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潘迪子, 李国军, 胡贵丽, 王玉诗, 张博, 贺喜. 活性酵母对脂多糖应激黄羽肉鸡肠道健康的影响[J]. 动物营养学报, 2017, 29(7): 2520-2534. 复制到剪切板

PAN Dizi, LI Guojun, HU Guili, WANG Yushi, ZHANG Bo, HE Xi. Effects of Active Yeast on Intestinal Health of Yellow-Feathered Broilers Challenged by Lipopolysaccharide[J]. Chinese Journal of Animal Nutrition, 2017, 29(7): 2520-2534. 复制到剪切板

活性酵母对脂多糖应激黄羽肉鸡肠道健康的影响

潘迪子¹, 李国军¹, 胡贵丽¹, 王玉诗¹, 张博², 贺喜¹

1. 湖南农业大学动物科学技术学院, 饲料安全与高效利用教育部工程研究中心, 湖南畜禽安全生产协同创新中心, 长沙 410128;
2. 法国乐斯福工业公司, 上海 200030

收稿日期: 2016-12-05

基金项目: 2014公益性行业（农业）科研专项项目（201403047）

作者简介: 潘迪子(1991—), 女, 湖南常德人, 硕士研究生, 从事单胃动物营养研究。E-mail:pandizy9188@163.com

通信作者: 贺喜, 教授, 硕士生导师, E-mail:hexi111@126.com

摘要: 本试验旨在研究活性酵母对脂多糖（LPS）应激黄羽肉鸡肠道健康的影响。选取480羽1日龄黄羽肉公鸡，随机分成6个组，分别为抗生素组（基础饲粮+0.025‰抗生素）、抗生素+LPS组（基础饲粮+0.025‰抗生素，注射LPS）、0.05%活性酵母组（基础饲粮+0.05%活性酵母）、0.05%活性酵母+LPS组（基础饲粮+0.05%活性酵母，注射LPS）、0.50%活性酵母组（基础饲粮+0.50%活性酵母）和0.50%活性酵母+LPS组（基础饲粮+0.50%活性酵母，注射LPS），每组8个重复，每个重复10只鸡。试验期56 d。抗生素+LPS组、0.05%活性酵母+LPS组和0.50%活性酵母+LPS组的试验鸡于21、23、25和27日龄每只鸡肌肉注射2 mL 0.2 mg/mL LPS，其余试验鸡肌肉注射等量生理盐水。于21和27日龄注射LPS或生理盐水后2、4、6、8、10、12和24 h测量试验鸡的直肠温度，并检测27和56日龄试验鸡的肠道细胞凋亡指数以及27、35和56日龄试验鸡的肠道食糜微生物数量和肠道形态结构。结果表明：1）与注射生理盐水相比，LPS刺激显著提高了21日龄注射后2 h和27日龄注射后2和4 h黄羽肉鸡的直肠温度（P < 0.05），显著降低了21日龄注射后12 h和27日龄注射后8 h黄羽肉鸡的直肠温度（P < 0.05）；饲粮中添加活性酵母对黄羽肉鸡的直肠温度无显著影响（P > 0.05）；饲粮中添加活性酵母与肌肉注射LPS对黄羽肉鸡的直肠温度无显著交互作用（P > 0.05）。2）与注射生理盐水相比，LPS应激显著提高了27日龄黄羽肉鸡的十二指肠和回肠细胞凋亡指数（P < 0.05）；饲粮中添加活性酵母对黄羽肉鸡的肠道细胞凋亡指数无显著影响（P > 0.05）；饲粮中添加活性酵母与肌肉注射LPS对黄羽肉鸡的肠道细胞凋亡指数无显著交互作用（P > 0.05）。3）与抗生素相比，饲粮中添加0.05%和0.50%的活性酵母显著提高了27、35和56日龄黄羽肉鸡回肠食糜酵母菌数量（P < 0.05）；与注射生理盐水相比，LPS应激对黄羽肉鸡回肠和盲肠食糜微生物数量无显著影响（P > 0.05）；饲粮中添加活性酵母与肌肉注射LPS对黄羽肉鸡肠道食糜微生物数量无显著交互作用（P > 0.05）。4）与抗生素相比，饲粮中添加0.50%活性酵母显著提高了35日龄黄羽肉鸡的空肠绒毛高度（P < 0.05）；与注射生理盐水相比，LPS应激显著提高了56日龄黄羽肉鸡的十二指肠隐窝深度（P < 0.05）；饲粮中添加活性酵母与肌肉注射LPS对黄羽肉鸡的肠道形态结构无显著交互作用（P > 0.05）。综上所述，LPS能成功诱导黄羽肉鸡的免疫应激反应，饲粮中添加活性酵母能够提高黄羽肉鸡肠道绒毛高度和食糜中酵母菌数量，改善肠道黏膜及菌群结构，但肌肉注射LPS与饲粮中添加活性酵母无显著交互作用。

关键词: 活性酵母黄羽肉鸡脂多糖肠道功能

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

PAN Dizi¹, LI Guojun¹, HU Guili¹, WANG Yushi¹, ZHANG Bo², HE Xi¹

1. Hunan Co-Innovation Center of Animal Production Safety, Engineering Research Center for Feed Safety and Efficient Utilization of Ministry of Education, College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China;
2. France Lesaffre Industrial Company, Shanghai 200030, China

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

Abstract: 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 the Saccharomycetes number 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 and Saccharomycetes number 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.

Key words: active yeast yellow-feathered broilers lipopolysaccharide intestinal function

现代集约化的畜牧生产环境导致有害微生物、内毒素和饲粮中的抗原分子极易引起动物机体的免疫应激反应，而动物长期处于免疫应激状态下，会导致肠道生长受阻，易激发肠炎，破坏肠道屏障功能^[1-2]，进而影响生长，给养殖业造成巨大的经济损失^[3-5]。活性酵母是一种单细胞真菌，属于兼性厌氧菌，是由鲜酵母经压榨干燥脱水而得到的干酵母制品，能耐受胃内的酸性环境，保持代谢活性。Yang等^[5]研究表明，饲粮中添加活性酵母能够显著缓解脂多糖(lipopolysaccharide, LPS)诱导的白羽肉仔鸡的炎症反应，但未从家禽肠道健康角度进行研究探讨。也有研究表明，饲粮中添加酵母能够增加肉鸡肠道中乳酸菌的数量、减少沙门氏菌数量，还能产生大量的氨基酸和B族维生素，促进肠道的消化与吸收功能，保证肠道健康^[6]。鉴于维持畜禽肠道健康的重要性，本研究以黄羽肉鸡为试验对象，探讨活性酵母对LPS应激黄羽肉鸡肠道健康的影响，为酵母菌制剂更好地在畜禽养殖中应用提供理论依据。

1 材料与方法 1.1 试验材料

活性酵母的菌株为啤酒酵母，经检测活性为1.2×10¹⁰ CFU/g，干物质含量为93.43%，粗蛋白质含量为45.76%，由法国乐斯福工业有限公司提供；抗生素为硫酸抗敌素(STY1506023)，硫酸黏菌素含量为10%，购自丽珠集团福州福兴医药有限公司；大肠杆菌(Escherichia coli)LPS，血清型为O127 : B8，购自美国Sigma公司，现用现配；灭菌生理盐水注射液浓度为500 μg/mL。

1.2 试验动物与试验设计

选取480羽1日龄新广黄K99黄羽肉公鸡，随机分成6个组，分别为抗生素组(基础饲粮+0.025‰抗生素)、抗生素+LPS组(基础饲粮+0.025‰抗生素，注射LPS)、0.05%活性酵母组(基础饲粮+0.05%活性酵母)、0.05%活性酵母+LPS组(基础饲粮+0.05%活性酵母，注射LPS)、0.50%活性酵母组(基础饲粮+0.50%活性酵母)和0.50%活性酵母+LPS组(基础饲粮+0.50%活性酵母，注射LPS)，每组8个重复，每个重复10只鸡，各组试验鸡初始体重无显著差异(P > 0.05)。抗生素+LPS组、0.05%活性酵母+LPS组和0.50%活性酵母+LPS组的试验鸡于21、23、25和27日龄08:00—09:00每只鸡肌肉注射2 mL 0.2 mg/mL LPS(LPS溶解在蒸馏水中)，其余试验鸡肌肉注射等量生理盐水。于21和27日龄注射LPS或生理盐水后2、4、6、8、10、12和24 h，每重复取1只鸡，用玻璃棒水银柱式兽用体温计插入试验鸡直肠约3 cm处，5 min后读数，用于直肠温度测定。常规饲养管理。试验期56 d。

1.3 试验饲粮

基础饲粮参照NRC(1994) 和《鸡饲养标准》(NY/T 33—2004) 推荐的营养水平配制，基础饲粮组成及营养水平见表 1。

表 1 基础饲粮组成及营养水平(风干基础) Table 1 Composition and nutrient levels of basal diets (air-dry basis)

1.4 测定指标与方法 1.4.1 肠道食糜微生物数量

于试验鸡27、35和56日龄时，每重复取1只鸡屠宰，取回肠的后1/2段及盲肠中食糜，用生理盐水冲食糜于铝盒中，再将处理好的样品放置于-20 ℃冰箱保存。采用平板涂布法测定回肠和盲肠食糜中微生物数量，酵母菌采用马铃薯葡萄糖琼脂(PDA)培养基有氧培养72 h进行计数，大肠杆菌采用麦康凯培养基有氧培养24 h进行计数，乳酸杆菌和双歧杆菌分别用乳酸细菌(MRS)培养基和溶菌肉汤(BL)培养基厌氧培养48 h进行计数。

1.4.2 肠道形态结构与肠道细胞凋亡指数

取27、35和56日龄试验鸡的十二指肠、空肠和回肠中段1 cm肠段，小心去除肠道内食糜后，固定于4%多聚甲醛溶液中，再将固定好的肠道组织经脱水→透明→浸蜡→包埋→修块→切片→展开→贴片等一系列处理后，用苏木精-伊红(HE)染色制成组织切片，在显微镜下测量每个组织切片上10根最长绒毛的高度，以其均值作为相应的绒毛高度(VH)和隐窝深度(CD)，并计算绒毛高度/隐窝深度(VH/CD)。肠道细胞凋亡采用Tunel原位凋亡检测法，其原理是细胞凋亡的发生使内源性核酸酶激活，DNA的一条链出现缺口，产生一系列3′-OH末端，在脱氧核糖核甘酸末端转移酶作用下，用生物素-dUTP标记组织细胞原位DNA切口。将包埋的肠道组织经脱蜡脱水→酶解→标记→信号转化和分析→复染等处理后，计算21和56日龄试验鸡的肠道细胞凋亡指数。

1.5 统计分析

采用SPSS 17.0统计软件中的双因素方差分析(two-way ANOVA)分析活性酵母和LPS 2个主效应，用GLM程序分析它们之间的互作关系。差异显著时用Duncan氏法进行多重比较，以P < 0.05作为差异显著性的判断标准。其中活性酵母和LPS为全模型的2个固定效应与互作效应，交互作用显著时，采用单因素方差分析(one-way ANOVA)进行分析。

2 结果 2.1 活性酵母对LPS应激黄羽肉鸡直肠温度的影响

由表 2可知，与注射生理盐水相比，LPS刺激显著提高了21日龄注射后2 h和27日龄注射后2和4 h黄羽肉鸡的直肠温度(P < 0.05)，显著降低了21日龄注射后12 h和27日龄注射后8 h黄羽肉鸡的直肠温度(P < 0.05)；饲粮中添加活性酵母对黄羽肉鸡的直肠温度无显著影响(P > 0.05)；饲粮中添加活性酵母与肌肉注射LPS对黄羽肉鸡的直肠温度无显著交互作用(P > 0.05)。

表 2 活性酵母对LPS应激黄羽肉鸡直肠温度的影响 Table 2 Effects of active yeast on the rectal temperature of yellow-feathered broilers challenged by LPS

℃
组别 Groups		21日龄注射后时间Time after injection aged at 21 days							27日龄注射后时间Time after injection aged at 27 days
组别 Groups		2 h	4 h	6 h	8 h	10 h	12h	24 h	2 h	4 h	6 h	8 h	10 h	12h	24 h
抗生素组 Antibiotic group		41.24	41.30	41.35	41.40	41.65	41.36	41.25	41.14	40.93	41.49	41.55	41.33	41.30	40.93
抗生素+LPS组 Antibiotic+LPS group		41.39	41.43	41.40	41.55	41.64	41.34	41.17	41.31	41.23	41.43	41.23	41.21	41.18	40.84
0.05%活性酵母组 0.05% active yeast group		41.24	41.33	41.20	41.52	41.30	41.43	41.33	41.25	40.83	41.56	41.44	41.25	41.20	40.83
0.05%活性酵母+LPS组 0.05% active yeast+LPS group		41.38	41.33	41.22	41.48	41.61	41.20	41.30	41.41	41.21	41.55	41.24	41.23	41.28	40.91
0.50%活性酵母组 0.50% active yeast group		41.22	41.30	41.27	41.26	41.38	41.38	41.16	41.09	40.86	41.34	41.49	41.28	41.38	40.86
0.50%活性酵母+LPS组 0.50% active yeast+LPS group		41.41	41.56	41.37	41.36	41.46	41.25	41.14	41.30	41.59	41.59	41.13	41.34	41.20	40.91
SEM		0.04	0.05	0.04	0.04	0.04	0.04	0.03	0.03	0.05	0.03	0.04	0.06	0.02	0.04
主效应Main effect
活性酵母Active yeast	抗生素Antibiotic	41.31	41.38	41.38	41.50	41.64	41.35	41.19	41.23	41.08	41.45	41.35	41.25	41.22	40.89
	0.05%	41.31	41.33	41.21	41.49	41.50	41.28	41.31	41.33	41.02	41.56	41.32	41.23	41.25	40.87
	0.50%	41.32	41.48	41.31	41.31	41.43	41.31	41.15	41.19	41.23	41.45	41.29	41.32	41.27	40.89
脂多糖	-	41.23^b	41.31	41.27	41.38	41.39	41.39^a	41.24	41.16^b	40.87^b	41.46	41.49^a	41.26	41.29	40.87
LPS	+	41.39^a	41.43	41.34	41.46	41.57	41.27^b	41.20	41.34^a	41.34^a	41.52	41.20^b	41.26	41.22	40.89
P值P-value
活性酵母Active yeast		0.991	0.664	0.205	0.158	0.055	0.631	0.099	0.058	0.107	0.386	0.664	0.616	0.644	0.981
脂多糖LPS		0.040	0.191	0.442	0.436	0.115	0.003	0.467	< 0.001	0.001	0.388	0.001	0.695	0.137	0.807
活性酵母×脂多糖Active yeast×LPS		0.961	0.546	0.883	0.664	0.238	0.093	0.888	0.906	0.085	0.145	0.655	0.467	0.105	0.649
同列数据肩标相同或无字母表示差异不显著(P＞0.05)，不同小写字母表示差异显著(P < 0.05)，不同大写字母表示差异极显著(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.

表 2 活性酵母对LPS应激黄羽肉鸡直肠温度的影响 Table 2 Effects of active yeast on the rectal temperature of yellow-feathered broilers challenged by LPS

2.2 活性酵母对LPS应激黄羽肉鸡肠道细胞凋亡指数的影响

由表 3可知，与注射生理盐水相比，LPS应激显著提高了27日龄黄羽肉鸡的十二指肠和回肠细胞凋亡指数(P < 0.05)；LPS应激对56日龄黄羽肉鸡的十二指肠、空肠和回肠细胞凋亡指数均无显著影响(P > 0.05)。饲粮中添加活性酵母对黄羽肉鸡的肠道细胞凋亡指数无显著影响(P > 0.05)。饲粮中添加活性酵母与肌肉注射LPS对黄羽肉鸡的肠道细胞凋亡指数无显著交互作用(P > 0.05)。

表 3 活性酵母对LPS应激黄羽肉鸡肠道细胞凋亡指数的影响 Table 3 Effects of active yeast on the apoptotic index of intestinal cell of yellow-feathered broilers challenged by LPS

组别 Groups		27日龄Aged at 27 days			56日龄Aged at 56 days
组别 Groups		十二指肠Duodenum	空肠Jejunum	回肠Ileum	十二指肠Duodenum	空肠Jejunum	回肠Ileum
抗生素组Antibiotic group		25.00	21.00	23.67	35.67	17.00	28.33
抗生素+LPS组Antibiotic+LPS group		36.67	18.00	36.00	33.67	17.33	32.00
0.05%活性酵母组0.05% active yeast group		29.67	19.00	32.33	27.67	20.33	38.67
0.05%活性酵母+LPS组 0.05% active yeast+LPS group		37.00	20.00	32.33	31.00	15.00	36.33
0.50%活性酵母组 0.50% active yeast group		25.00	18.33	29.67	32.00	17.67	36.33
0.50%活性酵母+LPS组 0.50% active yeast+LPS group		37.33	17.67	41.00	36.33	15.67	42.00
SEM		1.67	0.77	1.74	1.59	1.29	1.60
主效应Main effect
活性酵母 Active yeast	抗生素Antibiotic	30.83	19.50	29.83	34.67	17.17	30.17
	0.05%	33.33	19.50	32.33	29.33	17.67	37.50
	0.50%	31.17	18.00	35.33	34.17	16.67	39.17
脂多糖LPS	-	26.56^b	19.44	28.56^b	31.78	18.33	34.44
脂多糖LPS	+	37.00^a	18.56	36.44^a	33.67	16.00	36.78
P值P-value
活性酵母Active yeast		0.661	0.713	0.301	0.392	0.961	0.053
脂多糖LPS		0.001	0.610	0.014	0.586	0.437	0.427
活性酵母×脂多糖Active yeast×LPS		0.661	0.637	0.171	0.720	0.731	0.508

表 3 活性酵母对LPS应激黄羽肉鸡肠道细胞凋亡指数的影响 Table 3 Effects of active yeast on the apoptotic index of intestinal cell of yellow-feathered broilers challenged by LPS

2.3 活性酵母对LPS应激黄羽肉鸡肠道食糜微生物数量的影响

由表 4、5、6可知，与抗生素相比，饲粮中添加0.05%和0.50%的活性酵母显著提高了27、35和56日龄黄羽肉鸡的回肠食糜酵母菌数量(P < 0.05)；与注射生理盐水相比，LPS应激对黄羽肉鸡的回肠和盲肠食糜酵母菌、乳酸杆菌、双歧杆菌、大肠杆菌数量无显著影响(P > 0.05)；饲粮中添加活性酵母与肌肉注射LPS对黄羽肉鸡的肠道食糜微生物数量无显著交互作用(P > 0.05)。

表 4 活性酵母对LPS应激黄羽肉鸡27日龄肠道食糜微生物数量的影响 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)
组别 Groups		回肠Ileum				盲肠Cecum
组别 Groups		酵母菌Saccharo-mycetes	乳酸杆菌Lacto-bacillus	双歧杆菌Bifido-bacterium	大肠杆菌Escherichia coli	酵母菌Saccharo-mycetes	乳酸杆菌Lacto-bacillus	双歧杆菌Bifido-bacterium	大肠杆菌Escherichia coli
抗生素组 Antibiotic group		3.64	5.58	6.57	4.43	4.45	6.43	6.14	5.38
抗生素+LPS组 Antibiotic+LPS group		3.31	5.64	6.74	4.54	4.62	6.74	6.75	5.72
0.05%活性酵母组 0.05% active yeast group		4.65	5.54	6.53	4.56	4.48	6.61	6.41	5.83
0.05%活性酵母+LPS组 0.05% active yeast + LPS group		4.45	5.52	6.42	4.71	4.59	6.62	6.21	5.70
0.50%活性酵母组 0.50% active yeast group		4.71	5.61	6.86	4.86	4.44	6.73	6.27	5.36
0.50%活性酵母+LPS组 0.50% active yeast+ LPS group		4.69	5.47	6.50	4.66	4.74	6.40	6.47	5.65
SEM		0.10	0.06	0.06	0.06	0.06	0.06	0.08	0.05
主效应Main effect
活性酵母 Active yeast	抗生素Antibiotic	3.47^b	5.61	6.65	4.48	4.54	6.58	6.43	5.56
	0.05%	4.56^a	5.53	6.48	4.63	4.53	6.62	6.31	5.76
	0.50%	4.70^a	5.54	6.68	4.76	4.59	6.57	6.37	5.51
脂多糖LPS	-	4.32	5.58	6.65	4.62	4.47	6.59	6.28	5.52
LPS	+	4.14	5.54	6.55	4.63	4.65	6.59	6.47	5.69
P值P-value
活性酵母 Active yeast		0.001	0.842	0.276	0.115	0.941	0.942	0.771	0.074
脂多糖LPS		0.125	0.768	0.367	0.874	0.116	0.976	0.189	0.088
活性酵母×脂多糖 Active yeast×LPS		0.553	0.797	0.153	0.347	0.802	0.093	0.103	0.098

表 5 活性酵母对LPS应激黄羽肉鸡35日龄肠道食糜微生物数量的影响 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)
组别 Groups		回肠Ileum				盲肠Cecum
组别 Groups		酵母菌Saccharo-mycetes	乳酸杆菌Lacto-bacillus	双歧杆菌Bifido-bacterium	大肠杆菌Escherichia coli	酵母菌Saccharo-mycetes	乳酸杆菌Lacto-bacillus	双歧杆菌Bifido-bacterium	大肠杆菌Escherichia coli
抗生素组Antibiotic group		3.67	5.43	6.27	4.84	4.37	5.26	5.88	5.70
抗生素+LPS组 Antibiotic+LPS group		3.47	5.63	6.29	4.65	3.89	5.97	5.89	5.67
0.05%活性酵母组 0.05% active yeast group		4.51	5.20	6.60	4.79	4.49	5.82	5.97	5.51
0.05%活性酵母+LPS组 0.05% active yeast+ LPS group		4.59	5.52	6.16	4.67	4.47	5.44	6.04	4.91
0.50%活性酵母组 0.50% active yeast group		4.71	5.54	6.32	4.61	4.56	5.52	5.78	5.24
0.50%活性酵母+LPS组 0.50% active yeast+ LPS group		4.61	5.39	6.68	4.91	4.52	5.75	6.23	5.14
SEM		0.09	0.09	0.09	0.06	0.09	0.09	0.11	0.11
主效应Main effect
活性酵母Active yeast	抗生素 Antibiotic	3.56^b	5.53	6.28	4.74	4.13	5.59	5.88	5.69
	0.05%	4.55^a	5.36	6.38	4.73	4.48	5.60	6.00	5.21
	0.50%	4.66^a	5.46	6.50	4.75	4.54	5.64	6.01	5.19
脂多糖	-	4.31	5.39	6.40	4.74	4.47	5.50	5.88	5.48
LPS	+	4.22	5.51	6.38	4.73	4.29	5.71	6.05	5.22
P值P-value
活性酵母Active yeast		＜0.001	0.737	0.633	0.974	0.141	0.993	0.891	0.143
脂多糖LPS		0.430	0.495	0.921	0.969	0.329	0.278	0.459	0.276
活性酵母×脂多糖 Active yeast×LPS		0.451	0.529	0.225	0.207	0.512	0.053	0.724	0.526

表 6 活性酵母对LPS应激黄羽肉鸡56日龄肠道食糜微生物数量的影响 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)
组别 Groups		回肠Ileum				盲肠Cecum
组别 Groups		酵母菌Saccharo-mycetes	乳酸杆菌Lacto-bacillus	双歧杆菌Bifido-bacterium	大肠杆菌Escherichia coli	酵母菌Saccharo-mycetes	乳酸杆菌Lacto-bacillus	双歧杆菌Bifido-bacterium	大肠杆菌Escherichia coli
抗生素组 Antibiotic group		3.51	5.48	6.26	4.41	4.54	6.08	6.32	5.55
抗生素+LPS组 Antibiotic+LPS group		3.03	5.59	6.25	4.92	3.95	6.22	6.07	5.76
0.05%活性酵母组 0.05% active yeast group		4.46	5.51	6.53	4.12	4.46	6.39	6.26	5.47
0.05%活性酵母+LPS组 0.05% active yeast+ LPS group		4.43	5.36	6.40	4.67	4.30	6.28	6.11	5.51
0.50%活性酵母组 0.50% active yeast group		4.34	5.65	6.38	4.77	4.06	6.43	6.57	5.74
0.50%活性酵母+LPS组 0.50% active yeast+ LPS group		4.16	5.70	6.11	4.70	4.45	6.43	6.20	5.70
SEM		0.11	0.06	0.08	0.10	0.10	0.08	0.09	0.09
主效应Main effect
活性酵母 Active yeast	抗生素Antibiotic	3.27^b	5.54	6.26	4.66	4.25	6.15	6.19	5.65
	0.05%	4.45^a	5.44	6.46	4.40	4.38	6.34	6.19	5.49
	0.50%	4.25^a	5.68	6.25	4.73	4.26	6.43	6.38	5.72
脂多糖	-	4.10	5.55	6.39	4.43	4.35	6.30	6.38	5.59
LPS	+	3.87	5.55	6.25	4.76	4.23	6.31	6.13	5.65
P值P-value
活性酵母Active yeast		＜0.001	0.272	0.478	0.306	0.852	0.421	0.631	0.607
脂多糖LPS		0.156	0.999	0.397	0.078	0.577	0.951	0.182	0.731
活性酵母×脂多糖 Active yeast×LPS		0.529	0.669	0.800	0.309	0.178	0.842	0.895	0.874

2.4 活性酵母对LPS应激黄羽肉鸡肠道形态结构的影响

由表 7、8、9可知，与抗生素相比，饲粮中添加0.50%活性酵母显著提高了35日龄黄羽肉鸡的空肠绒毛高度(P < 0.05)；与注射生理盐水相比，LPS应激显著提高了56日龄黄羽肉鸡的十二指肠隐窝深度(P < 0.05)；饲粮中添加活性酵母与肌肉注射LPS对黄羽肉鸡的十二指肠、空肠和回肠绒毛高度、隐窝深度和绒毛高度/隐窝深度无显著交互作用(P > 0.05)。

表 7 活性酵母对LPS应激黄羽肉鸡27日龄肠道形态结构的影响 Table 7 Effects of active yeast on the intestinal morphology of yellow-feathered broilers aged at 27 days challenge by LPS

组别 Groups		十二指肠Duodenum			空肠Jejunum			回肠Ileum
组别 Groups		绒毛高度VH/μm	隐窝深度CD/μm	绒毛高度/隐窝深度VH/CD	绒毛高度VH/μm	隐窝深度CD/μm	绒毛高度/隐窝深度VH/CD	绒毛高度VH/μm	隐窝深度CD/μm	绒毛高度/隐窝深度VH/CD
抗生素组 Antibiotic group		382.1	293.3	1.3	249.6	207.38	1.2	211.8	135.0	1.9
抗生素+LPS组 Antibiotic+LPS group		399.2	301.2	1.2	278.2	209.95	1.4	213.9	158.1	1.4
0.05%活性酵母组 0.05% active yeast group		391.9	289.7	1.4	299.2	222.4	1.4	247.4	174.1	1.5
0.05%活性酵母+LPS组0.05% active yeast+LPS group		403.4	288.0	1.5	280.0	211.4	1.4	228.7	147.3	1.5
0.50%活性酵母组0.50% active yeast group		264.3	192.0	1.5	322.4	233.0	1.4	237.7	170.1	1.4
0.50%活性酵母+LPS组0.50% active yeast+LPS group		404.1	296.4	1.3	294.6	225.3	1.2	206.1	137.0	1.4
SEM		16.0	13.6	0.0	8.4	5.4	0.0	5.9	5.5	0.0
活性酵母Active yeast	抗生素Antibiotic	390.6	297.3	1.3	263.9	208.7	1.3	212.8	147.6	1.6
	0.05%	397.6	288.9	1.4	289.1	216.9	1.4	238.1	161.9	1.5
	0.50%	324.2	236.7	1.4	308.5	229.1	1.3	221.9	155.1	1.4
脂多糖	-	337.9	251.7	1.4	290.0	220.9	1.3	232.3	161.2	1.6
LPS	+	402.2	295.2	1.3	284.3	215.5	1.3	216.2	148.1	1.4
P值P-value
活性酵母Active yeast		0.148	0.191	0.096	0.096	0.328	0.721	0.207	0.555	0.346
脂多糖LPS		0.060	0.158	0.468	0.725	0.629	0.887	0.172	0.253	0.193
活性酵母×脂多糖Active yeast×LPS		0.126	0.175	0.546	0.329	0.872	0.474	0.488	0.075	0.215

表 8 活性酵母对LPS应激黄羽肉鸡35日龄肠道形态结构的影响 Table 8 Effects of active yeast on the intestinal morphology of yellow-feathered broilers aged at 35 days challenged by LPS

组别 Groups		十二指肠Duodenum			空肠Jejunum			回肠Ileum
组别 Groups		绒毛高度VH/μm	隐窝深度CD/μm	绒毛高度/隐窝深度VH/CD	绒毛高度VH/μm	隐窝深度CD/μm	绒毛高度/隐窝深度VH/CD	绒毛高度VH/μm	隐窝深度CD/μm	绒毛高度/隐窝深度VH/CD
抗生素组 Antibiotic group		388.6	273.9	1.3	286.5	204.9	1.3	208.0	132.6	1.8
抗生素+LPS组 Antibiotic+LPS group		375.1	284.7	1.4	242.5	213.9	1.3	226.4	160.6	1.5
0.05%活性酵母组 0.05% active yeast group		383.2	314.5	1.3	270.7	210.4	1.3	216.4	175.7	1.4
0.05%活性酵母+LPS组 0.05% active yeast+LPS group		405.4	328.6	1.2	302.3	231.9	1.3	207.7	142.7	1.5
0.50%活性酵母组 0.50% active yeast group		378.6	304.0	1.3	363.0	234.5	1.6	219.4	160.8	1.5
0.50%活性酵母+LPS组 0.50% active yeast+LPS group		366.2	269.8	1.3	302.5	215.2	1.4	227.0	135.7	1.7
SEM		7.7	8.3	0.0	10.7	9.8	0.0	3.7	7.3	0.1
活性酵母Active yeast	抗生素Antibiotic	380.5	278.2	1.3	266.5^b	209.4	1.3	217.2	146.6	1.6
	0.05%	394.3	322.2	1.3	286.5^ab	221.1	1.3	212.9	159.2	1.4
	0.50%	372.4	285.3	1.3	332.8^a	224.8	1.5	223.6	149.4	1.6
脂多糖	-	382.8	296.0	1.3	306.7	216.6	1.5	214.3	156.3	1.6
LPS	+	382.2	295.6	1.3	284.8	220.3	1.3	221.9	147.0	1.5
P值P-value
活性酵母Active yeast		0.526	0.091	0.344	0.019	0.820	0.060	0.503	0.744	0.713
脂多糖LPS		0.941	0.849	0.702	0.208	0.859	0.361	0.451	0.501	0.860
活性酵母×脂多糖Active yeast×LPS		0.588	0.369	0.818	0.116	0.717	0.310	0.352	0.193	0.505

表 9 活性酵母对LPS应激黄羽肉鸡56日龄肠道形态结构的影响 Table 9 Effects of active yeast on the intestinal morphology of yellow-feathered broilers aged at 56 days challenged by LPS

组别 Groups		十二指肠Duodenum			空肠Jejunum			回肠Ileum
组别 Groups		绒毛高度VH/μm	隐窝深度CD/μm	绒毛高度/隐窝深度VH/CD	绒毛高度VH/μm	隐窝深度CD/μm	绒毛高度/隐窝深度VH/CD	绒毛高度VH/μm	隐窝深度CD/μm	绒毛高度/隐窝深度VH/CD
抗生素组 Antibiotic group		1 180.5	177.3	6.7	1 166.6	196.9	6.1	972.7	174.3	5.6
抗生素+LPS组Antibiotic+LPS group		1 244.7	213.1	6.0	1 163.5	204.6	5.7	882.6	169.0	5.3
0.05%活性酵母组0.05% active yeast group		1 183.1	196.6	6.1	1 183.4	224.3	5.4	996.2	204.8	4.9
0.05%活性酵母+LPS组0.05% active yeast+LPS group		1 319.8	202.6	6.6	1 008.6	200.3	4.7	910.3	174.2	5.2
0.50%活性酵母组0.50% active yeast group		1 312.2	186.2	7.0	1 081.2	198.8	5.4	854.4	177.1	5.4
0.50%活性酵母+LPS组0.50% active yeast+LPS group		1 217.8	219.8	5.7	1 014.0	201.4	5.0	830.6	217.1	4.2
SEM		21.9	5.3	0.2	32.0	3.5	0.2	20.5	6.8	0.2
活性酵母Active yeast	抗生素Antibiotic	1 219.0	198.8	6.3	1 164.7	201.5	5.8	918.6	171.1	5.4
	0.05%	1 251.4	199.6	6.3	1 096.0	212.3	5.1	953.2	189.8	5.1
	0.50%	1 265.0	204.6	6.3	1 047.6	190.6	5.2	840.1	201.1	4.7
脂多糖	-	1 230.9	188.0^b	6.6	1 140.9	207.9	5.6	948.9	188.1	5.3
LPS	+	1 260.8	211.9^a	6.1	1 062.0	202.1	5.2	874.5	186.7	4.8
P值P-value
活性酵母Active yeast		0.613	0.833	0.999	0.254	0.259	0.170	0.069	0.312	0.267
脂多糖LPS		0.418	0.021	0.141	0.152	0.508	0.193	0.098	0.919	0.172
活性酵母×脂多糖Active yeast×LPS		0.085	0.411	0.108	0.457	0.139	0.913	0.748	0.104	0.117

3 讨论 3.1 活性酵母对LPS应激黄羽肉鸡直肠温度的影响

LPS作为细菌的一种内毒素，进入动物体后能够刺激产生内生致热原细胞，使其产生和释放内生致热原，进而影响体温调节中枢，最终引起体温升高^[7]。当动物机体接收到应激源刺激后，感受器会发出信号，通过神经传递到低级中枢，再向上传递到下丘脑；下丘脑分泌肾上腺皮质激素释放激素(CRH)，CRH经垂体门脉系统到达垂体前叶，刺激分泌促肾上腺皮质激素(ACTH)；ACTH进入血液循环系统后，最终肾上腺皮质分泌糖皮质激素来调节应激反应^[8]。肌肉注射LPS后2 h黄羽肉鸡的直肠温度显著升高，说明注射LPS成功引发了黄羽肉鸡的免疫应激反应；21日龄注射LPS后4 h黄羽肉鸡的直肠温度已恢复至正常水平，且在注射LPS后12 h出现了温度的逆转性生理调节反应，说明LPS的应激效果已经消除；而27日龄注射LPS后6 h黄羽肉鸡的直肠温度恢复至正常水平，注射LPS后8 h就出现了温度的逆转性生理调节反应，这可能是连续注射LPS导致黄羽肉鸡从急性应激转向了慢性应激，加快了对LPS抗原的清除和机体的调节反应时间轴。Liu等^[9]研究发现，每千克体重注射500 μg LPS后8 h鸡的体温达到最高，12 h后恢复正常。虽然在反应时间上与本试验有一定的差异，但都能达到体温升高的效果，LPS应激对体温的调节机制还有待进一步探究。另外，活性酵母在LPS的免疫应激过程中对黄羽肉鸡的直肠温度无显著影响，说明活性酵母只是作为一种饲料添加剂被肉鸡采食而不会刺激机体引起致热反应。

3.2 活性酵母对LPS应激黄羽肉鸡肠道细胞凋亡的影响

细胞凋亡是机体细胞在一定的病理或生理条件下由多种基因严格控制的细胞自主性死亡，是在细胞受到外界信号刺激后引发的一系列控制开关的开启或关闭的复杂生理过程。LPS是革兰氏阴性菌细胞壁外膜的主要成分，可引起机体的毒性病理活动，促进细胞因子的释放，诱导细胞异常增殖，从而导致细胞凋亡。本试验采用Tunel原位凋亡检测法标记肠道细胞DNA裂解片段末端，进行肠道细胞凋亡评估。试验结果表明，LPS应激显著提高了27日龄黄羽肉鸡的十二指肠和回肠细胞凋亡指数，证明试验成功建立了应激模型，LPS应激造成了肠道损伤。Williams等^[10]研究显示，在小鼠腹部按每千克体重注射LPS≥0.125 mg/kg能导致肠上皮细胞快速凋亡。本试验结果也表明，饲粮中添加活性酵母对黄羽肉鸡的肠道细胞凋亡指数无显著影响，可能是活性酵母维持了有益的肠道微生态环境，增强了肠道黏膜屏障的免疫功能^[11]。

3.3 活性酵母对LPS应激黄羽肉鸡肠道食糜微生物数量的影响

健康动物的肠道寄生着大量的菌群，稳定的微生态环境能协助动物产生免疫反应，对外袭菌群起着生物屏障作用。大肠杆菌、乳酸杆菌和双歧杆菌是肠道内的3种数量较多的正常菌群。本试验结果表明，饲粮中添加0.05%和0.50%的活性酵母显著提高了27、35和56日龄黄羽肉鸡的回肠食糜酵母菌数量，而对回肠和盲肠食糜乳酸杆菌、双歧杆菌和大肠杆菌数量无显著影响。陈生龙^[12]报道，断奶仔猪饲粮中添加2×10⁷ CFU/g的活酵母，可提高肠道内酵母菌的数量，并对肠道微生物菌群结构具有一定的改善作用，与本试验结果相一致。本试验结果也表明，LPS应激有增加56日龄黄羽肉鸡回肠食糜大肠杆菌数量的趋势，这与冯焱等^[13]的研究报道相似。胡友军等^[14]的研究结果表明，早期断奶仔猪饲粮中添加活性酵母可显著降低肠道内容物中大肠杆菌数量。

3.4 活性酵母对LPS应激黄羽肉鸡肠道形态结构的影响

动物肠道的绒毛高度、隐窝深度和绒毛高度/隐窝深度可反映其发育状况和肠道屏障功能^[15-16]，绒毛高度和隐窝深度的变化也是引起肠道功能和吸收机能改变的主要原因^[17-18]。本试验结果表明，LPS应激可显著提高56日龄黄羽肉鸡的十二指肠隐窝深度。赵珂立等^[19]研究报道，给小鼠按每千克体重腹腔注射0.8 mg/kg LPS，肠道隐窝深度显著增加，与本试验结果相一致。范伟等^[20]研究显示，给仔猪按每千克体重注射100 μg/kg LPS，十二指肠、空肠和回肠的隐窝深度均显著增加，表明LPS应激可造成肠道损伤。本试验结果发现，饲粮中添加0.50%活性酵母可显著提高35日龄黄羽肉鸡的空肠绒毛高度，并有提高绒毛高度/隐窝深度的趋势，表明活性酵母可促进肠道绒毛的发育，改善肠道形态结构。张爱武等^[21]研究结果显示，饲粮中添加1 g/kg的活性干酵母(2×10¹⁰ CFU/g)，可显著增加鹌鹑的空肠长度和空肠指数；Sachin等^[22]研究表明，仔猪饲粮中添加啤酒酵母[(2~3)×10⁶ CFU/g]显著提高了肠道绒毛高度与绒毛高度/隐窝深度，本试验结果与此结果相一致。

4 结论

LPS能成功诱导黄羽肉鸡的免疫应激反应；饲粮中添加活性酵母能够提高黄羽肉鸡肠道绒毛高度和食糜酵母菌数量，改善肠道黏膜及菌群结构，但肌肉注射LPS与饲粮中添加活性酵母无显著交互作用。

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