CN108157238B - 一种新的大黄鱼应激的监测方法 - Google Patents
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Abstract
本发明公开了一种新的大黄鱼应激的监测方法，方法基于大黄鱼个体左右肝叶对饥饿的不同响应来判断大黄鱼是否处于饥饿应激状态。有益效果为：本发明所用方法无需参考对照组，简单快捷，同时避免了污染组和对照组指示生物个体的差异对结果的影响，大大提高了监测的准确度和可靠性。
Description
技术领域
本发明涉及生物技术领域，尤其是涉及一种新的大黄鱼应激的监测方法。
背景技术
大黄鱼是我国重要的海水养殖鱼类。在养殖过程中，捕捞、分鱼、投喂等操作可能会对大黄鱼造成应激。应激的大黄鱼抗病能力减弱，很容易死亡。因此，时刻监测大黄鱼的应激状态十分重要。传统的监测方法为：比较正常和受应激的大黄鱼氧化应激相关指标，观察这些指标是否异常，指标异常则认定生物处于应激状态。这种方法必须考虑对照组的影响，因此具有很大的局限性。另一方面，污染组和正常组的指示生物来源于不同的个体，个体的差异势必会影响到组间的差异，因此降低了试验的重复性，从而影响评判结果的可靠性。
发明内容
本发明的目的在于提供一种新的大黄鱼应激的监测方法，该方法消除了对照组的背景差异，结果测定准确，使监测更加可靠。
本发明为实现上述目的所采取的技术方案为：一种新的大黄鱼应激的监测方法，方法基于大黄鱼个体左右肝叶对饥饿的不同响应来判断大黄鱼是否处于饥饿应激状态。与传统方法相比，该方法无需参考对照组，简单快捷，同时避免了污染组和对照组指示生物个体的差异对结果的影响，大大提高了监测的准确度和可靠性。
作为优选，监测方法具体过程为：获取大黄鱼的左右肝叶，分别测量谷胱甘肽还原酶的活性，若左右肝叶谷胱甘肽还原酶的活性差异在2.8U/mg以上，则大黄鱼处于饥饿应激状态。当大黄鱼处于非应激状态时，其左右肝叶谷胱甘肽还原酶的活性几乎没有差异，而处于饥饿状态时，其左右肝叶谷胱甘肽还原酶的活性具有显著差异，基于这种差异能够有效地判断大黄鱼是否处于饥饿应激状态。
作为优选，监测方法具体过程为：获取大黄鱼的左右肝叶，分别测量过氧化氢酶的活性，若左右边肝叶过氧化氢酶的活性差异在5.3U/mg以上，则大黄鱼处于饥饿应激状态。大黄鱼肝脏中的过氧化氢酶能专一地将细胞代谢产生的过氧化氢分解成水和氧气，避免过氧化氢在体内的累积，维持体内正常的活性氧水平，而外界环境的刺激具有诱导或抑制过氧化氢酶活性的作用，通过比较左右肝叶中过氧化氢酶的活性差异，能够准确地判断大黄鱼是否处于饥饿应激状态。
作为优选，监测方法具体过程为：获取大黄鱼的左右肝叶，分别测量粗脂肪含量，若左右肝叶粗脂肪含量差异在6％以上，则大黄鱼处于饥饿应激状态。通过比较左右肝叶的粗脂肪含量差异能够快速地监测大黄鱼是否处于应激状态，缩短了监测的时间，降低了成本，具有良好的经济效益。
与现有技术相比，本发明的有益效果为：本发明所用方法无需参考对照组，简单快捷，同时避免了污染组和对照组指示生物个体的差异对结果的影响，大大提高了监测的准确度和可靠性；当大黄鱼处于非应激状态时，其左右肝叶谷胱甘肽还原酶的活性几乎没有差异，而处于饥饿状态时，其左右肝叶谷胱甘肽还原酶的活性具有显著差异，基于这种差异能够有效地判断大黄鱼是否处于饥饿应激状态；通过比较左右肝叶中过氧化氢酶的活性差异，能够准确地判断大黄鱼是否处于饥饿应激状态；通过比较左右肝叶的粗脂肪含量差异能够快速地监测大黄鱼是否处于应激状态，缩短了监测的时间，降低了成本，具有良好的经济效益。
具体实施方式
以下结合实施例作进一步详细描述：
实施例1：一种新的大黄鱼应激的监测方法，包括以下步骤：大黄鱼取自当地渔场，室内维持在500L的塑料桶中，适应2周，每天投喂商业饲料(粗蛋白48％，粗脂肪11％)，一天投喂2次(早9点，下午4点)，实验开始时，选取体重为110-130g的大黄鱼饥饿4天，对照组正常投喂，其中水温25.9±2.7℃，光周期14L:10D，溶解氧6.43±0.56mg/L，pH值7.49±0.52，盐度32.6±1.5，硬度125mg/LCaCO3；获取大黄鱼的左右肝叶，分别测定其谷胱甘肽还原酶的活性，结果如表1所示，由表可知，当大黄鱼受到饥饿应激4天后，左右肝叶谷胱甘肽还原酶的活性具有显著差异，差异为2.8U/mg。
表1谷胱甘肽还原酶的活性
实施例2：一种新的大黄鱼应激的监测方法，包括以下步骤：大黄鱼取自当地渔场，室内维持在500L的塑料桶中，适应2周，每天投喂商业饲料(粗蛋白48％，粗脂肪11％)，一天投喂2次(早9点，下午4点)，实验开始时，选取体重为110-130g的大黄鱼饥饿4天，对照组正常投喂，其中水温25.9±2.7℃，光周期14L:10D，溶解氧6.43±0.56mg/L，pH值7.49±0.52，盐度32.6±1.5，硬度125mg/LCaCO3；获取大黄鱼的左右肝叶，分别测定其过氧化氢酶的活性，结果如表2所示，由表可知，当大黄鱼受到饥饿应激4天后，左右肝叶谷过氧化氢酶的活性具有显著差异，差异为5.3U/mg。
表2过氧化氢酶的活性
实施例3：一种新的大黄鱼应激的监测方法，包括以下步骤：大黄鱼取自当地渔场，室内维持在500L的塑料桶中，适应2周，每天投喂商业饲料(粗蛋白48％，粗脂肪11％)，一天投喂2次(早9点，下午4点)，实验开始时，选取体重为110-130g的大黄鱼饥饿4天，对照组正常投喂，其中水温25.9±2.7℃，光周期14L:10D，溶解氧6.43±0.56mg/L，pH值7.49±0.52，盐度32.6±1.5，硬度125mg/LCaCO3；获取大黄鱼的左右肝叶，测量粗脂肪含量，结果如表3所示，由表3可知，当大黄鱼受到饥饿应激4天后，大黄鱼右肝叶的粗脂肪含量显著低于左肝叶的粗脂肪含量，其差值为6％。
表3左右肝叶粗脂肪含量
实施例4：一种新的大黄鱼应激的监测方法，包括以下步骤：一种新的大黄鱼应激的监测方法，包括以下步骤：大黄鱼取自当地渔场，室内维持在500L的塑料桶中，适应2周，每天投喂商业饲料(粗蛋白48％，粗脂肪11％)，一天投喂2次(早9点，下午4点)，实验开始时，选取体重为110-130g的大黄鱼饥饿6天，对照组正常投喂，其中水温25.9±2.7℃，光周期14L:10D，溶解氧6.43±0.56mg/L，pH值7.49±0.52，盐度32.6±1.5，硬度125mg/LCaCO3；获取大黄鱼的左右肝叶，分别测定其过氧化氢酶的活性，结果如表4所示，由表可知，当大黄鱼受到饥饿应激6天后，左右肝叶谷过氧化氢酶的活性具有显著差异，差异为5.6U/mg。
表4过氧化氢酶的活性
其中，过氧化氢酶的活性测定方法为：分别称取1g左右肝叶，剪碎，加入9g浓度为0.155mol/L的KCl溶液，用研钵在冰浴中研磨5min，匀浆液在4℃、8000r/min下离心13min，取其上清液，最后用生理盐水稀释10倍，制得粗酶液，反应过程中按表5要求依次加入各试剂，加入基质液后置于25℃水浴5min，加入酶液后将所加过试剂的试管在25℃水浴60s，反应结束，立即向测定管中加入2.3g钼酸铵摇匀，再加入0.4μg绿原酸和0.63μg二苯基甲醇摇匀，10min后在405nm处以去离子水调零比色，记录各组吸光度，并计算酶活，所加入的绿原酸、二苯基甲醇与钾离子具有协同作用，增强了体系中残留的过氧化氢与钼酸铵所生成的黄色络合物的稳定性，提高了该络合物在405nm处所测的吸光度的精确度，从而使酶活性的测定结果更加准确，使监测方法更具可靠性。
表5可见分光光度法测定所需试剂的加样量
| 试剂 | 测定管 | 标准管 | 对照管 |
| 缓冲液(mL) | / | 0.2 | 1 |
| 基质液(mL) | 1 | 1 | 1 |
| 粗酶液(mL) | 0.2 | / | 0.2 |
以上实施方式仅用于说明本发明，而并非对本发明的限制，本领域的普通技术人员，在不脱离本发明的精神和范围的情况下，还可以做出各种变化和变型。因此，所有等同的技术方案也属于本发明的范畴，本发明的专利保护范围应由权利要求限定。
Claims (7)
1.一种大黄鱼应激的监测方法，其特征在于：所述方法基于大黄鱼个体左右肝叶对饥饿的不同响应来判断大黄鱼是否处于饥饿应激状态。
2.根据权利要求1所述的一种大黄鱼应激的监测方法，其特征在于：所述响应为肝叶的谷胱甘肽还原酶的活性。
3. 根据权利要求2所述的一种大黄鱼应激的监测方法，其特征在于：所述方法具体过程为：获取大黄鱼的左右肝叶，分别测量谷胱甘肽还原酶的活性，若左右肝叶谷胱甘肽还原酶的活性差异在2.8 U/mg以上，则大黄鱼处于饥饿应激状态。
4.根据权利要求1所述的一种大黄鱼应激的监测方法，其特征在于：所述响应为过氧化氢酶的活性。
5. 根据权利要求4所述的一种大黄鱼应激的监测方法，其特征在于：所述方法具体过程为：获取大黄鱼的左右肝叶，分别测量过氧化氢酶的活性，若左右边肝叶过氧化氢酶的活性差异在5.3 U/mg以上，则大黄鱼处于饥饿应激状态。
6.根据权利要求1所述的一种大黄鱼应激的监测方法，其特征在于：所述响应为肝叶的脂肪含量。
7.根据权利要求6所述的一种大黄鱼应激的监测方法，其特征在于：所述方法具体过程为：获取大黄鱼的左右肝叶，分别测量粗脂肪含量，若左右肝叶粗脂肪含量差异在6％以上，则大黄鱼处于饥饿应激状态。
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