CN118303532A - 哺乳动物来源的重组碱性磷酸酶用于提高家禽的生长性能与免疫功能的应用 - Google Patents
哺乳动物来源的重组碱性磷酸酶用于提高家禽的生长性能与免疫功能的应用 Download PDFInfo
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Abstract
本发明涉及农业养殖技术领域，具体涉及哺乳动物来源的重组碱性磷酸酶用于提高家禽的生长性能与免疫功能的应用。本发明的技术方案通过在小麦日粮中添加不同剂量bIAP，显著提高了肉鸡的生长性能，高剂量组的提升能力与抗生素相当。此外，其bIAP的添加提升了肉鸡免疫器官指数，提高了血清免疫球蛋白IgA和IgG等水平。
Description
技术领域
本发明涉及农业养殖技术领域，具体涉及哺乳动物来源的重组碱性磷酸酶用于提高家禽的生长性能与免疫功能的应用。
背景技术
饲用酶制剂已被证明在提升动物生产性能以及改善动物体健康方面具有巨大潜力。植酸酶的添加可以显著提高营养物质消化率并降低抗营养因子水平；木聚糖酶的添加可以提高饲料中植物细胞壁木聚糖的降解和营养释放效率，并产生木寡糖益生物质；蛋白酶、纤维素酶等针对营养物质的酶的添加也被证明有益于提高动物生产性能。在全面“禁抗”的今天，酶制剂已逐渐成为抗生素替代中的重要一员。
碱性磷酸酶( AKP) 是一种广泛分布于动物体各组织器官中的单脂磷酸水解酶，属于同源二聚体酶，包含四种同工酶，即组织非特异性碱性磷酸酶( TNAP，主要表达于肝、骨骼和肾) 、胎盘碱性磷酸酶 ( PLAP) 、生殖细胞碱性磷酸酶 (GCAP) 和肠道碱性磷酸酶（IAP），IAP 是一种经糖基磷脂酰肌醇键锚定于肠上皮细胞顶膜上的糖蛋白。在生理情况下，动物进食后后分泌的胰磷脂酶 A2 水解胆汁中卵磷脂所生成的溶血卵磷脂能特异性加速 IAP 经肠上皮细胞顶膜释放入肠腔。
IAP已被证明在维持动物肠道屏障健康、改善肠道菌群以及延缓肠道衰老方面有着积极作用。给予低剂量的外源碱性磷酸酶可以显著改善结肠炎小鼠的肠道屏障损伤，降低肠道通透性。此外，IAP 在十二指肠表面 pH 值调节中发挥着碱性化学传感器的作用，可以通过碳酸氢根分泌途径保护十二指肠黏膜免受胃酸对其造成的损伤，维护肠黏膜化学屏障的完整性。当肠腔中的IAP 催化脂多糖（LPS） 的去磷酸化反应，导致具有毒性的LPS 浓度降低，LPS对肠上皮细胞受体的刺激作用减弱，炎性相关基因表达水平下调。在调节肠道菌群方面，IAP可以通过降低肠道ATP水平降低肠道大肠杆菌的丰度，同时通过其他降低肠道内三磷酸核苷酸的浓度来促进肠道细菌生长，IAP 基因敲除小鼠粪便中需氧 菌和厌氧菌的种类和数量与野生型小鼠相比显著减少，口服 IAP 可促进肠道共生菌的生长，恢复因抗生素的使用所导致的肠道菌群紊乱，并能抑制肠道致病菌——鼠伤寒沙门菌的生长，从而优化肠道菌群组成。同时，外源IAP的添加，可以缓解大肠杆菌iF4 (K88)攻毒仔猪的感染症状。
虽然IAP对维系动物的肠道健康具有一定的有益作用，但是，总所周知，影响动物生长的因素更为复杂，遗传、营养、内分泌、疾病、环境等不同因素都可对动物的生长性状产生重大的影响，因此IAP是否可用于革兰氏阳性菌尚不得而知，而哺乳动物来源的重组IAP饲喂家禽后可否发挥和如小鼠的哺乳动物中相似的作用更不明确。
发明内容
本发明的目的是提供哺乳动物来源的重组碱性磷酸酶用于提高家禽的生长性能与免疫功能的应用。
根据本发明的技术方案，提供哺乳动物来源的重组碱性磷酸酶用于提高家禽的生长性能与免疫功能的应用。
其中，所述哺乳动物来源的重组碱性磷酸酶为牛重组碱性磷酸酶。
根据本发明的技术方案，其中，将牛来源的碱性磷酸酶bIAP添加于小麦豆粕日粮之中。
根据本发明的技术方案，其中，将牛来源的碱性磷酸酶bIAP添加于小麦豆粕日粮之中，用于饲喂肉鸡。
根据本发明的技术方案，其中，肉鸡小麦日粮中添加5000U/kg~10000U/kg bIAP。
根据本发明的方法，在步骤（1）中，因本实验所使用的bIAP为粗酶冻干粉，含有纤维素酶活性，因此分别设置阴性对照（NC）为常规日粮，阳性对照（PC）为纤维素酶组，PC组纤维素酶量与最高剂量bIAP组一致。
本发明的技术方案通过在小麦日粮中添加不同剂量bIAP，显著提高了肉鸡的生长性能，高剂量组的提升能力与抗生素相当。此外，其bIAP的添加提升了肉鸡免疫器官指数，提高了血清免疫球蛋白IgA和IgG等水平。
附图说明
图1显示21日龄bIAP对肉鸡免疫器官指数的提升作用；
图2为42日龄bIAP对肉鸡免疫器官指数测提升作用；
图3显示bIAP对肉鸡血清免疫球蛋白IgA、IgG和IgM含量的提升作用。
具体实施方式
下面结合具体实施例对本发明进行详细说明。
本发明所提供的牛肠碱性磷酸酶，为利用重组里氏木霉菌株发酵制得，其可达到提高肉鸡生长性能和免疫功能的作用，具体地，一方面可改善肉鸡的生长性能（日采食量、日增重和料肉比），另一方面还可改善免疫功能（胸腺指数、法氏囊指数、肝脏指数和脾脏指数）。
以下实施例中所使用基础饲料包括组分如下：小麦、豆粕、大豆油、磷酸氢钙、DL-蛋氨酸、L-赖氨酸、苏氨酸、石粉、食盐、多维预混料、微量元素预混料、氯化胆碱（50%）。所述养殖饲料中，所述复合维生素及矿物质中，包含：维生素A，维生素D，维生素E，维生素K3，维生素B1，维生素B2，维生素B6，维生素B12，烟酰胺，D-泛酸钙，叶酸，D-生物素，锌，铜，铁，锰，碘，硒。 按质量百分比计，其原料组成及占比如表1，基础日粮参照NRC(1994)肉鸡营养需要进行配制，见表2。
表1：基础日粮配方
。
表 2 基础日粮的营养成分含量
。
根据本申请的实施例，bIAP添加量（按酶活性单位计）分别2500 U/kg、5000 U/kg以及10000 U/kg，纤维素酶添加量与最高剂量bIAP组一致。进一步的，为与常规抗生素促生长剂进行比较，以盐酸土霉素作为抗生素类生长促进剂，添加量为0.3 g/kg。除添加酶与抗生素外的原料质量百分比之和为100％。
实施例1 在小麦日粮中添加bIAP提高肉鸡生长性能，降低料肉比
肉鸡的生长性能是衡量其经济效益的重要指标，可以通过测量平均日增重、平均日采食量、料肉比等反映。试验采用随机分组法，选取384只体重相近的健康AA肉仔鸡，随机分为6个处理组，每组8个重复（每个重复8羽)。如表3所示，实验分为阴性对照组饲喂基础日粮和阳性对照组添加纤维素酶组，PC组纤维素酶量与最高剂量bIAP组一致，添加低、中和高剂量的bIAP组以及抗生素组的饲粮，以掺入基础饲粮方式喂饲42天，分别命名为阴性对照组、阳性对照组、低剂量bIAP组、中剂量bIAP组、高剂量bIAP组和抗生素组，所述低剂量组、中剂量组和高剂量组分别添加bIAP冻干粉至终浓度为2500 U/kg，5000 U/kg以及10000 U/kg。肉鸡养殖于实验鸡舍，自由采食和饮水，24 h光照，定时清扫卫生，定期对舍内消毒，免疫及饲养管理遵照实验鸡场规定执行。
表3 实验分组
| 实验分组 | 处理 |
| NC | 基础日粮 |
| PC | 基础日粮＋10000 U/kg纤维素酶 |
| 基础日粮＋2500 U/kg bIAP | |
| 基础日粮＋5000 U/kg bIAP | |
| 基础日粮＋10000 U/kg bIAP | |
| 基础日粮＋0.3 g/kg 盐酸土霉素 |
。
实验第0、21、42日，分别以重复为单位对肉鸡进行空腹称重并记录，准确记录饲养期内肉鸡的给料量和撒料量，参照中华人民共和国农业行业标准（NY/T 823-2020-家禽生产性能名词术语和度量统计方法）测定肉鸡生产性能。计算公式为：日采食量（g/d/只）=全期耗料/饲养只日数；日均增重（g/d/只）=总增重/饲养只日数；耗料增重比=全程消耗饲料总量/总增重。不同添加bIAP对养殖肉鸡生长性能的比较结果见表4。
表4 添加bIAP对肉鸡生长性能的影响
。
由表4可知，在21日龄，与对照组相比，bIAP各剂量组肉鸡日增重显著升高(P ＜0.001)；平均日采食量随剂量增加而增加，但与空白对照组无显著性差异；料重比随bIAP添加量增加而降低，其中高剂量组料重比最低；在42日龄，bIAP低、中、高剂量组均显著提高了肉鸡平均日增重(P ＜ 0.05)，其中高剂量组效果最为明显，高剂量组肉鸡平均日采食量与空白对照组显著增加(P ＜ 0.05)，中、高剂量组的料重比显著低于对照组和低剂量组。结果表明，在肉仔鸡日粮中添加bIAP可显著提高肉鸡日增重，降低料重比。
实施例2 添加bIAP可以提高肉鸡的免疫功能
由实施例1可知，不同剂量组bIAP添加可显著提高肉鸡日增重，降低料重比，改善肉鸡的生长性能。免疫功能是反映肉鸡机体健康的重要标志，增强肉鸡免疫力可以防止外来细菌、病毒等的侵入。按照实施例1进行肉鸡养殖实验，各实验组分别在第21日龄和第42日龄时随机抽取16只鸡称重后，取脾脏、胸腺和法氏囊，用滤纸吸干水分后称重，计算免疫器官指数：免疫器官指数=免疫器官重量/体重。
各组于第21日龄与42日龄，分别随机抽取16只鸡，电击处死后取血，每只试鸡约5ml，静置后，经过高速离心机15min处理后，放置于-20℃下保存备用。血清中免疫球蛋白的测定采用相应的酶联免疫吸附法试剂盒测定。将试剂盒在室温中平衡20 min，取出待测血清，按照试剂盒说明书规范操作。通过测定特定波长的吸光度值，参照标准曲线即可算出血清中Ig G、Ig M、IgA 的含量。肝脏、胸腺、法氏囊和脾脏是家禽免疫系统重要的免疫器官，通过免疫器官指数可以初步衡量机体免疫功能。免疫球蛋白的含量与动物机体免疫功能也有密切的关系。所选取的样品鸡按照均匀随机原则，即与本重复平均体重接近的肉鸡中进行挑选，免疫器官称重时脂肪等其他组织剥离彻底，胸腺为单侧称重后复核为双侧重量。实验数据使用SPSS软件运行One-way ANOVA程序进行组间差异分析，P＜0.05为差异显著。
添加bIAP提升肉鸡免疫功能，如图1所示，bIAP对21日龄AA肉鸡免疫器官指数有提升作用，统计结果均为和对照NC组相比较，其中，“ns”为差异不显著，“*”为差异显著(P ＜0.05)；如图2所示，bIAP对42日龄AA肉鸡免疫器官指数有提升作用，统计结果均为和对照NC组相比较，其中，““ns”为差异不显著，“*”为差异显著(P ＜ 0.05)；如图3所示，bIAP对AA肉鸡血清免疫球蛋白含量有提升作用，统计结果均为和对照NC组相比较，其中，““ns”为差异不显著，“*”为差异显著(P ＜ 0.05)，“**”为差异极显著(P ＜ 0.01)，“***”为差异极显著(P ＜ 0.001)。
以上结果表明，在饲粮中添加bIAP能够提高肉鸡机体的免疫功能。其中，中和高的添加剂量的作用效果更为显著。
以上实施例有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
Claims (4)
1.哺乳动物来源的重组碱性磷酸酶用于提高家禽的生长性能与免疫功能的应用。
2.根据权利要求1所述的应用，其特征在于，所述哺乳动物来源的重组碱性磷酸酶为牛重组碱性磷酸酶。
3.根据权利要求1所述的应用，其特征在于，所述家禽为鸡。
4.根据权利要求1所述的应用，其特征在于，所述哺乳动物来源的重组碱性磷酸酶在家禽饲料中的添加量为5000U/kg~10000U/kg 。
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