CN110432404B - 降低促炎性响应与增强免疫应答的罗非鱼饲料添加剂及其制备方法和应用 - Google Patents
降低促炎性响应与增强免疫应答的罗非鱼饲料添加剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供降低促炎性响应与增强免疫应答的罗非鱼饲料添加剂及其制备方法和应用,属于水产养殖技术领域。降低促炎性响应与增强免疫应答的罗非鱼饲料添加剂,包括以下重量份的组分:刺五加茎25~35份、枇杷叶15~25份、雷公藤15~25份、山楂10~20份和桑叶10~20份。本发明还提供了一种包含所述罗非鱼饲料添加剂的罗非鱼饲料。罗非鱼饲料添加剂或所述罗非鱼饲料在罗非鱼养殖中的应用,所述应用能够有效的降低罗非鱼头肾组织与脾脏组织的促炎性响应,增强其免疫应答能力,从而提高其抵抗链球菌感染的能力。
Description
技术领域
本发明属于水产养殖技术领域,涉及养殖管理与饲料投喂技术领域,具体涉及降低促炎性响应与增强免疫应答的罗非鱼饲料添加剂及其制备方法和应用。
背景技术
流水槽循环水养殖是将传统池塘的“开放式散养”变为“集约化圈养”,使“静水”池塘实现了“流水”养鱼。通过机械造浪造流,在整个大池塘里形成环形水流,水流流经水槽,能在“跑道”内对水产品进行集中喂养,又能利用水流将排泄物集中到一个槽内统一处理,从而起到净化池塘水质的作用。环境应激改变了机体非特异性免疫力,导致鱼体出现炎症,损伤细胞。近年来,我国逐渐开展了循环水工厂化养殖模式的研究,为追求高产,人们往往采取高密度养殖,传统的充氧方式很难保证高密度养殖鱼类的健康成长,生产中常用补充液氧的方式来维持水体较高溶解氧水平。尽管高溶解氧环境可以提高鱼类的生长速度,但是在饱和溶氧条件下鱼体会产生的过多氧自由基会攻击生物细胞中蛋白质、脂肪和核酸等细胞结构及组织,导致机体损伤。同时,高密度养殖条件下,鱼体的活动范围有限,过量的营养会在鱼体肠道、肝脏与血液中沉积,导致鱼体过度肥胖。白色脂肪细胞的膨胀、缺氧和坏死,可能成为引发多种代谢性炎症。
罗非鱼是目前中国重要的淡水养殖鱼类之一,传统池塘与网箱养殖模式下,罗非鱼亩产在1500~3500斤。然而,罗非鱼夏季摄食旺盛,排泄物较多,造成水质污染较为严重。利用循环水进行工厂化养殖既能实现亩产10000~30000斤的高产量,又能实现养殖尾水的净化处理,符合绿色可持续发展要求。因此,如何降低高密度养殖下与高溶氧环境下罗非鱼促炎性响应,增加机体免疫应答,从而促进循环水工厂化养殖中罗非鱼健康养殖的目的已成为当前我们迫切需要解决的问题。
发明内容
有鉴于此,为了克服现有技术的缺陷,对罗非鱼流水槽循环水养殖中降低促炎性响应并增强其免疫应答能力,本发明提供了一种罗非鱼饲料添加剂及其制备方法和应用,能够有效的降低罗非鱼头肾组织与脾脏组织的促炎性响应,增强其免疫应答能力,从而提高罗非鱼抵抗链球菌感染的能力。
本发明提供了降低促炎性响应与增强免疫应答的罗非鱼饲料添加剂,包括以下重量份的组分:刺五加茎25~35份、枇杷叶15~25份、雷公藤15~25份、山楂10~20份和桑叶10~20份。
优选的,包括以下重量份的组分:刺五加茎28~32份、枇杷叶17~23份、雷公藤18~22份、山楂13~18份和桑叶14~18份。
优选的,包括以下重量份的组分:刺五加茎30份、枇杷叶20份、雷公藤19份、山楂16份和桑叶16份。
本发明提供了所述罗非鱼饲料添加剂的制备方法,包括以下步骤:
1)将刺五加茎、枇杷叶、雷公藤、山楂和桑叶混合,用水煎煮,得到煎煮液;
2)将所述煎煮液进行浓缩,得到的浓缩液进行醇沉,固液分离,收集液相得到饲料添加剂。
优选的,收集液相后还包括对所述液相依次进行减压浓缩和干燥处理,得到饲料添加剂干粉。
优选的,所述减压浓缩的温度为60~70℃,所述减压浓缩的压力为-0.08Mpa。
优选的,所述浓缩液和醇沉用溶剂的体积比为1:3~5;所述醇沉用溶剂为体积浓度90%~95%的乙醇水溶液。
本发明提供了一种具有降低促炎性响应与增强免疫应答功效的罗非鱼饲料,包括所述罗非鱼饲料添加剂或所述制备方法制备的罗非鱼饲料添加剂。
本发明提供了所述罗非鱼饲料添加剂、所述制备方法制备的罗非鱼饲料添加剂或所述罗非鱼饲料在罗非鱼养殖中的应用。
优选的,所述养殖包括在流水槽养殖。
本发明提供了降低促炎性响应与增强免疫应答的罗非鱼饲料添加剂,具有以下有益效果:(1)能够有效的降低水循环养殖中促炎性因子的表达,促进免疫应答,增强罗非鱼健康养殖;(2)复方中草药组合物属于绿色环保型制剂,鱼体残留以及对环境的负作用较小,符合绿色环保友好型饲料添加剂的要求;(3)复方中草药组合物鱼体吸收效率较好,肌肉无残留,鱼体的排泄物对环境无污染。
具体实施方式
本发明提供了降低促炎性响应与增强免疫应答的罗非鱼饲料添加剂,包括以下重量份的组分:刺五加茎25~35份、枇杷叶15~25份、雷公藤15~25份、山楂10~20份和桑叶10~20份。
在本发明中,所述罗非鱼饲料添加剂包括刺五加茎。按重量份计,所述刺五加茎优选为28~32份,更优选为30份。所述刺五加茎的作用是:富含皂苷、多糖、黄酮含量有助于提高鱼体的抗氧化能力,降低促炎性因子活性。
在本发明中,所述罗非鱼饲料添加剂包括枇杷叶。按重量份计,所述枇杷叶有选为17~23份,更优选为20份。所述枇杷叶的作用是:富含黄酮、总酚、多糖含量有助于促进脂肪代谢,提高鱼体的抗氧化能力,降低促炎性因子活性。
在本发明中,所述罗非鱼饲料添加剂包括雷公藤。按重量份计,所述雷公藤优选为18~22份,更优选为19份。所述雷公藤的作用是:富含二帖和三帖类化合物以及生物碱有助于鱼体增强抗炎作用与免疫促进作用。
在本发明中,所述罗非鱼饲料添加剂包括山楂。按重量份计,所述山楂优选为13~18份,更优选为16份。所述山楂的作用是:富含黄铜有助于促进鱼体的脂肪代谢与增强免疫调节作用。
在本发明中,所述罗非鱼饲料添加剂包括桑叶。按重量份计,所述桑叶优选为14~18份,更优选为16份。所述桑叶的作用是:富含生物碱、黄铜与多糖有助于提高鱼体的抗氧化能力,降低促炎性因子活性。
本发明对所述刺五加茎、枇杷叶、雷公藤、山楂和桑叶的来源没有特殊限制,采用本领域所熟知的中药来源即可。实验证明:刺五加茎、枇杷叶、雷公藤、山楂和桑叶按照上述比例复配,制备的罗非鱼饲料添加剂饲喂罗非鱼,罗非鱼血液白细胞数量与血清溶菌酶活力明显高于对照组。实验组头肾组织补体C3基因表达活力明显高于对照组;然而,白介素-1β、肿瘤坏死因子-α和干扰素-γ明显低于对照组。实验组链球菌感染后的累积死亡率明显低于对照组。饲料中添加饲料添加剂能明显调控免疫应答,降低促炎性因子的响应。
本发明提供了所述罗非鱼饲料添加剂的制备方法,包括以下步骤:
1)将刺五加茎、枇杷叶、雷公藤、山楂和桑叶混合,用水煎煮,得到煎煮液;
2)将所述煎煮液进行浓缩,得到的浓缩液进行醇沉,固液分离,收集液相得到饲料添加剂。
本发明将刺五加茎、枇杷叶、雷公藤、山楂和桑叶混合,用水煎煮,得到煎煮液。
在本发明中,中药材总质量和水的体积比优选为0.8~1.2g:0.8~1.2mL,更优选为1g:1mL。所述煎煮的温度优选为90~100℃,更优选为95℃。所述煎煮的时间优选为1~2h,更优选为1.5h。煎煮后,优选进行固液分离得到煎煮液。
得到煎煮液后,本发明将所述煎煮液进行浓缩,得到的浓缩液进行醇沉,固液分离,收集液相得到饲料添加剂。
本发明对所述浓缩的方法没有特殊限制,采用本领域所熟知的浓缩方案即可,例如采用减压浓缩,旋转蒸发或烘干等方法均可。所述浓缩的程度优选在55℃条件下烘干3h。所述浓缩液和醇沉用溶剂的体积比优选为1:3~5,更优选为1:4;所述醇沉用溶剂优选为体积浓度90%~95%的乙醇水溶液。所述醇沉的作用是除去杂质保留药物有效成分。所述固液分离的方法优选包括抽滤。抽滤用滤膜的孔径优选为0.1~5μm,更优选为0.5~3μm。
在本发明中,收集液相后优选还包括对所述液相依次进行减压浓缩和干燥处理,得到饲料添加剂干粉。所述减压浓缩的温度优选为60~70℃,更优选为65℃。所述减压浓缩的压力优选为-0.08Mpa。减压浓缩得到浸膏的相对密度优选为1.03~1.12。所述干燥处理的温度优选为45℃,所述干燥处理的时间优选为2h。
本发明提供了一种具有降低促炎性响应与增强免疫应答功效的罗非鱼饲料,包括所述罗非鱼饲料添加剂或所述制备方法制备的罗非鱼饲料添加剂。所述罗非鱼饲料还包括基础日粮。所述基础日粮中蛋白水平为26%,脂肪水平为7%。每100g基础日粮中添加0.1~0.4g罗非鱼饲料添加剂干粉,即所述罗非鱼饲料包含质量浓度0.1%~0.4%的罗非鱼饲料添加剂干粉。所述罗非鱼饲料的制备方法优选包括将基础日粮和罗非鱼饲料添加剂干粉充分混合即可。
本发明提供了所述罗非鱼饲料添加剂、所述制备方法制备的罗非鱼饲料添加剂或所述罗非鱼饲料在罗非鱼养殖中的应用。所述养殖优选包括在流水槽养殖。
在本发明中,所述罗非鱼养殖过程的饲料添加剂干粉的用量根据鱼体的重量不同而有所差异:鱼体规格为150g~350g,投喂添加含0.1%~0.3%饲料添加剂干粉的饲料;鱼体规格为350g以上,投喂0.2%~0.4%饲料添加剂干粉的饲料。投喂的时间为每日投喂二次,分别为7:00和16:00,投喂量为鱼体重的3%~5%。阴雨天气减少投喂次数与投喂量,或者不投喂。
下面结合实施例对本发明提供的降低促炎性响应与增强免疫应答的罗非鱼饲料添加剂及其制备方法和应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
降低流水槽养殖中罗非鱼成鱼促炎性响应与增强免疫应答的饲料添加剂的制备方法,包含以下步骤:
准确称量刺五加茎30g,枇杷叶20g,雷公藤20g,山楂15g和桑叶15g,混合后,按照每1kg中草药加1L水的比例加入水,在95℃蒸煮1.5h,固液分离,收集水相。使用烘箱55℃烘干水分3h,获取浓缩液。用95%乙醇对浓缩液进行醇沉。抽滤,滤液在60℃,-0.08Mpa条件下减压回收,得到浸膏。所得浸膏在45℃烘箱中干燥2h,粉碎成干粉。
实施例2
一套降低流水槽养殖中罗非鱼成鱼促炎性响应与增强免疫应答的饲料配伍及其实施方法,包含以下步骤:
准确称量刺五加茎30g,枇杷叶20g,雷公藤20g,山楂15g和桑叶15g。将上述中草药混合用水煎煮,每1kg中草药加入1L纯净水,100℃蒸煮1.0h。使用烘箱55℃条件下烘干水分3h,得到浓缩液。按照乙醇水溶液与浓缩液的体积比4:1,在醇沉罐中添加适量的95%乙醇对浓缩液进行醇沉。抽滤(抽滤用膜的孔径范围为3μm),滤液在65℃、-0.08Mpa条件下减压回收,得到相对密度为1.03~1.12的浸膏。所得浸膏在45℃烘箱中干燥2h,粉碎成干粉。
实施例3
饲料添加剂的制备方法
准确称量刺五加茎30g,枇杷叶20g,雷公藤20g,山楂15g,桑叶15g。按照实施例1的制备方法制备饲料添加剂干粉。
对比例1
准确称量枇杷叶29g,雷公藤29g,山楂21g,桑叶21g。按照实施例1的制备方法制备饲料添加剂干粉。
对比例2
准确称量五加皮30g,枇杷叶20g,雷公藤20g,山楂15g,桑叶15g。按照实施例1的制备方法制备饲料添加剂干粉。
实施例4
配制蛋白水平为28%,脂肪水平为7%的基础日粮饲料。按照每100g基础日粮中分别添加0.2g饲料添加剂干粉的比例将实施例1制备的饲料添加剂干粉与基础日粮混合,制备得到含有0.2%饲料添加剂干粉的罗非鱼饲料。
实施例5
在江苏扬中,选取6个800平方米的流水槽,每个流水槽放养15000尾罗非鱼,鱼体规格在180g左右。3个流水槽作为对照组,仅投喂基础日料饲料;3个流水槽作为实验组,投喂添加实施例3制备的罗非鱼饲料。投喂的时间为:每日投喂二次,分别为7:00和16:00,投喂量为鱼体重的5%。阴雨天气减少投喂次数与投喂量,或者不投喂。养殖周期为60天。血清溶菌酶含量采用双抗体夹心ELISA酶联免疫法定量测定。采用稀释法计数鱼类单位容积血液内的白细胞数。具体结果如表1所示。
表1饲料中添加饲料添加剂对罗非鱼成鱼生长与血液免疫指标的影响
指标 | 对照组 | 实验组 |
初始体重(g) | 183.28 | 185.91 |
终末体重(g) | 514.55 | 523.78 |
血清溶菌酶(U/L) | 874.52 | 1256.77 |
白细胞(×10<sup>9</sup>/L) | 201.45 | 237.82 |
为了明确饲料添加剂对罗非鱼中免疫指标与促炎性基因表达情况,采用RT-PCR方法检测多个基因的表达情况。用PrimeScript RT reagent Kit Perfect Real Time(TaKaRa,大连,中国)试剂盒进行反转录,然后采用ABI QuantStudio 5Real-Time PCRSystem的SYBR Green渗入法进行实时定量PCR扩增反应,以18s rRNA为内参。PCR反应体系(50μl)包括:高压灭菌去离子水19μl、SYBR Green PCR Master Mix(2×)25μl、正向及反向引物(10μmol/L)各2μl、cDNA工作液4μl。反应条件为:95℃5min,然后40个循环(95℃15S,60℃60S,读板记录荧光量),反应结束后制备溶解曲线,反应条件为:95℃15S,60℃15S,95℃15S。每个反应设3复孔。所有检测样品均包含1个无模板的阴性对照以排除假阳性结果。各基因的扩增用引物序列情况见表2,检测结果见表3。
表2引物序列
表3含饲料添加剂的饲料对罗非鱼头肾组织免疫指标与促炎性基因表达的影响
基因表达水平 | 对照组 | 实验组 |
补体C3(%18srRNA) | 1.45 | 2.57 |
白介素-1β(%18srRNA) | 1.52 | 0.67 |
肿瘤坏死因子-α(%18srRNA) | 1.24 | 0.69 |
干扰素-γ(%18srRNA) | 1.04 | 0.72 |
养殖试验结束后,随机选取20尾鱼作为一个实验组进行海豚链球菌感染实验,海豚链球菌的培养与计数、菌液注射感染等方法参照文献(QIANG J,YANG H,WANG H,etal.Interacting effects ofwater temperature and dietary protein level onhematological parameters in Nile tilapiajuveniles,Oreochromis niloticus(L.)and mortality under Streptococcus iniae infection[J].Fish and ShellfishImmunology,2013,34:8-16.)。本实验注射感染所用的海豚链球菌菌液浓度为6.15×106CFU/ml,计算192h时各实验组的累计死亡率。对照组与实验组各设置3组平行。结果见表4。
表4含饲料添加剂的饲料对罗非鱼海豚链球菌感染后的死亡率影响
对照组 | 实验组 | |
组1 | 55% | 30% |
组2 | 65% | 40% |
组3 | 65% | 45% |
从表1和表3可见,实验组罗非鱼血液白细胞数量与血清溶菌酶活力明显高于对照组。实验组头肾组织补体C3基因表达活力明显高于对照组;然而,白介素-1β,肿瘤坏死因子-α和干扰素-γ明显低于对照组。由表4可知,实验组链球菌感染后的累积死亡率明显低于对照组。饲料中添加本发明的复方中草药水提物能明显调控免疫应答,降低促炎性因子的响应。
实施例6
按照实施例3的方法配制基础日粮。每100g基础日粮中分别添加0.3g的实施例2制备的饲料添加剂干粉,配制成含0.3%的饲料添加剂的饲料。
实施例7
在江苏宜兴,选取6个1000平方米的流水槽,每个流水槽放养25000尾罗非鱼,鱼体规格在400g左右。将3个流水槽作为对照组,仅投喂基础日料饲料;另外3个流水槽作为实验组,投喂添加实施例5制备的饲料。投喂的时间为:每日投喂二次,分别为7:00和16:00,投喂量为鱼体重的3%。阴雨天气减少投喂次数与投喂量,或者不投喂。养殖周期为45天。按照实施例5的方法检测幼鱼生长与血液免疫响应的因素,采用实施例5中RT-PCR方法检测免疫指标与促炎性基因表达情况。
表5含饲料添加剂的饲料对罗非鱼幼鱼生长与血液免疫响应的影响
指标 | 对照组 | 实验组 |
初始体重(g) | 408.12 | 403.11 |
终末体重(g) | 578.24 | 591.35 |
血清溶菌酶(U/L) | 745.22 | 912.37 |
白细胞(×10<sup>9</sup>/L) | 206.71 | 235.29 |
表6含饲料添加剂的饲料对罗非鱼头肾组织免疫指标与促炎性基因表达的影响
对照组 | 实验组 | |
补体C3(%18srRNA) | 1.31 | 1.97 |
白介素-1β(%18srRNA) | 1.36 | 0.79 |
肿瘤坏死因子-α(%18srRNA) | 1.42 | 0.64 |
干扰素-γ(%18srRNA) | 1.23 | 0.77 |
实验组与对照组相比生长无显著差异,然后实验组头肾组织促炎性因子响应明显低于对照组,血液与头肾组织免疫指标明显高于对照组。
实施例8
将实施例3制备的饲料添加剂分别按照每100g基础日粮中添加0.2g的饲料添加剂的量加入基础日粮中充分混匀,配制成含0.2%的饲料添加剂的饲料。
对比例3~4
分别将对比例1~2制备的饲料添加剂分别按照每100g基础日粮中添加0.2g的饲料添加剂的量加入基础日粮中充分混匀,配制成含0.2%的饲料添加剂的饲料
实施例9
在江苏宜兴,选取9个800平方米的流水槽,每个流水槽放养20000尾罗非鱼,鱼体规格在250g左右。配制蛋白水平为26%,脂肪水平为7%的基础日粮饲料。3个流水槽作为实验组1,投喂添加实施例3制备的饲料添加剂的饲料;3个流水槽作为实验组2,投喂添加对比例1制备的饲料添加剂饲料;3个流水槽作为实验组3,投喂添加对比例2制备的饲料添加剂饲料。投喂的时间为:每日投喂二次,分别为7:00和16:00,投喂量为鱼体重的4%。阴雨天气减少投喂次数与投喂量,或者不投喂。养殖周期为60天。按照实施例5的方法测定初始体重、终末体重、血清溶菌酶活力和白细胞数量。同时按照实施例5中公开的RT-PCR的方法测定免疫指标和促炎性基因的表达情况。结果见表7和表8。
表7饲料中添加不同复方中草药水提物组合对罗非鱼幼鱼生长与血液免疫响应的影响
检测项目 | 实验组1 | 实验组2 | 实验组3 |
初始体重(g) | 254.17 | 259.22 | 252.35 |
终末体重(g) | 512.92 | 523.17 | 514.28 |
血清溶菌酶(U/L) | 834.56 | 728.11 | 738.14 |
白细胞(×10<sup>9</sup>/L) | 238.36 | 213.66 | 219.51 |
表8饲料中添加不同复方中草药水提物组合对罗非鱼头肾组织免疫指标与促炎性基因表达的影响
指标 | 实验组1 | 实验组2 | 实验组3 |
补体C3(%18srRNA) | 1.31 | 0.75 | 0.87 |
白介素-1β(%18srRNA) | 1.21 | 1.85 | 1.72 |
肿瘤坏死因子-α(%18srRNA) | 1.43 | 2.58 | 1.74 |
干扰素-γ(%18srRNA) | 1.36 | 2.84 | 2.71 |
以实验组1作为阳性对照组,罗非鱼摄食本发明提供的复方中草药(实验组1)后能够明显增强补体C3,降低促炎性因子:白介素-1β,肿瘤坏死因子-α和干扰素-γ的基因表达水平。而实验组2和实验组3结果表明,缺少或替换其中的组成成分无法起到促进免疫提高与降低促炎性因子表达,这说明本发明提供的中草药配方中各中药成分具有协同作用。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
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<120> 降低促炎性响应与增强免疫应答的罗非鱼饲料添加剂及其制备方法和应用
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Claims (10)
1.降低促炎性响应与增强免疫应答的罗非鱼饲料添加剂,由以下重量份的组分组成:刺五加茎25~35份、枇杷叶15~25份、雷公藤15~25份、山楂10~20份和桑叶10~20份;
所述罗非鱼饲料添加剂的制备方法,包括以下步骤:
1)将刺五加茎、枇杷叶、雷公藤、山楂和桑叶混合,用水煎煮,得到煎煮液;
2)将所述煎煮液进行浓缩,得到的浓缩液进行醇沉,固液分离,收集液相得到饲料添加剂。
2.根据权利要求1所述的罗非鱼饲料添加剂,其特征在于,由以下重量份的组分组成:刺五加茎28~32份、枇杷叶17~23份、雷公藤18~22份、山楂13~18份和桑叶14~18份。
3.根据权利要求1或2所述的罗非鱼饲料添加剂,其特征在于,由以下重量份的组分组成:刺五加茎30份、枇杷叶20份、雷公藤19份、山楂16份和桑叶16份。
4.权利要求1~3任意一项所述罗非鱼饲料添加剂的制备方法,其特征在于,包括以下步骤:
1)将刺五加茎、枇杷叶、雷公藤、山楂和桑叶混合,用水煎煮,得到煎煮液;
2)将所述煎煮液进行浓缩,得到的浓缩液进行醇沉,固液分离,收集液相得到饲料添加剂。
5.根据权利要求4所述制备方法,其特征在于,收集液相后还包括对所述液相依次进行减压浓缩和干燥处理,得到饲料添加剂干粉。
6.根据权利要求5所述制备方法,其特征在于,所述减压浓缩的温度为60~70℃,所述减压浓缩的压力为-0.08Mpa。
7.根据权利要求4所述制备方法,其特征在于,所述浓缩液和醇沉用溶剂的体积比为1:3~5;所述醇沉用溶剂为体积浓度90~95%的乙醇水溶液。
8.一种具有降低促炎性响应与增强免疫应答功效的罗非鱼饲料,其特征在于,包括权利要求1~3任意一项所述罗非鱼饲料添加剂或权利要求4~7任意一项所述制备方法制备的罗非鱼饲料添加剂。
9.权利要求1~3任意一项所述罗非鱼饲料添加剂、权利要求4~7任意一项所述制备方法制备的罗非鱼饲料添加剂或权利要求8所述罗非鱼饲料在罗非鱼养殖中的应用。
10.根据权利要求9所述的应用,其特征在于,所述养殖包括在流水槽养殖。
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