Ultralow-temperature preservation solution for Sinilabeo decorus tungting sperms and preparation method, preservation method and application of ultralow-temperature preservation solution

Abstract

The invention discloses ultralow-temperature preservation liquid for Sinilabeo decorus tungting sperms as well as a preparation method, a preservation method and application of the ultralow-temperature preservation liquid and belongs to the technical field of artificial breeding of Sinilabeo decorus tungting. The ultralow-temperature preservation liquid comprises a freezing diluent, an antifreeze agent glycol and an antioxidant, the antifreeze agent accounts for 8% of the volume of the ultralow-temperature preservation liquid, the antioxidant comprises reduced glutathione and alpha-lipoic acid, and the balance is the freezing diluent. According to the present invention, the formula compounding is different from the simple superposition of the traditional formula, the ratio and the combination mode of the components are precisely optimized, the combination has the synergistic effect, the resuscitation sperm activity is improved, the good oxidation resistance is provided, and the effect far better than the effect of other schemes is achieved. The method has remarkable advantages in efficient preservation of the sinilabeo decorus decorus decorus decorus decorus decorus decorus decorus decorus decorus decorus decorus decorus decorus decorus decorus decorus decorus decorus decorus decorus

Description

Technical Field

[0001] The invention relates to the technical field of artificial breeding of Xianghua ling, and more specifically to an ultra-low temperature preservation solution for sperm of Xianghua ling and a preparation method, a preservation method and an application thereof. Background Art

[0002] Bangana tungting is a wild fish endemic to Hunan Province, mainly distributed in the middle and upper reaches of the Xiang, Zi, Yuan and Li rivers. In the past 20 years, the population of Bangana tungting has decreased dramatically, and it has been designated as a key protected fish in the National Aquatic Germplasm Resources Conservation Area of ​​the Yuanshui River Basin. Since the implementation of the Yangtze River Protection Law, the phenomenon of people in the Yangtze River Basin eating wild fish will become history, and the development of high-quality aquaculture varieties has become an important demand. As a special fish with economic value, the protection and development of aquatic resources of Bangana tungting is of great significance. Previously, we achieved full artificial breeding of Bangana tungting. The promotion and optimization of breeding technology have improved the supply quality and quantity of Bangana tungting seedlings, met the market demand for high-quality aquatic products, and also helped to drive the development of local fisheries and promote the recovery of wild Bangana tungting resources. With the development of the Xianghua ling aquaculture industry, there is an urgent need for better seed preservation technology to improve the stable supply of semen, protect high-quality germplasm resources, enrich the source of parents, and at the same time reduce the breeding costs of enterprises. This is of great significance to the large-scale production of juveniles, selective breeding and related scientific research.

[0003] Ultra-low temperature cryopreservation of semen has become one of the main tools for protecting endangered or commercially important species, and can provide farms with a reliable means to preserve and utilize male parents with breeding value. By freezing and preserving sperm, high-quality germplasm resources can be obtained at any time for artificial insemination and population improvement, which is helpful for the selection and breeding of new varieties, and can achieve hybridization with distant species with different breeding times and geographical distributions, thus improving the innovation capacity of the seed industry. Sperm cryopreservation technology can serve as an important backup resource, providing effective response measures in the face of risks such as climate change, environmental mutations or disease outbreaks, and enhancing the risk resistance of enterprises.

[0004] Fish sperm cryopreservation solutions are species-specific because sperm from different species differ significantly in structure, physiological characteristics, cryosensitivity, antifreeze suitability, freezing procedures, and ecological adaptability. These differences require specific antifreeze formulations, concentrations, and freezing procedures for different species to ensure sperm survival and fertilization ability during freezing and thawing. Therefore, it is necessary to develop a suitable cryopreservation plan based on the physiological characteristics of Xianghua dace sperm to ensure preservation results.

[0005] Sperm motility is a major quality evaluation factor. It is generally believed that the higher the sperm motility and the longer the motility time, the stronger the fertilization ability. Computer-assisted sperm analysis (CASA) has more advantages than manual analysis, including more objective and repeatable measurement of many sperm cells, and has stronger statistical and analytical capabilities. In addition, whether it can achieve a physiological state similar to fresh sperm is the ultimate goal of cryopreserved sperm.

[0006] Therefore, how to develop a Xianghua ling sperm ultra-low temperature preservation solution and its preparation method, preservation method and application is a problem that technical personnel in this field urgently need to solve. Summary of the invention

[0007] In view of this, the present invention provides a Xianghua ling sperm ultra-low temperature preservation solution and a preparation method, a preservation method and an application thereof.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A Xianghua dace sperm ultra-low temperature preservation solution, comprising a freezing diluent, an antifreeze agent and an antioxidant, wherein the antifreeze agent accounts for 8% of the volume of the ultra-low temperature preservation solution, the antioxidant comprises reduced glutathione and α-lipoic acid, and in the ultra-low temperature preservation solution, the concentration of reduced glutathione is 0.1 mmol / L, the concentration of α-lipoic acid is 0.5 mmol / L, and the remainder is the freezing diluent;

[0010] The concentration of each component in the above-mentioned freezing diluent is: trehalose 150mmol / L, NaCl 75mmol / L, KCl 30mmol / L, Na2HPO4 1mmol / L, MgCl2 1mmol / L, CaCl2 1mmol / L, tris(hydroxymethyl)aminomethane 20mmol / L, bovine serum albumin accounts for 0.5% of the volume of the above-mentioned freezing diluent, and the solvent is ultrapure water;

[0011] The above antifreeze agent is ethylene glycol.

[0012] Beneficial effects of the present invention: the freezing diluent of the present invention has the functions of maintaining the pH of semen, maintaining the osmotic pressure and inhibiting the activation of sperm; trehalose, as a non-osmotic antifreeze agent, can reduce the concentration of extracellular solutes, dehydrate cells and reduce the formation of intracellular ice crystals, thereby reducing solute damage; bovine serum albumin contains a large amount of protein, which provides nutrients for sperm.

[0013] Ethylene glycol, as an osmotic antifreeze, can enter the interior of sperm, lower the freezing point of the cytoplasm, and prevent the formation of ice crystals; the combination of the non-osmotic antifreeze trehalose and the osmotic antifreeze ethylene glycol inside and outside the sperm improves the effect of ultra-low temperature preservation of semen, and the sperm still has a high vitality after thawing.

[0014] The present invention innovatively uses a combination of reduced glutathione and α-lipoic acid as antioxidants in the preservation of Xianghua ling semen, which can reduce freezing damage during freezing and thawing, maintain mitochondrial activity, maintain DNA integrity, and avoid oxidative damage, thereby ensuring normal development of the embryo after sperm thawing and fertilization.

[0015] The ultra-low temperature preservation solution of the present invention can protect the integrity of cell membranes, maintain mitochondrial function, reduce the damage of oxidative stress to sperm, and improve the survival rate and function of sperm.

[0016] The compound formula of the present invention is different from the simple superposition of the traditional formula. By accurately optimizing the proportion and combination of each component, the combination has a synergistic effect, improves the vitality of sperm and has good antioxidant capacity, achieving an effect far exceeding other solutions. The present invention has significant advantages in the efficient preservation of Xianghua ling semen. The thawed semen can meet the needs of artificial breeding, which is of great significance to the establishment and protection of Xianghua ling high-quality sperm bank.

[0017] The present invention has been verified through experiments that even if stored in liquid nitrogen for more than one month, the sperm survival rate and sperm motility after recovery are 69.19±6.55% and 87.74±6.48%, which is consistent with the ultra-low temperature cryopreservation effect of semen. The sperm DNA integrity and mitochondrial damage degree were analyzed by flow cytometry. The results showed that using the sperm cryopreservation solution and freezing scheme of the present invention, the sperm DNA integrity can reach 90.93±1.84%, and 75.5±5.36% of sperm maintain mitochondrial function. The sperm fertilization rate after recovery is 83.5±1.79%, which still has a high fertilization ability, which is of great significance to the protection and genetic improvement of Xianghua ling germplasm resources.

[0018] The present invention also provides a method for preparing the above-mentioned Xianghua ling sperm ultra-low temperature preservation solution, comprising the following steps:

[0019] (1) dissolving the above-mentioned trehalose, NaCl, KCl, Na2HPO4, MgCl2, CaCl2 and tris(hydroxymethyl)aminomethane in ultrapure water at room temperature to obtain a basic dilution solution, which is stored at 4°C for later use;

[0020] (2) adding bovine serum albumin to the basic diluent to obtain a freezing diluent, adding an antioxidant to the freezing diluent and mixing to obtain a sperm freezing preservation solution, and storing it in the dark for later use;

[0021] (3) Mixing the antifreeze agent with the sperm cryopreservation solution to obtain the above-mentioned Xianghua ling sperm ultra-low temperature preservation solution.

[0022] The present invention also provides a method for preserving semen using the Xianghua ling sperm ultra-low temperature preservation solution, comprising the following steps:

[0023] The semen of Xianghua ling was mixed with the above-mentioned cryopreservation solution and added to the freezing carrier, equilibrated on ice for 10-15 minutes, then placed in liquid nitrogen at a distance of 3-16 cm above the liquid nitrogen surface for 10 minutes, and finally completely immersed in liquid nitrogen for storage.

[0024] Beneficial effects of the present invention: The present invention optimizes the equilibrium distance, can protect the integrity of the cell membrane, maintain mitochondrial function, reduce the damage of oxidative stress to sperm, and improve the survival rate and function of sperm.

[0025] Furthermore, the volume ratio of Xianghua ling semen to the above-mentioned cryopreservation solution is 1:1.

[0026] Furthermore, the above-mentioned freezing carrier is a 2 mL cryovial or a 0.5 mL straw.

[0027] Preferably, the above-mentioned freezing carrier is a 2 mL cryovial.

[0028] The beneficial effects of adopting the above-mentioned further technical scheme are as follows: the present invention innovatively uses a combination of reduced glutathione and α-lipoic acid in the preservation of Xianghua ling semen, and optimizes the freezing carrier and its corresponding gas-phase liquid nitrogen equilibrium distance. Combining the above optimization means, it can protect the integrity of the cell membrane, maintain mitochondrial function, reduce the damage of oxidative stress to sperm, and improve the survival rate and function of sperm.

[0029] The present invention also provides a use of the ultra-low temperature preservation solution for sperm of Xianghua ling in preserving semen of Xianghua ling or in in vitro artificial insemination of Xianghua ling.

[0030] The invention mechanism of the present invention: During the cryopreservation process, sperm cells are exposed to low temperatures and are damaged by oxidative stress during freezing and thawing. Oxidative stress is mainly caused by reactive oxygen species (ROS), which can damage cell membranes, proteins and DNA, leading to cell damage or even death. Adding antioxidants during the cryopreservation process can neutralize ROS, reduce the impact of oxidative stress on sperm, and improve the survival rate and functionality of sperm after freezing. The cooling rate is a very critical factor in sperm cryopreservation. There is an obvious temperature gradient between the gas phase and the liquid phase of liquid nitrogen. The closer to the surface of liquid nitrogen, the lower the temperature. Samples placed at different heights will experience different cooling rates. At a position closer to the surface of liquid nitrogen, the sample will experience faster cooling, while at a farther position, the cooling rate will be slower. Too fast or too slow cooling may cause cell damage. Too fast cooling may cause a large number of small ice crystals to form in the cell to damage the internal structure of the cell, while too slow cooling may form large ice crystals outside the cell, causing mechanical damage to the cell membrane. By adjusting the equilibrium distance of the sample in the gas phase liquid nitrogen, the cooling rate can be controlled to adapt it to the tolerance of sperm and the effect of the cryoprotectant. Wheat straws are suitable for rapid cooling and thawing of small-volume samples, saving storage space and suitable for large-scale cryopreservation, but they are easily damaged and inconvenient to manage. Cryopreservation tubes are suitable for long-term storage of large-volume samples due to their large capacity, good sealing, easy labeling and resistance to mechanical damage, but they are slow to cool and thawing, increase the risk of ice crystal formation, and affect sperm survival rate. Therefore, it is necessary to weigh the cooling rate, storage space and sample management of different freezing carriers according to the needs of sperm samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Sperm motility after thawing under the protection of different types of antifreeze agents.

[0032] Figure 2 Figure A shows the sperm survival rate, sperm motility, and proportion of forward-moving sperm after resuscitation under the protection of different concentrations of ethylene glycol. Figure B shows the average linear movement speed of sperm after resuscitation under the protection of different concentrations of ethylene glycol.

[0033] Figure 3 Effects of different antioxidants, cryocarriers and gas phase liquid nitrogen equilibrium distance on sperm survival rate and sperm motility.

[0034] Figure 4The interactive effects of different antioxidants, freezing carriers and equilibrium distance of gas phase liquid nitrogen on sperm survival rate and sperm motility. Figure A shows the interactive effect of antifreeze and equilibrium distance under cryotube storage, Figure B shows the interactive effect of antifreeze and equilibrium distance under cryotube storage, Figure C shows the interactive effect of antifreeze and equilibrium distance under wheat straw storage, Figure D shows the interactive effect of antifreeze and equilibrium distance under wheat straw storage, Figure E shows the interactive effect of different freezing carriers and equilibrium distance, and Figure F shows the interactive effect of different freezing carriers and equilibrium distance.

[0035] Figure 5 Sperm DNA integrity under different combinations of antioxidants, cryotransfer agents and gas phase liquid nitrogen equilibrium distances. Figure A shows the recovery of sperm after equilibrium at 3 cm above the liquid nitrogen surface; Figure B shows the recovery of sperm after equilibrium at 16 cm above the liquid nitrogen surface.

[0036] Figure 6 Sperm mitochondrial membrane potential under different combinations of antioxidants, cryotransfer agents and gas phase liquid nitrogen equilibrium distances. Figure A shows the recovery of sperm after equilibrium at 3 cm above the liquid nitrogen surface; Figure B shows the recovery of sperm after equilibrium at 16 cm above the liquid nitrogen surface. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Example 1

[0039] (1) A method for preparing a Xianghua ling sperm cryopreservation solution, comprising the following steps:

[0040] (1) Dissolve trehalose, NaCl, KCl, Na2HPO4, MgCl2, CaCl2 and tris(hydroxymethyl)aminomethane) in ultrapure water at room temperature to obtain a basic dilution solution, which is stored at 4°C for later use;

[0041] (2) adding bovine serum albumin to the basic diluent to obtain a freezing diluent, wherein the concentrations of the components in the freezing diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na2HPO4 1 mmol / L, MgCl2 1 mmol / L, CaCl2 1 mmol / L, tris(hydroxymethyl)aminomethane 20 mmol / L, and bovine serum albumin accounts for 0.5% of the volume of the freezing diluent. Reduced glutathione and α-lipoic acid are added to the freezing diluent and mixed to obtain a sperm cryopreservation solution, which is stored away from light for later use;

[0042] (3) Ethylene glycol is mixed with sperm cryopreservation solution to obtain Xianghua ling sperm ultra-low temperature preservation solution, in which the antifreeze agent accounts for 8% of the volume of the ultra-low temperature preservation solution. In the ultra-low temperature preservation solution, the concentration of reduced glutathione is 0.1 mmol / L, the concentration of α-lipoic acid is 0.5 mmol / L, and the remainder is freezing diluent.

[0043] (ii) A method for preserving semen using a Xianghua ling sperm cryopreservation solution, comprising the following steps:

[0044] Select 10 male Xianghua ling with mature testes, wipe the area around the cloaca with a clean towel, gently press the belly of the fish from the head to the cloaca to collect the semen, and mix the semen of all 10 fish.

[0045] The semen of Xianghua ling and ultra-low temperature preservation solution were mixed in a volume ratio of 1:1 and added to 2 mL cryotubes. 1 mL of liquid was added to each tube, and the tubes were balanced on ice for 15 minutes, then placed in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 minutes, and finally completely immersed in liquid nitrogen for storage.

[0046] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 80 seconds until the semen mixture is completely melted.

[0047] Example 2

[0048] (1) A method for preparing a Xianghua ling sperm cryopreservation solution, comprising the following steps:

[0049] (1) Dissolve trehalose, NaCl, KCl, Na2HPO4, MgCl2, CaCl2 and tris(hydroxymethyl)aminomethane) in ultrapure water at room temperature to obtain a basic dilution solution, which is stored at 4°C for later use;

[0050] (2) adding bovine serum albumin to the basic diluent to obtain a freezing diluent, wherein the concentrations of the components in the freezing diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na2HPO4 1 mmol / L, MgCl2 1 mmol / L, CaCl2 1 mmol / L, tris(hydroxymethyl)aminomethane 20 mmol / L, and bovine serum albumin accounts for 0.5% of the volume of the freezing diluent. Reduced glutathione and α-lipoic acid are added to the freezing diluent and mixed to obtain a sperm cryopreservation solution, which is stored away from light for later use;

[0051] (3) Ethylene glycol is mixed with sperm cryopreservation solution to obtain Xianghua ling sperm ultra-low temperature preservation solution, in which the antifreeze agent accounts for 8% of the volume of the ultra-low temperature preservation solution. In the ultra-low temperature preservation solution, the concentration of reduced glutathione is 0.1 mmol / L, the concentration of α-lipoic acid is 0.5 mmol / L, and the remainder is freezing diluent.

[0052] (ii) A method for preserving semen using a Xianghua ling sperm cryopreservation solution, comprising the following steps:

[0053] Select 10 male Xianghua ling with mature testes, wipe the area around the cloaca with a clean towel, gently press the belly of the fish from the head to the cloaca to collect the semen, and mix the semen of all 10 fish.

[0054] The semen of Xianghua ling and ultra-low temperature preservation solution were mixed in a volume ratio of 1:1 and added to 2 mL cryotubes. 1 mL of liquid was added to each tube, and the tubes were balanced on ice for 15 minutes, then placed in liquid nitrogen at a distance of 3 cm above the liquid nitrogen surface for 10 minutes, and finally completely immersed in liquid nitrogen for storage.

[0055] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 80 seconds until the semen mixture is completely melted.

[0056] Example 3

[0057] (1) A method for preparing a Xianghua ling sperm cryopreservation solution, comprising the following steps:

[0058] (1) Dissolve trehalose, NaCl, KCl, Na2HPO4, MgCl2, CaCl2 and tris(hydroxymethyl)aminomethane) in ultrapure water at room temperature to obtain a basic dilution solution, which is stored at 4°C for later use;

[0059] (2) adding bovine serum albumin to the basic diluent to obtain a freezing diluent, wherein the concentrations of the components in the freezing diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na2HPO4 1 mmol / L, MgCl2 1 mmol / L, CaCl2 1 mmol / L, tris(hydroxymethyl)aminomethane 20 mmol / L, and bovine serum albumin accounts for 0.5% of the volume of the freezing diluent. Reduced glutathione and α-lipoic acid are added to the freezing diluent and mixed to obtain a sperm cryopreservation solution, which is stored away from light for later use;

[0060] (3) Ethylene glycol is mixed with sperm cryopreservation solution to obtain Xianghua ling sperm ultra-low temperature preservation solution, in which the antifreeze agent accounts for 8% of the volume of the ultra-low temperature preservation solution. In the ultra-low temperature preservation solution, the concentration of reduced glutathione is 0.1 mmol / L, the concentration of α-lipoic acid is 0.5 mmol / L, and the remainder is freezing diluent.

[0061] (ii) A method for preserving semen using a Xianghua ling sperm cryopreservation solution, comprising the following steps:

[0062] Select 10 male Xianghua ling with mature testes, wipe the area around the cloaca with a clean towel, gently press the belly of the fish from the head to the cloaca to collect the semen, and mix the semen of all 10 fish.

[0063] The semen of Xianghua ling and cryopreservation solution were mixed in a volume ratio of 1:1 and added to 0.5 mL wheat tubes. 0.5 mL of liquid was added to each tube, and the tubes were balanced on ice for 15 min, then placed in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 min, and finally completely immersed in liquid nitrogen for storage.

[0064] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 80 seconds until the semen mixture is completely melted.

[0065] Example 4

[0066] (1) A method for preparing a Xianghua ling sperm cryopreservation solution, comprising the following steps:

[0067] (1) Dissolve trehalose, NaCl, KCl, Na2HPO4, MgCl2, CaCl2 and tris(hydroxymethyl)aminomethane) in ultrapure water at room temperature to obtain a basic dilution solution, which is stored at 4°C for later use;

[0068] (2) adding bovine serum albumin to the basic diluent to obtain a freezing diluent, wherein the concentrations of the components in the freezing diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na2HPO4 1 mmol / L, MgCl2 1 mmol / L, CaCl2 1 mmol / L, tris(hydroxymethyl)aminomethane 20 mmol / L, and bovine serum albumin accounts for 0.5% of the volume of the freezing diluent. Reduced glutathione and α-lipoic acid are added to the freezing diluent and mixed to obtain a sperm cryopreservation solution, which is stored away from light for later use;

[0069] (3) Ethylene glycol is mixed with sperm cryopreservation solution to obtain Xianghua ling sperm ultra-low temperature preservation solution, in which the antifreeze agent accounts for 8% of the volume of the ultra-low temperature preservation solution. In the ultra-low temperature preservation solution, the concentration of reduced glutathione is 0.1 mmol / L, the concentration of α-lipoic acid is 0.5 mmol / L, and the remainder is freezing diluent.

[0070] (ii) A method for preserving semen using a Xianghua ling sperm cryopreservation solution, comprising the following steps:

[0071] Select 10 male Xianghua ling with mature testes, wipe the area around the cloaca with a clean towel, gently press the belly of the fish from the head to the cloaca to collect the semen, and mix the semen of all 10 fish.

[0072] The semen of Xianghua ling and cryopreservation solution were mixed in a volume ratio of 1:1 and added to 0.5 mL wheat tubes. 0.5 mL of liquid was added to each tube, and the tubes were balanced on ice for 15 min, then placed in liquid nitrogen at a distance of 3 cm above the liquid nitrogen surface for 10 min, and finally completely immersed in liquid nitrogen for storage.

[0073] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 80 seconds until the semen mixture is completely melted.

[0074] Effect experiment:

[0075] The other test examples differ from Example 1 in that they have different freezing carriers, equilibrium distances and antioxidants, and the other steps and parameters are the same as those of Example 1.

[0076] Sperm motility test: After the semen is thawed, use a pipette to draw 1 μL of diluted frozen semen and add sperm activation solution. The formula and concentration of sperm activation solution are: 45mM NaCl, 5mM KCl, 30mM Tris, the solvent is ultrapure water, pH: 8.0±0.2. Immediately after activation, use CASA to detect sperm motility and record sperm life span, and repeat each sample three times.

[0077] Sperm DNA integrity test: Wash sperm with Hank's solution, centrifuge at 1500rpm for 5 minutes, discard the supernatant, and repeat 2-3 times. Resuspend the precipitated sperm in acidic lysis buffer and lyse the sperm membrane in an ice bath for 30 seconds. Add 1mL of staining solution containing 50μg / mL acridine orange to each sample and incubate at room temperature for 5 minutes away from light. Analyze the stained sperm using a flow cytometer to measure the fluorescence signals of normal DNA and damaged DNA respectively.

[0078] Sperm mitochondrial membrane potential detection: Prepare Mitotracker RedCMXRos dye working solution at a concentration of 400nM. Wash the sperm with Hank's solution, centrifuge at 1500rpm for 5 minutes, discard the supernatant, and repeat 2-3 times. Resuspend the washed sperm with MitoTracker working solution and incubate in the dark for 30 minutes. After staining, centrifuge at 1500rpm for 5 minutes, discard the supernatant, and wash the sperm 1-2 times with Hank's solution to remove excess dye. Analyze the stained sperm using a flow cytometer to measure the sperm mitochondrial fluorescence signal.

[0079] After thawing, sperm survival rate and sperm motility under different freezing carriers, equilibrium distances and antioxidant protection Figure 3 Statistical analysis showed that the combination of antioxidants GSH and ALA (GSH+ALA) significantly increased the survival rate of Xianghua dace sperm compared with the use of GSH (reduced glutathione) or ALA (α-lipoic acid) alone, as shown in Table 1, indicating that the combined use of these two antioxidants has a synergistic effect and can improve sperm survival rate (e.g. Figure 4AD). In terms of sperm motility, there was a synergistic effect, especially when GSH+ALA was compared with ALA alone, with GSH+ALA showing significantly higher sperm motility (p<0.05, see Table 2). The combination of different equilibrium distances and cryocarriers also had a significant effect on sperm survival and sperm motility ( Figure 4 E, F). When considering the three factors of antioxidant, carrier type and equilibrium distance, sperm survival rate and sperm motility showed highly significant interactive effects. Specific antioxidants, carrier type and equilibrium distance all interacted to affect survival rate and sperm motility. Figure 5 , the sperm DNA integrity was significantly higher when GSH+ALA was added than when GSH and ALA were used alone (p<0.05), and the mitochondrial membrane potential was significantly higher than when GSH and ALA were used alone (p<0.05, Figure 6 ), indicating that the combination of antioxidants has a stronger antioxidant effect and maintains sperm DNA integrity. The above results prove that the combined use of antioxidants GSH+ALA can significantly prevent the damage to sperm caused by ultra-low temperature freezing and maintain sperm motility, indicating that GSH and ALA enhance each other's antioxidant capacity, and the two work on different oxidative stress pathways, thereby producing a greater protective effect. Fertilization experiments show that with 8% ethylene glycol as an antifreeze, the use of GSH+ALA and a compound solution of balancing in a cryopreservation tube at 16cm of liquid nitrogen, the fertilization rate of revived sperm can reach 83.5±1.79%. The formula compounding of the present invention is different from the simple superposition of traditional formulas. By accurately optimizing the proportions and combinations of each component, its combination has a synergistic effect and achieves an effect far exceeding other solutions.

[0080] Table 1 Sperm survival rate under different carriers, equilibrium distances and antioxidant protection (%)

[0081]

[0082] GSH: reduced glutathione; ALA: alpha lipoic acid. * indicates significant differences between different equilibrium distances (p<0.05); a, b, c indicate significant differences between different antioxidants (p<0.05).

[0083] Table 2 Sperm motility under different carriers, equilibrium distances and antioxidant protection (%)

[0084]

[0085] GSH: reduced glutathione; ALA: alpha lipoic acid. * indicates significant differences between different equilibrium distances (p<0.05); a, b, c indicate significant differences between different antioxidants (p<0.05).

[0086] Test Example 1

[0087] (1) A method for preparing a sperm preservation solution of Xianghua ling, comprising the following steps:

[0088] (1) Dissolve trehalose, NaCl, KCl, Na2HPO4, MgCl2, CaCl2 and tris(hydroxymethyl)aminomethane) in ultrapure water at room temperature to obtain a basic dilution solution, which is stored at 4°C for later use;

[0089] (2) adding bovine serum albumin to the basic diluent to obtain a frozen diluent, wherein the concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na2HPO4 1 mmol / L, MgCl2 1 mmol / L, CaCl2 1 mmol / L, tris(hydroxymethyl)aminomethane 20 mmol / L, and bovine serum albumin accounts for 0.5% of the volume of the frozen diluent, and the solution is stored away from light for future use;

[0090] (3) Ethylene glycol is mixed with the freezing diluent to obtain a Xianghua ling sperm preservation solution, wherein the antifreeze agent accounts for 10% of the volume of the preservation solution and the remainder is the freezing diluent.

[0091] (ii) A method for preserving semen using Xianghua ling sperm preservation fluid comprises the following steps:

[0092] Select 10 male Xianghua ling with mature testes, wipe the area around the cloaca with a clean towel, gently press the belly of the fish from the head to the cloaca to collect the semen, and mix the semen of all 10 fish.

[0093] The semen of Xianghua ling and the preservation solution were mixed in a volume ratio of 1:1 and added to 2 mL cryotubes. 1 mL of liquid was added to each tube, and the tubes were balanced on ice for 15 min, then placed in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 min, and finally completely immersed in liquid nitrogen for storage.

[0094] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 80 seconds until the semen mixture is completely melted.

[0095] Sperm motility test: After the semen is thawed, use a pipette to draw 1 μL of diluted frozen semen and add sperm activation solution. The formula and concentration of sperm activation solution are: 45mM NaCl, 5mM KCl, 30mM Tris (tris(hydroxymethyl)aminomethane), the solvent is ultrapure water, pH: 8.0±0.2. Immediately after activation, use CASA to detect sperm motility and record sperm life span, and repeat each sample three times.

[0096] Test Example 2

[0097] (1) A method for preparing a sperm preservation solution of Xianghua ling, comprising the following steps:

[0098] (1) Dissolve trehalose, NaCl, KCl, Na2HPO4, MgCl2, CaCl2 and tris(hydroxymethyl)aminomethane) in ultrapure water at room temperature to obtain a basic dilution solution, which is stored at 4°C for later use;

[0099] (2) adding bovine serum albumin to the basic diluent to obtain a frozen diluent, wherein the concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na2HPO4 1 mmol / L, MgCl2 1 mmol / L, CaCl2 1 mmol / L, tris(hydroxymethyl)aminomethane 20 mmol / L, and bovine serum albumin accounts for 0.5% of the volume of the frozen diluent, and the solution is stored away from light for future use;

[0100] (3) Methanol is mixed with the freezing diluent to obtain a Xianghua ling sperm preservation solution, wherein the antifreeze agent accounts for 10% of the volume of the preservation solution and the remainder is the freezing diluent.

[0101] (ii) A method for preserving semen using Xianghua ling sperm preservation fluid comprises the following steps:

[0102] Select 10 male Xianghua ling with mature testes, wipe the area around the cloaca with a clean towel, gently press the belly of the fish from the head to the cloaca to collect the semen, and mix the semen of all 10 fish.

[0103] The semen of Xianghua ling and the preservation solution were mixed in a volume ratio of 1:1 and added to 2 mL cryotubes. 1 mL of liquid was added to each tube, and the tubes were balanced on ice for 15 min, then placed in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 min, and finally completely immersed in liquid nitrogen for storage.

[0104] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 80 seconds until the semen mixture is completely melted.

[0105] Sperm motility test: After the semen is thawed, use a pipette to draw 1 μL of diluted frozen semen and add sperm activation solution. The formula and concentration of sperm activation solution are: 45mM NaCl, 5mM KCl, 30mM Tris, the solvent is ultrapure water, pH: 8.0±0.2. Immediately after activation, use CASA to detect sperm motility and record sperm life span, and repeat each sample three times.

[0106] Test Example 3

[0107] (1) A method for preparing a sperm preservation solution of Xianghua ling, comprising the following steps:

[0108] (1) Dissolve trehalose, NaCl, KCl, Na2HPO4, MgCl2, CaCl2 and tris(hydroxymethyl)aminomethane) in ultrapure water at room temperature to obtain a basic dilution solution, which is stored at 4°C for later use;

[0109] (2) adding bovine serum albumin to the basic diluent to obtain a frozen diluent, wherein the concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na2HPO4 1 mmol / L, MgCl2 1 mmol / L, CaCl2 1 mmol / L, tris(hydroxymethyl)aminomethane 20 mmol / L, and bovine serum albumin accounts for 0.5% of the volume of the frozen diluent, and the solution is stored away from light for future use;

[0110] (3) Dimethyl sulfoxide is mixed with the freezing diluent to obtain a Xianghua ling sperm preservation solution, wherein the antifreeze agent accounts for 10% of the volume of the preservation solution and the remainder is the freezing diluent.

[0111] (ii) A method for preserving semen using Xianghua ling sperm preservation fluid comprises the following steps:

[0112] Select 10 male Xianghua ling with mature testes, wipe the area around the cloaca with a clean towel, gently press the belly of the fish from the head to the cloaca to collect the semen, and mix the semen of all 10 fish.

[0113] The semen of Xianghua ling and the preservation solution were mixed in a volume ratio of 1:1 and added to 2 mL cryotubes. 1 mL of liquid was added to each tube, and the tubes were balanced on ice for 15 min, then placed in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 min, and finally completely immersed in liquid nitrogen for storage.

[0114] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 80 seconds until the semen mixture is completely melted.

[0115] Sperm motility test: After the semen is thawed, use a pipette to draw 1 μL of diluted frozen semen and add sperm activation solution. The formula and concentration of sperm activation solution are: 45mM NaCl, 5mM KCl, 30mM Tris, the solvent is ultrapure water, pH: 8.0±0.2. Immediately after activation, use CASA to detect sperm motility and record sperm life span, and repeat each sample three times.

[0116] like Figure 1As shown, in Comparative Examples 1-3, dimethyl sulfoxide, ethylene glycol, and methanol were used as antifreeze agents to freeze semen. After recovery and activation, the sperm survival rates were 22.42±11.27%, 30.33±9.68%, and 16.48±6.89%, respectively. The results of one-way ANOVA and Tukey HSD showed that when ethylene glycol was used as an antifreeze agent, the sperm survival rate after thawing was significantly higher than that of dimethyl sulfoxide and methanol (p=0.039<0.05). Among the surviving sperm, methanol as an antifreeze agent maintained a higher sperm motility (p=0.005<0.05). Among the viable sperm, there was no significant difference in the proportion of sperm forward movement preserved by the three antifreeze agents (p=0.078>0.05). In terms of rapid movement time, the protective effect of ethylene glycol as an antifreeze agent was significantly higher than that of other antifreeze agent groups (p=0.013<0.05). Comprehensively speaking, ethylene glycol as an antifreeze agent is better than dimethyl sulfoxide and methanol in protecting the sperm of Xianghua ling.

[0117] Test Example 4

[0118] (1) A method for preparing a sperm preservation solution of Xianghua ling, comprising the following steps:

[0119] (1) Dissolve trehalose, NaCl, KCl, Na2HPO4, MgCl2, CaCl2 and tris(hydroxymethyl)aminomethane) in ultrapure water at room temperature to obtain a basic dilution solution, which is stored at 4°C for later use;

[0120] (2) adding bovine serum albumin to the basic diluent to obtain a frozen diluent, wherein the concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na2HPO4 1 mmol / L, MgCl2 1 mmol / L, CaCl2 1 mmol / L, tris(hydroxymethyl)aminomethane 20 mmol / L, and bovine serum albumin accounts for 0.5% of the volume of the frozen diluent, and the solution is stored away from light for future use;

[0121] (3) Ethylene glycol is mixed with the freezing diluent to obtain a Xianghua ling sperm preservation solution, wherein the antifreeze agent accounts for 8% of the volume of the preservation solution and the remainder is the freezing diluent.

[0122] (ii) A method for preserving semen using Xianghua ling sperm preservation fluid comprises the following steps:

[0123] Select 10 male Xianghua ling with mature testes, wipe the area around the cloaca with a clean towel, gently press the belly of the fish from the head to the cloaca to collect the semen, and mix the semen of all 10 fish.

[0124] The semen of Xianghua ling and the preservation solution were mixed in a volume ratio of 1:1 and added to 2 mL cryotubes. 1 mL of liquid was added to each tube, and the tubes were balanced on ice for 15 min, then placed in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 min, and finally completely immersed in liquid nitrogen for storage.

[0125] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 80 seconds until the semen mixture is completely melted.

[0126] Sperm motility test: After the semen is thawed, use a pipette to draw 1 μL of diluted frozen semen and add sperm activation solution. The formula and concentration of sperm activation solution are: 45mM NaCl, 5mM KCl, 30mM Tris, the solvent is ultrapure water, pH: 8.0±0.2. Immediately after activation, use CASA to detect sperm motility and record sperm life span, and repeat each sample three times.

[0127] Test Example 5

[0128] (1) A method for preparing a sperm preservation solution of Xianghua ling, comprising the following steps:

[0129] (1) Dissolve trehalose, NaCl, KCl, Na2HPO4, MgCl2, CaCl2 and tris(hydroxymethyl)aminomethane) in ultrapure water at room temperature to obtain a basic dilution solution, which is stored at 4°C for later use;

[0130] (2) adding bovine serum albumin to the basic diluent to obtain a frozen diluent, wherein the concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na2HPO4 1 mmol / L, MgCl2 1 mmol / L, CaCl2 1 mmol / L, tris(hydroxymethyl)aminomethane 20 mmol / L, and bovine serum albumin accounts for 0.5% of the volume of the frozen diluent, and the solution is stored away from light for future use;

[0131] (3) Ethylene glycol is mixed with the freezing diluent to obtain a Xianghua ling sperm preservation solution, wherein the antifreeze agent accounts for 10% of the volume of the preservation solution and the remainder is the freezing diluent.

[0132] (ii) A method for preserving semen using Xianghua ling sperm preservation fluid comprises the following steps:

[0133] Select 10 male Xianghua ling with mature testes, wipe the area around the cloaca with a clean towel, gently press the belly of the fish from the head to the cloaca to collect the semen, and mix the semen of all 10 fish.

[0134] The semen of Xianghua ling and the preservation solution were mixed in a volume ratio of 1:1 and added to 2 mL cryotubes. 1 mL of liquid was added to each tube, and the tubes were balanced on ice for 15 min, then placed in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 min, and finally completely immersed in liquid nitrogen for storage.

[0135] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 80 seconds until the semen mixture is completely melted.

[0136] Sperm motility test: After the semen is thawed, use a pipette to draw 1 μL of diluted frozen semen and add sperm activation solution. The formula and concentration of sperm activation solution are: 45mM NaCl, 5mM KCl, 30mM Tris, the solvent is ultrapure water, pH: 8.0±0.2. Immediately after activation, use CASA to detect sperm motility and record sperm life span, and repeat each sample three times.

[0137] Test Example 6

[0138] (1) A method for preparing a sperm preservation solution of Xianghua ling, comprising the following steps:

[0139] (1) Dissolve trehalose, NaCl, KCl, Na2HPO4, MgCl2, CaCl2 and tris(hydroxymethyl)aminomethane) in ultrapure water at room temperature to obtain a basic dilution solution, which is stored at 4°C for later use;

[0140] (2) adding bovine serum albumin to the basic diluent to obtain a frozen diluent, wherein the concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na2HPO4 1 mmol / L, MgCl2 1 mmol / L, CaCl2 1 mmol / L, tris(hydroxymethyl)aminomethane 20 mmol / L, and bovine serum albumin accounts for 0.5% of the volume of the frozen diluent, and the solution is stored away from light for future use;

[0141] (3) Ethylene glycol is mixed with the freezing diluent to obtain a Xianghua ling sperm preservation solution, wherein the antifreeze agent accounts for 12% of the volume of the preservation solution and the remainder is the freezing diluent.

[0142] (ii) A method for preserving semen using Xianghua ling sperm preservation fluid comprises the following steps:

[0143] Select 10 male Xianghua ling with mature testes, wipe the area around the cloaca with a clean towel, gently press the belly of the fish from the head to the cloaca to collect the semen, and mix the semen of all 10 fish.

[0144] The semen of Xianghua ling and the preservation solution were mixed in a volume ratio of 1:1 and added to 2 mL cryotubes. 1 mL of liquid was added to each tube, and the tubes were balanced on ice for 15 min, then placed in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 min, and finally completely immersed in liquid nitrogen for storage.

[0145] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 80 seconds until the semen mixture is completely melted.

[0146] Sperm motility test: After the semen is thawed, use a pipette to draw 1 μL of diluted frozen semen and add sperm activation solution. The formula and concentration of sperm activation solution are: 45mM NaCl, 5mM KCl, 30mM Tris, the solvent is ultrapure water, pH: 8.0±0.2. Immediately after activation, use CASA to detect sperm motility and record sperm life span, and repeat each sample three times.

[0147] In test examples 4-6, the results of one-way ANOVA and Tukey post hoc test showed that the sperm motility after fresh semen activation was 88.26±1.73%, and the proportion of sperm motility in active sperm was 96.33±1.22%. After semen resuscitation and activation, when the antifreeze concentration was 8%, 10% or 12%, the sperm survival rate was 36.31±6.07%, 31.57±7.61% and 29.83±5.86%, respectively, and the sperm survival rate protected by 12% antifreeze was significantly lower than that of the other two groups (p=0.035<0.05). When the antifreeze is 8%, the sperm motility is 68.33±7.03%; when the antifreeze is 10%, the sperm motility is 61.82±4.63%; when the antifreeze is 12%, the sperm motility is 63.01±5.39%, and there is no significant difference between the three concentrations (p=0.178>0.05). The average linear motion velocity (VSL) of sperm between the 12% group and the 8% group showed significant differences (p=0.014<0.05). Considering multiple factors, 8% is the best. The technical solutions of Experimental Examples 1 and 5 are the same, but different batches of Xianghua ling semen are used, so the measurement results obtained are slightly different.

[0148] Sperm survival rate = (number of surviving sperm / total number of sperm) × 100%

[0149] Sperm motility = (number of motile sperm / number of viable sperm) × 100%

[0150] Statistical analysis:

[0151] One-way ANOVA: used to evaluate the effects of individual factors and combined factors on sperm motility parameters.

[0152] Two-way ANOVA: used to evaluate the effect of two factors on sperm motility parameters.

[0153] The synergistic effect index (CI) is calculated as follows (taking antioxidants as an example):

[0154] CI = (Effective dose of GSH combined with ALA) / (Effective dose of GSH alone) + (Effective dose of ALA alone)

[0155] If CI < 1, it indicates a synergistic effect.

[0156] Multiple comparison test: Tukey's test, used to further compare the differences between experimental groups.

[0157] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Xianghua ling sperm cryopreservation solution, characterized in that: It comprises a freezing diluent, an antifreeze agent and an antioxidant, wherein the antifreeze agent accounts for 8% of the volume of the ultra-low temperature preservation solution, the antioxidant comprises reduced glutathione and α-lipoic acid, and in the ultra-low temperature preservation solution, the concentration of reduced glutathione is 0.1 mmol / L, the concentration of α-lipoic acid is 0.5 mmol / L, and the remainder is the freezing diluent; The concentration of each component in the freezing diluent is: trehalose 150mmol / L, NaCl 75mmol / L, KCl 30mmol / L, Na2HPO4 1mmol / L, MgCl2 1mmol / L, CaCl2 1mmol / L, tris(hydroxymethyl)aminomethane 20mmol / L, bovine serum albumin accounts for 0.5% of the volume of the freezing diluent, and the solvent is ultrapure water; The antifreeze agent is ethylene glycol.

2. A method for preparing the Xianghua ling sperm cryopreservation solution according to claim 1, characterized in that: The steps include: (1) dissolving the trehalose, NaCl, KCl, Na2HPO4, MgCl2, CaCl2 and tris(hydroxymethyl)aminomethane) in ultrapure water at room temperature to obtain a basic dilution solution, which is stored at 4°C for later use; (2) adding bovine serum albumin to the basic diluent to obtain a freezing diluent, adding an antioxidant to the freezing diluent and mixing to obtain a sperm freezing preservation solution, and storing it in the dark for later use; (3) Mixing the antifreeze agent with the sperm cryopreservation solution to obtain the Xianghua ling sperm ultra-low temperature preservation solution.

3. A method for preserving semen using the Xianghua ling sperm cryopreservation solution according to claim 1, characterized in that: The steps include: The Xianghua ling semen and the ultra-low temperature preservation solution are mixed and added to the freezing carrier, balanced on ice for 10-15 minutes, then placed in liquid nitrogen at a distance of 3-16 cm above the liquid nitrogen surface for 10 minutes, and finally completely immersed in liquid nitrogen for storage.

4. The method for preserving semen using a Xianghua ling sperm cryopreservation solution according to claim 3, characterized in that: The volume ratio of Xianghua ling semen to the ultra-low temperature preservation solution is 1:

1.

5. The method for preserving semen using a Xianghua ling sperm cryopreservation solution according to claim 3, characterized in that: The freezing carrier is a 2 mL cryovial or a 0.5 mL straw.

6. The method for preserving semen using a Xianghua ling sperm cryopreservation solution according to claim 5, characterized in that: The freezing carrier is a 2 mL cryovial.

7. Use of the Xianghua ling sperm ultra-low temperature preservation solution according to claim 1 in preserving Xianghua ling semen or in in vitro artificial insemination of Xianghua ling.

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