Chinese Journal of Animal Nutrition >

2024 , Vol. 36 >Issue 8: 5039 - 5049

DOI: https://doi.org/10.12418/CJAN2024.429

RESEARCH PAPER

Effects of Eugenol and Glycerol Monolaurate Nanoemulsion on Growth Performance, Antioxidant Capacity and Intestinal Health of Broilers

  • LU Tinghuan ,
  • WANG Xingjie ,
  • WANG Luyi ,
  • ZHOU Xiangying ,
  • ZHAO Zhihong ,
  • MAO Qianqian ,
  • ZHANG Yan , *
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  • Qingdao Vland Biotech Company Limited/Qingdao Kangdien Animal Pharmaceutical Company Limited, Qingdao 266111, China
*E-mail:

Received date: 2024-03-04

  Online published: 2024-08-12

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Abstract

This experiment aimed to investigate the effects of eugenol (EUG) and glycerol monolaurate (GML) nanoemulsion added in drinking water on the growth performance, antioxidant capacity and intestinal health of broiler chickens. A total of 600 one-day-old white feathered broilers with similar body weight of (39.15±0.11) g were randomly divide into 5 groups with 6 replicates in each group and 20 chickens in each replicate. Broilers in all groups were fed a basal diet, which broilers in the control group were drinking water normally, and broilers in experimental groups were added 0.5, 1.0, 3.0 and 5.0 mL/L EUG and GML nanoemulsion in drinking water, respectively. The experiment lasted for 42 days. The results showed as follows: 1) compared with the control group, adding 1.0 and 3.0 mL/L EUG and GML nanoemulsion to drinking water significantly increased the body weight at 21 and 42 days of age and daily weight gain during 1 to 21 days of age, 22 to 42 days of age and 1 to 42 days of age (P<0.05), and significantly decreased the feed to gain ratio during 22 to 42 days of age and 1 to 42 days of age (P<0.05). 2) Compared with the control group, adding 1.0 and 3.0 mL/L EUG and GML nanoemulsion to drinking water significantly increased the serum total antioxidant capacity and glutathione peroxidase activity (P<0.05), and significantly decreased the serum malondialdehyde content (P<0.05); adding 3.0 mL/L EUG and GML nanoemulsion to drinking water significantly increased the serum superoxide dismutase activity (P<0.05). 3) Compared with the control group, adding 1.0, 3.0 and 5.0 mL/L EUG and GML nanoemulsion to drinking water significantly increased the duodenal and jejunal villus height (P<0.05), and significantly decreased the duodenal crypt depth (P<0.05); adding 1.0 and 3.0 mL/L EUG and GML nanoemulsion to drinking water significantly increased the duodenal and jejunal villus height/crypt depth (P<0.05). 4) Compared with the control group, adding 3.0 and 5.0 mL/L EUG and GML nanoemulsion to drinking water significantly decreased the gene relative expression levels of tumor necrosis factor-α and interleukin-1β in duodenum and jejunum (P<0.05), and adding 1.0 and 3.0 mL/L EUG and GML nanoemulsion to drinking water significantly increased the gene relative expression level of interleukin-10 in duodenum and jejunum (P<0.05). 5) There were no significant differences in the cecal microflora ACE, Chao1, Shannon and Simpson indexes among all groups (P>0.05). Compared with the control group, adding 3.0 mL/L EUG and GML nanoemulsion to drinking water increased the cecal Bacteroidetes relative abundance, and decreased the Firmicutes relative abundance. In summary, adding EUG and GML nanoemulsion to drinking water can enhance the antioxidant capacity, improve the intestinal morphology, inflammatory response and microbial structure, then increase the growth performance of broilers. In this experiment, the suitable added level of EUG and GML nanoemulsion in drinking water is 3.0 mL/L.

Key words： eugenol; glycerol monolaurate; broilers; growth performance; antioxidant capacity; inflammatory response; intestinal health

Cite this article

LU Tinghuan , WANG Xingjie , WANG Luyi , ZHOU Xiangying , ZHAO Zhihong , MAO Qianqian , ZHANG Yan . Effects of Eugenol and Glycerol Monolaurate Nanoemulsion on Growth Performance, Antioxidant Capacity and Intestinal Health of Broilers[J]. Chinese Journal of Animal Nutrition, 2024 , 36(8) : 5039 -5049 . DOI: 10.12418/CJAN2024.429

在畜牧业生产中,过度使用饲料抗生素导致畜禽细菌耐药性增强、肠道菌群失调以及肉类中药物残留的出现[1],对公共卫生和环境构成威胁。在畜禽生产中,常见的非抗生素饲料添加剂主要有植物提取物和精油、酸化剂、益生菌、酶制剂、抗菌肽、多糖等[2]。植物提取物和精油已被证明可以影响畜禽肠道信号分子、肠道微生物群组成、肠道完整性以及细胞炎症因子表达[3],同时能够提高畜禽生长性能、消化功能以及肉品质[4⇓⇓-7];酸化剂有助于维持最佳肠道pH,以实现有效的蛋白质水解消化酶活性和对肠道病原体的抗菌活性,从而减少亚临床感染[8]。
在植物性饲料添加剂中,丁香酚(eugenol,EUG)作为丁香油的主要成分,由于其抗氧化、抗菌和抗炎特性[9],对肉鸡生长性能和肠道健康有一定的积极影响[10];同时将EUG制成纳米乳液,可进一步提高其吸收效果[3]。月桂酸单甘油酯(glycerol monolaurate,GML)又名十二酸单甘油酯(C15H30O4),是一种中链脂肪酸酯,与传统的有机酸相比,具有更强的抑菌效果[8];同时具有减轻肠道炎症、改善肠道形态和屏障功能的有益作用[11]。近年来,人们逐渐发现混合添加剂相较于单一添加剂效果更好。有研究表明,有机酸-精油混合物提高了肉鸡的生长性能和改善了肠道形态[12],改善了肠道完整性和屏障功能[13]。
之前关于有机酸和精油的组合报道中,未发现EUG和GML联合应用的研究。因此,为研究EUG和GML联合应用对肉鸡生长是否起到更强的增效作用,本试验更全面地研究了EUG和GML联合应用在肉鸡饲养中的效果。由于EUG和GML存在稳定性低、溶解性差和生物利用率低等问题,我们通过超声和微射流的制剂手段,将EUG和GML制成纳米乳,通过在肉鸡饮水中添加不同水平的EUG和GML纳米乳,测定其对肉鸡生长性能、抗氧化能力和肠道健康的影响,为其在肉鸡生产中应用提供理论参考。

1 材料与方法

1.1 试验材料

EUG和GML纳米乳中EUG和GML含量均为10%,其余成分为吐温-80、无水乙醇和水等。

1.2 试验设计

选用1日龄、平均体重为(39.15±0.11) g的健康白羽肉鸡600只,随机分成5组,每组6个重复,每个重复20只。各组试验鸡均饲喂基础饲粮,其中对照组正常饮水,试验组分别在饮水中添加0.5、1.0、3.0、5.0 mL/L的EUG和GML纳米乳。试验期42 d。试验期内,所有组别自由采食和饮水。基础饲粮参照《鸡饲养标准》(NY/T 33—2004)配制,其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) %

项目
Items		 1~21日龄
1 to 21
days of age		 22~42日龄
22 to 42
days of age
原料Ingredients
玉米Corn 56.90 61.20
豆粕Soybean meal 35.50 30.20
大豆油Soybean oil 3.00 3.90
磷酸氢钙CaHPO4 1.45 1.65
石粉Limestone 1.05 1.05
食盐NaCl 0.30 0.30
预混料Premix1) 1.00 1.00
L-赖氨酸L-Lys (78%) 0.35 0.30
DL-蛋氨酸DL-Met (99%) 0.25 0.20
氯化胆碱Choline chloride 0.20 0.20
合计Total 100.00 100.00
营养水平Nutrient levels2)
代谢能ME/(MJ/kg) 12.19 12.54
粗蛋白质CP 21.25 20.36
钙Ca 0.92 0.96
磷P 0.48 0.50
赖氨酸Lys 1.22 1.09
蛋氨酸Met 0.59 0.48

1)预混料为每千克饲粮提供 The premix provided the following per kg of diets:VA 12 000 IU,VD 3 000 IU,VE 10 IU,VK 1 mg,VB1 1 mg,VB2 5 mg,VB6 1.5 mg,泛酸 pantothenic acid 10 mg,VB12 0.01 mg,烟酸 niacin 30 mg,叶酸 folic acid 1 mg,生物素 biotin 0.05 mg,Zn 60 mg,Mn 60 mg,Fe 30 mg,Cu 4 mg,I 0.3 mg,Se 0.1 mg。

2)代谢能为计算值,参考NY/T 33—2004,其余均为实测值。ME was a calculated value according to NY/T 33—2004, while the others were measured values.

1.3 测定指标及方法

1.3.1 饲粮营养成分

饲粮粗蛋白质、钙、磷和氨基酸含量分别采用GB/T 6432—2018、GB/T 6436—2018、GB/T 6437—2018和GB/T 18246—2019的方法检测。

1.3.2 生长性能

分别于试验第1、21和42天,以重复为单位对肉鸡空腹称重,并准确记录采食量。计算1~21日龄、22~42日龄、1~42日龄的平均日增重(ADG)、平均日采食量(ADFI)、料重比(F/G)。

1.3.3 血清抗氧化指标

于试验第42天,从每重复随机取2只肉鸡,进行翅静脉采血,3 500 r/min离心10 min,取上清液于离心管中,储存在-20 ℃待测。通过全自动生化分析仪和试剂盒测定血清总抗氧化能力(T-AOC)、丙二醛(MDA)含量和谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)活性,试剂盒均购自南京建成生物工程研究所。

1.3.4 肠道形态

于试验第42天,从每重复随机取2只肉鸡,进行屠宰,取十二指肠、空肠和回肠中部肠段各3 cm。用磷酸盐缓冲液(PBS)洗涤肠腔,将其固定在10%福尔马林的离心管中24 h,进行常规石蜡包埋,切片,用苏木精-伊红(HE)法进行染色。使用Carl Zeiss电子显微镜观察切片组织学结构,并测量肠道绒毛高度(VH)、隐窝深度(CD),并计算绒毛高度/隐窝深度(V/C)。

1.3.5 RNA的提取、cDNA的合成和实时荧光定量PCR

取1.3.4中肉鸡十二指肠、空肠和回肠肠黏膜,放入无RNA酶的试管中,立即置于液氮后转置-80 ℃储存。用组织/细胞RNA快速提取试剂盒(QIAGEN,德国)提取各样品的总RNA,进行RNA纯度测定。使用反转录试剂盒(TaKaRa,日本)获取cDNA模板。使用Light Cycler 480实时荧光定量PCR(Roche,美国)进行cDNA基因表达测定。以β-肌动蛋白(β-actin)为内参基因,PCR引物序列见表2,基因相对表达量采用2-ΔΔCt方法计算。
表2 PCR引物序列

Table 2 Primer sequences for PCR

基因
Genes		 登录号
Accession number		 引物序列
Primer sequences (5'—3')		 产物大小
Product size/bp
β-肌动蛋白
β-actin		 NM_205518.1 F:ATGTGGATCAGCAAGCAGGAGTA
R:TTTATGCGCATTTATGGGTTTTGT		 113
肿瘤坏死因子-α
TNF-α		 NM_204267.2 F:TGTTCTATGACCGCCCAGTT
R:TTCAGAGCATCAACGCAAAA		 169
白细胞介素-1β
IL-1β		 NM_204524.1 F:GGTCAACATCGCCACCTACA
R:CATACGAGATGCAAACCAGCAA		 86
白细胞介素-10
IL-10		 NM_001004414.2 F:GCTGTCACCGCTTCTTCACC
R:TCCCGTTCTCATCCATCTTCTC		 87

1.3.6 盲肠菌群结构和多样性

于试验第42天,从每重复随机取2只肉鸡,取右侧盲肠内容物,液氮速冻,立即转至-80 ℃超低温冰箱冻存,送至奥维森基因科技有限公司,采用16S rRNA高通量测序技术进行微生物学分析。

1.4 数据统计分析

所有数据均使用Excel 2016进行初步处理。使用SPSS 20.0软件进行单因素方差分析,采用Duncan氏法进行组间差异显著性检验。结果以平均值和均值标准误(SEM)表示,P<0.05表示差异显著。通过SPSS 20.0软件中的曲线估计对不同EUG和GML纳米乳添加水平下的生长性能与EUG和GML纳米乳添加水平进行二次曲线拟合。

2 结果

2.1 饮水中添加EUG和GML纳米乳对肉鸡生长性能的影响

由表3可知,与对照组相比,饮水中添加1.0和3.0 mL/L的EUG和GML纳米乳显著提高了肉鸡21、42日龄体重及1~21日龄、22~42日龄、1~42日龄ADG(P<0.05),显著降低了22~42日龄、1~42日龄F/G(P<0.05)。3.0 mL/L EUG和GML纳米乳组的21、42日龄体重及1~21日龄、22~42日龄、1~42日龄ADG显著高于其他试验组(P<0.05),22~42日龄F/G显著低于其他试验组(P<0.05)。
表3 饮水中添加EUG与GML纳米乳对肉鸡生长性能的影响

Table 3 Effects of EUG and GML nanoemulsion added in drinking water on growth performance of broilers

项目
Items		 EUG和GML纳米乳添加水平
EUG and GML nanoemulsion added level/(mL/L)		 SEM P值
P-value
0(对照
Control)		 0.5 1.0 3.0 5.0
1~21日龄
1 to 21
days of age		 1日龄体重
BW at 1 day of age/g		 39.48 39.03 38.85 39.15 39.22 0.11 0.455
平均日采食量
ADFI/(g/d)		 47.31 47.01 47.28 47.31 46.99 0.12 0.851
平均日增重
ADG/(g/d)		 41.67c 41.79c 42.45b 42.94a 41.76c 0.10 <0.001
料重比F/G 1.14a 1.12a 1.11ab 1.10b 1.13a 0.01 0.035
22~42日龄
22 to 42
days of age		 21日龄体重
BW at 21 days of age/g		 919.60c 921.69c 935.32b 945.92a 921.24c 2.00 <0.001
平均日采食量
ADFI/(g/d)		 113.95 113.63 112.30 111.66 111.92 0.57 0.645
平均日增重
ADG/(g/d)		 69.64c 69.43c 73.13b 76.72a 69.73c 0.58 <0.001
料重比F/G 1.64a 1.64a 1.54b 1.46c 1.61ab 0.02 <0.001
1~42日龄
1 to 42
days of age		 42日龄体重
BW at 42 days of age/g		 2 308.99c 2 310.34c 2 397.87b 2 483.62a 2 315.75c 13.05 <0.001
平均日采食量
ADFI/(g/d)		 80.38 80.01 79.98 81.22 80.86 0.35 0.782
平均日增重
ADG/(g/d)		 54.00c 53.95c 56.05b 58.00a 54.08c 0.33 <0.001
料重比F/G 1.49a 1.49a 1.43b 1.40b 1.50a 0.01 <0.001

同行数据肩标不同小写字母表示差异显著(P<0.05),相同或无字母表示差异不显著(P>0.05)。下表同。

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

2.2 饮水中EUG和GML纳米乳的适宜添加水平

在本试验条件下,EUG和GML纳米乳添加水平(X)与肉鸡21(Y1)、42日龄体重(Y2)以及1~21日龄(Y3)、22~42日龄(Y4)、1~42日龄F/G(Y5)之间存在二次关系,分别拟合二次曲线,建立的回归方程如下:
Y1=-4.2X2+22X+917 (R2=0.847,P<0.001);
Y2=-26.69X2+141X+2 280 (R2=0.778,P<0.001);
Y3=0.004 65X2-0.03X+1.14 (R2=0.329,P=0.050);
Y4=0.03X2-0.15X+1.66 (R2=0.536,P<0.001);
Y5=0.01X2-0.08X+1.43 (R2=0.510,P<0.001)。
由上述回归方程可知,21、42日龄体重以及1~21日龄、22~42日龄、1~42日龄F/G最佳时,肉鸡饮水中EUG和GML纳米乳的添加水平分别为2.62、2.64、3.23、2.50、4.00 mL/L。

2.3 饮水中添加EUG和GML纳米乳对肉鸡血清抗氧化指标的影响

由表4所知,与对照组相比,饮水中添加1.0和3.0 mL/L的EUG和GML纳米乳显著提高了血清T-AOC(P<0.05);饮水中添加1.0、3.0和5.0 mL/L的EUG和GML纳米乳显著提高了血清GSH-Px活性(P<0.05),同时显著降低了血清MDA含量(P<0.05);饮水中添加3.0 mL/L的EUG和GML纳米乳显著提高了血清SOD活性(P<0.05)。1.0 mL/L EUG和GML纳米乳组的血清GSH-Px活性显著高于其他试验组(P<0.05),3.0 mL/L EUG和GML纳米乳组的血清SOD活性显著高于其他试验组(P<0.05)。
表4 饮水中添加EUG与GML纳米乳对肉鸡血清抗氧化指标的影响

Table 4 Effects of EUG and GML nanoemulsion added in drinking water on serum antioxidant indexes of broilers

项目
Items		 EUG和GML纳米乳添加水平
EUG and GML nanoemulsion added level/(mL/L)		 SEM P值
P-value
0(对照
Control)		 0.5 1.0 3.0 5.0
总抗氧化能力T-AOC/(U/mL) 10.38a 11.01a 12.76b 13.47b 11.87ab 1.70 0.004
超氧化物歧化酶SOD/(U/mL) 120.42a 122.50a 132.36a 147.36b 123.25a 14.89 0.003
谷胱甘肽过氧化物酶GSH-Px/(U/mL) 503.91a 518.81ab 549.05c 537.41b 527.99b 21.50 0.001
丙二醛MDA/(nmol/L) 2.37c 2.27bc 2.11b 1.78a 1.81a 0.30 <0.001

2.4 饮水中添加EUG和GML纳米乳对肉鸡肠道形态的影响

由表5可知,与对照组相比,饮水中添加1.0、3.0和5.0 mL/L的EUG和GML纳米乳显著提高了十二指肠和空肠VH(P<0.05),显著降低了十二指肠CD(P<0.05);饮水中添加1.0和3.0 mL/L的EUG和GML纳米乳显著提高了十二指肠和空肠V/C(P<0.05)。3.0 mL/L EUG和GML纳米乳组的十二指肠和空肠VH和V/C显著高于其他试验组(P<0.05)。
表5 饮水中添加EUG与GML纳米乳对肉鸡肠道形态的影响

Table 5 Effects of EUG and GML nanoemulsion added in drinking water on intestinal morphology of broilers

项目
Items		 EUG和GML纳米乳添加水平
EUG and GML nanoemulsion added level/(mL/L)		 SEM P值
P-value
0(对照
Control)		 0.5 1.0 3.0 5.0
十二指肠Duodenum
绒毛高度VH/μm 1 596.82a 1 610.81ab 1 717.19b 1 837.86c 1 707.84b 19.59 0.009
隐窝深度CD/μm 240.53c 230.73bc 219.65ab 208.59a 219.82ab 2.43 0.039
绒毛高度/隐窝深度V/C 6.82a 7.06b 7.78c 8.92d 7.73c 0.10 <0.001
空肠Jejunum
绒毛高度VH/μm 938.62a 947.01b 1 020.17c 1 131.93d 948.09b 9.65 <0.001
隐窝深度CD/μm 235.43 233.96 235.13 233.77 233.69 0.62 0.881
绒毛高度/隐窝深度V/C 3.99a 4.05a 4.34b 4.85c 4.06a 0.04 <0.001
回肠Ileum
绒毛高度VH/μm 734.40 731.82 732.99 740.11 734.61 1.70 0.530
隐窝深度CD/μm 135.92 135.77 135.82 132.30 133.53 0.66 0.581
绒毛高度/隐窝深度V/C 5.41 5.40 5.41 5.60 5.51 0.03 0.305

2.5 饮水中添加EUG和GML纳米乳对肉鸡肠道炎症指标的影响

由表6可知,与对照组相比,饮水中添加3.0和5.0 mL/L的EUG和GML纳米乳显著降低十二指肠和空肠肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)基因相对表达量(P<0.05),饮水中添加1.0和3.0 mL/L的EUG和GML纳米乳显著提高了十二指肠和空肠白细胞介素-10(IL-10)基因相对表达量(P<0.05)。3.0 mL/L EUG和GML纳米乳组的十二指肠TNF-α基因相对表达量显著低于其他试验组(P<0.05),十二指肠IL-10基因相对表达量显著高于其他试验组(P<0.05)。
表6 饮水中添加EUG与GML纳米乳对肉鸡肠道炎症指标的影响

Table 6 Effects of EUG and GML nanoemulsion added in drinking water on intestinal inflammatory indexes of broilers %

项目
Items		 EUG和GML纳米乳添加水平
EUG and GML nanoemulsion added level/(mL/L)		 SEM P值
P-value
0(对照
Control)		 0.5 1.0 3.0 5.0
十二指肠Duodenum
肿瘤坏死因子-α TNF-α 1.00c 1.00c 0.88b 0.71a 0.86b 0.03 0.017
白细胞介素-1β IL-1β 1.00b 0.99b 0.93b 0.84a 0.84a 0.02 0.008
白细胞介素-10 IL-10 1.00a 1.05ab 1.22b 2.05d 1.83c 0.08 0.017
空肠Jejunum
肿瘤坏死因子-α TNF-α 1.00b 0.98b 0.86a 0.79a 0.81a 0.01 <0.001
白细胞介素-1β IL-1β 1.00b 0.95b 0.98b 0.87a 0.87a 0.03 <0.001
白细胞介素-10 IL-10 1.00a 1.14ab 1.21b 1.23b 1.06a 0.02 0.007
回肠Ileum
肿瘤坏死因子-α TNF-α 1.00 0.96 1.02 0.96 1.00 0.02 0.882
白细胞介素-1β IL-1β 1.00 1.03 1.02 1.08 1.01 0.01 0.533
白细胞介素-10 IL-10 1.00 0.99 0.92 1.04 1.03 0.02 0.384

2.6 饮水中添加EUG和GML纳米乳对肉鸡盲肠菌群结构和α多样性的影响

根据肉鸡生长性能、血清抗氧化指标、肠道形态以及炎症指标结果,判断出饮水中添加1.0和3.0 mL/L的EUG和GML纳米乳对肉鸡的影响较大。因此,选用1.0和3.0 mL/L EUG和GML纳米乳组进行盲肠菌群分析。

2.6.1 盲肠菌群共有物种分析和α多样性分析

由图1-a可知,对照组包含操作分类单元(OTU)数目2 190个,1.0 mL/L EUG和GML纳米乳组包含OTU数目2 263个,3.0 mL/L EUG和GML纳米乳组包含OTU数目2 269个,3组肉鸡盲肠菌群共有OTU数目1 178个。由图1-b可知,3组的盲肠菌群ACE、Chao1、Shannon和Simpson指数无显著差异(P>0.05)。
图1 肉鸡盲肠菌群OTU的Venn图和α多样性分析

A:对照组 control group;B:1.0 mL/L EUG和GML纳米乳组 1.0 mL/L EUG and GML nanoemulsion group;C:3.0 mL/L EUG和GML纳米乳组 3.0 mL/L EUG and GML nanoemulsion group。下图同 the same as below.

Fig.1 Venn diagram of OTU and α diversity analysis of cecal microflora of broilers

2.6.2 盲肠门水平菌群结构

由图2可知,在门水平上,盲肠厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidota)和变形菌门(Proteobacteria)总相对丰度占98.33%以上,且厚壁菌门占绝对优势,相对丰度超过78.00%。与对照组相比,3.0 mL/L EUG和GML纳米乳组盲肠拟杆菌门相对丰度有所提高,厚壁菌门相对丰度有所降低。
图2 肉鸡盲肠门水平菌群结构

Firmicutes:厚壁菌门;Bacteroidota:拟杆菌门;Proteobacteria:变形菌门;Actinobacteriota:放线菌门;Desulfobacterota:脱硫菌门;Cyanobacteria:蓝细菌门;Acidobacteriota:酸杆菌门;Campylobacterota:弯曲菌门;Verrucomicrobia:疣微菌门;Unclassified-Bacteria:未分类细菌;Others:其他。

Fig.2 Cecal microflora structure at phylum level of broilers

3 讨论

3.1 饮水中添加EUG和GML纳米乳对肉鸡生长性能的影响

开发安全有效的抗菌物质来取代饲料中的抗生素至关重要。Ibrahim等[3]研究表明,饲粮中添加400 mg/kg EUG纳米乳显著提高了肉鸡在整个饲养期间的ADFI、ADG和存活率,降低了F/G;Liu等[8]研究发现,GML有助于改善肉鸡中后期的ADFI和ADG;Yang等[14]研究表明,植物精油和有机酸的联合应用能够提高肉鸡生长性能。在本研究中,肉鸡的生长性能改善,一方面可归因于肠道形态的改善,其吸收生长所需营养物质的表面积增加[15];另一方面由于有机酸降低了消化酶的pH,增强了消化酶活性[16],从而提高了营养物质消化能力。植物精油中含有抗菌素、抗真菌素和抗氧化剂的活性成分,这些成分均对肉鸡的生长性能产生积极影响[17]。因此,通过试验结果和剂量效应分析,饮水中添加3.0 mL/L的EUG和GML纳米增加了肉鸡的体重和ADG,降低了F/G,提高了肉鸡的生长性能。

3.2 饮水中添加EUG和GML纳米乳对肉鸡血清抗氧化指标的影响

氧化应激主要表现为机体内氧化与抗氧化系统失调,其中MDA含量的高低直接或间接体现组织过氧化和细胞损伤程度,SOD和GSH-Px等抗氧化酶活性的高低则是反映机体抗氧化能力的重要指标[18]。研究表明,EUG具有清除自由基,抑制活性氧、活性氮的产生,增加细胞抗氧化潜力,保护微生物DNA和蛋白质的功能[19]。GML的抗氧化作用来源于降低机体脂质过氧化反应,调节脂类代谢基因的表达,促进肝脏脂类代谢和维持血脂平衡,进而降低机体氧化应激[20]。有研究证明,氧化应激与性能下降、采食量减少、腹泻和肝脏组织破坏有关[21];抗氧化能力的提高可以防止肠道绒毛受到自由基诱导的损伤,改善肠道形态和营养物质消化率[22],进一步提高生长性能。本试验结果表明,饮水中添加EUG和GML纳米乳能提高肉鸡血清SOD、GSH-Px活性和T-AOC,降低血清MDA含量,进而增强了肉鸡的抗氧化能力,有助于肉鸡生长性能的改善。

3.3 饮水中添加EUG和GML纳米乳对肉鸡肠道形态的影响

小肠是肉鸡消化吸收营养物质的重要部位。VH、CD和V/C是评估肠道发育的关键指标,较长的VH、较高的V/C和较低的CD导致更高的黏膜表面积,有助于提高肉鸡的消化能力[17]。研究表明,饲粮中添加GML显著增加了黄羽肉鸡空肠VH和十二指肠V/C[23];饮水中添加1%的EUG和3%的圣罗勒显著提高了肉鸡小肠的VH和绒毛表面积[24];在肉鸡饲粮中联合补充不同水平的GLM和牛至油,可改善小肠的CD和VH[17]。Deng等[25]研究表明,肠道健康的改善归因于短链脂肪酸作为肠上皮细胞的能量来源,进而增强了肉鸡的生长。本试验结果表明,饮水中添加EUG和GML纳米乳,肉鸡肠道形态的改善可能归因于植物精油可减少肠道中致病菌的数量,有利于VH和肠道表面积增加,同时GML可被肠细胞用作能量来源,进一步改善肉鸡肠道形态结构。

3.4 饮水中添加EUG和GML纳米乳对肉鸡肠道炎症指标的影响

细胞因子在肠道炎症反应中起着重要的调节作用。在细菌侵入肠上皮细胞时,胃肠道免疫细胞分泌的细胞因子,在针对病原体的免疫应答中发挥作用[26]。TNF-α和IL-1β是重要的促炎细胞因子,它们通过免疫细胞的分化和增殖、一氧化氮的产生和细胞凋亡来调节宿主对许多病原体的免疫应答[27]。同时,IL-10在抑制炎症和免疫应答中也起着至关重要的作用。有研究表明,饲粮中补充GML降低了脂多糖诱导的促炎趋化因子和细胞因子的表达,提高了回肠中抗炎因子IL-10的表达[28]。在针对大肠杆菌攻毒的肉鸡中,补充EUG纳米乳显著降低了促炎因子的表达,改善了由大肠杆菌引起的炎症反应,从而改善肉鸡肠道健康[3]。炎症与氧化应激密切相关,炎症反应可引发氧化应激,氧化应激又会加剧炎症,二者形成恶性循环。本试验研究中,EUG和GML纳米乳能够调节肉鸡肠道的炎症因子表达,抑制肠道过度炎症。EUG和GML纳米乳的抗炎作用一方面可能与其较高的抗氧化能力有关;另一方面EUG通过非特异性杀灭真菌、细菌、肿瘤细胞和寄生虫,从而降低病原体的负荷,从而增强机体的非特异性免疫[3],达到抗炎效果。

3.5 饮水中添加EUG和GML纳米乳对肉鸡盲肠菌群结构和α多样性的影响

肠道菌群有助于维持肠道的生理结构和功能,同时与宿主的肠道炎症、屏障功能和生长性能有关[29]。本试验中,通过α多样性分析发现,各组之间42日龄肉鸡盲肠菌群ACE、Chao1、Shannon和Simpson指数无显著差异,说明饮水中添加EUG和GML纳米乳不影响白羽肉鸡盲肠菌群的α多样性。基于门水平的分析结果表明,厚壁菌门、拟杆菌门和变形菌门是肉鸡盲肠的主要菌门,这与其他研究结果[30]一致。厚壁菌门和拟杆菌门同为肠道主要菌群之一,存在一种相互促进的共生关系,二者共同促进宿主吸收或储存能量[31]。研究表明,添加1 200 mg/kg的GML可增加盲肠拟杆菌门相对丰度,其主要促进难消化碳水化合物发酵为丁酸,并通过抗炎作用对黏膜屏障的完整性起到有利作用[32]。冯宇妍等[33]研究发现,肠道菌群中厚壁菌门和拟杆菌门比值降低,有助于改善肉鸡代谢、减少脂肪沉积。厚壁菌门和拟杆菌门比值的增加是肠道屏障受损的表现[34]。本试验中,饮水中添加5.0 mL/L EUG和GML纳米乳,一定程度上提高了盲肠拟杆菌门相对丰度,降低了厚壁菌门相对丰度,即降低了厚壁菌门和拟杆菌门比值,这可能是肉鸡肠道形态改善的潜在原因之一。

4 结论

饮水中添加EUG和GML纳米乳可增强肉鸡的抗氧化能力,改善肉鸡的肠道形态、炎症反应和菌群结构,进而提高肉鸡的生长性能。在本试验中,饮水中EUG和GML纳米乳的适宜添加水平为3.0 mL/L。

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