Effects of Dietary Zinc Deficiency on Growth Performance, Small Intestinal Morphology, Barrier Function and mRNA Expression of Tight Junction Proteins in Jejunum of Broilers during 22 to 42 Days of Age

WU Wei; HU Yangyang; GAO Feiyu; ZHANG Weiyun; WU Bingxin; ZHANG Liyang; CUI Xiaoyan; LI Tingting; WANG Shengchen; HU Yun; LUO Xugang

doi:10.12418/CJAN2024.022

2024 , Vol. 36 >Issue 1: 235 - 244

DOI: https://doi.org/10.12418/CJAN2024.022

RESEARCH PAPER

Effects of Dietary Zinc Deficiency on Growth Performance, Small Intestinal Morphology, Barrier Function and mRNA Expression of Tight Junction Proteins in Jejunum of Broilers during 22 to 42 Days of Age

WU Wei ^,¹ ,
HU Yangyang ¹^,^* ,
GAO Feiyu ¹ ,
ZHANG Weiyun ¹ ,
WU Bingxin ¹ ,
ZHANG Liyang ² ,
CUI Xiaoyan ¹ ,
LI Tingting ¹ ,
WANG Shengchen ¹ ,
HU Yun ^,¹^,^** ,
LUO Xugang ^,¹^,^**

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¹ Poultry Mineral Nutrition Laboratory, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
² Mineral Nutrition Research Division, Institute of Animal Science, Chinese Academy of Agricultural Science, Beijing 100193, China

** HU Yun, lecturer, E-mail: huyun@yzu.edu.cn;

LUO Xugang, professor, E-mail: wlysz@263.net

^*Contributed equally

Received date: 2023-07-07

Online published: 2024-01-12

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Abstract

This experiment was conducted to study the effects of dietary zinc deficiency on growth performance, small intestinal morphology, barrier function and mRNA expression of tight junction proteins in jejunum of broilers during 22 to 42 days of age. A total of 140 one-day-old male Arbor Acres broilers were fed the experimental diet with normal addition of zinc sulfate heptahydrate (ZnSO₄·7H₂O) during 1 to 21 days of age; at 22 days of age, a total of 112 broilers with similar body weight were selected and randomly divided into 2 groups (7 replicates in each group and 8 broilers in each replicate) according to a completely randomized design, broilers in the control group were fed the experimental diet which meet the zinc requirements of broilers (added 40 mg/kg zinc to the basal diet, and the measured zinc content was 64.36 mg/kg), and broilers in the zinc deficient group were fed the experimental diet without zinc addition (not added zinc to the basal diet, and the measured zinc content was 22.23 mg/kg). The experiment lasted for 42 days. The results showed that compared with the control group: 1) the feed to gain ratio of broilers during 22 to 42 days of age of zinc deficient group was significantly increased (P<0.05); 2) the crypt depth in duodenum and jejunum of broilers at 42 days of age of zinc deficient group was significantly increased (P<0.05), and the villus height/crypt depth ratio in duodenum, jejunum and ileum and villus height and villus surface area in jejunum of broilers at 42 days of age were significantly decreased (P<0.05); 3) the diamine oxidase activity and fluorescein isothiocyanate-dextran content in wing vein and hepatic portal vein serum of broilers at 42 days of age of zinc deficient group were significantly increased (P<0.05); 4) the mRNA expression levels of Claudin-1 in jejunum of broilers at 42 days of age of zinc deficient group was significantly decreased (P<0.05). In conclusion, dietary zinc deficiency can impair the intestinal mucosal integrity and barrier function of broilers possibly by decreasing the mRNA expression of Claudin-1, and thus decrease the feed conversion efficiency of broilers.

Key words： zinc deficiency; broilers; growth performance; intestinal barrier function; tight junction protein

Cite this article

WU Wei , HU Yangyang , GAO Feiyu , ZHANG Weiyun , WU Bingxin , ZHANG Liyang , CUI Xiaoyan , LI Tingting , WANG Shengchen , HU Yun , LUO Xugang . Effects of Dietary Zinc Deficiency on Growth Performance, Small Intestinal Morphology, Barrier Function and mRNA Expression of Tight Junction Proteins in Jejunum of Broilers during 22 to 42 Days of Age[J]. Chinese Journal of Animal Nutrition, 2024 , 36(1) : 235 -244 . DOI: 10.12418/CJAN2024.022

锌是动物必需的微量元素之一,具有重要而广泛的生物学功能,为体内300多种酶或功能蛋白质的必需组分或激活因子,广泛参与动物体内一系列代谢活动^[1]。研究表明,缺锌会导致动物生长缓慢、繁殖功能减退、肠道黏膜损伤及免疫系统疾病等一系列锌缺乏症^[2]。

肉仔鸡等畜禽肠道既是营养物质消化和吸收的关键部位,还是阻止外源病原微生物和有毒有害物质等侵入的第1道防线。完整的肠道屏障功能是动物机体消化和吸收营养物质的基本保证。动物肠道黏膜屏障主要由物理屏障、化学屏障、微生物屏障和免疫屏障组成。其中,物理屏障主要是肠黏膜上皮细胞及其紧密连接等构成的完整肠道上皮结构,在肠道黏膜屏障中尤为重要,是其他3种屏障的基础。小肠黏膜包括肠绒毛和隐窝,肠绒毛的高度受到成熟上皮细胞凋亡脱落、幼稚上皮细胞迁移和隐窝干细胞增殖及紧密连接蛋白mRNA表达等的共同影响^[3⇓-5]。小肠上皮细胞通过紧密连接蛋白、黏附连接蛋白及细胞桥粒等连接在一起,其中紧密连接蛋白是小肠上皮细胞最顶端的细胞连接,其在维持上皮细胞顶端和基底侧的极性及调节肠黏膜通透性等方面都有重要作用^[6-7]。紧密连接蛋白主要包括闭合蛋白(Claudin)、闭锁蛋白(Occludin)、闭合小环蛋白(zonula occluden,ZO)和连接黏附分子(junction adhesion molecular,JAM)等^[8]。由于受环境以及营养素缺乏等因素的影响,肠道黏膜完整性和屏障功能容易受到损伤,引起肠道平衡紊乱,进而引发各种疾病,降低动物生产性能,甚至导致死亡。在哺乳动物上的研究表明,饲粮锌缺乏可通过降低紧密连接蛋白Claudin-3和Occludin mRNA的表达进而引起肠道物理屏障功能损伤^[9]。然而,目前关于饲粮锌缺乏对肉仔鸡小肠形态结构、屏障功能及紧密连接蛋白mRNA表达的研究较少,有待进一步深入研究。

因此,本试验旨在通过研究饲粮锌缺乏对22~42日龄肉仔鸡生长性能、小肠形态、屏障功能及空肠中紧密连接蛋白mRNA表达的影响,为肉仔鸡生产中合理添加锌以及防治锌缺乏症提供科学依据。

1 材料与方法

1.1 试验设计

试验选用140只1日龄爱拔益加肉仔鸡公雏(购自江苏京海禽业有限公司),分2阶段饲养,1~21日龄统一饲喂正常添加七水合硫酸锌(ZnSO₄·7H₂O)的试验饲粮(实测锌含量为84.40 mg/kg);22日龄时,从中选取体重接近的112只肉仔鸡,采用单因子完全随机试验设计,随机分为2组(每组7个重复,每个重复8只鸡),对照组[肉仔鸡平均体重为(802.90±26.04) g]饲喂满足肉仔鸡锌需要量的试验饲粮(基础饲粮中添加40 mg/kg锌,以ZnSO₄·7H₂O的形式添加,实测锌含量为64.36 mg/kg),锌缺乏组[肉仔鸡平均体重为(801.14±30.84) g]饲喂不添加锌的试验饲粮(基础饲粮中不添加锌,实测锌含量为22.23 mg/kg)。试验期为42 d。

1.2 试验饲粮与饲养管理

参照NRC(1994)和《鸡饲养标准》(NY/T 33—2004)分别配制1~21日龄肉仔鸡的试验饲粮和22~42日龄肉仔鸡不同锌添加水平的试验饲粮,试验饲粮组成及营养水平见表1。试验用锌添加形式为ZnSO₄·7H₂O,购自于国药集团化学试剂有限公司,锌含量22.5%,纯度99.5%。饲养试验在扬州天一畜禽有限公司进行。饲养管理按《AA肉仔鸡饲养管理手册》进行,鸡只自由采食与饮水,24 h恒定光照,饲粮以粉料形式喂给。每日观察记录鸡舍环境温湿度以及鸡只健康状况、发病和死亡情况。

表1 试验饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of experimental 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
项目 Items	1~21日龄 1 to 21 days of age	对照组 Control group	锌缺乏组 Zinc deficient group
原料Ingredients
玉米Corn	54.75	58.78	58.78
豆粕Soybean meal	36.31	32.56	32.56
大豆油Soybean oil	4.95	5.15	5.15
磷酸氢钙CaHP $O 4 1)$	1.87	1.67	1.67
碳酸钙CaC $O 3 1)$	1.20	1.09	1.09
氯化钠NaCl¹⁾	0.30	0.30	0.30
DL-蛋氨酸DL-Met²⁾	0.31	0.15	0.15
预混料Premix³⁾	0.31	0.20	0.20
玉米淀粉+锌源Corn starch+zinc source⁴⁾		0.10	0.10
合计Total	100.00	100.00	100.00
营养水平Nutrient levels⁵⁾
代谢能ME/(MJ/kg)	12.75	12.98	12.98
粗蛋白质CP	21.44	20.08	20.08
赖氨酸Lys	1.10	1.01	1.01
蛋氨酸Met	0.61	0.44	0.44
L-苏氨酸L-Thr	0.80	0.75	0.75
色氨酸Trp	0.24	0.22	0.22
蛋氨酸+半胱氨酸Met+Cys	0.91	0.72	0.72
钙Ca	1.01	0.92	0.92
非植酸磷NPP	0.45	0.40	0.40
锌Zn/(mg/kg)	84.40	64.36	22.23

1)试剂级 Reagent grade。

2)饲料级 Feed grade。

3)预混料为每千克饲粮提供 Premix provided the following per kilogram of diets: 1~21日龄 1 to 21 days of age,VA 12 000 IU,VD₃ 4 500 IU,VE 33 IU,VK₃ 3 mg,VB₁ 3 mg,VB₂ 9.6 mg,VB₆ 4.5 mg,VB₁₂ 0.03 mg,泛酸钙 calcium pantothenate 15 mg,烟酸 niacin 54 mg,叶酸 folic acid 1.5 mg,生物素 biotin 0.15 mg,胆碱 choline 700 mg,Cu (CuSO₄·5H₂O) 6 mg,Fe (FeSO₄·H₂O) 40 mg,Zn (ZnSO₄·7H₂O) 60 mg,Mn (MnSO₄·H₂O) 110 mg,Se (Na₂SeO₃) 0.35 mg,I [Ca(IO₃)₂·H₂O] 0.35 mg;22~42日龄 22 to 42 days of age,VA 8 000 IU,VD₃ 3 000 IU,VE 22 IU,VK₃ 2 mg,VB₁ 2 mg,VB₂ 6.4 mg,VB₆ 3 mg,VB₁₂ 0.02 mg,泛酸钙 calcium pantothenate 10 mg,烟酸 niacin 36 mg,叶酸 folic acid 1.0 mg,生物素 biotin 0.10 mg,胆碱 choline 500 mg,Fe (FeSO₄·7H₂O) 30 mg,Mn (MnSO₄·H₂O) 80 mg,Se (Na₂SeO₃) 0.35 mg,I [Ca(IO₃)₂·H₂O] 0.35 mg。

4)按替代等重的玉米淀粉添加锌源。The addition of zinc source according to the replacement of equal weight corn starch.

5)粗蛋白质、钙、锌为实测值,其他为计算值。CP, Ca and Zn were measured values, while the others were calculated values.

1.3 样品采集与制备

饲粮配制时现场采集饲粮样品,用四分法取200 g饲粮样品,研磨过200目筛后置于自封袋中低温保存,用于测定其中粗蛋白质、钙和锌含量。

于试验的42日龄,在鸡只禁食不禁水12 h后,从每个重复选取接近平均体重的3只鸡,分别从翅静脉(反映肠道吸收后经过肝脏代谢后的情况)和肝门静脉(汇集肠静脉血液,反映肠道吸收情况)处抽取5 mL全血于非抗凝真空管中,离心后收集血清于-20 ℃冻存,以备测定血清内毒素含量和二胺氧化酶(diamine oxidase,DAO)活性。

鸡只采血后,取出其中1只鸡的小肠,分离十二指肠、空肠和回肠,切取小肠各段中间部分2 cm,置于4%多聚甲醛溶液中,以备分析小肠各段形态结构[绒毛高度(villus height,VH)、隐窝深度(crypt depth,CD)、VH/CD和绒毛表面积(villus surface area,VSA)];将空肠截取后的剩余部分肠段及剩下2只鸡的空肠用冰生理盐水冲洗干净后,用灭菌的载玻片刮取肠黏膜,置于灭菌离心管中,于-196 ℃液氮速冻后保存于-80 ℃冰箱,以备检测紧密连接蛋白(Claudin-1、Occludin、ZO-1、JAM-A)mRNA表达水平。

另从每个重复选取1只接近平均体重的鸡只,均按8.32 mg/kg体重的剂量灌服异硫氰酸荧光素-葡聚糖(fluorescein isothiocyanate-dextran,FITC-D)溶液,灌服1 h后,分别采集其翅静脉和肝门静脉血液5 mL于非抗凝真空管中,离心后收集血清于-20 ℃冻存,以备检测血清FITC-D含量。

1.4 指标测定

分别于22和42日龄,以重复为单元称肉仔鸡空腹体重,并统计耗料量,计算鸡只平均日增重(average daily gain,ADG)、平均日采食量(average daily feed intake,ADFI)和料重比(feed/gain,F/G)。

采用凯氏定氮法(GB/T 6432—1994)测定饲粮中粗蛋白质含量,采用5110型等离子体光学发射光谱仪(5110 ICP-OES,Agilent Technologies公司,澳大利亚)测定饲粮中钙、锌含量。

采用苏木精和伊红对固定好的十二指肠、空肠和回肠进行染色^[10-11],用显微镜观察,并使用图像处理和分析系统(Version 1,Leica Imaging Systems公司,英国)分析小肠各段形态学指标。

采用商业试剂盒测定血清内毒素含量(南京建成生物工程研究所,E039-1-1)和DAO活性(北京索莱宝生物科技有限公司,BC1285)。使用多模微板阅读器(Synergy HT,BioTek公司,美国)与428 nm激发波长和528 nm发射波长的双波长多功能酶标仪测量荧光,以检测血清FITC-D含量^[12]。

采用SYBR Green实时荧光定量PCR(RT-qPCR)法测定空肠黏膜中紧密连接蛋白(Claudin-1、Occludin、ZO-1、JAM-A)mRNA表达水平^[13-14],使用双内参法,2个内参基因分别为甘油醛-3-磷酸脱氢酶(GAPDH)和β-肌动蛋白(β-actin)。采用2^-△△Ct法计算各个目的基因的mRNA表达水平。

1.5 数据统计分析

试验数据采用SAS 9.4软件中的t检验进行统计分析,以每个重复作为1个试验单元,以P<0.05作为差异显著性检验水平,结果用平均值和集合标准误表示。

2 结果

2.1 饲粮锌缺乏对22~42日龄肉仔鸡生长性能和死亡率的影响

由表2可知,与对照组相比,锌缺乏组22~42日龄肉仔鸡ADG、ADFI和死亡率均无显著差异(P>0.05),但22~42日龄肉仔鸡F/G显著增加(P<0.05)。

表2 饲粮锌缺乏对22~42日龄肉仔鸡生长性能和死亡率的影响

Table 2 Effects of dietary zinc deficiency on growth performance and mortality of broilers during 22 to 42 days of age

项目 Items	平均日增重 ADG/g	平均日采食量 ADFI/g	料重比 F/G	死亡率 Mortality/%
对照组Control group	75.9	126	1.65^b	0.53
锌缺乏组Zinc deficient group	74.4	126	1.69^a	0.00
集合标准误Pooled SE	1.5	2	0.02	0.53
P值P-value	0.35	0.95	0.03	0.34

	同列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 column, 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 饲粮锌缺乏对42日龄肉仔鸡小肠形态的影响

由表3可知,与对照组相比,锌缺乏组42日龄肉仔鸡十二指肠VH和VSA以及回肠VH、CD和VSA均无显著差异(P>0.05),但42日龄肉仔鸡十二指肠和空肠CD显著增加(P<0.05),42日龄肉仔鸡十二指肠、空肠和回肠VH/CD以及空肠VH和VSA显著降低(P<0.05)。

表3 饲粮缺锌对42日龄肉仔鸡小肠形态的影响

Table 3 Effects of dietary zinc deficiency on small intestinal morphology of broilers at 42 days of age

项目 Items	十二指肠Duodenum				空肠Jejunum				回肠Ileum
项目 Items	绒毛高度 VH/ μm	隐窝深度 CD/ μm	绒毛高度/隐窝深度 VH/ CD	绒毛表面积 VSA/ mm²	绒毛高度 VH/ μm	隐窝深度 CD/ μm	绒毛高度/隐窝深度 VH/ CD	绒毛表面积 VSA/ mm²	绒毛高度 VH/ μm	隐窝深度 CD/ μm	绒毛高度/隐窝深度 VH/ CD	绒毛表面积 VSA/ mm²
对照组 Control group	1 971	171^b	11.56^a	1.44	1 730^a	157^b	10.87^a	1.17^a	1 089	170	7.20^a	0.77
锌缺乏组 Zinc deficient group	1 910	213^a	9.01^b	1.31	1 432^b	177^a	8.06^b	0.76^b	972	170	5.42^b	0.63
集合标准误 Pooled SE	94	5	0.59	0.08	88	1	0.53	0.05	76	5	0.58	0.09
P值 P-value	0.53	<0.01	<0.01	0.12	<0.01	<0.01	<0.01	<0.01	0.16	0.90	0.01	0.17

2.3 饲粮锌缺乏对42日龄肉仔鸡血清DAO活性及FITC-D和内毒素含量的影响

由表4可知,与对照组相比,锌缺乏组42日龄肉仔鸡肝门静脉血清内毒素含量无显著差异(P>0.05),但42日龄肉仔鸡翅静脉和肝门静脉血清DAO活性和FITC-D含量显著增加(P<0.05)。

表4 饲粮锌缺乏对42日龄肉仔鸡血清DAO活性及FITC-D和内毒素含量的影响

Table 4 Effects of dietary zinc deficiency on DAO activity, FITC-D and endotoxin contents in serum of broilers at 42 days of age

项目 Items	二胺氧化酶活性 DAO activity/(U/mL)		异硫氰酸荧光素-葡聚糖含量 FITC-D content/(ng/mL)		内毒素含量 Endotoxin content/ (EU/mL)
项目 Items	翅静脉血清 Wing vein serum	肝门静脉血清 Hepatic portal vein serum	翅静脉血清 Wing vein serum	肝门静脉血清 Hepatic portal vein serum	肝门静脉血清 Hepatic portal vein serum
对照组Control group	0.35^b	0.36^b	149.00^b	153.70^b	0.02
锌缺乏组Zinc deficient group	0.42^a	0.51^a	249.05^a	229.10^a	0.03
集合标准误Pooled SE	0.03	0.04	32.22	18.90	0.01
P值P-value	0.02	<0.01	<0.01	<0.01	0.24

2.4 饲粮锌缺乏对42日龄肉仔鸡空肠中紧密连接蛋白mRNA表达水平的影响

由表5可知,与对照组相比,锌缺乏组42日龄肉仔鸡空肠黏膜Occludin、JAM-A和ZO-1的mRNA表达水平均无显著差异(P>0.05),但42日龄肉仔鸡空肠黏膜Claudin-1的mRNA表达水平显著降低(P<0.05)。

表5 饲粮锌缺乏对42日龄肉仔鸡空肠紧密连接蛋白mRNA表达水平的影响

Table 5 Effects of dietary zinc deficiency on mRNA expression levels of tight junction proteins in jejunum of broilers at 42 days of age

项目 Items	闭合蛋白-1 Claudin-1	闭锁蛋白 Occludin	连接黏附分子-A JAM-A	闭合小环蛋白-1 ZO-1
对照组Control group	1.02^a	1.00	1.01	1.00
锌缺乏组Zinc deficient group	0.76^b	0.92	0.85	1.05
集合标准误Pooled SE	0.06	0.04	0.04	0.03
P值P-value	0.01	0.34	0.06	0.61

3 讨论

3.1 饲粮锌缺乏对22~42日龄肉仔鸡生长性能和死亡率的影响

饲粮锌缺乏会影响机体核酸的合成、氨基酸的利用以及蛋白质的合成,进而导致动物生长缓慢。NRC(1994)推荐22~42日龄肉仔鸡锌的需要量为40 mg/kg。然而关于肉仔鸡饲粮锌缺乏对生长性能影响的研究结果并不一致。黄金秀等^[15]研究发现,饲喂锌含量为10.1 mg/kg的低锌半纯合饲粮可显著降低肉仔鸡的ADFI,降低其生长速度。史彬林等^[16]研究发现,饲喂锌含量为4.67 mg/kg低锌半纯合饲粮可显著降低肉仔鸡的ADFI。然而,陈志敏等^[17]研究发现,饲粮锌缺乏(饲粮中锌含量为30 mg/kg)对1~21日龄肉仔鸡的ADG、ADFI以及F/G均无显著影响。本研究发现,饲粮锌缺乏对22~42日龄肉仔鸡的ADG和ADFI无显著影响,但显著提高了22~42日龄肉仔鸡的F/G。上述研究结果表明,饲粮锌缺乏对肉仔鸡生长性能的影响可能与肉仔鸡日龄、饲粮锌含量以及饲粮类型密切相关。本试验基础饲粮中锌含量为22.23 mg/kg,锌含量较高,能部分满足肉仔鸡的营养需要,因此对肉仔鸡ADFI和ADG无显著影响,该研究结果与陈志敏等^[17]研究结果相似。

3.2 饲粮锌缺乏对42日龄肉仔鸡小肠形态的影响

VH、CD、VH/CD及VSA是衡量小肠消化吸收功能的重要指标。VH和CD分别反映小肠的消化吸收能力和上皮细胞的成熟率,而VH/CD和VSA能够综合反映小肠的消化吸收功能状态,其数值下降,表明黏膜受损和消化吸收功能下降^[18]。谢正军等^[19]研究发现,与不添加锌的基础饲粮组相比,添加50 mg/kg的壳聚糖锌能够显著提高断奶仔猪十二指肠和空肠VH和VH/CD,显著降低CD。Ma等^[20]研究发现,与不添加锌的基础饲粮组相比,添加90 mg/kg的甘氨酸锌能够显著提高肉仔鸡十二指肠VH,显著降低回肠CD。Shah等^[21]研究发现,饲粮添加无机锌显著提高了热应激条件下肉仔鸡十二指肠、空肠和回肠VH、VSA及VH/CD。以上研究均表明,饲粮中添加适量锌可以改善肠道形态结构,提高肠道的消化和吸收功能。本研究发现,饲粮锌缺乏显著增加了肉仔鸡十二指肠和空肠CD,降低了十二指肠、空肠和回肠VH/CD以及空肠VH和VSA。这与以先前研究结果相一致,表明缺锌会影响肉仔鸡小肠的形态结构,降低小肠对营养物质的吸收能力。

3.3 饲粮锌缺乏对42日龄肉仔鸡血清DAO活性及内毒素和FITC-D含量的影响

肠黏膜通透性的改变可以准确反映肠黏膜的损伤程度,是反映肠道物理屏障功能的重要指标^[22]。内毒素是革兰氏阴性菌细胞壁中的脂多糖,在细菌代谢或死亡时释放,主要来源于肠道菌群代谢。结构完整的肠道黏膜会对细菌和内毒素构成屏障作用,内毒素难以进入血液。只有当肠道屏障受损导致黏膜通透性增加时,内毒素会通过肠黏膜进入血液循环中。因此,血清内毒素含量的增加是肠道通透性增加和肠道屏障功能受损的表现^[23-24]。DAO是动物肠黏膜上皮绒毛中具有高度活性的细胞内酶,当肠道黏膜屏障破坏时,肠上皮细胞释放大量DAO进入血液^[25],因此,通过测定血液中DAO活性可反映肠道黏膜通透性^[26-27]。此外,利用荧光标记的分子探针,如FITC-D,将荧光探针注入动物肠道后,通过测定血液中的荧光标记物FITC-D含量亦可反映肠道的通透性^[28]。谢正军等^[19]研究发现,与不添加锌的对照组相比,添加50 mg/kg的壳聚糖锌能够显著降低断奶仔猪血浆内毒素含量,而添加100 mg/kg的壳聚糖锌能够显著降低断奶仔猪血浆DAO活性。胡彩虹等^[29]研究发现,高剂量氧化锌(ZnO)可以显著降低断奶仔猪血浆内毒素含量和DAO活性,改善断奶仔猪肠道屏障功能。万妍等^[30]利用荧光标记的分子探针法研究发现,与饲粮添加120 mg/kg的硫酸锌组相比,饲粮不添加锌显著提高了21日龄肉仔鸡血清FITC-D含量,表明缺锌使得肉仔鸡肠道通透性增加和肠道屏障功能受损。此外,文敏^[31]采用同样的方法研究发现,与不添加锌的对照组相比,饲粮添加120 mg/kg硫酸锌显著降低了21日龄肉鸭血清FITC-D含量。上述研究表明,锌对维持畜禽肠道黏膜完整性和屏障功能具有重要的调控作用。与前人研究结果相似,本研究发现,饲粮锌缺乏显著增加了42日龄肉仔鸡翅静脉和肝门静脉血清DAO活性和FITC-D含量,表明缺锌增加了肉仔鸡肠道通透性,损伤了肠道的完整性和屏障功能。

3.4 饲粮锌缺乏对42日龄肉仔鸡空肠中紧密连接蛋白mRNA表达水平的影响

肠道物理屏障结构和功能的完整依赖于肠道上皮细胞之间紧密连接蛋白的表达。文敏^[31]研究发现,饲粮添加30和120 mg/kg的硫酸锌能够显著提高肉鸭空肠Claudin-1、Occludin、ZO-1和ZO-3的mRNA表达水平。万妍等^[30]研究发现,与不添加锌的对照组相比,肉仔鸡饲粮添加120 mg/kg的硫酸锌显著提高了回肠ZO-1的mRNA表达水平。Zhang等^[32]研究发现,饲粮添加锌可通过提高Claudin-1或Occludin的mRNA表达水平而改善肉仔鸡小肠屏障功能。Zhong等^[33]研究发现,饲粮添加锌能够抵抗酒精性诱导的大鼠肠道Claudin-1和ZO-1的mRNA表达水平的降低。Shao等^[34]研究发现,培养液中添加硫酸锌能上调Caco-2细胞的增殖和ZO-1的mRNA表达水平,从而改善肠细胞屏障功能。肉仔鸡十二指肠和空肠是营养物质吸收的主要场所。基于本研究结果中各个小肠段黏膜的损伤程度及Lei等^[35]研究的结果发现,锌缺乏对肉仔鸡空肠的肠道形态结构损伤最为明显。因此,本试验选择空肠作为目标肠段来分析紧密连接蛋白mRNA表达水平。与前人研究结果相似,饲粮锌缺乏显著降低了42日龄肉仔鸡空肠黏膜中紧密连接蛋白Claudin-1的mRNA表达水平,表明饲粮缺锌可能会通过降低肠道紧密连接蛋白的表达,损伤肠道的物理屏障功能。然而,本试验中饲粮锌缺乏对空肠Occludin和ZO-1的mRNA表达水平无显著影响,这可能是由于基础饲粮中锌含量较高。

4 结论

饲粮锌缺乏可能通过降低紧密连接蛋白Claudin-1的表达而损伤肉仔鸡小肠黏膜完整性和屏障功能,进而降低了其饲料转化效率。因此,保持肉仔鸡饲粮中适宜的锌添加有利于维持肉仔鸡小肠黏膜完整性及屏障功能,对于确保肉仔鸡的健康和高效生产具有重要意义。

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Abstract

Cite this article

1 材料与方法

1.1 试验设计

1.2 试验饲粮与饲养管理

表1 试验饲粮组成及营养水平(风干基础)

1.3 样品采集与制备

1.4 指标测定

1.5 数据统计分析

2 结果

2.1 饲粮锌缺乏对22~42日龄肉仔鸡生长性能和死亡率的影响

表2 饲粮锌缺乏对22~42日龄肉仔鸡生长性能和死亡率的影响

2.2 饲粮锌缺乏对42日龄肉仔鸡小肠形态的影响

表3 饲粮缺锌对42日龄肉仔鸡小肠形态的影响

2.3 饲粮锌缺乏对42日龄肉仔鸡血清DAO活性及FITC-D和内毒素含量的影响

表4 饲粮锌缺乏对42日龄肉仔鸡血清DAO活性及FITC-D和内毒素含量的影响

2.4 饲粮锌缺乏对42日龄肉仔鸡空肠中紧密连接蛋白mRNA表达水平的影响

表5 饲粮锌缺乏对42日龄肉仔鸡空肠紧密连接蛋白mRNA表达水平的影响

3 讨论

3.1 饲粮锌缺乏对22~42日龄肉仔鸡生长性能和死亡率的影响

3.2 饲粮锌缺乏对42日龄肉仔鸡小肠形态的影响

3.3 饲粮锌缺乏对42日龄肉仔鸡血清DAO活性及内毒素和FITC-D含量的影响

3.4 饲粮锌缺乏对42日龄肉仔鸡空肠中紧密连接蛋白mRNA表达水平的影响

4 结论

References