果顯示，飼糧中添加1 g/kg的活性干酵母(2×1010CFU/g)，可顯著增加鵪鶉的空腸長(zhǎng)度和空腸指數(shù)；Sachin等[22]研究表明，仔豬飼糧中添加啤酒酵母[(2～3)×106CFU/g]顯著提高了腸道絨毛高度與絨毛高度/隱窩深度，本試驗(yàn)結(jié)果與此結(jié)果相一致。

4 結(jié) 論

LPS能成功誘導(dǎo)黃羽肉雞的免疫應(yīng)激反應(yīng)；飼糧中添加活性酵母能夠提高黃羽肉雞腸道絨毛高度和食糜酵母菌數(shù)量，改善腸道黏膜及菌群結(jié)構(gòu)，但肌肉注射LPS與飼糧中添加活性酵母無(wú)顯著交互作用。

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*Corresponding author, professor, E-mail: hexi111@126.com

(責(zé)任編輯 李慧英)

Effects of Active Yeast on Intestinal Health of Yellow-Feathered Broilers Challenged by Lipopolysaccharide

PAN Dizi1LI Guojun1HU Guili1WANG Yushi1ZHANG Bo2HE Xi1*

(1.HunanCo-InnovationCenterofAnimalProductionSafety,EngineeringResearchCenterforFeedSafetyandEfficientUtilizationofMinistryofEducation,CollegeofAnimalScienceandTechnology,HunanAgriculturalUniversity,Changsha410128,China; 2.FranceLesaffreIndustrialCompany,Shanghai200030,China)

This experiment was conducted to study the effects of active yeast on intestinal health of yellow-feathered broilers challenged by lipopolysaccharide (LPS). Four hundred and eighty one-day-old male yellow-feathered broilers were randomly allotted to six groups with eight replicates per group and ten broilers per replicate. The broilers in antibiotic group were fed with the basal diet supplemented with 0.025‰ antibiotic, that in antibiotic+LPS group were fed with the basal diet supplemented with 0.025‰ antibiotic and injected with LPS, that in 0.05% active yeast group were fed with the basal diet supplemented with 0.05% active yeast, that in 0.05% active yeast+LPS group were fed with the basal diet supplemented with 0.05% active yeast and injected with LPS, that in 0.50% active yeast group were fed with the basal diet supplemented with 0.50% active yeast, and that in 0.50% active yeast+LPS group were fed with the basal diet supplemented with 0.50% active yeast and injected with LPS. The experiment lasted for 56 d. Broilers aged at 21, 23, 25 and 27 days in antibiotic+LPS group, 0.05% active yeast+LPS group and 0.50% active yeast+LPS group were injected intramuscularly with 2 mL 0.2 mg/mL LPS per broiler, and the others were injected intramuscularly with same volume saline. The rectal temperature of broilers aged at 21 and 27 days were measured at 2, 4, 6, 8, 10, 12 and 24 h after the injection of LPS or saline, and the apoptotic index of intestinal cell of broilers aged at 27 and 56 days, microflora number of intestinal chyme and intestinal morphology of broilers aged at 27, 35 and 56 days were detected. The results showed as follows: 1) compared with saline injection, LPS challenged significantly increased the rectal temperature of yellow-feathered broilers at 2 h after injection aged at 21 days and at 2 h and 4 h after injection aged at 27 days (P<0.05), and significantly decreased the rectal temperature of yellow-feathered broilers at 12 h after injection aged at 21 days and at 8 h after injection aged at 27 days (P<0.05). Dietary active yeast had no significant effect on the rectal temperature of yellow-feathered broilers (P>0.05), and dietary active yeast and intramuscular injection of LPS had no significant interaction on the rectal temperature of yellow-feathered broilers (P>0.05). 2) Compared with saline injection, LPS challenged significantly increased the apoptotic index of duodenum and ileum of yellow-feathered broilers aged at 27 days (P<0.05). Dietary active yeast had no significant effect on the apoptotic index of intestinal cell of yellow-feathered broilers (P>0.05), and dietary active yeast and intramuscular injection of LPS had no significant interaction on the apoptotic index of intestinal cell of yellow-feathered broilers (P>0.05). 3) Compared with antibiotic, dietary 0.05% and 0.50% active yeast significantly increased theSaccharomycetesnumber of ileal chyme of yellow-feathered broilers aged at 27, 35 and 56 days (P<0.05). Compared with saline injection, LPS challenged had no significant effect on the microflora number of ileal and cecal chyme of yellow-feathered broilers (P>0.05). Dietary active yeast and intramuscular injection of LPS had no significant interaction on the microflora number of intestinal chyme of yellow-feathered broilers (P>0.05). 4) Compared with antibiotic, dietary 0.50% active yeast significantly increased the villus height of jejunum of yellow-feathered broilers aged at 35 days (P<0.05). Compared with saline injection, LPS challenged significantly increased the crypt depth of duodenum of yellow-feathered broilers aged at 56 days (P<0.05). Dietary active yeast and intramuscular injection of LPS had no significant interaction on the intestinal morphology of yellow-feathered broilers (P>0.05). In conclusion, the immune stress of yellow-feathered broilers is successfully induced by LPS, and dietary active yeast can increase the intestinal villus height andSaccharomycetesnumber of chyme, improve the intestinal mucosa and microflora structure of yellow-feathered broilers, but intramuscular injection of LPS and dietary active yeast have no significant interaction.[ChineseJournalofAnimalNutrition, 2017, 29(7)：2520-2534]

active yeast; yellow-feathered broilers; lipopolysaccharide; intestinal function

10.3969/j.issn.1006-267x.2017.07.037

2016-12-05

2014公益性行業(yè)(農(nóng)業(yè))科研專項(xiàng)項(xiàng)目(201403047)

潘迪子(1991—)，女，湖南常德人，碩士研究生，從事單胃動(dòng)物營(yíng)養(yǎng)研究。E-mail: pandizy9188@163.com

*通信作者:賀 喜，教授，碩士生導(dǎo)師，E-mail: hexi111@126.com

S831

A

1006-267X(2017)07-2520-15