Effects of Plant Essential Oil and Bile Salt Compound Preparation on Intestinal Barrier Function of Goats

GAO Ruiling; LI Shengli; JIN Lu; ZHANG Chunhua; ZHANG Chongzhi; ZHANG Jiacheng; SUN Haizhou

doi:10.12418/CJAN2023.295

Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 5: 3174 - 3182

DOI: https://doi.org/10.12418/CJAN2023.295

RESEARCH PAPER

Effects of Plant Essential Oil and Bile Salt Compound Preparation on Intestinal Barrier Function of Goats

GAO Ruiling ,
LI Shengli ,
JIN Lu ,
ZHANG Chunhua ,
ZHANG Chongzhi ,
ZHANG Jiacheng ,
SUN Haizhou ^,^*

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Inner Mongolia Key Laboratory of Herbivores Nutrition, Animal Nutrition and Feed Research Institution, Inner Mongolia Academy of Agriculture and Animal Husbandry, Hohhot 010031, China

*professor, E-mail: sunhaizhou@china.com

Received date: 2022-11-07

Online published: 2023-05-11

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Abstract

This experiment was conducted to investigate the effects of plant essential oil and bile salt compound preparation on intestinal barrier function of goats. Twenty-four 5-month-old Alxa white cashmere goat rams with body weight of (21.46±2.07) kg were randomly divided into 4 groups with 6 replicates in each group and 1 goat in each replicate. The control group was fed a basal diet, and the experimental groups were fed basal diet supplemental with 5 g/kg compound preparation with medical stone as carrier; among them, the compound preparation in experimental group 1 (T1 group) contained 500 mg plant essential oil (60% artemisia oil, 40% carvacrol+cinnamaldehyde+oregano oil), the compound preparation in experimental group 2 (T2 group) contained 500 mg plant essential oil (60% artemisia oil, 40% carvacrol+cinnamaldehyde+oregano oil) and 500 mg bile salt, and the compound preparation in experimental group 3 (T3 group) contained 500 mg bile salt. The pre-feeding period was 10 days and the feeding period was 60 days. The results showed as follows: 1) compared with the control group, the average daily feed intake of T1 group and T2 group was significantly increased (P<0.05); the feed to gain ratio of T1 group was decreased by 28%, and the feed to gain ratio of T2 group was decreased by 10%. 2) Compared with the control group, the villous height and villous height/crypt depth in jejunum of T1 and T2 groups were significantly increased (P<0.05), and the crypt depth in jejunum of T1 group was significantly decreased (P<0.05); the villous height and villous height/crypt depth in ileum of T1 group were significantly increased (P<0.05), and the crypt depth in ileum of T1 and T2 groups was significantly decreased (P<0.05). 3) Compared with the control group, the mRNA relative expression level of zonula occluden-1 (ZO-1) in cecal mucosa of T1 group was significantly increased (P<0.05); the mRNA relative expression level of Occludin in rumen of T2 and T3 groups was significantly increased (P<0.05), and the mRNA relative expression level of Occludin in ileal mucosa of T1 and T2 groups was significantly increased (P<0.05); the mRNA relative expression level of Claudin-1 in cecal mucosa of T1 and T3 groups was significantly increased (P<0.05); the mRNA relative expression level of farnesoid X receptor (FXR) in liver of T1, T2 and T3 groups was significantly increased (P<0.05). In conclusion, the addition of compound plant essential oil in diet has the best effect on average daily feed intake, feed to gain ratio and intestinal tissue morphology development of goats, and can promote the mRNA expression of intestinal tight junction proteins. The addition of plant essential oil and bile salt compound preparation in diet can increase average daily feed intake and decrease feed to weight ratio to a certain extent of goats, and has a positive effect on the healthy development of the intestine.

Key words： young cashmere goats; plant essential oil; bile salt; intestinal barrier

Cite this article

GAO Ruiling , LI Shengli , JIN Lu , ZHANG Chunhua , ZHANG Chongzhi , ZHANG Jiacheng , SUN Haizhou . Effects of Plant Essential Oil and Bile Salt Compound Preparation on Intestinal Barrier Function of Goats[J]. Chinese Journal of Animal Nutrition, 2023 , 35(5) : 3174 -3182 . DOI: 10.12418/CJAN2023.295

幼龄绒山羊消化系统发育尚不完全,肠道屏障功能不完善,随着粗饲料和固体饲料摄入增加,可能会产生应激,引起食欲衰减、消化不良、生长迟滞、饲料利用率低、抵抗力低下等问题,严重时引起肠道的损伤^[1]。肠道屏障是肠道上皮有效阻止有毒有害物质侵入的重要方式^[2]。当前,植物精油是众多替代抗生素候选物中公认的较有应用前景的替代品,具有抗菌活性,通过破坏细菌细胞膜保护机体免受肠道内生长的革兰氏阳性菌的侵害,作为一种防御机制来对抗感染性病原体和毒素^[3]。胆汁酸可作为脂肪乳化剂,而且还是一种信号分子,通过激活核受体和G蛋白偶联受体释放信号分子,形成复杂的网络调节结构,调节糖脂代谢和能量平衡,尤其是在肠道黏膜屏障稳态中发挥关键作用^[4]。在肠道中,受胆汁酸激活的核受体法尼醇X受体(FXR)可以通过上调紧密连接蛋白封闭蛋白-1(Claudin-1)、闭锁蛋白(Occludin)、闭锁小带蛋白-1(ZO-1)的表达以修复损伤的回肠黏膜上皮,而且FXR的激活可以抑制肿瘤坏死因子-α(TNF-α)诱导的核因子-κB(NF-κB)的转录活性,对肠道上皮细胞具有免疫调节作用^[5]。因此,胆汁酸的添加对肠道屏障的保护也具有重要意义。目前,复合植物精油的使用已经在畜禽生产中有一定研究,胆盐也在包括水产动物、单胃动物等方向上开展过相关研究^[6],但二者在山羊饲粮中组合添加的研究较少。因此,本试验通过添加植物精油和胆盐复合制剂,测定其对幼龄绒山羊胃肠道组织形态、肠道屏障功能相关基因和胆汁酸受体表达的影响,探究其对山羊胃肠道物理屏障的影响。

1 材料与方法

1.1 试验动物及试验设计

选择24只5月龄体重为(21.46±2.07) kg的阿拉善白绒山羊公羊,随机分为4组,每组6个重复,每个重复1只。对照组饲喂基础饲粮;试验组均在基础饲粮中额外添加5 g/kg以麦饭石为载体的复合制剂,其中试验1组(T1组)复合制剂中含500 mg植物精油(60%沙蒿油、40%香芹酚+肉桂醛+牛至油),试验2组(T2组)复合制剂中含500 mg植物精油(60%沙蒿油、40%香芹酚+肉桂醛+牛至油)和500 mg胆盐,试验3组(T3组)复合制剂中含500 mg胆盐。

1.2 基础饲粮

基础饲粮参考NRC(2007)饲养标准配制,其组成及营养水平见表1。

表1 基础饲粮组成及营养水平表(风干基础)

Table 1 Composition and nutrient levels of the basal diet (air-dry basis) %

项目Items	含量Content
原料Ingredients
玉米秸Corn stalk	48.00
苜蓿Alfalfa	12.00
玉米Corn	23.80
麸皮Bran	7.40
豆粕Soybean meal	3.00
棉籽粕Cottonseed meal	2.00
干酒糟及其可溶物DDGS	1.50
碳酸钙CaCO₃	0.75
磷酸氢钙CaHPO₄	0.25
预混料Premix¹⁾	0.80
碳酸钠NaCO₃	0.25
食盐NaCl	0.25
合计Total	100.00
营养水平Nutrient levels²⁾
代谢能ME/(MJ/kg)	9.89
粗蛋白质CP	9.83
粗脂肪EE	2.43
钙Ca	0.61
磷P	0.40
中性洗涤纤维NDF	47.09
非纤维性碳水化合物NFC	32.54
非纤维性碳水化合物/中性洗涤纤维 NFC/NDF	0.69

1)每千克预混料含有 One kilogram of premix contained:Fe (FeSO₄·7H₂O) 170 g,Cu (CuSO₄·5H₂O)70 g,Mn (MnSO₄·5H₂O) 290 g,Zn (ZnSO₄·7H₂O) 240 g,Co (CoCl₂·6H₂O) 510 mg,KI 200 mg,NaSeO₃ 130 mg,VA 1 620 000 IU,VD₃ 324 000 IU,VE 540 IU,VK₃ 150 mg,VB₁₂ 0.9 mg,VB₅ 450 mg。

2)计算值 Calculated values。

1.3 饲养管理

试验山羊单笼饲养,预试期10 d,正试期50 d,每日09:00和18:00各饲喂1次,先粗后精,自由饮水。预试期前1天统一免疫驱虫。

1.4 样品采集

饲养试验结束时进行屠宰,采集新鲜十二指肠、空肠、回肠组织,将组织切成约5 cm×5 cm的小块,4%多聚甲醛固定,用于石蜡切片的制作。另外选取瘤胃上皮组织、十二指肠、回肠、盲肠分离黏膜,分装至冻存管中,置于-80 ℃冰箱冻存待测。

1.5 测定指标与方法

1.5.1 生长性能

在正式试验开始前第1天清晨空腹对试验羊称重,试验期内每日记录采食量,并计算试验羊的平均日增重(ADG)、平均日采食量(ADFI)及料重比(F/G),计算公式如下:

平均日增重=试验期增重/(试验天数×试验动物数);

平均日采食量=试验期采食量/(试验天数×试验动物数);

料重比=试验期采食量/试验期增重。

1.5.2 苏木精-伊红染色及上皮组织形态结构检测

十二指肠、空肠和回肠固定样由上海达为科生物科技有限公司制作,肠道组织切片制作完成后,使用NIKON公司ECLIPSE Ci正置显微镜下放大100倍对绒毛结构观察、拍照。每张切片随机选取绒毛结构清晰且走向平直完整的3个不同视野,使用Image-Pro-Plus 5.0图像处理分析软件随机选取5根完整的绒毛,测定绒毛高度(villous height)、隐窝深度(crypt depth),并计算绒毛高度/隐窝深度(villous height/crypt depth,V/C)。

1.5.3 紧密连接蛋白基因mRNA相对表达量测定

1.5.3.1 总RNA的提取与反转录

使用RNA提取试剂盒RNAiso Plus(TaKaRa)提取羔羊瘤胃、十二指肠、回肠、盲肠上皮组织中总RNA,并使用反转录试剂盒Prime Script RT reagent Kit with gDNA Eras(TaKaRa)将其反转录成cDNA,于-20 ℃冰箱保存待测。

1.5.3.2 引物设计和实时荧光定量PCR(RT-PCR)

根据NCBI GenBank中相关基因序列设计引物。测定试验采用TB Green Premix Ex Taq^TM Ⅱ(No:DRR041A)试剂盒,详细操作按说明书。上机使用BIO-RAD荧光定量PCR仪。PCR反应体系总体积为20 μL,其中包括TB Green Premix Ex Taq^TM Ⅱ 10 μL,上游引物、下游引物(25 μmol/L)各0.4 μL,cDNA 2 μL,ddH₂O 7.2 μL。PCR过程为:预变性95 ℃ 30 s;变性95 ℃ 3 s,退火60 ℃ 30 s,共40个循环。阴性对照用无酶水代替cDNA模板,每个样品做3个技术重复,对定量结果进行溶解曲线分析,检测扩增片段的特异性,使用油醛-3-磷酸脱氢酶(GAPDH)作为内参基因,mRNA相对表达量采用2^-△△Ct法进行相对定量数据分析。

表2 引物序列

Table 2 Primer sequences

基因 Genes	引物序列 Primer sequence (5'—3')	产物大小 Product size/bp	退火温度 Tm/℃
闭锁小带蛋白-1 ZO-1	F:ATTTGGGGCTGATGAGG R:TGGCTGGTATTTTAACGGA	109	58
封闭蛋白-1 Claudin-1	F:CAGGCCTTCTCGTGGTTAGG R:ATGGAACAGGGTGCCAACAA	93	56
闭锁蛋白 Occludin	F:GGAAAACATAGACGGCAGA R:CGGGTTAGAGCCCAAAG	106	60
法尼醇X受体 FXR	F:TCTGAAACTGAATCTGAGCGT R:CCTGAAACACTTCTGCCTCT	128	56
油醛-3-磷酸脱氢酶 GAPDH	F:CCTGCCACACTGCAAGA R:AGGGCTGTCACCAAACTG	70	58

1.6 数据分析

试验数据使用Excel 2016初步分析,采用SPSS 20.0软件进行进一步数据处理和分析,数据符合正态分布时,组间比较用进行ANOVA进行分析,两两比较用Bonferroni法进行分析;当方差不齐时,组间比较采用Dunnett’s T3法进行近似方差分析。数据以平均值±标准差表示,P<0.05表示有统计学差异,P>0.05表示无统计学差异。

2 结果

2.1 植物精油和胆盐复合制剂对山羊生长性能的影响

从表3可以看出,与对照组相比,T1组T2组的平均日采食量显著提高(P<0.05)。与对照组相比,T1组T2组的平均日增重提高,但差异不显著(P>0.05)。与对照组相比,T1组的料重比降低了28%,T2组的料重比降低了10%。

表3 植物精油和胆盐复合制剂对山羊生长性能的影响

Table 3 Effects of plant essential oil and bile salt compound preparation on growth performance of goats

项目 Items	组别Groups				P值 P-value
项目 Items	对照Control	T1	T2	T3	P值 P-value
初始体重IBW/kg	21.10±2.02	21.69±1.21	21.92±2.39	21.68±4.20	0.958
终末体重FBW/kg	25.48±2.21	28.53±1.51	27.21±4.13	26.00±3.39	0.319
平均日采食量ADFI/g	736.67±68.36^b	826.42±18.10^a	798.66±39.59^a	781.01±45.20^ab	0.023
平均日增重ADG/g	87.50±17.57	136.83±31.11	105.83±44.52	86.33±44.64	0.086
料重比F/G	8.42	6.04	7.55	9.05

	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 植物精油和胆盐复合制剂对山羊肠道上皮组织形态结构的影响

由图1和表4可以看出,各组之间十二指肠绒毛高度、隐窝深度、绒毛高度/隐窝深度均差异不显著(P>0.05)。与对照组相比,T1、T2组的空肠绒毛高度、绒毛高度/隐窝深度显著升高(P<0.05),T1组的空肠隐窝深度显著降低(P<0.05)。与对照组相比,T1组的回肠绒毛高度和绒毛高度/隐窝深度显著升高(P<0.05),T1、T2组的回肠隐窝深度显著降低(P<0.05)。

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图1 植物精油和胆盐复合制剂对山羊小肠组织形态结构的影响

CK:对照组 control group;T1:T1组 T1 group;T2:T2组 T2 group;T3:T3组 T3 group。

Fig.1 Effects of plant essential oil and bile salt compound preparation on morphological structure of small intestine of goats

表4 植物精油和胆盐复合制剂对山羊肠道上皮组织形态结构的影响

Table 4 Effects of plant essential oil and bile salt compound preparation on morphological structure of intestinal epithelium of goats

项目 Items		组别Groups				P值 P-value
项目 Items		对照Control	T1	T2	T3	P值 P-value
十二指肠 Duodenum	绒毛高度Villous height/μm	500.50±17.28	512.55±20.14	502.02±19.18	514.25±12.04	0.265
	隐窝深度Crypt depth/μm	171.26±20.79	163.21±9.44	165.06±20.12	158.03±15.21	0.189
	绒毛高度/隐窝深度V/C	2.45±0.21	2.63±0.18	2.45±0.21	2.34±0.14	0.231
空肠 Jejunum	绒毛高度Villous height/μm	510.06±11.70^b	540.25±19.35^a	538.26±19.20^a	524.30±15.21^ab	0.047
	隐窝深度Crypt depth/μm	200.19±20.27^a	180.23±22.17^b	192.42±20.02^ab	194.20±20.12^ab	0.022
	绒毛高度/隐窝深度V/C	2.54±0.24^b	2.98±0.15^a	2.81±0.11^a	2.68±0.35^ab	0.021
回肠 Ileum	绒毛高度Villous height/μm	360.19±24.41^b	388.70±20.62^a	362.21±24.32^b	360.32±23.36^b	0.013
	隐窝深度Crypt depth/μm	208.75±18.13^a	180.21±14.31^b	178.55±17.14^b	192.21±11.25^ab	0.024
	绒毛高度/隐窝深度V/C	1.78±0.33^b	2.14±0.15^a	2.08±0.12^b	1.86±0.23^ab	0.002

**2.3 植物精油和胆盐复合制剂对山羊胃肠黏膜上皮紧密连接蛋白和FXR mRNA表达的影响**

由表5可以看出,与对照组相比,T1组的盲肠黏膜ZO-1的mRNA相对表达量显著提高(P<0.05)。与对照组相比,T2和T3组的瘤胃Occludin的mRNA相对表达量显著提高(P<0.05),T1和T2组的回肠黏膜Occludin的mRNA相对表达量显著提高(P<0.05)。与对照组相比,T1和T3组的盲肠黏膜Claudin-1的mRNA相对表达量显著提高(P<0.05)。与对照组相比,T1、T2和T3组的肝脏FXR的mRNA相对表达量显著提高(P<0.05)。

**表5 植物精油和胆盐复合制剂对山羊胃肠黏膜上皮紧密连接蛋白和FXR mRNA表达的影响**

Table 5 Effects of plant essential oil and bile salt compound preparation on expression of tight junction protein and FXR mRNA in gastrointestinal mucosal epithelium of goats

项目 Items		组别Groups				P值 P-value
项目 Items		对照Control	T1	T2	T3	P值 P-value
闭锁小带蛋白-1 ZO-1	瘤胃Rumen	0.96±0.08	1.52±0.21	1.01±0.02	1.18±0.28	0.401
	十二指肠黏膜Duodenal mucosa	1.02±0.20	1.03±0.15	0.98±0.04	1.12±0.06	0.801
	回肠黏膜Ileal mucosa	1.05±0.04	1.32±0.01	1.29±0.05	1.20±0.26	0.705
	盲肠黏膜Cecal mucosa	0.90±0.01^b	1.25±0.15^a	1.08±0.10^ab	0.88±0.03^b	0.020
闭锁蛋白 Occludin	瘤胃Rumen	0.76±0.22^c	0.97±0.07^bc	1.74±0.03^a	1.28±0.24^b	0.011
	十二指肠黏膜Duodenal mucosa	0.95±0.21	0.99±0.25	1.04±0.06	1.20±0.30	0.514
	回肠黏膜Ileal mucosa	1.00±0.05^b	1.66±0.22^a	1.52±0.04^a	1.13±0.22^b	0.025
	盲肠黏膜Cecal mucosa	0.82±0.11	1.31±0.01	1.28±0.32	1.53±0.50	0.125
封闭蛋白-1 Claudin-1	瘤胃Rumen	0.67±0.12	1.11±0.05	1.03±0.03	1.28±0.39	0.114
	十二指肠黏膜Duodenal mucosa	1.10±0.30	1.03±0.05	0.98±0.21	0.96±0.03	0.726
	回肠黏膜Ileal mucosa	0.79±0.13	1.44±0.19	1.17±0.19	1.22±0.26	0.091
	盲肠黏膜Cecal mucosa	0.78±0.02^b	1.30±0.25^a	0.80±0.07^b	1.14±0.21^a	0.029
法尼醇X 受体 FXR	肝脏Liver	0.98±0.02^b	1.15±0.70^a	1.20±0.01^a	1.05±0.08^a	0.009
	结肠Colon	0.97±0.01	0.95±0.01	0.96±0.35	0.96±0.28	0.159
	回肠Ileum	0.98±0.15	0.96±0.04	0.95±0.12	0.96±0.07	0.269

3 讨论

本研究中,饲喂植物精油和胆盐复合制剂的山羊,在生长性能中表现平均日采食量、平均日增重升高,料重比降低。卫玲娟等^[7]在家禽中饲喂以胆汁酸为主要成分的乳化剂,一定程度上提高了采食量并降低了料重比;毛婷^[8]研究不同水平的植物精油对仔猪生长性能的影响,也得到了类似结果。

肠道屏障功能是指肠道上皮有效阻止有毒有害物质的侵入^[9]。上皮细胞完整性是影响肠物理屏障的重要因素,由肠上皮细胞、肠内分泌细胞、杯状细胞、Paneth细胞和微折叠细胞等组成^[10]。这些细胞共同形成一个连续的极化单层,将管腔与固有层分开。肠黏膜上皮细胞以及上皮细胞间的紧密连接是物理屏障的重要组成部分,紧密连接主要由跨膜蛋白[如封闭蛋白(Claudins)、Occludin]、外周膜蛋白[如ZO-1、闭锁小带蛋白-2(ZO-2)]和调节蛋白组成,能有效地阻止细菌及脂多糖(LPS)等有毒有害物质透过胃肠黏膜进入血液循环^[11]。

目前,香芹酚、百里香酚和肉桂醛等植物精油是公认的替抗产品。在影响畜禽肠道屏障功能方面具有重要意义。张燕利^[12]研究表明,饲粮中添加植物精油能够显著提高十二指肠、回肠、空肠的绒毛高度/隐窝深度,改善肉鸡肠道形态,一定程度上提高空肠屏障基因的表达。香芹酚和百里香酚通过多种机制调控并增强肠道功能完整性^[13⇓-15]。周洪彬等^[16]研究发现,肉桂醛(10%)与百里香酚(10%)植物精油组合添加和能显著提高肉仔鸡空肠绒毛高度。Lei等^[17]研究发现,胡椒提取的精油可显著提高空肠绒毛高度,降低隐窝深度,增大绒毛表面积。宋转等^[18]和王蕾蕾等^[19]研究发现,在LPS攻毒的条件下,饲粮中添加复合精油可显著提高仔猪空肠、回肠的绒毛高度和空肠的绒毛高度/隐窝深度。本试验结果与前人研究结果基本一致,说明幼龄山羊饲粮中添加复合植物精油可能对肠道发育有一定改善作用。

王改玲^[20]研究结果显示,饲粮中添加胆汁酸可以显著提高黄羽肉雏鸡肠道绒毛高度。曾本和等^[21]研究发现,饲喂胆汁酸可以显著提高齐口裂腹鱼肠管壁厚度及肠绒毛高度。朱传忠等^[22]研究发现,在吉富罗非鱼幼鱼饲料中添加胆汁酸,一定程度上增加了肠道绒毛的个数和肌层厚度。隐窝深度增加可以提高肠上皮细胞成熟率,使肠黏膜上皮细胞生长加快,具有增强修复肠道损伤的作用。本研究也得到相同结果,表明饲粮中添加植物精油和胆盐复合制剂能促进幼龄山羊的肠道组织形态发育。

陈磊等^[23]研究表明,给猪饲喂25 mg/kg牛至油(含81%香芹酚)4周后,空肠绒毛高度及Occludin和ZO-1的蛋白相对表达量显著增加,表明牛至油可提高猪肠道的屏障功能。王亚男^[24]研究发现,包被植物精油替代硫酸粘杆菌素极显著提高了仔猪空肠黏膜Occludin的mRNA相对表达量。FXR能被初级胆汁酸直接激活,鹅脱氧胆酸(CDCA)是人FXR的生理配体。使用FXR激动剂可以通过上调紧密连接蛋白Claudin-1、Occludin、ZO-1的表达,来修复损伤的回肠黏膜上皮。本研究中,与对照组相比,T2组的瘤胃及回肠黏膜Occludin的mRNA相对表达量显著提高,且肝脏中FXR的mRNA相对表达也显著提高,与前人研究结果基本一致,表明饲粮中添加植物精油和胆盐复合制剂在幼龄山羊胃肠道上皮屏障功能的发育中起到积极作用。

4 结论

饲粮中添加复合植物精油对山羊平均日采食量、料重比以及肠道发育的改善效果最佳,对肠道紧密连接蛋白的mRNA表达有促进作用;饲粮中添加植物精油和胆盐复合制剂也能在一定程度上提高山羊的平均日采食量、降低料重比,且在肠道发育过程中有积极影响。

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