Ultralow-temperature preservation solution for Sinilabeo malacatus sperms as well as preparation method, preservation method and application of ultralow-temperature preservation solution

Abstract

The invention discloses a semilabeo nolabeo sperm ultralow-temperature preservation solution as well as a preparation method, a preservation method and application thereof, and belongs to the technical field of semilabeo nolabeo artificial breeding. The ultralow-temperature preservation liquid comprises a freezing diluent, an antifreeze agent 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; and the antifreeze agent is dimethyl sulfoxide. 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 on efficient preservation of the seminal fluid of the semilabeo nolabeo, the unfrozen seminal fluid can meet artificial improved variety breeding, and the method has important significance on establishment and protection of an excellent seminal fluid bank of the semilabeo nolabeo.

Description

Technical Field

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

[0002] Cirrhinus mrigala, commonly known as mrigala, belongs to the Cyprinidae, Abaloneidae, Cirrhinus. Recently, mrigala is mainly used as live bait for carnivorous species such as mandarin fish and Channa chub, and has important economic value. Mrigala has a mixed diet and has the advantages of high population yield, short breeding cycle, and tolerance to dense breeding. However, mrigala has poor adaptability to low temperatures. It stops feeding when the water temperature is below 15℃, and suffers from cold shock when the water temperature is below 10℃. It will die after 1-2 days under such low temperature conditions. Therefore, it is difficult to promote large-scale breeding in central and western my country. In recent years, the winter water temperature in Guangdong and Guangxi has been continuously low, and a large number of farmed mrigala have frozen to death, threatening the reproduction and industrial application of mrigala.

[0003] With the development of the aquaculture industry of the Merigala ling, 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 reduce the breeding costs of enterprises. This is of great significance to the large-scale production of juveniles, selective breeding and related scientific research. Ultra-low temperature cryopreservation technology of semen has become one of the main tools for protecting endangered or important commercial 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 conducive to the selection and breeding of new varieties, and can achieve hybridization with distant species with differences in reproduction time and geographical distribution, and enhance 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 sperm from Merrigala 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 sperm ultra-low temperature preservation solution for Merigala ling 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 Merrigala 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 cryopreservation solution for sperm of Merrigala dace, comprising a freezing diluent, an antifreeze agent and an antioxidant, wherein the antifreeze agent accounts for 8% of the volume of the cryopreservation solution, the antioxidant comprises reduced glutathione and α-lipoic acid, and in the cryopreservation 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 concentrations of the components in the freezing diluent are: trehalose 150mmol / L, NaCl 75mmol / L, KCl 30mmol / L, Na 2 HPO 4 1mmol / L, MgCl 2 1mmol / L, CaCl 2 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;

[0011] The antifreeze agent is dimethyl sulfoxide.

[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] Dimethyl sulfoxide, methanol and ethylene glycol are osmotic antifreeze agents that 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 agent trehalose and the osmotic antifreeze agent 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 the semen of the Mailui Gala ling. The thawed semen can meet the needs of artificial breeding, which is of great significance to the establishment and protection of the excellent sperm bank of the Mailui Gala ling.

[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 ratio after recovery are 69.34±6.21% and 88.81±6.01%, 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 77.93±8.84%, and 82.45±3.27% of sperm maintain mitochondrial function. The sperm fertilization rate after recovery is 78.52±2.99%, which still has a high fertilization ability, which is of great significance to the protection and genetic improvement of the germplasm resources of Mailigala.

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

[0019] (1) The above trehalose, NaCl, KCl, Na 2 HPO 4 MgCl 2 , CaCl 2 and tris(hydroxymethyl)aminomethane) were dissolved in ultrapure water at room temperature to obtain a basic dilution solution, which was 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 Merrigala sperm ultra-low temperature preservation solution.

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

[0023] The semen of Merigala dace is mixed with the ultra-low temperature preservation solution and added to a freezing carrier, balanced on ice for 10-15 minutes, and then directly and completely immersed in liquid nitrogen for storage, or placed in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for balancing for 10 minutes or 20 minutes, and finally completely immersed in liquid nitrogen for storage.

[0024] Beneficial effects of the present invention: The present invention optimizes the equilibrium time, 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 the semen of the Merrigala ling and 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 Merigala semen, and optimizes the freezing carrier and its corresponding gas-phase liquid nitrogen equilibrium time. Combining the above optimization measures, 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 above-mentioned Mai Rui Gala sperm ultra-low temperature preservation solution in preserving Mai Rui Gala sperm or in in vitro artificial insemination of Mai Rui Gala.

[0030] The inventive mechanism of the present invention is that during the cryopreservation process, sperm cells are exposed to low temperatures and are damaged by oxidative stress during the freezing and thawing process. Oxidative stress is mainly caused by reactive oxygen species (ROS), which can damage the lipid membrane, protein and DNA of sperm cells, thereby reducing the vitality, motility and fertilization ability of sperm. Adding antioxidants is a key measure to improve the cryopreservation effect of fish sperm, reducing the oxidative stress damage caused by freezing and thawing, protecting the integrity of cell membranes and organelles, and improving sperm survival rate and fertilization success rate.

[0031] 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 factors such as the cooling rate, storage space and sample management of different freezing carriers according to the needs of sperm samples. Appropriate liquid nitrogen surface equilibration time is an important factor in ensuring the freezing effect. Too long or too short equilibration time will lead to improper cooling, thereby affecting the survival rate and functionality of sperm. The optimal equilibration time allows the sample to gradually adapt to the low temperature environment and reduce the damage to sperm caused by ice crystals and osmotic pressure changes. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Sperm motility after thawing under the protection of different types of antifreeze agents.

[0033] Figure 2 Sperm survival rate, sperm motility, proportion of forward-moving sperm and rapid motility time after resuscitation under the protection of different concentrations of dimethyl sulfoxide.

[0034] Figure 3 Effects of different antioxidants, cryocarriers and gas-phase liquid nitrogen equilibrium time on sperm survival rate and sperm motility.

[0035] Figure 4 Interactive effects of different antioxidants on sperm motility.

[0036] Figure 5 Sperm DNA integrity under different combinations of antioxidants, cryocarriers and vapor-phase liquid nitrogen equilibration time.

[0037] Figure 6 Sperm mitochondrial membrane potential under different combinations of antioxidants, freezing carriers and gas-phase liquid nitrogen equilibration time. DETAILED DESCRIPTION

[0038] 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.

[0039] Example 1

[0040] (1) A method for preparing a sperm cryopreservation solution of Merrigala ling, comprising the following steps:

[0041] (1) Trehalose, NaCl, KCl, Na 2 HPO 4 MgCl 2 , CaCl 2 and tris(hydroxymethyl)aminomethane) were dissolved in ultrapure water at room temperature to obtain a basic dilution solution, which was stored at 4°C for later use;

[0042] (2) Add bovine serum albumin to the basic diluent to obtain a frozen diluent. The concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na 2 HPO 4 1mmol / L, MgCl 2 1mmol / L, CaCl 2 1mmol / L, tris(hydroxymethyl)aminomethane 20mmol / L, bovine serum albumin accounts for 0.5% of the volume of the freezing diluent, and reduced glutathione and α-lipoic acid are added to the freezing diluent to obtain sperm cryopreservation solution, which is stored away from light for later use;

[0043] (3) Dimethyl sulfoxide is mixed with sperm cryopreservation solution to obtain Merigala sperm cryopreservation solution, wherein dimethyl sulfoxide accounts for 8% of the volume of the cryopreservation solution, the concentration of reduced glutathione in the cryopreservation solution is 0.1 mmol / L, the concentration of α-lipoic acid is 0.5 mmol / L, and the remainder is freezing diluent.

[0044] (ii) A method for preserving semen using a sperm ultra-low temperature preservation solution of a dace, comprising the following steps:

[0045] Mix the semen of Merigala dace and cryopreservation solution in a volume ratio of 1:1 and add to 2 mL cryotubes, add 1 mL of liquid to each tube, balance on ice for 15 minutes, then place in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 20 minutes, and finally completely immerse 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 10 seconds until the semen mixture is completely melted.

[0047] Example 2

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

[0049] (1) Trehalose, NaCl, KCl, Na 2 HPO 4 MgCl 2 , CaCl 2 and tris(hydroxymethyl)aminomethane) were dissolved in ultrapure water at room temperature to obtain a basic dilution solution, which was stored at 4°C for later use;

[0050] (2) Add bovine serum albumin to the basic diluent to obtain a frozen diluent. The concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na 2 HPO 4 1mmol / L, MgCl 2 1mmol / L, CaCl 2 1mmol / L, tris(hydroxymethyl)aminomethane 20mmol / L, bovine serum albumin accounts for 0.5% of the volume of the freezing diluent, and reduced glutathione and α-lipoic acid are added to the freezing diluent to obtain sperm cryopreservation solution, which is stored away from light for later use;

[0051] (3) Dimethyl sulfoxide is mixed with sperm cryopreservation solution to obtain Merigala sperm cryopreservation solution, wherein dimethyl sulfoxide accounts for 8% of the volume of the cryopreservation solution, the concentration of reduced glutathione in the cryopreservation solution 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 sperm ultra-low temperature preservation solution of a dace, comprising the following steps:

[0053] Mix the semen of Merigala dace and cryopreservation solution in a volume ratio of 1:1 and add to 2 mL cryotubes, add 1 mL of liquid to each tube, balance on ice for 15 minutes, then place in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 minutes, and finally completely immerse in liquid nitrogen for storage.

[0054] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 10 seconds until the semen mixture is completely melted.

[0055] Example 3

[0056] (1) A method for preparing a sperm cryopreservation solution of Merrigala ling, comprising the following steps:

[0057] (1) Trehalose, NaCl, KCl, Na 2 HPO 4 MgCl 2 , CaCl 2 and tris(hydroxymethyl)aminomethane) were dissolved in ultrapure water at room temperature to obtain a basic dilution solution, which was stored at 4°C for later use;

[0058] (2) Add bovine serum albumin to the basic diluent to obtain a frozen diluent. The concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na 2 HPO 4 1mmol / L, MgCl 2 1mmol / L, CaCl 2 1mmol / L, tris(hydroxymethyl)aminomethane 20mmol / L, bovine serum albumin accounts for 0.5% of the volume of the freezing diluent, and reduced glutathione and α-lipoic acid are added to the freezing diluent to obtain sperm cryopreservation solution, which is stored away from light for later use;

[0059] (3) Dimethyl sulfoxide is mixed with sperm cryopreservation solution to obtain Merigala sperm cryopreservation solution, wherein dimethyl sulfoxide accounts for 8% of the volume of the cryopreservation solution, the concentration of reduced glutathione in the cryopreservation solution is 0.1 mmol / L, the concentration of α-lipoic acid is 0.5 mmol / L, and the remainder is freezing diluent.

[0060] (ii) A method for preserving semen using a sperm ultra-low temperature preservation solution of a dace, comprising the following steps:

[0061] Mix the semen of the Merrigala dace and the cryopreservation solution in a volume ratio of 1:1 and add them to 0.5 mL wheat tubes. Add 0.5 mL of liquid to each tube, balance on ice for 15 minutes, then place in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 20 minutes, and finally completely immerse in liquid nitrogen for storage.

[0062] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 10 seconds until the semen mixture is completely melted.

[0063] Example 4

[0064] (1) A method for preparing a sperm cryopreservation solution of Merrigala ling, comprising the following steps:

[0065] (1) Trehalose, NaCl, KCl, Na 2 HPO 4MgCl 2 , CaCl 2 and tris(hydroxymethyl)aminomethane) were dissolved in ultrapure water at room temperature to obtain a basic dilution solution, which was stored at 4°C for later use;

[0066] (2) Add bovine serum albumin to the basic diluent to obtain a frozen diluent. The concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na 2 HPO 4 1mmol / L, MgCl 2 1mmol / L, CaCl 2 1mmol / L, tris(hydroxymethyl)aminomethane 20mmol / L, bovine serum albumin accounts for 0.5% of the volume of the freezing diluent, and reduced glutathione and α-lipoic acid are added to the freezing diluent to obtain sperm cryopreservation solution, which is stored away from light for later use;

[0067] (3) Dimethyl sulfoxide is mixed with sperm cryopreservation solution to obtain Merigala sperm cryopreservation solution, wherein dimethyl sulfoxide accounts for 8% of the volume of the cryopreservation solution, the concentration of reduced glutathione in the cryopreservation solution is 0.1 mmol / L, the concentration of α-lipoic acid is 0.5 mmol / L, and the remainder is freezing diluent.

[0068] (ii) A method for preserving semen using a sperm ultra-low temperature preservation solution of a dace, comprising the following steps:

[0069] Mix the semen of the Merrigala dace and the cryopreservation solution in a volume ratio of 1:1 and add them to 0.5 mL wheat tubes. Add 0.5 mL of liquid to each tube, balance on ice for 15 minutes, then place in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 minutes, and finally completely immerse in liquid nitrogen for storage.

[0070] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 10 seconds until the semen mixture is completely melted.

[0071] Effect experiment:

[0072] The other test examples differ from Example 1 in that they use different freezing carriers, gas-phase liquid nitrogen equilibration time and antioxidants, and the other steps and parameters are the same as those of Example 1.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] After thawing, sperm survival rate and sperm motility under different carriers, equilibrium time and antioxidant protection Figure 3 . Statistical analysis showed that freezing carrier, equilibration time and antioxidants had significant effects on cell viability and motility, respectively (p<0.001). Compared with the use of GSH (reduced glutathione) or ALA (α-lipoic acid) alone, the combination of antioxidants GSH and ALA (GSH+ALA) significantly increased the sperm survival rate of Merrigala dace (Table 1), indicating that the combined use of these two antioxidants has a synergistic effect and can improve sperm survival rate ( Figure 4 ). In terms of sperm motility, there was a synergistic effect, especially when GSH+ALA was compared with ALA alone, and GSH+ALA showed significantly higher sperm motility (p<0.05, Table 2). Different equilibration times and freezing carrier combinations also had a significant effect on sperm survival and sperm motility. In terms of sperm survival and sperm motility, there was a significant interaction of freezing carrier × equilibration time × antioxidant (p<0.05), indicating that the effect of antioxidants is not isolated, but is affected by the carrier and treatment time. Specific antioxidants as well as carrier type and equilibration time will interact 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, which may work through different mechanisms and enhance each other when used in combination. Fertilization experiments show that the fertilization rate of revived sperm can reach 79.44±3.98% when 8% dimethyl sulfoxide is used as an antifreeze, GSH+ALA is used, and the compounding scheme of balancing the cryopreservation tube above liquid nitrogen for 20 minutes. 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 schemes.

[0077] Table 1 Sperm survival rate under different carriers, equilibrium time and antioxidant protection (%)

[0078]

[0079]

[0080] GSH: reduced glutathione; ALA: alpha lipoic acid. * indicates significant differences between different equilibrium times (p<0.05); a, b, c indicate significant differences between different antioxidants (p<0.05).

[0081] Table 2 Sperm motility under different carriers, equilibrium time and antioxidant protection (%)

[0082]

[0083] GSH: reduced glutathione; ALA: alpha lipoic acid. * indicates significant differences between different equilibrium times (p<0.05); a, b, c indicate significant differences between different antioxidants (p<0.05).

[0084] Test Example 1

[0085] (1) A method for preparing a sperm preservation solution of a dace, comprising the following steps:

[0086] (1) Trehalose, NaCl, KCl, Na 2 HPO 4 MgCl 2 , CaCl 2 and tris(hydroxymethyl)aminomethane) were dissolved in ultrapure water at room temperature to obtain a basic dilution solution, which was stored at 4°C for later use;

[0087] (2) Add bovine serum albumin to the basic diluent to obtain a frozen diluent. The concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na 2 HPO 4 1mmol / L, MgCl 2 1mmol / L, CaCl 2 1mmol / L, tris(hydroxymethyl)aminomethane 20mmol / L, bovine serum albumin 0.5% of the volume of the frozen diluent, stored in the dark for later use;

[0088] (3) Dimethyl sulfoxide is mixed with the freezing diluent to obtain a sperm preservation solution for Merigala dace, wherein the antifreeze agent accounts for 10% of the volume of the preservation solution.

[0089] (ii) A method for preserving semen using the Merrigala sperm preservation solution comprises the following steps:

[0090] Select 10 male Merrigala dace 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.

[0091] Mix the semen of Merigala dace and the preservation solution in a volume ratio of 1:1 and add them to 2 mL cryotubes. Add 1 mL of liquid to each tube, balance on ice for 15 minutes, then place in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 minutes, and finally completely immerse in liquid nitrogen for storage.

[0092] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 10 seconds until the semen mixture is completely melted.

[0093] 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.

[0094] Test Example 2

[0095] (1) A method for preparing a sperm preservation solution of a dace, comprising the following steps:

[0096] (1) Trehalose, NaCl, KCl, Na 2 HPO 4 MgCl 2 , CaCl 2and tris(hydroxymethyl)aminomethane) were dissolved in ultrapure water at room temperature to obtain a basic dilution solution, which was stored at 4°C for later use;

[0097] (2) Add bovine serum albumin to the basic diluent to obtain a frozen diluent. The concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na 2 HPO 4 1mmol / L, MgCl 2 1mmol / L, CaCl 2 1mmol / L, tris(hydroxymethyl)aminomethane 20mmol / L, bovine serum albumin 0.5% of the volume of the frozen diluent, stored in the dark for later use;

[0098] (3) Methanol is mixed with the freezing diluent to obtain sperm preservation solution of Merigala dace, wherein the antifreeze agent accounts for 10% of the volume of the preservation solution.

[0099] (ii) A method for preserving semen using the Merrigala sperm preservation solution comprises the following steps:

[0100] Select 10 male Merrigala dace 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.

[0101] Mix the semen of Merigala dace and the preservation solution in a volume ratio of 1:1 and add them to 2 mL cryotubes. Add 1 mL of liquid to each tube, balance on ice for 15 minutes, then place in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 minutes, and finally completely immerse in liquid nitrogen for storage.

[0102] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 10 seconds until the semen mixture is completely melted.

[0103] 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.

[0104] Test Example 3

[0105] (1) A method for preparing a sperm preservation solution of a dace, comprising the following steps:

[0106] (1) Trehalose, NaCl, KCl, Na 2 HPO 4 MgCl 2, CaCl 2 and tris(hydroxymethyl)aminomethane) were dissolved in ultrapure water at room temperature to obtain a basic dilution solution, which was stored at 4°C for later use;

[0107] (2) Add bovine serum albumin to the basic diluent to obtain a frozen diluent. The concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na 2 HPO 4 1mmol / L, MgCl 2 1mmol / L, CaCl 2 1mmol / L, tris(hydroxymethyl)aminomethane 20mmol / L, bovine serum albumin 0.5% of the volume of the frozen diluent, stored in the dark for later use;

[0108] (3) Ethylene glycol is mixed with the freezing diluent to obtain a sperm preservation solution for Merigala dace, wherein the antifreeze agent accounts for 10% of the volume of the preservation solution.

[0109] (ii) A method for preserving semen using the Merrigala sperm preservation solution comprises the following steps:

[0110] Select 10 male Merrigala dace 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.

[0111] Mix the semen of Merigala dace and the preservation solution in a volume ratio of 1:1 and add them to 2 mL cryotubes. Add 1 mL of liquid to each tube, balance on ice for 15 minutes, then place in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 minutes, and finally completely immerse in liquid nitrogen for storage.

[0112] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 10 seconds until the semen mixture is completely melted.

[0113] 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.

[0114] like Figure 1As shown, in Experimental Examples 1-3, dimethyl sulfoxide, ethylene glycol, and methanol were used as cryoprotectants to freeze semen. After recovery and activation, the sperm survival rates were 36.90% ± 7.59%, 29.71% ± 13.27%, and 17.48% ± 7.36%, respectively. The results of the Kruskal-Wallis H test and Dunn's multiple comparisons showed that when dimethyl sulfoxide was used as an antifreeze agent, the sperm survival rate and forward motility ratio were the highest, indicating that the use of DMSO as a cryoprotectant can better maintain the survival of sperm and that sperm is more likely to be successfully fertilized. MeOH performed best in terms of motility, but its survival rate was lower. EG performed best in terms of rapid forward motility, but was relatively weak in terms of survival rate and forward motility. Overall, dimethyl sulfoxide is the best choice among the three cryoprotectants, especially in maintaining sperm survival rate and forward motility rate.

[0115] Test Example 4

[0116] (1) A method for preparing a sperm preservation solution of a dace, comprising the following steps:

[0117] (1) Trehalose, NaCl, KCl, Na 2 HPO 4 MgCl 2 , CaCl 2 and tris(hydroxymethyl)aminomethane) were dissolved in ultrapure water at room temperature to obtain a basic dilution solution, which was stored at 4°C for later use;

[0118] (2) Add bovine serum albumin to the basic diluent to obtain a frozen diluent. The concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na 2 HPO 4 1mmol / L, MgCl 2 1mmol / L, CaCl 2 1mmol / L, tris(hydroxymethyl)aminomethane 20mmol / L, bovine serum albumin 0.5% of the volume of the frozen diluent, stored in the dark for later use;

[0119] (3) Dimethyl sulfoxide is mixed with sperm freezing diluent to obtain sperm preservation solution of Merigala ling, wherein the antifreeze agent accounts for 8% of the volume of the preservation solution.

[0120] (ii) A method for preserving semen using the Merrigala sperm preservation solution comprises the following steps:

[0121] Select 10 male Merrigala dace 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.

[0122] Mix the semen of Merigala dace and the preservation solution in a volume ratio of 1:1 and add them to 2 mL cryotubes. Add 1 mL of liquid to each tube, balance on ice for 15 minutes, then place in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 minutes, and finally completely immerse in liquid nitrogen for storage.

[0123] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 10 seconds until the semen mixture is completely melted.

[0124] 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.

[0125] Test Example 5

[0126] (1) A method for preparing a sperm preservation solution of a dace, comprising the following steps:

[0127] (1) Trehalose, NaCl, KCl, Na 2 HPO 4 MgCl 2 , CaCl 2 and tris(hydroxymethyl)aminomethane) were dissolved in ultrapure water at room temperature to obtain a basic dilution solution, which was stored at 4°C for later use;

[0128] (2) Add bovine serum albumin to the basic diluent to obtain a frozen diluent. The concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na 2 HPO 4 1mmol / L, MgCl 2 1mmol / L, CaCl 2 1mmol / L, tris(hydroxymethyl)aminomethane 20mmol / L, bovine serum albumin 0.5% of the volume of the frozen diluent, stored in the dark for later use;

[0129] (3) Dimethyl sulfoxide is mixed with the freezing diluent to obtain a sperm preservation solution for Merigala dace, wherein the antifreeze agent accounts for 10% of the volume of the preservation solution.

[0130] (ii) A method for preserving semen using the Merrigala sperm preservation solution comprises the following steps:

[0131] Select 10 male Merrigala dace 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.

[0132] Mix the semen of Merigala dace and the preservation solution in a volume ratio of 1:1 and add them to 2 mL cryotubes. Add 1 mL of liquid to each tube, balance on ice for 15 minutes, then place in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 minutes, and finally completely immerse in liquid nitrogen for storage.

[0133] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 10 seconds until the semen mixture is completely melted.

[0134] 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.

[0135] Test Example 6

[0136] (1) A method for preparing a sperm preservation solution of Merrigala ling, comprising the following steps:

[0137] (1) Trehalose, NaCl, KCl, Na 2 HPO 4 MgCl 2 , CaCl 2 and tris(hydroxymethyl)aminomethane) were dissolved in ultrapure water at room temperature to obtain a basic dilution solution, which was stored at 4°C for later use;

[0138] (2) Add bovine serum albumin to the basic diluent to obtain a frozen diluent. The concentrations of the components in the frozen diluent are: trehalose 150 mmol / L, NaCl 75 mmol / L, KCl 30 mmol / L, Na 2 HPO 4 1mmol / L, MgCl 2 1mmol / L, CaCl 2 1mmol / L, tris(hydroxymethyl)aminomethane 20mmol / L, bovine serum albumin 0.5% of the volume of the frozen diluent, stored in the dark for later use;

[0139] (3) Dimethyl sulfoxide is mixed with the freezing diluent to obtain a sperm preservation solution for Merigala dace, wherein the antifreeze agent accounts for 12% of the volume of the preservation solution.

[0140] (ii) A method for preserving semen using the Merrigala sperm preservation solution comprises the following steps:

[0141] Select 10 male Merrigala dace 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.

[0142] Mix the semen of Merigala dace and the preservation solution in a volume ratio of 1:1 and add them to 2 mL cryotubes. Add 1 mL of liquid to each tube, balance on ice for 15 minutes, then place in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for 10 minutes, and finally completely immerse in liquid nitrogen for storage.

[0143] (III) Sperm thawing: Take out the cryotube from liquid nitrogen and shake it continuously in a 37°C water bath for 10 seconds until the semen mixture is completely melted.

[0144] 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.

[0145] Test examples 4-6, the results show that after semen resuscitation activation, when the antifreeze concentration is 8%, 10% or 12%, the sperm survival rate is 39.63% ± 4.30%, 40.33% ± 3.87% and 37.62% ± 2.62%, respectively. The Kruskal-Wallis H test shows that there is no significant difference in the sperm survival rate under the three concentrations, but 8% and 10% help to improve the sperm motility rate, and 12% concentration has an adverse effect on the motility rate. Overall, the 8% concentration of cryoprotectant performs best in improving sperm motility, rapid forward movement and linear speed.

[0146] Sperm survival rate = (number of surviving sperm / total number of sperm) × 100%

[0147] Sperm motility = (number of motile sperm / number of viable sperm) × 100%

[0148] Statistical analysis:

[0149] The nonparametric Kruskal-Wallis H test was used to compare the differences between multiple groups. If the Kruskal-Wallis test results showed a significant difference (p < 0.05), Dunn's multiple comparison test was further performed, and the Bonferroni correction was used to determine which specific two groups had significant differences.

[0150] The synergistic effect index (CI) is calculated as follows (taking antioxidants as an example):

[0151] CI = (Effective dose of GSH combined with ALA) / (Effective dose of GSH alone) + (Effective dose of ALA alone)

[0152] If CI < 1, it indicates a synergistic effect.

[0153] Multiple comparison test: Tukey's test, used to further compare the differences between experimental groups.

[0154] 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 cryopreservation solution for sperm of Melugala ling, 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 dimethyl sulfoxide.

2. A method for preparing the Merrigala 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 sperm ultra-low temperature preservation solution of the Merrigala ling sperm.

3. A method for preserving semen using the Merrigala sperm cryopreservation solution according to claim 1, characterized in that: The steps include: The semen of Merigala dace is mixed with the ultra-low temperature preservation solution and added to a freezing carrier, balanced on ice for 10-15 minutes, and then directly and completely immersed in liquid nitrogen for storage, or placed in liquid nitrogen at a distance of 16 cm above the liquid nitrogen surface for balancing for 10 minutes or 20 minutes, and finally completely immersed in liquid nitrogen for storage.

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

1.

5. The method for preserving semen using the Merrigala 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 the Merrigala sperm cryopreservation solution according to claim 3, characterized in that: The freezing carrier is a 2 mL cryovial.

7. Use of the Mai Rui Gala sperm ultra-low temperature preservation solution according to claim 1 in preserving Mai Rui Gala sperm or in in vitro artificial insemination of Mai Rui Gala.

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