阅读说明：本技术 一种裸盖鱼***冷冻保存的方法 (Method for preserving sperm of naked-cap fish by freezing ) 是由 王伟 罗珺 张赛赛 李梦瑶 李雪洁 成智丽 于 2019-06-18 设计创作，主要内容包括：本发明公开了一种裸盖鱼精子冷冻保存的方法,包括精子的获取、培养液的制备、精子的冻存过程、精子的复苏与激活过程；针对性成熟的裸盖鱼,采用人工麻醉的方法获取精子；根据裸盖鱼的精子渗透压,制备精子培养液；将采集到的精液与培养液按1∶2的体积比混合制得混合液,然后加入混合液体积10％的二甲基亚砜制成精子冻存混合液；将混匀后的精子冻存混合液样品置于4℃冰箱中15min,接着于液氮罐瓶口处静置5min后向下移,液氮液面上方再置5min,液氮液面再置5min,然后浸入液氮中冷冻保存。本发明方法使裸盖鱼精子保存工作更加便捷有效,避免了裸盖鱼精子在体外快速失活和死亡,提高了精子的保存质量,延长了保存时间,使裸盖鱼精子在实际生产中随用随取。(The invention discloses a method for preserving sperm of a naked gesso by freezing, which comprises the steps of obtaining the sperm, preparing a culture solution, freezing the sperm, and recovering and activating the sperm; obtaining sperms of the sexually mature naked capers by adopting an artificial anesthesia method; preparing a sperm culture solution according to the sperm osmotic pressure of the bare cap fish; mixing the collected semen and the culture solution according to the volume ratio of 1: 2 to prepare a mixed solution, and then adding dimethyl sulfoxide with the volume of 10% of the mixed solution to prepare a sperm cryopreservation mixed solution; placing the mixed sperm frozen mixed solution sample in a refrigerator at 4 deg.C for 15min, standing at the bottle mouth of a liquid nitrogen tank for 5min, moving downwards, placing above the liquid level of the liquid nitrogen for 5min, placing the liquid level of the liquid nitrogen for 5min, and soaking in liquid nitrogen for freezing. The method of the invention leads the preservation work of the sperm of the naked caper to be more convenient and effective, avoids the rapid inactivation and death of the sperm of the naked caper in vitro, improves the preservation quality of the sperm, prolongs the preservation time and leads the sperm of the naked caper to be taken at any time in the actual production.)

1. A method for preserving sperm of naked-cover fish by freezing is characterized in that: the method mainly comprises the steps of sperm acquisition, preparation of a culture solution, a sperm cryopreservation process, and a sperm recovery and activation process;

the method comprises the following specific steps:

s1, obtaining sperms by adopting an artificial anesthesia method aiming at the sexually mature naked caper;

s2, preparing a sperm culture solution according to the sperm osmotic pressure of the bare cap fish;

s3, mixing the collected semen and the culture solution according to the volume ratio of 1: 2 to prepare a mixed solution, and then adding dimethyl sulfoxide with the volume of 10% of the mixed solution to prepare a sperm cryopreservation mixed solution;

s4, placing the mixed sperm cryopreservation mixed solution sample in a refrigerator at 4 ℃ for 15min, standing at the bottleneck of a liquid nitrogen tank for 5min, moving downwards, placing above the liquid level of the liquid nitrogen for 5min, placing the liquid level of the liquid nitrogen for 5min, and then immersing in the liquid nitrogen for cryopreservation;

s5, after being frozen and stored for 24 hours, taking out the frozen sperm stock solution sample from liquid nitrogen, unfreezing the frozen sperm stock solution sample for 15 minutes at room temperature, unfreezing the frozen sperm stock solution sample for 15 minutes at 10 ℃, activating the sperm by using the volume ratio of the culture solution to the frozen sperm stock solution of 2: 1, staining the sperm by using trypan blue staining solution and the activated sperm stock solution according to the volume ratio of 1: 9, observing and evaluating the sperm motility under a microscope, and counting by using a blood counting chamber.

2. The method of claim 1, wherein the preservation of sperm in a naked hood fish is performed by: the formula of the culture solution is as follows: 0.8-1.7 g of sodium chloride, 0.06-0.07 g of sodium bicarbonate, 0.064-0.069 g of anhydrous calcium chloride, 1.4-1.7 g of glucose, 0.04-0.06 g of Tris alkali, 0.4-0.5 g of fetal calf serum and 0.12-0.13 g of glutathione.

Technical Field

The invention relates to the field of biological sperm preservation, in particular to a fish sperm preservation method.

Background

The naked gay fish belongs to the subclasses of the radiata finfish, the order of sebastes, the family of sebastes and the genus of naked gay fish, and is distributed on the coasts of the Pacific ocean. As the economic fish inhabiting in the deep water, the naked canopy fish is large, white and tender in meat quality, rich in nutrition and extremely high in economic value. In fact, there have been reports of commercial exploitation of naked-cap fish as early as 1800 years. Currently, Okins, Japan, Canada and the United states all regard naked-cap fish as an important fishery resource, and research on its basic biology is gradually being conducted. The adult naked-cap fish is reported to have a length of 50-70 cm, a life of over 100 years (known as maximum 114 years), a rapid growth time of 5-7 years, wherein about 50% of individuals grow to sexual maturity, and 10-15 years of the adult naked-cap fish all grow to sexual maturity and the growth tends to be stopped. At present, researchers in the countries of America, Canada and the like develop related researches on the aspects of resource distribution, feeding habits, reproduction behaviors, nutritional requirements, chromosome karyotypes, environmental adaptability, industrial culture and the like, preliminarily master the reproduction habits of the bare-capped fish, and break through artificial breeding technologies of the bare-capped fish. The nude gehead fishes are introduced into China in 2013, and are successfully cultured in coastal areas in northern China, so that good raw materials are provided for the research of the fish species resources, and a good variety is provided for the industrial culture of marine fishes in China.

As is known, no report is found on a sperm preservation method of the naked-cap fish in China, and no suitable sperm cryopreservation method of the naked-cap fish exists, which is one of the technical bottlenecks for limiting artificial breeding of the naked-cap fish.

Disclosure of Invention

The invention aims to provide a method for preserving sperm of a bare cap fish by freezing, activating and reviving the sperm of the bare cap fish to obtain high-quality sperm, thereby providing technical support for further carrying out the breeding work of the bare cap fish, solving the problem of difficult sperm preservation in the breeding process of the bare cap fish and breaking through the bottleneck of artificial breeding of the bare cap fish.

In order to solve the technical problems, the method mainly comprises the steps of sperm acquisition, preparation of a culture solution, a sperm cryopreservation process, and a sperm recovery and activation process;

the method comprises the following specific steps:

s1, obtaining sperms by adopting an artificial anesthesia method aiming at the sexually mature naked caper;

s2, preparing a sperm culture solution according to the sperm osmotic pressure of the bare cap fish;

s3, mixing the collected semen and the culture solution according to the volume ratio of 1: 2 to prepare a mixed solution, and then adding dimethyl sulfoxide with the volume of 10% of the mixed solution to prepare a sperm cryopreservation mixed solution;

s4, placing the mixed sperm cryopreservation mixed solution sample in a refrigerator at 4 ℃ for 15min, standing at the bottleneck of a liquid nitrogen tank for 5min, moving downwards, placing above the liquid level of the liquid nitrogen for 5min, placing the liquid level of the liquid nitrogen for 5min, and then immersing in the liquid nitrogen for cryopreservation;

s5, after freezing and storing for 24h, taking out the sperm freezing solution sample from liquid nitrogen, unfreezing for 15min at room temperature, unfreezing for 15min in water at 10 ℃, activating the sperm by using the volume ratio of the culture solution to the frozen semen of 2: 1, staining the sperm by trypan blue staining solution and the activated semen according to the volume ratio of 1: 9, observing and evaluating the sperm motility under a microscope, and counting by using a blood counting chamber.

Further, the formula of the culture solution is as follows: 0.8-1.7 g of sodium chloride, 0.06-0.07 g of sodium bicarbonate, 0.064-0.069 g of anhydrous calcium chloride, 1.4-1.7 g of glucose, 0.04-0.06 g of Tris alkali, 0.4-0.5 g of fetal calf serum and 0.12-0.13 g of glutathione.

Compared with the prior art, the method has the beneficial effects that:

1. the preservation work of the sperm of the naked caper is more convenient and effective, the suitable preservation method avoids the rapid inactivation and death of the sperm of the naked caper in vitro, improves the preservation quality of the sperm, prolongs the preservation time and ensures that the sperm of the naked caper can be taken at any time in the actual production. Further perfects and promotes the breeding work of the bare-capped fish, and provides basic guarantee for the yield, biological diversity and sustainable development of fishery.

2. The long-term cryopreservation of the sperms has a decisive influence on the preservation of the germplasm of the sperms. Through the screening of multiple preservation solution formulas, the selected preservation solution formula enables the survival rate of the frozen and revived sperms to reach 60%.

3. In order to protect germ plasm resources of the bare-capped fish, the long-term cryopreservation technology of sperms of the bare-capped fish is explored and researched, so that the method has important significance for further understanding of basic life processes and artificial breeding of the bare-capped fish.

Detailed Description

Embodiments of the present invention are described in further detail below. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The method mainly comprises the steps of sperm acquisition, preparation of a culture solution, a sperm cryopreservation process, and a sperm recovery and activation process;