CN104304236A - 翘嘴红鲌精液冷冻保存液 - Google Patents
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Abstract
本发明涉及一种翘嘴红鲌精液冷冻保存液,该冷冻保存液以稀释液为基质,每1000mL冷冻保存液中含有34.23g海藻糖和2.9g谷氨酰胺,并含有8%(体积百分数)乙二醇。采用本发明所述的冷冻保存液,翘嘴红鲌精液解冻后,精子的活率在70%以上,经过冷冻保存及复苏后的冻精人工授精得到的受精卵与鲜精人工授精得到的受精卵相比,平均孵化率无显著差异。
Description
技术领域
本发明涉及一种翘嘴红鲌精液冷冻保存液,属于水产养殖技术领域。
背景技术
精子在液氮超低温(-196℃)条件下细胞的代谢基本停止,处于假死状态,可以长期保存。鱼类精子的低温冷冻保存对于鱼类繁殖育种、资源保护和遗传改良都具有重要意义。一些鱼类物种特异的精子冷冻方案已经建立。鱼类精子冷冻首先要将精液稀释于冷冻保存液中,平衡一段时间后再进行降温冷冻。冷冻保存液除了无机盐(如NaCl、KCl、NaHCO3、KHCO3和柠檬酸钠等)和渗透性防冻剂之外,往往还会添加一些非渗透性抗冻剂。非渗透性抗冻剂是一类不能透过细胞膜的大分子物质,主要有糖类,如葡萄糖、果糖、蔗糖、海藻糖、棉籽塘、麦芽糖等;脂类,如磷脂、胆固醇等;蛋白质类,如卵黄、脱脂奶粉、小牛血清、抗冻蛋白等,以及其他添加剂,如氨基酸、抗生素、抗氧化剂等。冷冻保存液的成分到目前为止仍然没有一个统一的定论,一般认为应根据不同鱼类精子特性进行逐个试验筛选。而不同的非渗透性抗冻剂及不同使用浓度,对不同物种精子冷冻保存,具有不同的作用。
翘嘴红鲌是我国淡水水域常见的一种大型淡水经济鱼类。其肉洁白鲜嫩,营养价值较高,是肉食性名贵鱼类,具有很高的经济价值和生态价值。有关翘嘴红鲌精子冷冻保存过程中各个环节的研究在国内外未见报道,本发明涉及的翘嘴红鲌精液冷冻保存液也是无意中的发现,从而完善了翘嘴红鲌的精子冷冻保存方案,具有实际应用和推广价值。
发明内容
本发明的目的是克服现有技术的不足,提供一种可以降低冷冻对精子的损伤同时可以提高冻精活率的翘嘴红鲌精液冷冻保存液。
本发明解决所述技术问题的方案是:一种翘嘴红鲌精液冷冻保存液,所述冷冻保存液中含有海藻糖和谷氨酰胺。
作为进一步的技术方案,所述的冷冻保存液以稀释液为基质,每1000mL冷冻保存液中含有34.23g海藻糖和2.9g谷氨酰胺。
作为进一步的技术方案,所述冷冻保存液中,含有8%(体积百分数)乙二醇。
作为进一步的技术方案,所述的稀释液为HBSS稀释液。
作为进一步的技术方案,所述稀释液的组成为,
每1000mL稀释液中含有
本发明的有益效果:采用本发明所述的冷冻保存液,翘嘴红鲌精液解冻后,精子的活率在70%以上,经过冷冻保存及复苏后的冻精人工授精得到的受精卵与鲜精人工授精得到的受精卵相比,平均孵化率无显著差异。
具体实施方式
实施例1
一、配制含海藻糖和谷氨酰胺的翘嘴红鲌精液冷冻保存液
配制稀释液:准确称取氯化钠7.896g、氯化钾0.396g、七水合硫酸镁0.195g、十二水合磷酸氢二钠0.072g、磷酸二氢钾0.054g、碳酸氢钠0.345g3、葡萄糖0.99g溶于1000mL蒸馏水中,充分混匀,分装至离心管中,放到冰箱里冷冻备用。
配制冷冻保存液:在配制好的HBSS稀释液中加入防冻液乙二醇至终浓度为8%(体积百分数),海藻糖和谷氨酰胺的终浓度分别为34.23g/L和2.9g/L。即翘嘴红鲌精液冷冻保存液每1000mL组成如下:
乙二醇 80mL
海藻糖 34.23g
谷氨酰胺 2.9g
适用于翘嘴红鲌精液冷冻保存液的稀释液即HBSS稀释液补足至1000mL。
为说明该冷冻保存液的效果,配置3种用于对比的冷冻保存液。
第一种对比的冷冻保存液每1000mL组成如下:
乙二醇 80mL
第二种对比的冷冻保存液每1000mL组成如下:
乙二醇 80mL
海藻糖 34.23g
第三种对比的冷冻保存液每1000mL组成如下:
乙二醇 80mL
谷氨酰胺 2.9g
二、获取可用的精液
选取性成熟的雄性翘嘴红鲌亲鱼,轻压腹部使白色精液流出,精液采集后应立即放在4℃冰箱内,防止精子活力下降,同时对采集的多管精液进行精子活力鉴定,具体步骤为在载玻片上滴10μl纯水,取5μl鲜精于纯水中,立即用显微镜观察其活力,精子活力在90%以上的精液用于精子冷冻。
三、精液的冷冻
将步骤一得到的4种冷冻保存液分别装至4个试管中,放在4℃冰箱中平衡30min,平衡时将步骤二得到的精液平均分装到4个试管中,然后再把每个管中管中的精液平均的打入到六根麦管中,待30min的平衡时间过去后,同时将麦管放于离液氮表面7cm处熏蒸10min。熏蒸结束后,把麦管丢到液氮里保存备用。
四、精液的解冻
为了检测本发明专利的实施效果,我们将冷冻的麦管置于37℃水浴锅中解冻6s,解冻后在显微镜下检测。实验证明仅添加乙二醇的对照组精子的活率为47.50%,添加了海藻糖和乙二醇的实验组,精子的活率为60.50%,添加了谷氨酰胺和乙二醇的实验组49.17%,而同时添加了乙二醇、海藻糖和谷氨酰胺的实验组却高达72.33%,具体如下:
事实证明含同时海藻糖和谷氨酰胺的翘嘴红鲌精液冷冻保存液的确有很好的效果。
五、利用解冻的精液进行人工授精
采取干法授精,将鲜精、经过冷冻保存及复苏后的冻精分别与翘嘴红鲌鱼卵充分混合,加入适量池塘水激活,搅拌1min进行人工授精,黄泥浆脱粘后用清水漂洗2遍,然后将受精卵置于斑马鱼养殖系统1.5L养殖盒,在水温26℃条件下孵化。重复3次,每次随机选取100颗受精卵,统计孵化率,结果如下:
翘嘴红鮊鲜精和冷冻精液人工授精的孵化率实验结果
结果表明:经过冷冻保存及复苏后的冻精人工授精的平均孵化率为49.19%,与鲜精工授精的受精卵的平均孵化率50.83%,无显著差异,证明本方案可行。
Claims (5)
1.一种翘嘴红鲌精液冷冻保存液,其特征在于:所述冷冻保存液中含有海藻糖和谷氨酰胺。
2.如权利要求1所述的翘嘴红鲌精液冷冻保存液,其特征在于:所述的冷冻保存液以稀释液为基质,每1000mL冷冻保存液中含有34.23g海藻糖和2.9g谷氨酰胺。
3.如权利要求2所述的翘嘴红鲌精液冷冻保存液,其特征在于:所述冷冻保存液中,含有8%(体积百分数)乙二醇。
4.如权利要求2所述的翘嘴红鲌精液冷冻保存液,其特征在于:所述的稀释液为HBSS稀释液。
5.如权利要求4所述的翘嘴红鲌精液冷冻保存液,其特征在于:所述稀释液的组成为,
每1000mL稀释液中含有
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN105494314A (zh) * | 2015-12-17 | 2016-04-20 | 武汉先锋水产科技有限公司 | 一种黑尾近红鲌精子保存方法 |
CN113558044A (zh) * | 2021-08-28 | 2021-10-29 | 上海海洋大学 | 一种翘嘴鲌精子冷冻保存技术 |
CN113647381A (zh) * | 2021-08-31 | 2021-11-16 | 上海海洋大学 | 一种蒙古鲌精子冷冻保存技术 |
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JP2005261413A (ja) * | 2004-03-19 | 2005-09-29 | Yamaguchi Technology Licensing Organization Ltd | 動物胚凍結保存液とそれを用いた動物胚凍結保存法 |
JP2005270006A (ja) * | 2004-03-25 | 2005-10-06 | Kyudo Kk | 精子凍結保存剤 |
EP1082006B1 (en) * | 1998-05-26 | 2006-02-01 | Lifecell Corporation | Cryopreservation of human red blood cells |
CN101720753A (zh) * | 2009-12-09 | 2010-06-09 | 中国人民解放军第四军医大学 | 组织工程产品的低温保存液及其使用方法 |
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2014
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EP1082006B1 (en) * | 1998-05-26 | 2006-02-01 | Lifecell Corporation | Cryopreservation of human red blood cells |
JP2005261413A (ja) * | 2004-03-19 | 2005-09-29 | Yamaguchi Technology Licensing Organization Ltd | 動物胚凍結保存液とそれを用いた動物胚凍結保存法 |
JP2005270006A (ja) * | 2004-03-25 | 2005-10-06 | Kyudo Kk | 精子凍結保存剤 |
CN101720753A (zh) * | 2009-12-09 | 2010-06-09 | 中国人民解放军第四军医大学 | 组织工程产品的低温保存液及其使用方法 |
Cited By (3)
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CN105494314A (zh) * | 2015-12-17 | 2016-04-20 | 武汉先锋水产科技有限公司 | 一种黑尾近红鲌精子保存方法 |
CN113558044A (zh) * | 2021-08-28 | 2021-10-29 | 上海海洋大学 | 一种翘嘴鲌精子冷冻保存技术 |
CN113647381A (zh) * | 2021-08-31 | 2021-11-16 | 上海海洋大学 | 一种蒙古鲌精子冷冻保存技术 |
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