Enterobacter cloacae Z0206富硒多糖对动物生长性能和免疫功能的影响及其作用机制研究
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摘要
本课题组前期从自然界分离、筛选并驯化得到一株高产胞外多糖细菌菌株Enterobacter cloacae Z0206,该菌株不仅多糖产量高,而且对硒有较强的耐受能力,在含有无机硒的培养基上生长可以将无机硒转化为硒多糖(Se-ECZ-EPS), Se-ECZ-EPS含硒量为535.7mg/kg.初步生物学功能研究发现,Se-ECZ-EPS可以提高小鼠的细胞和体液免疫功能,改善环磷酰胺引起的小鼠免疫抑制,在细胞水平上发现Se-ECZ-EPS对H2O2引起的小鼠巨噬细胞RAW264.7的氧化损伤具有保护作用。
本研究拟在前期研究Se-ECZ-EPS可以改善小鼠免疫功能的基础上,探讨Se-ECZ-EPS在动物养殖生产中的免疫调节作用,为将其开发成潜在的动物免疫调节制剂奠定基础。本研究设计了四个试验：试验一,首先采用小鼠急性经口毒性试验进行了Se-ECZ-EPS在动物中使用的的安全性评价。试验二,在确定了Se-ECZ-EPS在动物上的使用安全性后,为了拓展其在水产及畜禽养殖动物上的应用,选择水产甲壳类的理想模式动物-螫虾为试验对象,研究了Se-ECZ-EPS对螯虾的免疫调节功能及对感染白斑病毒后的保护作用；试验三,在水产螯虾研究基础上深入探讨了Se-ECZ-EPS对家禽动物-肉仔鸡的促生长及免疫调节作用；试验四,进一步选择断奶仔猪为试验对象,深入研究了Se-ECZ-EPS对断奶仔猪生长性能、免疫功能、肠道形态结构及肠道微生物多样性的影响并探讨其生物学机制；通过以上研究为Se-ECZ-EPS在动物中的应用奠定了坚实理论基础。
试验一、Z0206富硒多糖的安全性评价
为了奠定Z0206富硒多糖在水产及畜禽养殖动物上应用的基础,本研究首先采用小鼠急性经口毒性试验验证其在动物上的使用安全性。试验结果显示,受试小鼠急性经口毒性试验雌、雄两性别最大耐受量均大于20.0g/kg体重,依据GB15193.4-2003《食品安全性毒理学评价程序和检验方法-急性毒性试验》表明Se-ECZ-EPS无毒,为其在水产动物、畜禽动物上的应用提供了可能性。
试验二、Z0206富硒多糖对水产动物螯虾免疫功能的影响
在确定Se-ECZ-EPS在动物上的使用安全性基础上,以水产甲壳类的理想模式动物-克氏原螯虾(Procambarus clarkii (Girard))为动物模型,通过注射1μgg-1Se-ECZ-EPS,测定免疫相关指标和基因表达,同时探讨Se-ECZ-EPS对螯虾感染白斑病毒后的保护作用。研究结果显示,在注射Se-ECZ-EPS24,48和72h后,螯虾血液中酚氧化酶(PO),呼吸爆发(RB)和超氧化物歧化酶(sOD)活性显著升高(P<0.05)；基因表达结果显示,在注射Se-ECZ-EPS24和48h后,丝氨酸蛋白酶、HSP70和Mn-SOD基因表达显著上调,而酚氧化酶在24到72h后无显著影响；14天的攻毒试验表明,注射Se-ECZ-EPS后能够显著提高螯虾感染WSSV后的存活率(对照组：0%、Se-ECZ-EPS组：59.7%)。综上结果揭示,Se-ECZ-EPS可以提高克氏原螯虾的免疫功能,是一种有效的免疫调节剂。
试验三、Z0206富硒多糖对肉仔鸡生长性能、免疫功能及肠道形态结构的影响
在研究了Se-ECZ-EPS对螯虾的免疫调节功能及感染白斑病毒后的保护作用基础上,继而深入探讨Se-ECZ-EPS在家禽养殖业中占有重要地位的另一种动物-肉仔鸡上的促生长及免疫调节作用。
3.1.Z0206富硒多糖对肉仔鸡生长性能、胴体品质的影响
试验选取1日龄艾维茵肉仔鸡240羽,随机分为5个处理,每个处理4个重复,每个重复12羽。对照组在基础日粮中以亚硒酸钠的形式添加0.15mg/kg的硒水平,四个试验组在基础日粮中分别添加280、560、840、1120mg/kg的Z0206Se-ECZ-EPS(四个试验组硒含量分别为0.15、0.30、0.45、0.60mg/kg)。结果显示：与对照组相比,添加840、1120mg/kg的Se-ECZ-EPS可以显著提高肉仔鸡0-6周平均日增重,显著降低料重比(P<0.05)；添加560mg/kg.840mg/kg、1120mg/kg Se-ECZ-EPS则显著降低了胸肌、腿肌的滴水损失(P<0.05),而对肌肉pH无显著性影响。结果表明Se-ECZ-EPS可以降低鸡肉的滴水损失,有效提高肉仔鸡的生长性能。
3.2.E. cloacae Z0206富硒多糖对肉仔鸡免疫功能的影响
肉仔鸡免疫功能研究结果显示：血清抗氧化指标方面：与对照组和280mg/kg Se-ECZ-EPS组相比,添加840mg/kg和1120mg/kg的Se-ECZ-EPS可显著增加抗氧化酶GSH-Px、SOD的活性,显著降低MDA的含量(P<0.05)。细胞因子含量方面：添加280mg/kg、560mg/kg、840mg/kg和1120mg/kg的Se-ECZ-EPS均能显著提高血清TNF-a和IL-2的浓度(P<0.05),同时,560mg/kg和1120mg/kg Se-ECZ-EPS组血清IL-6的水平亦显著提高(P<0.05)。抗体水平方面：添加840mg/kg和1120mg/kg的Se-ECZ-EPS显著提高了21、35日龄血清新城疫HI水平(P<0.05),而在42日龄,只有840mg/kg组新城疫HI水平显著升高(P<0.05)；此外,添加1120mg/kg的Se-ECZ-EPS能显著提高免后7d、14d血清禽流感抗体水平(P<0.05)。提示Se-ECZ-EPS可以通过增强抗氧化酶活性、促进血清细胞因子分泌、提高血清抗体水平来增强免疫功能,进而提高肉仔鸡的生长性能。
3.3.E. cloacae Z0206富硒多糖对肉仔鸡肠道形态结构的影响
肉仔鸡肠道形态结构研究结果显示：添加560mg/kg和840mg/kg的Se-ECZ-EPS显著降低了十二指肠隐窝深度,显著提高了十二指肠绒毛高度和隐窝深度比VCR(P<0.05),而添加1120mg/kg的Se-ECZ-EPS显著提高了空肠绒毛高度(P<0.05)。提示Se-ECZ-EPS改善了肉仔鸡肠道的形态结构,有利于养分物质的吸收,进而提高肉仔鸡的生长性能。
试验四、Z0206富硒多糖对断奶仔猪生长性能、免疫功能、肠道形态结构及肠道微生物多样性的影响
在研究Se-ECZ-EPS对肉仔鸡促生长、增强免疫功能、改善肠道形态结构的基础上,进一步选择在畜牧养殖业中占有重要地位的断奶仔猪为试验对象,探讨Se-ECZ-EPS对断奶仔猪生长性能、免疫功能、肠道形态结构及肠道微生物多样性的影响及其作用机制。
4.1.Z0206富硒多糖对断奶仔猪生长性能的影响
试验选择150头28日龄“杜×长×大”三元杂交断奶仔猪,随机分成5组：对照组(基础日粮)、Na2Se03组(基础日粮+0.30mg/kg Na2Se03)、Na2SeO3+APS组(基础日粮,+0.30mg/kg Na2SeO3+560mg/kg黄芪多糖)、Na2Se03+EPS组(基础日粮+0.30mg/kg Na2SeO3+560mg/kg Z0206多糖)、Se-ECZ-EPS组(基础日粮+560mg/kg Z0206富硒多糖)。结果显示,日粮中添加Na2SeO3+APS、 Na2SeO3+EPS、Se-ECZ-EPS显著提高断奶仔猪平均日增重(P<0.05),且Se-ECZ-EPS组显著高于Na2Se03组(P<0.05),而与Na2Se03+APS、Na2Se03+EPS组无显著性差异；Na2SeO3+EPS、Se-ECZ-EPS组料重比显著低于对照组和Na2SeO3组(P<0.05),与Na2SeO3+EPS组无显著性差异。结果提示：Se-ECZ-EPS可以促进断奶仔猪的生长性能,使用效果与黄芪多糖APS相当。
4.2.Z0206富硒多糖对断奶仔猪免疫功能的影响
断奶仔猪免疫功能结果显示,在抗氧化功能方面：与对照组相比,日粮中添力口Na2SeO3+EPS、Se-ECZ-EPS显著提高仔猪血清总抗氧化力(T-AOC)(P<0.05)、显著降低了血清丙二醛(MDA)含量(P<0.05)。此外,Se-ECZ-EPS组血清谷胱甘肽还原酶(GSH-Px)、超氧化物歧化酶(sOD)活性显著提高了(P<0.05),而与Na2SeO3+APS、Na2SeO3+EPS组相比没有显著性差异。细胞因子方面：日粮中添加Na2SeO3+EPS、Se-ECZ-EPSS显著提高了仔猪血清TNF-α、IL-2、IL-6含量(P<0.05),而与Na2SeO3+APS组相比没有显著性差异。免疫球蛋白方面：相比于对照组和Na2SeO3组,日粮中添加Se-ECZ-EPS显著提高了仔猪血清IgG、IgM含量,且Se-ECZ-EPS组IgM含量显著高于Na2SeO3+APS组(P<0.05)。组织抗菌肽表达方面Na2SeO3+APS组、Na2SeO3+EPS组、Se-ECZ-EPS组显著提高了肝脏抗菌肽基因PR-39、Hepcidin的表达(P<0.05)；且Se-ECZ-EPS显著提高了十二指肠中pBD-1的基因表达,提高了空肠和回肠中pBD-2的基因表达(P<0.05)；而Na2SeO3+EPS提高了空肠和回肠中pBD-2的基因表达(P<0.05),Na2SeO3+APS只提高了回肠中pBD-2的基因表达(P<0.05)。结果提示Se-ECZ-EPS可以通过提高血清免疫球蛋白含量、促进细胞因子分泌、提高肝脏和肠道抗菌肽基因表达来增强免疫功能。
4.3.Z0206富硒多糖对断奶仔猪肠道形态结构及肠道微生物多样性的影响
断奶仔猪肠道形态结构及肠道微生物多样性研究结果显示：断奶仔猪日粮中添加Na2SeO3+EPS、Se-ECZ-EPS显著提高了十二指肠、空肠绒毛高度和隐窝深度比VCR<0.05),明显增加空肠绒毛高度(P<0.05)。肠道微生物多样性分析显示,Se-ECZ-EPS可以提高结肠、盲肠内容物中微生物多样性指数(Shannon指数),与Na2SeO3组相比,添加Se-ECZ-EPS能够增加结肠、盲肠中革兰氏阳性菌(厚壁菌门)比例,减少革兰氏阴性菌(拟杆菌门)比例。结果提示：Z0206富硒多糖可以改善肠道结构、调节菌群平衡,进而提高断奶仔猪的生长性能。
Enterobacter cloacae Z0206(E. cloacae Z0206), a recent discovered bacterial strain, could produce huge amount of exopolysaccharides. Our previous studies found that E. cloacae Z0206could accumulate selenium (Se) in the form of Se-enriched exopolysaccharide (Se-ECZ-EPS) efficiently in medium enriched with selenium. Se-ECZ-EPS contained535.7mg/kg Se. Se-ECZ-EPS has been shown to improve the innate, cellular and humoral immune responses in immunosuppressed mice induced by cyclophosphamide and protect RAW264.7cells from oxidative damage and apoptosis induced by H2O2.
However, little was known about the bioactive activities of Se-ECZ-EPS in crayfish, broilers and weaning piglets. Thus the present study was conducted to investigate the effects of Se-ECZ-EPS on growth performance, antioxidant status, immune function, intestinal morphology and intestinal microbial diversity in crayfish, broilers and weaning piglets. To develop Se-ECZ-EPS into a potential immunomodulator in animals, the acute toxicity of Se-ECZ-EPS in rat was investigated. The researches provide the basis for the application of Se-ECZ-EPS in husbandry.
Exp.l. Study on the acute toxicity of Se-ECZ-EPS in rat.
To develop Se-ECZ-EPS into a potential immunomodulator in animals such as crayfish, broilers and weaning piglets, this study was conducted to investigate the acute toxicity of Se-ECZ-EPS in rat according to GB15193.4-2003. The acute oral maximum-tolerated dose (MTD) of Se-ECZ-EPS were greater than20.0g/kg in male and female rats, and Se-ECZ-EPS was classified as non-toxic substance. This study provide the basis for the application of Se-ECZ-EPS in husbandry.
Exp.2. Effects of Se-ECZ-EPS on immune function in crayfish.
To investigate the modulation of immunity in crustaceans, we examined phenoloxidase (PO) activity, respiratory burst (RB) ability, superoxide dismutase (SOD) level and several immune-related gene expression profile in red swamp crayfish [Procambarus clarkii (Girard)] that were individually induced with Se-ECZ-EPS at1μg g-1.The protection of crayfish against the white spot syndrome virus (WSSV) by Se-ECZ-EPSwas also investigated.The results showed that PO, RB and SOD activities of crayfish increased significantly (P<0.05) at24,48and72h after being injected with Se-ECZEPS. Among the genes examined, the mRNA expressions of serine proteinase, HSP70and Mn-SOD were significantly up-regulated at24and48h after the Se-ECZ-EPS treatment, while no significant changes in the expression in proPO were observed from24to72h. The survival rate of crayfish that received Se-ECZ-EPS was significantly higher than that of the control group after14days (59.7%and0%respectively). Taken together, it can be concluded that Se-ECZ-EPS is an efficient immunostimulant and can improve the immunity of crayfish.
Exp.3. Effects of Se-ECZ-EPS on growth performance, immune function and intestinal morphology in broilers.
3.1Effects of Se-ECZ-EPS on growth performance and carcass quality in broilers.
This study was conducted to investigate the effects of Se-ECZ-EPS on growth performance and carcass quality in broilers. Two hundred and forty Avian broiler chickens were allocated randomly into five groups with four replicates in each group and twelve chickens in each replicate. Control group was fed with basal diet containing0.15mg/kg Se (Na2SeO3), and the another four groups were fed with diets supplemented with Se-ECZ-EPS at the level of280mg/kg (0.15mg/kg Se),560mg/kg (0.30mg/kg Se),840mg/kg (0.45mg/kg Se) and1120mg/kg (0.60mg/kg Se), respectively. The results showed that average daily gain and feed conversion ratio in840and1120mg/kg groups were significantly enhanced from0to42days compared to the control group (P<0.05). Drip loss of muscles in chest and leg were significantly decreased in560、840and1120mg/kg groups(P<0.05).These results implicated that Se-ECZ-EPS can decrease drip loss of muscles and improve the growth performance in broilers.
3.2Effects of Se-ECZ-EPS on immune function in broilers.
This study was conducted to investigate the effects of Se-ECZ-EPS on immune function in broilers. Administration of Se-ECZ-EPS at the level of560,840and1120mg/kg significantly increased activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and decreased malondialdehyde (MDA) production compared with the control and280mg/kg group(P<0.05). Compared with the control, the serum concentrations of TNF-a and IL-2showed positive responses in broilers fed with Se-ECZ-EPS at the levels of280、560、840and1120mg/kg (P<0.05). Significant increases in the level of IL-6were observed only in the serum of birds treated with Se-ECZ-EPS at560and1,120mg/kg as compared with the control group (P<0.05). At21days and35days of age, the HI titers against NDV in groups supplied with Se-ECZ-EPS at840and1,120mg/kg were significantly higher than that in the control (P<0.05) At42days of age, a significant increase in HI titer was observed only in birds treated with Se-ECZ-EPS at840mg/kg as compared with the normal control (P<0.05) And1120mg/kg group significantly improve avian influenza antibody levels in7d and14d after immunization (P<0.05). These results implicated that Se-ECZ-EPS can improve the growth performance through enhancing immune functions in broilers.
3.3Effects of Se-ECZ-EPS on intestinal morphology in broilers.
This study was conducted to investigate the effects of Se-ECZ-EPS on intestinal morphology in broilers. Duodenum crypt depth was shallow and villus height and crypt depth ratio (VCR) was significantly high in560and840mg/kg groups (P <0.05). And jejunum villous height in1120mg/kg group was significantly increased (P<0.05).These results implicated that Se-ECZ-EPS can improve the growth performance through enhancing intestinal absorption function in broilers. Exp.4. Effects of Se-ECZ-EPS on growth performance, immune function, intestinal morphology and intestinal microbial diversity in weaning piglets.4.1Effects of Se-ECZ-EPS on growth performance in weaning piglets.
This study was conducted to investigate the effects of Se-ECZ-EPS on growth performance in weaning piglets. A total of150piglets [(Landrace×Yorkshire)×Duroc] weaned at28d of age with an initial BW of8.11±0.12kg were randomly allocated into5groups of3replicates each. The5treatments were: basal control diet (CON), Na2SeO3diet (CON+0.30mg/kg Na2Se03), Na2SeO3+astragalus polysaccharide (APS) diet (CON+0.30mg/kg Na2SeO3+560mg/kg APS), Na2SeO3+EPS diet (CON+0.30mg/kg Na2SeO3+560mg/kg EPS) and Se-ECZ-EPS diet (CON+560mg/kg Se-ECZ-EPS). Compared with CON group, ADG was significantly enhanced in the Na2Se03+APS, Na2Se03+EPS and Se-ECZ-EPS groups and F:G was significantly decreased(P<0.05). There were no differences in ADG and F:G between pigs fed the Na2SeO3+APS, Na2Se03+EPS and Se-ECZ-EPS diets. These results implicated that Se-ECZ-EPS can improve the growth performance in weaning piglets.
4.2. Effects of Se-ECZ-EPS on immune function in weaning piglets.
This study was conducted to investigate the effects of Se-ECZ-EPS on immune function in weaning piglets. The Na2SeO3+EPS and Se-ECZ-EPS groups had higher serum T-AOC and lower MDA than the CON group (P<0.05). GSH-Px and SOD activities in Se-ECZ-EPS group were significantly increased compared with CON group (P<0.05). There were no differences in T-AOC、GSH-Px、SOD and MDA between pigs fed the Na2SeO3+APS, Na2Se03+EPS and Se-ECZ-EPS diets. Increases in serum TNF-a, IL-2and IL-6were observed in the Na2SeO3+EPS and Se-ECZ-EPS groups compared with the CON (P<0.05), and there were no difference in the TNF-a, IL-2and IL-6levels between pigs fed Na2SeO3+APS, Na2SeO3+EPS and Se-ECZ-EPS diets. The Se-ECZ-EPS group had higher serum IgG than the CON and Na2SeO3groups (P<0.05), and higher serum IgM than the CON, Na2Se03and Na2Se03+APS groups (P<0.05). Pigs fed with Na2Se03+APS, Na2SeO3+EPS and Se-ECZ-EPS diets had higher PR-39and Hepcidin gene expression levels in liver (P <0.05). The Se-ECZ-EPS group had higher pBD-1gene expression in duodenum and pBD-2gene expression in jejunum and ileum compared with the CON group (P <0.05). The Na2Se03+EPS group had higher pBD-2gene expression in jejunum and ileum, and the Na2SeO3+APS group had higher pBD-2gene expression in ileum compared with the CON group (P<0.05). These results implicated that Se-ECZ-EPS can improve the growth performance through enhancing immune functions in weaning piglets.
4.3Effects of Se-ECZ-EPS on intestinal morphology and intestinal microbial diversity in weaning piglets.
This study was conducted to investigate the effects of Se-ECZ-EPS on intestinal morphology and intestinal microbial diversity in weaning piglets. Villous height in jejunum and villus height and crypt depth ratio (VCR) in duodenum and jejunum were significantly increased in Na2Se03+EPS and Se-ECZ-EPS groups (P<0.05). Gut microbial diversity display that Se-ECZ-EPS can increase microbial diversity index (Shannon index) in colon and cecum. Compared with Na2SeO3group, Se-ECZ-EPS can increase Gram-positive bacteria (Firmicutes) and decrease gram-negative bacteria (Bacteroides door) in colon and cecum. These results implicated that Se-ECZ-EPS can improve the growth performance through regulating the balance of intestinal flora in weaning piglets.
本研究拟在前期研究Se-ECZ-EPS可以改善小鼠免疫功能的基础上,探讨Se-ECZ-EPS在动物养殖生产中的免疫调节作用,为将其开发成潜在的动物免疫调节制剂奠定基础。本研究设计了四个试验：试验一,首先采用小鼠急性经口毒性试验进行了Se-ECZ-EPS在动物中使用的的安全性评价。试验二,在确定了Se-ECZ-EPS在动物上的使用安全性后,为了拓展其在水产及畜禽养殖动物上的应用,选择水产甲壳类的理想模式动物-螫虾为试验对象,研究了Se-ECZ-EPS对螯虾的免疫调节功能及对感染白斑病毒后的保护作用；试验三,在水产螯虾研究基础上深入探讨了Se-ECZ-EPS对家禽动物-肉仔鸡的促生长及免疫调节作用；试验四,进一步选择断奶仔猪为试验对象,深入研究了Se-ECZ-EPS对断奶仔猪生长性能、免疫功能、肠道形态结构及肠道微生物多样性的影响并探讨其生物学机制；通过以上研究为Se-ECZ-EPS在动物中的应用奠定了坚实理论基础。
试验一、Z0206富硒多糖的安全性评价
为了奠定Z0206富硒多糖在水产及畜禽养殖动物上应用的基础,本研究首先采用小鼠急性经口毒性试验验证其在动物上的使用安全性。试验结果显示,受试小鼠急性经口毒性试验雌、雄两性别最大耐受量均大于20.0g/kg体重,依据GB15193.4-2003《食品安全性毒理学评价程序和检验方法-急性毒性试验》表明Se-ECZ-EPS无毒,为其在水产动物、畜禽动物上的应用提供了可能性。
试验二、Z0206富硒多糖对水产动物螯虾免疫功能的影响
在确定Se-ECZ-EPS在动物上的使用安全性基础上,以水产甲壳类的理想模式动物-克氏原螯虾(Procambarus clarkii (Girard))为动物模型,通过注射1μgg-1Se-ECZ-EPS,测定免疫相关指标和基因表达,同时探讨Se-ECZ-EPS对螯虾感染白斑病毒后的保护作用。研究结果显示,在注射Se-ECZ-EPS24,48和72h后,螯虾血液中酚氧化酶(PO),呼吸爆发(RB)和超氧化物歧化酶(sOD)活性显著升高(P<0.05)；基因表达结果显示,在注射Se-ECZ-EPS24和48h后,丝氨酸蛋白酶、HSP70和Mn-SOD基因表达显著上调,而酚氧化酶在24到72h后无显著影响；14天的攻毒试验表明,注射Se-ECZ-EPS后能够显著提高螯虾感染WSSV后的存活率(对照组：0%、Se-ECZ-EPS组：59.7%)。综上结果揭示,Se-ECZ-EPS可以提高克氏原螯虾的免疫功能,是一种有效的免疫调节剂。
试验三、Z0206富硒多糖对肉仔鸡生长性能、免疫功能及肠道形态结构的影响
在研究了Se-ECZ-EPS对螯虾的免疫调节功能及感染白斑病毒后的保护作用基础上,继而深入探讨Se-ECZ-EPS在家禽养殖业中占有重要地位的另一种动物-肉仔鸡上的促生长及免疫调节作用。
3.1.Z0206富硒多糖对肉仔鸡生长性能、胴体品质的影响
试验选取1日龄艾维茵肉仔鸡240羽,随机分为5个处理,每个处理4个重复,每个重复12羽。对照组在基础日粮中以亚硒酸钠的形式添加0.15mg/kg的硒水平,四个试验组在基础日粮中分别添加280、560、840、1120mg/kg的Z0206Se-ECZ-EPS(四个试验组硒含量分别为0.15、0.30、0.45、0.60mg/kg)。结果显示：与对照组相比,添加840、1120mg/kg的Se-ECZ-EPS可以显著提高肉仔鸡0-6周平均日增重,显著降低料重比(P<0.05)；添加560mg/kg.840mg/kg、1120mg/kg Se-ECZ-EPS则显著降低了胸肌、腿肌的滴水损失(P<0.05),而对肌肉pH无显著性影响。结果表明Se-ECZ-EPS可以降低鸡肉的滴水损失,有效提高肉仔鸡的生长性能。
3.2.E. cloacae Z0206富硒多糖对肉仔鸡免疫功能的影响
肉仔鸡免疫功能研究结果显示：血清抗氧化指标方面：与对照组和280mg/kg Se-ECZ-EPS组相比,添加840mg/kg和1120mg/kg的Se-ECZ-EPS可显著增加抗氧化酶GSH-Px、SOD的活性,显著降低MDA的含量(P<0.05)。细胞因子含量方面：添加280mg/kg、560mg/kg、840mg/kg和1120mg/kg的Se-ECZ-EPS均能显著提高血清TNF-a和IL-2的浓度(P<0.05),同时,560mg/kg和1120mg/kg Se-ECZ-EPS组血清IL-6的水平亦显著提高(P<0.05)。抗体水平方面：添加840mg/kg和1120mg/kg的Se-ECZ-EPS显著提高了21、35日龄血清新城疫HI水平(P<0.05),而在42日龄,只有840mg/kg组新城疫HI水平显著升高(P<0.05)；此外,添加1120mg/kg的Se-ECZ-EPS能显著提高免后7d、14d血清禽流感抗体水平(P<0.05)。提示Se-ECZ-EPS可以通过增强抗氧化酶活性、促进血清细胞因子分泌、提高血清抗体水平来增强免疫功能,进而提高肉仔鸡的生长性能。
3.3.E. cloacae Z0206富硒多糖对肉仔鸡肠道形态结构的影响
肉仔鸡肠道形态结构研究结果显示：添加560mg/kg和840mg/kg的Se-ECZ-EPS显著降低了十二指肠隐窝深度,显著提高了十二指肠绒毛高度和隐窝深度比VCR(P<0.05),而添加1120mg/kg的Se-ECZ-EPS显著提高了空肠绒毛高度(P<0.05)。提示Se-ECZ-EPS改善了肉仔鸡肠道的形态结构,有利于养分物质的吸收,进而提高肉仔鸡的生长性能。
试验四、Z0206富硒多糖对断奶仔猪生长性能、免疫功能、肠道形态结构及肠道微生物多样性的影响
在研究Se-ECZ-EPS对肉仔鸡促生长、增强免疫功能、改善肠道形态结构的基础上,进一步选择在畜牧养殖业中占有重要地位的断奶仔猪为试验对象,探讨Se-ECZ-EPS对断奶仔猪生长性能、免疫功能、肠道形态结构及肠道微生物多样性的影响及其作用机制。
4.1.Z0206富硒多糖对断奶仔猪生长性能的影响
试验选择150头28日龄“杜×长×大”三元杂交断奶仔猪,随机分成5组：对照组(基础日粮)、Na2Se03组(基础日粮+0.30mg/kg Na2Se03)、Na2SeO3+APS组(基础日粮,+0.30mg/kg Na2SeO3+560mg/kg黄芪多糖)、Na2Se03+EPS组(基础日粮+0.30mg/kg Na2SeO3+560mg/kg Z0206多糖)、Se-ECZ-EPS组(基础日粮+560mg/kg Z0206富硒多糖)。结果显示,日粮中添加Na2SeO3+APS、 Na2SeO3+EPS、Se-ECZ-EPS显著提高断奶仔猪平均日增重(P<0.05),且Se-ECZ-EPS组显著高于Na2Se03组(P<0.05),而与Na2Se03+APS、Na2Se03+EPS组无显著性差异；Na2SeO3+EPS、Se-ECZ-EPS组料重比显著低于对照组和Na2SeO3组(P<0.05),与Na2SeO3+EPS组无显著性差异。结果提示：Se-ECZ-EPS可以促进断奶仔猪的生长性能,使用效果与黄芪多糖APS相当。
4.2.Z0206富硒多糖对断奶仔猪免疫功能的影响
断奶仔猪免疫功能结果显示,在抗氧化功能方面：与对照组相比,日粮中添力口Na2SeO3+EPS、Se-ECZ-EPS显著提高仔猪血清总抗氧化力(T-AOC)(P<0.05)、显著降低了血清丙二醛(MDA)含量(P<0.05)。此外,Se-ECZ-EPS组血清谷胱甘肽还原酶(GSH-Px)、超氧化物歧化酶(sOD)活性显著提高了(P<0.05),而与Na2SeO3+APS、Na2SeO3+EPS组相比没有显著性差异。细胞因子方面：日粮中添加Na2SeO3+EPS、Se-ECZ-EPSS显著提高了仔猪血清TNF-α、IL-2、IL-6含量(P<0.05),而与Na2SeO3+APS组相比没有显著性差异。免疫球蛋白方面：相比于对照组和Na2SeO3组,日粮中添加Se-ECZ-EPS显著提高了仔猪血清IgG、IgM含量,且Se-ECZ-EPS组IgM含量显著高于Na2SeO3+APS组(P<0.05)。组织抗菌肽表达方面Na2SeO3+APS组、Na2SeO3+EPS组、Se-ECZ-EPS组显著提高了肝脏抗菌肽基因PR-39、Hepcidin的表达(P<0.05)；且Se-ECZ-EPS显著提高了十二指肠中pBD-1的基因表达,提高了空肠和回肠中pBD-2的基因表达(P<0.05)；而Na2SeO3+EPS提高了空肠和回肠中pBD-2的基因表达(P<0.05),Na2SeO3+APS只提高了回肠中pBD-2的基因表达(P<0.05)。结果提示Se-ECZ-EPS可以通过提高血清免疫球蛋白含量、促进细胞因子分泌、提高肝脏和肠道抗菌肽基因表达来增强免疫功能。
4.3.Z0206富硒多糖对断奶仔猪肠道形态结构及肠道微生物多样性的影响
断奶仔猪肠道形态结构及肠道微生物多样性研究结果显示：断奶仔猪日粮中添加Na2SeO3+EPS、Se-ECZ-EPS显著提高了十二指肠、空肠绒毛高度和隐窝深度比VCR<0.05),明显增加空肠绒毛高度(P<0.05)。肠道微生物多样性分析显示,Se-ECZ-EPS可以提高结肠、盲肠内容物中微生物多样性指数(Shannon指数),与Na2SeO3组相比,添加Se-ECZ-EPS能够增加结肠、盲肠中革兰氏阳性菌(厚壁菌门)比例,减少革兰氏阴性菌(拟杆菌门)比例。结果提示：Z0206富硒多糖可以改善肠道结构、调节菌群平衡,进而提高断奶仔猪的生长性能。
Enterobacter cloacae Z0206(E. cloacae Z0206), a recent discovered bacterial strain, could produce huge amount of exopolysaccharides. Our previous studies found that E. cloacae Z0206could accumulate selenium (Se) in the form of Se-enriched exopolysaccharide (Se-ECZ-EPS) efficiently in medium enriched with selenium. Se-ECZ-EPS contained535.7mg/kg Se. Se-ECZ-EPS has been shown to improve the innate, cellular and humoral immune responses in immunosuppressed mice induced by cyclophosphamide and protect RAW264.7cells from oxidative damage and apoptosis induced by H2O2.
However, little was known about the bioactive activities of Se-ECZ-EPS in crayfish, broilers and weaning piglets. Thus the present study was conducted to investigate the effects of Se-ECZ-EPS on growth performance, antioxidant status, immune function, intestinal morphology and intestinal microbial diversity in crayfish, broilers and weaning piglets. To develop Se-ECZ-EPS into a potential immunomodulator in animals, the acute toxicity of Se-ECZ-EPS in rat was investigated. The researches provide the basis for the application of Se-ECZ-EPS in husbandry.
Exp.l. Study on the acute toxicity of Se-ECZ-EPS in rat.
To develop Se-ECZ-EPS into a potential immunomodulator in animals such as crayfish, broilers and weaning piglets, this study was conducted to investigate the acute toxicity of Se-ECZ-EPS in rat according to GB15193.4-2003. The acute oral maximum-tolerated dose (MTD) of Se-ECZ-EPS were greater than20.0g/kg in male and female rats, and Se-ECZ-EPS was classified as non-toxic substance. This study provide the basis for the application of Se-ECZ-EPS in husbandry.
Exp.2. Effects of Se-ECZ-EPS on immune function in crayfish.
To investigate the modulation of immunity in crustaceans, we examined phenoloxidase (PO) activity, respiratory burst (RB) ability, superoxide dismutase (SOD) level and several immune-related gene expression profile in red swamp crayfish [Procambarus clarkii (Girard)] that were individually induced with Se-ECZ-EPS at1μg g-1.The protection of crayfish against the white spot syndrome virus (WSSV) by Se-ECZ-EPSwas also investigated.The results showed that PO, RB and SOD activities of crayfish increased significantly (P<0.05) at24,48and72h after being injected with Se-ECZEPS. Among the genes examined, the mRNA expressions of serine proteinase, HSP70and Mn-SOD were significantly up-regulated at24and48h after the Se-ECZ-EPS treatment, while no significant changes in the expression in proPO were observed from24to72h. The survival rate of crayfish that received Se-ECZ-EPS was significantly higher than that of the control group after14days (59.7%and0%respectively). Taken together, it can be concluded that Se-ECZ-EPS is an efficient immunostimulant and can improve the immunity of crayfish.
Exp.3. Effects of Se-ECZ-EPS on growth performance, immune function and intestinal morphology in broilers.
3.1Effects of Se-ECZ-EPS on growth performance and carcass quality in broilers.
This study was conducted to investigate the effects of Se-ECZ-EPS on growth performance and carcass quality in broilers. Two hundred and forty Avian broiler chickens were allocated randomly into five groups with four replicates in each group and twelve chickens in each replicate. Control group was fed with basal diet containing0.15mg/kg Se (Na2SeO3), and the another four groups were fed with diets supplemented with Se-ECZ-EPS at the level of280mg/kg (0.15mg/kg Se),560mg/kg (0.30mg/kg Se),840mg/kg (0.45mg/kg Se) and1120mg/kg (0.60mg/kg Se), respectively. The results showed that average daily gain and feed conversion ratio in840and1120mg/kg groups were significantly enhanced from0to42days compared to the control group (P<0.05). Drip loss of muscles in chest and leg were significantly decreased in560、840and1120mg/kg groups(P<0.05).These results implicated that Se-ECZ-EPS can decrease drip loss of muscles and improve the growth performance in broilers.
3.2Effects of Se-ECZ-EPS on immune function in broilers.
This study was conducted to investigate the effects of Se-ECZ-EPS on immune function in broilers. Administration of Se-ECZ-EPS at the level of560,840and1120mg/kg significantly increased activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and decreased malondialdehyde (MDA) production compared with the control and280mg/kg group(P<0.05). Compared with the control, the serum concentrations of TNF-a and IL-2showed positive responses in broilers fed with Se-ECZ-EPS at the levels of280、560、840and1120mg/kg (P<0.05). Significant increases in the level of IL-6were observed only in the serum of birds treated with Se-ECZ-EPS at560and1,120mg/kg as compared with the control group (P<0.05). At21days and35days of age, the HI titers against NDV in groups supplied with Se-ECZ-EPS at840and1,120mg/kg were significantly higher than that in the control (P<0.05) At42days of age, a significant increase in HI titer was observed only in birds treated with Se-ECZ-EPS at840mg/kg as compared with the normal control (P<0.05) And1120mg/kg group significantly improve avian influenza antibody levels in7d and14d after immunization (P<0.05). These results implicated that Se-ECZ-EPS can improve the growth performance through enhancing immune functions in broilers.
3.3Effects of Se-ECZ-EPS on intestinal morphology in broilers.
This study was conducted to investigate the effects of Se-ECZ-EPS on intestinal morphology in broilers. Duodenum crypt depth was shallow and villus height and crypt depth ratio (VCR) was significantly high in560and840mg/kg groups (P <0.05). And jejunum villous height in1120mg/kg group was significantly increased (P<0.05).These results implicated that Se-ECZ-EPS can improve the growth performance through enhancing intestinal absorption function in broilers. Exp.4. Effects of Se-ECZ-EPS on growth performance, immune function, intestinal morphology and intestinal microbial diversity in weaning piglets.4.1Effects of Se-ECZ-EPS on growth performance in weaning piglets.
This study was conducted to investigate the effects of Se-ECZ-EPS on growth performance in weaning piglets. A total of150piglets [(Landrace×Yorkshire)×Duroc] weaned at28d of age with an initial BW of8.11±0.12kg were randomly allocated into5groups of3replicates each. The5treatments were: basal control diet (CON), Na2SeO3diet (CON+0.30mg/kg Na2Se03), Na2SeO3+astragalus polysaccharide (APS) diet (CON+0.30mg/kg Na2SeO3+560mg/kg APS), Na2SeO3+EPS diet (CON+0.30mg/kg Na2SeO3+560mg/kg EPS) and Se-ECZ-EPS diet (CON+560mg/kg Se-ECZ-EPS). Compared with CON group, ADG was significantly enhanced in the Na2Se03+APS, Na2Se03+EPS and Se-ECZ-EPS groups and F:G was significantly decreased(P<0.05). There were no differences in ADG and F:G between pigs fed the Na2SeO3+APS, Na2Se03+EPS and Se-ECZ-EPS diets. These results implicated that Se-ECZ-EPS can improve the growth performance in weaning piglets.
4.2. Effects of Se-ECZ-EPS on immune function in weaning piglets.
This study was conducted to investigate the effects of Se-ECZ-EPS on immune function in weaning piglets. The Na2SeO3+EPS and Se-ECZ-EPS groups had higher serum T-AOC and lower MDA than the CON group (P<0.05). GSH-Px and SOD activities in Se-ECZ-EPS group were significantly increased compared with CON group (P<0.05). There were no differences in T-AOC、GSH-Px、SOD and MDA between pigs fed the Na2SeO3+APS, Na2Se03+EPS and Se-ECZ-EPS diets. Increases in serum TNF-a, IL-2and IL-6were observed in the Na2SeO3+EPS and Se-ECZ-EPS groups compared with the CON (P<0.05), and there were no difference in the TNF-a, IL-2and IL-6levels between pigs fed Na2SeO3+APS, Na2SeO3+EPS and Se-ECZ-EPS diets. The Se-ECZ-EPS group had higher serum IgG than the CON and Na2SeO3groups (P<0.05), and higher serum IgM than the CON, Na2Se03and Na2Se03+APS groups (P<0.05). Pigs fed with Na2Se03+APS, Na2SeO3+EPS and Se-ECZ-EPS diets had higher PR-39and Hepcidin gene expression levels in liver (P <0.05). The Se-ECZ-EPS group had higher pBD-1gene expression in duodenum and pBD-2gene expression in jejunum and ileum compared with the CON group (P <0.05). The Na2Se03+EPS group had higher pBD-2gene expression in jejunum and ileum, and the Na2SeO3+APS group had higher pBD-2gene expression in ileum compared with the CON group (P<0.05). These results implicated that Se-ECZ-EPS can improve the growth performance through enhancing immune functions in weaning piglets.
4.3Effects of Se-ECZ-EPS on intestinal morphology and intestinal microbial diversity in weaning piglets.
This study was conducted to investigate the effects of Se-ECZ-EPS on intestinal morphology and intestinal microbial diversity in weaning piglets. Villous height in jejunum and villus height and crypt depth ratio (VCR) in duodenum and jejunum were significantly increased in Na2Se03+EPS and Se-ECZ-EPS groups (P<0.05). Gut microbial diversity display that Se-ECZ-EPS can increase microbial diversity index (Shannon index) in colon and cecum. Compared with Na2SeO3group, Se-ECZ-EPS can increase Gram-positive bacteria (Firmicutes) and decrease gram-negative bacteria (Bacteroides door) in colon and cecum. These results implicated that Se-ECZ-EPS can improve the growth performance through regulating the balance of intestinal flora in weaning piglets.
引文
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