Cryopreservation liquid and cryopreservation method for Sichuan parabramis pekinensis sperms

Abstract

The invention discloses a cryopreservation solution and a cryopreservation method for Sichuan parabramis pekinensis sperms, and belongs to the technical field of sperm freezing. According to the invention, barley beta-glucan is used as a precursor, barley beta-glucan-based carbon complex particles are prepared by a hydrothermal method, and then the barley beta-glucan-based carbon complex particles are mixed with glycerol and a diluent to obtain the cryopreservation liquid. The Sichuan parabramis pekinensis frozen sperms have large-scale production and application values, redundant sperms are preserved by adopting the cryopreservation liquid prepared by the invention in the prosperous period, so that resource waste is avoided, and when the seminal fluid of reverse-season male fishes is insufficient, the sperms can be directly unfrozen and applied to actual production, and reverse-seasonal breeding is facilitated. The sperms are preserved to prevent germplasm decline, heritable variation and other phenomena caused by inbreeding, and meanwhile, hybridization among different species can be carried out. Reference is provided for ultralow-temperature cryopreservation of sperms of other rare fishes through ultralow-temperature cryopreservation research of Sichuan parabramis pekinensis sperms, insight of injury mechanisms and exploration of preservation methods and technologies.

Description

Technical Field

[0001] The invention belongs to the technical field of sperm freezing, and in particular relates to a freezing preservation solution and a freezing preservation method for sperm of Sichuan bream. Background Art

[0002] Sichuan bream belongs to the genus Sinobream of the subfamily Cyprinidae of the order Cypriniformes. It is a rare and unique small economic fish in the upper reaches of the Yangtze River. It can only reproduce 1-2 times a year in the wild. Due to the serious ecological damage to the waters where Sichuan bream is distributed in the Minjiang River section of the upper reaches of the Yangtze River, the intensity of fishery operations is high, and Sichuan bream faces continuous high-intensity fishing pressure, while the population replenishment is slow, resulting in a continuous decline in resources and miniaturization of individuals. Therefore, it is urgent to carry out artificial breeding of Sichuan bream, and how to improve the mating success rate is a major problem.

[0003] In aquaculture production, with the development of artificial insemination technology and the need for germplasm resource protection, the research on sperm in vitro preservation technology has received more and more attention. Ultra-low temperature freezing preservation technology is the most widely used method for long-term preservation of animal germplasm resources, and is widely used in human assisted reproduction and animal husbandry breeding. The long-term preservation of sperm can ensure the stability of genes for a long time, while also avoiding the cost required for raising parents, greatly improving the feasibility of breeding, and also providing a technical platform for sperm metabolism and physiological research. Among the currently disclosed technologies for animal sperm preservation, most are sperm preservation technologies for mammals (cattle, goats, etc.). Since sperm of different types of animals have different biological characteristics, the sperm preservation methods disclosed in the prior art are not suitable for the preservation of Sichuan bream. And sperm is very likely to produce various damages during cooling and freezing preservation, including physical, chemical and biological damages. Therefore, the study of low temperature freezing preservation of Sichuan bream sperm and the extension of the use time and application range of sperm are not only of great significance for the protection of its germplasm resources, but also have become a problem that must be solved in the process of artificial breeding. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a freezing preservation solution and a freezing preservation method for sperm of Sichuan bream, so as to solve the technical problem that the survival rate of sperm is not high after freezing preservation.

[0005] To achieve the above object, the technical solution adopted by the present invention is: to provide a method for preparing a freezing preservation solution for sperm of Sichuan bream, comprising the following steps:

[0006] S1. Using barley β-glucan as a precursor, barley β-glucan-based carbon complex microparticles are prepared by a hydrothermal method; barley β-glucan is dissolved in water, and heat-treated at 30-50PSI and 170-190°C for 3-5h. Under high temperature and high pressure conditions, the sugar molecules in the barley polysaccharide undergo glycosylation, cross-linking and other processes to eventually form carbon nanoparticles or microparticles; then centrifugation is performed, and the precipitate is discarded to remove unreacted substances and impurities; then dialysis is performed to further remove unreacted organic precursors and small molecular impurities in the supernatant, and barley β-glucan-based carbon complex microparticles (barley β-glucan-CCPs) are obtained after freezing;

[0007] S2. Dissolve NaCl, CaCl2, KCl and NaHCO3 in water to obtain a diluent; the ratio of NaCl, CaCl2, KCl, NaHCO3 and water is 7-8g: 0.15-0.25g: 0.1-0.3g: 1-3g: 900-1100mL;

[0008] S3. Mix an osmotic cryoprotectant and a diluent in a volume ratio of 0.5-1.5:7-9, then add barley β-glucan-based carbon complex particles (barley β-glucan-CCPs) to make the concentration of barley β-glucan-based carbon complex particles in the mixed solution 40-160 mmol / L, and mix well to obtain a cryopreservation solution; the osmotic cryoprotectant is glycerol.

[0009] On the basis of the above technical solution, the present invention can also be improved as follows:

[0010] Further, the preparation process of barley β-glucan is as follows:

[0011] A1. Crush the barley and then place it at 80-90℃ to inactivate the enzyme for 20-40 minutes;

[0012] A2, dispersing the enzyme-inactivated barley flour in water, adjusting the pH value of the dispersion to 4-5, and then performing enzymolysis to obtain an extract;

[0013] A3. Concentrate the extract, dry it, and freeze it to obtain barley β-glucan.

[0014] Barley Polysaccharides (BPS) is a natural polysaccharide derived from plants, mainly containing polysaccharide components such as β-glucan, with good biocompatibility, degradability and certain pharmacological activity. The synthesis of carbon complex particles (CCPs) using barley β-glucan is achieved by converting barley β-glucan into particles with carbon complex properties, which can effectively inhibit the formation and growth of ice crystals, thereby reducing damage to frozen biological samples.

[0015] Furthermore, the material-water ratio of barley flour to water in A2 is 0.5-1.5:18-22.

[0016] Furthermore, the enzymatic hydrolysis in A2 is specifically as follows: first add 50-60U / mL of cellulase, and bathe in water at 50-60°C for 1.5-2.5h; then add 20-40U / mL of high temperature resistant Q-amylase, and bathe in water at 90-110°C for 20-40min; finally add 100-120U / mL of neutral protease, and bathe in water at 50-60°C for 5-7h.

[0017] Furthermore, before A3 is dried, the concentrate is mixed with 450-550 wt % ethanol, and precipitated overnight to remove small molecular impurities by alcohol precipitation.

[0018] Furthermore, the drying temperature is 50-90°C and the drying time is 8-12h.

[0019] Furthermore, the freezing temperatures in S1 and A3 are both -90 to -80°C, and the freezing times are both 1.5 to 2.5 hours.

[0020] Furthermore, the centrifugal speed is 3800-4200 r / min, and the time is 8-12 min.

[0021] Furthermore, the molecular weight cutoff of the dialysis membrane used for dialysis is 10000-14000Da, and the dialysis time is 0.5-2h.

[0022] Furthermore, the mass fraction of glycerol is 4-6wt%.

[0023] The invention also discloses a freezing preservation solution for sperm of Sichuan bream prepared by the preparation method.

[0024] The invention also discloses a freezing preservation method of the freezing preservation solution of Sichuan bream sperm, comprising the following steps: mixing semen and the freezing preservation solution in a volume ratio of 0.5-1.5:8-10, first balancing at 3-5°C for 3-8 minutes, then balancing at 8-12 cm above the liquid nitrogen surface for 8-12 minutes, and finally putting into liquid nitrogen for preservation.

[0025] The beneficial effects of the present invention are as follows: the sperm of Sichuan bream is cryopreserved by using the cryopreservation solution and the cryopreservation method disclosed in the present invention, and the added barley β-glucan-based carbon complex can effectively reduce the damage to the cryopreservation of the sperm of Sichuan bream, improve the survival rate and utilization rate of the sperm, and prevent the phenomena of germplasm decline and genetic variation caused by inbreeding; and the method of the present invention is easy to implement and is suitable for large-scale breeding and breeding of Sichuan bream. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The effect of the type and concentration of non-penetrating cryoprotectants on the survival rate of frozen sperm of Sichuan bream. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are described below to facilitate the understanding of the present invention by those skilled in the art. If the specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

[0028] The cellulase, thermostable Q-amylase and neutral protease used below were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0029] Example 1

[0030] A method for preparing a freezing preservation solution for sperm of Sichuan bream comprises the following steps:

[0031] S1. Grind the barley, pass it through a 40-mesh sieve, and place it at 85°C to inactivate the enzyme for 30 minutes;

[0032] S2, dispersing the enzyme-killed barley flour in water at a material-water ratio of 1:20, and then adjusting the pH value of the dispersion to 4.5; then adding cellulase to make the concentration of cellulase in the solution 55U / mL, and water bathing at 55°C for 2h; then adding high-temperature resistant Q-amylase to make the concentration of high-temperature resistant Q-amylase in the solution 30U / mL, and water bathing at 100°C for 30min; finally adding neutral protease to make the concentration of neutral protease in the solution 110U / mL, and water bathing at 55°C for 6h to obtain an extract;

[0033] S3, concentrating the extract to 1 / 3 of the original volume, then adding 5 times the volume of 95% ethanol to precipitate overnight, drying the clear solution at 70°C for 10 hours after impurities removal, and then freezing it at -86°C for 2 hours to obtain barley β-glucan;

[0034] S4, 0.5 g of wheat β-glucan was dissolved in 50 mL of water, and then the solution was transferred to an autoclave, heat treated at 40 PSI and 180°C for 4 h, and then the mixed solution was centrifuged at 4000 r / min for 10 min; then dialyzed using a dialysis membrane with a molecular weight cutoff of 12000 Da for 1 h, and finally the dialyzate was frozen at -86°C for 2 h to obtain barley β-glucan-based carbon complex particles (barley β-glucan-CCPs);

[0035] S5, dissolving 0.78 g NaCl, 0.021 g CaCl2, 0.02 g KCl and 0.2 g NaHCO3 in 100 mL water to obtain a diluent;

[0036] S6. Mix 5 wt% of glycerol and a diluent in a volume ratio of 1:8, and then add a non-permeable antifreeze agent, barley β-glucan-based carbon complex particles, so that the concentration of the barley β-glucan-based carbon complex particles in the mixed solution is 40 mmol / L. After mixing evenly, a cryopreservation solution is obtained.

[0037] Example 2

[0038] A method for preparing a freezing preservation solution for sperm of Sichuan bream comprises the following steps:

[0039] S1. Grind the barley, pass it through a 40-mesh sieve, and place it at 80°C to inactivate the enzyme for 40 minutes;

[0040] S2, dispersing the enzyme-killed barley flour in water at a material-water ratio of 0.5:18, and then adjusting the pH value of the dispersion to 4; then adding cellulase to make the concentration of cellulase in the solution 50U / mL, and water bathing at 50°C for 2.5h; then adding high-temperature resistant Q-amylase to make the concentration of high-temperature resistant Q-amylase in the solution 40U / mL, and water bathing at 110°C for 20min; finally adding neutral protease to make the concentration of neutral protease in the solution 100U / mL, and water bathing at 60°C for 5h to obtain an extract;

[0041] S3, concentrating the extract to 1 / 3 of the original volume, then adding 4 times the volume of 95% ethanol to precipitate overnight, drying the clear solution at 50°C for 12h after impurities removal, and then freezing it at -90°C for 1.5h to obtain barley β-glucan;

[0042] S4, 0.5 g of wheat β-glucan was dissolved in 50 mL of water, and then the solution was transferred to an autoclave, heat treated at 30 PSI and 170°C for 5 h, and then the mixed solution was centrifuged at 3800 r / min for 12 min; then dialyzed for 2 h using a dialysis membrane with a molecular weight cutoff of 10000 Da, and finally the dialyzate was frozen at -80°C for 2.5 h to obtain barley β-glucan-based carbon complex particles (barley β-glucan-CCPs);

[0043] S5, dissolving 0.7 g NaCl, 0.015 g CaCl2, 0.03 g KCl and 0.3 g NaHCO3 in 110 mL water to obtain a diluent;

[0044] S6. Mix 4 wt% of glycerol and a diluent in a volume ratio of 0.5:7, and then add a non-permeable antifreeze agent, barley β-glucan-based carbon complex particles, so that the concentration of the barley β-glucan-based carbon complex particles in the mixed solution is 80 mmol / L. After mixing evenly, a cryopreservation solution is obtained.

[0045] Example 3

[0046] A method for preparing a freezing preservation solution for sperm of Sichuan bream comprises the following steps:

[0047] S1. Grind the barley, pass it through a 40-mesh sieve, and place it at 90°C to inactivate the enzyme for 20 minutes;

[0048] S2, dispersing the enzyme-killed barley flour in water at a material-water ratio of 1.5:22, and then adjusting the pH value of the dispersion to 5; then adding cellulase to make the concentration of cellulase in the solution 60U / mL, and water bathing at 60°C for 1.5h; then adding high-temperature resistant Q-amylase to make the concentration of high-temperature resistant Q-amylase in the solution 20U / mL, and water bathing at 90°C for 40min; finally adding neutral protease to make the concentration of neutral protease in the solution 120U / mL, and water bathing at 50°C for 7h to obtain an extract;

[0049] S3, concentrating the extract to 1 / 3 of the original volume, then adding 6 times the volume of 95% ethanol to precipitate overnight, drying the clear solution at 90°C for 8h after impurities removal, and then freezing it at -80°C for 2.5h to obtain barley β-glucan;

[0050] S4, 0.5 g of wheat β-glucan was dissolved in 50 mL of water, and then the solution was transferred to an autoclave, heat treated at 50 PSI and 190°C for 3 h, and then the mixed solution was centrifuged at 4200 r / min for 8 min; then dialyzed using a dialysis membrane with a molecular weight cutoff of 14000 Da for 0.5 h, and finally the dialyzate was frozen at -90°C for 1.5 h to obtain barley β-glucan-based carbon complex particles (barley β-glucan-CCPs);

[0051] S5, dissolving 0.8 g NaCl, 0.025 g CaCl2, 0.01 g KCl and 0.1 g NaHCO3 in 90 mL water to obtain a diluent;

[0052] S6. Mix 6 wt% of glycerol and a diluent in a volume ratio of 1.5:9, then add a non-permeable antifreeze agent, barley β-glucan-based carbon complex particles, so that the concentration of the barley β-glucan-based carbon complex particles in the mixed solution is 160 mmol / L, and mix well to obtain a cryopreservation solution.

[0053] Experimental example

[0054] The types of non-permeable cryoprotectants were set to sucrose, glucose, trehalose and barley β-glucan-based carbon complex, and the concentrations were set to 40, 80 and 160 mmol / L, respectively. The target cryopreservation solution was prepared according to the preparation method of Example 1 to explore the effects of different types and concentrations of non-permeable cryoprotectants on the sperm survival rate of Sichuan bream sperm after cryopreservation. The experimental process is as follows: semen and cryopreservation solution are mixed evenly at a volume ratio of 1:9 to obtain an experimental sample solution, the experimental sample solution is first placed at 4°C for 5 minutes, then placed 10 cm above the liquid nitrogen surface for 10 minutes, and finally placed in liquid nitrogen for 7 days; the experimental sample solution is taken out, placed at 37°C for thawing for 30 seconds, and the survival rate of Sichuan bream sperm in the sample solution is detected.

[0055] The results are as follows Figure 1 As shown in the figure, it can be seen that when the concentration of barley β-glucan-based carbon complex in the cryopreservation solution is 40mmol / L, the survival rate of frozen sperm of Sichuan bream is the highest, and the sperm survival rate is 56.75%±0.88%.

Claims

1. A method for preparing a freezing preservation solution for sperm of Sichuan bream, characterized in that: The following steps are involved: S1, dissolving barley β-glucan in water, heat-treating at 30-50 PSI and 170-190° C. for 3-5 hours, then centrifuging, dialyzing, and freezing to obtain barley β-glucan-based carbon complex microparticles; S2, dissolving NaCl, CaCl2, KCl and NaHCO3 in water to obtain a diluent; the ratio of NaCl, CaCl2, KCl, NaHCO3 and water is 7-8g: 0.15-0.25g: 0.1-0.3g: 1-3g: 900-1100mL; S3. Mix an osmotic antifreeze agent and a diluent in a volume ratio of 0.5-1.5:7-9, then add barley β-glucan-based carbon complex particles to make the concentration of barley β-glucan-based carbon complex particles in the mixed solution 40-160 mmol / L, and mix well to obtain a cryopreservation solution; the osmotic antifreeze agent is glycerol.

2. The method for preparing the freezing preservation solution of Sichuan bream sperm according to claim 1, characterized in that: The preparation process of the barley β-glucan is as follows: A1. Crush the barley and then place it at 80-90℃ to inactivate the enzyme for 20-40 minutes; A2, dispersing the enzyme-inactivated barley flour in water, adjusting the pH value of the dispersion to 4-5, and then performing enzymolysis to obtain an extract; A3. Concentrate the extract, dry it, and freeze it to obtain barley β-glucan.

3. The method for preparing the freezing preservation solution of Sichuan bream sperm according to claim 2, characterized in that, The material-water ratio of barley flour to water in A2 is 0.5-1.5:18-22.

4. The method for preparing the freezing preservation solution of Sichuan bream sperm according to claim 2, characterized in that, The enzymatic hydrolysis in A2 is specifically as follows: first add 50-60U / mL of cellulase, and bathe in water at 50-60°C for 1.5-2.5h; then add 20-40U / mL of high temperature resistant Q-amylase, and bathe in water at 90-110°C for 20-40min; finally add 100-120U / mL of neutral protease, and bathe in water at 50-60°C for 5-7h.

5. The method for preparing the freezing preservation solution of Sichuan bream sperm according to claim 2, characterized in that, Before the A3 is dried, the concentrated solution is mixed with 450-550 wt % ethanol and precipitated overnight.

6. The method for preparing the freezing preservation solution of Sichuan bream sperm according to claim 2, characterized in that: The drying temperature is 50-90° C. and the drying time is 8-12 hours.

7. The method for preparing the freezing preservation solution of Sichuan bream sperm according to claim 2, characterized in that: The freezing temperatures in S1 and A3 are both -90 to -80°C, and the freezing time is both 1.5 to 2.5 hours.

8. The method for preparing the freezing preservation solution of Sichuan bream sperm according to claim 1, characterized in that: The molecular weight cutoff of the dialysis membrane used for the dialysis is 10000-14000Da, and the dialysis time is 0.5-2h.

9. A freezing preservation solution for sperm of Sichuan bream, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 8.

10. The method for cryopreservation of the Sichuan bream sperm cryopreservation solution according to claim 9, characterized in that: The method comprises the following steps: mixing semen and cryopreservation solution in a volume ratio of 0.5-1.5:8-10, first equilibrating at 3-5°C for 3-8 minutes, then equilibrating at 8-12 cm above the liquid nitrogen surface for 8-12 minutes, and finally storing in liquid nitrogen.

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