葡萄糖氧化酶对大肠杆菌攻毒肉鸭生长性能、免疫功能及肠道健康的影响

doi:10.3864/j.issn.0578-1752.2021.22.017

摘要/Abstract

摘要：

【目的】探究饲粮中添加葡萄糖氧化酶（GOD）对大肠杆菌攻毒肉鸭生长性能、免疫功能和肠道健康的影响及其作用机制,为寻求预防肉鸭大肠杆菌病的抗生素替代品研究提供思路。【方法】选用144只1日龄健康北京公鸭,随机分为3组,每组6个重复,每重复8只鸭。对照组饲喂基础饲粮,两个试验组分别在基础饲粮中添加30 mg·kg^-1维吉尼亚霉素（抗生素组）或200 U·kg^-1 GOD,于试验第7天分两次对所有鸭口腔灌服0.2 mL大肠杆菌 O₈₈（3×10⁹CFU / mL）,两次攻毒间隔8 h。试验期28 d。【结果】（1）与对照组相比,饲粮中添加抗生素和GOD显著提高攻毒肉鸭1—14日龄平均日增重和平均日采食量（P<0.05）。（2）GOD和抗生素显著降低28日龄攻毒肉鸭血液中白细胞数（P<0.05）,提高血液中淋巴细胞百分比（P<0.05）。此外,GOD显著降低血液中红细胞含量（P<0.05）。（3）GOD和抗生素显著降低14日龄肉鸭血清MDA和28日龄CAT的含量（P<0.05）; GOD显著提高28日龄肉鸭血清T-AOC（P<0.05）,且有降低14日龄CAT的趋势（P=0.087）。（4）抗生素和GOD显著降低14、28日龄肉鸭血清内毒素含量（P<0.05）。（5）GOD和抗生素显著降低肉鸭14、28日龄空肠IL-1β和IL-6浓度（P<0.05）,以及28日龄空肠TNF-α浓度（P<0.05）,两组间无显著差异（P>0.05）。（6）抗生素和GOD显著降低大肠杆菌攻毒肉鸭14、28日龄血清DAO活性和D-LA含量（P<0.05）,两组间无显著差异（P>0.05）。（7）GOD增加了回肠特有OTUs的数量,降低了大肠杆菌含量,提高了乳酸杆菌和双歧杆菌等益生菌的相对丰度。【结论】饲粮中添加GOD通过平衡大肠杆菌攻毒肉鸭的肠道菌群结构、减少细菌内毒素的产生以及降低氧化应激对细胞的损伤来维持肠黏膜的完整性、避免因内毒素进入血液后激活炎症信号通路而引起炎症反应的发生,从而改善肉鸭肠道健康、促进肉鸭生长。GOD能够作为抗生素替代物用于预防或减轻肉鸭的大肠杆菌病。

关键词: 葡萄糖氧化酶, 肉鸭, 大肠杆菌攻毒, 生长性能, 免疫功能, 肠道健康

Abstract:

【Objective】 This experiment was conducted to study the effects of glucose oxidase (GOD) on growth performance, immune functions and intestinal health of ducks challenged by Escherichia coli (E. coli) O₈₈and its mechanism to find a substitute for antibiotics to prevent duck colibacillosis_.【Method】 A total of 144 one-day-old healthy male lean Peking ducklings were selected and randomly divided into three groups with six replicates and eight ducks per replicate. Ducks were fed the three diets supplemented with nothing (Control group), 30 mg·kg^-1virginiamycin (Antibiotic group) and 200 U·kg^-1 GOD in basal diet, respectively. On day 7, all ducks were orally taken 0.2 mL E. coli O₈₈(3×10⁹ CFU/mL) twice, 8 hours apart. The experiment lasted for 28 days. 【Result】The results showed as follows: 1) Compared with the control group, adding antibiotic or GOD in the diet significantly increased the average daily gain and average daily feed intake of 1 to 14-day-old attacking ducks (P<0.01). 2) The number of serum white blood cell of 28-day-old attacking ducks was significantly reduced and the percentage of serum lymphocyte was significantly increased by adding antibiotic and GOD in diet (P<0.01), and the number of serum red blood cell of 28-day- old attacking ducks was significantly reduced by adding GOD in diet (P<0.05). 3) Serum MDA at day 14 (P<0.05) and CAT contents at day 28 (P<0.01) were significantly reduced by adding antibiotic and GOD, serum T-AOC at day 28 was significantly increased (P<0.05), and CAT at day 14 showed a tendency to decrease by adding GOD (P=0.087). 4) Supplementation of antibiotic and GOD significantly decreased endotoxin of 14 and 28-day-old attacking ducks (P<0.01). 5) Adding antibiotic or GOD significantly decreased the concentration of IL-1β and IL-6 of ducks at day 14 and 28 and TNF-α at d 28 (P<0.05), no significant differences between the GOD and the antibiotic groups（P>0.05）. 6) The concentration of serum diamine oxidase and D-lactic of 14 and 28-day-old attacking ducks were significantly reduced by adding antibiotic and GOD (P<0.05), no significant differences between the GOD and the antibiotic groups (P>0.05). 7) The addition of GOD increased the number of unique OTUs in ileum, reduced the contents of E. coli, and increased the relative abundance of probiotics such as Lactobacillus and Bifidobacterium. 【Conclusion】 In conclusion, addition of GOD in the diet can balance the structure of intestinal flora, reduce the production of bacterial endotoxin and decrease oxidative stress, and then maintain the integrity of intestinal mucosa and prevent activation of inflammatory signal pathway by endotoxin and occurrence of inflammatory reaction, so as to improve intestinal health and growth of ducks infected with E. coli. The GOD can be used as antibiotics substitute to prevent or alleviate colibacillosis of ducks.

Key words: glucose oxidase, duck, Escherichia coli challenge, growth performance, immune function, intestinal health

刘娇,陈志敏,郑爱娟,刘国华,蔡辉益,常文环. 葡萄糖氧化酶对大肠杆菌攻毒肉鸭生长性能、免疫功能及肠道健康的影响[J]. 中国农业科学, 2021, 54(22): 4917-4930.

LIU Jiao,CHEN ZhiMin,ZHENG AiJuan,LIU GuoHua,CAI HuiYi,CHANG WenHuan. Effects of Glucose Oxidase on Growth Performance, Immune Functions and Intestinal Health of Ducks Challenged by Escherichia coli[J]. Scientia Agricultura Sinica, 2021, 54(22): 4917-4930.

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组别Groups		攻毒剂量Dosage (mL)	菌液接种量 Inoculation quantity(CFU)	攻毒次数Time
T1	LB肉汤培养基	1	-	1
T2	大肠杆菌O88 E.coli O88	0.05	0.5×10⁸	1
T3	大肠杆菌O88 E.coli O88	0.05	0.5×10⁸	2
T4	大肠杆菌O88 E.coli O88	0.10	1×10⁸	1
T5	大肠杆菌O88 E.coli O88	0.10	1×10⁸	2
T6	大肠杆菌O88 E.coli O88	0.20	3×10⁸	1
T7	大肠杆菌O88 E.coli O88	0.20	3×10⁸	2
T8	大肠杆菌O88 E.coli O88	0.60	6×10⁸	1
T9	大肠杆菌O88 E.coli O88	0.60	6×10⁸	2
T10	大肠杆菌O88 E.coli O88	1.00	1×10⁹	1
T11	大肠杆菌O88 E.coli O88	1.00	1×10⁹	2

项目 Items	1—14 日龄 1 to 14 days of age (%)	15—28 日龄 15 to 28 days of age (%)
原料 Ingredients
玉米 Corn	58.90	67.98
豆粕 Soybean meal	32.42	22.78
棉籽粕 Cottonseed meal	2.00	4.00
大豆油 Soybean oil	2.28	1.10
石粉 Limestone	1.07	1.14
磷酸氢钙 CaHPO₄	1.71	1.49
食盐 NaCl	0.30	0.30
L-赖氨酸 L-Lys	0.17	0.09
DL-蛋氨酸 DL-Met	0.14	0.12
维生素预混料 Vitamin premix¹⁾	0.03	0.03
微量元素预混料 Microelement premix²⁾	0.10	0.10
沸石粉 Zeolite powder	0.87	0.87
合计 Total	100.00	100.00
营养水平 Nutrient level
代谢能 ME (MJ·kg^-1)	12.14	12.14
粗蛋白质 CP	20.00	17.50
钙 Ca	0.90	0.85
总磷 TP	0.65	0.60
有效磷 AP	0.45	0.40
赖氨酸 Lys	1.10	0.85
蛋氨酸 Met	0.45	0.40
蛋氨酸+半胱氨酸 Met + Cys	0.80	0.71

目标基因 Gene name	引物序列（5′-3′） Primers sequence (5′-3′) ^*
大肠杆菌 Escherichia coli	F: GTTAATACCTTTGCTCATTGA
大肠杆菌 Escherichia coli	R: ACCAGGGTATCTAATCCTGTT

项目 Items	对照组 Control group	抗生素组 Antibiotic group	葡萄糖氧化酶组 Glucose oxidase group	标准误 SEM	P值 P-value
1—14 日龄 1 to 14 days of age
14日龄体重BW at 14 days (kg)	0.60	0.62	0.64	0.006	0.178
平均日增重ADG (g)	38.17^Bb	41.17^Aa	42.27^Aa	0.558	<0.001
平均日采食量ADFI (g)	50.21^Bb	54.32^Aa	54.93^Aa	0.691	<0.001
料重比F / G	1.32	1.32	1.30	0.009	0.674
14—28日龄14 to 28 days of age
平均日增重ADG (g)	77.96	80.11	81.26	1.528	0.697
平均日采食量ADFI (g)	137.67	140.62	139.81	1.847	0.817
料重比F / G	1.77	1.76	1.73	0.016	0.614
1—28 日龄 1 to 28 days of age
28日龄体重BW at 28 days (kg)	1.70	1.74	1.77	0.025	0.525
平均日增重ADG (g)	58.94	60.32	61.43	0.889	0.549
平均日采食量ADFI (g)	92.20	94.92	94.36	1.043	0.560
料重比F / G	1.57	1.58	1.54	0.011	0.404

项目 Items	对照组 Control group	抗生素组 Antibiotic group	葡萄糖氧化酶组 Glucose oxidase group	标准误 SEM	P值 P-value
14日龄 14 days of age
总抗氧化能力T-AOC (U·mL^-1)	23.27	18.96	20.17	0.875	0.118
总超氧化物歧化酶T-SOD (U·mL^-1)	107.02	111.92	114.58	2.199	0.394
丙二醛MDA (nmol·mL^-1)	5.79a	5.12b	5.13b	0.117	0.030
过氧化氢酶CAT (U·mL^-1)	4.27	4.01	2.94	0.276	0.087
28日龄 28 days of age
总抗氧化能力T-AOC (U·mL^-1)	16.68b	19.76ab	22.57a	0.887	0.015
总超氧化物歧化酶T-SOD (U·mL^-1)	116.58	117.80	121.43	2.615	0.790
丙二醛MDA (nmol·mL^-1)	4.75	4.24	4.65	0.164	0.440
过氧化氢酶CAT (U/·mL^-1)	5.14Aa	3.59Bb	2.62Bb	0.335	0.004