CN108576403A - 硒在提高黄颡鱼生长性能、抗氧化能力、抗低温应激能力中的应用 - Google Patents
硒在提高黄颡鱼生长性能、抗氧化能力、抗低温应激能力中的应用 Download PDFInfo
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- CN108576403A CN108576403A CN201810290856.5A CN201810290856A CN108576403A CN 108576403 A CN108576403 A CN 108576403A CN 201810290856 A CN201810290856 A CN 201810290856A CN 108576403 A CN108576403 A CN 108576403A
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
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- Inorganic Chemistry (AREA)
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- Feed For Specific Animals (AREA)
Abstract
本发明公开了硒在提高黄颡鱼生长性能、抗氧化能力、抗低温应激能力中的应用。本发明通过实验证实,在黄颡鱼饲料中添加硒,可提高黄颡鱼幼鱼的增重率、鱼体和肌肉硒的沉积以及肝脏抗氧化酶活性;低温应激时,黄颡鱼血清甘油三酯含量显著升高,能更加有效地启动黄颡鱼脂肪代谢以抵御低温应激;同时,饲料中添加硒可使低温应激的黄颡鱼血糖消耗相对较慢,有利于低温应激后的恢复。因此,可将硒制备黄颡鱼的饲料添加剂,用于提高黄颡鱼生长性能、抗氧化能力或抗低温应激能力,为黄颡鱼的健康养殖和耐寒研究提供了新的方法。
Description
技术领域
本发明属于饲料添加剂技术领域,更具体地说,本发明涉及将硒用于提高黄颡鱼的生长性能、抗氧化能力和抗低温应激能力中,及相应的饲料添加剂。
背景技术
饲料补硒是弥补动物从自然环境中获取硒不足的主要途径。饲料中补充硒制剂的形式主要有两种,即无机硒和有机硒。无机硒的添加形式主要是亚硒酸钠,而有机硒则是硒半胱氨酸(Sec)、硒蛋氨酸(Se-Met)和酵母硒。有机硒在胃肠道是主动吸收,而无机硒是被动扩散,它必须先进入肝脏转化成生物硒方可利用。动物胃肠道对有机硒的吸收效率高于无机硒,亚硒酸盐中硒吸收率在80%以上,而硒化蛋氨酸中的硒吸收率则超过90%。
温度作为一项重要的生态因子,可引起水产动物消化吸收、耗氧率与呼吸率、代谢频率、行为行态、血液学指标等的变化。研究发现,低温可以引起遮目鱼(Chanos chanos)、罗非鱼、七带石斑鱼鱼体血清总蛋白、血糖、甘油三酯等血液生化指标的变化;鲤鱼、大黄鱼、黄颡鱼、奥尼罗非鱼、谷丙转氨酶、谷草转氨酶等酶类以及皮质醇、儿茶酚胺等激素的变化;罗非鱼、大西洋鲑、蓝鳍金枪鱼(Thunnusorientalis)肝脏HSP70基因表达的变化。黄颡鱼属温水性鱼类,最佳生长温度25~28℃,不耐受低温。近年来,我国南方水产养殖业因寒潮造成了重大的危害和巨大的经济损失,水产动物的抗低温胁迫引起了研究者和行业人员的广泛关注。
发明内容
本发明目的在于:提供了将硒用于提高黄颡鱼生长性能、抗氧化能力和抗低温应激能力中,以及相应的饲料添加剂。
为了实现上述发明目的,本发明将硒添加到黄颡鱼饲料中,测试其对黄颡鱼的影响。实验结果表明,黄颡鱼饲料中添加硒,可提高黄颡鱼的生长性能、抗氧化能力和抗低温应激能力。
优选地,所述硒包括无机硒和有机硒。
更进一步地,所述无机硒为亚硒酸钠;所述有机硒包括硒半胱氨酸、硒蛋氨酸、酵母硒。
本发明还提供了一种饲料添加剂,其包括无机硒和有机硒,其中,所述无机硒为亚硒酸钠;所述有机硒包括硒半胱氨酸、硒蛋氨酸、酵母硒。
本发明上述饲料添加剂添加至黄颡鱼饲料中,可提高黄颡鱼生长性能、抗氧化能力或抗低温应激能力。
相对于现有技术,本发明具有如下有益效果:
本发明通过实验证实,在黄颡鱼饲料中添加硒,可提高黄颡鱼幼鱼的增重率、鱼体和肌肉硒的沉积以及肝脏抗氧化酶活性;低温应激时,黄颡鱼血清甘油三酯含量显著升高,能更加有效地启动黄颡鱼脂肪代谢以抵御低温应激;同时,饲料中添加硒可使低温应激的黄颡鱼血糖消耗相对较慢,有利于低温应激后的恢复。低温条件下,0.30mg/kg酵母硒组相比亚硒酸钠能够使黄颡鱼血糖保持在较高的水平,有利于黄颡鱼抗低温应激,与0.25mg/kg亚硒酸钠相比,饲料中添加0.30mg/kg酵母硒能够提高黄颡鱼的抗低温应激能力。因此,可将硒制备黄颡鱼的饲料添加剂,用于提高黄颡鱼生长性能、抗氧化能力或抗低温应激能力,为黄颡鱼的健康养殖和耐寒研究提供了新的方法。
附图说明
图1为亚硒酸钠和酵母硒对低温应激下黄颡鱼幼鱼血清甘油三酯含量的影响结果。
图2为亚硒酸钠和酵母硒对低温应激下血糖含量的影响结果。
图3为亚硒酸钠和酵母硒对低温应激下黄颡鱼幼鱼血清尿素氮含量的影响结果。
图4为亚硒酸钠和酵母硒对低温应激下黄颡鱼肝脏HSP70mRNA表达的影响结果。
具体实施方式
为了使本发明的目的、技术方案和有益技术效果更加清晰,以下结合实施例,对本发明进行进一步详细说明。应当理解的是,本说明书中描述的实施例仅仅是为了解释本发明,并非为了限定本发明,实施例的参数、比例等可因地制宜做出选择而对结果并无实质性影响。
实验饲料:
本发明实施例使用的实验饲料是以酪蛋白为蛋白质源,鱼油和磷脂油(4:1)为脂肪源,高筋面粉为糖源配制成粗蛋白质含量为39.96%,粗脂肪含量为9.93%的基础饲料,配方见表1。分别以亚硒酸钠和酵母硒(美国奥特奇公司)作为硒的来源配置配制4种实验饲料,硒的添加量为0.23mg/kg。所有饲料原料均粉碎并过60目筛,采用逐级扩大法将亚硒酸钠、维生素和微量元素等微量成分混匀,然后加入鱼油、磷脂油和水混匀,使用SLX-80型双螺旋杆挤压机将其制成直径为2.5mm的颗粒饲料,50℃烘干,自然冷却后装入密封袋,-20℃冰箱中保存备用。
采用氢化物发生-原子吸收光谱法(AOAC,1995)测得最终4种实验饲料中硒的含量分别为0.028mg/kg(对照组)、0.25mg/kg(亚硒酸钠组)、0.30mg/kg(酵母硒组)和0.52mg/kg(酵母硒组),将4组实验饲料依次标记为对照组、Na2SeO3、Se-Y1和Se-Y2组。
表1基础饲料配方组成及营养组成
1由广州飞禧特水产科技有限公司,广东省农业科学院动物科学研究所提供。
2由广州飞禧特水产科技有限公司,广东省农业科学院动物科学研究所提供。维生素组成(/kg饲料):VA,2000IU;VD3,700IU;VE,10mg;VK3,2.5mg;VB1,2.5mg;VB2,5mg;VB6,3mg;VB12,0.01mg;烟酰胺,17.5mg;D-泛酸钙,10mg;叶酸,0.8mg;生物素,0.045mg;肌醇,25mg。
3由广州飞禧特水产科技有限公司,广东省农业科学院动物科学研究所提供。微量元素组成(/kg饲料):MgSO4.H2O,280mg;CuSO4.5H2O,6mg;FeSO4.H2O,30mg;MnSO4.H2O,4.9mg;FeSO4.H2O,30mg;ZnSO4.H2O,86.95mg;饲料级碘酸钙(5%),15mg;饲料级硫酸钴(5%),3mg。
实验鱼与饲养管理:
本发明实施例实验用黄颡鱼幼鱼购于广东省清远市黄沙渔业基地,购回后暂养于广东省农业科学院动物科学研究所水产研究室的室内循环水养殖系统中,每天用商品饲料(粗蛋白,42.24%;粗脂肪,7.06%;灰分,10.06%;水分,9.42%;硒,0.46mg/kg)饱食投喂2次(09:00和16:00),驯养2周。养殖系统由12个容量为350L(直径80cm,高70cm)的圆柱形玻璃纤维缸组成,养殖实际水容量为300~320L实验开始时,挑选出体格健壮大小均匀平均体重2.12±0.01g的黄颡鱼幼420尾,分配于12个养殖缸中,每缸放养35尾。将12个养殖缸随机分为4组(每组3个重复),分别投喂对应的实验饲料,每天投喂2次(09:00和16:00),投饲量为体重的4%~6%,并根据摄食和生长情况调节,每天记录投饲量,死亡情况以及水温,全天24h不间断曝气,光照为自然光源,水温,水温22~25℃,pH 7.3~7.9,氨氮<0.2mg/L,实验期为8周。8周后将采样剩余的实验鱼按上述同样的分组方法转移到12个容量为150L的圆柱形玻璃纤维缸组成的温控水循环养殖系统(大连汇新钛设备开发有限公司),待实验鱼稳定1天后,正式开始实验。降温前测得水温22℃,pH 7.5,氨氮<0.2mg/L,连续充气,自然光周期,利用温控系统设计水体的降温速度为1℃/1h,最低温度为13℃,降温期间禁食。
样品采集:
饲养实验结束时,禁食24h后,计数、称重。每缸随机取3尾鱼存放于-20℃冰箱中保存,用于测定鱼体硒含量,再随机取6尾鱼,用MS-222(苏州信永生物医药技术有限公司)60mg/L进行麻醉,取鱼的肌肉、肝脏和肠道,6尾鱼的肌肉、肝脏和肠道分别合并为一个样品,迅速投入液氮中速冻,保存于-78℃冰箱用于测定肌肉硒含量、肝脏GPx活性、SOD活性、MDA含量以及肠道消化酶。
水温为22℃和13℃时,分别采样每缸随机取3用MS-222(苏州信永生物医药技术有限公司)60mg/L进行麻醉,尾静脉取血于抗凝管(BD公司)中混匀,于4℃4000r/min离心10min,制备血清样品,-78℃保存,用于血清相关指标测定。然后在冰盘上解剖采血后鱼的肝脏,3尾鱼肝脏合并为一个样品,迅速投入液氮中速冻,保存于-78℃冰箱用于HSP70基因表达分析。
样品分析:
饲料和肌肉组成样品中水分含量采用105℃常压干燥法、粗蛋白质含量采用凯氏定氮法、粗脂肪含量采用乙醚抽提法、灰分含量采用550℃灼烧法(AOAC1995)进行测定。采用氢化物发生-原子吸收光谱法(AOAC,1995)测定鱼体和肌肉硒含量。MDA含量、GPx活性、SOD活性的测定均采用市售的南京建成生物工程研究所的实剂盒,按照使用说明进行操作。肠道粗酶液制备:称取一定量新鲜组织(胃、前肠、肝胰脏),滤纸吸干组织表面水分后用10倍生理盐水(1g组织:9ml生理盐水)在冰水浴中匀浆,匀浆液在4℃3000r/min离心10min,取上清液为粗酶液。蛋白酶单位定义:在pH 8.0,37℃条件下,每毫克蛋白中含有的胰蛋白酶每分钟使吸光度变化0.003即为一个酶活力单位(U/mg)。脂肪酶单位定义:在37℃条件下,每克组织蛋白在本反应体系中与底物反应1min,每消耗1μmol底物为一个酶活力单位(U/g)。淀粉酶单位定义:组织中每毫克蛋白在37℃与底物作用30min,水解10mg淀粉为1个淀粉酶活力单位(U/mg)。血清葡萄糖(glucose,GLU)、总蛋白(totalprotein,TP)、胆固醇(cholesterol,CHO)和甘油三酯(triglyceride,TG)含量采用日立全自动生化分析仪(HITACHI 7170A)进行测定。取出-78℃中保存的鱼肝脏组织,在液氮中充分研磨成粉末状后,取实剂盒说明书建议用量加入裂解液中,并按照Takara RNA小量提取实剂盒操作步骤进行RNA的抽提。实时荧光定量PCR测定瓦氏黄颡鱼幼鱼肝脏中的HSP70mRNA的相对表达量,以β-肌动蛋白为内参基因。根据瓦氏黄颡鱼HSP70mRNA序列利用Primer5.0软件设计荧光定量PCR特异引物HSP70(JN579124.1)、Actin(EU161066.1)(表2)。使用的主要仪器为定量PCR仪器:Biorad CFX connect,分光光度计:Thermo Scientific NanoDrop ND2000。将2μL模版cDNA,4μL引物对混合液,4μL ddH2O和10μL SYBR Green qPCR kit(All-in-OneTMmiRNAqRT-PCR Detection Kit)构成20μL反应体系。每个样品做三个重复,以不加模版的PCR反应样品作为阴性对照。反应循环参数为:95℃预变性12min;95℃变性10s,58℃退火10s,72℃延伸10s,40个循环。使用Option Monitor software 2.03version(MJ Research,Cambridge,MA)软件分析溶解曲线。按照2-△△Ct法进行表达量差异分析。
表2本实验中实时荧光定量PCR使用的引物
指标计算:
增重率(weight gain rate,WGR,%)=100×(终末体重+死亡体重-初始体重)/初始体重;饲料系数(feed coefficient,FC)=投饲总量/(终末体重+死亡体重-初始体重);存活率(survival rate,SR,%)=100×终末尾数/初始尾数。
数据统计与分析:
验数据用平均值±标准差(n=3)表示。采用SPSS11.5软件进行统计分析。首先对数据进行方差齐性检验,若满足方差齐性则用单因素方差分析(one-way ANOVA)分析数据,差异显著者再用LSD检验方法进行多重比较,若不满足方差齐性则采用Dunnett’s T3检验法进行多重比较。P<0.05表示差异显著。
实施例1亚硒酸钠和酵母硒对黄颡鱼生长性能和硒沉积的影响
如表3所示,添加亚硒酸钠和酵母硒组黄颡鱼幼鱼的增重率高于对照组但差异不显著(P>0.05),与对照组相比亚硒酸钠组增重率的生物效价为101.12%,0.30mg/kg酵母硒组和0.52mg/kg酵母硒组的生物效价分别为106.41%和102.23%。各组之间增重率、饲料系数和存活率差异不显著(P>0.05)。与对照组相比添加亚硒酸钠和酵母硒能够显著提高黄颡鱼幼鱼体硒含量和肌肉硒含量(P<0.05),这进一步说明饲料中适量的添加硒对动物获得较好的生长是必需的。添加0.30mg/kg酵母硒组肌肉硒含量显著高于0.25mg/kg亚硒酸钠组(P<0.05),0.25mg/kg亚硒酸钠组和0.30mg/kg酵母硒组与对照组相比,硒的沉积效率分别为200%和267%。比较0.25mg/kg亚硒酸钠组和0.30mg/kg酵母硒组黄颡鱼幼鱼的增重率发现,0.30mg/kg酵母硒组的增重率高于0.25mg/kg亚硒酸钠组,说明在适宜的添加范围内,同剂量的酵母硒比亚硒酸钠对黄颡鱼的促生长效果更好,生物效率更高。
表3亚硒酸钠和酵母硒对生长性能的影响
注:同行数据肩标不同小写字母表示差异显著(P<0.05),下同。
实施例2亚硒酸钠和酵母硒对黄颡鱼抗氧化能力的影响
如表4所示,与对照组相比添加亚硒酸钠和酵母硒能够显著提高黄颡鱼幼肝脏GPx活性和SOD活性(P<0.05),添加0.30mg/kg酵母硒组GPx活性和SOD活性显著高于0.25mg/kg亚硒酸钠组(P<0.05)。0.25mg/kg亚硒酸钠组和0.30mg/kg酵母硒组分别与对照组相比,GPx活性的生物效价为:129.24%和168.18%;SOD活性的生物效价为143.11%和183.54%。
表4亚硒酸钠和酵母硒对黄颡鱼幼鱼抗氧化能力的影响
实施例3亚硒酸钠和酵母硒对低温应激黄颡鱼血清甘油三酯含量的影响
如图1所示,22℃时,Na2SeO3和酵母硒对黄颡鱼血清甘油三酯无显著影响(P>0.05),13℃时,Na2SeO3组和Se-Y1组黄颡鱼血清甘油三酯含量显著升高(P<0.05)。这一结果表明机体开始动用体内存贮的脂肪以维持生存代谢,添加Na2SeO3组和Se-Y1组更能有效地启动黄颡鱼脂肪代谢以抵御低温应激。
实施例4亚硒酸钠和酵母硒对低温应激黄颡鱼血糖含量的影响
如图2所示,22℃条件下,酵母硒组黄颡鱼血糖含量显著高于Na2SeO3组(P<0.05),以Se-Y1组最高。13℃条件下,黄颡鱼血糖含量较22℃出现降低,酵母硒组黄颡鱼血糖含量显著高于亚硒酸组(P<0.05)。一般认为,血糖含量较高时,鱼类表现为摄食积极,健康状况良好,这说明酵母硒与亚硒酸相比利于葡萄糖的生成,获得最佳生长。在13℃条件下,黄颡鱼血糖含量较22℃出现明显降低,说明此时黄颡鱼已经消耗了大量的血糖以抵御低温应激,低温下黄颡鱼首先利用糖类作为能量来源。同时,发现在13℃条件下,酵母硒组黄颡鱼血糖含量显著高于亚硒酸组,说明亚硒酸钠组血糖的消耗要比酵母硒组的快,血糖的大量消耗易造成营养匮乏,不利于黄颡鱼低温应激后的恢复。
实施例5亚硒酸钠和酵母硒对低温应激黄颡鱼血清尿素氮含量的影响
如图3所示,22℃条件下,Na2SeO3和酵母硒对黄颡鱼血清尿素氮含量无显著影响(P>0.05),13℃条件下,Se-Y1组黄颡鱼血清尿素氮含量较22℃条件下降低,并显著低于Na2SeO3组(P<0.05)。这可能是在低温状况下,鱼类通过降低蛋白质的合成分解速率,降低耗能过程,抵御温度应激。
实施例6亚硒酸钠和酵母硒对低温应激黄颡鱼FAS mRNA表达量的影响
如图4所示,22℃条件下,Se-Y1组黄颡鱼肝脏FAS mRNA表达量显著高于其他组(P<0.05),13℃条件下,各组黄颡鱼肝脏FAS mRNA表达量无显著差异(P>0.05)但各组表达量比22℃时显著降低。这说明包括亚硒酸钠在内的其余各组黄颡鱼对低温应激表现的更为敏感,Se-Y1能使黄颡鱼保持相对稳定的状态。
根据上述说明书的揭示和指导,本发明所属领域的技术人员还可以对上述实施方式进行适当的变更和修改。因此,本发明并不局限于上面揭示和描述的具体实施方式,对本发明的一些修改和变更也应当落入本发明的权利要求的保护范围内。此外,尽管本说明书中使用了一些特定的术语,但这些术语只是为了方便说明,并不对本发明构成任何限制。
Claims (7)
1.硒在提高黄颡鱼生长性能中的应用。
2.硒在提高黄颡鱼抗氧化能力中的应用。
3.硒在提高黄颡鱼抗低温应激能力中的应用。
4.根据权利要求1或2或3所述的应用,其特征在于,所述硒包括无机硒和有机硒。
5.根据权利要求4所述的应用,其特征在于,所述无机硒为亚硒酸钠;所述有机硒包括硒半胱氨酸、硒蛋氨酸、酵母硒。
6.一种饲料添加剂,其特征在于,包括无机硒和有机硒,其中,所述无机硒为亚硒酸钠;所述有机硒包括硒半胱氨酸、硒蛋氨酸、酵母硒。
7.权利要求6所述饲料添加剂在提高黄颡鱼生长性能、抗氧化能力或抗低温应激能力中的应用。
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