A cryopreservation solution and a cryopreservation method for pseta alges sperm

Abstract

The application provides a large yellow croaker sperm freezing solution and a method for cryopreservation of large yellow croaker sperm, and belongs to the technical field of sperm freezing. The large yellow croaker sperm freezing solution comprises Hanks buffer solution, ergothioneine, glycerol and trehalose. The application adopts a step-by-step cooling method, first cools the mixed solution to-5 to-3 DEG C, and then cools to-197 to-195 DEG C after cold equilibrium, and then is transferred into a liquid nitrogen tank for freezing storage. Before use, thawing and recovery are carried out in a 36 to 38 DEG C water bath. The large yellow croaker sperm freezing solution can effectively protect sperm, and can significantly improve the survival rate of recovered sperm in the subsequent sperm recovery process, thereby providing a new idea for the freezing of large yellow croaker sperm.

Description

TECHNICAL FIELD

[0001] The present application relates to the sperm freezing technical field, especially to a large yellow croaker sperm freezing solution and a method for cryopreservation of large yellow croaker sperm. BACKGROUND

[0002] Large yellow croaker, belonging to Perciformes, Sciaenidae, Pseudosciaena, is commonly known as yellow croaker, red melon, yellow croaker, yellow fish, etc. It is a warm-temperate near-shore fish with a migratory habit, mainly distributed in the southern Yellow Sea, the East China Sea and the South China Sea. Its meat is delicious, and it is one of the traditional valuable marine economic fish in China. Since the 1980s, artificial breeding and seedling have been successful, and now more than 100,000 tons of large yellow croaker are produced annually, with a value of more than 7 billion yuan. Large yellow croaker has become one of the largest marine cage culture fish and eight major export aquaculture products in China.

[0003] The preservation technology of sperm is of great significance for the aquaculture, breeding of superior strains, resource protection and biological research of aquatic animals. The preservation time of large yellow croaker sperm is relatively short after being discharged from the body. For example, at 4℃, the survival rate can be maintained at 80% within 3h, and the survival rate is only 50% within 7h. In the process of distant hybridization breeding, the effective breeding time is short or the hybridization survival rate is low due to the difference in breeding time of different species. The use of sperm preservation solution to preserve large yellow croaker sperm can reduce the loss and dosage of parents, accurately grasp the sperm motility, improve the fertilization rate and hatching rate, prolong the preservation time of sperm and the breeding time of distant hybridization, and improve the survival rate of distant hybridization breeding to large-scale breed new varieties or new strains.

[0004] Although the existing sperm freezing method can achieve certain preservation effect, the motility, survival rate and fertilization ability of frozen sperm are still difficult to be effectively guaranteed due to oxidative stress, cell membrane damage and ice crystal formation caused by freezing in the freezing process. Therefore, there is an urgent need for a new technical scheme to improve the quality of large yellow croaker frozen sperm. SUMMARY

[0005] The purpose of the present application is to provide a large yellow croaker sperm freezing solution and a method for cryopreservation of large yellow croaker sperm to solve the problem of reduced sperm motility after freezing of large yellow croaker sperm.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical scheme:

[0007] The present application provides a large yellow croaker sperm freezing solution, which uses Hanks buffer solution as a solvent and comprises the following components at the following concentrations: ergothioneine 4-12 μmol / L, glycerol 4-6% v / v, and trehalose 0.01-0.02 g / mL.

[0008] Preferably, the cryopreservation solution for large yellow croaker sperm uses Hanks buffer as a solvent and includes the following components at the following concentrations: ergothioneine 8 μmol / L, glycerol 5% v / v, and trehalose 0.015 g / mL.

[0009] The present invention also provides the application of the aforementioned large yellow croaker sperm cryopreservation solution in the preservation of large yellow croaker sperm.

[0010] This invention also provides a method for preserving the sperm of large yellow croaker, comprising the following steps:

[0011] (1) Collect sperm from large yellow croaker;

[0012] (2) Mix the large yellow croaker sperm with the large yellow croaker sperm cryopreservation solution at a volume ratio of 0.8 to 1.2:1 to obtain a mixture;

[0013] (3) Cool the mixture to -5 to -3℃, perform cold equilibrium, cool it down to -197 to -195℃, and then transfer it to a liquid nitrogen tank for freezing storage.

[0014] Preferably, the cold equilibrium time is 10 to 15 minutes.

[0015] Preferably, the cooling rate of the mixture during the cooling period from -5 to -3°C is 0.8 to 1.2°C / min, and the cooling rate of the mixture during the cooling period from -197 to -195°C is -32 to -28°C / min.

[0016] Preferably, the concentration of large yellow croaker sperm in the mixture is 0.8–1.2 × 10⁻⁶. 9 per mL.

[0017] Preferably, the process includes a purification step before mixing the large yellow croaker sperm with the cryopreservation solution.

[0018] Preferably, the frozen sperm from large yellow croaker obtained by the method is thawed and revived at 36–38°C.

[0019] Preferably, the thawing and recovery time is 2.5 to 3.5 minutes.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects: The large yellow croaker sperm cryopreservation solution of the present invention includes Hanks buffer, ergothioneine, glycerol, and trehalose. The present invention employs a stepwise cooling method, first cooling the mixture to -5 to -3°C, then, after cold equilibration, further cooling to -197 to -195°C, and then transferring it to a liquid nitrogen tank for cryopreservation. Before use, it is thawed and recovered in a water bath at 36 to 38°C. The large yellow croaker sperm cryopreservation solution of the present invention can effectively protect sperm and significantly improve the sperm survival rate after recovery during the subsequent sperm recovery process, providing a new approach to the cryopreservation of large yellow croaker sperm. Detailed Implementation

[0021] The application provides a Pseudosciaena crocea sperm freezing solution, which is prepared by using Hanks buffer as a solvent and comprises the following components in the following concentrations: ergothioneine 4-12 μmol / L, glycerol 4-6 % v / v, and trehalose 0.01-0.02 g / mL; the concentration of ergothioneine in the Pseudosciaena crocea sperm freezing solution is further preferably 6-10 μmol / L, and more preferably 8 μmol / L; the concentration of glycerol is further preferably 4.5-5.5 % v / v, and more preferably 5 % v / v; and the concentration of trehalose is further preferably 0.013-0.017 g / mL, and more preferably 0.15 g / mL.

[0022] In the application, ergothioneine can effectively remove free radicals and reduce the damage caused by oxidative stress to cells; ergothioneine protects the integrity of the membrane by interacting with lipids in the cell membrane, thereby providing the survival rate of Pseudosciaena crocea sperm.

[0023] The application further provides application of the Pseudosciaena crocea sperm freezing solution to preservation of Pseudosciaena crocea sperm.

[0024] The application further provides a preservation method of Pseudosciaena crocea sperm, which comprises the following steps:

[0025] (1) collecting Pseudosciaena crocea sperm;

[0026] (2) mixing the Pseudosciaena crocea sperm with the Pseudosciaena crocea sperm freezing solution according to a volume ratio of 0.8-1.2:1 to obtain a mixed solution;

[0027] (3) cooling the mixed solution to-5--3 ℃ for cold equilibrium, cooling to-197--195 ℃, and then transferring into a liquid nitrogen tank for frozen storage.

[0028] The application first takes Pseudosciaena crocea in the breeding period to obtain fresh sperm. After obtaining the fresh sperm of Pseudosciaena crocea, impurities are removed and washing is performed. The differential centrifugation method is used to remove impurities, first, the sperm is placed in a centrifuge tube, and centrifugation is performed at a low speed (300-500 g, further preferably 350-450 g, and more preferably 400 g) for 3-5 min. In this process, the impurities with a higher density are precipitated at the bottom of the centrifuge tube, and the sperm is mainly concentrated in the liquid at the upper layer of the centrifuge tube. Then, the upper layer liquid is carefully sucked to remove most of the impurities. PBS buffer is used for washing, and the sperm is mixed with PBS according to a volume ratio of 1:3-1:4, and the centrifuge tube is gently inverted to mix them fully. Then, centrifugation is performed at a centrifugal force of 500-700 g for 4-8 min, and the supernatant is discarded to repeat the washing operation. The purpose of PBS washing is to remove enzymes and other chemical substances that may affect the activity of sperm, and to help maintain the normal physiological function of sperm.

[0029] The application mixes the large yellow croaker sperm after impurity removal and washing with the large yellow croaker sperm freezing solution to obtain a mixed solution; the volume ratio of the large yellow croaker sperm to the large yellow croaker sperm freezing solution is 0.8-1.2:1, further preferably 0.9-1.1, and more preferably 1:1. In the mixed solution, the concentration of the large yellow croaker sperm is 0.8-1.2×10 9 / mL, further preferably 0.9-1.1×10 9 / mL, and more preferably 1×10 9 / mL. The application first cools the mixed solution to-5--3℃ for cold equilibration, further preferably-4.5--3.5℃, and more preferably-4℃; the cooling rate during the process is 0.8-1.2℃ / min, further preferably 0.9-1.1℃ / min, and more preferably 1℃ / min; the time for the cold equilibration is 10-15min, further preferably 12-14min, and more preferably 13min. Then the mixed solution is cooled to-197--195℃, further preferably-196.5--195.5℃, and more preferably-196℃, and is then transferred into a liquid nitrogen tank for frozen storage; the cooling rate during the process is-32--28℃ / min, further preferably-31--29℃ / min, and more preferably 30℃ / min.

[0030] The application also provides a method for resuscitating the sperm in the large yellow croaker sperm freezing solution, and the large yellow croaker frozen sperm obtained by the method is thawed and resuscitated at 36-38℃, further preferably 36.5-37.5℃, and more preferably 37℃; the time for the thawing and resuscitation is 2.5-3.5min, and more preferably 3min.

[0031] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0032] Example 1

[0033] The large yellow croaker sperm freezing solution uses Hanks buffer as a solvent, and the concentrations of the components are as follows: ergothioneine 4μmol / L, glycerol 4%v / v, and trehalose 0.01g / mL.

[0034] The method for preserving the sperm of Pseudosciaena crocea comprises the following steps: selecting male Pseudosciaena crocea parent fish with good gonadal development, smooth body surface and strong vitality, injecting a labor-inducing agent from the base of the pectoral fin after the fish is anesthetized by eugenol, and collecting the sperm of the male parent fish within 12 hours after the injection. The collected sperm of Pseudosciaena crocea is subjected to impurity removal and washing. First, the sperm is placed in a centrifuge tube and centrifuged at a centrifugal force of 300 g for 5 min. Then, the upper liquid is carefully sucked to obtain the sperm with impurities removed. The sperm with impurities removed is mixed with PBS at a volume ratio of 1:3, and the centrifuge tube is gently inverted to mix the sperm and PBS thoroughly. Then, the sperm is centrifuged at a centrifugal force of 500 g for 8 min, and the supernatant is discarded. The washing is repeated once.

[0035] The sperm with impurities removed and washed is mixed with the prepared freezing diluent at a ratio of 1:1 to obtain a mixed solution, and the concentration of the sperm of Pseudosciaena crocea in the mixed solution is 0.8×10 9 The temperature is lowered at a rate of 0.8 ℃ / min to -5 ℃, and cold equilibrium is maintained for 12 min. Then, the temperature is lowered at a rate of -32 ℃ / min to -197 ℃, and the sperm is stored in a liquid nitrogen tank.

[0036] Example 2

[0037] The freezing solution of the sperm of Pseudosciaena crocea uses Hanks buffer as a solvent, and the concentrations of the components are as follows: 8 μmol / L of ergothioneine, 5% v / v of glycerol, and 0.015 g / mL of trehalose.

[0038] The method for preserving the sperm of Pseudosciaena crocea comprises the following steps: selecting male Pseudosciaena crocea parent fish with good gonadal development, smooth body surface and strong vitality, injecting a labor-inducing agent from the base of the pectoral fin after the fish is anesthetized by eugenol, and collecting the sperm of the male parent fish within 12 hours after the injection. The collected sperm of Pseudosciaena crocea is subjected to impurity removal and washing. First, the sperm is placed in a centrifuge tube and centrifuged at a centrifugal force of 400 g for 4 min. Then, the upper liquid is carefully sucked to obtain the sperm with impurities removed. The sperm with impurities removed is mixed with PBS at a volume ratio of 1:3, and the centrifuge tube is gently inverted to mix the sperm and PBS thoroughly. Then, the sperm is centrifuged at a centrifugal force of 600 g for 6 min, and the supernatant is discarded. The washing is repeated once.

[0039] The sperm with impurities removed and washed is mixed with the prepared freezing diluent at a ratio of 1:1 to obtain a mixed solution, and the concentration of the sperm of Pseudosciaena crocea in the mixed solution is 1×10 9 The temperature is lowered at a rate of 1 ℃ / min to -4 ℃, and cold equilibrium is maintained for 12 min. Then, the temperature is lowered at a rate of -30 ℃ / min to -196 ℃, and the sperm is stored in a liquid nitrogen tank.

[0040] Example 3

[0041] The sperm freezing solution of Pseudosciaena crocea uses Hanks buffer as solvent, and the concentration of each component is as follows: ergothioneine 12 μmol / L, glycerol 6% v / v, trehalose 0.02 g / mL.

[0042] The sperm preservation method of Pseudosciaena crocea has the following steps: selecting male parent fish of Pseudosciaena crocea with good gonadal development, smooth body surface and strong vitality, injecting a labor-inducing agent from the base of the pectoral fin after being anesthetized by eugenol, and collecting the semen of the male parent fish within 12 hours after injection. The collected semen of Pseudosciaena crocea is decontaminated and washed, first, the semen is placed in a centrifuge tube and centrifuged at a centrifugal force of 500 g for 3 min. Then, the upper liquid is carefully sucked to remove the impurities. The decontaminated semen is washed with PBS buffer, and the decontaminated semen and PBS are mixed at a volume ratio of 1:4, and the centrifuge tube is gently inverted to mix thoroughly. Then, the centrifuge tube is centrifuged at a centrifugal force of 700 g for 4 min, and the supernatant is discarded and washed again.

[0043] The decontaminated and washed sperm is mixed with the pre-prepared freezing diluent at a ratio of 1:1 to obtain a mixed solution, and the concentration of Pseudosciaena crocea sperm in the mixed solution is 1.2×10 9 The temperature is lowered at a rate of 1.2 ℃ / min to -3 ℃, cold equilibrium for 12 min, and then lowered at a rate of -28 ℃ / min to -195 ℃, and then transferred to a liquid nitrogen tank for frozen storage.

[0044] Comparative Example 1

[0045] The sperm is frozen without using ergothioneine according to the traditional freezing method. The composition of the freezing solution is as follows: sodium chloride 13.5 g / L, potassium chloride 0.6 g / L, calcium chloride 0.25 g / L, magnesium chloride 0.35 g / L, sodium bicarbonate 0.2 g / L, glycerol 6% v / v, and the solvent is distilled water. The decontaminated and washed semen of Pseudosciaena crocea is mixed with the freezing solution at a volume ratio of 1:3 to prepare a mixed solution, which is loaded into a freezing tube. The freezing tube is pre-cooled in an environment of -20 ℃ to -50 ℃ for 10 min to 15 min. After pre-cooling, the freezing tube is placed in liquid nitrogen at -180 ℃ for storage.

[0046] Experimental Example 1

[0047] The sperm of Pseudosciaena crocea is frozen according to the methods of Example 1, Example 2, Example 3 and Comparative Example 1, respectively. After being frozen for 30 days, the sperm is thawed in a water bath at 37 ℃ for 3 min, and then the sperm survival rate is determined by using a computer-aided sperm analysis system (CASA).

[0048] Then, female Pseudosciaena crocea with mature gonadal development are selected, and ovulation is induced by a suitable labor-inducing method. The abdomen is squeezed by hand, and the eggs are collected in a clean container. The thawed sperm is slowly added to the container containing the eggs, and the ratio of sperm to eggs is controlled at 1×105 ~5x10 5 Sperm / egg. At the same time, gently stir the egg and sperm mixture to make full contact, using a feather to stir gently, stirring time is about 1-3 min, to promote fertilization. Then add an appropriate amount of salinity of 30 ‰ artificial seawater, to make the zygote suspended in the solution, to avoid mutual adhesion.

[0049] The zygote is transferred to the hatching container, and the hatching water is filtered and disinfected to remove impurities, bacteria and parasites. The water temperature is controlled at 18-22℃, the salinity is maintained at 28-32%, the pH value is between 7.8-8.2, and the dissolved oxygen content is maintained at 5-8mg / L. During the embryonic development, the morphological changes and development process of the embryo are observed regularly using a microscope. The survival rate of sperm, fertilization rate and embryonic development of different groups of large yellow croaker are shown in Table 1.

[0050] Table 1 Survival rate of sperm, fertilization rate and embryonic development of different groups of large yellow croaker

[0051]

[0052] Experimental example 2

[0053] The thawing temperature is compared in the range of 20℃, 30℃ and 40℃ to see the effect of different temperatures on the thawing effect of sperm.

[0054] Test results: 20℃: poor freezing effect, survival rate: 45%. 30℃: survival rate: 60%. 40℃: survival rate: 75% (close to the optimal freezing rate). It is shown that the optimal freezing temperature should be controlled between 30℃ and 40℃.

[0055] From the above examples, the present application provides a large yellow croaker sperm freezing solution and a cold storage freezing method of large yellow croaker sperm. The freezing method of the present application can effectively protect the large yellow croaker sperm, and significantly improve the survival rate of the recovered sperm in the subsequent sperm recovery process.

[0056] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A frozen solution of Pseudosciaena crocea sperm, characterized in that, The sperm freezing solution of the large yellow croaker uses Hanks buffer as solvent and comprises the following components at the following concentrations: ergothioneine 4-12 μmol / L, glycerol 4-6% v / v, and trehalose 0.01-0.02 g / mL.

2. The P. major sperm freezing solution of claim 1, characterized in that, The sperm freezing solution of the large yellow croaker uses Hanks buffer as solvent and comprises the following components at the following concentrations: ergothioneine 8 μmol / L, glycerol 5% v / v, and trehalose 0.015 g / mL.

3. Use of the sperm freezing solution of the large yellow croaker according to claim 1 or 2 in preserving sperm of the large yellow croaker.

4. A method for preserving the sperm of Larimichthys crocea, characterized in that, The method comprises the following steps: (1) collecting sperm of the large yellow croaker; (2) mixing the sperm of the large yellow croaker with the sperm freezing solution of the large yellow croaker according to claim 1 or 2 at a volume ratio of 0.8-1.2:1 to obtain a mixed solution; (3) cooling the mixed solution to -5 to -3 ℃ for cold equilibration, cooling to -197 to -195 ℃, and then transferring into a liquid nitrogen tank for frozen storage.

5. The preservation method according to claim 4, characterized in that, The cold equilibration time is 10-15 min.

6. The preservation method according to claim 5, characterized in that, The cooling rate during cooling the mixed solution to -5 to -3 ℃ is 0.8-1.2 ℃ / min, and the cooling rate during cooling the mixed solution to -197 to -195 ℃ is -32 to -28 ℃ / min.

7. The preservation method according to claim 6, characterized in that, The concentration of the P. major sperm in the mixed solution is 0.8-1.2×10 9 / mL.

8. The preservation method according to claim 7, characterized in that, The method further comprises a step of removing impurities before mixing the sperm of the large yellow croaker with the sperm freezing solution.

9. A method for recovering sperm in a large yellow croaker sperm freezing preservation solution, characterized in that, The frozen sperm of the large yellow croaker obtained by the method according to any one of claims 4-8 is thawed and recovered at 36-38 ℃.

10. The resuscitation method of claim 9, wherein, The thawing and recovering time is 2.5-3.5 min.

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