Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 8: 5069 - 5084

DOI: https://doi.org/10.12418/CJAN2023.470

RESEARCH PAPER

Effects of Mulberry Leaf Water Extract on Growth Performance, Antioxidant Capacity and Intestinal Health of Broilers Challenged with Escherichia coli

  • LIU Chunming , 1 ,
  • HUANG Hui 1 ,
  • LI Yanwu 2 ,
  • CHEN Yulian 1 ,
  • MENG Tiantian 1 ,
  • ZHONG Ruyi 1 ,
  • ZHOU Yingjun 3 ,
  • ZOU Shengwen 4 ,
  • HE Wenxiang 1 ,
  • XIAO Dingfu , 1, *
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  • 1 College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China
  • 2 College of Animal Science and Technology, Shandong Veterinary and Animal Husbandry Vocational College, Weifang 261061, China
  • 3 Hunan Geneham Pharmaceutical Co., Ltd., Changsha 410014, China
  • 4 Hunan Wenfeng Agricultural and Animal Husbandry Technology Co., Ltd., Loudi 417700, China
*professor, E-mail:

Received date: 2023-02-13

  Online published: 2023-08-10

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Abstract

This experiment was conducted to investigate the effects of dietary supplementation of mulberry leaf water extract on growth performance, antioxidant capacity, intestinal health of broilers challenged with Escherichia coli. Two hundred and fifty-six 8-day-old female yellow-feathered broilers were randomly divided into 4 groups with 8 replicates per group and 8 broilers per replicate. Broilers in control group (CON group) and Escherichia coli challenge group (EC group) were fed a basal diet; broilers in mulberry leaf water extract group (MWE group) and mulberry leaf water extract+Escherichia coli challenge group (ECMWE group) were fed the basal diet supplemented with 12 00 mg/kg mulberry leaf water extract. The experimental period was 28 days (8 to 35 days of age). At 33 to 35 days of age, broilers in the EC group and ECMWE group were given 2 mL Escherichia coli K88 bacterial solution (1.0×109 CFU/mL), while broilers in the CON group and MWE group were given 2 mL sterile saline. The results showed as follows: 1) after the test broilers were challenged with Escherichia coli, the average daily feed intake (ADFI) of EC group was significantly decreased (P<0.05), and the diarrhea rate was significantly increased (P<0.05). Adding mulberry leaf water extract could extremely significantly increase the average daily gain (ADG) (P<0.01), and extremely significantly reduce the feed/gain ratio (F/G) and diarrhea rate of broilers (P<0.01). Adding mulberry leaf water extract and Escherichia coli challenge showed a extremely significant interaction on ADFI of broilers (P<0.01), and showed a trend of interaction on diarrhea rate of broilers (P=0.07). 2) After Escherichia coli challenge, the activity of glutathione peroxidase (GSH-Px) in serum was significantly decreased (P<0.05), and the activity of catalase (CAT) in serum was extremely significantly decreased (P<0.01). Adding mulberry leaf water extract tended to increase serum CAT activity of broilers (P=0.09). 3) Adding mulberry leaf water extract significantly increased the jejunal villus height/crypt depth (V/C) ratio of broilers (P<0.05). 4) Adding mulberry leaf water extract and Escherichia coli challenge both could obviously change the cecal microflora structure of broilers. At the phylum level, adding mulberry leaf water extract tended to reduce the relative abundance of Firmicutes (P=0.09), and tended to increase the relative abundance of Proteobacteria (P=0.08); at the genus level, adding mulberry leaf water extract could significantly increase the relative abundances of Alistipes, Streptococcus and Lachnospiraceae_UCG-004 (P<0.05), and extremely significantly decrease the relative abundances of Ruminococcaceae_UCG-011 and Ruminococcaceae_UCG-013 (P<0.01). There was a significant interaction between adding mulberry leaf water extract and Escherichia coli challenge in the relative abundances of Shuttleworthia, Peptococcus and Lachnospiraceae_UCG-004 (P<0.05). In summary, it can be seen that adding 1 200 mg/kg mulberry leaf water extract to the diet of broilers can reduce the diarrhea rate, improve the growth performance, enhance the antioxidant capacity, improve the intestinal morphology and cecal microflora structure of broilers, and then effectively alleviate the adverse effects of Escherichia coli challenge on broilers.

Key words： mulberry leaf water extract; Escherichia coli; broilers; growth performance; antioxidant capacity; intestinal microflora

Cite this article

LIU Chunming , HUANG Hui , LI Yanwu , CHEN Yulian , MENG Tiantian , ZHONG Ruyi , ZHOU Yingjun , ZOU Shengwen , HE Wenxiang , XIAO Dingfu . Effects of Mulberry Leaf Water Extract on Growth Performance, Antioxidant Capacity and Intestinal Health of Broilers Challenged with Escherichia coli[J]. Chinese Journal of Animal Nutrition, 2023 , 35(8) : 5069 -5084 . DOI: 10.12418/CJAN2023.470

由大肠杆菌引起的禽类大肠杆菌病是一种重要的感染性疾病,在集约化肉鸡养殖业中尤为常见[1]。这种细菌可以通过受污染的饲料、水和粉尘传播,使肉鸡生长性能下降和死亡率增加,给养殖业造成重大的经济损失[2]。抗生素通常用于治疗肉鸡细菌性疾病,而随着抗药性细菌的出现以及动物产品中抗生素残留的问题,限制了抗生素的使用[3]。因此,一些中草药提取物替代抗生素的研究为畜牧业发展带来了新的突破点。桑叶作为一种重要的药食同源植物资源,具有无残留、低毒副作用和无耐药性等特点,富含蛋白质、碳水化合物、维生素和矿物质等营养物质,具有抗氧化、抗炎、降糖等作用[4⇓-6]。桑叶是一种优质的蛋白饲料资源,在饲粮中添加可增强畜禽的免疫力[7],调节脂肪代谢[8],改善畜产品质量[9]。研究发现,由于桑叶中较高的粗纤维含量以及单宁等抗营养因子的存在,过量添加会影响畜禽的生产性能和健康[10]。而桑叶经提取加工后不仅能够有效降低桑叶中的粗纤维含量,还可以极大程度地保留其生物活性成分[11]。为探究桑叶水提物对肉鸡的影响,本试验以黄羽肉鸡为试验对象,研究桑叶水提物对大肠杆菌攻毒肉鸡生长性能、抗氧化能力和肠道健康的影响,旨在为桑叶水提物作为新型绿色的潜在替抗饲料添加剂的开发与应用提供科学研究基础。

1 材料与方法

1.1 试验材料

桑叶水提物由湖南某药业有限公司提供,主要成分为桑叶黄酮(≥20%)、多糖(≥25%)、植物蛋白(≥25%)、生物碱(0.03%~0.50%)、叶绿素(3%~5%)、甾醇(≥2%)。桑叶水提物制备方法:将采摘到的新鲜桑叶晒干、剪碎,称取150 kg的干燥桑叶加入到提取罐中,然后加入750 kg的水,蒸汽加热,回流提取1.5 h,过滤;滤渣再次加入600 kg的水,回流提取1.5 h,过滤;将2次的提取滤液浓缩,经喷雾干燥制成桑叶水提取干粉,保存备用。攻毒采用大肠杆菌K88菌液,浓度为1.0×109 CFU/mL。

1.2 试验动物与试验设计

选取健康状况良好、体重为(105.0±2.0) g的8日龄黄羽肉鸡(全母)256羽,随机分为4组,每组8个重复,每个重复8羽。对照组(CON组)和大肠杆菌攻毒组(EC组)饲喂基础饲粮;桑叶水提物组(MWE组)和桑叶水提物+大肠杆菌攻毒组(ECMWE组)在基础饲粮中添加1 200 mg/kg桑叶水提物。桑叶水提物的添加量是根据由前期试验结果[12]确定。试验期28 d(8~35日龄),在33~35日龄进行为期3 d的大肠杆菌攻毒试验。
大肠杆菌攻毒:借鉴本团队前期攻毒肉鸡试验的方法[13],在33~35日龄给需进行攻毒的EC组和ECMWE组鸡群灌服2 mL浓度为1.0×109 CFU/mL的大肠杆菌K88菌液,不攻毒的CON组和MWE组鸡群每天灌服2 mL无菌生理盐水。

1.3 基础饲粮

本试验所用基础饲粮参照《黄羽肉鸡营养需要量》(NY/T 647—2020)进行配制,为粉状饲料,其组成及营养水平见表1。
表1 基础饲粮组成及营养水平(干物质基础)

Table 1 Composition and nutrient levels of basal diets (DM basis) %

项目
Items		 8~28日龄
8 to 28
days of age		 29~35日龄
29 to 35
days of age
原料Ingredients
玉米Corn 62.18 67.49
豆粕Soybean meal 28.00 28.00
膨化大豆Extruded soybean 6.00
大豆油Soybean oil 0.10 1.18
DL-蛋氨酸DL-Met 0.13 0.10
磷酸氢钙CaHPO4 1.48 1.31
石粉Limestone 1.21 1.12
氯化胆碱Choline chloride 0.10
氯化钠NaCl 0.30 0.30
预混料Premix1) 0.50 0.50
合计Total 100.00 100.00
营养水平Nutrient levels2)
代谢能ME/(MJ/kg) 12.40 12.54
粗蛋白质CP 20.49 18.80
赖氨酸Lys 1.13 1.00
蛋氨酸Met 0.46 0.40
项目
Items		 8~28日龄
8 to 28
days of age		 29~35日龄
29 to 35
days of age
钙Ca 1.00 0.90
有效磷AP 0.45 0.40

1)预混料为每千克饲粮提供 The premix provided the following per kg of diets:Cu 10 mg,Fe 90 mg,Mn 90 mg,Zn 50 mg,I 0.4 mg,Se 0.2 mg,Co 0.4 mg,VA 5 000 IU,VD 3 500 IU,VE 10 IU,VB1 1.5 mg,VB6 1.5 mg,VB2 6.0 mg,VB12 10 μg,烟酸nicotinic acid 35 mg,叶酸 folic acid 0.8 mg,泛酸 pantothenic acid 12 mg,生物素biotin 0.8 mg。

2)营养水平均为计算值。Nutrient levels were all calculated values.

1.4 饲养管理

本试验采用4层式立体笼养,将256羽黄羽肉鸡置于同一饲养环境中。鸡舍定时消毒和清洁,保持通风,鸡舍温度保持在(24±2) ℃,肉鸡自由采食和饮水,鸡群按常规程序免疫接种。

1.5 样品采集与处理

在35日龄试验结束时,称取每个重复肉鸡体重。每个重复随机选取1只健康的肉鸡,颈静脉采血20 mL于一次性试管中,37 ℃斜放静置1 h,待血清析出后于3 500 r/min离心10 min,分离血清,装入1.5 mL试管后于-80 ℃保存备用;采血后将肉鸡屠宰,打开腹腔取空肠、回肠肠段中间部位各1~2 cm(尽量控制在相同位置),用0.9%生理盐水轻轻地冲洗后置于4%多聚甲醛溶液固定48 h,对肠道组织制作石蜡切片进行常规苏木精-伊红(HE)染色,用于肠道组织形态分析;取盲肠内容物于2 mL冻存管中,用液氮速冻之后置于-80 ℃保存备用。

1.6 测定指标与方法

1.6.1 生长性能和腹泻率

于肉鸡8、29和35日龄空腹12 h(停料不停水)后测定肉鸡体重,每天统计每个重复的采食量,分别8~28日龄和29~35日龄的计算平均日增重(ADG)、平均日采食量(ADFI)和料重比(F/G)。大肠杆菌攻毒期间,每天记录每组鸡群的腹泻情况,参照赵宪臣[14]的方法,通过观察肉鸡肛门的肮脏程度和粪盘中的拉稀占比,计算每组每天的腹泻率,最终取7 d腹泻率平均值进行统计分析。

1.6.2 血清生化指标

按相应试剂盒(南京建成生物工程研究所)说明书的方法,采用迈瑞BS200全自动生化分析仪检测血清中甘油三酯(TG,单试剂GPO-PAP法)、总胆固醇(TC,单试剂GPO-PAP法)、总蛋白(TP,考马斯亮蓝法)、葡萄糖(GLU,己糖激酶法)、白蛋白(ALB,溴甲酚绿法)、尿素(UREA,脲酶法)、高密度脂蛋白-胆固醇(HDL-C,双试剂直接法)、低密度脂蛋白-胆固醇(LDL-C,双试剂直接法)的含量以及碱性磷酸酶(ALP,微量酶标法)、乳酸脱氢酶(LDH,微板法)、天门冬氨酸氨基转移酶(AST,赖氏法)、丙氨酸氨基转移酶(ALT,赖氏法)的活性。

1.6.3 血清抗氧化指标

按相应试剂盒(南京建成生物工程研究所)说明书的方法,采用酶标仪测定血清中丙二醛(MDA)、一氧化氮(NO)的含量以及总超氧化物歧化酶(T-SOD)、过氧化物酶(POD)、总一氧化氮合成酶(NOS)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)活性与总抗氧化能力(T-AOC)。

1.6.4 肠道组织形态

肠道组织经石蜡包埋、HE染色后,显微镜观察组织切片,在随机多个非连续性视野中选取具有代表性的视野拍成图片,使用YLE-21DY显微成像系统的自带图像分析软件IMAGEEX测量肠道绒毛高度(VH)、隐窝深度(CD),并计算绒毛高度与隐窝深度的比值(V/C)。

1.6.5 盲肠菌群结构和多样性

将-80 ℃保存的肉鸡盲肠内容物送至奥维森科技公司,采用16S rRNA高通量测序技术进行微生物学分析。

1.7 数据处理与分析

试验原始数据用Excel 2010进行预处理,采用统计软件SPSS 21.0通过一般线性模型进行单因素方差分析,然后再进行2×2双因素分析,如存在交互作用和主效应显著,则采用Duncan氏法进行多重比较,盲肠内容物中差异显著菌群使用Metastats分析,结果均以平均值和均值标准误(SEM)表示,P<0.05为差异显著,P<0.01为差异极显著,0.05≤P<0.10为差异有显著趋势。

2 结果与分析

2.1 桑叶水提物对大肠杆菌攻毒肉鸡生长性能的影响

由表2可知,8~28日龄时,与CON组相比,饲粮中添加桑叶水提物能显著提高肉鸡的ADFI(P<0.05),且具有提高肉鸡ADG(P=0.09)和降低F/G的趋势(P=0.07)。
表2 桑叶水提物对8~28日龄肉鸡生长性能的影响

Table 2 Effects of MWE on growth performance of broilers from 8 to 28 days of age

项目
Items		 组别Groups SEM P值
P-value
CON MWE
初始体重Initial BW/g 105.29 105.03 0.35 0.47
终末体重Final BW/g 629.05 645.94 10.64 0.27
平均日采食量ADFI/g 43.50b 44.73a 0.36 0.03
平均日增重ADG/g 24.46 25.75 0.51 0.09
料重比F/G 1.82 1.72 0.04 0.07

同行数据肩标无字母或相同字母者表示差异不显著(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 row, values with no letter or the same letter superscripts mean no significant difference (P>0.05), while with different small letter superscripts mean significant difference (P<0.05), and with different capital letter superscripts mean extremely significant difference (P<0.01). The same as below.

由表3可知,在33~35日龄灌服大肠杆菌对EC组和ECMWE组肉鸡进行攻毒,29~35日龄时,与CON组相比,EC组的ADFI显著降低(P<0.05),腹泻率显著增高(P<0.05)。与EC组相比,添加桑叶水提物可以显著降低大肠杆菌攻毒肉鸡的腹泻率(P<0.05)。添加桑叶水提物极显著提高了肉鸡的ADG(P<0.01),极显著降低了肉鸡的F/G和腹泻率(P<0.01);添加桑叶水提物与大肠杆菌攻毒对肉鸡的ADFI表现出极显著的交互作用(P<0.01),对肉鸡的腹泻率表现出存在交互作用的趋势(P=0.07),提示在大肠杆菌攻毒的情况下,添加桑叶水提物可以提高肉鸡的ADFI并降低腹泻率,提高大肠杆菌攻毒下肉鸡的生长性能。
表3 桑叶水提物对大肠杆菌攻毒肉鸡29~35日龄生长性能和腹泻率的影响

Table 3 Effects of MWE on growth performance and diarrhea rate from 29 to 35 days of age of broilers challenge with Escherichia coli

项目
Items		 组别Groups SEM P值P-value
CON EC MWE ECMWE MWE EC MWE×EC
终末体重Final BW/g 836.19b 831.00b 868.04a 852.86ab 11.42 0.03 0.38 0.67
平均日采食量ADFI/g 83.26a 79.01b 81.19ab 83.50a 0.88 0.88 0.28 <0.01
平均日增重ADG/g 28.40c 27.53bc 35.32a 32.25ab 1.20 <0.01 0.19 0.46
料重比F/G 2.88a 2.94a 2.29b 2.61ab 0.13 <0.01 0.15 0.42
腹泻率Diarrhea rate/% 18.30bc 34.36a 14.29c 22.32b 4.70 <0.01 <0.01 0.07

MWE:添加桑叶水提物效应;EC:大肠杆菌攻毒效应;MWE×EC:添加桑叶水提物与大肠杆菌攻毒的交互作用。下表同。

MWE: the effect of adding MWE; EC: the effect of Escherichia coli challenge; MWE×EC: the incteraction of adding MWE and Escherichia coli challenge. The same as below.

2.2 桑叶水提物对大肠杆菌攻毒肉鸡血清生化指标的影响

由表4可知,添加桑叶水提物有提高肉鸡血清中AST活性的趋势(P=0.05);大肠杆菌攻毒有增加肉鸡血清中UREA含量的趋势(P=0.06),并有降低血清中LDL-C含量的趋势(P=0.08);此外,添加桑叶水提物与大肠杆菌攻毒对肉鸡血清中TC、GLU的含量表现出存在交互作用的趋势(P=0.06),提示在大肠杆菌攻毒的情况下,添加桑叶水提物可能会升高肉鸡血清中TC的含量,降低血清中GLU的含量。大肠杆菌攻毒和添加桑叶水提物对肉鸡血清其余生化指标无显著影响且二者无显著交互作用(P>0.05)。
表4 桑叶水提物对大肠杆菌攻毒肉鸡血清生化指标的影响

Table 4 Effects of MWE on serum biochemical indices of broilers challenged with Escherichia coli

项目
Items		 组别Groups SEM P值P-value
CON EC MWE ECMWE MWE EC MWE×EC
丙氨酸氨基转移酶
ALT/(U/L)		 4.39 4.62 4.50 4.26 0.56 0.99 0.81 0.66
白蛋白ALB/(g/L) 12.78 13.34 13.08 12.95 0.49 0.94 0.72 0.56
尿素UREA/(mmol/L) 0.21 0.24 0.26 0.31 0.08 0.17 0.06 0.97
甘油三酯TG/(mmol/L) 0.29 0.26 0.33 0.23 0.04 0.89 0.19 0.48
总胆固醇TC/(mmol/L) 3.96 3.51 3.37 3.61 0.17 0.17 0.56 0.06
总蛋白TP/(g/L) 29.80 31.18 28.72 28.88 1.36 0.24 0.59 0.67
天门冬氨酸氨基转移酶
AST/(U/L)		 235.32 212.87 245.15 244.77 9.70 0.05 0.26 0.27
碱性磷酸酶ALP/(U/L) 1 671.00 1 822.77 1 509.43 1 651.42 249.94 0.53 0.58 0.99
葡萄糖GLU/(mmol/L) 14.42 13.78 14.32 13.56 0.52 0.24 0.93 0.06
高密度脂蛋白-胆固醇
HDL-C/(mmol/L)		 2.37 2.17 2.06 2.25 0.10 0.48 0.17 0.26
低密度脂蛋白-胆固醇
LDL-C/(mmol/L)		 0.89 0.64 0.78 0.75 0.07 0.94 0.08 0.16
乳酸脱氢酶LDH/(U/L) 1 055.77 1 407.40 1 400.95 1 443.02 150.84 0.39 0.18 0.15

2.3 桑叶水提物对大肠杆菌攻毒肉鸡血清抗氧化指标的影响

由表5可知,大肠杆菌攻毒显著降低了血清中GSH-Px的活性(P<0.05),极显著降低了血清中CAT的活性(P<0.01);添加桑叶水提物有提高肉鸡血清中CAT活性的趋势(P=0.09);添加桑叶水提物与大肠杆菌攻毒对肉鸡血清各抗氧化指标均无显著的交互作用(P>0.05)。多重比较分析显示,MWE组血清中GSH-Px和CAT的活性最高,且显著高于EC组(P<0.05)。
表5 桑叶水提物对大肠杆菌攻毒肉鸡血清抗氧化指标的影响

Table 5 Effects of MWE on serum antioxidant indices of broilers challenged with Escherichia coli

项目
Items		 组别Groups SEM P值P-value
CON EC MWE ECMWE MWE EC MWE×EC
超氧化物歧化酶
SOD/(U/mL)		 115.6 97.39 97.66 102.77 5.96 0.30 0.28 0.06
过氧化物酶POD/(U/mL) 4.73 4.35 4.10 4.82 0.44 0.86 0.71 0.23
总一氧化氮合成酶
NOS/(U/mL)		 15.58 14.73 15.69 16.39 0.82 0.29 0.93 0.35
一氧化氮NO/(nmol/L) 3.55 3.30 3.13 3.11 0.44 0.50 0.75 0.79
总抗氧化能力
T-AOC/(U/mL)		 0.67 0.66 0.67 0.68 0.02 0.42 0.88 0.53
谷胱甘肽过氧化物酶
GSH-Px/(U/mL)		 2 878.44ab 2 697.58b 3 004.72a 2 750.44ab 78.20 0.26 0.01 0.64
丙二醛MDA/(nmol/mL) 3.68 4.83 2.17 3.90 1.06 0.26 0.18 0.79
过氧化氢酶CAT/(U/mL) 4.05ab 2.42c 5.21a 3.04bc 0.51 0.09 <0.01 0.60

2.4 桑叶水提物对大肠杆菌攻毒肉鸡肠道组织形态的影响

35日龄时各组肉鸡空肠、回肠组织形态学如图1所示。由表6可知,大肠杆菌攻毒对肉鸡空肠和回肠绒毛高度、隐窝深度以及绒毛高度与隐窝深度的比值无显著影响(P>0.05),且与添加桑叶水提物在这些指标上无显著交互作用(P>0.05),而添加桑叶水提物则显著增加了肉鸡空肠绒毛高度与隐窝深度的比值(P<0.05),同时有增加空肠绒毛高度(P=0.06)和降低空肠与回肠隐窝深度的趋势(P=0.08)。
图1 35日龄肉鸡空肠、回肠组织形态学(HE染色)

CON:CON组 CON group;EC:EC组 EC group;MWE:MWE组 MWE group;ECMWE:ECMWE组 ECMWE group。下图同 the same as below。

Fig.1 Histomorphology of jejunum and ileum of 35-day-old broilers (HE staining, 40×)

表6 桑叶水提物对大肠杆菌攻毒肉鸡肠道组织形态的影响

Table 6 Effects of MWE on intestinal morphology of broilers challenged with Escherichia coli

项目
Items		 组别Groups SEM P值P-value
CON EC MWE ECMWE MWE EC MWE×EC
空肠Jejunum
绒毛高度Villus height/μm 1 127.11 1 152.59 1 227.48 1 336.84 65.28 0.06 0.39 0.63
隐窝深度Crypt depth/μm 193.22 205.39 155.10 172.96 19.23 0.08 0.45 0.88
绒毛高度与隐窝深度的比值
V/C		 5.95b 6.20b 7.67ab 8.10a 0.82 0.04 0.68 0.91
回肠Ileum
绒毛高度Villus height/μm 1 101.21 1 062.21 1 121.26 1 160.42 144.51 0.69 0.99 0.79
隐窝深度Crypt depth/μm 193.22 205.39 155.10 172.72 19.23 0.08 0.45 0.88
绒毛高度与隐窝深度的比值
V/C		 5.92 5.24 6.83 7.26 1.00 0.17 0.90 0.59

2.5 桑叶水提物对大肠杆菌攻毒肉鸡盲肠菌群的影响

2.5.1 基于操作分类单元(OTU)的Venn图

由图2可知,在97%相似性水平下,各组肉鸡盲肠菌群所共有的OTU有943个;CON组盲肠菌群OTU数目是1 490个,特有OTU数目为86个;EC组盲肠菌群OTU数目为1 444个,特有OTU数目为59个;ECMWE组盲肠菌群OTU数目为1 457个,特有OTU数目为42个;MWE组盲肠菌群OTU数目为1 157个,特有OTU数目为21个。上述结果说明添加桑叶水提物和大肠杆菌攻毒改变了肉鸡肠道菌群结构,产生了特异性菌群。
图2 盲肠菌群OTU的Venn图

Fig.2 Venn diagram of OTU of cecal microflora

2.5.2 盲肠菌群α多样性

由表7可知,添加桑叶水提物能极显著降低肉鸡盲肠菌群PD_whole_tree指数(P<0.01),有降低Observed_species指数的趋势(P=0.06);添加桑叶水提物与大肠杆菌攻毒对肉鸡盲肠菌群α多样性指数中的Chao1指数、Observed_species指数和PD_whole_tree指数存在显著的交互作用(P<0.05),提示在大肠杆菌攻毒的情况下,添加桑叶水提物可以改变肉鸡盲肠菌群的α多样性。
表7 盲肠菌群α多样性指数
项目
Items		 组别Groups SEM P值P-value
CON EC MWE ECMWE MWE EC MWE×EC
Chao1指数Chao1 index 1 110.01a 1 024.14ab 881.41b 1 072.72a 54.29 0.12 0.35 0.02
Observed_species指数
Observed_species index		 841.60a 805.63a 814.80a 685.85b 36.86 0.06 0.22 0.04
PD_Whole_tree指数
PD_Whole_tree index		 43.87Aa 38.02ABab 31.79Bb 37.20ABb 2.06 <0.01 0.91 0.01
Shannon指数Shannon index 5.78 5.73 5.42 6.00 0.22 0.85 0.25 0.17

Table 7 α diversity indexes of cecal microflora

2.5.3 盲肠菌群β多样性

图3的主成分分析(PCA)和图4的非加权UniFrac距离矩阵热图显示各组肉鸡的盲肠菌群分布均匀集中,但不同组间菌群结构存在明显差异,通过Anoism分析证实,基于Bray-Curtis距离,添加桑叶水提物后肉鸡盲肠菌群结构发生显著变化(P=0.003,R=0.226;表8)。通过LEfSe分析,找到组间具有显著差异的菌群标志物,并评估每个差异显著物种丰度对差异效果的影响力。图5(LDA分布柱状图)和图6(LDA进化分支图)显示了肉鸡盲肠菌群中最丰富的分类单元,不同组间盲肠菌群中具有显著差异的物种不同。
图3 盲肠菌群主成分分析

Fig.3 Principal component analysis of cecal microflora

图4 盲肠菌群非加权UniFrac距离矩阵热图

CON_1、CON_2、CON_3、CON_4、CON_5和CON_6为CON组的6个样本,EC_1、EC_2、EC_3、EC_4、EC_5和EC_6为EC组的6个样本,ECMLE_1、ECMLE_2、ECMLE_3、ECMLE_4、ECMLE_5和ECMLE_6为ECMLE组的6个样本,MLE_1、MLE_2、MLE_3、MLE_4、MLE_5和MLE_6为MLE组的6个样本。

Fig.4 Heatmap of unweighted UniFrac distance matrix of cecal microflora

CON_1, CON_2, CON_3, CON_4, CON_5 and CON_6 were six samples from CON group, EC_1, EC_2, EC_3, EC_4, EC_5 and EC_6 were six samples from EC group, ECMLE_1, ECMLE_2, ECMLE_3, ECMLE_4, ECMLE_5 and ECMLE_6 were six samples from ECMLE group, and MLE_1, MLE_2, MLE_3, MLE_4, MLE_5 and MLE_6 were six samples from MLE group.

表8 肉鸡盲肠菌群Anoism分析

Table 8 Anosim analysis of cecal microflora

R值
R-value		 P值
P-value		 排列数量
Number of permutations		 组间比较
Comparison among groups
0.240 7 0.047 999 CON vs. EC
0.185 2 0.099 999 CON vs. ECMWE
0.288 9 0.010 999 CON vs. MWE
0.201 9 0.079 999 EC vs. ECMWE
0.114 8 0.169 999 EC vs. MWE
-0.024 1 0.521 999 ECMWE vs. MWE
0.173 7 0.022 999 CON vs. EC vs. MWE vs. ECMWE
图5 盲肠菌群LDA分布柱状图

k_:界 kingdom;p_:门 phylum;c_:纲 class;o_:目 order;f_:科 family;g_:属genus;s_:种 species。Bifidobacteriales:双歧杆菌目;Bifidobacteriaceae:双歧杆菌科;Bifidobacterium:双歧杆菌属;Actinobacteria:放线菌纲;gut_metagenome:肠道微生物组;Bacteria:细菌界;Actinobacteria:放线菌门;Bacteroides_fragilis:脆弱拟杆菌;Ruminococcaceae_UCG-011:瘤胃球菌科UCG-011;Ruminiclostridium_5:瘤胃梭菌属5;Methanobacteriales:甲烷杆菌目;Archaea:古菌界;Methanobacteria:甲烷杆菌纲;Methanobacteriaceae:甲烷杆菌科;Methanobrevibacter:甲烷短球菌属;Ruminiclostridium_9:瘤胃梭菌属9;Cyanobacteria:蓝藻门;Ruminococcaceae_UCG-005:瘤胃球菌科UCG-005;Acidobacteria:酸杆菌门;Catenulispora:细链孢菌属;Catenulisporaceae:细链孢菌科;Rhizomicrobium:根霉菌属;Chloroflexi:绿弯菌门;Rhizobiales_incertae_sedis:根瘤菌目分类未定科。下图同 the same as below。

Fig.5 LDA distribution histogram of cecal microflora

图6 盲肠菌群LDA进化分支图

Fig.6 LDA cladogram of cecal microflora

2.5.4 门水平下盲肠菌群结构

由表9可知,添加桑叶水提物有降低肉鸡盲肠菌群中厚壁菌门(Firmicutes)相对丰度的趋势(P=0.09),有升高变形菌门(Proteobacteria)相对丰度的趋势(P=0.08),而对拟杆菌门(Bacteroidetes)、软壁菌门(Tenericutes)和放线菌门(Actinobacteria)的相对丰度无显著影响(P>0.05),且添加桑叶水提物与大肠杆菌攻毒在放线菌门的相对丰度上存在交互作用的趋势(P=0.07)。
表9 盲肠菌群在门水平上相对丰度排名前5的物种

Table 9 Top 5 species in relative abundance of cecal microflora at phylum level

项目
Items		 组别Groups SEM P值P-value
CON EC MWE ECMWE MWE EC MWE×EC
厚壁菌门Firmicutes 73.00 69.24 64.03 56.68 5.94 0.09 0.36 0.77
拟杆菌门Bacteroidetes 21.82 23.04 23.25 26.61 12.50 0.65 0.67 0.84
软壁菌门Tenericutes 2.72 2.55 1.95 3.06 0.55 0.81 0.40 0.26
放线菌门Actinobacteria 1.41 3.06 6.56 2.04 1.62 0.22 0.39 0.07
变形菌门Proteobacteria 0.38 0.41 0.68 0.52 0.11 0.08 0.55 0.37

2.5.5 属水平下盲肠菌群结构

基于肉鸡盲肠菌群属水平97%的相对丰度和通过Kruskal-wallis及Metastats分析筛选出组间存在显著差异的菌群进行统计学分析,结果如表10所示。添加桑叶水提物与大肠杆菌攻毒在沙特尔沃思菌属(Shuttleworthia)、消化球菌属(Peptococcus)和毛螺菌科UCG-004(Lachnospiraceae_UCG-004)的相对丰度上存在显著的交互作用(P<0.05)。主效应分析显示,添加桑叶水提物能显著提高另枝菌属(Alistipes)、链球菌属(Streptococcus)和毛螺菌科UCG-004的相对丰度(P<0.05),极显著降低瘤胃球菌科UCG-011(Ruminococcaceae_UCG-011)和瘤胃球菌科UCG-013(Ruminococcaceae_UCG-013)的相对丰度(P<0.01),并有提高扭链瘤胃球菌组(Ruminococcus torques_group)(P=0.08)和双歧杆菌属(Bifidobacterium)相对丰度(P=0.07)以及降低瘤胃球菌科UCG-005(Ruminococcaceae_UCG-005)相对丰度的趋势(P=0.050);大肠杆菌攻毒可显著降低多尔氏菌属(Dorea)的相对丰度(P<0.05),极显著提高瘤胃梭菌属5(Ruminiclostridium_5)的相对丰度(P<0.01),并有升高拉克氏梭状芽胞杆菌属(Lachnoclostridium)(P=0.06)和消化球菌属相对丰度(P=0.09)以及降低毛螺菌科UCG-004相对丰度的趋势(P=0.06)。
表10 盲肠菌群在属水平上具有显著差异的物种

Table 10 Species with significant difference at genus level of cecal microflora

项目
Items		 组别Groups SEM P值P-value
CON EC MWE ECMWE MWE EC MWE×EC
扭链瘤胃球菌组
Ruminococcus torques_group		 7.17ab 5.55b 10.76a 7.39ab 1.47 0.08 0.11 0.56
多尔氏菌属Dorea 2.01a 0.86b 1.26ab 0.93b 0.28 0.25 0.02 0.17
瘤胃球菌科UCG-005
Ruminococcaceae_UCG-005		 1.50a 1.17ab 1.03ab 0.62b 0.25 0.05 0.15 0.88
沙特尔沃思菌属
Shuttleworthia		 1.26a 0.84ab 0.61b 1.12ab 0.18 0.32 0.79 0.02
拉克氏梭状芽胞杆菌属
Lachnoclostridium		 1.09ab 1.22ab 1.04b 1.71a 0.20 0.28 0.06 0.19
另枝菌属Alistipes 0.69b 0.72b 1.93ab 3.13a 0.76 0.03 0.43 0.44
瘤胃球菌科UCG-011
Ruminococcaceae_UCG-011		 0.62a 0.67a 0.13b 0.32ab 0.14 <0.01 0.40 0.64
链球菌属Streptococcus 0.34b 0.95ab 1.76a 1.48ab 0.43 0.04 0.71 0.32
消化球菌属Peptococcus 0.32b 1.09a 0.96a 0.84ab 0.18 0.28 0.09 0.02
瘤胃梭菌属5
Ruminiclostridium_5		 0.20b 0.38a 0.29ab 0.37a 0.04 0.35 <0.01 0.28
瘤胃球菌科UCG-013
Ruminococcaceae_UCG-013		 0.15Aa 0.19AaBb 0.10Bb 0.10Bb 0.02 <0.01 0.35 0.31
毛螺菌科UCG-004
Lachnospiraceae_UCG-004		 0.03Bb 0.01Bb 0.02Bb 0.08Aa 0.01 0.01 0.06 <0.01
双歧杆菌属Bifidobacterium 0.10 1.05 1.95 1.98 0.70 0.07 0.50 0.52

3 讨论

3.1 桑叶水提物对大肠杆菌攻毒肉鸡生长性能的影响

大肠杆菌能刺激肠道分泌内毒素,改变小肠水分和电解质的含量,引起肉鸡肠道内稳态失衡,从而引起肉鸡腹泻[15-16]。有研究表明,桑叶中的活性物质具有抗炎活性,能有效缓解肠道损伤,降低腹泻率[17-18]。本试验发现,经大肠杆菌攻毒后,肉鸡腹泻率显著增加,饲料中添加1 200 mg/kg的桑叶水提物则能显著降低肉鸡的腹泻率,这可能是桑叶中黄酮、多糖等活性物质发挥了抗菌、抗炎作用。有研究报道,桑叶中的黄酮类化合物可抑制脂多糖(LPS)诱导的巨噬细胞中的炎症反应和自噬途径,有效抑制大肠杆菌等有害菌的活性[19]。桑叶多糖可以通过调节肠道Toll样受体4(TLR4)表达,激活p65、髓样细胞分化因子88(MyD88)信号通路,增强肠黏膜免疫屏障[20]。桑叶中的酚类化合物新绿原酸(nCGA)可通过激活AMP活化蛋白激酶(AMPK)/核因子E2相关因子2(Nrf2)信号通路来减弱炎症反应,起到消炎作用[21]。本试验结果显示,大肠杆菌攻毒后肉鸡的ADFI显著降低,而在饲粮中添加1 200 mg/kg的桑叶水提物时肉鸡的ADFI显著升高。此外,从试验全期来看,饲料中添加1 200 mg/kg的桑叶水提物显著提高了肉鸡的ADFI和ADG,降低了F/G。宋琼莉等[22]在矮脚黄鸡基础饲粮中分别添加0.2%、0.5%和0.8%的桑叶提取物,发现桑叶提取物组肉鸡的ADFI和ADG均显著提高,且F/G有降低的趋势,这与本试验结果一致。有研究表明,黄酮类化合物可以通过刺激肠道有益菌增殖、降低有害菌增殖来提高营养物质的消化利用率[23-24]。由此推测,桑叶中的活性物质可能是通过影响肠道菌群来提高肉鸡对营养物质的消化利用;也可能是桑叶黄酮通过促进肠道绒毛发育,增加肠道消化吸收的面积[25],从而增强肉鸡对营养物质的消化和吸收。综上可知,大肠杆菌攻毒对肉鸡的生长性能有负面影响,而桑叶水提物具有提高攻毒后肉鸡ADFI和降低腹泻率的作用,提示桑叶水提物能缓解大肠杆菌对肉鸡造成的负面影响,促进肉鸡生长发育。

3.2 桑叶水提物对大肠杆菌攻毒肉鸡血清生化指标的影响

血液指标的变化通常反映机体脏器功能损伤情况及物质代谢状态[26]。研究表明,大肠杆菌会使肉鸡脏器组织或细胞发生损伤,相应的转氨酶则可进入血液引起血清内转氨酶如ALT、AST活性的升高[27]。另有研究报道,桑叶提取物能通过降低肝脏损伤小鼠血清中ALT、AST的活性修复肝脏氧化损伤[28-29]。但Kojima等[30]研究发现,不同的桑叶提取物添加剂量和试验周期,在一定情况下会使血清中AST的活性有增加的趋势。本试验结果与上述研究结果基本相似,饲粮中添加1 200 mg/kg桑叶水提物使肉鸡血清中AST的活性升高,但与CON组相比没有显著差异。与上述结果不同的是,大肠杆菌攻毒对肉鸡血清中ALT、AST的活性并没有产生显著影响,其产生不同结果的原因可能是肉鸡日龄以及大肠杆菌添加剂量的不同。血清中UREA的含量是检测肾脏功能的重要指标,可反映机体的氮代谢情况[31]。有研究表明,大肠杆菌攻毒会使大鼠血清中UREA的含量显著升高[32]。LDL-C和TC合成储存于肝脏,其在血清中的含量是反映机体脂肪代谢的重要指标。GLU是机体重要的能力来源,反映机体的营养代谢情况[33]。当机体受损时,血清中LDL-C、TC的含量会升高,GLU的含量会降低。本试验结果显示,大肠杆菌攻毒有升高血清中UREA含量和降低LDL-C含量的趋势;大肠杆菌攻毒的情况下在饲粮中添加1 200 mg/kg桑叶提取物具有提高肉鸡血清中TC含量以及降低血清中GLU含量的趋势。总体来讲,饲粮中添加1 200 mg/kg桑叶水提物后大肠杆菌攻毒未对肉鸡血清生化指标带来明显的不利影响。

3.3 桑叶水提物对大肠杆菌攻毒肉鸡抗氧化能力的影响

抗氧化能力能够反映机体的健康情况。GSH-Px、SOD和CAT等酶构成了一线抗氧化防御系统,保护机体器官组织和细胞免受自由基带来的有害影响[34]。研究发现,肉鸡在感染大肠杆菌后容易引起氧化应激[35]。而桑叶水提物因含有黄酮类化合物、多糖类化合物、生物碱类化合物和多酚类化合物等活性物质,具有很强的抗氧化能力,能提高GSH-Px、SOD和CAT等酶的活性[36-37]。GSH-Px可以清除机体内的自由基,阻止生成脂质过氧化物[38]。CAT通过消解超氧化物抵御氧基和催化内源性超氧化物基对过氧化氢(H2O2)的抑制来清除自由基[39]。有研究表明,黄酮类化合物槲皮素可以缓解氧化损伤细胞中CAT和SOD活性的变化[40]。本试验研究显示,在饲粮中添加1 200 mg/kg桑叶水提物能显著提高肉鸡血清中GSH-Px和CAT的活性,而大肠杆菌攻毒后肉鸡血清中GSH-Px和CAT的活性显著下降,且添加桑叶水提物的攻毒组上述酶的活性要高于基础饲粮攻毒组。这提示桑叶水提物可以增强肉鸡的抗氧化能力,减少氧化损伤。其原因可能是桑叶中的黄酮类化合物能够通过清除自由基和螯合金属而产生抗氧化活性[41]。综上所述,大肠杆菌能造成肉鸡一定程度的氧化损伤,而桑叶水提物中的活性成分能通过增强抗氧化酶活性提高肉鸡的抗氧化能力;此外,肠道菌群对营养物质的消化吸收也是促进机体健康的关键原因之一[42],因此我们进一步探讨了桑叶水提物对大肠杆菌攻毒肉鸡肠道形态和盲肠菌群的影响。

3.4 桑叶水提物对大肠杆菌攻毒肉鸡肠道形态的影响

肠道的形态与其消化功能和对营养成分的吸收密切相关,肠黏膜结构和肠绒毛结构会影响肠道对营养成分的吸收能力[43]。肉鸡感染大肠杆菌后,可导致肠黏膜出现炎症反应,肠道绒毛高度降低,消化吸收能力下降[44]。有研究报道,黄酮类化合物可透过增强肠上皮细胞中碱性磷酸酶活性,促进肠上皮细胞的生长和增殖,增强肠上皮屏障,并促进肠绒毛组织的发育[45]。张金龙等[46]研究发现,在饲粮中添加2%的发酵桑叶粉可以显著提高肉鸡空肠和回肠绒毛高度。本试验结果发现,大肠杆菌攻毒未对肉鸡的空肠和回肠组织形态造成不利影响,其可能是大肠杆菌的浓度或攻毒天数不足所导致的。此外,在大肠杆菌攻毒情况下,饲粮中添加1 200 mg/kg桑叶水提物能显著增加肉鸡空肠绒毛高度与隐窝深度的比值,且空肠绒毛高度有升高的趋势,空肠和回肠隐窝深度有下降的趋势。这与上述研究结果一致,推测可能是桑叶中的黄酮类化合物发挥了抗氧化功能和免疫功能,调节了肠道菌群结构,从而改善了肠道形态。综上可知,饲粮中添加1 200 mg/kg桑叶水提物能促进肉鸡肠道绒毛发育,改善肠道形态结构,增加肠道对营养物质的消化吸收面积,从而提高肉鸡的生长性能。

3.5 桑叶水提物对大肠杆菌攻毒对肉鸡盲肠菌群的影响

家禽胃肠道的微生物群落是由细菌主导的许多不同种类的微生物组成的复杂群落。这种多样化的菌群为宿主提供了广泛的酶和基质,在提高营养物质消化和吸收、促进动物生长和增强免疫能力等方面发挥着重要作用[47]。本试验结果显示,饲粮中添加1 200 mg/kg桑叶水提物能显著降低肉鸡盲肠菌群α多样性。同时,通过Venm图、PCA、LEfSe分析和Anosim分析进一步证实这一结果,说明桑叶水提物能明显改变肉鸡盲肠菌群结构。
有研究报道,肉鸡肠道中的优势菌群在门水平上主要是厚壁菌门、拟杆菌门和变形菌门[48],本研究结果与此一致。厚壁菌门一般占据肠道近端,该菌门下的许多菌种可以参与宿主的物质能量代谢过程,能产生促进肠上皮细胞发育、具有抗炎作用的丁酸盐[49]。变形菌门是一类致病性病原菌[50],其相对丰度与肠道菌群稳定程度呈负相关,在机体胃肠道受到应激时,肠道中变形菌门的比例迅速增加[51]。本试验结果显示,大肠杆菌攻毒对肉鸡盲肠中门水平下菌群的相对丰度没有产生显著影响,而添加桑叶水提物有降低厚壁菌门相对丰度和升高变形菌门相对丰度的趋势。Yao等[52]观察到肥胖小鼠肠道中厚壁菌门的比例显著增加,采用桑叶茶饲喂小鼠进行调控,结果发现肥胖小鼠肠道中厚壁菌门的比例显著下降。Li等[53]研究也得到相同的结果。以上研究与本试验结果一致,说明桑叶水提物能降低肉鸡肠道中厚壁菌门的比例。Sheng等[54]研究证实,用桑叶饲喂糖尿病大鼠能使肠道中变形菌门的比例显著增加,推测可能与治疗糖尿病的机制有关。
有研究报道,多尔氏菌属可以维持肠道形态,产生抗炎性挥发性脂肪酸(SCFAs)[55]。在某些疾病中Lachnoclostridium的相对丰度增加,会导致肠道疾病易感性[56]。双歧杆菌是人类和动物肠道内的一种主要益生菌,具有增强免疫力、缓解结肠炎、抗肿瘤和促进肠道健康等功能,与人类和动物健康密切相关[57]。毛螺菌科某些菌属可以降解非淀粉多糖并产生乙酸和丁酸[58],是上皮细胞生长的主要来源,可增强上皮屏障的完整性,并抑制肠道炎症反应[59]。此外,毛螺菌科的某些菌属还与肉鸡的料重比增加和体重增加相关[60],这与在肉鸡生长性能和腹泻率上所得结果一致。链球菌属是一种致病菌,能引起肉鸡呼吸道和胃肠道疾病[61-62]。另枝菌属是拟杆菌门的一种有益菌,能分解膳食纤维产生乙酸[63]。瘤胃球菌属属于厚壁菌门,能分泌纤维素酶和半纤维素酶来水解木质纤维素,产生大量的乙酸和丁酸[64]。本研究结果显示,大肠杆菌攻毒后能显著降低肉鸡盲肠中多尔氏菌属的相对丰度,极显著提高瘤胃梭菌属5的相对丰度,对毛螺菌科UCG-004的相对丰度有降低趋势,对Lachnoclostridium和消化球菌属的相对丰度有升高趋势;而添加桑叶水提物能显著提高大肠杆菌攻毒后肉鸡盲肠中另枝菌属、链球菌属和毛螺菌科UCG-004的相对丰度,能极显著降低瘤胃球菌科UCG-011和瘤胃球菌科UCG-013的相对丰度,并有提高扭链瘤胃球菌组和双歧杆菌属相对丰度以及降低瘤胃球菌科相对丰度的趋势。综上所述,桑叶水提物能通过增加肉鸡肠道中有益菌的数量,减少有害菌的数量来改善肠道菌群结构,促进肠道健康。这与桑叶水提物能改善肉鸡生长性能的结论一致,说明良好的生长性能与肠道有益菌的增殖关系十分密切,保持肠道健康是提高肉鸡生长性能的重要因素。

4 结论

大肠杆菌攻毒对肉鸡的生长性能有负面影响,而在饲粮中添加1 200 mg/kg桑叶水提物能提高大肠杆菌攻毒肉鸡的ADFI,并能降低腹泻率和F/G;此外,桑叶水提物还能提高肉鸡血清中抗氧化酶GSH-Px和CAT的活性,增加肉鸡空肠绒毛高度与隐窝深度的比值,改善肠道形态和菌群结构,缓解大肠杆菌攻毒对肉鸡肠道健康带来的不利影响。上述结果说明桑叶水提物具有作为绿色饲料添加剂应用于肉鸡生产的潜力。

References

Publishing order | Descend order by publishing year | Descend order by cited within
[1]
ZHANG L, CAO G T, ZENG X F, et al. Effects of Clostridium butyricum on growth performance,immune function,and cecal microflora in broiler chickens challenged with Escherichia coli K88[J]. Poultry Science, 2014, 93(1):46-53.

DOI

[2]
ATEYA A I, ARAFAT N, SALEH R M, et al. Intestinal gene expressions in broiler chickens infected with Escherichia coli and dietary supplemented with probiotic,acidifier and synbiotic[J]. Veterinary Research Communications, 2019, 43(2):131-142.

DOI

[3]
WANG S, PENG Q, JIA H M, et al. Prevention of Escherichia coli infection in broiler chickens with Lactobacillus plantarum B1[J]. Poultry Science, 2017, 96(8):2576-2586.

DOI

[4]
XV Z C, HE G L, WANG X L, et al. Mulberry leaf powder ameliorate high starch-induced hepatic oxidative stress and inflammation in fish model[J]. Animal Feed Science and Technology, 2021, 278:115012.

DOI

[5]
刘纯铭, 钟如意, 肖定福. 青贮桑叶的生产工艺、营养作用及其在反刍动物上的应用效果研究进展[J]. 中国饲料, 2022(7):126-130.

LIU C M, ZHONG R Y, XIAO D F. Production technology and nutritional function of silage mulberry leaves and its application effect in ruminants[J]. China Feed, 2022(7):126-130. (in Chinese)

[6]
MAQSOOD M, ANAM SAEED R, SAHAR A, et al. Mulberry plant as a source of functional food with therapeutic and nutritional applications:a review[J]. Journal of Food Biochemistry, 2022, 46(11):e14263.

[7]
OH N S, LEE J Y, LEE J M, et al. Mulberry leaf extract fermented with Lactobacillus acidophilus A4 ameliorates 5-fluorouracil-induced intestinal mucositis in rats[J]. Letters in Applied Microbiology, 2017, 64(6):459-468.

DOI

[8]
LEE M R, KIM J E, YUN W B, et al. Lipolytic effect of novel extracts from mulberry (Morus alba) leaves fermented with Cordyceps militaris in the primary adipocytes derived from SD rats[J]. Laboratory Animal Research, 2017, 33(3):270-279.

DOI

[9]
ZHANG T, ZHANG X Q, HAN K P, et al. Genome-wide analysis of lncRNA and mRNA expression during differentiation of abdominal preadipocytes in the chicken[J]. G3 Genes Genomes Genetics, 2017, 7(3):953-966.

DOI

[10]
SRIVASTAVA S, KAPOOR R, THATHOLA A, et al. Nutritional quality of leaves of some genotypes of mulberry (Morus alba)[J]. International Journal of Food Sciences and Nutrition, 2006, 57(5/6):305-313.

DOI

[11]
PRZYGOŃSKI K, WOJTOWICZ E. The optimization of extraction process of white mulberry leaves and the characteristic bioactive properties its powder extract[J]. Herba Polonica, 2019, 65(1):12-19.

DOI

[12]
黄慧, 夏鑫, 周应军, 等. 不同提取方法的桑叶提取物对小鼠生长性能、抗氧化和免疫功能的作用研究[J]. 动物营养学报, 2022, 34(4):2702-2711.

DOI

HUANG H, XIA X, ZHOU Y J, et al. Effects of mulberry leaf extracts with different extraction methods on growth performance,antioxidant and immune function in mice[J]. Chinese Journal of Animal Nutrition, 2022, 34(4):2702-2711. (in Chinese)

[13]
贺文祥, 邓菊婴, 黄慧, 等. 迷迭香提取物对大肠杆菌攻毒肉鸡生长性能、血清生化指标、抗氧化能力和肠道形态的影响[J]. 动物营养学报, 2022, 34(12):7735-7746.

DOI

HE W X, DENG J Y, HUANG H, et al. Effects of rosemary extract on growth performance,serum biochemical indices,antioxidant capacity and intestinal morphology of broilers challenged with Escherichia coli[J]. Chinese Journal of Animal Nutrition, 2022, 34(12):7735-7746. (in Chinese)

[14]
赵宪臣. 鸡腹泻的初步诊断、中西疗法与预防措施[J]. 现代畜牧科技, 2019(10):115-116.

ZHAO X C. Preliminary diagnosis,Chinese and western therapy and preventive measures of chicken diarrhea[J]. Modern Animal Husbandry Science&Technology, 2019(10):115-116. (in Chinese)

[15]
BIN P, TANG Z Y, LIU S J, et al. Intestinal microbiota mediates enterotoxigenic Escherichia coli-induced diarrhea in piglets[J]. BMC Veterinary Research, 2018, 14(1):385.

DOI

[16]
BI Y L, YANG C T, DIAO Q Y, et al. Effects of dietary supplementation with two alternatives to antibiotics on intestinal microbiota of preweaned calves challenged with Escherichia coli K99[J]. Scientific Reports, 2017, 7(1):5439.

DOI

[17]
LIN Z W, GAN T T, HUANG Y Z, et al. Anti-inflammatory activity of mulberry leaf flavonoids in vitro and in vivo[J]. International Journal of Molecular Sciences, 2022, 23(14):7694.

DOI

[18]
CHEN G S, SHUI S Z, CHAI M J, et al. Effects of paper mulberry (Broussonetia papyrifera) leaf extract on growth performance and fecal microflora of weaned piglets[J]. BioMed Research International, 2020, 2020:6508494.

[19]
LEE M R, KIM J E, PARK J J, et al. Protective role of fermented mulberry leave extract in LPS-induced inflammation and autophagy of RAW264.7 macrophage cells[J]. Molecular Medicine Reports, 2020, 22(6):4685-4695.

DOI

[20]
陈晓兰, 宣嘉颖, 王婧, 等. 桑叶多糖对免疫抑制小鼠肠道损伤和微生物多样性的调节作用[J]. 动物营养学报, 2022, 34(3):1996-2008.

DOI

CHEN X L, XUAN J Y, WANG J, et al. Regulating effects of mulberry leaf polysaccharide on intestinal damage and microbial diversity of immunosuppressed mice[J]. Chinese Journal of Animal Nutrition, 2022, 34(3):1996-2008. (in Chinese)

[21]
GAO X H, ZHANG S D, WANG L T, et al. Anti-inflammatory effects of neochlorogenic acid extract from mulberry leaf (Morus alba L.) against LPS-stimulated inflammatory response through mediating the AMPK/Nrf2 signaling pathway in a549 cells[J]. Molecules, 2020, 25(6):1385.

DOI

[22]
宋琼莉, 周泉勇, 韦启鹏, 等. 桑叶提取物对矮脚黄鸡生长性能、屠宰性能及肉品质的影响[J]. 动物营养学报, 2018, 30(1):191-201.

SONG Q L, ZHOU Q Y, WEI Q P, et al. Effects of mulberry leaf extract on growth performance,slaughter performance and meat quality of yellow dwarf chickens[J]. Chinese Journal of Animal Nutrition, 2018, 30(1):191-201. (in Chinese)

[23]
PEI R S, LIU X C, BOLLING B. Flavonoids and gut health[J]. Current Opinion in Biotechnology, 2020, 61:153-159.

DOI PMID

[24]
SHORTT C, HASSELWANDER O, MEYNIER A, et al. Systematic review of the effects of the intestinal microbiota on selected nutrients and non-nutrients[J]. European Journal of Nutrition, 2018, 57(1):25-49.

DOI PMID

[25]
DING Y N, JIANG X D, YAO X F, et al. Effects of feeding fermented mulberry leaf powder on growth performance,slaughter performance,and meat quality in chicken broilers[J]. Animals, 2021, 11(11):3294.

DOI

[26]
ZHANG H, QI R L, ZENG Y H, et al. Chinese sweet leaf tea (Rubus suavissimus) mitigates LPS-induced low-grade chronic inflammation and reduces the risk of metabolic disorders in a C57BL/6J mouse model[J]. Journal of Agricultural and Food Chemistry, 2020, 68(1):138-146.

DOI

[27]
BAO J L, ZHANG Y, ZHANG L C, et al. Therapeutic effect of schisandrin A on avian colibacillosis through gut-liver axis[J]. Poultry Science, 2021, 100(10):101371.

DOI

[28]
鱼晓敏, 安馨, 鲁慧, 等. 桑叶水提物与醇提物对糖尿病小鼠的协同降血糖效应[J]. 卫生研究, 2018, 47(3):432-436.

YU X M, AN X, LU H, et al. Hypoglycemic effects of mulberry leaf extracts on diabetic mice[J]. Journal of Hygiene Research, 2018, 47(3):432-436. (in Chinese)

[29]
王玲, 邹莉芳, 黄先智, 等. 桑叶水提物对高脂饮食小鼠脂代谢的调控机制初探[J]. 中国食品学报, 2019, 19(10):23-33.

WANG L, ZOU L F, HUANG X Z, et al. Regulation mechanism of water extract of mulberry leaf on lipid metabolism in high-fat fed mice[J]. Journal of Chinese Institute of Food Science and Technology, 2019, 19(10):23-33. (in Chinese)

[30]
KOJIMA Y, KIMURA T, NAKAGAWA K, et al. Effects of mulberry leaf extract rich in 1-deoxynojirimycin on blood lipid profiles in humans[J]. Journal of Clinical Biochemistry and Nutrition, 2010, 47(2):155-161.

DOI PMID

[31]
BIGOT A, TCHAN M C, THOREAU B, et al. Liver involvement in urea cycle disorders:a review of the literature[J]. Journal of Inherited Metabolic Disease, 2017, 40(6):757-769.

DOI

[32]
RAŠKOVIĆ A, MILANOVIĆ I, PAVLOVIĆ N, et al. Antioxidant activity of rosemary (Rosmarinus officinalis L.) essential oil and its hepatoprotective potential[J]. BMC Complementary and Alternative Medicine, 2014, 14:225.

DOI

[33]
RICHARDS M P, ROSEBROUGH R W, COON C N, et al. Feed intake regulation for the female broiler breeder:in theory and in practice[J]. Journal of Applied Poultry Research, 2010, 19(2):182-193.

DOI

[34]
SZCZUBIAŁ M, KANKOFER M, WAWRON W, et al. The dynamics of changes in erythrocyte glutathione peroxidase activity and serum selenium content during the periparturient period in sows[J]. Polish Journal of Veterinary Sciences, 2004, 7(1):21-26.

PMID

[35]
DA ROSA G, ALBA D F, SILVA A D, et al. Impact of Escherichia coli infection in broiler breeder chicks:the effect of oxidative stress on weight gain[J]. Microbial Pathogenesis, 2020, 139:103861.

DOI

[36]
LI M W, HASSAN F U, TANG Z H, et al. Mulberry leaf flavonoids improve milk production,antioxidant,and metabolic status of water buffaloes[J]. Frontiers in Veterinary Science, 2020, 7:599.

DOI

[37]
CHEN W, LU Y, HU D W, et al. Black mulberry (Morus nigra L.) polysaccharide ameliorates palmitate-induced lipotoxicity in hepatocytes by activating Nrf2 signaling pathway[J]. International Journal of Biological Macromolecules, 2021, 172:394-407.

DOI

[38]
CICHOSKI A J, ROTTA R B, SCHEUERMANN G, et al. Investigation of glutathione peroxidase activity in chicken meat under different experimental conditions[J]. Food Science and Technology, 2012, 32(4):661-667.

DOI

[39]
ZHANG L H, HE T F, HU J X, et al. Effects of normal and low calcium and phosphorus levels and 25-hydroxycholecalciferol supplementation on performance,serum antioxidant status,meat quality,and bone properties of broilers[J]. Poultry Science, 2020, 99(11):5663-5672.

DOI

[40]
CHEN Z G, YUAN Q L, XU G R, et al. Effects of quercetin on proliferation and H2O2-induced apoptosis of intestinal porcine enterocyte cells[J]. Molecules, 2018, 23(8):2012.

DOI

[41]
RODRIGUEZ-SANCHEZ R, TRES A, SALA R, et al. Evolution of lipid classes and fatty acid digestibility along the gastrointestinal tract of broiler chickens fed different fat sources at different ages[J]. Poultry Science, 2019, 98(3):1341-1353.

DOI

[42]
GALLEANO M, VERSTRAETEN S V, OTEIZA P I, et al. Antioxidant actions of flavonoids:thermodynamic and kinetic analysis[J]. Archives of Biochemistry and Biophysics, 2010, 501(1):23-30.

DOI

[43]
BONOS E, GIANNENAS I, SIDIROPOULOU E, et al. Effect of Bacillus pumilus supplementation on performance,intestinal morphology,gut microflora and meat quality of broilers fed different energy concentrations[J]. Animal Feed Science and Technology, 2021, 274:114859.

DOI

[44]
MAHDAVI A H, RAHMANI H R, NILI N, et al. Effects of dietary egg yolk antibody powder on growth performance,intestinal Escherichia coli colonization,and immunocompetence of challenged broiler chicks[J]. Poultry Science, 2010, 89(3):484-494.

DOI

[45]
LIU F, ZHANG X S, JI Y. Total flavonoid extract from hawthorn (Crataegus pinnatifida) improves inflammatory cytokines-evoked epithelial barrier deficit[J]. Medical Science Monitor, 2020, 26:e920170.

[46]
张金龙, 张宁, 杨雪, 等. 发酵桑叶粉对肉鸡生长性能、血清生化指标及肠道组织形态结构的影响[J]. 饲料工业, 2017, 38(20):38-42.

ZHANG J L, ZHANG N, YANG X, et al. Effects of fermented product of mulberry leaf on growth performance,serum biochemical indexes and intestinal morphology of broiler chickens[J]. Feed Industry, 2017, 38(20):38-42. (in Chinese)

[47]
STANLEY D, GEIER M S, DENMAN S E, et al. Identification of chicken intestinal microbiota correlated with the efficiency of energy extraction from feed[J]. Veterinary Microbiology, 2013, 164(1/2):85-92.

DOI

[48]
OH J K, PAJARILLO E A B, CHAE J P, et al. Protective effects of Bacillus subtilis against Salmonella infection in the microbiome of Hy-Line Brown layers[J]. Asian-Australasian Journal of Animal Sciences, 2017, 30(9):1332-1339.

DOI

[49]
LOUIS P, YOUNG P, HOLTROP G, et al. Diversity of human colonic butyrate-producing bacteria revealed by analysis of the butyryl-CoA:acetate CoA-transferase gene[J]. Environmental Microbiology, 2010, 12(2):304-314.

DOI PMID

[50]
孙伦. 天然功能植物保障肉兔肠道健康与提高生长性能的研究及应用[D].硕士学位论文. 贵阳: 贵州大学, 2019.

SUN L. Research and application of natural functional plants in securing intestinal health and improving growth performance of meat rabbits[D].Master’s Thesis. Guiyang: Guizhou University, 2019. (in Chinese)

[51]
SHIN N R, WHON T W, BAE J W. Proteobacteria:microbial signature of dysbiosis in gut microbiota[J]. Trends in Biotechnology, 2015, 33(9):496-503.

DOI

[52]
SHENG Y, ZHENG S J, ZHANG C H, et al. Mulberry leaf tea alleviates diabetic nephropathy by inhibiting PKC signaling and modulating intestinal flora[J]. Journal of Functional Foods, 2018, 46:118-127.

DOI

[53]
LI Q, LIU F, LIU J, et al. Mulberry leaf polyphenols and fiber induce synergistic antiobesity and display a modulation effect on gut microbiota and metabolites[J]. Nutrients, 2019, 11(5):1017.

DOI

[54]
SHENG Y, ZHENG S J, MA T S, et al. Mulberry leaf alleviates streptozotocin-induced diabetic rats by attenuating NEFA signaling and modulating intestinal microflora[J]. Scientific Reports, 2017, 7(1):12041.

DOI PMID

[55]
FERNÁNDEZ J, DE LA FUENTE V G, GARCÍA M T F, et al. A diet based on cured acorn-fed ham with oleic acid content promotes anti-inflammatory gut microbiota and prevents ulcerative colitis in an animal model[J], 2020, 19(1):28.

[56]
ZHOU Y, CHEN L, SUN G F, et al. Alterations in the gut microbiota of patients with silica-induced pulmonary fibrosis[J]. Journal of Occupational Medicine and Toxicology, 2019, 14:5.

DOI PMID

[57]
SCHIAVI E, GLEINSER M, MOLLOY E, et al. The surface-associated exopolysaccharide of Bifidobacterium longum 35624 plays an essential role in dampening host proinflammatory responses and repressing local TH17 responses[J]. Applied and Environmental Microbiology, 2016, 82(24):7185-7196.

DOI

[58]
GUO J R, DONG X F, LIU S, et al. High-throughput sequencing reveals the effect of Bacillus subtilis CGMCC 1.921 on the cecal microbiota and gene expression in ileum mucosa of laying hens[J]. Poultry Science, 2018, 97(7):2543-2556.

DOI

[59]
FUKUDA S, TOH H, HASE K, et al. Bifidobacteria can protect from enteropathogenic infection through production of acetate[J]. Nature, 2011, 469(7331):543-547.

DOI

[60]
STANLEY D, HUGHES R J, GEIER M S, et al. Bacteria within the gastrointestinal tract microbiota correlated with improved growth and feed conversion:challenges presented for the identification of performance enhancing probiotic bacteria[J]. Frontiers in Microbiology, 2016, 7:187.

[61]
RIISE G C, QVARFORDT I, LARSSON S, et al. Inhibitory effect of N-acetylcysteine on adherence of Streptococcus pneumoniae and Haemophilus influenzae to human oropharyngeal epithelial cells in vitro[J]. Respiration, 2000, 67(5):552-558.

DOI

[62]
PNG C W, LINDÉN S K, GILSHENAN K S, et al. Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria[J]. The American Journal of Gastroenterology, 2010, 105(11):2420-2428.

DOI

[63]
HU R K, ZENG F, WU L X, et al. Fermented carrot juice attenuates type 2 diabetes by mediating gut microbiota in rats[J]. Food&Function, 2019, 10(5):2935-2946.

[64]
DONG L L, CAO G L, WU J W, et al. Reflux of acidizing fluid for enhancing biomethane production from cattle manure in plug flow reactor[J]. Bioresource Technology, 2019, 284:248-255.

DOI PMID

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