CN107333689B - 一种提高卤虫生长和epa含量的营养强化方法 - Google Patents
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Abstract
本发明公开了一种提高卤虫生长和EPA含量的营养强化方法。在卤虫发育至3龄期间,持续投喂扁藻可有效提高卤虫生长和EPA含量。本发明所述方法能够提高藻类饵料的利用率,提高了卤虫的生长质量,为水产养殖提供了优质的饵料。
Description
技术领域
本发明属于水产养殖业和生物研究领域,具体涉及一种提高卤虫生长和EPA含量的营养强化方法。
背景技术
卤虫(Artemia sp.)隶属于甲壳纲鳃足亚纲无甲目,具有广泛地理分布和耐盐耐温特性。因其具有良好的适口性以及较高的营养价值,卤虫幼体和成体被作为水产动物苗种生产中的主要饵料。与卤虫卵营养成分不同,孵化后的卤虫幼体和成体的蛋白含量和脂肪酸含量随着发育的行进发生变化。该营养成分因个体发育的自然改变,影响其在水产动物苗种生产中提高存活力和产量的稳定性。因此,人们尝试不同强化饵料以期提高卤虫的蛋白质和脂肪酸含量。
卤虫初孵无节幼体的蛋白含量在40%以上,脂肪含量在20%以上(干重),因不同品种和不同的测定方法略有差异。卤虫蛋白组成中必须氨基酸约占总氨基酸含量的0.48%,但其中的蛋氨酸含量较少。虽然卤虫不饱和脂肪酸含量较高,但孵化后的无节幼体均含少量的18:3n-3和20:4n-6,并缺乏22:6n-3。与其它饵料动物相比,卤虫幼体粗蛋白含量适中,低于短额刺糠虾,但高于强壮藻钩虾等。虽然卤虫幼体的总脂肪含量较高,但其中的长链不饱和脂肪酸含量远远低于其它饵料动物,尤其是EPA和DHA含量,原因是卤虫幼体体内易于合成短链不饱和脂肪酸。因此,生产中常常采用营养强化方式补充卤虫幼体中的蛋白质和长链不饱和脂肪酸含量。但生化检测结果表明,不同饵料的强化饲养,导致卤虫幼体体内蛋白质、脂肪(酸)和碳水化合物的含量发生非均衡性变化。文献表明,微藻强化投喂后卤虫蛋白含量、脂肪酸含量以及碳水化合物含量之间存在负相关性变化(Fábregas J,Otero A,Domínguez A,Growth rate of the microalga tetraselmis suecicachanges the biochemical composition of Artemia species[J].MarineBiotechnology(New York),2001,3(3):256-263)。因此采用合适的饵料有针对性地提高卤虫幼体体内某一成分是营养强化卤虫的根本途径。
发明内容
本发明考察了微藻强化投喂对卤虫生长和营养价值的影响,选取了三种常用微藻对孵化卤虫进行了投喂,并对其生物学长度、蛋白质含量、EPA(二十碳五烯酸)和DHA(二十二碳六烯酸)含量进行了分析,提供了一种提高卤虫生长和EPA含量的营养强化方法。
本发明的具体技术方案如下:
一种提高卤虫生长和EPA含量的营养强化方法,在卤虫发育至3龄期间(培养时间72h),投喂扁藻。所述扁藻优选亚心形扁藻Platymonas subcordiformis。
优选投喂微藻密度:15×104cells/mL。
优选微藻饵料初次投喂量:0.5×104cells/mL。
本发明的另一目的在于提供扁藻在制备提高卤虫生长和EPA含量的营养强化饲料中的应用。优选扁藻为亚心形扁藻Platymonas subcordiformis。
可在卤虫发育至3龄期间,持续投喂含有扁藻的营养强化饲料。
本发明优点:
本发明所述方法能够提高藻类饵料的利用率,提高了卤虫的生长质量,为水产养殖提供了优质的饵料。
附图说明
图1为微藻营养强化对卤虫体长的影响。
图2为微藻营养强化对卤虫蛋白质含量的影响。
图3为微藻营养强化对卤虫EPA含量的影响。
具体实施方式
在本发明中所使用的术语,除非另有说明,一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例并参照数据进一步详细描述本发明。应理解,该实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。
在以下实施例中,未详细描述的各种过程和方法是本领域中公知的常规方法。下述实例中所用的材料、试剂、装置、仪器、设备等,如无特殊说明,均可从商业途径获得。
实施例1
本发明所用卤虫(Artemia sp.)由渤海湾盐田卤虫卵孵化。试验所用湛江叉鞭金藻(Dicrateria zhanjiangensis)、亚心形扁藻(Platymonas subcordiformis)和小球藻(Chlorella pyenoidosa)取自江苏省海洋水产研究所。
湛江叉鞭金藻、亚心形扁藻和小球藻培养基参照文献《生物饵料培养》(陈明耀.北京:中国农业出版社,1995:27-37),采用自然光照,水温24℃-26℃。经过4天培养,密度分别达到300×104cells/mL、15×104cells/mL和500×104cells/mL,作为微藻饵料。
卤虫孵化
100L孵化桶中加入盐度30‰的海水。准确称取卤虫卵10g放入孵化桶中充气孵化,孵化温度28℃。
卤虫接种
卤虫孵化后,分离并选出健康的无节幼体用于营养强化试验,卤虫初始接种密度为1000ind/L,置于40L白色塑料桶内进行培育,每天50%换水,实验过程采取微充气。
强化投喂
卤虫发育至3龄期(培养时间72h),采用不同微藻饵料营养强化试验。试验设4种饵料处理,以不投喂饵料组为对照,各设3个平行组。4种饵料分别为江叉鞭金藻、亚心形扁藻、小球藻、混合藻(湛江叉鞭金藻+亚心形扁藻+小球藻)。微藻饵料初次投喂量湛江叉鞭金藻3-4×104cells/mL;亚心形扁藻0.5×104cells/mL;小球藻8-10×104cells/mL;混合藻按照单种藻的1/3来投喂。定时测定饵料密度,及时补加微藻饵料。
1.卤虫的体长测量
每试验样品随机取30只卤虫,镜检测量幼体体长,计算平均体长。
2.蛋白质、EPA和DHA含量的测定
采收不同时间点的卤虫幼体,0.1M的PBS(pH7.4)冲洗3次,-20℃保存,用于后续检测。蛋白质含量测定采用凯氏定氮法(GB 5009.5-2010)。EPA、DHA含量测定:甲醇(3%浓硫酸)、正己烷与样品体积比例(10:10:1)加入反应器,70℃冷凝回流反应2h。冷却,倒入分液漏斗静置,取正己烷层,60℃旋转蒸发。用色谱纯正己烷定容,过膜进样。气相条件:色谱柱:HP-5MS(30m×0.25mm×0.25μm)。进样口温度:250℃,进样量1.0μL,不分流。载气:He,流速:1.2mL/min。升温程序:100℃(保持5min)以4℃/min升至240℃(保持20min)。MSD传输线250℃。质谱条件:电离源,EI离子源,离子源温度230℃,四极杆温度150℃,电离能量70eV,全扫描模式,m/z:50~500。定量方法:峰面积百分比法。
将测得的数据进行方差分析,并进行多重比较(P<0.01表示差异极显著,P<0.05表示差异显著)。
实施例2卤虫幼体不同发育时期体长化
卤虫的发育分期多以龄(instar)表示,性成熟前每蜕皮一次即为1龄,孵化卤虫在3龄期消化道才具备生理功能开始摄食。在卤虫发育一龄早期(培养24h)、一龄后期(培养34h)、三龄早期(培养54h)、三龄后期(培养72h)、培养96h、培养120h等6个时间点,测量幼体平均体长。结果表明,幼体在培养前3天体长增加较快,由0.432mm增加到0.808mm,平均日增长0.125mm;4天后增长幅度减小,达到体长平台期,结果如表1所示。
表1卤虫幼体不同时期体长
实施例3微藻营养强化对卤虫体长的影响
卤虫孵化发育至3龄期,采用湛江叉鞭金藻、亚心形扁藻和小球藻强化投喂卤虫24h和48h。保持饵料密度分别为3-4×104、0.5×104和8-10×104cells/mL。强化投喂24h后,卤虫幼体体长与对照组相比(未投喂组)略有增加,但没有显著差异(p>0.05);投喂48h后,卤虫幼体生长速度明显加快,体长显著增加,各投喂组体长极显著大于对照(p<0.01),在3种强化藻类中,投喂扁藻对卤虫幼体生长的促进作用最为明显,平均体长达到0.935mm;其次是小球藻,为0.882mm结果如表2和图1所示。结果提示,扁藻强化投喂对卤虫幼体生长促进作用最好。
表2微藻营养强化对卤虫体长的影响
实施例4微藻营养强化对卤虫蛋白质含量的影响
采用同样方法微藻强化投喂卤虫24h后,除扁藻组外,其他各组卤虫幼体中的蛋白含量略有下降,金藻组下降最为显著(p<0.05),而小球藻组和混合组则没有显著差异;投喂48h后,各组卤虫幼体中蛋白质含量除扁藻组外,下降均较为显著(p<0.05),结果如表3和图2所示。
表3微藻营养强化对卤虫蛋白质含量的影响
实施例5微藻营养强化对卤虫EPA和DHA含量的影响
不同微藻饵料投喂卤虫24h后,卤虫幼体中EPA含量发生变化,小球藻组卤虫幼体中EPA含量显著高于对照(p<0.05),而扁藻组显著低于对照(p<0.05),混合藻组极显著低于对照(p<0.01);微藻饵料投喂48h后,卤虫幼体中EPA含量显著增加,各投喂组EPA含量均极显著高于对照(p<0.01),其中扁藻组含量最高,为3.46%,其次是小球藻组,为3.36%,而混合藻组EPA含量升高幅度较低,为2.86%,结果如表4和图3所示。结果提示扁藻微藻营养强化有利于促进卤虫幼体EPA含量的增加。
表4微藻营养强化对卤虫EPA含量的影响
本发明研究表明,在促进卤虫幼体生长方面,效果为扁藻组>小球藻组>混合藻组>金藻组(48h);在提高EPA含量方面,扁藻组>小球藻组>金藻组>混合藻组(48h)。投喂上述饵料均会导致卤虫粗蛋白含量的降低。因此,在微藻营养强化卤虫幼体过程中,可适量补充高蛋白含量的其它饵料。近年来,随着水产养殖业的快速发展,对卤虫的需求量不断增加,本发明一种提高卤虫生长和EPA含量的营养强化方法。
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1.一种提高卤虫生长和EPA含量的营养强化方法,在卤虫发育至3龄期间,持续投喂亚心形扁藻Platymonas subcordiformis,投喂扁藻密度为15×104 cells/mL,初次投喂量为0.5×104cells/mL。
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