动物营养学报    2021, Vol. 33 Issue (8): 4779-4790    PDF    
低锌饲粮添加载锌蒙脱石对肉鸡免疫功能和肝脏含锌酶活性的影响
王海波 , 秦士贞 , 李金录 , 武真邑 , 龚莉媛 , 裴文刚 , 车育彦 , 杨敏敏 , 史兆国     
甘肃农业大学动物科学技术学院, 兰州 730070
摘要: 本试验旨在研究低锌饲粮添加载锌蒙脱石(Zn-MMT)对肉鸡免疫功能、肝脏含锌酶活性的影响。随机选择1日龄健康科宝公雏240只,按体重随机分成5组,每组6个重复,每个重复8只仔鸡。对照组(CK组)饲喂玉米-豆粕型基础饲粮,试验组分别在基础饲粮中替代载体添加20、40、60和80 mg/kg Zn-MMT(均以锌含量计算),自由采食饮水,试验期为42 d,测定空肠黏膜免疫球蛋白含量、肝脏含锌酶活性、血清细胞因子含量、脾脏免疫相关基因表达。结果表明:1)相比CK组,添加40和60 mg/kg的Zn-MMT显著提高肉鸡血清中干扰素-γ(IFN-γ)的含量(P < 0.05),对血清白细胞介素-1β(IL-1β)、白细胞介素-2(IL-2)、白细胞介素-4(IL-4)的含量有升高趋势(0.05 < P < 0.10),但对血清中免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的含量无显著影响(P>0.05)。2)相比CK组,添加Zn-MMT对肉鸡21和42日龄空肠黏膜的免疫球蛋白G(IgG)、分泌型免疫球蛋白(sIgA)含量均无显著影响(P>0.05)。3)相比CK组,添加Zn-MMT显著提高肉鸡21和42日龄肉鸡肝脏中铜锌超氧化物歧化酶(Cu-Zn SOD)活性,但对碱性磷酸酶(ALP)、乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)活性无显著影响(P>0.05)。4)相比CK组,添加40 mg/kg的Zn-MMT显著提高肉鸡脾脏核转录因子-κBp65(NF-κBp65)、肿瘤坏死因子受体相关因子-2(TRAF-2)、肿瘤坏死因子受体超家族1B(TNFRSF1B)、核因子κB抑制蛋白-α(IκB-α)、IL-1βIL-4、IL-6、白细胞介素-10(IL-10)基因相对表达量(P < 0.05),但对转化生长因子-β4(TFG-β4)、IFN-γ基因相对表达量无显著影响(P>0.05)。综上所述,低锌饲粮添加Zn-MMT可显著提高脾脏免疫相关基因的表达,增强机体免疫力,增强含锌酶活性。
关键词: 载锌蒙脱石    肉鸡    免疫功能    含锌酶    
Effects of Adding Zinc-Montmorillonite in Low Zinc Diet on Immune Function and Zinc-Containing Enzyme Activities in Liver of Broilers
WANG Haibo , QIN Shizhen , LI Jinlu , WU Zhenyi , GONG Liyuan , PEI Wengang , CHE Yuyan , YANG Minmin , SHI Zhaoguo     
College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China
Abstract: This experiment was conducted to investigate the effects of zinc-montmorillonite (Zn-MMT)on immune function and zinc-containing enzyme activities of broilers fed a low zinc diet. A total of 240 one-day-old male Cob broiler were randomly assigned to 5 groups with 6 replicates per group and 8 birds per replicate. The birds were fed a corn-soybean meal basal diet (CK group) or one of the four Zn-MMT diets, which were supplemented with 20, 40, 60, 80 mg/kg Zn-MMT(Zinc content measurement) based on the basal diet. The broilers were provided the diets in ad libitum basis and the experiment lasted for 42 days. The activities of immunoglobulin in jejunal mucosa and Zn-related enzymes in liver were measured. Meanwhile, serum cytokine contents and the expression levels of genes related to spleen immunity was measured. The results showed as follows: 1) compared with CK group, adding 40 and 60 mg/kg Zn-MMT significantly increased the content of interferon-γ in broiler serum (P < 0.05), and the contents of interleukin-1, interleukin-2 and interleukin-4 showed an increasing trend (0.05 < P < 0.10), but had no significant influence on the contents of immunoglobulin A, immunoglobulin M, tumor necrosis factor-α and interleukin-6 in broiler serum (P>0.05). 2) Compared with CK group, adding Zn-MMT had no significant effect on immunoglobulin G and secretory immunoglobulin contents of jejunal mucosa of broilers aged 21 and 42 days (P>0.05). 3) Compared with CK group, adding Zn-MMT significantly increased the activity of Cu-Zn superoxide dismutase (P < 0.05), but had no significant effect on the activities of alkaline phosphatase, lactate dehydrogenase and malate dehydrogenase in the liver (P>0.05). 4) Compared with CK group, adding 40 mg/kg Zn-MMT significantly increased the expression level of nuclear transcription factor-κBp65, tumor necrosis factor receptor-related factors 2, tumor necrosis factor receptor superfamily 1B, nuclear factor κB inhibits protein α, interleukin-1β, interleukin-4, interleukin-6, interleukin-10 genes in the spleen at 42 days of age (P < 0.05), but had no significant effect on the expression levels of transformed growth factor-β4 and interferon-γ genes (P>0.05). It is concluded that dietary supplementation with Zn-MMT can significantly improve the expression of spleen immune-related genes, enhance the immune function, and enhance the activity of zinc-containing enzymes in the low zinc diet.
Key words: zinc loaded montmorillonite    broilers    immune function    zinc-containing enzymes    

锌作为家禽维持机体正常生理和生化代谢所必需的第一限制性微量元素,为六大酶类的组分或辅助因子,在免疫系统的发育、监视、应答中发挥调控功能,维持淋巴细胞、T细胞及自然杀伤细胞等免疫细胞的结构和功能,参与特异性或非特异性免疫反应及金属酶的催化调控发挥重要功能[1-5]。但饲粮中常以硫酸锌(ZnSO4)为锌源,其吸收利用率低,过度排放到环境中易造成锌污染[3]。蒙脱石属于2∶1型层状硅铝酸盐矿物,每层结构由2个共顶联接的硅氧四面体片中间夹着1个共棱联接的铝氧八面体片构成,已探明其储量位居世界第一,且具有表面积大及强离子交互性,畜牧生产实践中常用来抗腹泻、治疗肠道疾病,还可以作为控释载体、吸附剂[6-7]。研究显示,通过离子交换对沸石、蒙脱石等黏土矿物富集微量元素,可使微量元素在动物肠道内合理释放,持久被机体利用,提高微量元素的利用率,减少微量元素对环境的污染[8],改善肠道形态与结构,提高肠黏膜的免疫功能[5],缓解细菌造成氧化应激[9],降低肉鸡沙门氏菌和大肠杆菌的数量,提高沙门氏菌感染肉鸡肠道的免疫能力[10]。同时,载钙蒙脱石能有效扭转黄曲霉毒素B1(AFB1)对肉鸭造成的免疫抑制,提高免疫器官指数及血清中免疫球蛋白G(IgG)、免疫球蛋白M(IgM)的含量[11]。前期的研究发现,锌和载锌沸石增强肉鸡、仔猪的免疫功能[10, 12]。然而,脾脏作为动物机体最大的免疫器官,参与产生抗体和初始免疫球蛋白、防御素、肿瘤坏死因子(tumor necrosis factor, TNF)等细胞因子,在先天性免疫和适应性免疫发挥应答调控[13-14]。添加载锌沸石可改善肉鸡微量元素代谢与生产性能[5];本试验前期研究发现,肉鸡玉米-豆粕型基础饲粮中添加40 mg/kg(以锌含量计)的载锌蒙脱石(zinc-montmorillonite, Zn-MMT)对肉鸡42日龄平均日增重无显著影响,但显著降低肉鸡料重比,提高十二指肠、空肠、回肠的绒毛高度(VH)和绒毛高度/隐窝深度(V/C),促进肠道发育[15];且低蛋白质水平饲粮添加Zn-MMT相比添加ZnSO4,可显著提高肉鸡免疫器官指数,提高小肠VH和V/C,降低隐窝深度(CD),促进肉鸡肠道发育[16]。Zn-MMT作为饲料添加剂对肉鸡免疫调控及含锌酶活性的研究相对较少。因此,本试验旨在研究低锌饲粮添加Zn-MMT对肉鸡免疫功能及肝脏含锌酶活性的影响,为Zn-MMT作为饲料添加剂在肉鸡生产中的应用提供参考理论依据。

1 材料与方法 1.1 试验材料

试验用载锌蒙脱石经兰州中检科测试技术有限公司检测,锌含量为10 408.80 mg/kg。

1.2 试验设计

采用单因子完全随机试验设计,选取1日龄健康科宝公雏240只,按体重随机分成5组,每组6个重复,每个重复8只仔鸡。对照组饲喂玉米-豆粕型基础饲粮,4个试验组分别在基础饲粮中替代预混料载体(次粉)添加20、40、60和80 mg/kg Zn-MMT (均以锌含量计算)。

1.3 饲养管理

2019年10月26日至2019年12月7日在甘肃农业大学动物科学技术学院实训中心进行饲养试验,试验期为42 d。试验采用3层笼养。饲养管理和常规免疫按照《科宝肉仔鸡饲养管理手册》进行。采用24 h恒定光照,鸡只自由采食、饮水。

1.4 基础饲粮

基础饲粮参照《鸡饲养标准》(NY/T 33—2004)及NRC(1994)配制为玉米-豆粕型粉料,其组成及营养水平见表 1

表 1 基础饲粮组成及营养水平(风干基础) Table 1 Composition and nutrient levels of basal diets (air-dry basis)  
1.5 测定指标与方法 1.5.1 血清免疫球蛋白及细胞因子含量的测定

在试验第42天时,每组选取6只接近平均体重的肉仔鸡进行翅下静脉采血5 mL,离心管分装,-20 ℃保存,并送于北京华英生物技术研究所用免疫比浊法测定血清免疫球蛋白(Ig)A和IgM含量,并通过酶联免疫吸附法测定血清细胞因子白细胞介素(IL)-6、IL-4、IL-2、IL-1β、干扰素-γ(IFN-γ)含量。

1.5.2 空肠黏膜IgG、分泌型免疫球蛋白A(sIgA)含量的测定

在试验第21和42天时,每组选取6只接近平均体重的肉仔鸡收集空肠黏膜,液氮速冻后,在-80 ℃冰箱中保存,并送甘肃硕联生物技术有限公司采用酶联免疫吸附法测定空肠黏膜IgG、sIgA含量。

1.5.3 肝脏含锌酶活性的测定

在试验第21和42天时,每组选取6只接近平均体重的肉仔鸡采集肝脏组织,液氮速冻后,在-80 ℃冰箱中保存,并送甘肃硕联生物技术有限公司采用酶联免疫吸附法测定肝脏铜锌超氧化物歧化酶(Cu-Zn SOD)、碱性磷酸酶(ALP)、乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)活性。

1.5.4 脾脏免疫相关基因表达

在试验第42天,从每组中选择6只接近平均体重的肉鸡,迅速屠宰取出脾脏液氮速冻,在-80 ℃冰箱中保存直至分析。使用TRIZOL(北京全式金生物技术有限公司)法,提取脾脏组织总RNA,经微量紫外可见分光光度计(NanoDrop-2000)测定RNA浓度和纯度,参照反转录试剂盒(PrimeScriptTM RT reagent Kitwith gDNA Eraser)说明,经去除基因组DNA反应(5×gDNA Eraser Buffer 2.0 μL、gDNA Eraser 1.0 μL、Total RNA 1.0 μL、RNase Free dH2O 6 μL)后,对总RNA进行反转录合成cDNA(加入10 μL的去除基因组反应液、1.0 μL的PrimeScript RT Enzyme Mix I、1.0 μL的RT Primer Mix、4.0 μL的5×PrimeScript Buffer 2和4.0 μL的RNase Free dH2O)将上述总体系溶液置于37 ℃恒温水浴15 min,85 ℃恒温水浴5 s得到cDNA,储存在-20 ℃冰箱中备用。并利用Light Cycler ®480ⅡSystems荧光定量基因扩增仪,采用SYBR®GreenⅠ染料法,以β-肌动蛋白(β-actin)为内参基因,参照荧光定量试剂盒说明书(SYBR®Green Pro Taq HS预混型qPCR试剂盒)进行实时荧光定量PCR扩增,用2-△△Ct法计算基因相对表达量。目的基因(杨家新[17]、Song等[18]、张柏林等[19]、夏亿[20]、邓美玉[21])及内参基因引物序列见表 2,引物由苏州金唯智生物科技有限公司合成。

表 2 目的基因及内参基因引物 Table 2 Primers of target genes and reference gene
1.6 数据统计分析

数据经Excel 2013处理后,采用SPSS 26.0进行单因素方差分析(one-way ANOVA),用Duncan氏法进行多重比较,试验结果均以平均值±标准差(SD)表示,P<0.05表示显著水平。

2 结果与分析 2.1 低锌饲粮添加Zn-MMT对肉鸡血清免疫球蛋白及细胞因子含量的影响

表 3可见,低锌饲粮添加Zn-MMT显著影响血清中IFN-γ的含量(P<0.05),但对血清中IgA、IgM、TNF-α、IL-1β、IL-2、IL-4、IL-6的含量无显著影响(P>0.05)。相比CK组,添加40和60 mg/kg的Zn-MMT显著提高肉鸡血清中IFN-γ的含量(P<0.05)。

表 3 低锌饲粮添加Zn-MMT对42日龄肉鸡血清免疫球蛋白及细胞因子含量的影响 Table 3 Effects of adding Zn-MMT in low zinc diet on serum immunoglobulin and cytokines contents of 42-day-old broilers
2.2 低锌饲粮添加Zn-MMT对肉鸡空肠黏膜免疫球蛋白含量的影响

表 4可见,相比CK组,低锌饲粮添加Zn-MMT对肉鸡21和42日龄空肠黏膜的IgG、sIgA的含量均无显著影响(P>0.05)。

表 4 低锌饲粮添加Zn-MMT对肉鸡空肠黏膜免疫球蛋白含量的影响 Table 4 Effects of adding Zn-MMT in low zinc diet on immunoglobulin contents in jejunal mucosa of broilers
2.3 低锌饲粮添加Zn-MMT对肉鸡肝脏含锌酶活性的影响

表 5可见,相比CK组,低锌饲粮添加Zn-MMT显著影响肉鸡21和42日龄肉鸡肝脏中Cu-Zn SOD活性(P<0.05),但对ALP、LDH、MDH活性均无显著影响(P>0.05)。相比CK组,低锌饲粮添加Zn-MMT显著提高肉鸡21和42日龄肉鸡肝脏中Cu-Zn SOD活性(P<0.05)。

表 5 低锌饲粮添加Zn-MMT对肉鸡肝脏含锌酶活性的影响 Table 5 Effects of adding Zn-MMT in low zinc diet on zinc-containing enzyme activities in liver of broilers
2.4 低锌饲粮添加Zn-MMT对肉鸡脾脏免疫功能的影响

表 6可见,低锌饲粮添加Zn-MMT显著影响肉鸡42日龄脾脏核转录因子-κBp65(NF-κBp65)、肿瘤坏死因子受体相关因子-2(TRAF-2)、肿瘤坏死因子受体超家族1(TNFRSF1B)、核因子κB抑制蛋白-α(IκB-α)、转化生长因子-β4(TFG-β4)、IFN-γIL-1βIL-4、IL-6、IL-10基因相对表达量(P<0.05)。相比CK组,低锌饲粮添加40 mg/kg的Zn-MMT显著提高肉鸡脾脏NF-κBp65、TRAF-2、TNFRSF1BIκB-αIL-1βIL-4、IL-6、IL-10基因相对表达量(P<0.05),但对TFG-β4、IFN-γ基因相对表达无显著影响(P>0.05)。同时,低锌饲粮添加20 mg/kg Zn-MMT显著提高肉鸡脾脏TNFRSF1B基因相对表达量(P<0.05),且添加80 mg/kg Zn-MMT显著提高肉鸡脾脏TRAF-2、IL-1βIL-6基因相对表达量(P<0.05)。

表 6 低锌饲粮添加Zn-MMT对肉鸡脾脏免疫相关基因表达的影响 Table 6 Effects of adding Zn-MMT in low zinc diet on spleen immune-related gene expression of broilers
3 讨论 3.1 低锌饲粮添加Zn-MMT对肉鸡血清免疫球蛋白及细胞因子含量的影响

机体许多器官和系统都受到锌缺乏的负面影响。在体内,几乎先天免疫和适应性免疫的所有方面都受到锌的影响,缺锌能改变中性粒细胞、单核细胞、自然杀伤细胞、T细胞和B细胞的数量和功能,这表明锌在免疫防御方面具有重要意义[22]。免疫球蛋白参与介导体液免疫,其功能随含量升高而增强[23-24]。细胞因子在神经内分泌系统和免疫系统之间起到双向通信的作用的可溶性小分子多肽[25-26],由Thl细胞分泌的IL-2、IFN-γ及TNF-α等协助T细胞的增殖,启动细胞介导的免疫反应[27];而Th2细胞分泌的IL-4、IL-6、IL-10等细胞因子,刺激B细胞增殖并产生抗体,参与体液免疫[28];以及活化的巨噬细胞分泌的IL-1、IL-2、IL-6、IFN-γ和TNF-α等多种细胞因子[29],共从参与机体的免疫调控。詹小立等[23]研究发现,改性蒙脱石对断奶仔猪血清IgG、IgA和IgM含量无显著差异;但血清中IL-2含量的升高,能够促进IFN-γ的产生[30]。在本试验中,相比对照组,低锌饲粮添加40和60 mg/kg的Zn-MMT对血清IL-2的含量有提高的趋势,且添加40和60 mg/kg的Zn-MMT显著提高肉鸡血清中IFN-γ的含量,鸡IFN-γ可针对不同的病原感染与相应的细胞因子协同作用进行免疫调节,完成免疫系统的防御功能[31],有效提高免疫功能。

3.2 低锌饲粮添加Zn-MMT对肉鸡空肠黏膜免疫球蛋白含量的影响

家禽肠道中的sIgA能与微生物和黏膜中的抗原结合,限制微生物和抗原进入肠道,IgM参与机体原发性免疫反应,使可溶性抗原发生沉淀,提高机体吞噬、清除抗原的能力,维持肠道免疫屏障功能[32-34]。研究证实,铝硅酸盐可提高小鼠白细胞的吞噬活性,刺激脾脏B淋巴细胞分化,指导免疫球蛋白合成[35]。载锌沸石[36]、凹凸棒石[37]、凹凸棒石和沸石混合物[38]等均能提高空肠黏膜中IgG、sIgA含量,增强肠道免疫力。但本试验研究发现,低锌饲粮添加Zn-MMT对肉鸡空肠黏膜免疫球蛋白含量无显著差异,这可能与Zn-MMT覆盖在健康肠道黏膜表面,减少了抗原对黏膜表面的直接刺激,使肠道免疫系统处于较低的免疫激活状有关[39]

3.3 低锌饲粮添加Zn-MMT对肉鸡肝脏含锌酶活性的影响

锌在动物体内抗氧化系统中发挥重要作用,适量补锌可影响动物体内抗氧化酶活性和金属硫蛋白表达,进而保护动物机体免受自由基损伤,其中最具有代表性的为酶催抗氧化Cu-ZnSOD和糖代谢中的ALP[3, 5, 40];并缓解机体因重金属中毒、细菌、球虫感染导致的氧化应激,增加肝脏中Cu-ZnSOD活性[10, 41-42],且对MDH活性[43]有提高趋势,但对LDH活性影响表现出先升高后下降的趋势。饲粮中添加0.11%~0.46%载锌沸石可增加蛋鸡肝脏ALP、MDH活性,且LDH活性在0.23%时最高,随后呈下降趋势[40]。本试验结果表明,低锌饲粮添加Zn-MMT对肝脏ALP、LDH、MDH活性均无显著影响,这与马芳[44]的研究不同锌水平对肉鸡ALP、LDH活性无显著影响的结果相似。但与李林枫[40]在蛋鸡中添加载锌沸石提高肝脏ALP、LDH、MDH活性的研究结果出现差异。其原因可能是基础饲粮中的锌能够满足肉鸡ALP、LDH、MDH对锌的正常需求,且刘泽辉[43]报道发现,锌水平对肉鸡肝脏MDH活性无显著影响。陈志敏等[45]的研究表明,饲粮中添加硫酸锌显著影响肝脏Cu-ZnSOD活性。本试验发现,低锌饲粮添加Zn-MMT显著提高肉鸡21和42日龄肉鸡肝脏中Cu-ZnSOD活性,这与陈刚耀[3]在肉鸡中添加载锌沸石提高肝脏中Cu-ZnSOD活性研究结果相似。硅酸盐黏土中金属离子能在动物胃肠道内缓慢释放,持久维持组织细胞中锌含量,并参与维持Cu-ZnSOD结构稳定,抑制有害菌增殖,防止蛋白质或氨基酸巯基的氧化,减少机体在代谢过程中产生的羟自由基[10, 46-48]

3.4 低锌饲粮添加Zn-MMT对肉鸡脾脏免疫功能的影响

锌对维持动物机体的免疫器官的结构和功能具有重要作用,缺锌或锌过量会直接造成免疫器官萎缩或病变、免疫细胞数减少、抗体含量降低[46]。脾脏作为动物机体最大的免疫器官,参与机体免疫调控,经T淋巴细胞以及B淋巴细胞主导的NF-κB通路调节获得性免疫反应,同时参与胚胎发育、淋巴细胞生成及调控骨形成;NF-κB通常被隔离在未受刺激的细胞的细胞质中,与IκB结合形成一个保守的三聚体复合物p50-p65-IκB,掩盖了核定位序列,核定位序列暴露后能与IκB-α核导出序列结合,使IKB-α与NF-κB二聚体的复合物在细胞质和细胞核内穿梭运动,转移到核内与其相应的DNA序列结合从而促进靶基因的转录。另外,TRAF能直接或间接与受体结合,介导NF-κB通路信号转导[49-51]。IL-4和IL-10通过Toll样受体4(TLR4)、NF-κB和其他信号通路调节免疫反应以维持肠道健康的抗炎因子[52-54]。陈娜娜等[55]研究结果表明,适量的蛋氨酸锌可显著增加蛋鸡盲肠内容物中双歧杆菌、乳杆菌数量,显著减少大肠杆菌数量,但1 400 mg/kg蛋氨酸锌反而极显著增加了大肠杆菌数量,导致肠道菌群失调,生物屏障遭到破坏;且双歧杆菌[56]能促进B淋巴细胞的分化成熟,诱导T淋巴细胞的增殖并参与T淋巴细胞、自然杀伤细胞及LAK的细胞分化,促进机体免疫器宫的生长、发育、成熟。罗有文[46]研究发现,一定量的载锌凹土可以抑制肉鸡空肠大肠杆菌的增殖,并适当增加乳酸杆菌的数量。本试验研究表明,相比CK组,肉鸡低锌饲粮添加40 mg/kg的Zn-MMT显著提高肉鸡脾脏NF-κBp65、TRAF-2、TNFRSF1BIκB-αIL-1βIL-4、IL-6、IL-10基因相对表达量。体内的微生态系统与动物免疫之间的关系极为密切。Zn-MMT进入动物肠道后,可以吸附其中的氨、氨气等极性分子,为嗜酸性的乳酸杆菌提供更优良的生长环境,促进了有益菌的增殖,改善了肠道微生态系统,维持机体免疫功能。

4 结论

① 低锌玉米-豆粕型基础饲粮中添加Zn-MMT可提高21和42日龄肉鸡肝脏Cu-ZnSOD活性,增强肝脏抗氧化能力;但对42日龄肉仔鸡血清和空肠黏膜中免疫球蛋白含量无显著影响。

② 低锌玉米-豆粕型基础饲粮中添加40 mg/kg的Zn-MMT可显著提高脾脏免疫相关基因的表达。

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