阅读说明：本技术 大鳞副泥鳅***超低温冷冻保存方法 (Ultralow-temperature cryopreservation method for paramisgurnus dabryanus semen ) 是由 陆专灵 韦友传 王大鹏 唐章生 乔瑞峰 高爽爽 雷坤 谢业扬 侯树鉴 卢智发 钟 于 2020-06-28 设计创作，主要内容包括：本发明公开了大鳞副泥鳅精液超低温冷冻保存方法,包括以下步骤：步骤一、将NaCl、KCl、CaCl<Sub>2</Sub>·2H<Sub>2</Sub>O、MgCl<Sub>2</Sub>·6H<Sub>2</Sub>O、NaHCO<Sub>3</Sub>按照浓度比依次为80:50:5:2:50进行混合,配置成稀释液,置于4℃保存,备用；步骤二、将大鳞副泥鳅精液与稀释液按照体积比为1:2～4进行混合,再加入体积分数为7.5％～12.5％的DMSO混匀,得到精液抗冻混合液,并分装于冻存管中；步骤三、将步骤二的冻存管先在液氮面上方6cm处平衡10min,接着在液氮面上平衡5min,最后投入液氮中冷冻保存。本发明使得精子活力,精子的寿命,细胞膜的完整率都得到提高,成为有效、持久的保存方法。(The invention discloses an ultralow temperature cryopreservation method for paramisgurnus dabryanus semen, which comprises the following steps: step one, adding NaCl, KCl and CaCl 2 ·2H 2 O、MgCl 2 ·6H 2 O、NaHCO 3 Mixing according to the concentration ratio of 80:50:5:2:50 in sequence to prepare a diluent, and storing at 4 ℃ for later use; mixing the paramisgurnus dabryanus semen with the diluent according to the volume ratio of 1: 2-4, adding DMSO with the volume fraction of 7.5% -12.5%, uniformly mixing to obtain a semen anti-freezing mixed solution, and sub-packaging in a freezing storage tube; and step three, balancing the freezing tube in the step two at a position 6cm above the liquid nitrogen surface for 10min, balancing on the liquid nitrogen surface for 5min, and finally putting into liquid nitrogen for freezing and preserving. The invention improves the sperm motility, the service life of the sperm and the integrity rate of the cell membrane, and becomes an effective and durable preservation method.)

1. The ultralow-temperature cryopreservation method for the paramisgurnus dabryanus semen is characterized by comprising the following steps of:

step one, adding NaCl, KCl and CaCl₂·2H₂O、MgCl₂·6H₂O、NaHCO₃Mixing according to the concentration ratio of 80:50:5:2:50 in sequence to prepare a diluent, and storing at 4 ℃ for later use;

mixing the paramisgurnus dabryanus semen with the diluent according to the volume ratio of 1: 2-4, adding DMSO with the volume fraction of 7.5% -12.5%, uniformly mixing to obtain a semen anti-freezing mixed solution, and sub-packaging in a freezing storage tube;

and step three, balancing the freezing tube in the step two at a position 6cm above the liquid nitrogen surface for 10min, balancing on the liquid nitrogen surface for 5min, and finally putting into liquid nitrogen for freezing and preserving.

2. The ultralow-temperature cryopreservation method of the paramisgurnus dabryanus semen as claimed in claim 1, further comprising the following steps:

and step four, taking the semen anti-freezing mixed solution out of the liquid nitrogen, balancing the semen anti-freezing mixed solution at a liquid nitrogen opening for 5min, and then unfreezing the semen anti-freezing mixed solution in a water bath at 37 ℃ until the semen anti-freezing mixed solution is melted.

3. The ultralow-temperature cryopreservation method for the paramisgurnus dabryanus semen according to claim 1, wherein in the second step, the volume ratio of the paramisgurnus dabryanus semen to the diluent is 1:3, and the volume fraction of DMSO is 10%.

4. The ultralow-temperature cryopreservation method of the paramisgurnus dabryanus semen as claimed in claim 1, wherein in the second step, the paramisgurnus dabryanus semen is collected in the following way: the male loach scalper is anesthetized by using MS-222 solution, the water of the fish body is wiped off by using a towel, and the abdomen is squeezed to collect semen.

5. The ultralow temperature cryopreservation method of the paramisgurnus dabryanus semen as claimed in claim 1, wherein before the paramisgurnus dabryanus semen is mixed with the diluent in the second step, the paramisgurnus dabryanus semen is placed in a centrifuge tube, the temperature is kept for 10min in a constant temperature water bath at 20 ℃, penicillin with the concentration of 50IU/mL is added and mixed, then the centrifugal rotation speed is controlled at 160rpm, the centrifugation treatment is carried out for 4min, the lower layer liquid is retained, glucose and glycerol are added into the lower layer liquid and mixed, and finally the mixed solution is mixed with the diluent, wherein 0.05g of glucose and 0.5mL of glycerol are added into each 10mL of the lower layer liquid.

6. The ultralow temperature cryopreservation method for the sperm of Paramisgurnus dabryanus as claimed in claim 1, wherein in the fourth step, during the thawing process, the cryopreservation tube in the water bath is fixed, and the water in the water bath is stirred at the rotating speed of 300 r/min.

Technical Field

The invention relates to the technical field of ultralow-temperature cryopreservation of semen, in particular to an ultralow-temperature cryopreservation method for paramisgurnus dabryanus semen.

Background

Paramisgurnus dabryanus (Paramisgurnus dabryanus) belongs to the genus of Paramisgurnus dabryanus belonging to the order of Cyprinidae, and is naturally distributed in Yangtze river, Jialingjiang river, Minjiang river system, Liaohe river middle and lower reaches, yellow river and Heilongjiang areas, the Paramisgurnus dabryanus has delicious meat quality and rich vitamins and unsaturated fatty acids, and the Paramisgurnus dabryanus has the functions of enriching blood and replenishing qi, tonifying yang and inducing diuresis according to the records of traditional Chinese medicine, and becomes an important freshwater aquaculture variety in China. With the increasing expansion of the culture scale and the fry requirement of the paramisgurnus dabryanus, the fry culture requirement can be met only by artificial breeding. However, most of artificial breeding is inbred, which easily causes problems of germplasm degeneration, genetic diversity reduction, disease resistance and stress resistance reduction, and the like, and seriously affects the sustainable and healthy development of the breeding industry of the paramisgurnus dabryanus.

The methods for cryopreservation of semen of different fishes are different. As an important economic cultured fish, the normal-temperature (20-24 ℃) and ultralow-temperature freezing (-196 ℃) storage of the paramisgurnus dabryanus semen is researched, and the paramisgurnus dabryanus semen is short in storage time at normal temperature and can be stored for 132 hours at most; in the ultra-low temperature freezing, the storage time is prolonged, and since various proposals have not been studied, it is impossible to determine whether there is any optimum proposal. The existing preservation method has low sperm motility, sperm life and cell membrane integrity rate of the paramisgurnus dabryanus, and is not beneficial to effective and durable preservation.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

The invention also aims to provide an ultralow-temperature cryopreservation method for the paramisgurnus dabryanus semen, which improves the sperm motility, the sperm service life and the integrity rate of cell membranes and becomes an effective and durable preservation method.

To achieve these objects and other advantages in accordance with the present invention, there is provided an ultralow temperature cryopreservation method of paramisgurnus dabryanus semen, comprising the steps of:

step one, adding NaCl, KCl and CaCl₂·2H₂O、MgCl₂·6H₂O、NaHCO₃Mixing according to the concentration ratio of 80:50:5:2:50 in sequence to prepare a diluent, and storing at 4 ℃ for later use;

mixing the paramisgurnus dabryanus semen with the diluent according to the volume ratio of 1: 2-4, adding DMSO with the volume fraction of 7.5% -12.5%, uniformly mixing to obtain a semen anti-freezing mixed solution, and sub-packaging in a freezing storage tube;

and step three, balancing the freezing tube in the step two at a position 6cm above the liquid nitrogen surface for 10min, balancing on the liquid nitrogen surface for 5min, and finally putting into liquid nitrogen for freezing and preserving.

Preferably, the method further comprises the following steps:

and step four, taking the semen anti-freezing mixed solution out of the liquid nitrogen, balancing the semen anti-freezing mixed solution at a liquid nitrogen opening for 5min, and then unfreezing the semen anti-freezing mixed solution in a water bath at 37 ℃ until the semen anti-freezing mixed solution is melted.

Preferably, in the second step, the volume ratio of the paramisgurnus dabryanus semen to the diluent is 1:3, and the volume fraction of DMSO is 10%.

Preferably, the paramisgurnus dabryanus semen in the second step is collected in the following mode: the male loach scalper is anesthetized by using MS-222 solution, the water of the fish body is wiped off by using a towel, and the abdomen is squeezed to collect semen.

Preferably, before mixing the paramisgurnus dabryanus semen and the diluent in the second step, the paramisgurnus dabryanus semen is contained in a centrifuge tube, the temperature is kept for 10min in a constant-temperature water bath at 20 ℃, penicillin with the concentration of 50IU/mL is added and mixed evenly, then the centrifugal rotation speed is controlled to be 160rpm, centrifugal treatment is carried out for 4min, the lower layer liquid is kept, glucose and glycerol are added into the lower layer liquid and mixed evenly, and finally the mixed solution is mixed with the diluent, wherein 0.05g of glucose and 0.5mL of glycerol are added into each 10mL of the lower layer liquid.

Preferably, in the fourth step, during the thawing process, the freezing tube in the water bath is fixed, and the water in the water bath is stirred at a rotating speed of 300 r/min.

The invention at least comprises the following beneficial effects:

the semen ultra-low temperature cryopreservation technology can effectively and durably preserve sperms, is convenient for transportation between seedling farms, is beneficial to developing hybridization of fishes of different varieties or strains and different geographic populations, exerts the heterosis, and is one of effective means for solving the problem of inbreeding.

The sperm viability of the paramisgurnus dabryanus semen obtained by the semen ultralow temperature cryopreservation technology reaches 50%, the service life of the sperm reaches 69s, the integrity rate of the cell membrane reaches 82%, the sperm viability after centrifugation and other treatments and water bath thawing reaches 62%, the service life of the sperm reaches 75s, and the integrity rate of the cell membrane reaches 87%.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a graph of the effect of DMSO at different volume fractions on sperm motility;

FIG. 2 is a graph showing the effect of different semen to diluent ratios on sperm motility.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

< example 1>

The invention provides an ultralow temperature cryopreservation method for paramisgurnus dabryanus semen, which comprises the following steps:

step one, adding NaCl, KCl and CaCl₂·2H₂O、MgCl₂·6H₂O、NaHCO₃Mixing according to the concentration ratio of 80:50:5:2:50 in sequence to prepare a diluent, and storing at 4 ℃ for later use; wherein the concentration of NaCl can be 80mmol/L, the concentration of KCl can be 50mmol/L, CaCl₂·2H₂The concentration of O can be 5mmol/L, MgCl₂·6H₂The concentration of O can be 2mmol/L, NaHCO₃The concentration of (b) can be 50 mmol/L;

mixing the paramisgurnus dabryanus semen with the diluent according to the volume ratio of 1:2, adding DMSO with the volume fraction of 7.5%, uniformly mixing to obtain a semen anti-freezing mixed solution, and subpackaging in a freezing storage tube;

and step three, balancing the freezing tube in the step two at a position 6cm above the liquid nitrogen surface for 10min, balancing on the liquid nitrogen surface for 5min, and finally putting into liquid nitrogen for freezing and preserving.

And step four, taking the semen anti-freezing mixed solution out of the liquid nitrogen, balancing the semen anti-freezing mixed solution at a liquid nitrogen opening for 5min, and then unfreezing the semen anti-freezing mixed solution in a water bath at 37 ℃ until the semen anti-freezing mixed solution is melted.

And in the second step, the paramisgurnus dabryanus semen is collected in the following mode: the male loach scalper is anesthetized by using MS-222 solution, the water of the fish body is wiped off by using a towel, and the abdomen is squeezed to collect semen.

< example 2>

The invention provides an ultralow temperature cryopreservation method for paramisgurnus dabryanus semen, which comprises the following steps:

step one, adding NaCl, KCl and CaCl₂·2H₂O、MgCl₂·6H₂O、NaHCO₃Mixing according to the concentration ratio of 80:50:5:2:50 in sequence to prepare a diluent, and storing at 4 ℃ for later use; wherein the concentration of NaCl can be 80mmol/L, the concentration of KCl can be 50mmol/L, CaCl₂·2H₂The concentration of O can be 5mmol/L, MgCl₂·6H₂The concentration of O can be 2mmol/L, NaHCO₃The concentration of (b) can be 50 mmol/L;

mixing the paramisgurnus dabryanus semen with the diluent according to the volume ratio of 1:3, adding 10% of DMSO (dimethyl sulfoxide) by volume fraction, mixing uniformly to obtain an antifreeze semen mixed solution, and subpackaging the antifreeze semen mixed solution in a freezing storage tube;

and step three, balancing the freezing tube in the step two at a position 6cm above the liquid nitrogen surface for 10min, balancing on the liquid nitrogen surface for 5min, and finally putting into liquid nitrogen for freezing and preserving.

And step four, taking the semen anti-freezing mixed solution out of the liquid nitrogen, balancing the semen anti-freezing mixed solution at a liquid nitrogen opening for 5min, and then unfreezing the semen anti-freezing mixed solution in a water bath at 37 ℃ until the semen anti-freezing mixed solution is melted.

And in the second step, the paramisgurnus dabryanus semen is collected in the following mode: the male loach scalper is anesthetized by using MS-222 solution, the water of the fish body is wiped off by using a towel, and the abdomen is squeezed to collect semen.

< example 3>

The invention provides an ultralow temperature cryopreservation method for paramisgurnus dabryanus semen, which comprises the following steps:

step one, adding NaCl, KCl and CaCl₂·2H₂O、MgCl₂·6H₂O、NaHCO₃Mixing according to the concentration ratio of 80:50:5:2:50 in sequence to prepare a diluent, and storing at 4 ℃ for later use; wherein the concentration of NaCl can be 80mmol/L, the concentration of KCl can be 50mmol/L, CaCl₂·2H₂The concentration of O can be 5mmol/L, MgCl₂·6H₂The concentration of O can be 2mmol/L, NaHCO₃The concentration of (b) can be 50 mmol/L;

mixing the paramisgurnus dabryanus semen with the diluent according to the volume ratio of 1:4, adding DMSO with the volume fraction of 12.5%, uniformly mixing to obtain semen anti-freezing mixed liquid, and subpackaging in a freezing storage tube;

and step three, balancing the freezing tube in the step two at a position 6cm above the liquid nitrogen surface for 10min, balancing on the liquid nitrogen surface for 5min, and finally putting into liquid nitrogen for freezing and preserving.

And step four, taking the semen anti-freezing mixed solution out of the liquid nitrogen, balancing the semen anti-freezing mixed solution at a liquid nitrogen opening for 5min, and then unfreezing the semen anti-freezing mixed solution in a water bath at 37 ℃ until the semen anti-freezing mixed solution is melted.

And in the second step, the paramisgurnus dabryanus semen is collected in the following mode: the male loach scalper is anesthetized by using MS-222 solution, the water of the fish body is wiped off by using a towel, and the abdomen is squeezed to collect semen.

< example 4>

The invention provides an ultralow temperature cryopreservation method for paramisgurnus dabryanus semen, which comprises the following steps:

step one, adding NaCl, KCl and CaCl₂·2H₂O、MgCl₂·6H₂O、NaHCO₃Mixing according to the concentration ratio of 80:50:5:2:50 in sequence to prepare a diluent, and storing at 4 ℃ for later use; wherein the concentration of NaCl can be 80mmol/L, the concentration of KCl can be 50mmol/L, CaCl₂·2H₂The concentration of O can be 5mmol/L, MgCl₂·6H₂The concentration of O can be 2mmol/L, NaHCO₃The concentration of (b) can be 50 mmol/L;

mixing the paramisgurnus dabryanus semen with the diluent according to the volume ratio of 1:3, adding 10% of DMSO (dimethyl sulfoxide) by volume fraction, mixing uniformly to obtain an antifreeze semen mixed solution, and subpackaging the antifreeze semen mixed solution in a freezing storage tube;

and step three, balancing the freezing tube in the step two at a position 6cm above the liquid nitrogen surface for 10min, balancing on the liquid nitrogen surface for 5min, and finally putting into liquid nitrogen for freezing and preserving.

And step four, taking the semen anti-freezing mixed solution out of the liquid nitrogen, balancing the semen anti-freezing mixed solution at a liquid nitrogen opening for 5min, and then unfreezing the semen anti-freezing mixed solution in a water bath at 37 ℃ until the semen anti-freezing mixed solution is melted.

And in the second step, the paramisgurnus dabryanus semen is collected in the following mode: the male loach scalper is anesthetized by using MS-222 solution, the water of the fish body is wiped off by using a towel, and the abdomen is squeezed to collect semen.

Before mixing the paramisgurnus dabryanus semen and the diluent in the second step, putting the paramisgurnus dabryanus semen into a centrifuge tube, preserving the heat for 10min in a constant-temperature water bath at 20 ℃, adding penicillin with the concentration of 50IU/mL, uniformly mixing, controlling the centrifugal speed to be 160rpm, centrifuging for 4min, retaining the lower-layer liquid, adding glucose and glycerol into the lower-layer liquid, uniformly mixing, and finally mixing with the diluent, wherein 0.05g of glucose and 0.5mL of glycerol are added into each 10mL of the lower-layer liquid.

In the fourth step, in the thawing process, the freezing tube in the water bath is fixed, and the water in the water bath is stirred at the rotating speed of 300 r/min.

Using the preparation method of the present invention, the range of volume fractions of DMSO used in the present invention was obtained by comparing sperm motility after cryopreservation (sperm motility after one week of storage). As can be seen from FIG. 1, the final sperm motility reaches more than 40% due to the DMSO with the volume fraction of 7.5% -12.5%. The different letters in figure 1 represent significant differences (P <0.05),

with the preparation method of the present invention, the range of the ratio of semen to diluent used in the present invention was obtained by comparing the sperm motility after cryopreservation (sperm motility after one week of storage). As can be seen from FIG. 2, the ratio of semen to diluent is in the range of 1: 2-4, so that the final sperm motility reaches more than 50%. The different letters in figure 2 represent significant differences (P <0.05),

< comparative test >

Comparative example 1: the diluent in example 2 was replaced with the fish ringer's solution: NaCl 0.78g, CaCl₂0.021g，KCl 0.02g，NaHCO₃0.2g, adding double distilled water to 100mL, wherein the pH value is 8.0;

comparative example 2: the diluent in example 2 was replaced with Hanks sperm preservation solution consisting of solution A and solution B (solution A: NaCl 9.6g, KCl 0.48g, CaCl)₂0.168g，MgSO₄·7H₂0.274g of O and 60mL of distilled water; solution B of NaHCO₃0.42g，Na₂HPO₄·H₂O 0.208g，KH₂PO₄0.072g，C₆H₁₂O₆·H₂O1.2 g and distilled water 60 mL;

comparative example 3: the diluent in example 2 was replaced with 2.90g of glucose, 0.05g of KCl, CaCl₂0.02g，NaHCO₃0.21g, 100mL of distilled water;

comparative example 4: the diluent in example 2 was replaced with NaCl 0.75g, KCl 0.03g, glucose 0.3g, NaHCO₃0.04g, and distilled water was further added to make 100 mL.

As shown in the following Table 1, the quality of the thawed sperm of the examples 1-4 and the comparative examples 1-4 after being preserved for one week is shown, which illustrates that the sperm motility of the paramisgurnus dabryanus semen treated by the diluent of the invention and the antifreeze DMSO (the final concentration is 10%) is up to 50%, the sperm lifetime is up to 69s, the cell membrane integrity is up to 82%, and the sperm motility after being treated by centrifugation and the like and being thawed by water bath is up to 62%, the sperm lifetime is up to 75s, and the sperm cell membrane integrity is up to 87%.

TABLE 1

	Motility of sperm	Longevity of sperm	Integrity rate of cell membrane
				Example 1	47％	67s	80％
Example 2	50％	69s	82％
				Example 3	48％	68s	79％
Example 4	62％	75s	87％
				Comparative example 1	25％	42s		50％
Comparative example 2	28％	46s	54％
				Comparative example 3	16％	34s	43％
Comparative example 4	22％	39s	46％

The paramisgurnus dabryanus used in the examples and comparative examples of the present invention was 2-year-old, 20.3 + -2.4 cm in body length and 78 + -5 g in body mass, which was given from nama base of Guangxi aquatic science institute, and was temporarily kept in a laboratory at a water temperature of 26 + -2 deg.C for two weeks, and the commercial pellet feed was fed 1 time per day, and was used in the subsequent experiments after confirming that the health status was good.

While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.