CN114747674A - 一种预防羔羊腹泻和母羊乳房炎的开口料 - Google Patents
一种预防羔羊腹泻和母羊乳房炎的开口料 Download PDFInfo
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Abstract
本发明公开了一种预防羔羊腹泻和母羊乳房炎的开口料,该开口料包括混合料以及混合料重量0.001%~0.1%的复合纳米抗菌肽,所述混合料按重量百分数计包括膨化大豆、膨化玉米、膨化小麦、乳清粉、预混料、食盐、小苏打以及奶香调味剂;所述复合纳米抗菌肽包括天蚕素复合物。所述开口料可作为强制补饲技术中的关键饲料。
Description
技术领域
本发明涉及家畜养殖,具体涉及羔羊开口料及其使用方法。
背景技术
肉羊养殖产业经过多年的品种改良,湖羊、小尾寒羊等品种的繁殖率得到了显著提高,每胎可以产2只以上羔羊。但由于母羊产羔后乳汁分泌不足,导致羔羊腹泻发病率超过30%,每年新生羔羊死亡率达到20%,并且羔羊吸允乳汁时造成的机械损伤引起母羊乳房炎发病率高达15%以上,造成严重的经济损失。采取羔羊早期断奶结合强制补饲能从根本上解决以上导致优良肉羊品种养殖效益提升困难的问题。
随着药物饲料添加剂的全面禁止使用,在肉羊各阶段饲料中都不再允许添加促生长和抗菌、保健作用的抗生素,这对于采取羔羊早期断奶及强制补饲的肉羊养殖技术造成的影响包括:羔羊成活率显著降低、生长速度降低,以及养殖效益下降。为了消除以上影响,探索新的替代饲料势在必行。
中国专利CN111685226A提出了一种不含抗生素的教槽料配方。但是该教槽料配方中必须包括酸类添加剂、酶类添加剂及菌群添加剂等复杂原料组成,导致成本高,并且对制作工艺要求较高,对推广应用不利。而且该教槽料的优势仅仅在于轻微降低了弱仔猪的比例,解决不了幼龄仔畜普遍存在的应激、腹泻等问题。同时,相比单胃动物,反刍动物消化生理更加复杂,食物首先经过瘤胃发酵,然后流入小肠进行化学性消化。人为在饲喂羊等反刍动物过程中添加酶类和益生菌的效果并不好,因为瘤胃pH值极低,多数酶类和益生菌不能存活,更不能发挥作用,同时在瘤胃环境中这些被添加的益生菌只有需氧菌与厌氧菌达到一定比例时才能发挥作用,否则无法存活,因此对于羊等反刍动物而言,饲料中添加各类益生菌和酶通常起不到应有的功效。此外,中国专利CN111685226A中是通过改变饲料成份调控胃肠道环境,从而抑制致病菌繁殖,但该专利通过环境变化抑制致病菌的繁殖存在很大的变数,是否能完全抑制致病菌的繁殖没有相关信息。
目前尚缺乏与强制补饲措施有效配合的羔羊开口料,以含有益生素的开口料进行强制补饲,无法在40日龄断奶后立即饲喂育肥或育成饲料,并且羔羊腹泻及母羊乳房炎的发生率仍较高。
发明内容
本发明的目的在于提供一种预防羔羊腹泻和母羊乳房炎的开口料。
为达到上述目的,本发明采用了以下技术方案:
一种羔羊开口料,该开口料包括混合料以及混合料重量0.001%~0.1%的复合纳米抗菌肽,所述混合料按重量百分数计包括以下组分:25%~35%的膨化大豆、45%~60%的膨化玉米、8%~18%的膨化小麦、1%~5%的乳清粉、1%~5%的预混料、0.1%~1.0%的食盐、0.2%~0.8%的小苏打以及0.005%~0.05%的奶香调味剂;所述复合纳米抗菌肽包括天蚕素复合物。
优选的,所述复合纳米抗菌肽为混合料重量的0.005%~0.01%。
优选的,所述混合料按重量百分数计包括以下组分:30%的膨化大豆、52%的膨化玉米、13%的膨化小麦、2%的乳清粉、2%的预混料、0.5%的食盐、0.49%的小苏打以及0.01%的奶香调味剂。
优选的,所述预混料包括维生素和微量元素。
上述羔羊开口料的制备方法,包括以下步骤:
将所述膨化大豆、膨化玉米、膨化小麦、乳清粉、预混料、食盐、小苏打、奶香调味剂以及复合纳米抗菌肽与水拌合后升温至70℃~80℃进行制粒,然后进行干燥。
一种羔羊的诱导采食方法,包括以下步骤:
1)羔羊自5~10日龄开始,间断性将羔羊与母羊分离,在分离期间供给羔羊清洁饮水和上述开口料;
2)羔羊自30~40日龄开始断奶,即将羔羊与母羊分栏饲养,此时羔羊开始饲喂育成饲料或育肥饲料,不再饲喂上述开口料。
优选的,羔羊自10~20日龄开始,逐渐增大采食上述开口料的时间(延长母子分开时间),同时诱导羔羊采食干草,最大程度缓解羔羊断奶应激。
本发明的有益效果体现在:
本发明提出的羔羊开口料采用具有抑菌、促生长、提高免疫力的复合纳米抗菌肽作为添加剂,与开口料中其他组分配伍,可作为强制补饲技术中的关键饲料,从而为实现羔羊早期断奶提供保障,应用在湖羊等肉羊品种的规模化饲养中,并取得了阶段性的进展(有效避免羔羊腹泻,提高生长速度,降低母羊乳房炎的发生,同时,羔羊可以在40日龄断奶后立即进入育肥或育成饲养阶段)。
附图说明
图1为不同开口料对湖羊增重的影响。
图2为Scaffold长度分布;横轴Sequence Length表示组装产生的scaffold的长度,纵轴Number of Sequence表示产生的某一长度的scaffold对应的reads数量。
图3为Gene长度分布;纵轴Number of genes表示gene catalogue中对应read的数目;横轴Gene length表示gene catalogue中基因的长度。
图4为基因数目韦恩图分析;图中:每个圈代表一个样品,圈和圈重叠部分的数字代表样品之间共有的基因个数,没有重叠部分的数字代表样品的特有基因个数。
图5为注释物种数目及相对丰度聚类分析;图中仅仅展示种(species)这一层级,横轴表示样品,纵轴表示注释到某类型的物种的相对比例,各区块对应的物种类别见图例。
图6为不同分类水平物种数目及丰度聚类热图;图中每一行代表一个物种,每一列代表一个样品,对应的值为物种相对丰度;图左侧的聚类树为物种聚类树;图上部的聚类树为样品聚类树;未分类的部分(Unclassified)未纳入图中。
具体实施方式
下面结合附图和实施例对本发明做进一步详细说明。所述实施例仅用于解释本发明,而非对本发明保护范围的限制。
(一)羔羊开口料(颗粒)的制备
1.配方
1.1开口料实例1
混合料(质量分数):膨化大豆30%、膨化玉米52%、膨化小麦13%、乳清粉2%、羊预混料2%、食盐0.5%、小苏打0.49%以及奶香调味剂0.01%;
添加剂:混合料重量0.01%的复合纳米抗菌肽(经医药级别的纳米材料包被的天蚕素A与D的复合物,该复合物是利用基因工程菌表达的仅含有天蚕素A与D的融合蛋白,纳米材料为纳米多孔淀粉,复合纳米抗菌肽的复合物载药量为10%)。
其中,羊预混料的组成为:维生素A(≥120KIU/kg)、维生素D3(≥60KIU/kg)、维生素E(≥750IU/kg)、铜(≥350mg/kg)、铁(≥1500mg/kg)、锰(≥1500mg/kg)、锌(≥1200mg/kg)、钙(≥18%)、赖氨酸(≥5%)。
1.2开口料实例2
混合料(质量分数):膨化大豆30%、膨化玉米52%、膨化小麦13%、乳清粉2%、羊预混料2%、食盐0.5%、小苏打0.49%以及奶香调味剂0.01%;
添加剂:混合料重量0.005%的复合纳米抗菌肽(即实例1中经医药级别的纳米材料包被的天蚕素A与D的复合物);
其中,羊预混料与实例1相同。
1.3开口料对照例
按质量分数计,由膨化大豆30%、膨化玉米58%、麸皮5%、乳清粉3%、全脂奶粉1.1%、羊预混料2%、食盐0.65%、反刍动物专用酶制剂0.03%、奶香调味剂0.02%及0.2%的益生素组成;
其中,反刍动物专用酶制剂由10%葡萄糖氧化酶、10%木聚糖酶、15%纤维素酶、5%酸性蛋白酶、10%α-淀粉酶及50%酿酒酵母培养物组成;
益生素(购自山东明发兽药股份有限公司)由20%低聚果糖、15%菊粉、10%低聚半乳糖、15%大豆低聚糖及40%辅料(葡萄糖)组成。
2.羔羊颗粒料制作工艺
按上述开口料配方(实例1、实例2)中的配比称取混合料组分于颗粒料制作机中初步混合,再将一定比例的复合纳米抗菌肽(参照实例1、实例2)加入颗粒料制作机中,继续拌匀,同时额外添加2%(颗粒料制作机中物料总重的2%)的水,然后将温度调节到70℃,边搅拌边制作颗粒料,出料后晾晒干燥,所得颗粒开口料袋装保存。
对照例颗粒的制作工艺与以上工艺相似。
(二)羔羊早期断奶中强制补饲的效果
试验一:不同开口料对羔羊生长发育的影响
1.1试验流程
1.1.1强制补饲
湖羊羔羊出生后,自由吮乳。从7日龄开始强制补饲,在补饲栏放置开口料和饮水碗。每天清晨在羔羊采食过母乳后,诱导其采食开口料,即人为地将羔羊与母羊分离后供给羔羊清洁饮水和上述实例1、实例2或对照例的开口料,2小时后将羔羊放回母羊身边,供其自由采食母乳;羔羊自14日龄开始,逐渐增大羔羊采食开口料的时间,每天8:00-12:00、15:00-19:00定时与母羊分离,诱导羔羊采食开口料;羔羊自30日龄开始,进一步增大羔羊与母羊分离时间,即除了每天两次定时采食母乳外,其他时间都与母羊分离。
自从强制补饲开始,通过1周的适应,自羔羊14日龄开始后,羔羊就形成了条件反射,采食母乳后就自动去采食饲料,期间逐渐补充少量优质干草。所有试验羊(母羔、公羔)于40日龄断奶,母羔进入育成羊、公羔进入育肥羊饲喂阶段,不再饲喂开口料。
1.1.2检测
羔羊出生后,每隔7天称重一次,制作生长曲线。
羔羊进行强制补饲的第7天开始,统计各组(饲喂不同开口料)母羊乳房炎发病情况,统计时间段为7日龄至120日龄。
羔羊进行强制补饲的第7天开始,统计各组(饲喂不同开口料)羔羊腹泻发病情况,统计时间段为7日龄至120日龄。
1.2结果与分析
1.2.1不同开口料对羔羊生长速度的影响
对采用强制补饲措施(开口料实例1、开口料实例2、开口料对照例)的羔羊进行对比,称重后将公羔、母羔混合计算,用平均数标准差表示,利用SPSS 18.0统计软件进行单因素方差分析,P<0.05为差异显著。
从图1可以看出,开口料实例1与开口料实例2的饲喂效果(指每周的增重)在28日龄内无显著性差异。但之后开口料实例1的饲喂效果逐渐优于开口料实例2的效果,并且随着饲喂时间延长,效果差异越来越明显。而开口料实例1与开口料实例2的饲喂效果在14日龄时与开口料对照例无显著性差异,但是从21日龄后,开口料实例1与开口料实例2的饲喂效果显著优于开口料对照例,并且随着时间的延长,效果差异越发明显。因此,选用开口料实例1作为湖羊羔羊的开口料更为有利。
1.2.2不同开口料预防母羊乳房炎发病效果
乳房炎的判断标准为:母羊乳房发热、红肿,触摸变硬或者局部颜色发紫,或有乳房外伤。试验在两个万只湖羊养殖场进行,将发生乳房炎的母羊数除以试验用母羊总数,即为乳房炎发病率,结果见表1。
表1.母羊乳房炎发病率统计结果
从表1可知,开口料实例1组与实例2组的母羊乳房炎发病率显著低于对照例。
1.2.3不同开口料预防羔羊腹泻效果
羔羊腹泻判断标准:羔羊排出稀软或水样粪便,粪便酸臭,或尾根被粪便污染,排粪时表现腹痛,体温正常或升高等。试验在两个万只湖羊养殖场进行,将发生腹泻羔羊数除以试验用羔羊总数,即为羔羊腹泻发病率,结果见表2。
表2.羔羊腹泻发病率统计结果
从表2可知,开口料实例1组与实例2组羔羊腹泻发病率显著低于对照例。
试验二:不同开口料对羔羊瘤胃微生物区系的影响
2.1试验流程
2.1.1样品准备
从强制补饲开口料实例1、开口料实例2、开口料对照例的不同试验分组中分别随机挑选3只羊进行屠宰(40日龄),取瘤胃内容物,用四层无菌纱布过滤,收集滤液于离心管中,置于液氮中运回实验室。最后在0℃条件下发往北京天根生物科技有限公司进行宏基因组测序分析(提取细菌基因组DNA、文库构建、库检及上机测序)。
2.1.2测序数据质量控制
测序得到的原始序列(Raw reads)含有带接头的、低质量的reads,会增加后续分析复杂度。为了保证信息分析质量,需要对Raw reads进行精细过滤,得到clean reads,后续分析都基于clean reads进行。数据处理的步骤为:去除带接头(adapter)的reads对,当单端测序read中N(N表示无法确定碱基信息)的比例大于10%时,需要去除此对reads,当单端测序read中含有的低质量(低于5)碱基数超过该条read长度比例的50%时,去除此对reads。DNA-Seq的接头信息如下:
5’Adapter:5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
3’Adapter:5’-ATCGGAAGAGCACACGTCTGAACTCCAGTCACATCTCGTATGCCGTCTTCTGCTTG-3’
2.2结果与分析
2.2.1测序数据统计
表3.原始测序数据质量情况汇总
注:Sample列为样品名称(BU11表示强制补饲开口料实例1试验中的第1只羊,其余类推;强制补饲开口料实例2的试验羊的命名以BU2开头,强制补饲开口料对照例的试验羊的命名以BU3开头),Raw Reads列为原始数据reads数,Bases列为数据量大小,GC列为原始数据中GC占总碱基数的比例,Q20列为原始数据中碱基质量大于20的占比,Q30列为原始数据中碱基质量大于30的占比。
从表3中可以看出,测序获得的原始序列都在4千万以上,碱基数量在6千兆以上,按照Q30的控制控制标准,说明每个碱基的准确度非常高,完全符合试验的需要。
2.2.2Metagenome组装
经过预处理后得到clean reads,使用megaHit进行组装分析,组装时选取K-mer=21到141之间的多个参数分别进行组装,然后选择最优结果为最终的组装结果,混合组装生成的Scaffold,保留长度500bp以上的序列,并进行统计分析和后续基因预测。混合组装后的序列数量为504725,数据量大小为870526505,组装后序列N50长度有2827,序列N90长度有647,组装后序列长度最大值为528151,最小值为500,平均值为1724.754。从组装结果统计Scaffold长度的分布(图2)。
2.2.3基因预测及丰度分析
从混合组装的Scaffold(>=500bp)出发,采用prodigal进行ORF(Open ReadingFrame)预测,对混合组装的ORF预测结果,采用CD-HIT软件进行去冗余,以获得非冗余的初始gene catalogue,默认以identity 95%、coverage 90%进行聚类,并选取最长的序列为代表性序列;采用Bowtie2将各样品的clean reads比对至初始gene catalogue,计算得到基因在各试验羊样品中比对上的reads数目,从比对上的reads数目及基因长度出发,计算得到各基因在各样品中的丰度信息,并在此基础上进行基本信息统计。Gene catalogue基本信息统计发现,混合组装后的序列数量为763503,数据量大小为538127571,组装后序列长度最小值为90,平均值为704。从图3中可以看出,获得的基因长度都在100到2000个碱基之间,数量在几百到十万个之间。
2.2.4基因数目差异分析
为了考察指定样品(组)间的基因数目分布情况,分析不同样品(组)之间的基因共有、特有信息,绘制了韦恩图(Venn Graph),结果见图4(五组以上仅展示其中五组的venn图)。
2.2.5物种注释及丰度分析
①物种注释基本步骤
利用DIAMOND软件将去冗余后基因(指gene catalogue)与从NCBI的NR(Version:2018.01)数据库中抽提出的细菌(Bacteria)序列进行比对,对于每一条序列的比对结果,选取evalue<=最小evalue*10的比对结果进行后续分析,由于每一条序列可能会有多个比对结果,得到多个不同的物种分类信息,为了保证其生物意义,采取LCA算法(应用于MEGAN软件的系统分类),将出现第一个分支前的分类级别,作为该序列的物种注释信息,从LCA注释结果及基因丰度表出发,获得各个样品在各个分类层级(界、门、纲、目、科、属、种)上的丰度信息,对于某个物种在某个样品中的丰度等于注释为该物种的基因丰度的加和,从LCA注释结果及基因丰度表出发,获得各个样品在各个分类层级上的基因数目表,对于某个物种在某个样品中的基因数目,等于在注释为该物种的基因中丰度不为0的基因数目。
②注释物种数目及相对丰度聚类分析
从不同分类层级的相对丰度表出发,利用各个层级的所有样品中的丰度信息绘制柱状图及热图,并从物种层面进行聚类,便于结果展示和信息发现,从而找出样品中聚集较多的物种,结果见图5及图6。
从图5可以看出,图最下部分是目前没有了解清楚的瘤胃微生物,而图的最上部分是在分类学上未知的瘤胃微生物,除此之外,常见的瘤胃微生物,比如杆菌、球菌和弧菌等仅仅占瘤胃微生物的10%左右。
从图6可以看出,通过对瘤胃常见的35种细菌进行比较,只有长双歧杆菌(Bifidobacterium longum)、溶糊精琥珀酸弧菌(Lysodextrin Vibrio succinate)和白色瘤胃球菌(Ruminococcus albus)的数量在强制补饲开口料实例1的试验羊中最高,而瘤胃新月单胞菌和黄色瘤胃球菌(Ruminococcusflavefaciens)在强制补饲开口料实例2的试验羊中最高,其他种类的瘤胃细菌,例如产琥珀酸丝状杆菌(Fibrobacter succinogenes)、牛链球菌(Streptococcus bovis)、布氏密螺旋体(Treponema bryantii)、溶纤维丁酸弧菌(Butyrivibriofibrisolvens)、布氏瘤胃球菌(Ruminococcusbromii)、布氏普雷沃氏菌(Prevotellabryantii)、小杆菌(Dialistersuccinatiphilus)和月形单胞菌属(Selenomonasbovis)在强制补饲不同开口料的试验羊中差异不显著。
对湖羊而言,通过不同的开口料进行强制补饲,并利用宏基因组测序进行高通量分析后发现,强制补饲不同开口料(开口料实例1、开口料实例2及开口料对照例)的试验羊瘤胃微生物具有明显的差别:长双歧杆菌能够改善瘤胃功能,增强免疫力;瘤胃新月单胞菌与溶糊精琥珀酸弧菌能促进淀粉降解,白色瘤胃球菌与黄色瘤胃球菌具有消化纤维素的功能,因此,根据细菌的种类、数量及功能等角度,强制补饲开口料实例1能显著促进湖羊瘤胃微生物区系的形成,从而对饲草料在瘤胃这一消化阶段产生明显的差异(提高纤维素、半纤维素物质的消化率,促进部分维生素的合成效率,提高饲料消化吸收比例)。
试验三:不同开口料对羔羊免疫力的影响
用酶联免疫分析(ELISA)试剂盒(R&D公司)检测血清中IgG、IFNα、IFNγ、IL 1β、IL2、IL 5、IL 6、IL 12 8个免疫指标的水平。具体操作根据ELISA试剂盒说明书进行。同一检测指标取三个样品的平均值,采用单因素方差分析进行显著性检验,结果见表4。
表4.血清免疫指标测定
注:同一行不同字母上标表示差异显著(P<0.05)。
通过对8个代表性的免疫力相关指标进行检测,发现血清中抗体IgG水平在强制补饲开口料实例1、开口料实例2的两组试验羊中差异不显著,但是显著高于强制补饲开口料对照例的试验羊;IL2的水平在强制补饲开口料实例1、开口料实例2的两组试验羊中差异不显著,尽管低于强制补饲开口料对照例的试验羊,但是处于正常水平范围之内;其他各血清免疫指标在强制补饲不同开口料的试验羊差异不显著(表4)。
动物机体的抗体是由免疫系统,包括中枢免疫器官、免疫细胞生产的,是动物机体保持体内、外环境平衡的一种生理学反应,正常情况下,抗体水平越高,对抵抗各种疾病的能力越强。动物外周血液抗体量的增加和减少可反映机体的健康水平,这些抗体具有免疫调节和抗病毒、抗肿瘤的作用,能直接反映动物正常状态下的免疫状态。IL2是一种白介素,是免疫系统中的一类细胞生长因子,能调控免疫系统中白血球的细胞活性,参与抗体反应、造血和肿瘤监视。免疫球蛋白IgG在机体免疫防护中起着主要的作用,能够抵抗细菌、病毒、毒素等致病因素。因此,从表4所示结果可以确定,强制补饲开口料实例1、开口料实例2的试验羊在抗病力方面要优于强制补饲开口料对照例的试验羊。
经过以上试验,并从对湖羊羔羊的生长速度、羔羊瘤胃微生物区系和羔羊免疫力相关指标进行综合评价的结果来看,开口料实例1优于开口料实例2,而开口料实例2优于开口料对照例。
总之,本发明通过开发营养价值高、适口性好、容易消化利用的开口料,与采取的母子定时分栏饲养、羔羊诱导采食等措施配合,实现了羔羊早开食,使其瘤胃尽快发育完全、采食量和饲料消化利用率得到明显提高,并明显降低羔羊腹泻发病率,进而达到提高羔羊成活率和生长速度、降低羔羊的断奶应激反应,同时减少因羔羊营养不足而撕咬母羊乳房导致的母羊乳房炎的发生几率(明显降低母羊乳房炎发病率),从而明显提高湖羊的繁育效率和养殖效益。
Claims (8)
1.一种羔羊开口料,其特征在于:该开口料包括混合料以及混合料重量0.001%~0.1%的复合纳米抗菌肽,所述混合料按重量百分数计包括以下组分:25%~35%的膨化大豆、45%~60%的膨化玉米、8%~18%的膨化小麦、1%~5%的乳清粉、1%~5%的预混料、0.1%~1.0%的食盐、0.2%~0.8%的小苏打以及0.005%~0.05%的奶香调味剂;所述复合纳米抗菌肽包括天蚕素。
2.根据权利要求1所述一种羔羊开口料,其特征在于:所述复合纳米抗菌肽为混合料重量的0.005%~0.01%。
3.根据权利要求1所述一种羔羊开口料,其特征在于:所述混合料按重量百分数计包括以下组分:30%的膨化大豆、52%的膨化玉米、13%的膨化小麦、2%的乳清粉、2%的预混料、0.5%的食盐、0.49%的小苏打以及0.01%的奶香调味剂,复合纳米抗菌肽为混合料重量的0.005%~0.01%。
4.根据权利要求1所述一种羔羊开口料,其特征在于:所述复合纳米抗菌肽具体包括天蚕素A与D的复合物。
5.根据权利要求1所述一种羔羊开口料,其特征在于:所述预混料包括维生素和微量元素。
6.一种如权利要求1所述的羔羊开口料的制备方法,其特征在于:包括以下步骤:
将所述膨化大豆、膨化玉米、膨化小麦、乳清粉、预混料、食盐、小苏打、奶香调味剂以及复合纳米抗菌肽与水拌合后升温至70℃~80℃进行制粒,然后进行干燥。
7.一种羔羊诱导采食方法,其特征在于:包括以下步骤:
1)羔羊自5~10日龄开始,间断性将羔羊与母羊分离,在分离期间供给羔羊清洁饮水和如权利要求1所述的羔羊开口料;
2)羔羊自30~40日龄开始断奶,即将羔羊与母羊分栏饲养,此时羔羊开始饲喂育成饲料或育肥饲料。
8.根据权利要求7所述一种羔羊诱导采食方法,其特征在于:羔羊自10~20日龄开始,逐渐增大采食所述开口料的时间,同时诱导羔羊采食干草。
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