CN111057676A - 利于镜下观察鱼类胚胎发育过程的受精卵处理方法 - Google Patents
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Abstract
本发明公开一种利于镜下观察鱼类胚胎发育过程的受精卵处理方法，按照如下步骤进行：将多个鱼受精卵暂养于培养皿中；取透明液加入烧杯后加水搅拌均匀得稀释液，透明液与水的体积比为3：5，所述透明液是用硬度低于8的软化水与表面活性剂混合而成，软化水与表面活性剂的用量比为10:1；取1～3个暂养的鱼受精卵放到另一培养皿中，倒入稀释液淹没鱼受精卵，静置30～60s。操作简单，条件可控，受外界环境影响小，经本发明处理的拉式鱥、唇䱻及北方须鳅等鱼受精卵的卵膜变得透明清晰、无灰尘且胚胎不易破碎，可以在显微镜下清晰地观察到完整胚胎具体的发育分期并得到对应的鱼类胚胎发育图。
Description
技术领域
本发明属于鱼类胚胎发育观察技术领域，尤其是一种利于镜下观察鱼类胚胎发育过程的受精卵处理方法。
背景技术
鱼类胚胎发育一直是发育生物学、组织胚胎学、鱼类养殖学等学科的重点研究内容。深入细致地研究鱼类胚胎发育过程，有助于理解和认知鱼类的发育及进化过程等，便于在生产上指导鱼类的人工繁殖和孵化。目前，对于鱼类胚胎发育过程的观察方法通常是将内置于受精卵的培养皿置于显微镜或解剖镜等物镜下，先用4倍物镜找到要观察的鱼受精卵，然后换10倍物镜仔细观察，同时可通过与显微镜相连的专业照相系统得到各个时期及完整的鱼类胚胎发育图。然而，由于许多鱼类的卵（例如拉式鱥、唇䱻、北方须鳅等）具有卵膜厚以及黏性（卵膜表面容易粘附灰尘）等特点，无法直接在显微镜下清楚地观察到胚胎发育过程。为此，有的方法是在镜下观察前用胰蛋白酶等处理受精卵，但是胚胎极易破碎。
发明内容
本发明是为了解决现有技术所存在的上述技术问题，提供一种利于镜下观察鱼类胚胎发育过程的受精卵处理方法。
本发明的技术解决方案是：一种利于镜下观察鱼类胚胎发育过程的受精卵处理方法，其特征在于按照如下步骤进行：
a. 将多个鱼受精卵暂养于培养皿中；
b. 取透明液加入烧杯后加水搅拌均匀得稀释液，透明液与水的体积比为3：5，所述透明液是用硬度低于8的软化水与表面活性剂混合而成，软化水与表面活性剂的用量比为10:1
c. 取1～3粒暂养的鱼受精卵放到另一培养皿中，倒入稀释液淹没鱼受精卵，静置30～60s。
所述表面活性剂应能使溶液体系的界面状态发生明显变化，可以采用直链烷基苯磺酸钠（LAS）、脂肪醇聚氧乙烯醚硫酸钠（AES）、脂肪醇聚氧乙烯醚硫酸铵（AESA）、月桂醇硫酸钠（K12或SDS）中的至少一种。
本发明操作简单，条件可控，受外界环境影响小，经本发明处理的拉式鱥、唇䱻及北方须鳅等鱼受精卵的卵膜变得透明清晰、无灰尘且胚胎不易破碎，可以在显微镜下清晰地观察到完整胚胎具体的发育分期并得到对应的鱼类胚胎发育图。
附图说明
图1是本发明对比例的拉式鱥鱼胚胎镜下图。
图2是本发明实施例的拉式鱥鱼胚胎镜下图。
具体实施方式
本发明的利于镜下观察鱼类胚胎发育过程的受精卵处理方法，按照如下步骤进行：
a. 将多个鱼受精卵暂养于培养皿中：
在本溪市桓仁县养殖拉式鱥，待雌性卵巢发育成熟后将卵挤出，同时挤出成熟雄鱼的精液，进行人工受精。将受精完成的受精卵放在培养皿中，加入适量的水冲开，一个培养皿中放置适量（不可过多）的鱼卵，且需要经常换水保证水质健康，使得胚胎发育状况良好。
b. 取30ml透明液加入烧杯后加入50ml水搅拌均匀得稀释液，透明液是用硬度低于8的软化水与表面活性剂-直链烷基苯磺酸钠（LAS）混合而成，软化水与直链烷基苯磺酸钠（LAS）的用量比为10:1。
c. 取3个暂养的鱼受精卵放到另一培养皿中，倒入稀释液淹没鱼受精卵，静置40s，此时肉眼可以看到培养皿中鱼受精卵由之前的灰色变得光滑透明。
实验：
取4个a步骤暂养于培养皿中受精卵放到另一培养皿中并倒入与稀释液等量的纯水淹没作为对比例。
将对比例的受精卵培养皿及本发明处理后的受精卵培养皿分别置于显微镜下观察，先用4倍显微镜找到要观察的鱼卵，然后换10倍显微镜仔细观察。对比例10倍显微镜镜下图如图1所示，本发明实施例10倍显微镜镜下图如图2所示。从图1、图2可以看出，未经本发明处理的拉式鱥鱼受精卵在10倍显微镜下，只能看的卵膜表面模糊的形状，看不到内部胚胎发育情况；而经过本发明处理的拉式鱥鱼受精卵在10倍显微镜下，可以清晰看到内部胚胎发育情况且胚胎完整。
Claims (2)
1.一种利于镜下观察鱼类胚胎发育过程的受精卵处理方法，其特征在于按照如下步骤进行：
a. 将多个鱼受精卵暂养于培养皿中；
b. 取透明液加入烧杯后加水搅拌均匀得稀释液，透明液与水的体积比为3：5，所述透明液是用硬度低于8的软化水与表面活性剂混合而成，软化水与表面活性剂的用量比为10:1；
c. 取1～3个暂养的鱼受精卵放到另一培养皿中，倒入稀释液淹没鱼受精卵，静置30～60s。
2.根据权利要求1所述的利于镜下观察发育过程的鱼类胚胎处理方法，其特征在于所述表面活性剂为直链烷基苯磺酸钠、脂肪醇聚氧乙烯醚硫酸钠、脂肪醇聚氧乙烯醚硫酸铵和月桂醇硫酸钠中的至少一种。
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| CN113201485A (zh) * | 2021-05-10 | 2021-08-03 | 浙江省淡水水产研究所 | 一种利于镜下观察胚胎发育的鱼类粘性受精卵处理方法 |
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| SU733588A1 (ru) * | 1977-12-14 | 1980-05-15 | Всесоюзный научно-исследовательский институт прудового рыбного хозяйства | Способ обесклеивани оплодотворенной икры рыб |
| CN108251356A (zh) * | 2017-12-29 | 2018-07-06 | 中山大学 | 一种抗鱼类神经坏死病毒的鱼卵清洗液 |
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| SU733588A1 (ru) * | 1977-12-14 | 1980-05-15 | Всесоюзный научно-исследовательский институт прудового рыбного хозяйства | Способ обесклеивани оплодотворенной икры рыб |
| CN108251356A (zh) * | 2017-12-29 | 2018-07-06 | 中山大学 | 一种抗鱼类神经坏死病毒的鱼卵清洗液 |
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