CN114868842B - 一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂及其制备方法和应用 - Google Patents
一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂及其制备方法和应用 Download PDFInfo
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Abstract
一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂及其制备方法和应用。本发明属于饲料添加剂领域。本发明的目的是为了解决仔猪对微量元素需求大但吸收率低以及添加过高的铁易引起腹泻的技术问题。本发明的饲料添加剂由二氧化硅、乳酸、延胡索酸、氨基乙酰丙酸、柠檬酸、维生素B族、磷酸酯化维生素C、鱼粉、甘氨酸螯合铁、L‑精氨酸‑α‑酮戊二酸盐、低聚糖、丙酮酸肌酸和米糠粕制备而成。本发明的添加剂主要通过干涉机体防御、平衡和免疫系统三大生理功能,来提高机体免疫机能,本发明的添加剂能够明显改善仔猪日增重,降低料肉比,有效预防仔猪腹泻现象发生,提高经济价值。
Description
技术领域
本发明属于饲料添加剂领域,具体涉及一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂及其制备方法和应用。
背景技术
初生仔猪消化器官虽已形成,但其体积和重量比较小,很不发达,在早期仔猪消化道的发育尚未完善的情况下,如何促进早期仔猪最佳的消化吸收的能力,提高仔猪免疫力,保证仔猪营养需要,平稳的度过断奶应激,挖掘最大生长潜能,是当前首要难题。
仔猪由于摄食不足、营养缺乏,而在此阶段其体重和血液红细胞体积迅速增加,因此,如果不能及时、有效地补充体内所需的铁,就会导致生长缓慢、饲料效率降低、免疫力降低,严重时甚至会引起仔猪死亡。在当前饲料中为满足仔猪对铁的需求,日粮中往往还需添加额外铁源。但仔猪对微量元素的需要量高但吸收率低,添加过高的铁又增加了仔猪肠道有害菌的繁殖,引起腹泻率的增加,这对外加铁源和合理性的应用提出了更高的要求。
发明内容
本发明的目的是为了解决仔猪对微量元素需求大但吸收率低以及添加过高的铁易引起腹泻的技术问题,而提供了一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂及其制备方法和应用。
本发明的一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂按质量份数由二氧化硅19.0~19.3份、乳酸27.0~27.5份、延胡索酸1.50~2.50份、氨基乙酰丙酸0.80~1.20份、柠檬酸0.80~1.20份、维生素B族0.04~0.05份、磷酸酯化维生素C 0.40~0.60份、鱼粉4.50~5.50份、甘氨酸螯合铁1.50~2.50份、L-精氨酸-α-酮戊二酸盐6.50~7.50份、低聚糖9.0~11.0份、丙酮酸肌酸90~110份和米糠粕800~850份制备而成。
进一步限定,所述维生素B族为维生素B6、维生素B1、维生素B12中的一种或几种按任意比的混合物。
进一步限定,所述低聚糖为低聚壳聚糖、果寡糖、甘露寡糖、低聚木糖中的一种或几种按任意比的混合物。
本发明的一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂的制备方法按以下步骤进行:
步骤1:将二氧化硅与乳酸混合,然后加入粉碎过筛后的延胡索酸、氨基乙酰丙酸和柠檬酸继续混合,再加入聚维酮溶液,依次经过挤出、滚圆、造粒、干燥、过筛,得到微丸,将微丸置于沸腾流化床中进行包衣,得到包被颗粒;
步骤2:将米糠粕粉碎后,先将一部分粉碎后的米糠粕投入到混合机中,然后加入维生素B族、磷酸酯化维生素C和初筛后的鱼粉进行混合,混合均匀后再加入另一部分米糠粕、甘氨酸螯合铁、L-精氨酸α-酮戊二酸盐和低聚糖继续混合,混合均匀后加入剩余米糠粕、丙酮酸肌酸和步骤1得到的包被颗粒,继续混合至均匀,得到预防仔猪贫血促进免疫机能的包被营养饲料添加剂。
进一步限定,步骤1中所述微丸尺寸为35~45目。
进一步限定,步骤1中所述二氧化硅、乳酸、延胡索酸、氨基乙酰丙酸、柠檬酸的质量之和与聚维酮溶液的体积的比为7mg:3mL。
进一步限定,步骤1中所述包衣是采用聚丙烯酸树脂的乙醇溶液进行包衣。
进一步限定,所述聚丙烯酸树脂为聚丙烯酸树脂Ⅱ、聚丙烯酸树脂Ⅳ中的一种或两种。
进一步限定,所述聚丙烯酸树脂的乙醇溶液中聚丙烯酸树脂的质量浓度2%~7%。
进一步限定,步骤2中所述一部分粉碎后的米糠粕是指米糠粕总质量的30%~50%。
进一步限定,步骤2中所述另一部分米糠粕是指米糠粕总质量的30%~50%。
进一步限定,步骤2中所述初筛是指过40目筛。
进一步限定,步骤2中所述混合均采用三维混合机进行。
进一步限定,步骤2中加入维生素B族、磷酸酯化维生素C和初筛后的鱼粉混合15min~20min,加入另一部分米糠粕、甘氨酸螯合铁、L-精氨酸α-酮戊二酸盐和低聚糖继续混合15min~20min,加入剩余米糠粕、丙酮酸肌酸和步骤1得到的包被颗粒,继续混合15min~20min。
本发明的预防仔猪贫血促进免疫机能的包被营养饲料添加剂作为饲料添加剂应用,所述包被营养饲料添加剂在饲料中的添加量为0.8‰~1.2‰。
本发明相比现有技术的优点如下:
1)本发明的添加剂主要通过干涉机体防御、平衡和免疫系统三大生理功能,来提高机体免疫机能,首先,胸腺对于仔猪前期阶段是最重要的免疫器官,氨基乙酰丙酸和精氨酸均可以增加胸腺的重量,促进胸腺细胞和T细胞的生长而起到优异的免疫功能改善作用。
2)肌酸能够显著提高线粒体功能的正常性,丙酮酸降低了肝细胞自由基的产生,减轻了肝细胞的氧化损伤,但是单独的丙酮酸比较活跃,不容易单独添加,丙酮酸肌酸可以兼有丙酮酸和肌酸的双重生理作用,并且非常容易添加和储存。
3)a-酮戊二酸也具有一定的抗氧化作用,a-酮戊二酸在清除氧自由基方面具有一定作用,有利于增强仔猪的抗氧化能力。a-酮戊二酸可以缓解其肝脏丙二醛含量升高和超氧化物歧化酶活性降低,降低仔猪肝脏抗氧化损伤的作用,其在仔猪日粮中的添加可以保护肝脏的正常机能。
4)本发明通过柠檬酸和延胡索酸促进肠道有益菌的繁殖,抑制有害菌的生长,提高其空肠和盲肠中乳酸杆菌的数量,降低其中大肠杆菌的数量,减少仔猪下痢的发生,能够有效的预防仔猪腹泻,提高仔猪成活率。
5)本发明的添加剂中氨基乙酰丙酸、维生素C和甘氨酸螯合铁联合使用,在动物体内通过活化血球细胞和肌红蛋白,提高生物体内的氧气输送、营养素的输送及新陈代谢效率,进而提高饲料中铁元素和其他营养元素的利用率,增加吸收率和代谢速度,具有显著的生理效果。添加剂中延胡索酸同样也可以促进矿物元素Ca2+和Zn2+的吸收和蓄积。延胡索酸与饲料中高水平硫酸铜之间具有协同作用,两者共同使用可以使饲料利用率得到提高。
6)本发明的添加剂通过将酸性饲粮、维生素C等还原性物质复配使用,以促进铁的吸收,在酸度减弱的小肠中仍保持可溶状态,有助于铁穿过小肠进入血浆,此外,本申请添加剂中的甘氨酸鳌合铁其水溶液总铁保留率保持在98%左右,油脂催化氧化作用极小。
7)包被酸化剂可以起到分段释放,克服了普通酸化剂在胃中过多吸收、无法到达肠道的缺点,包被后产品质量更为稳定、无腐蚀性、不吸湿;不破坏饲料中的其它成份,易于流动、混合均匀。
具体实施方式
实施例1:本实施例的一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂按质量份数由二氧化硅19.12份、乳酸27.36份、延胡索酸2.00份、氨基乙酰丙酸1.00份、柠檬酸1.00份、维生素B60.02份、维生素B10.025份、维生素B120.0002份、磷酸酯化维生素C0.50份、鱼粉5.00份、甘氨酸螯合铁2.00份、L-精氨酸-α-酮戊二酸盐7.00份、低聚壳聚糖2.00份、果寡糖2.00份、甘露寡糖1.00份、低聚木糖5.00份、丙酮酸肌酸100份和米糠粕825份制备而成。
制备实施例1的预防仔猪贫血促进免疫机能的包被营养饲料添加剂的方法按以下步骤进行:
步骤1:将二氧化硅与乳酸混合5min,然后加入粉碎过筛后的延胡索酸(40目)、氨基乙酰丙酸(40目)和柠檬酸(40目)继续混合,再加入2.15mL聚维酮k30溶液,依次经过挤出、滚圆、造粒、干燥、过40目筛,得到微丸,将微丸置于沸腾流化床中进行包衣,得到包被颗粒;所述包衣是采用浓度为5%的聚丙烯酸树脂Ⅱ的乙醇溶液进行包衣,所述乙醇质量浓度为95%;
步骤2:将米糠粕粉碎后,先将40wt%的粉碎后的米糠粕投入到三维混合机中,然后加入维生素B族、磷酸酯化维生素C和初筛后的鱼粉混合20min,混合均匀后再加入30wt%的米糠粕、甘氨酸螯合铁、L-精氨酸α-酮戊二酸盐和低聚糖继续混合20min,混合均匀后加入剩余米糠粕、丙酮酸肌酸和步骤1得到的包被颗粒,继续混合20min至均匀,得到预防仔猪贫血促进免疫机能的包被营养饲料添加剂。
实施例2:本实施例的一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂按质量份数由二氧化硅19.12份、乳酸27.36份、延胡索酸2.00份、氨基乙酰丙酸1.00份、柠檬酸1.00份、维生素B60.02份、维生素B10.025份、维生素B120.0002份、磷酸酯化维生素C0.50份、鱼粉5.00份、甘氨酸螯合铁2.00份、L-精氨酸-α-酮戊二酸盐7.00份、低聚壳聚糖2.00份、果寡糖2.00份、甘露寡糖1.00份、低聚木糖5.00份、丙酮酸肌酸100份和米糠粕825份制备而成。
制备实施例2的预防仔猪贫血促进免疫机能的包被营养饲料添加剂的方法按以下步骤进行:
步骤1:将二氧化硅与乳酸混合5min,然后加入粉碎过筛后的延胡索酸(40目)、氨基乙酰丙酸(40目)和柠檬酸(40目)继续混合,再加入2.15mL聚维酮k30溶液,依次经过挤出、滚圆、造粒、干燥、过40目筛,得到微丸,将微丸置于沸腾流化床中进行包衣,得到包被颗粒;所述包衣是采用浓度为5%的聚丙烯酸树脂Ⅳ的乙醇溶液进行包衣,所述乙醇质量浓度为95%;
步骤2:将米糠粕粉碎后,先将40wt%的粉碎后的米糠粕投入到三维混合机中,然后加入维生素B族、磷酸酯化维生素C和初筛后的鱼粉混合20min,混合均匀后再加入30wt%的米糠粕、甘氨酸螯合铁、L-精氨酸α-酮戊二酸盐和低聚糖继续混合20min,混合均匀后加入剩余米糠粕、丙酮酸肌酸和步骤1得到的包被颗粒,继续混合20min至均匀,得到预防仔猪贫血促进免疫机能的包被营养饲料添加剂。
实施例3:本实施例的一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂按质量份数由二氧化硅19.12份、乳酸27.36份、延胡索酸2.00份、氨基乙酰丙酸1.00份、柠檬酸1.00份、维生素B60.02份、维生素B10.025份、维生素B120.0002份、磷酸酯化维生素C0.50份、鱼粉5.00份、甘氨酸螯合铁2.00份、L-精氨酸-α-酮戊二酸盐7.00份、低聚壳聚糖2.00份、果寡糖2.00份、甘露寡糖1.00份、低聚木糖5.00份、丙酮酸肌酸100份和米糠粕825份制备而成。
制备实施例3的预防仔猪贫血促进免疫机能的包被营养饲料添加剂的方法按以下步骤进行:
步骤1:将二氧化硅与乳酸混合5min,然后加入粉碎过筛后的延胡索酸(40目)、氨基乙酰丙酸(40目)和柠檬酸(40目)继续混合,再加入2.15mL聚维酮k30溶液,依次经过挤出、滚圆、造粒、干燥、过40目筛,得到微丸,将微丸置于沸腾流化床中进行包衣,得到包被颗粒;所述包衣的具体过程为:其中40%的微丸采用浓度为2%的聚丙烯酸树脂Ⅱ的乙醇溶液进行包衣,其中60%的微丸采用浓度为5%的聚丙烯酸树脂Ⅳ的乙醇溶液进行包衣,所述乙醇质量浓度为95%;
步骤2:将米糠粕粉碎后,先将40wt%的粉碎后的米糠粕投入到三维混合机中,然后加入维生素B族、磷酸酯化维生素C和初筛后的鱼粉混合20min,混合均匀后再加入30wt%的米糠粕、甘氨酸螯合铁、L-精氨酸α-酮戊二酸盐和低聚糖继续混合20min,混合均匀后加入剩余米糠粕、丙酮酸肌酸和步骤1得到的包被颗粒,继续混合20min至均匀,得到预防仔猪贫血促进免疫机能的包被营养饲料添加剂。
(一)验证试验:本发明的包被营养添加剂对不同生长阶段仔猪的生长性能、防腹泻、增强免疫力及预防仔猪贫血的影响试验
试验一:针对21-25日龄的断奶仔猪的试验过程如下:
1、试验所用饲料组成及营养水平
饲料中的营养物质含量及能量的测定参照杨胜主编的《饲料分析及饲料质量检测技术》进行。饲料配方按照1998年NRC标准及中华人民共和国农业行业标准猪饲养标准(NY/T65-2004)进行配制,试验日粮组成及营养水平见表1。
表1:饲料组成及营养水平
注:表1中所述预混料为每kg配合饲料提供Cu≥5g,Fe≥130mg,Zn≥5g,Mn≥1.8g,Se≥15g,I≥20mg,钴≥25mg,钙≥160-270g,铁≥4.6g,磷≥38g,水分≤10%,维生素A(KIU)≥253,维生素D3(KIU)≥47,维生素E(IU)≥662,维生素K≥53mg,维生素B1≥58mg,维生素B2≥75mg,维生素B6≥67mg,维生素B12≥0.8mg,叶酸≥27mg,生物素≥1.1mg,烟酸≥975mg,泛酸钙≥750mg,氯化胆碱≥5g。
2、试验动物与试验分组
试验选择体格健壮、发育良好的21-25日龄断奶的二元(长×大)仔猪72头,随机分为4组,组间体重差异不显著(P>0.05),每组设置3个重复,每个重复6头,分别为试验Ⅰ组喂食添加了实施例1的营养添加剂的饲料(如表1所示),试验Ⅱ组喂食添加了实施例2的营养添加剂的饲料(如表1所示),试验Ⅲ组喂食添加了实施例3的营养添加剂的饲料(如表1所示),各试验组每吨配合饲料中添加上述各实施例营养添加剂1kg,对照组仅喂食表1饲料,试验场所在大北农集团唐山玉田种猪场进行。
3、试验仔猪饲养管理
试验仔猪全部饲养于同一猪舍,由专人管理,断奶时间为21天,仔猪进舍前3d对猪舍进行严格清洗、消毒,预饲期饲喂各组日粮7天,试验期10天,自由采食、自由饮水,仔猪进行正常免疫和驱虫,对患病仔猪给予适当治疗,试验仔猪置于高床单拦饲养,以乳头式自动饮水器提供充足、清洁饮水,舍温控制在22~24℃,自由采食,每日清粪2次,保持舍内清洁,随时观察仔猪生长发育、采食及腹泻等情况,做好记录。
4、试验数据统计分析
试验数据统计利用SAS9.0软件包中的平衡实验设计ANOVA过程进行方差分析,采用Duncan法进行均值的多重比较。经济效益数据的统计分析利用Office应用软件中的Microsoft Excel进行。
试验二:针对40日龄保育阶段仔猪的试验过程如下:
1、试验所用饲料组成及营养水平
饲料中的营养物质含量及能量的测定参照杨胜主编的《饲料分析及饲料质量检测技术》进行。饲料配方按照1998年NRC标准及中华人民共和国农业行业标准猪饲养标准(NY/T65-2004)进行配制,试验日粮组成及营养水平见表2。
表2:饲料组成及营养水平
注:表2中所述预混料为每kg配合饲料提供Cu≥5g,Fe≥130mg,Zn≥5g,Mn≥1.8g,Se≥15g,I≥20mg,钴≥25mg,钙≥160-270g,铁≥4.6g,磷≥38g,水分≤10%,维生素A(KIU)≥253,维生素D3(KIU)≥47,维生素E(IU)≥662,维生素K≥53mg,维生素B1≥58mg,维生素B2≥75mg,维生素B6≥67mg,维生素B12≥0.8mg,叶酸≥27mg,生物素≥1.1mg,烟酸≥975mg,泛酸钙≥750mg,氯化胆碱≥5g。
2、试验动物与试验分组
试验选择体格健壮、发育良好的40日龄保育阶段二元(长×大)仔猪72头,随机分为4组,组间体重差异不显著(P>0.05),每组设置3个重复,每个重复6头,分别为试验Ⅳ组喂食添加了实施例1的营养添加剂的饲料(如表2所示),试验Ⅴ组喂食添加了实施例2的营养添加剂的饲料(如表2所示),试验Ⅵ组喂食添加了实施例3的营养添加剂的饲料(如表2所示),各试验组每吨配合饲料中添加上述各实施例营养添加剂1kg,对照组仅喂食表2饲料,试验场所在大北农集团唐山玉田种猪场进行。
3、试验仔猪饲养管理
试验仔猪全部饲养于同一猪舍,由专人管理,断奶时间为21天,仔猪进舍前3d对猪舍进行严格清洗、消毒,预饲期饲喂各组日粮7天,试验期10天,自由采食、自由饮水,仔猪进行正常免疫和驱虫,对患病仔猪给予适当治疗,试验仔猪置于高床单拦饲养,以乳头式自动饮水器提供充足、清洁饮水,舍温控制在20~22℃,自由采食,每日清粪2次,保持舍内清洁,随时观察仔猪生长发育、采食及腹泻等情况,做好记录。
4、试验数据统计分析
试验数据统计利用SAS9.0软件包中的平衡实验设计ANOVA过程进行方差分析,采用Duncan法进行均值的多重比较。经济效益数据的统计分析利用Office应用软件中的Microsoft Excel进行。
试验三:针对65-70日龄仔猪的试验过程如下:
1、试验所用饲料组成及营养水平
饲料中的营养物质含量及能量的测定参照杨胜主编的《饲料分析及饲料质量检测技术》进行。饲料配方按照1998年NRC标准及中华人民共和国农业行业标准猪饲养标准(NY/T65-2004)进行配制,试验日粮组成及营养水平见表3。
表3:饲料组成及营养水平
注:表3中所述预混料为每kg配合饲料提供Cu≥3g,Fe≥100mg,Zn≥3g,Mn≥1.8g,Se≥15g,I≥20mg,钴≥25mg,钙≥120-270g,铁≥4.6g,磷≥30g,水分≤10%,维生素A(KIU)≥253,维生素D3(KIU)≥47,维生素E(IU)≥662,维生素K≥53mg,维生素B1≥58mg,维生素B2≥75mg,维生素B6≥67mg,维生素B12≥0.8mg,叶酸≥27mg,生物素≥1.1mg,烟酸≥975mg,泛酸钙≥750mg,氯化胆碱≥5g。
2、试验动物与试验分组
试验选择体格健壮、发育良好的65-70日龄、体重在28-30kg的二元(长×大)仔猪72头,随机分为4组,组间体重差异不显著(P>0.05),每组设置3个重复,每个重复6头,分别为试验Ⅶ组喂食添加了实施例1的营养添加剂的饲料(如表3所示),试验Ⅷ组喂食添加了实施例2的营养添加剂的饲料(如表3所示),试验Ⅸ组喂食添加了实施例3的营养添加剂的饲料(如表3所示),各试验组每吨配合饲料中添加上述各实施例营养添加剂1kg,对照组仅喂食表3饲料,试验场所在大北农集团唐山玉田种猪场进行。
3、试验仔猪饲养管理
试验仔猪全部饲养于同一猪舍,由专人管理,断奶时间为21天,仔猪进舍前3d对猪舍进行严格清洗、消毒,预饲期饲喂各组日粮7天,试验期10天,自由采食、自由饮水,仔猪进行正常免疫和驱虫,对患病仔猪给予适当治疗,试验仔猪置于高床单拦饲养,以乳头式自动饮水器提供充足、清洁饮水,舍温控制在16~20℃,自由采食,每日清粪2次,保持舍内清洁,随时观察仔猪生长发育、采食及腹泻等情况,做好记录。
4、试验数据统计分析
试验数据统计利用SAS9.0软件包中的平衡实验设计ANOVA过程进行方差分析,采用Duncan法进行均值的多重比较。经济效益数据的统计分析利用Office应用软件中的Microsoft Excel进行。
(二)试验结果与分析
1.添加剂对仔猪ADFI、ADG和F:G(料肉比)及腹泻率的影响
在正式试验期的第一天和最后一天早晨进行空腹称重,并记录耗料量和腹泻次数,计算日采食量(ADFI)、日增重(ADG)、料重比(F:G)、腹泻率及仔猪增重的经济效益,计算方法如下:
试验一结果如表4所示,由表4可知:本发明的添加剂可在一定程度上改善仔猪的生长性能,减少仔猪腹泻,在ADFI方面,试验Ⅰ、Ⅱ组显著高于对照组和试验Ⅲ组(P<0.05);其余各组之间均无显著差异(P>0.05)。在ADG方面,添加剂试验组均高于对照组,且试验Ⅰ组最高。在F:G方面,试验Ⅰ组表现最好,各试验组均低于对照组;腹泻率以试验Ⅱ组最佳,对照组效果极差,其他各试验组效果均表现较好。
表4添加剂对仔猪生长性能及腹泻率的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05);
试验二结果如表5所示,由表5可知:本试验所用添加剂可在一定程度上改善保育仔猪的生长性能,减少保育仔猪腹泻。在ADFI方面,试验Ⅴ组显著高于对照组、Ⅳ、Ⅵ组(P<0.05);试验Ⅳ、Ⅵ组显著高于对照组,但试验Ⅳ、Ⅵ组间差异不显著(P>0.05)。在ADG方面,添加剂试验组均高于对照组,且试验Ⅳ组最高。在F:G方面,试验Ⅳ组表现最好;腹泻率以试验Ⅵ组表现最佳,对照组效果极差,其他各试验组效果均表现较好。
表5添加剂对保育仔猪生长性能及腹泻率的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05);
试验三结果如表6所示,由表6可知:本试验所用添加剂可在一定程度上改善仔猪的生长性能,减少仔猪腹泻。在ADFI方面,试验Ⅷ组显著高于对照组、试验Ⅶ、Ⅸ组(P<0.05);试验Ⅶ、Ⅸ组显著高于对照组,但试验Ⅶ、Ⅸ组间差异不显著(P>0.05)。在ADG方面,添加剂试验组均高于对照组,且试验Ⅶ组最高。在F:G方面,试验Ⅶ组表现最好;腹泻率以试验Ⅶ组表现最佳,对照组效果较差,其他各试验组效果均表现较好。
表6添加剂对仔猪生长性能及腹泻率的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05);
2、添加剂对仔猪免疫力的影响
2.1、T-淋巴细胞转化率的测定
(1)血样采集与制备
在正式试验期最后一天,所有试验用仔猪早晨空腹前腔静脉采血2ml,置于加有ACD抗凝的试管内,4℃条件下保存备用。
(2)测定方法
淋巴细胞转化试验采用形态学检查法,应用非特异性有丝分裂原植物血凝素(PHA)作为刺激物,取抗凝血0.1mL直接加入装有1mLRPMI 1640培养液(RPMI 1640培养基、胎牛血清20%、青霉素100U/mL、PHA 50μg/mL)的淋巴细胞转化管中,用橡皮塞塞紧管口,摇匀,置37℃培养72h,每天振摇2次,终止培养后,离心(1000r/min,5~10min),去上清,使细胞分散,滴于载玻片上,用滴管前端将细胞推开,使其分布均匀,干燥后瑞特氏染色,油镜观察计数200个淋巴细胞,计算转化百分率,按如下公式计算:
试验一结果如表7所示,由表7可知:试验所用添加剂可提高LTR,各试验组比对对照组分别提高4.23%、4.79%、1.38%,且试验Ⅰ、Ⅱ组显著高于对照组、Ⅲ组(P<0.05)。
表7添加剂对仔猪血液淋巴细胞转化率的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05)。
试验二结果如表8所示,由表8可知:试验所用添加剂可提高保育仔猪LTR,且试验Ⅳ、Ⅴ组显著高于对照组和试验Ⅵ组(P<0.05)。
表8添加剂对保育仔猪血液淋巴细胞转化率的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05)。
试验三结果如表9所示,由表9可知:试验各组与对照组之间均无明显差异,但可以看出,以试验Ⅶ组有明显增加趋势。
表9添加剂对仔猪血液淋巴细胞转化率的影响
2.2、血清IgG、IgM、IgA和补体C3、C4的测定
(1)血样采集与制备
在正式试验期最后一天,所有试验用仔猪早晨空腹前腔静脉采血5ml,在室温下静置半小时,待血清析出后于2000-2500r/min离心10min,制得血清样品置于-20℃条件下保存备用。
(2)测定指标及方法
测定指标主要包括血清中IgG、IgM、IgA及补体C3、C4,均通过免疫透射比浊法测定,测试用试剂盒购自哈尔滨伊利康生物科技有限公司,免疫球蛋白IgG、IGM、IGA、C3、C4测定试剂盒,具体试验参数、操作步骤参照试剂盒说明书。
(3)试验数据统计分析
试验数据统计利用SAS9.0软件包中的平衡实验设计ANOVA过程进行方差分析,采用Duncan法进行均值的多重比较。
试验一结果见表10和表11,由表10可知:试验所用添加剂可在一定范围内提高仔猪血清免疫球蛋白含量,在IgG方面,试验组显著高于对照组(P<0.05),以试验Ⅱ组效果最好;在IgM方面,各试验组显著高于对照组(P<0.05),以试验Ⅱ组效果最好,试验Ⅰ组和试验Ⅱ组显著高于试验Ⅲ组(P<0.05);IgA各试验组显著高于对照组(P<0.05),以试验Ⅰ组效果最好,试验Ⅰ组和试验Ⅱ组显著高于试验Ⅲ组(P<0.05);由表11可知:试验所用添加剂可提高血清补体含量,在补体C3方面,试验Ⅰ、Ⅱ组表现较好。在补体C4方面,试验Ⅰ、Ⅱ组表现较好(P<0.05);对照组、试验Ⅲ组间均无显著差异(P>0.05)。
表10添加剂对仔猪血清免疫蛋白的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05);
表11添加剂对仔猪血清补体含量的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05);
试验二结果见表12和表13,由表12可知:试验所用添加剂可在一定范围内提高仔猪血清免疫球蛋白含量,在IgG方面,试验组显著高于对照组(P<0.05),以试验Ⅵ组效果最好;在IgM方面,各试验组高于对照组(P<0.05),以试验Ⅳ组效果最好,以试验Ⅳ组效果最好;血清中IgG、IgM、IgA含量均以试验Ⅳ组最高,效果最佳。由表13可知:试验所用添加剂可提高血清补体含量,在补体C3方面,试验Ⅳ、Ⅴ组表现较好。在补体C4方面,试验Ⅳ、Ⅴ组表现较好(P<0.05);对照组和试验Ⅵ组间均无显著差异(P>0.05)。
表12添加剂对保育仔猪血清免疫蛋白的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05);
表13添加剂对保育仔猪血清补体含量的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05);
试验三结果见表14和表15,由表14可知:试验所用添加剂可在一定范围内提高仔猪血清免疫球蛋白含量,在IgG方面,试验Ⅷ组显著高于对照组(P<0.05),其余各组间差异不显著,以试验Ⅷ组效果最好;在IgM方面,各组间差异不显著,但以试验Ⅶ组效果最好;由表15可知:试验所用添加剂可提高血清补体含量,在补体C3方面,试验Ⅶ组表现较好,在补体C4方面,试验Ⅶ、Ⅷ组表现较好;但各组间均无显著差异(P>0.05)。
表14添加剂对仔猪血清免疫蛋白的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05);
表15添加剂对仔猪血清补体含量的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05);
2.3、添加剂对仔猪预防贫血效果的影响
(1)试验材料
双氧水,0.1mol/LHN03,离心机,10mL离心管,血球分析仪,冰箱,高压灭菌锅冷冻机,高速粉碎机,原子吸收分析仪,微波消化仪,微波清洗器,乙炔气,空气压缩机,电子天平,鼓风干燥箱,粉碎机,消化管,容量瓶,移液管,陶瓷刀。
(2)样品采集和处理
试验结束后每栏挑选2头健康仔猪静脉采抗凝血(肝素钠)10mL,其余血液3000转/分离心15分钟后分装血浆到1ml Eddendorf管中-80℃冰箱保存。组织采样及处理:试验10天后每组选4头生长状况相近的仔猪屠宰,采取肝脏、脾脏、肾脏放入塑料袋中-20℃冰箱中冻存,剥去组织表皮,用陶瓷刀切取组织的皮质部放于培养皿中,冻干24小时后,用粉碎机粉碎后过筛,装入塑料袋中在-20℃冰箱中冻存。
粪便采取与处理:采取每组仔猪饲料100g左右,放入封口袋封口保存,回到试验后,粉碎过筛,装于封口袋中-20℃冰箱中冻存;试验的第10天上午7:30-8:00,以栏为单位,收集新鲜粪样,取100g左右放入封口袋封口保存,回到试验室后,鼓风干燥箱中65℃烘干,粉碎过筛,装于塑料袋中-20℃冰箱中冻存。抗凝剂的配制:用0.9%生理盐水配制1000IU/ml的肝素钠抗凝剂,采血前一天,用量取10u1注入离心管,100℃烘干。
(3)仔猪组织、粪便中铁含量的测定
标准工作液的配制:吸取5ml浓度1000ug/mlFe标准液于50mL容量瓶中,用0.1mol/LHNO3定容得浓度为100mg/L的标准中间液,分别用吸取0mL、0.25mL、0.5mL、1.0mL、1.5mL、2mL、2.5mL、3mL标准中间液于50mL容量瓶中,用0.lmol/mL HNO3定容,配制成0mL、0.5mL、1.0mL、2.0mL、3.0mL、4.0mL、5.0mL、6.0mg/mL所需要的标准溶液测定组织铁含量。量取1mL、2mL、3mL、4mL、5mL标准中间液于50mL容量瓶中,用0.lmol/mL HNO3定容,配制成2mg/mL、4mg/mL、6mg/mL、8mg/mL、10mg/mL所需要的标准溶液测定粪中铁含量。
样品称重:分析天平精确称取饲料、肝、肾0.5g左右,脾0.3g左右,粪0.2g左右。
样品消化:称取样品放入消化管中,分别加4mL硝酸(工业分析纯)、1mL双氧水、5mL水,放入微波消化炉中消化,每次消化样品时都要做相应的空白对照。
溶液定容:消化好的样品,转移到50mL容量瓶中,用蒸馏水定容后过滤转移到50mL试管中,-4℃保存。
样品铁元素的测定,将铁标准溶液导入原子化器,绘制标准曲线,验证标准曲线的相关系数,相关系数大99%的条件下,按照提示依次导入空白溶液和样品所有试验器皿,使用前用3:1硝酸泡12小时,烘箱65℃烘干备用。
(4)仔猪血液生化指标的测定
细胞色素P450氧化还原酶(CPR)酶、细胞色素氧化酶(CCO),铁蛋白(SF)、肌红蛋白(MB)采用生物素双抗体夹心酶联免疫吸附法(ELISA)测定,血浆总铁结合力(TIBC)试剂盒购自上海鑫乐公司。
(5)数据统计分析
试验数据采用Excel进行初步处理,用PASS 18.0软件进行统计。
试验一结果如表16所示,由表16可知:试验组仔猪粪便中铁含量与对照组差别不显著,但粪便中铁含量与对照组相比有下降趋势(P>0.05);与对照组相比试验各组仔猪肝脏中铁含量显著提高(P<0.05),试验Ⅰ、Ⅱ组与试验Ⅲ组间差异显著(P<0.05);仔猪肾脏含铁量各试验组间差异不显著(P>0.05),但有上升趋势。血红蛋白试验Ⅰ、Ⅱ、Ⅲ组与对照组有明显增加,差异显著(P<0.05)。
表16添加剂对仔猪预防贫血病的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05);
试验二结果如表17所示,由表17可知:试验组仔猪粪便中铁含量与对照组差别显著(P<0.05),试验组粪便中铁含量与对照组相比有下降趋势;对照组相比试验各组仔猪肝脏中铁含量显著提高(P<0.05),试验Ⅳ、Ⅴ组与试验Ⅵ组间差异显著(P<0.05);保育阶段仔猪肾脏含铁量各试验组间差异不显著(P>0.05),但有上升趋势。血红蛋白试验Ⅳ、Ⅴ、Ⅵ组与对照组有明显增加,差异显著(P<0.05)。
表17添加剂对保育仔猪预防贫血病的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05);
试验三结果如表18所示,由表18可知:试验组仔猪粪便中铁含量与对照组有明显的降低趋势,差异显著(P<0.05),以试验Ⅶ组效果最佳;仔猪肝脏中铁含量,试验Ⅶ效果最佳,差异显著(P<0.05),试验Ⅷ、Ⅸ组与对照组有提高趋势但不显著;仔猪肾脏含铁量各试验组间差异不显著(P>0.05),但有上升趋势。血红蛋白试验Ⅶ、Ⅷ、Ⅸ组与对照组有增加趋势,但差异不显著(P>0.05)。
表18添加剂对仔猪预防贫血病的影响
注:同行中小写字母有不相同者表示差异显著(P<0.05);
2.4、添加剂对仔猪经济效益的影响
(1)测定方法
仔猪增重收入=平均增重×仔猪活重单价;
饲料成本=饲料单价×平均耗料量;
仔猪平均利润=仔猪增重收入-饲料成本-添加剂成本。
(2)添加剂对仔猪经济效益的影响
试验一结果如表19所示,由表19可知:在相同饲养条件和饲料价格无差异的情况下,添加剂可促进仔猪生长,提高经济效益。试验所用添加剂Ⅱ、Ⅲ、Ⅳ组中以试验Ⅱ组效果最佳,断奶后10天平均每头多增收21.48元,净效益增收提高13.9%,对于1个500头基础母猪的猪场,仅此仔猪阶段使用该添加剂,年效益增收就可多达到20多万元。
表19不同处理对仔猪的影响
试验二结果如表20所示,由表20可知:在相同饲养条件和饲料价格无差异的情况下,添加剂可促进保育仔猪生长,提高经济效益。试验所用添加剂Ⅳ、Ⅴ、Ⅵ组中以试验Ⅳ组经济效益最佳,保育阶段仔猪在试验10天期间每头多增收10.6元。
表20不同处理对仔猪的影响
试验三结果如表21所示,由表21可知:在相同饲养条件和饲料价格无差异的情况下,添加剂可促进仔猪生长,提高经济效益。试验所有对照组、Ⅶ、Ⅷ、Ⅸ组中以试验Ⅶ组经济效益最佳,仔猪在试验20天期间每头多增收11元,增收效益明显。
表21不同处理对仔猪的影响
根据以上试验数据分析得出,该添加剂能够明显改善仔猪日增重,降低料肉比,有效预防仔猪腹泻现象发生,提高经济价值。
铁是仔猪必需的微量元素,影响仔猪的生长、发育、代谢过程和生理防御机能。肝是它主要的贮存部位。吸收的铁首先在肝等部位贮存,当机体需要时由贮存部位释放进入血浆。血浆中的铁几乎不反向转移到肠道内。体内的各种含铁类物质降解时所释放的铁几乎全部被机体再利用。粪中的铁绝大部分是饲料中未被吸收的铁。体内降解的血小板少量随胆汁和肠粘膜细胞脱落而由体内排出。仔猪平均血红蛋白浓度在100g/L以上,就可以满足仔猪对铁的需要,平均血红蛋白浓度在80g/L时表示仔猪临近贫血。各试验组仔猪的平均血红蛋白浓度都高于对照组(P<0.05),通过以上试验数据分析结果,说明这个该添加剂可以有效地提高仔猪对铁的吸收能力,有效的预防仔猪贫血病的发生。
总蛋白包括白蛋白、免疫球蛋白等,除了免疫球蛋白的免疫作用外,白蛋白在机体内也具有维持血浆胶体渗透压、维持血浆正常PH值、运输、催化、营养等功能。免疫球蛋白是猪体受抗原刺激后体内产生的能与抗原特异性相互作用的一类球蛋白,亦称抗体。免疫球蛋白具有结合抗原、激活补体和调节作用等生物学功能。其中IgG、IgM、IgA是免疫球蛋白中重要的三大类,IgG是血清中含量最高的免疫球蛋白,是介导体液免疫的主要抗体,其在体内发挥抗菌、抗病毒、抗毒素等免疫学活性,在抗肿瘤免疫中也是必不可少的;IgM是动物机体初次体液免疫反应最早产生的免疫球蛋白,但持续时间短,其具有抗菌、抗病毒、中和毒素等免疫活性,且亦有抗肿瘤作用;IgA同样具有前二者的免疫活性,另外还对机体呼吸道、消化道等局部粘膜免疫起着重要的“屏障”防御作用。因此,仔猪阶段血清IgA、IgM、IgG的提高,在一定程度上可以减少仔猪疾病的发生。以上试验结果显示:各处理组IgA、IgM、IgG含量较对照一组均有不同程度的提高。这说明以上试验所采用的添加剂对仔猪血清IgA、IgM、IgG含量有提高的作用,在生产上即表现为腹泻率降低,抗病力增强。
补体是特异性免疫的主要组成成分,是类似酶活性的一组蛋白质,参与机体保护性的免疫应答和自身稳定功能,协助抗体和吞噬细胞杀灭病原微生物和加强细胞免疫功能,担负机体非特异性抗感染作用。以上试验显示:各组补体C3、C4均高于对照一组,说明以上试验所用添加剂均能提高仔猪血液补体C3、C4含量。通过血液免疫指标:添加剂对LTR、血清IgG、IgM、IgA、补体C3、C4水平有显著提高,即提高了仔猪的免疫力和抗病力,其中各项免疫指标均以试验Ⅰ、Ⅱ组(Ⅳ、Ⅴ组andⅦ、Ⅷ组)较佳,综合仔猪各阶段饲养特点和经济效益分析,该添加剂以试验Ⅰ组(ⅣandⅦ组)为最佳。
Claims (5)
1.一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂,其特征在于,该饲料添加剂按质量份数由二氧化硅19.0~19.3份、乳酸27.0~27.5份、延胡索酸1.50~2.50份、氨基乙酰丙酸0.80~1.20份、柠檬酸0.80~1.20份、维生素B族0.04~0.05份、磷酸酯化维生素C 0.40~0.60份、鱼粉4.50~5.50份、甘氨酸螯合铁1.50~2.50份、L-精氨酸-α-酮戊二酸盐6.50 ~7.50份、低聚糖9.0~11.0份、丙酮酸肌酸90~110份和米糠粕800~850份制备而成;
所述包被营养饲料添加剂的制备方法按以下步骤进行:
步骤1:将二氧化硅与乳酸混合,然后加入粉碎过筛后的延胡索酸、氨基乙酰丙酸和柠檬酸继续混合,再加入聚维酮溶液,依次经过挤出、滚圆、造粒、干燥、过筛,得到微丸,将微丸置于沸腾流化床中进行包衣,得到包被颗粒;
其中,所述微丸尺寸为35~45目;所述包衣是采用聚丙烯酸树脂的乙醇溶液进行包衣,所述聚丙烯酸树脂为聚丙烯酸树脂Ⅱ、聚丙烯酸树脂Ⅳ中的一种或两种,所述聚丙烯酸树脂的乙醇溶液中聚丙烯酸树脂的质量浓度2%~7%;
步骤2:将米糠粕粉碎后,先将一部分粉碎后的米糠粕投入到混合机中,然后加入维生素B族、磷酸酯化维生素C和初筛后的鱼粉进行混合,混合均匀后再加入另一部分米糠粕、甘氨酸螯合铁、L-精氨酸α-酮戊二酸盐和低聚糖继续混合,混合均匀后加入剩余米糠粕、丙酮酸肌酸和步骤1得到的包被颗粒,继续混合至均匀,得到预防仔猪贫血促进免疫机能的包被营养饲料添加剂。
2.根据权利要求1所述的一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂,其特征在于,所述维生素B族为维生素B6、维生素B1、维生素B12中的一种或几种的混合物。
3.根据权利要求1所述的一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂,其特征在于,所述低聚糖为低聚壳聚糖、果寡糖、甘露寡糖、低聚木糖中的一种或几种的混合物。
4.根据权利要求1所述的一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂,其特征在于,步骤1中所述二氧化硅、乳酸、延胡索酸、氨基乙酰丙酸、柠檬酸的质量之和与聚维酮溶液的体积的比为7g:3mL。
5.根据权利要求1所述的一种预防仔猪贫血促进免疫机能的包被营养饲料添加剂,其特征在于,步骤2中所述一部分粉碎后的米糠粕是指米糠粕总质量的30%~50%,步骤2中所述另一部分米糠粕是指米糠粕总质量的30%~50%,步骤2中所述初筛是指过40目筛,步骤2中所述混合均采用三维混合机进行,步骤2中加入维生素B族、磷酸酯化维生素C和初筛后的鱼粉混合15min~20min,加入另一部分米糠粕、甘氨酸螯合铁、L-精氨酸α-酮戊二酸盐和低聚糖继续混合15min~20min,加入剩余米糠粕、丙酮酸肌酸和步骤1得到的包被颗粒,继续混合15min~20min。
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