Pseudosciaena crocea sperm cryopreservation liquid and cryopreservation freezing method of pseudosciaena crocea sperms

Abstract

The invention provides a pseudosciaena crocea sperm cryopreservation solution and a cryopreservation freezing method of pseudosciaena crocea sperms, and belongs to the technical field of sperm cryopreservation. The large yellow croaker sperm cryopreservation solution comprises a Hanks buffer solution, ergothioneine, glycerol and trehalose. A step-by-step cooling method is adopted, firstly, mixed liquid is cooled to-5 DEG C to-3 DEG C, after cold balance, the mixed liquid is cooled to-197 DEG C to-195 DEG C, and then the mixed liquid is transferred into a liquid nitrogen tank to be frozen and stored. And unfreezing and resuscitating in a water bath at 36-38 DEG C before use. The cryopreservation liquid for the sperms of the large yellow croakers can effectively protect the sperms, can remarkably improve the survival rate of the recovered sperms in the subsequent sperm recovery process, and provides a new idea for cryopreservation of the sperms of the large yellow croakers.

Description

Technical Field

[0001] The invention relates to the technical field of sperm cryopreservation, in particular to a large yellow croaker sperm cryopreservation solution and a large yellow croaker sperm cold preservation freezing method. Background Art

[0002] Yellow croaker, belonging to the order Perciformes, family Sciaenidae, genus Pseudocroaker, commonly known as cucumber fish, red melon, yellow croaker, yellow croaker, etc., is a warm-temperate near-shore fish that migrates in groups. It is mainly distributed in the southern Yellow Sea, the East my country Sea and the South China Sea. Its meat is delicious and it is one of my country's traditional precious marine economic fish. Since the 1980s, artificial breeding and seedling cultivation have been successful. At present, my country has formed a yellow croaker industry with an annual output of more than 100,000 tons of cultivated yellow croaker and an output value of more than 7 billion yuan. Yellow croaker is now among the largest marine cage-cultured fish in my country and one of the eight major export aquaculture products.

[0003] Sperm preservation technology is of great significance to the breeding, breeding of fine varieties, resource protection and biological research of aquatic animals. The storage time of yellow croaker sperm is short after being discharged from the body. For example, under 4°C conditions, the survival rate can be maintained at 80% within 3 hours, and the survival rate is only 50% within 7 hours. In the process of distant hybridization of yellow croaker, due to the differences in the reproduction time of different species, the effective breeding time is short or the hybrid survival rate is low. Using sperm preservation fluid to preserve yellow croaker sperm can first reduce parental loss and dosage; second, accurately grasp sperm vitality, improve fertilization rate and hatching rate; third, extend sperm storage time and distant hybridization breeding time, improve distant hybridization breeding survival rate, and cultivate new varieties or new strains on a large scale.

[0004] Although the existing sperm cryopreservation methods can achieve a certain preservation effect, the vitality, survival rate and fertilization ability of the frozen sperm are still difficult to be effectively guaranteed due to the oxidative stress, cell membrane damage and ice crystal formation caused by freezing during the cryopreservation process. Therefore, a new technical solution is urgently needed to improve the quality of frozen sperm of large yellow croaker. Summary of the invention

[0005] The invention aims to provide a large yellow croaker sperm freezing solution and a large yellow croaker sperm cold preservation freezing method, so as to solve the problem of reduced sperm motility after large yellow croaker sperm freezing.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

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

[0009] The invention also provides application of the large yellow croaker sperm freezing solution in preserving large yellow croaker sperm.

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

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

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

[0013] (3) The mixed solution is cooled to -5 to -3°C, cold balanced, cooled to -197 to -195°C, and then transferred to a liquid nitrogen tank for frozen storage.

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

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

[0016] Preferably, in the mixed solution, the sperm concentration of large yellow croaker is 0.8-1.2×10 9 Pieces / mL.

[0017] Preferably, a step of removing impurities is also included before mixing the large yellow croaker sperm with the freezing solution.

[0018] Preferably, the frozen sperm of 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 scheme, 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 adopts a gradual cooling method, first cooling the mixed solution to -5 to -3°C, cooling it to -197 to -195°C after cold equilibrium, and then transferring it to a liquid nitrogen tank for frozen storage. Thaw and revive in a water bath at 36 to 38°C before use. The large yellow croaker sperm cryopreservation solution of the present invention can effectively protect sperm, and in the subsequent sperm revival process, can significantly improve the sperm survival rate after revival, providing a new idea for the cryopreservation of large yellow croaker sperm. DETAILED DESCRIPTION

[0021] The invention provides a large yellow croaker sperm freezing solution. The large yellow croaker sperm freezing solution uses Hanks buffer as a solvent and comprises components with the following concentrations: 4-12 μmol / L of ergothioneine, 4-6% v / v of glycerol, and 0.01-0.02 g / mL of trehalose. The concentration of ergothioneine in the large yellow croaker 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 present invention, ergothioneine can effectively remove free radicals and reduce the damage caused by oxidative stress to cells. Ergothioneine interacts with lipids in cell membranes to protect the integrity of the membranes, thereby increasing the survival rate of large yellow croaker sperm.

[0023] The invention also provides application of the large yellow croaker sperm freezing solution in preserving large yellow croaker sperm.

[0024] The present invention also provides a method for preserving sperm of large yellow croaker, comprising the following steps:

[0025] (1) Collecting sperm from large yellow croaker;

[0026] (2) mixing the large yellow croaker sperm with the large yellow croaker sperm freezing solution at a volume ratio of 0.8 to 1.2:1 to obtain a mixed solution;

[0027] (3) The mixed solution is cooled to -5 to -3°C, cold balanced, cooled to -197 to -195°C, and then transferred to a liquid nitrogen tank for frozen storage.

[0028] The present invention first takes a large yellow croaker in the breeding period to obtain its fresh semen. After obtaining the fresh semen of the large yellow croaker, the present invention removes impurities and washes it. Differential centrifugation is used for impurity removal. First, the semen is placed in a centrifuge tube and centrifuged for 3 to 5 minutes at a relatively low speed (300 to 500 g, more preferably 350 to 450 g, and more preferably 400 g). In this process, impurities with higher density will settle to the bottom of the centrifuge tube, while sperm are mainly concentrated in the liquid on the upper layer of the centrifuge tube. Then, the upper layer of liquid is carefully sucked to remove most of the impurities. PBS buffer is used for washing, and the semen and PBS are mixed in a volume ratio of 1:3 to 1:4, and the centrifuge tube is gently inverted to mix them fully. After that, centrifuge at a centrifugal force of 500 to 700 g for 4 to 8 minutes, and the supernatant is poured out and the washing operation is repeated. The purpose of PBS washing is to remove enzymes and other chemicals that may affect sperm motility, which helps to maintain the normal physiological function of sperm.

[0029] The present invention mixes the impurity-removed and washed large yellow croaker sperm 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 to 1.2:1, more preferably 0.9 to 1.1, and more preferably 1:1. In the mixed solution, the concentration of the large yellow croaker sperm is 0.8 to 1.2×10 9 pcs / mL, more preferably 0.9 to 1.1×10 9 / mL, more preferably 1×10 9 / mL. The present invention first cools the mixed solution to -5 to -3°C for cold balance, further preferably to -4.5 to -3.5°C, and more preferably to -4°C; the cooling rate during this period is 0.8 to 1.2°C / min, further preferably to 0.9 to 1.1°C / min, and more preferably to 1°C / min; the time for the cold balance is 10 to 15min, further preferably to 12 to 14min, and more preferably to 13min. The mixed solution is then cooled to -197 to -195°C, further preferably to -196.5 to -195.5°C, and more preferably to -196°C, and then transferred to a liquid nitrogen tank for frozen storage; the cooling rate during this period is -32 to -28°C / min, further preferably to -31 to -29°C / min, and more preferably to 30°C / min.

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

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] Example 1

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

[0034] The method for preserving sperm of yellow croaker is as follows: select male yellow croaker broodstock with well-developed gonads, smooth body surface and strong vitality, inject oxytocin from the base of the pectoral fin after anesthesia with eugenol, and collect semen from male broodstock within 12 hours after injection. Remove impurities and wash the collected yellow croaker semen. First, put the semen into a centrifuge tube and centrifuge it at a centrifugal force of 300g for 5 minutes. Then, carefully aspirate the upper liquid to remove impurities from the semen. Wash with PBS buffer, mix the semen after impurities removal with PBS at a volume ratio of 1:3, and gently invert the centrifuge tube to mix it thoroughly. Then centrifuge at a centrifugal force of 500g for 8 minutes, pour out the supernatant and repeat the washing once.

[0035] The sperm after impurity removal and washing was mixed with the pre-prepared cryopreservation diluent in a ratio of 1:1 to obtain a mixed solution. The concentration of sperm of yellow croaker in the mixed solution was 0.8×10 9 The temperature was lowered to -5°C at a rate of 0.8°C / min, and the mixture was cooled for 12 minutes. The mixture was then cooled to -197°C at a rate of -32°C / min, and then transferred to a liquid nitrogen tank for frozen storage.

[0036] Example 2

[0037] The large yellow croaker sperm cryopreservation solution uses Hanks buffer as solvent, and the concentrations of each component are as follows: ergothioneine 8 μmol / L, glycerol 5% v / v, and trehalose 0.015 g / mL.

[0038] The method for preserving sperm of yellow croaker is as follows: select male yellow croaker broodstock with well-developed gonads, smooth body surface and strong vitality, inject oxytocin from the base of the pectoral fin after anesthesia with eugenol, and collect semen from male broodstock within 12 hours after injection. Remove impurities and wash the collected yellow croaker semen. First, put the semen into a centrifuge tube and centrifuge it at a centrifugal force of 400g for 4 minutes. Then, carefully aspirate the upper liquid to remove impurities from the semen. Wash with PBS buffer, mix the semen after impurity removal with PBS at a volume ratio of 1:3, and gently invert the centrifuge tube to mix it thoroughly. Then centrifuge at a centrifugal force of 600g for 6 minutes, pour out the supernatant and repeat the washing once.

[0039] The sperm after impurity removal and washing was mixed with the pre-prepared cryopreservation diluent in a ratio of 1:1 to obtain a mixed solution. The concentration of sperm of yellow croaker in the mixed solution was 1×10 9 The temperature was lowered to -4°C at a rate of 1°C / min, and then the sample was cooled to -196°C at a rate of -30°C / min, and then transferred to a liquid nitrogen tank for frozen storage.

[0040] Example 3

[0041] The large yellow croaker sperm cryopreservation solution uses Hanks buffer as solvent, and the concentrations of each component are as follows: ergothioneine 12 μmol / L, glycerol 6% v / v, and trehalose 0.02 g / mL.

[0042] The method for preserving sperm of yellow croaker is as follows: select male yellow croaker broodstock with well-developed gonads, smooth body surface and strong vitality, inject oxytocin from the base of the pectoral fin after anesthesia with eugenol, and collect semen from male broodstock within 12 hours after injection. Remove impurities and wash the collected yellow croaker semen. First, put the semen into a centrifuge tube and centrifuge it at a centrifugal force of 500g for 3 minutes. Then, carefully aspirate the upper liquid to remove impurities from the semen. Wash with PBS buffer, mix the semen after impurity removal with PBS at a volume ratio of 1:4, and gently invert the centrifuge tube to mix it thoroughly. Then centrifuge at a centrifugal force of 700g for 4 minutes, pour out the supernatant and repeat the washing once.

[0043] The sperm after impurity removal and washing was mixed with the pre-prepared cryopreservation diluent in a ratio of 1:1 to obtain a mixed solution. The sperm concentration of yellow croaker in the mixed solution was 1.2×10 9 The temperature was lowered to -3°C at a rate of 1.2°C / min, and the mixture was cooled for 12 minutes. The mixture was then cooled to -195°C at a rate of -28°C / min, and then transferred to a liquid nitrogen tank for frozen storage.

[0044] Comparative Example 1

[0045] Ergothioneine was not used, and sperm was cryopreserved according to the traditional cryopreservation method. The freezing solution consists of 13.5 g / L sodium chloride, 0.6 g / L potassium chloride, 0.25 g / L calcium chloride, 0.35 g / L magnesium chloride, 0.2 g / L sodium bicarbonate, 6% v / v glycerol, and the solvent is distilled water. The yellow croaker semen after impurities removal and washing was mixed with the freezing solution in a volume ratio of 1:3 and loaded into the cryopreservation tube. The cryopreservation tube was placed in an environment of -20°C to -50°C for pre-cooling for 10 to 15 minutes. After pre-cooling, the cryopreservation tube was stored in liquid nitrogen at -180°C.

[0046] Experimental Example 1

[0047] The sperm of large yellow croaker was 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 was thawed in a 37° C. water bath for 3 minutes. After thawing, the sperm survival rate was determined using a computer-assisted sperm analysis system (CASA).

[0048] Then, female yellow croakers with mature gonads were selected and ovulated by appropriate induced ovulation methods. The abdomen was squeezed manually and the eggs were collected in a clean container. The thawed sperm was slowly dripped into the container containing the eggs, and the ratio of sperm to eggs was controlled at 1×105 ~5×10 5 Sperm / egg. At the same time, gently stir the egg and sperm mixture to make them fully contact, and use a feather to gently stir for about 1 to 3 minutes to promote fertilization. Then add an appropriate amount of artificial seawater with a salinity of 30‰ to suspend the fertilized eggs in the solution to avoid mutual adhesion.

[0049] The fertilized eggs were transferred to the incubation container, and the incubation water was filtered and disinfected to remove impurities, bacteria and parasites. The water temperature was controlled at 18-22°C, the salinity was maintained at 28-32%, the pH value was between 7.8 and 8.2, and the dissolved oxygen content was maintained between 5 and 8 mg / L. During embryonic development, the morphological changes and development progress of the embryos were regularly observed under a microscope. The survival rate, fertilization rate and embryonic development of sperm in different groups of large yellow croaker are shown in Table 1.

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

[0051]

[0052] Experimental Example 2

[0053] Compare the thawing temperatures within the range of 20℃, 30℃ and 40℃ to see the impact of different temperatures on sperm thawing results.

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

[0055] As can be seen from the above embodiments, the present invention provides a large yellow croaker sperm cryopreservation solution and a large yellow croaker sperm cryopreservation freezing method. The freezing method of the present invention can effectively protect large yellow croaker sperm and significantly improve the sperm survival rate after recovery in the subsequent sperm recovery process.

[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

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

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

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

4. A method for preserving sperm of large yellow croaker, characterized in that: The following steps are involved: (1) Collecting sperm from yellow croaker; (2) mixing the large yellow croaker sperm with the large yellow croaker sperm freezing solution according to claim 1 or 2 at a volume ratio of 0.8 to 1.2:1 to obtain a mixed solution; (3) The mixed solution is cooled to -5 to -3°C, cold balanced, cooled to -197 to -195°C, and then transferred to a liquid nitrogen tank for frozen storage.

5. The storage method according to claim 4, characterized in that: The cold balance time is 10 to 15 minutes.

6. The storage method according to claim 5, characterized in that: The cooling rate during the period when the mixed liquid is cooled to -5 to -3°C is 0.8 to 1.2°C / min, and the cooling rate during the period when the mixed liquid is cooled to -197 to -195°C is -32 to -28°C / min.

7. The storage method according to claim 6, characterized in that: In the mixed solution, the concentration of sperm of large yellow croaker is 0.8-1.2×10 9 Pieces / mL.

8. The storage method according to claim 7, characterized in that: The process also includes a step of removing impurities before mixing the large yellow croaker sperm with the freezing solution.

9. A method for resuscitating sperm in a sperm cryopreservation solution of large yellow croaker, characterized in that: The frozen sperm of large yellow croaker obtained by the method according to any one of claims 4 to 8 is thawed and revived at 36 to 38°C.

10. The resuscitation method according to claim 9, characterized in that: The thawing and recovery time is 2.5 to 3.5 minutes.

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